2nd International Factor XIII Workshop
September 28 - October 2, 2016
Program and Abstract Book
2nd International Factor XIII Workshop
September 28 - October 2, 2016
Program and Abstract Book
Golden sponsor
Sponsors
2nd International Factor XIII Workshop
Welcome
Dear Participants,
On behalf of the Organizing Committee I would like to welcome you at the 2nd International Factor XIII Workshop in
Hévíz, Hungary between September 28 and October 2, 2016. The 1st International Factor XIII Workshop was organized
four years ago in Debrecen, Hungary. Many participants of the 1st Workshop appreciated the high scientific value of
the meeting and the friendly atmosphere. Such favorable reactions and discussions with prominent FXIII researchers
prompted us to cope with the hard task of organizing the FXIII Workshop for the second time. It is a great pleasure to
us that you who attended the first meeting and also first time participants accepted our invitation to this, hopefully
successful Workshop.
Over 60 scientists registered from 4 continents (14 countries) and 43 abstracts were accepted for oral presentations. The
scientific sessions include 20+5 min invited lectures and 10+5 min regular oral communications on the following topics:
FXIII: structure and activation
Inhibition of FXIIIa, FXIII and fibrinolysis
Cellular FXIII
FXIII: methodological aspects
Function of FXIII
FXIII deficiency
FXIII polymorphisms
FXIII in diseases
In addition an industrial session is also part of the scientific program.
We expect informal open scientific discussions with friendly atmosphere that would broaden existing collaborations and
help in building new ties among research groups.
We are indebted to the sponsors, their contribution was a tremendous help in the organization and without such help
we could not support young scientists in attending the Workshop.
We thank for the hospitality of Hévíz town and feel honored for opening the Workshop by the mayor of Hévíz. Hévíz is a
world famous town in Western Hungary with the largest natural thermal lake in Europe. The venue of the meeting is the
Danubius Health Spa Resort Hévíz is an excellent four-star superior hotel. The hotel is just a short walk away from the
thermal lake and during the meeting we will find time for the participants to have a dip in the lake.
An enjoyable social program is also offered, which includes a boat trip on the picturesque lake Balaton with visiting
the surrounding wine producing area and a farewell dinner in the Festetics Palace of Keszthely, a close-by small town.
We hope that you will have a fruitful scientific meeting and a pleasant stay in Hévíz.
With best wishes,
Prof. László Muszbek MD, PhD
Chairman of the Organizing Committee
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Program
2nd International Factor XIII Workshop
Program, General Overview
September 28, Wednesday
12:00-22:00
ARRIVAL
REGISTRATION IN THE HOTEL
Welcome drink, dinner
19:00
September 29, Thursday
8:00- 9:00
REGISTRATION IN THE HOTEL
9:00- 9:30
OPENING
9:30-12:45
SCIENTIFIC PROGRAM
12:45-15:00
Lunch break
15:00-17:40
SCIENTIFIC PROGRAM
Dinner
19:00
September 30, Friday
9:00-11:50
11:50-14:00
SCIENTIFIC PROGRAM
Lunch break
14:00-15:00 INDUSTRIAL SESSION
16:00
Excursion
October 1, Saturday
9:00-11:50
11:50-13:00
SCIENTIFIC PROGRAM
Lunch break
13:00-17:10 SCIENTIFIC PROGRAM
18:00 Farewell dinner
October 2, Sunday
4
DEPARTURE
2nd International Factor XIII Workshop
Program
Day 1 (September 29, Thursday)
OPENING
9:00- 9:30
GREETING BY GÁBOR PAPP THE MAYOR OF HÉVÍZ
GREETING BY THE CHAIRMAN OF THE ORGANIZING COMMITTEE, GENERAL INSTRUCTIONS
SCIENTIFIC PROGRAM
T OPIC 1: FXIII STRUCTURE AND ACTIVATION
Chairpersons: Imhof D (Germany), Kolev K (Hungary)
9:30- 9:45
T1/1
REVISITING THE MECHANISM OF COAGULATION FACTOR XIII ACTIVATION AND ITS REGULATION
FROM A STRUCTURE/FUNCTIONAL PERSPECTIVE
Gupta S (Germany), Biswas A Germany), Krettler C (Germany), Reinhart C (Germany),
Dodt J (Germany), Philippou H (UK), Ivaskevicius V (Germany), Oldenburg J (Germany)
9:45-10:05
T1/2
THE FIRST EVENTS IN BLOOD COAGULATION FXIII-A2 ACTIVATION;
MODELING THE THROMBIN-FXIII-A2 COMPLEX AND THE EFFECT OF Ca2+ IONS ON
THE DYNAMICS OF FXIII-A2’
Fekete A, Muszbek L, Komáromi I (Hungary)
10:05-10:20
T1/3
MASS-SPECTROMETRY STUDY OF NON-OXIDIZED AND OXIDIZED HUMAN PLASMA
FIBRIN-STABILIZING FACTOR
Bychkova AV, Vasylieva AD, Danilova TA, Shchegolikhin AN, Bugrova AE, Kononikhin AS,
Indeykina MI, Nikolaev EN, and Rosenfeld MA (Russia)
10:20-10:35
T1/4
THE TRANSGLUTAMINASE REACTION OF BLOOD COAGULATION FACTOR XIII; A MODEL
FOR THE FIRST (ACYL-ENZYME FORMATION) STEP BASED ON HYBRID QM/MM CALCULATIONS
Balogh G and Komáromi I (Hungary)
10:35-10:50
T1/5
EXPRESSION ANALYSIS REVEALS VARYING DEGREES OF EFFECTIVENESS FOR HETEROZYGOUS
MISSENSE MUTATIONS REPORTED FROM PATIENTS WITH MILD FXIII DEFICIENCY
Biswas A (Germany), Thomas A (Germany), Dodt J (Germany), Philippou H (UK),
Hethershaw E (Germany), Juergen HE (Germany), Ivaskevicius V (Germany), Oldenburg J (Germany)
10:50-11:15 Coffee break
T OPIC 2: INHIBITION OF FXIIIa, FXIII AND FIBRINOLYSIS
Chairpersons: Pease RJ (UK), Bagoly Z (Hungary)
11:15-11:30
T2/1
TARGETED SYNTHESIS AND CHARACTERIZATION OF DIFFERENT ISOMERS OF
FACTOR XIIIA INHIBITOR TRIDEGIN
Bäuml CA, Loef I, Steinmetzer T, Biswas A, and Imhof D (Germany)
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Program
2nd International Factor XIII Workshop
11:30-11:55
T2/2
STRUCTURE-ACTIVITY RELATIONSHIP STUDIES OF DISULFIDE ISOMERS OF
THE FACTOR XIIIA INHIBITOR TRIDEGIN
Bäuml CA, Loef I, Hardes K, Steinmetzer T, Biswas A, and Imhof D (Germany)
11:55-12:20
T2/3
COAGULATION CHANGES IN DIABETES WITH EMPHASIS ON FIBRIN AND FIBRINOLYSIS
Grant PJ (UK)
12:20-12:45
T2/4
FIBRINOLYSIS: THE MAIN ENEMY OF FACTOR XIII
Kolev K (Hungary)
12:45-15.00 Lunch break, visiting the lake
TOPIC 3: CELLULAR FXIII
Chairpersons: Wolberg A (USA), Grant P (UK)
15:00-15:25
T3/1
RESIDENT TISSUE MACROPHAGES SECRETE PLASMA FXIII-A AND MAINTAIN THE PLASMA POOL
Beckers CML (UK), Simpson KR (UK), Griffin KJ (UK), Brown JM (UK), Cordell PA (UK), Smith KA (UK),
Kearney MT (UK), Vacher J (Canada), Alexander W (Australia), Grant PJ (UK) and Pease RJ (UK)
15:25-15:45
T3/2
TRANSGLUTAMINASES IN HUMAN CORNEA AND LENS
Muszbek L, Orosz ZZ, Bárdos H, Shemirani AH, Vereb G, Hidasi V, Nagy B, Kappelmayer J,
Ádány R, Berta A, Facskó A (Hungary)
15:45-16:00
T3/3
FACTOR XIII IN TEARS AND ITS POSSIBLE ROLE IN CORNEAL WOUND HEALING
Orosz ZZ, Szöôr Á, Veréb Z, Hassan Z, Katona É, Vereb G, Facskó A, Muszbek L (Hungary)
16:00-16:25
Tea break
TOPIC 4: FXIII: METHODOLOGICAL ASPECTS
Chairpersons: Menegatti M (Italy), Kappelmayer J (Hungary)
16:25-16:45 T4/1
THE ACTION OF FXIII MADE VISIBLE IN A MICROVASCULAR FLOW MODEL
Jenny L, Kohler HP, Schroeder V (Switzerland)
16:45-17:10
T4/2
FACTOR XIII LABORATORY TESTING: LESSONS FROM THE ECAT EQA PROGRAMME
Meijer P (The Netherlands)
17:10-17:25
T4/3
CORRELATION OF FXIII ACTIVITY WITH LABORATORY PARAMETERS THAT ASSESS THROMBIN
GENERATION AND CLINICAL PARAMETERS IN HEMOPHILIA A PATIENTS
Milos M, Coen Herak D, Zupancic-Salek S, Zadro R (Croatia)
17:25-17:40
T4/4
MONOCLONAL ANTIBODY DIRECTED AGAINST THE CROSSLINKED FIBRIN NEOEPITOPE; “DD-XLINK-MAB”
Pasternack R (Germany)
Dinner over the lake
6
19:00
2nd International Factor XIII Workshop
Program
Day 2 (September 30, Friday)
TOPIC 5: FUNCTION OF FXIII
Chairpersons: Ariens RAS (UK), Pieters M (South Africa)
9:00- 9:25
T5/1
PLASMA FXIII, BUT NOT PLATELET FXIII, PROMOTES RED BLOOD CELL RETENTION
IN CONTRACTED CLOTS
Kattula S (USA), Byrnes JR (USA), Bagoly Z (Hungary), Muszbek L (Hungary),
and Wolberg AS (USA)
9:25- 9:50
T5/2
FACTOR XIII ACTIVITY PLAYS A ROLE IN THE RATE OF FIBRIN FORMATION
Ali M (UK), Pease R (UK), Hardy L (UK), Howell G (UK), Law G (UK), La Corte AC (UK),
Hethershaw E (UK), Duval C (UK), Ridger V (UK), Weisel JW (USA), Grant PJ (UK), Ariëns RAS (UK),
Philippou H (UK)
9:50-10:15
T5/3
FACTOR XIII ACTIVATION PEPTIDE – A SHORT PEPTIDE WITH MULTIPLE FUNCTIONS
Kohler HP (Switzerland)
10:15-11:45 Coffee break
10:45-11:00
T5/4
FACTOR XIII AND VASCULAR SMOOTH MUSCLE CELLS
Bogáti R, Katona É, Balogh E, Jeney V, Muszbek L (Hungary)
11:00-11:20
T5/5
FXIII-A AND TRANSGLUTAMINASE 2 JOINTLY PROTECT THE MYOCARDIUM AGAINST FIBROSIS
Beckers CM, Newell LM, Drinkhill MJ, Griffin KJ, Cheah L, Simpson KR, Brown JM, Jackson CL,
Pease RJ and Grant PJ (UK)
11:20-11:35
T5/6
FACTOR XIII DEFICIENCY ENHANCES THROMBIN GENERATION DUE TO IMPAIRED FIBRIN
POLYMERIZATION - AN EFFECT CORRECTED BY FACTOR XIII REPLACEMENT
Pitkänen H H, Jouppila A, Lemponen M, Ilmakunnas M, Ahonen J, Lassila R (Finland)
11:35-11:50
T5/7
PROLONGED WOUND HEALING DUE TO MODERATE FXIII DEFICIENCY IN A PATIENT WITH
FEMORAL FRACTURE
Bronic A (Croatia)
11:50-14.00 Lunch break, visiting the lake
INDUSTRIAL SESSION
Chairperson: Muszbek L (Hungary)
14:00-15:00
OVERVIEW ON rFXIII AND THE CLINICAL PROGRAM
Driessler F
Novo Nordisk Health Care AG, Zürich, Switzerland
Boat excursion on the Lake Balaton
16:00
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Program
2nd International Factor XIII Workshop
Day 3 (October 1, Saturday)
TOPIC 6: FXIII DEFICIENCY
Chairpersons: Köhler HP (Switzerland), Meijer P (The Netherlands)
9:00- 9:25
T6/1
CONGENITAL FACTOR XIII DEFICIENCY – UNRESOLVED PROBLEMS REGARDING NOMENCLATURE,
DIAGNOSIS, AND THERAPY
Schroeder V (Switzerland)
9:25- 9:40
T6/2
IRANIAN COHORT, FIRST REPORT OF THE FACTOR XIII DEFICIENCY IN SISTAN AND
BALOUCHESTAN PROVINCE OF IRAN
Naderi M, Hosseini S, Eshghi P, Malek F, Dorgalaleh A (Iran)
9:40- 9:55
T6/3
CLINICAL AND MOLECULAR CHARACTERISTICS OF PATIENTS AFFECTED BY CONGENITAL
FXIII DEFICIENCY IN PAKISTAN
Borhany M, Fatima N, Abid M, Shamsi TS (Pakistan)
9:55:10:10
T6/4
PROSPECTIVE EVALUATION OF BLEEDING INCIDENCE IN FACTOR XIII DEFICIENCY
(PRO-RBDD STUDY)
Palla R (Italy), Menegatti M (Italy), Boscarino M (Italy), Halimeh S (Germany),
Lachamann B (Germany), Borhany M (Pakistan), Naveena F (Pakistan), Ozdemir N (Turkey),
Siboni SM (Italy), Mikovic D (Serbia), Saracevic M (Serbia), Mumford A (UK), Harvey A (UK),
Schutgens REG (The Netherland), van Haaften-Spoor M (The Netherland), Chapin J (USA),
Hsu F (USA), Platokouki H (Greece), Pergantou H (Greece), Yilmaz A (Turkey), Shapiro AD (USA),
Williamson A (USA), De Moerloose P (Switzerland), Casini A (Switzerland), Payne J (UK),
Muszbek L (Hungary), Peyvandi F (Italy)
10:10-10:30
T6/5
MINIMUM FACTOR XIII CLOTTING LEVEL TO PREVENT MAJOR SPONTANEOUS BLEEDINGS:
RESULTS FROM THE PROSPECTIVE DATABASE ON FACTOR XIII DEFICIENCY (PRO-RBDD)
Menegatti M (Italy), Palla R (Italy), Boscarino M (Italy), Halimeh S (Germany),
Lachamann B (Germany), Borhany M (Pakistan), Naveena F (Pakistan), Ozdemir N (Turkey),
Siboni SM (Italy), Mikovic D (Serbia), Saracevic M (Serbia), Mumford A (UK), Harvey A (UK),
Schutgens REG (The Netherland), van Haaften-Spoor M (The Netherland), Chapin J (USA),
Hsu F (USA), Platokouki H (Greece), Pergantou H (Greece), Yilmaz A (Turkey), Shapiro AD (USA),
Williamson A (USA), De Moerloose P (Switzerland), Casini A (Switzerland), Payne J (UK),
Muszbek L (Hungary), Peyvandi F (Italy)
10:30-10:55 Coffee break
10:55-11:10
T6/6
RECOMBINANT FXIII FOR PROPHYLAXIS AND TREATMENT IN CONGENITAL FXIII DEFICIENCY:
A CASE REPORT
Schlammadinger Á, Árokszállási A, Kerényi A, Katona É, Bereczky Zs, Muszbek L, Boda Z (Hungary)
11:10-11:25
T6/7
BLEEDING EPISODES IN HETEROZYGOTES OF FACTOR XIII DEFICIENCY
Naderi M, Eshghi P, Malek M, Karbasian F, Dorgalaleh A (Iran)
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2nd International Factor XIII Workshop
11:25-11:50
Program
T6/8
THE DIAGNOSIS AND CHARACTERIZATION OF ALLO-, OR AUTOANTIBODIES AGAINST
FACTOR XIII SUBUNITS
Muszbek L (Hungary), Pénzes K (Hungary), Katona É Hungary), Bereczky Z (Hungary),
Kerényi A (Hungary), Rázsó K (Hungary), Kun M (Hungary), Boda Z (Hungary), Vezina C (Canada),
Rivard GE (Canada)
11:50-13:00 Lunch break
TOPIC 7: FXIII POLYMORHISMS
Chairpersons: Schroeder V (Switzerland), Philippou H (UK)
13:00-13:15
T7/1
REGULATION OF PLASMA FXIII ANTIGEN LEVELS IN HEALTHY INDIVIDUALS
Katona É, Mezei ZA, Kállay J, Bereczky Z, Kovács B, Ajzner É, Muszbek L (Hungary)
13:15-13:40
T7/2
FUNCTIONAL VARIANTS OF FXIII - WHAT HAVE WE LEARNED SO FAR
Ariëns RAS (UK)
13:40-14:05
T7/3
THE INFLUENCE OF THE FXIII VAL34LEU POLYMORPHISM ON PLASMA CLOT STRUCTURE
IN AFRICANS
Pieters M (South Africa)
14:05-14:20
T7/4
GENDER SPECIFIC EFFECT OF THE FXIII-A VAL34LEU POLYMORPHISM ON THE RISK
FOR CHILDHOOD AND PERINATAL ARTERIAL ISCHEMIC STROKE
Coen Herak D, Ceri A, Grzunov A, Lenicek Krleza J, Radic Antolic M, Horvat I, Djuranovic V,
Barisic Nina, Zrinski Topic R, Zadro R (Croatia)
14:20-14:35
T7/5
THE EFFECT OF FXIII LEVELS AND FXIII-B POLYMORPHISMS ON THE RISK OF
VENOUS THROMBOEMBOLISM
Mezei ZA, Katona É, Kállay J, Bereczky Z, Somodi L, Kovács B, Miklós T, Ajzner É,
Muszbek L (Hungary)
14:35-14:50
T7/6
EFFECT OF FACTOR XIII LEVELS AND POLYMORPHISMS ON THE RISK OF MYOCARDIAL INFARCTION
IN YOUNG PATIENTS
Bereczky Z, Mezei ZA, Balogh L, Katona É, Fiatal S, Ádány R, Miklós T, Kovács B, Ajzner É,
Édes I, Muszbek L (Hungary)
14:50-15:20 Tea break
TOPIC 8: FXIII IN DISEASES
Chairpersons: Bereczky Z (Hungary), Biswas A (Germany)
15:20-15:35
T8/1
FXIII LEVELS IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS AND ANTIPHOSPHOLIPID
SYNDROME
Bagoly Z, Tóth NK, Tarr T, Soltész P, Katona E, Mezei ZA, Muszbek L (Hungary)
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Program
2nd International Factor XIII Workshop
15:35-15:50
T8/2
THE EFFECT OF HEMODIAFILTRATION AND HEMODIALYSIS ON COAGULATION PARAMETERS
IN PATIENTS WITH END-STAGE RENAL DISEASE
Pénzes K, Becs G, Hurják B, Molnár E, Katona É, Balla J and Muszbek L (Hungary)
15:50-16:15
T8/3
INTRACELLULAR FACTOR XIII-A IS A FLOW CYTOMETRIC DIAGNOSTIC TEST
IN ACUTE LEUKEMIAS
Kappelmayer J (Hungary)
16:15-16:30
T8/4
FACTOR XIII-A POSITIVE MACROPHAGES AND CD11c POSITIVE DENDRITIC CELLS IN
MALIGNANT MELANOMA
Bárdos H, Töröcsik D, Emri E, Dezsô B, Emri G, Remenyik E, Balázs M, Ádány R (Hungary)
16:30-16:45
T8/5
EXPRESSION OF BLOOD COAGULATION FACTOR XIII-A IN LYMPHOBLASTS PREDICTS
FAVOURABLE OUTCOME IN PEDIATRIC PRECURSOR B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA
Kárai B, Szánthó E, Csáthy L, Ujfalusi A, Gyurina K, Szegedi I, Kappelmayer J, Kiss Cs,
Hevessy Zs (Hungary)
16:45-17:10
T8/6
IMMUNOHISTOCHEMICAL DETECTION OF FACTOR XIII SUBUNIT A AS
A DIAGNOSTIC TOOL IN DERMATOPATHOLOGY: BELIEFS AND DISBELIEFS
Törôcsik D, Bárdos H, Ádány R (Hungary)
Visiting the Festetics Palace in Keszthely and farewell dinner
10
18:00
2nd International Factor XIII Workshop
Abstracts
Abstracts
T1/1
Revisiting the mechanism of coagulation Factor XIII activation and its regulation from a
structure/functional perspective
Gupta S1 and Biswas A1, Krettler C2, Reinhart C2, Dodt J3, Philippou H4, Ivaskevicius V1, Oldenburg J1
University Clinic Bonn, Institute of Experimental Hematology and Transfusion Medicine, Bonn, Germany
Max Planck institute of Biophysics, Frankfurt, Germany
3
Paul Ehrlich Institute, Langen, Germany
4
Leeds Institute for Cardiovascular and Metabolic Medicine, Leeds, United Kingdom
1
2
Aim: To apply a theoretical approach towards understanding and explaining the concerted model of FXIII activation and regulation.
Method: A combination of alignment based structural comparison, molecular dynamic (MD) simulation, steered molecular dynamic
(SMD) simulation, transition state modeling, threading/homology modeling, constrained symmetric/blind docking have been used
as part of a theoretical exercise to study the structure and functional aspects of FXIII activation and regulation. FXIIIA2B2 purified
from Fibrogammin P (CSL Behring) was treated with different concentrations of Calcium/Thrombin and resolved with size exclusion
chromatography to study the sequential dissociation of the heterotetramer in vitro. Activated FXIIIA (FXIIIAa) generation assay was
used to study the effect of B subunit on FXIIIA activation.
Results and conclusions: The perspective generated from this study puts the FXIII activation and regulation process on an integrated
platform. The main outcomes from this study are: a) Similarities and differences between FXIIIA and TG2 suggest evolution of specific
regions with similar functions but altered binding partners b) the first calcium binding site of FXIIIA subunit imparts a stabilizing/
zymogenic constraint/influence on the zymogenic FXIIIA c) The calcium binding to the three sites on FXIIIA subunit follows a
chronological sequence essential for the correct and directed conformational opening of the FXIIIA subunit as well as the timely
dissociation of the FXIIIB subunit d) FXIIIB subunit mediates its regulatory influence (i.e. accelerating FXIIIA activation) partly by
its interaction with the activation peptide e) Non-proteolytic activation of FXIIIA leads to the generation of a weak heterodimer and
is therefore a reversible process while proteolytic activation results in FXIIIA subunit monomerization which contributes to its further
down regulation and therefore is an irreversible process f) FXIIIAa down regulation is mediated by sequential protease cleavage that
is dictated by exposure of critical protease sites during FXIIIA subunit conformational transition to FXIIIAa as well as dissociation
of the FXIIIB subunit.
11
Abstracts
2nd International Factor XIII Workshop
T1/2
THE FIRST EVENTS IN BLOOD COAGULATION FXIII-A2 ACTIVATION; MODELING THE THROMBIN-FXIII-A2
COMPLEX AND THE EFFECT OF CA2+ IONS ON THE DYNAMICS OF FXIII-A2’
Fekete A, Muszbek L, Komáromi I
Division of Clinical Laboratory Sciences, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen,
Hungary
In the inactive dimeric (“closed”) structure of blood coagulation factor FXIII A subunits (FXIII-A) the catalytic centers are buried.
Active (“open”) conformations have been proposed both on the basis of theoretical modeling [1] and from X-ray diffraction experiments
[2]. The normal physiological activation of FXIII-A2 requires the concerted action of thrombin and Ca2+ ions, but neither the structure
of the FXIII-A2:thrombin Michaelis complex nor the effect of Ca2+ on the dynamics of FXIII-A2’ (the proteolytically cleaved but still
inactive FXIII-A2) are known.
In order to derive a theoretical model for the FXIII-A2: thrombin complex, protein-protein docking experiments were carried out and
the results were compared with the X-ray structure of the thrombin-activation peptide (AP) fragment complex [3]. Although the
structure elements we obtained had certain similarity to the corresponding elements derived from the X-ray analysis, remarkable
differences can be observed. The most notable one is that according to our results, in contrast to the results of X-ray analysis,
thrombin can accommodate the AP fragment conformation, which exists in non-cleaved FXIII-A2.
Molecular dynamics simulations were performed on FXIII-A2’ and FXIII-A2 in the presence and absence of Ca2+ ions. The larger root
mean square deviations and larger radius of gyration we observed in the first case indicates increased flexibility for the FXIII-A2’
system. Simulation carried out on FXIII-A2 at high ionic strength also demonstrated increased flexibility. Large amplitude – low
frequency modes derived from dynamics trajectory indicate the spatial separation of b-barrel domains from the catalytic one and even
showed the separation of the two FXIII-A domains. Sampling snapshots from the molecular dynamics trajectory allow the localization
of Ca2+ binding sites on the surface of FXIII-A2’.
In conclusion, the atomic details of the first events of FXIII-A2 activation outlined above are in a good accordance with the
experimental data disclosed so far, and provide valuable complementary information.
References:
[1] Komaromi I, et al. J Thromb Haemost 2011;9:9-20.
[2] Stieler M, et al. Angew Chem Int Ed Engl 2013;52:11930-11934.
[3] Sadasivan C, and Yee VC. J Biol Chem 2000;275:36942-36948.
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2nd International Factor XIII Workshop
Abstracts
T1/3
MASS-SPECTROMETRY STUDY OF NON-OXIDIZED AND OXIDIZED HUMAN PLASMA FIBRIN-STABILIZING
FACTOR
Bychkova AV1, Vasylieva AD1, Danilova TA1, Shchegolikhin AN1, Bugrova AE1, Kononikhin AS1,2,3, Indeykina MI1, Nikolaev EN1,2,3,
Rosenfeld MA1
N. M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia
Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Moscow, Russia
3
Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia
1
2
For the first time mass-spectrometry study of plasma fibrin-stabilizing factor (pFXIII) has been carried out. Non-oxidized and ozoneoxidized pFXIII at different stages of its enzymatic activation has been investigated: zymogen (pFXIII), pFXIII activated by calcium
ions, FXIIIa. pFXIII was isolated from human plasma. Protein hydrolysis was performed by GluC followed by tryptic digestion for
mass-spectrometry analysis. LC-MS/MS analysis was carried out with the system consistent of Agilent 1100 chromatograph (Agilent
Technologies Inc., Santa-Clara, USA) and hybrid LTQ-FT Ultra mass-spectrometer (Thermo, Bremen, Germany). Mascot and PEAKS
search engines were used for peptide and protein identifications including oxidative modifications sites. In the oxidized pFXIII the
followings were revealed: three modifications in the β-sandwich (52% coverage), 18 modifications in the catalytic core (49% coverage), no modifications in the barrel 1 (64% coverage), 3 modifications in the barrel 2 (89% coverage). Calcium treatment led to
appearance of the additional modifications in all structural elements of the pFXIII subunit A. On the basis of data we have obtained
earlier regarding functioning of oxidized plasma and cellular FXIII [1, 2], the correlation between oxidative sites involved and enzymatic activity is discussed.
The study was supported by RFBR, research project No. 15-15-04-08188a. The part of research related to peptides and PTM identification
by high-resolution mass spectrometry measurements was supported by the Russian Science Foundation No. 16-14-00181.
References:
[1] Rosenfeld MA, et al. Biochim Biophys Acta - Proteins and Proteomics 2013;1834:2470-2479.
[2] Rosenfeld MA, et al. Doklady Biochemistry and Biophysics 2016;467:128-131.
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Abstracts
2nd International Factor XIII Workshop
T1/4
THE TRANSGLUTAMINASE REACTION OF BLOOD COAGULATION FACTOR XIII; A MODEL FOR THE FIRST
(ACYL-ENZYME FORMATION) STEP BASED ON HYBRID QM/MM CALCULATIONS
Balogh G and Komáromi I
Division of Clinical Laboratory Science, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen,
Hungary
The main physiological role of the activated blood coagulation factor XIII (FXIIIa) is to covalently crosslink peptide/protein chains
bearing appropriate glutamine and lysine residues through (iso)peptide bond. A reverse cysteine protease mechanism was proposed
for this transglutaminase reaction. However, solid reaction kinetics and/or theoretical proofs on this mechanism are still lacking in
the literature. Our aim was to propose a plausible reaction path for the reaction of FXIIIa and to characterize the stationary points
on its potential energy surface by means of hybrid QM/MM methods. We started from the recently resolved X-ray structure of nonenzymatically activated FXIIIa (FXIIIAº) [1]. A dodecapeptide substrate was docked to the active site of the enzyme and then simulated annealing protocol was used to find its binding position. Both QM/MM dynamics using the recently introduced DFTB3 method
[2,3] and ONIOM type static calculations [4] using dispersion corrected density functional methods (M06-2X, ωB97XD) were applied
to reveal the fine details of the reaction. In addition to the unbiased QM/MM dynamics the umbrella sampling protocol was applied to
disclose the dynamic properties of the reaction path. It was found that the Cys-His catalytic dyad exist in ion-pair form both in the
free enzyme and in the Michaelis complex. The proton transfer has low barrier (ONIOM calculations) or it can be a spontaneous event
(QM/MM dynamics). The acyl-enzyme formation takes place via a zwitterionic intermediate where the glutamine side chain amide is
protonated and the amide bond is still unbroken, just as the (cysteine)S-C(glutamine side chain carbonyl) bond. The combination of
the QM/MM dynamics based on DFTB3 method and the high level static ONIOM calculation using 2nd derivatives to prove the existence
of stationary points was found to be a powerful combination in identifying the possible local minima and in disclosing the reaction
paths which connect them.
References:
[1] Stieler M, et al. Angew Chem Int Edit 2013;52:11930-11934.
[2] Gaus M, et al. J Chem Theory Comput 2011;7:931-948.
[3] Kubar T, et al. J Comput Chem 2015;36:1978-1989.
[4] Vreven T, et al. J Chem Theory Comput 2006;2:815-826.
14
2nd International Factor XIII Workshop
Abstracts
T1/5
EXPRESSION ANALYSIS REVEALS VARYING DEGREES OF EFFECTIVENESS FOR HETEROZYGOUS MISSENSE
MUTATIONS REPORTED FROM PATIENTS WITH MILD FXIII DEFICIENCY
Biswas A1 and Thomas A1, Dodt J2, Philippou H3, Hethershaw E3, Juergen HE4, Ivaskevicius V1, Oldenburg J1
Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, 53127 Bonn, Germany
Paul Ehrlich Institut, 63225 Langen, Germany.
3
Leeds Institute for Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, UK.
4
Nees Institute for Biodiversity of Plants, University of Bonn, Bonn, Germany.
1
2
Background: Mild coagulation Factor XIII deficiency in comparison to its severe form is a more frequent event but is difficult to
detect since the patients are often asymptomatic and develop bleeding symptoms only when exposed to trauma. Over the past five
years we have reported in excess of 20 mutations in F13A1 and F13B genes from patients with mild Factor XIII deficiency.
Aim: To heterologously express and evaluate the causality of heterozygous missense mutations reported in F13A1 and F13B genes
reported from patients with mild Factor XIII deficiency
Methods: The reported F13A1 and F13B missense mutations were cloned in mammalian expression vectors carrying F13A1 and F13B
cDNAs and transfected into Cos-1 and HEK293T cell lines respectively. The intracellular-lysates/extracellular-medium were collected
after a transient transfection period and tested with a variety of assays reflecting the various functional/antigenic aspects of FXIII.
Confocal microscopy was performed for FXIIIB subunit mutations to detect any secretion related defects. Structural modeling was
performed with available crystals structures of FXIIIA subunit and homology based models of FXIIIB subunit to determine their putative structural impact.
Results and Discussion: Only one FXIIIB subunit mutation i.e. p.Cys5Arg showed a true secretion related defect. The remaining
FXIIIB subunit mutations showed either antigenic instability or lack of interaction with FXIIIA subunit. FXIIIA subunit mutations
could be categorized into mild moderate and severe forms based on their in vitro expression phenotype. The biggest category of mutations belonged to the severe category having severe and direct implications for antigenic stability and hence also activity levels.
Two substitutions at the thrombin cleavage site belonged to the rare category of mutations that showed severely reduced activity
levels but only mild reduction in antigen. All the FXIIIA missense mutations also showed concurrent but varied (depending on their
severity) effects on alpha-2-antiplasmin incorporation, clot thickness and fibrinogen cross-linking.
15
Abstracts
2nd International Factor XIII Workshop
T2/1
TARGETED SYNTHESIS AND CHARACTERIZATION OF DIFFERENT ISOMERS OF FACTOR XIIIA INHIBITOR
TRIDEGIN
Bäuml CA1, Loef I1, Steinmetzer T2, Biswas A3, Imhof D1
Pharmaceutical Chemistry I, Institute of Pharmacy, University of Bonn, Germany
Department of Pharmacy, Institute of Pharmaceutical Chemistry, University of Marburg, Germany
3
Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Germany
1
2
Blood coagulation factor XIIIa (FXIIIa) is an interesting target for antithrombotic and thrombolytic therapy as it catalyzes the last
step of the blood coagulation cascade, i.e. the cross-linking of fibrin monomers.[1] Furthermore, studies in the past decades pointed
out its participation in several other physiological processes such as pregnancy, wound healing, and inflammation.[2] So far, the
66mer peptide tridegin, which was originally isolated from the salivary gland of the giant Amazon leech Haementeria ghilianii in 1997,
depicts the only potent and specific natural peptide inhibitor of FXIIIa.[3] Inhibition of FXIIIa by tridegin is mediated by its flexible
C-terminal region, while the N-terminal part is stabilized by three disulfide bridges and plays a decisive role in inhibitor binding.
[4,5] Former studies on the cysteine connectivity of the synthetically produced peptide by enzymatic digestion and subsequent MS/
MS analysis revealed the presence of three distinct isomers in an oxidative self-folding approach in buffer. Molecular modeling of
these isomers and successive molecular docking studies with FXIIIa suggested different binding modes and distinctive activities for
the respective isomers. However, corresponding experimental data is lacking due to the fact that separation of the three disulfidebridged isomers by the currently available techniques was not possible so far. Hence, synthesis of the three identified isomers via a
protection group strategy is required in order to characterize their potency and inhibition mode as individually isolated compounds.
In addition, structural analysis of the isolated isomers ought to be performed. The envisaged structure-activity studies will lead to a
better understanding of the inhibitor-target interaction. Our present studies will further increase the comprehension of the natural
FXIIIa inhibitor tridegin and thus provide the scientific community with a valuable research tool as well as a potential lead structure
for the development of new FXIIIa inhibitors.
References:
[1] Reed GL, et al. Circulation 1999;99:299-304.
[2] Fadoo Z, et al. J Blood Med 2013;4:65-7193.
[3] Finney S, et al. Biochem J 1997;324:797-805.
[4] Böhm M, et al. Chem Med Chem 2012;7:326-333.
[5] Böhm M, et al. J Med Chem 2014;57:10355-10365.
16
2nd International Factor XIII Workshop
Abstracts
T2/2
STRUCTURE-ACTIVITY RELATIONSHIP STUDIES OF DISULFIDE ISOMERS OF THE FACTOR XIIIA INHIBITOR
TRIDEGIN
Bäuml CA1, Loef I1, Hardes K2, Steinmetzer T2, Biswas A3, Imhof D1
Pharmaceutical Chemistry I, Institute of Pharmacy, University of Bonn, Germany
Department of Pharmacy, Institute of Pharmaceutical Chemistry, University of Marburg, Germany
3
Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Germany
1
2
The cysteine-rich 66mer peptide tridegin from the giant amazon leech Haementeria ghilianii is a potent and specific native peptide
inhibitor of the transglutaminase factor XIIIa (FXIIIa).[1,2] FXIIIa catalyzes the last step in the blood coagulation cascade and is
thus a potential target for antithrombotic and thrombolytic therapy.[3] In our previous studies we focused on the production and
in-depth study of tridegin and several analogues using different strategies. Apart from the finding that a glutamine residue (Q52) in
the C-terminal region of tridegin interacts with the active site it was shown that the cysteine-rich N-terminal of the polypeptide also
seems to play a major role in the inhibition.[4] This part of the molecule increased inhibitor potency about 2-fold, and moreover the
presence of the disulfide-linked N-terminus was found to stabilize tridegin in the presence of FXIIIa. Bioanalytical studies, however,
revealed that regardless of the production strategy used a mixture of at least three disulfide isomers was obtained and tested.[5] In
order to analyze each individual isomer, which could be elucidated using tandem mass spectrometry techniques, we recently focused
on the targeted synthesis of several of the possible conformational isomers. In addition, structural investigations of the inhibitor were so far hampered by the use of an isomer mixture, i.e. a molecular modeling and docking approach therefore needed to be
employed.[5] However, by providing the individual pure isomers SAR studies will shed light on the suitability of tridegin as potential
lead compound for further FXIIIa inhibitors. The progress of these investigations will be reported.
References:
[1] Finney S, et al. Biochem J 1997;324;797-805.
[2] Shebuski RJ, et al. Blood 1990;75:1455
[3] Reed GL, et al. Circulation 1999;99, 299-304.
[4] Böhm M, et al. Chem Med Chem 2012;7:326-333.
[5] Böhm M, et al. J Med Chem 2014;57:10355-10365.
17
Abstracts
2nd International Factor XIII Workshop
T2/3
COAGULATION CHANGES IN DIABETES WITH EMPHASIS ON FIBRIN AND FIBRINOLYSIS
Grant PJ
Division of Cardiovascular and Diabetes Research, School of Medicine, University of Leeds, UK
The fibrinolytic system consists of a series of activators and inhibitors that regulate the production and activity of plasmin.
Fibrinolytic activity has important roles in clot lysis and remodelling and tissue repair and effects in the cardiovascular and central
nervous systems, malignancy and diabetes. Subjects with diabetes have a 3-4 fold overall risk of CV disease and the pathophysiology
of coronary artery disease involves the development of inflammatory atherothrombotic changes in the vessel wall, leading to plaque
rupture, occlusive thrombus formation and acute coronary syndromes. Studies of pre-diabetes indicates that major disturbances
of haemostatic mechanisms occur prior to the development of diabetes, with alterations in fibrinolysis dominating the observed
changes. Plasminogen activator inhibitor-1 (PAI-1) levels are elevated in insulin resistant, non-diabetes subjects, with evidence
that PAI-1 may be regulated through gene specific effects associated with underlying insulin resistance. Higher levels of FXIIIA also
associate with insulin resistance, although the implications for thrombotic risk are unclear. Complement C3 interacts with the fibrin network to produce a structure resistant to lysis which emphasises the importance of inflammatory thrombotic interactions in CV
risk. As β-cell failure progresses and diabetes develops, there are further increases in both PAI-1 and tissue plasminogen activator
(tPA) and clinical studies of metformin (an insulin sensitising agent) demonstrated marked and persistent falls in both PAI-1 and
tPA to support the importance of insulin resistance in these processes. As glycaemia progresses, post translational modifications to
fibrin(ogen) generate a fibrin network with a denser structure and thinner fibers that is intrinsically resistant to lysis whilst at the
same time diminishing plasmin(ogen) binding to fibrin and plasmin generation on the clot surface. Plasminogen similarly undergoes
post-translational modifications by glycation affecting the lysine residues responsible for fibrin binding and cleavage of plasminogen
to plasmin. The importance of glucose/glycation in these processes is revealed by amelioration of depressed fibrinolytic activity by
improving glycaemic control.
In summary, inhibition of clot lysis in diabetes occurs due to elevated PAI-1, inhibition of plasmin generation and alterations in fibrin structure all changes related to the underlying metabolic milieu. Multiple factors contribute to these findings and work is required
to understand the implications of these changes on the multiple processes in which the fibrinolytic system impacts.
Further reading:
1. Anfosso F, et al. J Clin Invest 1993;91(5):2185-93.
2. Mansfield MW, et al. Circulation 1996; 94: 2171-2176.
3. Juhan-Vague I, Alessi MC. Thromb Haemost 1997;78(1):656-60.
4. Dunn EJ, et al. Diabetologia 2006; 49(5):1071-80.
5. Hess K, et al. Diabetologia 2012;55(4):1103-13.
6. Ajjan RA, et al. Blood 2013;122(1):134-42.
18
2nd International Factor XIII Workshop
Abstracts
T2/4
FIBRINOLYSIS: THE MAIN ENEMY OF FACTOR XIII
Kolev K
Semmelweis University, Budapest, Hungary
Following cleavage of fibrinogen by thrombin, the generated fibrin monomers are assembled in longitudinal protofibrils, lateral
clusters and three-dimensional fiber network. The geometry at each of these multiscale levels is of primary importance for the
proper interpretation of the action of plasmin as a major protease that degrades the fibrin matrix. Plasmin cleaves the aC-domains
at first, followed by removal of a peptide from the N-terminal of the b-chain and cleavage of the coiled coil connector of the E- and
D-domains. It is important to appreciate that efficient disintegration of the fibrin meshwork requires this last cleavage to occur in the
same cross-section at all levels of protein chain assembly: all three polypeptide chains of the triple helical structure within a fibrin
monomer, both adjacent monomers within a protofibril and in all adjacent protofibrils within a fiber. Proteolytic resistance conferred
to fibrin by chemical modifications can be traced back to changes in clot morphology. Factor XIIIa (FXIIIa) is a transglutaminase of
plasma or platelet origin known to introduce g-glutamyl-lysine cross-links between gC and aC domains of adjacent fibrin monomers.
This covalent cross-linking results in a significant reduction (by about 20 %) of the fiber diameter without any change in the number
of protofibrils in a fiber, implying that FXIIIa tightens the lateral attachment of protofibrils and decreases the volume of the vacant
fluid space within the fibers. Such structural changes appear to be unfavourable for the action of plasmin limiting the possibility for
intrafiber diffusion and altering the optimal distances between plasmin binding sites and susceptible peptide bonds with consequent
impaired lysis of cross-linked fibrin. The primary role of the steric structural factors in the lytic resistance of cross-linked fibrin is
supported by the differences in the fibrinolytic potency of full-length plasmin and its truncated variants miniplasmin that lacks
the K1-K4 domains and microplasmin that is composed of the catalytic domain only. The size and shape of the plasmin molecule is
optimal for lysing non-crosslinked fibrin resulting in a 2-4-fold higher catalytic efficiency than the truncated variants. The altered
structure of cross-linked fibrin, however, is more favourable for the smaller miniplasmin, which shows 2-fold higher efficiency than
plasmin on the FXIIIa-modified substrate. The single kringle domain of miniplasmin is essential to maintain its fibrinolytic potency,
because its loss in microplasmin is coupled to a 5-fold lower activity on cross-linked fibrin. An additional structural feature underlying
the lytic resistance of cross-linked fibrin is a 2-fold reduction of the pore size in the three-dimensional fiber network. In plasma
environment additional factors take over the leading role as a determinant of the lytic resistance conferred by FXIIIa. a2-Plasmin inhibitor can serve as a substrate of FXIIIa and its covalent attachment to fibrin appears to be indispensable for the lytic stabilization of
plasma clots. Depletion of a2-plasmin inhibitor eliminates the anti-fibrinolytic effect of FXIIIa and lysis rates are inversely correlated
with the amount of a2-plasmin inhibitor cross-linked to fibrin in plasma clots. In view of these effects of FXIII it is not surprising that
hyperfibrinolysis contributes to the bleeding trend in FXIII deficiency states. Improved understanding of the structural background
of the lytic resistance of crosslinked fibrin could provide grounds for the development of novel therapeutic strategies in bleeding and
thrombotic conditions.
19
Abstracts
2nd International Factor XIII Workshop
T3/1
RESIDENT TISSUE MACROPHAGES SECRETE PLASMA FXIII-A AND MAINTAIN THE PLASMA POOL
Beckers CML1, Simpson KR1, Griffin KJ1, Brown JM1, Cordell PA1, Smith KA1, KearneyMT1, Vacher J2, Alexander W3, Grant PJ1, Pease RJ1
Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds LS2 9JT, United Kingdom
Clinical Research Institute of Montreal, Department of Medicine, McGill University, Montreal, Quebec, Canada
3
Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
1
2
While it has been assumed that plasma (p)FXIII-A is secreted from the megakaryocyte/platelet lineage, pFXIII-A is conserved in
the Mpl-/- (thrombopoietin receptor knockout) mouse in which platelets and their precursors are depleted. To identify the FXIII-A
secretory cells, cre-recombinase expressing mice were crossed with mice floxed in coding exon 7 of the F13a gene. FXIII-A was
determined by the biotin-pentylamine assay and by immunoblotting. FXIII-A mRNA was assayed by RT-PCR. Floxed and recombined
F13a alleles in genomic (g)DNA were quantified by qPCR.
The myeloid-cre mouse lines, LysM-cre and CD11b-cre caused depletion of pFXIII-A, but had minimal effect on platelet FXIII-A,
suggesting a macrophage origin for pFXIII-A. Pf4-cre depleted platelet FXIII-A, but, unexpectedly, it also extensively depleted
pFXIII-A and FXIII-A mRNA in heart, aorta and brain. FXIII-A mRNA in these tissues was depleted in the myeloid cre mice but
unaffected in the Mpl-/- mouse, confirming that the Pf4-cre/FXIII-A-expressing cells were not of megakaryocytic origin, but were
macrophages. Assay of mRNA from heart, cultured macrophages and isolated heart cells showed that the endogenous platelet factor
4 mRNA is expressed in macrophages, while assay of the recombined FXIII-A allele in gDNA indicated that Pf4-cre recombined within
≤5% of cardiac cells, which is again consistent with Pf4-targeting to macrophages. The pattern of depletion of pFXIII-A in the cre-lox
crosses mirrored the depletion of FXIII-A mRNA in tissues, particularly aorta, suggesting that cells resembling aortic macrophages
secrete pFXIII-A.
Transplantation of FXIII-A-positive bone marrow into irradiated FXIII-A-knockout mice restored pFXIII-A, to near physiological levels
within 10 weeks, and resulted in transcription of FXIII-A mRNA in heart and aorta. Transplantation of FXIII-A-positive bone marrow
into FXIII-A wild-type mice caused no increase in pFXIII-A above normal levels, which suggests that there is a fixed number of niches
within which the FXIII-A secretory macrophages reside.
20
2nd International Factor XIII Workshop
Abstracts
T3/2
TRANSGLUTAMINASES IN HUMAN CORNEA AND LENS
Muszbek L1,2, Orosz ZZ1,3, Bárdos H4, Shemirani AH2, Vereb G5, Hidasi V3, Nagy B6, Kappelmayer J6, Ádány R4, Berta A7, Facskó A3
University of Debrecen, Debrecen, Hungary: 1Division of Clinical Laboratory Science, 2Vascular Medicine, Thrombosis and Hemostasis
Research Group of the Hungarian Academy of Sciences, 4Department of Preventive Medicine, 5Department of Biophysics and Cell
Biology, 6Department of Laboratory Medicine, 7Department of Ophthalmology; University of Szeged, Szeged, Hungary: 3Department of
Ophthalmology
Transglutaminases (TGs) catalyze an acyl transfer reaction in which a peptide bound glutamine is the acyl donor and a primary amine
is the acyl acceptor. If the primary amine is the ε-amino group of a peptide bound lysine residue the end result is the cross-linking of
two peptide chains. Although the major role of active TGs is the cross-linking of proteins they very likely also have other functions,
independent of protein cross-linking. Among the 8 known TGs factor XIII (FXIII), TG-1 and TG-3 present primarily in keratinocytes,
and the rather ubiquitous TG-2 are the most well characterized enzymes. In this study the occurrence of TGs was investigated in
human cornea and lens. In the cornea the cellular form of coagulation factor XIII (cFXIII; FXIII-A2), was detected in stromal CD34+
keratocytes by immunofluorescence. Neither other investigated TGs nor the carrier B subunit of plasma FXIII was present in these
cells. Flow cytometry revealed that 84% of isolated corneal cells were CD34+ keratocytes and two third of them were FXIII-A+. These
cells were particularly abundant in the subepithelial region of corneal stroma. cFXIII was of cytoplasmic localization, occasionally
it also appeared in the nucleus. 2.87 ng cFXIII/mg corneal protein was measured by quantitative Western blotting. The synthesis
of cFXIII by these cells was confirmed by RT-qPCR. Recombinant cFXIII promoted the proliferation of corneal epithelial cells, which
suggests that cFXIII released from keratocytes is involved in corneal wound healing. Another TG (TG-2), but not FXIII was detected
in human lens. It was localized to the epithelial cell layer on the anterior lens surface and to a thin stripe between the capsule and
the peripheral cortex on the posterior surface. It is suggested that TG-2 strengthens the lens tissue by covalent cross-links.
21
Abstracts
2nd International Factor XIII Workshop
T3/3
FACTOR XIII IN TEARS AND ITS POSSIBLE ROLE IN CORNEAL WOUND HEALING
Orosz ZZ1,2, Szöôr Á3, Veréb Z1, Hassan Z4, Katona É2, Vereb G3, Facskó A1, Muszbek L2,5
Department of Ophthalmology, University of Szeged, Szeged, Hungary
Division of Clinical Laboratory Science, Department of Laboratory Medicine, University of Debrecen, Debrecen, Hungary
3
Department of Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary
4
Orbident Health and Lasercenter, Debrecen, Hungary
5
Vascular Biology and Thrombosis, Hemostasis Research Group of the Hungarian Academy of Sciences, University of Debrecen,
Debrecen, Hungary
1
2
Purpose. The effect of different factor XIII (FXIII) concentrations was investigated on wound healing, in vitro on corneal epithelial
cells and in vivo in tears of patients following corneal surgeries with different types of wound: phacoemulsification, penetrating
keratoplasty (PKP) and photo-refractive keratectomy (PRK).
Methods. Scratch-wound assay, proliferation and migration assays were applied to detect the effect of FXIII on wound healing of
immortalized corneal epithelial cells. Using a hypersensitive chemiluminescent ELISA method, developed in our laboratory, FXIII
complex and subunits were detected in tears of patients before and after different surgical interventions of the cornea, and post
surgical angiogenesis and re-epithelization was observed.
Results. The addition of recombinant cellular FXIII (cFXIII, rFXIII-A2) resulted in a concentration dependent faster healing of the
scratch wound. rFXIII-A2 promoted the proliferation of corneal epithelial cells, but no effect on migration was observed. After corneal
surgeries FXIII complex and subunit concentrations increased in tears, then decreased reaching the normal interval at different
times after the surgical intervention. After cataract surgery, FXIII concentrations correlated with the inflammation of the eye and
the corneal oedema. Slower re-epithelisation of the corneal surface after PRK associated with lower FXIII concentrations. Extremely
high FXIII concentrations measured in a few cases after PKP was associated with neovascularization of the normally avascular cornea.
Conclusion. According to our in vitro and in vivo investigations, FXIII present in tear proteome has a beneficial effect on corneal
re-epithelisation, which results in decreased period of complaints caused by the corneal erosion. FXIII might be considered as an
additional therapy in the treatment of corneal erosions, but long exposition to high FXIII concentrations in tears might induce
undesired angiogenesis of the cornea.
22
2nd International Factor XIII Workshop
Abstracts
T4/1
THE ACTION OF FXIII MADE VISIBLE IN A MICROVASCULAR FLOW MODEL
Jenny L1, Kohler HP1,2, Schroeder V1
1
2
Department of Clinical Research, University of Bern, Bern, Switzerland
Department of Internal Medicine, Tiefenauspital, Inselgruppe, Bern, Switzerland
Background: In vitro laboratory assays are important to characterize biochemical processes and to investigate their underlying
mechanisms, but they do not reflect well the in vivo situation. We have set up a microvascular flow model that better reflects
physiological conditions. At the same time the model is flexible enough to test a variety of triggers and inhibitors of coagulation
activation and other factors possibly influencing clot formation. The model combines the following features: endothelial cells, whole
blood, vessel structure with a diameter of 50 μm (similar to human arterioles), and physiological shear rates. It allows to study clot
formation and vessel occlusion in real time under the microscope under conditions close to physiological.
Aim: Our aim is to visualize the crosslinking actions of FXIIIa during clot formation and identify relevant influencing factors.
Methods: We manufacture the chip devices from poly-dimethyl-siloxane. The channels are coated with fibronectin, seeded with
endothelial cells and perfused with endothelial cell growth medium for 24-48 hours, until the cells have grown to a confluent
monolayer. For a perfusion experiment, the channels are perfused with freshly drawn, recalcified citrated whole blood containing
fluorescent labelled antibodies and/or labelled fibrinogen and labelled FXIII peptide substrates. Clot formation is observed in realtime using a confocal fluorescent microscope.
Results and conclusions: After recalcification of the whole blood, clot formation starts after 15-20 min. The forming fibrin and
incorporation of specific FXIII peptide substrates can be observed. Details in clot structure as well as the breakdown of the clot by
the fibrinolytic system can also be detected. Experiments with a FXIII inhibitor and labelled antibodies to detect incorporation of
physiological FXIII substrates into the clot are underway. This microvascular flow model is suitable to study in real-time the multiple
functions of FXIII during clot formation.
23
Abstracts
2nd International Factor XIII Workshop
T4/2
FACTOR XIII LABORATORY TESTING: LESSONS FROM THE ECAT EQA PROGRAMME
Meijer P
ECAT Foundation, Voorschoten, The Netherlands
Since 2009 the ECAT Foundation, an international EQA provider in the field of thrombosis and haemostasis, has provided on a regular
basis surveys for Factor XIII (FXIII), both functional and immunological testing. Currently the number of participants in this survey
is approximately 145 laboratories. For functional testing the majority of participants (approx. 75%) perform a quantitative test, while
20-25% perform a qualitative test. Only a minority of the participants (approx. 20%) perform immunological testing.
For qualitative functional testing up to 25% of the participants are not able to detect an abnormal FXIII level in a sample with less
than 2% FXIII. This lack of sensitivity shows that qualitative FXIII testing is unsuitable for the detection of severe FXIII deficiencies.
For quantitative functional testing the vast majority (> 90%) identified a sample with a FXIII level > 50% as normal. On the other
hand, samples with a FXIII level < 50% are identified by the vast majority (> 95%) as abnormal.
The between-laboratory variation varied from 10 - 100%, depending on the level of FXIII in the sample. A major problem is the
accurate measurement of samples with a FXIII level < 2%. Results up to 20% FXIII are reported for these type of samples. Also a
substantial number of participants use a lower limit of detection up to 15%, which makes it impossible to measure severe deficiencies.
For quantitative functional testing the Siemens Berichrom assay is the predominantly used method. It is well known that this method
needs an additional blank correction to measure accurate in the low range. In our surveys we ask the participants whether they apply
blank correction or not. Unfortunately we could not observe any improvement in the accuracy of measurement when participants
indicate to use blank correction.
Also for immunological testing a wide range in the between-laboratory variation is observed. However, these tests are much more
accurate in testing samples with a very low FXIII level. The mean value for samples with a FXIII level < 2% is indeed 2% or less.
Although ECAT is now providing these external quality control surveys for almost 7 years no improvement in the accuracy of functional
FXIII testing is observed. The lack of accurate measurement in the very low range may hamper the proper diagnosis of severe FXIII
deficiencies.
24
2nd International Factor XIII Workshop
Abstracts
T4/3
CORRELATION OF FXIII ACTIVITY WITH LABORATORY PARAMETERS THAT ASSESS THROMBIN GENERATION
AND CLINICAL PARAMETERS IN HEMOPHILIA A PATIENTS
Milos M1, Coen Herak D1, Zupancic-Salek S2, Zadro R1,3
Department of Laboratory Diagnostics, University Hospital Centre Zagreb, Croatia
Department of Medicine, University Hospital Centre Zagreb, Croatia
3
Faculty of Pharmacy and Biochemistry, Zagreb, Croatia
1
2
Although hemophilia A (HA) patients usually form initial hemostatic plugs in response to injury, these primary clots are not able to
maintain hemostasis and lead to extended bleeding. FXIII, the activation of which closely depends on thrombin generation, is one
of the most important parameters that influences fibrin clot structure and stability. The purpose of this study was to find out if FXIII
activity significantly correlates with laboratory parameters that assess thrombin generation and with the most important clinical
parameters (age at first joint bleed, number of joints with hemophilic arthropathy, number of annual joint bleeds and annual FVIII
consumption) in order to get information of possible FXIII influence on presentation of bleeding. The study group comprised of 81 HA
patients, 37 severe and 44 non-severe HA patients. Clinical parameters were collected from medical records. FXIII activity (Berichrom
FXIII) and Endogenous Thrombin Potential Test (setting ETP-C) were performed on BCS-XP, whereas aPTT waveform analysis (Actin FS)
was performed on BCS. Prothrombin fragment F1+2 concentrations (PF1+2) were measured by ELISA (Enzygnost F1+2). All reagents and
analyzers were from Siemens Healthcare Diagnostics (Marburg, Germany). Significantly lower FXIII activities (P=0.014) were identified
in severe (90.0±21.5%) than in non-severe HA patients (103.8±27.2%). No correlation was found between FXIII and ETP-C (P>0.05
for all ETP parameters) and waveform parameters (P>0.05 for all waveform parameters) as well as PF1+2 (P=0.228). Correlation study
with clinical parameters revealed only weak correlation of FXIII activity with age at first joint bleed (r=0.231, P=0.043). The observed
lower FXIII activities in severe HA patients can contribute to accelerated fibrin clot lysis resulting in more pronounced fibrinolysis and
bleeding phenotype. No correlation of FXIII activity with laboratory parameters that assess thrombin generation can be explained by
the type of FXIII measurement and, to obtain more informative data, a method that indicates activation of FXIII should be applied.
25
Abstracts
2nd International Factor XIII Workshop
T4/4
MONOCLONAL ANTIBODY DIRECTED AGAINST THE CROSSLINKED FIBRIN NEOEPITOPE; “DD-XLINK-MAB”
Pasternack R
ZEDIRA, Darmstadt, Germany
Since decades, coagulation factor XIII (FXIII) is considered as a scientific qualified target for the development of novel anticoagulants.
Inhibiting FXIII may lower the current bleeding risk in anticoagulation therapy since neither the thrombin level nor the platelet
activation is affected.
Zedira develops small molecule inhibitors to modulate FXIII activity by structure-assisted drug design. To assess the efficacy in blood,
we aimed to generate a monoclonal antibody directed against the isopeptide bond (“crosslink”) within fibrin as preferred biomarker.
Monoclonal “D-dimer” antibodies (e.g. DD-3B6/22) are commercially available and are used in In Vitro Diagnostics (IVD) to exclude
thromboembolic events. However, these monoclonals do not detect the crosslink itself but address a portion of polypeptides within
the D-domain after plasmin degradation that are conformationally reactive. This hinders for example standardization and comparison
of data obtained by different assays. Therefore, we developed a monoclonal antibody (DD-XLink-mab) which directly recognizes the
cross-linked fibrin neo-epitope.
26
2nd International Factor XIII Workshop
Abstracts
T5/1
PLASMA FXIII, BUT NOT PLATELET FXIII, PROMOTES RED BLOOD CELL RETENTION IN CONTRACTED CLOTS
Kattula S1, Byrnes JR1, Bagoly Z2, Muszbek L2, Wolberg AS1
Department of Pathology and Laboratory Medicine and McAllister Heart Institute, University of North Carolina at Chapel Hill, USA
Division of Clinical Laboratory Science, Department of Laboratory Medicine and Thrombosis, Haemostasis and Vascular Biology Research
Group of the Hungarian Academy of Sciences, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
1
2
Background: Venous thrombi have a characteristic fibrin- and red blood cell (RBC)-rich composition. We previously demonstrated that
factor XIII (FXIII) promotes RBC retention in contracted clots by crosslinking fibrin. Delayed or inhibited FXIII activation reduces
clot RBC retention and consequently, thrombus size. However, the relative contributions of FXIII found in plasma and cells, including
platelets, to clot contraction and RBC retention is unknown. Moreover, the plasma FXIII Val34Leu polymorphism, which results in
accelerated FXIII activation, has not been studied in a whole blood system.
Methods: Murine RBCs were reconstituted with FXIII-sufficient or -deficient plasma and platelets. Human RBCs and platelets were
reconstituted in plasma with or without Leu34 polymorphism. Clotting was triggered and clot weights were analyzed.
Results: Presence or absence of FXIII in platelets did not alter the weight of contracted whole blood clots. Irrespective of the platelet
FXIII compartment, clots formed in the absence of plasma FXIII were smaller than clots formed in the presence of plasma FXIII.
In reactions containing FXIIIVal/Val in plasma, increased age, FXIII activity, and thrombin generation parameters were positively
associated with clot weight. However, presence of the Leu allele mitigated this effect. In the presence of FXIIIVal/Val, activated
partial thromboplastin time and prothrombin time were negatively associated with clot weight; presence of the Leu allele diminished
this effect on the activated partial thromboplastin time, but not the prothrombin time.
Conclusions: Plasma FXIII, but not platelet FXIII, promotes RBC retention in contracted whole blood clots. The Leu34 polymorphism
influences whole blood clot weight through complex gene-plasma environment interactions.
27
Abstracts
2nd International Factor XIII Workshop
T5/2
FACTOR XIII ACTIVITY PLAYS A ROLE IN THE RATE OF FIBRIN FORMATION
Ali M1, Pease R1, Hardy L1, Howell G2, Law G3, La Corte AC1, Hethershaw E1, Duval C1, Ridger V4, Weisel JW5, Grant PJ1, Ariëns RAS1,
Philippou H1
Division of Cardiovascular and Diabetes Research, MCRC, Leeds Institute of Cardiovascular & Metabolic Medicine, University of Leeds,
Leeds, UK
2
Leeds Bio-imaging and Flow Cytometry Group, University of Leeds, UK
3
Division of Biostatistics, Leeds Institute of Cardiovascular & Metabolic Medicine, University of Leeds, Leeds, UK
4
Department of Cardiovascular Science, Faculty of Medicine, Dentistry and Health, University of Sheffield, Sheffield, UK
5
University of Pennsylvania, Philadelphia, USA
1
The transglutaminase factor XIII (FXIII) once activated by thrombin is known to crosslink fibrin and alpha-2-antiplasmin to fibrin, to
make it more stable and resistant to fibrin breakdown by fibrinolysis. Our work has shown that FXIII activity also plays a role in the
rate at which fibrin forms in vitro and in vivo. We have established a human whole blood flow model of thrombosis where we monitor fibrin development in real time, and we have demonstrated delayed fibrin deposition in the presence of a small molecule inhibitor of FXIII activity. Furthermore, using an in vivo model of thrombosis we demonstrate that mice deficient of FXIII A subunit show
delayed fibrin clot formation that is corrected to normal rates upon the infusion of recombinant FXIII A subunit. Our data indicate
that FXIII activity plays an important role in the development phase of the forming fibrin clot.
28
2nd International Factor XIII Workshop
Abstracts
T5/3
FACTOR XIII ACTIVATION PEPTIDE – A SHORT PEPTIDE WITH MULTIPLE FUNCTIONS.
Kohler HP
Department of Clinical Research, University of Bern, and Department of Internal Medicine, Spital Netz Bern Hospitals, Tiefenauspital, Bern,
Switzerland
The factor XIII (FXIII) activation peptide (AP-FXIII) raised a lot of interest when we first showed that the common FXIII Val34Leu
polymorphism, located in AP-FXIII three amino acids upstream the thrombin cleavage site at Arg37, was associated with a lower risk
of myocardial infarction.
Nevertheless, the role of AP-FXIII was disputed for some time since analyses of the crystal structures of zymogen and activated
FXIII-A gave conflicting results on whether AP-FXIII release would occur during or even be necessary for the activation of FXIII-A.
With specific monoclonal antibodies we have shown that AP-FXIII is indeed released into plasma upon cleavage by thrombin and
we could quantify free AP-FXIII in plasma samples from patients with acute stroke. Free AP-FXIII, whether circulating in plasma or
associated with a fibrin clot, may also have regulatory effects by reducing further FXIII activation and fibrin crosslinking. In addition,
AP-FXIII may be even more important for the stability of the FXIII-A2 dimer than anticipated. FXIII-A variants with deletions of the
first 11 amino acids or longer were not expressed on protein level. In silico calculations indicated that AP-FXIII plays a substantial
role in inter-subunit interactions in FXIII-A2 homodimers. These results point towards a major role of AP-FXIII in dimer stability. This
is also supported by the finding of several mutations leading to congenital FXIII-A deficiency that potentially disturb the correct
positioning of AP-FXIII and thereby may weaken or even disrupt FXIII-A2 homodimer interactions. Taken together, the presence of
AP-FXIII has both structural and functional implications for FXIII stability and activation.
29
Abstracts
2nd International Factor XIII Workshop
T5/4
FACTOR XIII AND VASCULAR SMOOTH MUSCLE CELLS
Bogáti R1, Katona É1, Balogh E2, Jeney V2, Muszbek L1
Division of Clinical Laboratory Science, Department of Laboratory Medicine, Faculty of Medicine
Department of Internal Medicine, Clinical Centre, University of Debrecen, Debrecen, Hungary
1
2
Background: Factor XIII (FXIII), a protransglutaminase, circulates in the plasma as heterotetramer of FXIII-A and FXIII-B subunits
(FXIII-A2B2; pFXIII). The dimer of FXIII-A (cellular FXIII; cFXIII) is also present in platelets, monocytes, macrophages, osteoblasts
and chondrocytes. pFXIII plays an important role in blood coagulation, angiogenesis, wound healing and maintaining pregnancy.
cFXIII has been implicated in phagocytosis, cell differentiation, formation and assembly of extracellular matrix. Its role in the
mineralization process has also been suggested. During atherosclerosis vascular smooth muscle cells can proliferate and go through
osteoblastic transformation.
Aims: 1/ To investigate if cFXIII is expressed during the osteoblastic transformation of human aorta derived smooth muscle cells
(HAoSMC). 2/ To investigate the effect of extracellular FXIII on vascular smooth muscle cells.
Methods: Osteoblastic transformation of HAoSMC was induced by Pi and Ca2+ containing medium. FXIII-A in the cell lysate was
investigated by ELISA and Western blot. Cell proliferation and migration were measured by EZ4U or CCK-8 cell proliferation assay kits
and CytoSelect 24-Well Wound Healing Assay, respectively. Cell migration was monitored by Juli Stage Real Time Cell History Recorder.
Thrombospondin-1 levels were measured by ELISA.
Results: FXIII-A could not be detected in differentiated HAoSMCs. Activated recombinant cFXIII (rFXIIIa), but not the non-activated
form, increased cell proliferation in concentration dependent manner. Its effect in the wound healing assay was even more considerable.
The time to reach 30% and 80% confluence was decreased to less than 1/7 and 1/3 by the addition of 20 µg/mL cFXIIIa. In parallel
highly significant decreases in thrombospondin-1 concentration was measured.
Conclusion: During the course of plaque hemorrhage FXIII can be activated and exert its effect on vascular smooth muscle cells. This
effect might be important in the pathogenesis of atherosclerotic plaques.
30
2nd International Factor XIII Workshop
Abstracts
T5/5
FXIII-A AND TRANSGLUTAMINASE 2 JOINTLY PROTECT THE MYOCARDIUM AGAINST FIBROSIS
Beckers CM1, Newell LM2, Drinkhill MJ1, Griffin KJ1, Cheah L1, Simpson KR1, Brown JM1, Jackson CL2, Pease RJ1, Grant PJ1
Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds LS2 9JT, United Kingdom
Bristol Heart Institute, Bristol, United Kingdom
1
2
Despite postulated roles for FXIII-A and transglutaminase 2 in cardiovascular protection, we have observed only minor changes in
CaCl2-induced aortic aneurysm development in TG2-/-/FXIII-A-/- double knockout mice, and no exacerbation of sinus or brachiocephalic
atherosclerosis in fat-fed male apoE-/-/TG2-/-/FXIII-A-/- triple knockout mice. However the fat-fed triple knockouts developed extensive
myocardial fibrosis, in contrast to apoE-/-/TG2-/- or apoE-/-/FXIII-A-/- double knockout mice. Myocardial fibrosis in ageing male FXIII-A-/mice was previously ascribed to extravasation of blood resulting from injury.
To characterize the accelerated, spontaneous fibrosis in transglutaminase-deficient mice, collagen and hemosiderin deposition were
quantified in C57BL/6 wild-type, FXIII-A-/- knockout, TG2-/- knockout and FXIII-A-/-/TG2-/- double knockout mice. Hearts were examined
at baseline (8 weeks) and following 12 weeks of a normal chow or high-fat diet. Cardiac fibrosis was already apparent in FXIII-A-/-/
TG2-/- double knockout mice at baseline, showing that early fibrosis was not due to increased vessel permeability previously associated
with apoE deficiency. Fibrosis progressed at a similar rate in fat-fed or chow-fed male double knockout mice, and was present, but
reduced, in female double knockout mice. Fibrosis was greatly reduced in FXIII-A-/- single knockout mice and essentially absent in
thrombocytopenic (Mpl-/-) mice, despite their comparable clotting deficiency. Hemosiderin deposition showed similar differences
between genotypes. Mice remained apparently healthy and cardiac pressure-volume parameters after were essentially normal except
for a small increase in left ventricular stiffness, consistent with the onset of fibrosis.
Deficiencies in specific clotting factors cause myocardial fibrosis, independent of their effect on haemostasis, suggesting that these
factors, including FXIII-A, stabilize the microvasculature against leakage or local rupture. TG2 has been proposed to be pro-fibrotic
but in this context protects against fibrosis, possibly by further stabilizing the microvasculature. In summary, the double knockout
model of fibrosis is tolerable and may prove suitable for developing therapies to reverse cardiac fibrosis in human disease.
31
Abstracts
2nd International Factor XIII Workshop
T5/6
FACTOR XIII DEFICIENCY ENHANCES THROMBIN GENERATION DUE TO IMPAIRED FIBRIN POLYMERIZATION
- AN EFFECT CORRECTED BY FACTOR XIII REPLACEMENT
Pitkänen H H1,2, Jouppila A1, Lemponen M3, Ilmakunnas M2, Ahonen J2,4, Lassila R3
Helsinki University Hospital Research Institute
Helsinki University, Division of Anaesthesiology, Department of Anaesthesiology, Intensive Care and Pain Medicine, University of
Helsinki and Helsinki University Hospital
3
Coagulation Disorders Unit, Department of Haematology, Comprehensive Cancer Center, and HUSLAB and Laboratory Services HUSLAB,
University of Helsinki and Helsinki University Hospital
4
Maternity Hospital, Helsinki University Hospital, Helsinki, Finland
1
2
Introduction: Factor XIII (FXIII) cross-links fibrin completing blood coagulation. Congenital FXIII deficiency is managed with
plasma-derived FXIII (pdFXIII) or recombinant FXIII (rFXIII) concentrates.
Aim: As the mechanisms protecting patients with low FXIII levels (<5 IU/dL) from spontaneous bleeds remain unknown we assessed
the interplay between thrombin generation (TG), fibrin formation and clot kinetics before and after FXIII administration in three
patients with FXIII deficiency.
Methods: Patients received either rFXIII (35 IU/kg, A-subunit) or standard pdFXIII at 1250 IU or 2500 IU (12-30 IU/kg) monthly.
Thrombin generation, thromboelastometry (ROTEM), prothrombin fragments F1+2, fibrinogen and FXIII activity were measured at
baseline and after a one-hour recovery.
Results: FXIII:C was at the target level of 20±6 IU/dL at the 4-week trough. rFXIII corrected FXIII:C to 98±15 IU/dL and high-dose
pdFXIII to a level of 90±6 IU/dL, whereas low-dose/half dose pdFXIII reached 45±4 IU/dL. Although fibrinogen (Clauss Method)
was normal, FIBTEM was impaired, however FXIII administration showed a tendency to correct this impairment. Fibrinolysis was not
enhanced, whereas F1+2 levels exceeded normal levels. Simultaneously CAT measured 1.6- to 1.9-fold enhanced TG, which normalized
after FXIII administration. Inhibition of fibrin polymerization by the Gly-Pro-Arg-Pro peptide mimicked FXIII deficiency in CAT and
enhanced TG both in control and FXIII recovery plasma.
Conclusion: Overall baseline FIBTEM implied a lack of fibrinogen, but was improved by FXIII, consistent with its role of stabilizing
fibrin in whole blood. The fibrin polymerization defect associated with enhanced TG, possibly aiding haemostasis in FXIII deficiency.
32
2nd International Factor XIII Workshop
Abstracts
T5/7
PROLONGED WOUND HEALING DUE TO MODERATE FXIII DEFICIENCY IN A PATIENT WITH FEMORAL
FRACTURE
Bronic A
Clinical Institute of Chemistry, Medical School University Hospital “Sestre Milosrdnice”, Zagreb, Croatia
The deficiency of FXIII, the final enzyme of a clotting cascade, can be congenital or acquired due to numerous reasons: liver disease,
autoimmune disorders, disseminated intravascular coagulation (DIC), etc. It is not understood whether FXIII low activity associated
with some of the above conditions actually contribute to clinical bleeding. A wound healing complications were reported in a case
following femoral fracture surgery.
A 91 year old male, long standing cardiac patient with femoral fracture was referred to our hospital. Upon admission, platelet count
(Plt), fibrinogen (3.9 g/L), prothrombin time (PT:0.88) and activated partial thromboplastin time (aPTT:26.5 sec) were found to be
normal. His current therapy was amended by pacemaker and after stabilisation of the vital functions surgery under total anaesthesia
was performed. Due to circulatory instability and blood loss, the patient was compensated by RBC concentrate (1820 ml), 6% HAES
and crystalloids. In spite of careful monitoring, in the early postoperative period decline in Plt count, prolongation of coagulation
times (PT:0.51; aPTT:34.3 sec), subcutaneous haematoma and profuse wound bleeding occurred and the patient developed DIC.
However, compensation of RBC, albumin and antithrombin were replenished to mitigate the loss and hemodynamic parameters were
stabilized on the 14th day. Although coagulation times were recovered (PT:0.61; APTT: 29.5 sec), wound healing was still slow and
spontaneous bleeding occurred at two points within 1 cm distance. Therefore, coagulation factor activities were determined and low
FXIII activity (0.55 IU/mL) was recorded. Following 5-day administration (200-300 mL of FFP/24h) an increase in FXIII activity (0.82
IU/mL) was recorded as well as a substantial reduction of the hematoma’s size and wound bleeding.
A moderate reduction of FXIII secondary to DIC led to wound healing complications in this case. Preoperative assessment of factors
related to wound repair such as FXIII may be important in surgery patients, especially those with certain comorbidities.
33
Abstracts
2nd International Factor XIII Workshop
T6/1
CONGENITAL FACTOR XIII DEFICIENCY – UNRESOLVED PROBLEMS REGARDING NOMENCLATURE, DIAGNOSIS,
AND THERAPY
Schroeder V
Department of Clinical Research, University of Bern, Bern, Switzerland.
The present recommendations published on behalf of the Factor XIII and Fibrinogen subcommittee by Kohler et al. J Thromb Haemost
2011 contain the classification of FXIII deficiencies and the recommended diagnostic algorithm with a quantitative functional assay
as first-line screening test. However, this publication is difficult to find amidst the abundance of publications on this topic, and it
does not contain any recommendations regarding management and treatment. The biggest problem, however, is that the recommended
diagnostic algorithm cannot be implemented in many countries with restricted healthcare resources which have the highest incidence
of congenital FXIII deficiency. As a consequence, clot solubility assays are still widely used despite of their problems and even genetic
analyses are performed for diagnosis, which are not recommended as first-line tests. Efforts are underway to put together a team of
FXIII experts and clinicians, including clinicians from countries with restricted healthcare resources, to develop novel guidelines that
offer several diagnostic options and also include recommendations on management of FXIII deficiencies in various clinical situations.
Special efforts should be made to actively disseminate the novel guidelines by approaching haemophilia organisations and clinicians
(including haematologists, pediatricians, gynecologists, surgeons).
34
2nd International Factor XIII Workshop
Abstracts
T6/2
IRANIAN COHORT, FIRST REPORT OF THE FACTOR XIII DEFICIENCY IN SISTAN AND BALOUCHESTAN
PROVINCE OF IRAN
Naderi M1, Hosseini S2, Eshghi P3, Malek F4, Dorgalaleh A5
Departement Of Pediatrics Hematology & Oncology, Ali Ebn-e Abitaleb Hospital Research Center For Children And Adolescents Health
[RCCAH], Zahedan University of Medical Sciences, Zahedan, IR Iran
2
Aliasghar Children Hospital, Laboratory, Iran University of Medical Science Tehran, Iran
3
Pediatric Congenital Hematologic Disorders Research Center, Mofid Children’s Hospital, Shahid Beheshti University of Medical Sciences,
Tehran, Iran
4
Pediatric Respiratory Diseases Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti
University of Medical Sciences, Tehran, Iran
5
Hematology and Blood Transfusion Department of Hematology and Blood Transfusion School of Allied Medicine Iran University of Medical
Sciences, Tehran, Iran
1
Congenital FXIII deficiency (FXIIID) is a rare bleeding disorder. Hence, this cohort study was designed to assess the prevalence of
FXIIID, molecular basis as well as clinical manifestations of FXIIID in Sistan and Baluchistan Province in Iran. Hereby we report the
first comprehensive cohort study report about the demographic information of FXIIID in the S&B province. All laboratory phenotype
and genotype data, and also the clinical and treatment information on all patients of FXIIID diagnosed and followed-up were
collected from 2010 to 2016.
Data on 410 patients with FXIII deficiency were collected. The mean age of patients was 12 years (range: 1 months to 55 years),
with 67% of patients less than age 30. Patients were divided equally between both sexes. 380 patients were on prophylaxis with 10
IU/Kg of Fibrogammin with 28 days interval, noteworthy to mention, none of the patients experienced any life-threatening bleeding
event. Sixty-three bleeding episodes in patients on prophylaxis were detected, 70% was lower extremity ecchymoses, 25% was oral
cavity bleeding and 5% of bleeding manifestations was menorrhagia. Prophylaxis interval of 5 patients was reduced to 21 days due to
bleeding events. All patients had p.Trp187Arg mutation, 35% of patients had an affected relative. Factor level activity in all patients
was less than 5% and more to add all patients were categorized in level 3 of bleeding severity.
Seventy patients underwent minor and major surgery without any significant event. Furthermore, 32 successful deliveries were
reported, that consisted of 17 cases of normal delivery and 14 cases of Caesarian section.
Factor XIII deficiency is a rare hemorrhagic disorder, with the highest worldwide prevalence in Sistan and Baluchestan Province in
southeast of Iran detecting Trp187Arg mutation in all patients reveals a valuable demographic point.
35
Abstracts
2nd International Factor XIII Workshop
T6/3
CLINICAL AND MOLECULAR CHARACTERISTICS OF PATIENTS AFFECTED BY CONGENITAL FXIII DEFICIENCY
IN PAKISTAN
Borhany M, Fatima N, Abid M, Shamsi TS
National Institute of Blood Diseases and Bone Marrow Transplantation, Karachi, Pakistan
Introduction: Deficiency of coagulation factor XIII (FXIII) belongs to the rare bleeding disorders and its incidence is higher in
populations with consanguineous marriages. The aim of this study was to characterize patients and relatives from twelve families with
suspected FXIII deficiency from Pakistan and to identify the underlying mutations.
Material and Methods: FXIII deficient patients, enrolled at National Institute of Blood diseases and Bone Marrow Transplantation
were included in the study. Informed consent was obtained from all patients and relatives according to a protocol approved by the
local ethics committees. The patients’ medical histories were recorded in a questionnaire. As a first indicator of FXIII deficiency, a 5M
urea clot solubility test was used. Plasma FXIII A- and B-subunit antigen levels were determined by ELISA. FXIII activity was measured
with an amine incorporation assay. Sequencing of all exons and intron/exon boundaries of F13A was performed.
Results: We analyzed 12 families in which 20 patients were FXIII deficient. In these patients we identified 7 missense mutations
[(c.1126 T>C, p.Trp375Arg), (c.1040C>A, p.Ala346Asp), (c.1241 C>T, p.Ser413Leu), (c.782G>A, p.Arg260His), (c.562T>C, p.Trp187Arg),
(c.749T>C, p.Leu249Pro), (c.732C>A, p.Asp243Glu)]. 4 splicing mutations [(c.2045 G>A), (c.1460+1G>A), (IVS11+1G>A)] and one
nonsense mutation (c.567T>A, p.Cys188X). Wound healing, bleeding after injury (80%), umbilical cord bleeding (55%), abortions and
menorrhagia in females (40%) were the main clinical manifestations followed by haematoma, bruises (35%), circumcision (29%), bum
bleeding (25%) and joint bleeding (20%).
Conclusion: A Cohort of 39 individuals from 12 families in which 20 were severely FXIII deficient and 10 were FXIII deficiency carrier
was analyzed. We identified 10 mutations in these families leading to congenital FXIII deficiency.
36
2nd International Factor XIII Workshop
Abstracts
T6/4
PROSPECTIVE EVALUATION OF BLEEDING INCIDENCE IN FACTOR XIII DEFICIENCY (PRO-RBDD STUDY)
Palla R1, Menegatti M1, Boscarino M2, Halimeh S3, Lachamann B3, Borhany M4, Naveena F4, Ozdemir N5, Siboni SM2, Mikovic D6,
Saracevic M6, Mumford A7, Harvey A7, Schutgens REG8, van Haaften-Spoor M8, Chapin J9, Hsu F9, Platokouki H10, Pergantou H10,
Yilmaz A11, Shapiro AD12, Williamson A12, De Moerloose P13, Casini A13, Payne J14, Muszbek L15, Peyvandi F1,2
Department of Pathophysiology and Transplantation, Università degli Studi di Milano, and Luigi Villa Foundation, Milan, Italy
A. Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy
3
Coagulation Center Rhine Ruhr, Duisburg, Germany
4
National Institute of Blood Disease & Bone Marrow Transplantation, Karachi, Pakistan
5
Department of Pediatric Hematology, Cerrahpasa Medical Faculty of Istanbul University, Istanbul, Turkey
6
Haemostasis Department, Blood Transfusion Institute of Serbia, Belgrade, Serbia
7
Bristol Haematology and Oncology Centre, Bristol, United Kingdom
8
Van Creveldkliniek, Universitair Medisch Centrum, Utrecht, The Netherland
9
Weill Cornell Medical College, New York Presbyterian Hospital, New York, NY, USA
10
Haemophilia Centre/Haemostasis Unit, Agia Sofia Children’s Hospital, Athens, Greece
11
Dr. Behcet Uz Children’s Hospital, Izmir, Turkey
12
Indiana Hemophilia and Thrombosis Center, Indianapolis, IN, USA
13
Division of Angiology and Haemostasis, University Hospitals of Geneva, Geneva, Switzerland
14
Department of Haematology, Sheffield Childrens Hospital, Sheffield, United Kingdom.
15
Division of Clinical Laboratory Science, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen,
Hungary
1
2
Background: The PRO-RBDD registry was designed to prospectively collect data on patients with factor (F)XIII deficiency. Data
retrieved at patient enrollment identified a new FXIII activity cut-off of 15% with a very high sensitivity and a promising capability
to identify patients who may develop a spontaneous major bleeding (results under submission).
Aims: The aim of the analysis herein reported was to evaluate the benefits and complication of current treatment regimens.
Methods: Since February 2013, 13 Hemophilia Treatment Centers collected data on 60 FXIII-deficient patients (32 females; 28 males),
updating data at least biannually over three years. Bleeding incidence was calculated. A survival analysis was used to evaluate the
cumulative incidence of the first bleeding treated with replacement therapy.
Results: Patients were followed up for a median of 528 days (IQR: 185-790). Bleeding incidence of 1.19 patient-year-1 in on-demand
therapy and 0.14 in prophylaxis (9-59 U/Kg/month FXIII concentrate) was calculated, with a significant reduction of bleedings in
prophylaxis (IR=0.12, IC95% 0.09-0.15). A clear decrease of bleeding cumulative incidence using prophylaxis was observed (46%,
95%CI 13-68 on demand vs 5%, 95%CI 0-10 on prophylaxis).
Similarly, in patients with FXIII activity <15%, the bleeding incidence was 1.94 patient-year-1 in on-demand therapy and 0.15 in
prophylaxis, with a significant reduction of bleedings in prophylaxis (IR=0.08, IC95% 0.06-0.10). The risk of bleeding requiring
replacement therapy is significantly lower in patients on prophylaxis (cumulative incidence: 67%, 95%CI 20-84 on demand vs 5%,
95%CI 0-15 on prophylaxis).
No adverse events were observed.
Conclusion: The PRO-RBDD is the first prospective registry in the field of RBDs collecting data on a large group of FXIII deficiency
patients observed in a long follow-up period. The beneficial role and the safety of FXIII prophylaxis to prevent bleeding episodes is
clearly confirmed, making this treatment strongly recommendable, in particular in severe deficiency.
37
Abstracts
2nd International Factor XIII Workshop
T6/5
MINIMUM FACTOR XIII CLOTTING LEVEL TO PREVENT MAJOR SPONTANEOUS BLEEDINGS: RESULTS FROM
THE PROSPECTIVE DATABASE ON FACTOR XIII DEFICIENCY (PRO-RBDD)
Marzia Menegatti1, Roberta Palla1, Marco Boscarino2, Paolo Bucciarelli2, Susan Halimeh3, Britta Lachmann3, Christoph Bidlingmaier4,
Helen Platokouki5, Helen Pergantou5, Simona M. Siboni2, Roger E.G. Schutgens6, Munira Borhany7, Naveena Fatima7, Danijela Mikovic8,
Marko Saracevic8, Philippe De Moerloose9, Alessandro Casini9, Nihal Ozdemir10, Yilmaz Ay11, Andrew Mumford12, Andrew Harvey12,
Jeanette Payne13, Amy D. Shapiro14, Adrianna Williamson14, John Chapin15, Fraustina Hsu15, Laszlo Muszbek16, Flora Peyvandi1, 2
Department of Pathophysiology and Transplantation, Università degli Studi di Milano, and Luigi Villa Foundation, Milan, Italy;
Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy;
3
Coagulation Center Rhine Ruhr, Duisburg, Germany; 4Pediatric Hemophilia and Thrombosis Centre, Dr von Hauner’s Children’s University
Hospital, Munich, Germany; 5Haemophilia Centre/Haemostasis Unit, Agia Sofia Children’s Hospital, Athens, Greece; 6Van Creveldkliniek,
Universitair Medisch Centrum, Utrecht, Netherlands; 7National Institute of Blood Disease & Bone Marrow Transplantation, Karachi,
Pakistan; 8Haemostasis Department, Blood Transfusion Institute of Serbia, Belgrade, Serbia; 9Division of Angiology and Haemostasis,
University Hospitals of Geneva, Geneva, Switzerland; 10Department of Pediatric Hematology, Cerrahpasa Medical Faculty of Istanbul
University, Istanbul, Turkey; 11Dr. Behcet Uz Children’s Hospital, Izmir, Turkey; 12Bristol Haematology and Oncology Centre, Bristol,
United Kingdom; 13Department of Haematology, Sheffield Childrens Hospital, Sheffield, United Kingdom; 14Indiana Hemophilia and
Thrombosis Center, Indianapolis, IN, USA; 15Comprehensive Center for Hemophilia and Coagulation Disorders, Weill Cornell Medicine,
New York Presbyterian Hospital, New York, NY, USA; 16Division of Clinical Laboratory Science, Faculty of Medicine, University of Debrecen,
Debrecen, Hungary
1
2
Congenital factor XIII (FXIII) deficiency (FXIII CD) is a rare bleeding disorder associated with a significant bleeding phenotype;
patients with the severe deficiency suffer from early umbilical bleeding, delayed wound healing, soft tissue or subcutaneous bleeding,
spontaneous central nervous system bleeding and recurrent abortion in women. From 5 to 7% of FXIII coagulant activity levels have
been previously reported as a target trough level to obtain a satisfactory hemostasis. The European Network of Rare Bleeding Disorders
(EN-RBD) has described a method for classifying FXIII CD according to bleeding severity and FXIII activity and has reported a strong
association between these two parameters; in addition this study described patients with bleeding history despite FXIII activity
levels between 5 to 30%. Given the clinical heterogeneity in such patients, exhibiting a wide range of FXIII activity levels, more data
are needed to define which minimum FXIII activity level is required to prevent spontaneous major bleedings. One of the aims of the
PRO-RBDD was to identify a cut-off of FXIII activity level which discriminates patients with severe bleeding manifestations (spontaneous major bleeding) from those with minor or no bleeding, in a real-life representative patient population. With a cross-sectional
design, baseline data were analyzed in 64 patients affected with FXIII CD; patients were divided in four groups with different bleeding
severity according to EN-RBD (asymptomatic, grade I, grade II and grade III). For the purpose of this study, the first 3 groups were
combined in a single one, which was compared to the grade III group (patients with spontaneous major bleeding). The relationship between FXIII activity levels (kept continuous) and grade III bleeding (outcome) was assessed by a logistic regression model,
adjusting for sex and age. A restricted cubic spline function was used to check whether there were non-linear relationships between
FXIII activity and bleeding severity. In addition, ROC curve analysis was used to assess the optimal cut-off of FXIII activity level as a
predictive value to prevent spontaneous major bleeding. The results confirmed that FXIII coagulant activity is correlated with clinical
severity defined as age at first bleeding, age at diagnosis and severity of bleeding history. In addition, 15 IU/dl of FXIII activity was
identified as the minimum hemostatic FXIII level that should be achieved to prevent spontaneous major bleeding.
38
2nd International Factor XIII Workshop
Abstracts
T6/6
RECOMBINANT FXIII FOR PROPHYLAXIS AND TREATMENT IN CONGENITAL FXIII DEFICIENCY: A CASE
REPORT
Schlammadinger Á1, Árokszállási A1, Kerényi A2, Katona É3, Bereczky Zs3, Muszbek L3, Boda Z1
Thrombosis and Haemostasis Centre, Institute of Medicine, University of Debrecen, Debrecen, Hungary
Institute of Laboratory Medicine, University of Debrecen, Debrecen, Hungary
3
Clinical Research Centre, University of Debrecen, Debrecen, Hungary
1
2
Congenital FXIII deficiency is a rare but severe bleeding disorder, associated with high risk of intracranial haemorrhage. Once the
diagnosis is established, prophylaxis is indicated. Recombinant FXIII-A (rFXIII-A) is a new option for prophylaxis in FXIII-A
deficiency. However, data about its therapeutic application are limited.
A young man was diagnosed with severe congenital FXIII-A deficiency at the age of 19. His case history included umbilical stump
bleeding, gastrointestinal bleeding, traumatic soft palate bleed, subdural haematoma and repeated joint and intramuscular bleeds.
Plasma FXIII activity and FXIII-A antigen were <1% (normal range: 69-143% and 64-136%, respectively). rFXIII-A prophylaxis was
planned. In November 2013, before the initiation of prophylaxis, a huge haematoma in his right thigh developed causing intense
pain and decreased mobility. rFXIII-A was applied in the dose recommended for prophylaxis: 35 IU/kg. FXIII activity raised to 70%
and the clinical symptoms significantly improved by the next day. He regained his full mobility by the fourth day. A painful tooth was
extracted on the sixth day (FXIII activity 32%) without additional factor supplementation.
Since December 2013 he is on regular prophylaxis with rFXIII in a dose of 35 IU/kg administered every four weeks. During this time
no spontaneous bleeding occurred. A traumatic left knee bleed was successfully treated with a dose of rFXIII. In the past 2.5 years
no inhibitor developed. FXIII through levels are between 3-9%, peak levels are 64-96%.
Recombinant FXIII-A is safe and efficacious for the prophylaxis and treatment of congenital FXIII-A deficiency. More data are needed
to determine the exact dose needed for the treatment of the different bleeding events.
39
Abstracts
2nd International Factor XIII Workshop
T6/7
BLEEDING EPISODES IN HETEROZYGOTES OF FACTOR XIII DEFICIENCY
Naderi M1, Eshghi P2, Malek M3, Karbasian F2, Dorgalaleh A4
Departement Of Pediatrics Hematology & Oncology, Ali Ebn-e Abitaleb Hospital Research Center For Children And Adolescents Health
[RCCAH], Zahedan University of Medical Sciences, Zahedan, IR Iran
2
Pediatric Congenital Hematologic Disorders Research Center, Mofid Children’s Hospital, Shahid Beheshti University of Medical Sciences,
Tehran, Iran
3
Pediatric Respiratory Diseases Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti
University of Medical Sciences, Tehran, Iran
4
Hematology and Blood Transfusion Department of Hematology and Blood Transfusion School of Allied Medicine Iran University of Medical Sciences, Tehran, Iran
1
Introduction. The aim of this study was to assess the occurrence of bleeding episodes and explore the relationship between factor
activity and bleeding severity in the heterozygous carriers of FXIII deficiency (FXIIID).
Method: In this prospective study from 2013 to 2016, we assessed 50 females heterozygous for FXIII deficiency based on genetic
analysis. Twelve of 50 cases had recurrent abortions so, data included demographic, coagulation factor activity level, and lifelong
bleeding histories, including type of bleeding, and bleeding scores were retrieved.
Result: The mean age of patients at data collection was 32.67 (SD=7.25) years (range from 22 to 45 years). Five patients had
1 abortion, 6 patients had 2 and 1 patient had 3 abortions respectively. Seven patients had 1 affected child and 5 patients had
2 affected children. All patients except one had positive family history, 6 patients (50%) had one affected relative, 4 of them had 2
and 1 had 3 affected relatives respectively.
All patients except one had positive consanguinity history. Factor activity levels of patients were from 35% to 60% with mean of
49.4%. The age of the first bleeding episode was between at birth to 18 years old. Bleeding score of patients was between 3 to 6 with
4.42 mean (SD=0.79). There was no correlation between factor activity level and the age of when first bleeding score was noticed
(P=0.673). Strong association between bleeding score and factor activity level were detected (P=0.001).
Conclusion: Examining clinical data revealed that 12 patients had bleeding symptoms, manifesting as postpartum hemorrhage,
menorrhagia, recurrent abortions and umbilical bleeding. Our data indicate that as a challenging finding the heterozygotes of FXIIID,
could experience bleeding episodes upon provocation.
40
2nd International Factor XIII Workshop
Abstracts
T6/8
THE DIAGNOSIS AND CHARACTERIZATION OF ALLO-, OR AUTOANTIBODIES AGAINST FACTOR XIII SUBUNITS
Muszbek L1,2, Pénzes K1, Katona É1, Bereczky Z1, Kerényi A3, Rázsó K4, Kun M1, Boda Z4, Vezina C5, Rivard GE5
University of Debrecen, Debrecen, Hungary: 1Division of Clinical Laboratory Science, 2Vascular Medicine, Thrombosis and Hemostasis
Research Group of the Hungarian Academy of Sciences, 3Department of Laboratory Medicine, 4Department of Internal Medicine,
Debrecen, Hungary;
5
CHU Sainte-Justine, Montréal, QC, Canada
Antibodies against a coagulation factor may develop in patients deficient for the respective clotting factor (alloantibody) or in
patients without factor deficiency (autoantibody). In both cases the management of patients becomes extremely difficult and fatal
outcome is not rare. The antibodies can be classified as neutralizing, non-neutralizing and combined type. The former one interferes
with the process of factor activation or with the activity of activated clotting factor or exerts multiple inhibitory effects. The nonneutralizing antibody simply binds to the protein and accelerates its elimination. In the case of FXIII the antibody might be directed
against either of the FXIII subunits. Evidently, anti-FXIII-B antibody could only be of non-neutralizing type. During the last years
we diagnosed four patients with anti-FXIII antibodies and characterized the antibodies. Based on this experience the following
methodology is recommended. For non-neutralizing antibodies the binding of patient’s isolated IgG to purified FXIII species (FXIIIA2B2, FXIII-A2 and FXIII-B2) should be assessed using the following techniques: surface plasmon resonance, thermophoresis, ELISA,
Western/dot blotting. The advantage of the former two techniques is the determination of affinity constant. These binding assays
should also be used in the case of neutralizing antibodies. In addition to the establishment of the binding, the accelerated clearance
of administered FXIII concentrate should also be demonstrated. For the detection of neutralizing antibody a mixing study, and for
its semi-quantitative measurement a Bethesda/Nijmegen type inhibitor assay is recommended. The strength of the inhibitory effect
should be quantitatively assessed by IC50 determination. The effect of patient’s IgG on the thrombin cleavage of FXIII-A is monitored
by Western blotting. The inhibitory effect of the patient’s IgG on Ca2+ induced activation and on fully activated FXIII (FXIIIa) is
established by activity measurement of thrombin cleaved FXIII and FXIIIa, respectively. Characterization of the antibodies might lead
to new therapeutic approaches.
41
Abstracts
2nd International Factor XIII Workshop
T7/1
REGULATION OF PLASMA FXIII ANTIGEN LEVELS IN HEALTHY INDIVIDUALS
Katona É1, Mezei ZA1, Kállay J1, Bereczky Zs1, Kovács B1,2, Ajzner É3, Muszbek L1
Division of Clinical Laboratory Science, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
Borsod-Abaúj-Zemplén County Hospital and University Teaching Hospital, Miskolc, Hungary; 3András Jósa Szabolcs-Szatmár-Bereg County
Hospital and University Teaching Hospital, Nyíregyháza, Hungary
1
2
Background: Coagulation factor XIII (FXIII) is a tetramer of two catalytic FXIII-A and two protective/inhibitory FXIII-B subunits
(FXIII-A2B2). FXIII-A and FXIII-B are synthesized at different locations, they form 1:1 complex in the plasma. Ninety-nine % of
FXIII-A subunits are in complex, while FXIII-B is present both in complex and in free form. Complex formation significantly prolongs
the half-life of FXIII-A in the circulation and prevents its spontaneous activation. Most recently we developed new ELISA methods
for the determination of total and free FXIII-B (tFXIII-B, fFXIII-B), which together with the FXIII-A2B2 ELISA allow the measurement
of different FXIII forms.
Aims: To determine the reference interval of tFXIII-B and fFXIII-B. To investigate the effect of FXIII-B p.His95Arg and FXIII-B nt
29756 C>G polymorphisms in intron K on the level of FXIII-A2B2, total and free FXIII-B levels in plasma samples of healthy individuals.
Materials and methods: Two-hundred sixty-eight apparently healthy Hungarians were enrolled in the study. FXIII-A2B2, tFXIII-B and
fFXIII-B concentrations were measured by one-step sandwich ELISA methods. FXIII-B polymorphisms were determined on LightCycler
480.
Results: The reference interval was 15-30 mg/L (67-133%) for tFXIII-B and 5.5-14.9 mg/L for fFXIII-B. FXIII-A2B2 and tFXIII-B
levels showed positive correlation with age (r=0.356, and r=0.351 respectively, p<0.0001), while there was no significant correlation
between fFXIII-B and age. In carriers of FXIII-B p.His95Arg polymorphism tFXIII-B (107.9±18.1% vs. 114.0±17.4%; p=0.007) and
FXIII-A2B2 levels (103.8±23.3% vs. 114.5±25.0%; p=0.001) were elevated, while fFXIII-B levels were not influenced. In the plasma
of carriers for FXIII-B intron K C>G polymorphism FXIII-A2B2, tFXIII-B and fFXIII-B levels were significantly decreased as compared
to wild type individuals (108.8±24.3% vs. 96.9±18%, p<0.0001; 111.6±17.3% vs. 101.0±15.8%, p<0.0001; 9.16±0.18 mg/L vs.
8.29±0.30 mg/L, p=0.010).
Conclusions: Polymorphisms of FXIII-B subunit considerably modify the level of complexed and free FXIII-B levels and the ratio of
bound to free FXIII-B in the plasma.
42
2nd International Factor XIII Workshop
Abstracts
T7/2
FUNCTIONAL VARIANTS OF FXIII - WHAT HAVE WE LEARNED SO FAR
Ariëns RAS
Thrombosis and Tissue Repair Group, Division of Cardiovascular and Diabetes Research, University of Leeds, Leeds, United Kingdom
FXIII is one of the most polymorphic proteins, with both the A- and B-subunits containing a number of commonly occurring mutations.
Polymorphisms that change the primary amino acid sequence have been reported for FXIII-A and FXIII-B, and polymorphisms
associated with splice variation have been reported for FXIII-B. Some of these polymorphisms have a clear effect on the function
of FXIII. In addition, associations with thrombosis and bleeding disorders have been reported. However, clear causal links between
polymorphisms, biochemical effect and clinical outcome remain elusive. One of the FXIII-A polymorphisms, Val34Leu, which has the
clearest functional effect of increasing FXIII activation by thrombin, has been associated with altered risk of myocardial infarction,
venous thrombosis, coronary artery disease and other diseases associated with haemostasis and thrombosis, but not in the way as
expected based on its biochemical effects. A recent murine study confirmed its biochemical phenotype also in vivo. Also, a recent
study showed synergism between Val34Leu and FXIII-B intron K nt29756 polymorphism (associated with splice variation), involving
a reduction in FXIII plasma levels. Further studies are required to investigate the functional effects of this polymorphism further,
including studies involving other substrates of FXIII. An example of a FXIII-B subunit polymorphism involves His95Arg. This common
mutation occurs in the second Sushi domain of the B-subunit and has been reported to increase subunit dissociation while associating
with vascular disease and thrombosis. Again, clear causal relationships remain elusive. In conclusion, while we have learned a great
deal with regards to enzyme structure and function from the genetic polymorphisms of FXIII, the clinical significance remains unclear.
It is likely that the FXIII polymorphisms represent molecular evolution in progress, but there are currently no indications for using
these markers in a clinical setting.
43
Abstracts
2nd International Factor XIII Workshop
T7/3
THE INFLUENCE OF THE FXIII VAL34LEU POLYMORPHISM ON PLASMA CLOT STRUCTURE IN AFRICANS
Pieters M
Centre of Excellence for Nutrition, North-West University, Potchefstroom, South Africa
FXIII Val34Leu has been demonstrated to alter fibrin clot structure, in a fibrinogen concentration dependent manner, due to the
enhanced rate of activation peptide cleavage in the presence of the Leu34 allele. Its effect on clot structure is furthermore thought
to be influenced by gene-gene and gene-environment interactions. The influence of a common splice variant, fibrinogen g’, on the
effect of the FXIII Val34Leu on clot structure also remains to be determined. To this end we investigated the effect of FXIII Val34Leu
on plasma clot properties measured by turbidity in a population-based study of 2000 Africans. Interactions with fibrinogen gene
SNP’s, total and g’ fibrinogen concentration and other environmental factors were considered. Lagtime, slope, max absorbance and
clot lysis time were calculated from the turbidity curves. FXIII Val34Leu together with 2 coding fibrinogen SNP’s (FGAThr312Ala
and FGB Arg448Lys) were genotyped. Additional co-variates were obesity, metabolic syndrome, age, gender, HbA1c, HDL-C, Hcy
and CRP based on independent associations with clot properties. In unadjusted models, Leu34 allele carriers had significantly
longer lagtimes than Val34 homozygotes (p=0.02), also after adjusting for total and g’ fibrinogen respectively. FXIII Val34Leu
had a significant interaction with both absolute g’ fibrinogen and the g’ ratio in determining maximum absorbance. At lower g’
fibrinogen concentration, Leu34 homozygotes had lower maximum absorbance than Val34 homozygotes, but at higher concentrations
(above 0.4g/L) the Leu34 homozygotes had higher maximum absorbances. Compared to Val34 homozygotes, Leu34 homozygotes also
demonstrated a significantly steeper decline in max absorbance as the g’ ratio increased. This could be a result of increased local
FXIII concentration due to the binding of the FXIIIB subunit by g’ fibrinogen as well as combined effects on the intrafibrillar structure
of fibrin. FXIII Val34Leu also showed an interaction with the FGAThr312Ala SNP in determining lagtime. Carriers of the Leu34 allele
had longer lagtimes than Val34 homozygotes in the presence of Thr312 homozygotes and heterozygotes, but in the presence of the
ALA312 homozygotes, they had shorter lagtimes. This could be related to the FGAThr312Ala SNP being in the FXIII binding region on
the αC-domain with resultant increased α-chain crosslinking. Our data demonstrates that in a population-based study, FXIII Val34Leu
interacts with plasma g’ concentration and FGAThr312Ala in determining plasma clot properties.
44
2nd International Factor XIII Workshop
Abstracts
T7/4
GENDER SPECIFIC EFFECT OF THE FXIII-A VAL34LEU POLYMORPHISM ON THE RISK FOR CHILDHOOD AND
PERINATAL ARTERIAL ISCHEMIC STROKE
Coen Herak D1, Ceri A2, Grzunov A3, Lenicek Krleza J3, Radic Antolic M1, Horvat I1, Djuranovic V4, Barisic Nina5, Zrinski Topic R6,
Zadro R1,2
Department of Laboratory Diagnostics, University Hospital Centre Zagreb
Faculty of Pharmacy and Biochemistry, Zagreb
3
Department of Laboratory Diagnostics, Children’s Hospital Zagreb
4
Department of Neuropediatrics, Children’s Hospital Zagreb
5
Division of Neuropediatrics, Department of Pediatrics, University Hospital Centre Zagreb
6
Department of Clinical Laboratory Diagnostics, Children’s Hospital Srebrnjak, Zagreb, Croatia
1
2
Introduction: Although recent meta-analysis indicated the lack of evidence for the association between FXIII-A Val34Leu polymorphism and ischemic stroke in adults, studies evaluating the relationship of this polymorphism with arterial ischemic stroke (AIS) in
children are scarce. Moreover, it has been consistently reported that AIS in children occurs more frequently in boys than in girls,
with no explanation up to date. The aim of this study was to investigate the gender-specific effect of FXIII-A Val34Leu on the risk
for childhood and perinatal AIS.
Material and methods: The study group comprised of 52 children (35 boys, 17 girls) with childhood AIS, 43 children (24 boys, 19
girls) with perinatal AIS aged ≤18 years, and 125 age- and sex-matched (84 boys, 41 girls) control subjects. A genotype analysis
of the FXIII-A Val34Leu polymorphism was performed using a commercial multilocus genotyping assay (CVD StripAssay, ViennaLab
Diagnostics, Vienna, Austria). Genotype and allele frequencies obtained in gender-specific AIS subgroups (childhood and perinatal)
were compared with frequencies obtained in gender-specific control subjects by using a dominant model.
Results: A gender-specific difference of FXIII-A Val34Leu genotype frequencies was found in children with AIS (P=0.044), whereas
this was not the case in control subjects (P=0.447). Besides, a statistically significant difference between FXIII-A Val34Leu genotype
distributions was observed in boys with childhood AIS compared to boys from control subjects (P=0.045), resulting in a modest
association of the presence of at least one FXIII-A Leu34 allele (OR=2.44; 95% CI=1.07-5.53). In contrast, this association was
found neither in boys with perinatal AIS (P=0.594; OR=1.27; 95% CI=0.51-3.15), nor in girls with childhood (P=0.265; OR=0.55; 95%
CI=0.17-1.72) or perinatal AIS (P=0.083; OR=0.36; 95% CI=0.11-1.14).
Conclusion: This case-control study has revealed the gender-specific effect of the presence of FXIII-A Leu34 allele, that is associated
with increased risk for childhood AIS in boys only.
45
Abstracts
2nd International Factor XIII Workshop
T7/5
THE EFFECT OF FXIII LEVELS AND FXIII-B POLYMORPHISMS ON THE RISK OF VENOUS THROMBOEMBOLISM
Mezei ZA1, Katona É1, Kállay J1, Bereczky Z1, Somodi L1, Kovács B1,2, Miklós T3, Ajzner É3, Muszbek L1
Division of Clinical Laboratory Science, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen,
Hungary
2
Borsod-Abaúj-Zemplén County Hospital and University Teaching Hospital, Miskolc, Hungary
3
András Jósa Szabolcs-Szatmár-Bereg County Hospital and University Teaching Hospital, Nyíregyháza, Hungary
1
Introduction: As FXIII plays an essential role in the protection of newly formed fibrin its association with the risk of thrombotic diseases
has been a subject of intensive research. However, the association of plasma FXIII level with the risk of venous thromboembolism
(VTE) is still controversial and the effect of the recently discovered polymorphism in FXIII-B Intron K (F13B:c.1952+144 C>G) leading
to a novel splice acceptor site and 25 new amino acids at the C-terminal end has not been investigated in this respect. In this study
we explored the association of FXIII levels and FXIII-B polymorphisms with the risk of VTE.
Subjects and methods: Two-hundred and twenty-eight VTE patients and 227 age and gender matched controls were enrolled in the
study. There was at least three months between the acute thrombotic event and the investigation. FXIII activity, FXIII-A2B2 antigen,
FXIII-B antigen levels and fibrinogen were measured and FXIII-B p.His95Arg and FXIII-B Intron K polymorphisms were determined.
Results: Adjusted FXIII activity and FXIII-A2B2 antigen levels were significantly higher in the VTE group than in controls. In contrast,
FXIII-B levels were significantly lower in the control group. The Arg95 allele was associated with elevated FXIII activity, FXIII-A2B2
and FXIII-B antigen levels in the control but not in the VTE group. FXIII-B Intron K G allele significantly lowered the FXIII activity,
FXIII-A2B2 and FXIII-B antigen levels in both groups. FXIII activity and FXIII-A2B2 antigen levels in the upper tertile as compared to
the lower tertile significantly increased the risk of VTE (adjusted OR:1.81 CI:1.12-2.93 and OR: 2.30 CI:1.41-3.74, respectively) the
increased risk was more pronounced in women than in men. FXIII-B polymorphisms did not influence the risk of VTE.
Conclusion: Elevated FXIII levels could be considered as risk factor of VTE. The FXIII-B polymorphisms are not associated with
VTE risk.
46
2nd International Factor XIII Workshop
Abstracts
T7/6
EFFECT OF FACTOR XIII LEVELS AND POLYMORPHISMS ON THE RISK OF MYOCARDIAL INFARCTION IN
YOUNG PATIENTS
Bereczky Z1, Mezei ZA1, Balogh L2, Katona É1, Fiatal S3, Ádány R3, Miklós T4, Kovács B5, Ajzner É4, Édes I2, Muszbek L1,6
Division of Clinical Laboratory Science, Department of Laboratory Medicine and 2Department of Cardiology, Faculty of Medicine,
Department of Preventive Medicine, Faculty of Public Health, University of Debrecen, Debrecen
4
András Jósa Szabolcs-Szatmár-Bereg County Hospital and University Teaching Hospital, Nyiregyháza,
5
Borsod-Abaúj-Zemplén County Hospital and University Teaching Hospital, Miskolc,
6
Vascular Biology, Thrombosis and Haemostasis Research Group of the Hungarian Academy of Sciences, Hungary
1
3
In our previous studies elevated factor XIII (FXIII) levels were associated with increased risk of myocardial infarction (MI) in females,
FXIII-A p.Val34Leu and IntronK (nt29756C>G; c.1952+144 C>G) polymorphism (IK) of FXIII-B subunit gene were protective against
MI in patients with elevated fibrinogen. All these investigations were carried out in elderly patients. The role of elevated FXIII levels,
FXIII-A and FXIII-B subunit polymorphisms in young MI patients has not been investigated, yet.
In this study FXIII activity and FXIII antigen concentration, the above-mentioned polymorphisms and FXIII-B p.His95Arg were
determined in 112 MI patients (MI+) below the age of 40 and in two age-matched control groups (n=100 clinical controls with
negative coronarography result, MI- and n=112 healthy controls, HC).
FXIII activity (MI+, 119±27%, MI-, 111±25% and HC, 108±24%) and FXIIIA2B2 antigen (24.3±6.0 mg/L, MI-, 22.6±5.2 mg/L and
HC, 21.2±5.0 mg/L) were significantly elevated in MI+ patients compared to both controls. After adjustments for fibrinogen and
smoking these differences were not significant. The minor allele frequencies of the FXIII-A p.Val34Leu, p.His95Arg and IK did not
differ markedly among the different study groups and were in good agreement with data obtained from the HapMap database. FXIII-A
p.Val34Leu did not influence FXIII activity and FXIIIA2B2 antigen concentration. Polymorphisms in the B-subunit, however, had
significant effects on both FXIII activity and FXIIIA2B2 antigen in the total group (n=324) even after adjustments. FXIII-B p.His95Arg
increased FXIII activity (112±25% and 119±25% for wild type and carriers, respectively), IK decreased it (117±25% and 105±24% for
wild type and carriers, respectively). FXIII-B IK decreased the risk of MI (OR MI+ vs MI-, 0.22, 95%CI, 0.06-0.84) in patients with
elevated fibrinogen level (above 3.7 g/L).
Our results confirm that the risk of MI is modified by IK even in young individuals with high fibrinogen and FXIII levels are influenced
by FXIII-B subunit polymorphisms.
47
Abstracts
2nd International Factor XIII Workshop
T8/1
FXIII LEVELS IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS AND ANTIPHOSPHOLIPID SYNDROME
Bagoly Z1, Tóth NK1, Tarr T2, Soltész P2, Katona E1, Mezei ZA1, Muszbek L1
Division of Clinical Laboratory Sciences, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen,
Hungary
2
Department of Clinical Immunology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
1
Background: Patients with systemic lupus erythematosus (SLE) are subject to significant morbidity and mortality due to atherosclerotic diseases, which cannot be fully explained by traditional risk factors. Antiphospholipid syndrome (APS) is characterized by
recurrent thrombosis and pregnancy morbidity in the presence of antiphospholipid antibodies (aPL). APS can be primary or secondary,
often associated with SLE.
Aims: To find out whether levels of factor XIII (FXIII) might differ in patients with SLE and/or APS as compared to healthy individuals
and if levels are associated with thrombotic episodes.
Patients and Methods: One-hundred and twenty-two patients with SLE and/or APS and 140 age and sex-matched healthy controls
were enrolled. Patients were grouped: SLE without APS (n=60), SLE with APS (n=16), primary APS (n=26), SLE with aPL without clinical symptoms of APS (n=16), aPL without clinical symptoms of APS (n=4). FXIII activity, FXIII-A2B2 antigen levels, FXIII-B subunit,
FVIII activity, von Willebrand factor (VWF) antigen levels were measured. Clinical parameters including age, sex, BMI, smoking habit,
traditional risk factors, thrombotic history and disease activity were registered.
Results: FXIII activity and FXIII-A2B2 antigen levels were significantly elevated in APS patients (primary or secondary) as compared
to controls. In SLE patients, regardless of the presence of APS, FXIII-B levels were significantly elevated. FXIII-A2B2 and FXIII-B levels
significantly correlated with C4 complement levels. As compared to controls, FVIII and VWF levels were highly elevated in all patient
groups except for aPL without clinical symptoms of APS. Among SLE patients, FXIII A2B2 levels were significantly elevated in those
with history of atherothrombosis, while no such association was found for FXIII-B levels. A FXIII level in the upper tertile conferred
a significant risk for arterial but not venous thrombotic events in SLE patients (OR:4.48; 95% CI:1.153-17.44, p=0.035).
Conclusions: Elevated FXIII levels are associated with increased atherothrombotic risk in SLE patients.
48
2nd International Factor XIII Workshop
Abstracts
T8/2
THE EFFECT OF HEMODIAFILTRATION AND HEMODIALYSIS ON COAGULATION PARAMETERS IN PATIENTS
WITH END-STAGE RENAL DISEASE
Pénzes K1, Becs G2, Hurják B1, Molnár E1, Katona É1, Balla J2, Muszbek L1
Division of Clinical Laboratory Science;
Debrecen, Debrecen, Hungary
1
2
Division of Nephrology, Department of Internal Medicine, Faculty of Medicine, University of
Changes in the hemostatic system might contribute to the highly increased risk of atherothrombotic complications in end-stage renal
disease (ESRD). Our aim was to test the level of certain hemostatic factors, which might be associated with the risk of thrombosis
in these patients. Thirty patients being on hemodiafiltration (HDF) treatment for at least one year were enrolled in the study and
their hemostasis parameters were measured before as well as one and four hours after the initiation of an actual HDF. Then, the
treatment was switched to hemodialysis (HD) for 2 weeks, after which period hemostasis parameters was measured according to
the same protocol. Factor VIII (FVIII) activity, antithrombin (AT) activity, and alpha2-plasmin inhibitor activity were determined
by chromogenic assays. Factor XIII (FXIII) activity and FXIII-A2B2 antigen were measured by ammonia release method and ELISA,
respectively. Fibrinogen was determined by Clauss method, immunoturbidimetry was used for the measurement of C reactive protein
(CRP). FVIII activity was above the reference interval in 46% and 23% of the patients being on HDF and HD treatment, respectively.
FVIII levels poorly correlated with CRP concentrations. Twenty-six % of HDF patients showed elevated FXIII levels and 17% of them
had decreased AT activity. Following HD treatment 43% of the patients had increased FXIII activity. The AT activity was decreased in
27% of the patients. Alpha2-plasmin inhibitor activity remained in the reference interval. Fibrinogen levels were slightly/moderately
elevated in half of the patients. When the above parameters were corrected for albumin only FXIII activities showed significant
elevation during the 4-h course of HDF or HD treatment. The elevated FVIII and FXIII levels and the decreased AT activity might
contribute to the increased atherothrombotic risk of ESRD patients. Evaluation of specific hemostatic parameters in these patients
could contribute to a more effective prevention of atherothrombotic events.
49
Abstracts
2nd International Factor XIII Workshop
T8/3
Intracellular factor XIII-A is a FLOW CYTOMETRIC diagnostic test in acute leukemias
Kappelmayer J
Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Hungary
The first identification of intracellular FXIII-A was described in platelets as early as 1955 and this was followed by the demonstration
of FXIII-A in monocytes 30 years later. The potential usefulness of FXIII-A as a marker for leukemias was suggested already soon
after this discovery, however the state-of-the art method is multi-color flow cytometry that requires suitably labelled monoclonal
antibodies.
In our flow cytometric studies in the past 12 years we utilized monoclonal antibodies that were successfully used for measuring
cellular FXIII-A content in cell lysates. First, we investigated the sensitivity and specificity of the FXIII-A labelling by studying
cytoplasmic staining of normal peripheral blood leukocytes and magnetically separated non-leukemic precursor cells. Cytoplasmic
FXIII-A was negative in lymphocytes and normal B-cell precursors and was positive in monocytes intracellularly but not on the cell
surface. In a series of studies when 3-8 color flow cytometry was used for the analysis of leukemic blasts, we introduced the directly
FITC-conjugated anti-FXIII-A antibody in our acute leukemia flow panel. FXIII-A positivity proved more sensitive in identifying
monocytic leukemias when compared to surface CD14 (63% versus 25%). In addition to this lineage-restricted expression, FXIII-A
was found as an aberrantly expressed marker and thus was suitable to be introduced as a leukemia associated immune phenotype in
a part of the acute promyelocytic leukemias, where FXIII-A negativity proved to be an unfavorable prognostic marker. Furthermore,
in 40% of acute lymphoblastic leukemic (ALL) blasts from pediatric ALL cases we could also identify FXIII-A by flow cytometry and
these results were confirmed by western-blot and confocal laser scanning cytometry. In these lymphoblasts the FXIII-A content was
found to be 3 fg/blast, a much lower value than obtained for monocytes and platelets. These results may further our understanding,
as the knowledge about the role of intracellular FXIII is rather limited.
50
2nd International Factor XIII Workshop
Abstracts
T8/4
FACTOR XIII-A POSITIVE MACROPHAGES AND CD11c POSITIVE DENDRITIC CELLS IN MALIGNANT MELANOMA
Bárdos H1, Törôcsik D2, Emri E2, Dezsô B3, Emri G2, Remenyik E2, Balázs M 1,4, Ádány R1,4
Department of Preventive Medicine, Faculty of Public Health, University of Debrecen, Debrecen, Hungary.
Department of Dermatology, Faculty of Medicine, Medical and Health Science Centre, University of Debrecen, Debrecen, Hungary
3
Department of Pathology, Faculty of Medicine, Research Center for Molecular Medicine, University of Debrecen, Debrecen, Hungary
4
MTA-DE Public Health Research Group of the Hungarian Academy of Sciences, University of Debrecen, Debrecen, Hungary
1
2
Factor XIIIA has been widely used as a dermal dendrocyte marker for histiocytic phenotyping of capillary hemangiomas, dermatofibromas
and tumors of the central nervous system. Moreover, recent studies have shown that FXIII-A positivity also clearly distinguishes
macrophages (CD163+/FXIII-A+) from dendritic cells (CD11c+/FXIII-A-) in the normal dermis just as in non-infectious, non-malignant
pathological conditions of the skin such as granuloma annulare and necrobiosis lipoidica.
The aim of the present study was to characterize and compare the distribution of macrophages and dendritic cells in benign nevi
(n=6), primary cutaneous malignant melanomas - non-metastatic (n=23) and with hematogenous metastasis (n=23) according to a
5 year follow up - with normal skin samples (n=6) by tissue microarray analysis using immunohistochemical detection for FXIII-A,
CD163 and CD11c on serial sections of tissue multiblocks derived from paraffin embedded normal skin samples.
Our results showed that while in the normal skin FXIII-A+/CD163+ cells were found scattered in the papillary and reticular dermis and
the presence of CD11c+ cells were minimal, the number of FXIII-A+/CD163+ but not of CD11c+ cells increased in the close vicinity
of nevi. In primary malignant melanomas a markedly strong proliferation of FXIII-A+ macrophages but not of CD11c+ dendritic cells,
were observed in the peritumoral stroma, in a strong correlation with the melanoma thickness. Importantly, thick melanomas (>1.00
mm) with hematogenous spread showed the strongest positivity for FXIII-A+ macrophages, which were detected mostly at the site of
invasion adjacent to tumor cells. Moreover, with an increase in size of primary malignant melanomas, FXIII-A+ macrophages appeared
intratumorally in association with tumor microvasculature.
These results suggest that the detection of FXIIIA+ macrophages in melanomas is a sensitive marker for tumor invasiveness and
angiogenesis, in which both the tumor environment as well as malignant melanocytes could play an important role.
51
Abstracts
2nd International Factor XIII Workshop
T8/5
EXPRESSION OF BLOOD COAGULATION FACTOR XIII-A IN LYMPHOBLASTS PREDICTS FAVOURABLE OUTCOME
IN PEDIATRIC PRECURSOR B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA
Kárai B1, Szánthó E1, Csáthy L1, Ujfalusi A1, Gyurina K2, Szegedi I2, Kappelmayer J1, Kiss Cs2, Hevessy Zs1
1
Department of Laboratory Medicine, 2Department of Pediatric Hematology and Oncology, Faculty of Medicine, University of Debrecen,
Hungary
Previously we identified B cell-lineage leukemic lymphoblasts as a new expression site for subunit A of blood coagulation factor XIII
(FXIII-A). The exact role of FXIII-A in malignant cells is still unclear but this aberrant expression may suggest the existence of a
prognostically different subgroup of B cell precursor acute lymphoblastic leukemia (BCP-ALL) upon the FXIII-A expression profile.
Fifty-five children with BCP-ALL were included in the study. Bone marrow samples were obtained by aspiration and the presence
of FXIII-A was detected by flow cytometry. G-banding and fluorescence in situ hybridization was performed according to standard
procedures. Survival analyses were done using the Kaplan-Meier survival estimator, survival curves were compared by log-rank test.
Hazard ratio (HR) and 95% CI were estimated by Cox proportional hazard model analysis.
The 10-year event-free survival (EFS) of FXIII-A positive and FXIII-A negative patients showed significant difference (84% vs. 61%,
respectively; p=0.031). This difference was more pronounced in overall survival (OS) (89% vs. 61%; p=0.008). Of all the parameters
examined, there was correlation only between FXIII-A expression and ‘B-other’ genetic subgroup. The multivariate logistic regression
analysis confirmed the association between the FXIII-A characteristics and the ‘B-other’ group (OR: 7.3; 1.8–29.9; p=0.007). Further
multivariate Cox regression analysis of ‘B-other’ group and FXIII-subtype adjusted for gender, age and WBC upon diagnosis identified
FXIII-A negative characteristic as an independent predictor for poor outcome in BCP-ALL (HR: 4.8; 1.2–19.7; p=0.03). The poor
outcome associated with FXIII-A negativity was not merely the result of its association with the ‘B-other’ characteristic, whose
negative effect on survival is well-known.
We found an excellent correlation between long-term survival and FXIII-A positive phenotype of lymphoblasts at presentation. The
results seem to be convincing enough to suggest a possible role of FXIII-A expression in prognostic grouping of childhood BCP-ALL
patients.
52
2nd International Factor XIII Workshop
Abstracts
T8/6
IMMUNOHISTOCHEMICAL DETECTION OF FACTOR XIII SUBUNIT A AS A DIAGNOSTIC TOOL IN DERMATOPATHOLOGY: BELIEFS AND DISBELIEFS
Törôcsik D1, Bárdos H2, Ádány R2
Departments of Dermatology and
Preventive Medicine, University of Debrecen, Debrecen, Hungary
1
2
Immunohistochemical detection of Factor XIII subunit A (FXIII-A) is used as a diagnostic marker in a wide range of dermatological
diseases ranging from inflammatory lesions to malignancies. Historically, in addition to monocyte-derived tissue histiocytes, dendritic
cells, the key antigen-presenting cells of the immune system were also considered to be positive for FXIII-A. However, keratinocytes
and sebocytes were also reported to express it. The aim of the presentation is therefore to provide a guide on the still controversial
interpretation of the dermal cell types expressing FXIII-A and the mechanism(s) behind the local accumulation of FXIII-A+ cells
both in physiological and pathological conditions of the human skin. On the basis of our findings it will also be demonstrated that
the presence of FXIII-A in skin lesions is not a disease-specific marker but indicates a possible common mechanism of macrophage
activation in various dermatological diseases.
53
2nd International Factor XIII Workshop
Participants
PARTICIPANTS
Name
COUNTRY
Ariëns, RAS
UK
Bádogos, Á
Hungary
Bagoly, Z
Hungary
Balogh, G
Hungary
Baráth, B
Hungary
Bárdos, H
Hungary
Bäuml, CA
Germany
Beck, A
Germany
Bereczky, Z
Hungary
Berezina, T
USA
Biswas, A
Germany
Bogáti, R
Hungary
Borhany, M
Pakistan
Bronic, A
Croatia
Bychkova, AV
Russia
Coen Herak, D
Croatia
de Lange, Z
South Africa
Driessler, F
Switzerland
Dudás, N
Hungary
Grant, PJ
UK
Gupta, S
Germany
Hevessy, Z
Hungary
Hiremath, VV
India
Hurják, B
Hungary
Imhof, D
Germany
Kappelmayer, J
Hungary
Kárai, B
Hungary
Katona, É
Hungary
Kerényi, A
Hungary
Kohler, HP
Switzerland
Kolev, K
Hungary
Komáromi, I
Hungary
Kun, M
Hungary
Marin, I
Switzerland
Meijer, P
The Netherlands
Menegatti, M
Italy
Mezei, Z
Hungary
Milos, M
Croatia
Muszbek, L
Hungary
Naderi, M
Iran
Orosz, ZZ
Hungary
Palla, R
Italy
Pasternack, R
Germany
Pease, RJ UK
Pénzes, K
Hungary
Philippou, H
UK
54
Presentation No.
T5/2, T7/2
T5/1, T8/1
T1/4
T3/2, T8/4, T8/6
T2/1, T2/2
T6/6, T6/8, T7/1, T7/5, T7/6
T1/1, T1/5, T2/1, T2/2,
T5/4
T6/3, T6/4, 6/5
T5/7
T1/3
T4/3, T7/4
Industrial session
T2/3, T3/1, T5/2, T5/5,
T1/1
T8/5
T8/2
T2/1, T2/2
T3/2, T8/3, T8/5
T8/5
T3/3, T5/4, T6/6, T6/8, T7/1, T7/5, T7/6, T8/1, T8/2
T6/6, T6/8
T4/1, T5/3
T2/4
T1/2, T1/4
T6/8
T4/2
T6/4, 6/5
T7/1, T7/5, T7/6, T8/1
T4/3
T1/2, T3/2, T3/3, T5/1, T5/4, T6/4, 6/5, T6/6,
T6/8, T7/1, T7/5, T7/6, T8/1, T8/2
T6/2, T6/7
T3/2, T3/3
T6/4, 6/5
T4/4
T3/1, T5/2, T5/5
T6/8, T8/2
T1/1, T1/5, T5/2
2nd International Factor XIII Workshop
Pieters, M
Pitkänen, HH
Schlammadinger, Á
Schroeder, V
Scully, M
Shemirani, AH Somodi, L
Stencel, D
Szabó, M
Törôcsik, D
Vasylieva, AD
Wolberg, A
Zadro, R
Zaets, S Zak, M
South Africa
Finland
Hungary
Switzerland
Canada
Hungary
Hungary
Switzerland
Hungary
Hungary
Russia
USA
Croatia
USA
Denmark
Participants
T7/3
T5/6
T6/6
T4/1, T6/1
T3/2
T7/5
T8/4, T8/6
T1/3
T5/1
T4/3, T7/4
55
Notes
56
2nd International Factor XIII Workshop