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Thrombin Cleavage Enhances Exposure of a Heparin Binding Domain in the
N-Terminus of the Fibrin p Chain
By Tatjana M. Odrljin, John R. Shainoff, Sarah 0. Lawrence, and Patricia J. Simpson-Haidaris
Thrombin (Ila)-cleavageof fibrinogen(FBG)t o form polymerized fibrin promotes
endothelial cellspreading, proliferation,
and vonWillebrand factor release, requiring theexposure of
the p15-42 domain. Studies reported here indicate that Ilacleavage of fibrinopeptide B enhances exposure of a heparin
bindingdomainatthe
/315-42 neo-N-terminusof fibrin.
Crossed immunoelectrophoresis showed heparin-induced
mobility shifts indicative of complexing with FBG and with
N-terminal CNBr fragments of FBG (NDSK) and offibrin (IlaNDSK), but no evidence of heparin complexing with FBG
lacking Bp1-42 or with FBG fragments D and E was seen.
Elution fromheparin-agarose with a linear gradient of NaCl
showed that bound portions
of both intact FBG and D fragments eluted below physiologic
salt
concentrations,
whereas E3 fragments lacking BPI-53 did not bind. NDSK
bound with higher affinity than did intact FBG, whereas
binding of Ila-NDSK was maximal in this system. Binding
of fibrin(ogen1 t o heparin agarose was saturable as well as
inhibitable in a dose-dependent manner with both FBG and
heparin. Scatchard analysis indicated a single class of binding site, with dissociation constants (kd) of 0.3 pmol/L for
Ila-NDSK, 0.8 pmol/L for NDSK, and 18 pmol/L for FBG.
Immobilized fibrin had twofold more heparin binding sites
than did immobilized FBG and required a 5.5-fold higher
concentration of heparin t o inhibit by 50% the binding of
labeled heparin. Together, the results indicatethat Ila-cleavage results in enhanced exposure of two heparin binding
domains (HBDs)with approximately threefold higher affinity
in fibrin thanin FBG. Synthetic peptidep15-42 showed highest binding t o heparin-agarose followed byBpl-42, whereas
peptides p18-31, p18-27, and p24-42 did not bind. Thus, the
primary structure ofp15-42 is required for specificity ofheparin binding. Basic residues within the p15-32 region segregate primarily to one side of an a-helix in a helical wheel
diagram, as is typical for
authentic HBDs. Desulfated heparin
and heparan sulfate bound morefibrin(ogen) than did other
proteoglycans; however, heparin bound sixfold more IlaNDSK than NDSK. Theseresults confirm thatfibrin binds t o
heparin with higher affinity than does FBG and that fibrin
binding is not solely dependent on charge interactions of
p15-42 with the negatively charged glycosaminoglycan.
0 1996 by The American Society of Hematology.
H
observations. First, thrombin (Ira)-cleavage of FBG to form
fibrin promotes endothelial cell (EC) spreading,"' proliferation," andvon Willebrand factor release.'* These interactions require the exposure of the p15-42 domain. Second,
both the primary structureI3 and secondary c~nformation'~
of p1S-42 are highly conserved across species, whereas the
fibrinopeptide B primary structures are not.I3 Third, an EC
surface glycoprotein of 130 kD was identified that binds to
synthetic peptide p IS-42, suggesting the presence of a receptor for fibrin on EC." Fourth, the C-terminal FN HBD interacts with heparansulfate proteoglycan syndecan 4 to mediate
focal adhesion formation by cells that are prespread on substrates with RGD-cell binding domains.*,' Finally, y-thrombin cleavage of the matrix protein vitronectin (VN) alters its
conformation to expose a masked HBD.".I7
Heparin has important clinical application in the prevention of thrombosis by modulating various enzymes of the
clotting and fibrinolytic
Moreover, heparan sulfate proteoglycans are essential cofactors in receptor-growth
factor interactions, cell-cell recognition systems, and cellmatrix adhesion processes.'~7.'6.'7,26-zK
Thus, knowledge regarding the location of an HBD exposed on fibrin may have
significant impact on the interpretation of studies regarding
the interaction of heparin with fibrin during clotting, fibrinolysis, or fibrin-cell interactions. The data presented here
support the hypothesis thatfibrin p15-42 HBDsplayan
active role in heparin or heparan sulfate modulation of the
hemostatic balance, cell-matrix interactions, andwound
healing.
EPARIN BINDING domains (HBDs) are found in a
number of adhesive glycoproteins involved in extracellular matrix-cell interactions, including fibronectin (FN),'"
laminin: thrombospondin,5*6and von Willebrand f a ~ t o r . ~
Specific heparin binding peptides of these glycoproteins promote cell adhesion, spreading, and focal contact assembly.'"
Only C-terminal BPIy chain fragments of fibrinogen (FBG)
were purported to contain HBDs.~We hypothesized that
HBDs reside in the central, N-terminal domain of fibrin(ogen) due to the clustering of basic residues within Bpl42 displaying a positive charge density similar to the HBD
consensus sequence.' Mohri et a1' were unable to detect an
HBD within Bpl-42, perhaps due to the presence of a V8
protease cleavage site between pGlu*' and PAlaZ6,the cleavage of which would destroy the integrity of a Bpl-42 HBD.
Therefore, we sought to determine whether the central domain of fibrin(ogen), particularly residues p15-42, constitutes a HBD.
The rationale for the present study was based on several
From the Hematology Unit, Department oj Medicine, University
of Rochester, School of Medicine and Dentistry, Rochester, NY; and
The Cleveland Clinic Foundation, Cleveland, OH.
Submitted October 16, 1995; accepted May 2, 1996.
Supported by Grants No. HL-50615, HL-30616, and HL-16361
from the National Institutes of Health (Bethesda, MD) and by a
grant from the University of Rochester Strong Children's Research
Center.
Address reprint requests to Patricia J . Simpson-Haidaris, PhD,
Hematology Unit, Department of Medicine, PO Box 610, 601 Elmwood Ave, Rochester, NY 14642.
The publication costsof this article were defrayed in part by page
chargepayment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8806-00I7$3.00/0
2050
MATERIALS AND METHODS
Materials. Human FBG was purchased from Kabi Vitrum
(Franklin, OH). Purified FBG lacking residues 1-42 of the BD chain
(desB@l-42-FBG,also known as FBG-325) was kindly supplied by
Dr A. Budzynski (Temple University, Philadelphia, PA). FBG plasmic fragments D and E were from Crystal Chemicals (Chicago,
Blood, Vol 88,No 6 (September 15), 1996 pp 2050-2061
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CRYPTICHEPARIN
BINDING DOMAINOF
2051
FIBRIN
Table 1. Structure of Different Fibrin(ogen1Fragments
Fragment
Chain Composition
Au1-610, FBG-325
NDSK
Ila-NDSK
71-411
hl-51,
h17-51,
hl-104,
Aa20-78,
Au105-206,
Aa105-206,
h105-206,
El
E3
Dl
D2
D3
6043-461,
Bp1-118,
Bp15-118,
661-133.
6054-133,
Bo134-461,
Bp134-461,
Bp134-461,
yl-78
71-78
71-62
71-53
786-406
786-356
786-302
Data from Hantgan et al.''
IL). See Table 1 for a summary ofthe structures of fibrin(ogen)
preparations and Table 2 for the fibrin(ogen)-specific monoclonal
antibodies (MoAbs) used in this study. Thrombin (human plasma,
3,554 NIH Ulmg) was from CalBiochem (San Diego, CA). Heparinagarose beads (750 to 1,OOO mg heparidml agarose, type II),heparin
sodium salt (porcine intestinal mucosa, grade 11, 162 USP U/mg),
heparin-bovine serum albumin (BSA)-biotin, heparan sulfate sodium
salt (bovine intestinal mucosa), greater than 99% de-N-sulfated heparin sodium salt, chondroitin sulfate C (shark cartilage), chondroitin
sulfate A (bovine trachea), and keratan sulfate (bovine cornea) were
all purchased from Sigma (St Louis, MO). Fibrin(ogen) BP chain
peptides FBG p24-42 (>99% purity) were from Bachem (Torrance,
CA); peptide p15-42 (>99% purity) was synthesized and purified
by Cornell Biotechnology Facility (Ithaca, NY); and peptides Bp1 42 (>95% purity), p18-27, and,BM-31 (each 100% purity) were
synthesized and purified by Louisiana State UMC (New Orleans,
LA). Laminin heparin binding peptide F9 (97% purity) was purchased from Sigma.
CNBr cleavage of jbrin(ogen). N-terminal disulfide knot
(NDSK) and IIa-cleaved NDSK (IIa-NDSK) fragments were prepared as de~cribed.~'IIa-NDSK was used to mimic soluble fibrin.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was performed as described."
Crossed immunoelectrophoresis (CIEP). Trace antibodies to FN
and to other serum proteins were removed from polyclonal antibody
(PoAb) antihuman FBG by affinity chromatography over a column
of human serum proteins supplemented with purified human FN
coupled to CNBr-Sepharose CL-4B. After protein A-Sepharose
chromatography, thepurified
IgG was designated PoAb antiFBG'").
Agarose gels (1% in 25 mmol/L Tricinel80 mmol/L Tris,
pH 8.6/1% calcium lactate/O.O2% sodium azide) were poured on
GelBond film (FMC BioProducts, Rockland, ME). Each well was
loaded with fibrin(ogen) preincubated for 60 minutes at room temperature (RT) with or without heparin (as described in the figure
legend^).^." Electrophoresis in the first dimension was at 250 V
constant voltage in 0.02 mol/L barbital buffer, pH 8.6, for 1 hour.
Each sample lane cut from the first dimension gel was applied to
the cathode side of the second dimension gel consisting of 1% agarose with PoAb anti-FBG'"'
at l mg/mL gel. The proteins were
electrophoresed into the second dimension at 250 V for 20 hours.
Saturation and inhibition of binding of fibrin(ogen) to heparinagarose. FBG, NDSK, and IIa-NDSK were labeled with canierfree Na[lZ51](Dupont-NEN, Boston, MA) using Iodo-Gen (Pierce
Chemical, Rockford, IL).33The specific activity varied between 2.5
and 92.4 pCi/nmol. The binding assay was performed essentially as
de~cribed.'~
Nonspecific binding was determined in the presence of
100 molar excess heparin or unlabeled FBG. Specific binding was
expressed after the subtraction of nonspecific binding from the total
binding: free ligand was calculated by subtracting the amount bound
from total ligand. The best fit of experimental points was obtained
using LIGAND from Biosoft (Ferguson, MO). Competitive inhibition of 150 nmol/L FBG binding to heparin-agarose was performed
using heparin from 0.032 to 100 pmol/L.
Saturation and inhibition of binding of heparin to immobilized
jbrin(ogen). FBG and fibrin were immobilized on microtiter plate
wells as described,'" except that the FBG (FN-free) solution was l
mg/mL FBG instead of 3 mg/mL. After washing and blocking in
5% ovalbumin/PBS for 1 hour at 37°C. increasing amounts of heparin-BSA-biotin were added to wells and incubated 1 hour at RT.
After washing, the amount of heparin-BSA-biotin bound was detected with a streptavidin-horseradish peroxidase system. Equal
amounts of FBG and fibrin were bound in the solid phase as determined by enzyme-linked immunosorbent assay (ELISA) with PoAb
antihuman FBG. Efficiency of IIa-cleavage was detected by ELISA
with antifibrin p15-42 MoAb T2G1 compared with anti-FBG Bpl42 MoAb 18C6. Competitive inhibition of heparin-BSA-biotin bind-
Table 2. Epitope Distribution of Fibrinlogen) Fragments Used for ClEP and Heparin-AgaroseAffinity Chromatography as Determined by
ELISA and Wastarn Blotting
Antibody and Specificity
Fibrin(ogen)
Derivative
FBG-325
D
E3
FBG
NDSK
Ha-NDSK
peak
ClEP Shift Factor5
18C6' B p - 4 2
1.o
1.o
1.14
+
2.0
1st peak
2nd
1.15
1.57
-
+++
+++
+++ +
-
015-42
T2Glf
~
-
+++
+++
PoAbt Human FBG
+++
+++
+++
+++
+++
+++
313+ FBG D
ND
+++
-
ND
-
ND
ND
Symbols denote the following Am ranges: -, <0.050; +, 0.100 to 0.250; +++. >0.750.
Abbreviation: ND, not determined.
MoAbs to FBG Bpl-42 (18C6)and to fibrin p15-42 (T2G1)were from Accurate Chemical CO (Westbury, NJ).
t Anti-human FBG PoAb was obtained from Dako Corp (Capinteria,CA).
MoAb (313)for FBG fragment D was from American Bioproducts (Pursippany, NJ).
5 ClEP shift factor was determined from the cross-immunoelectrophoresis data shown in Fig 3A through C. The relative distance of migration
(R,) of the peak (center) from the origin (well)was determined by dividing the distance of peak migration by the total distance of migration of
the dye front in the gel. The ClEP shift factor represents the degree of shift in anodal migration due to protein complexing with heparin, and
was calculated as (R, + heparin)/(R,- heparin). No shift in migration in the presence of heparin results in a factor of 1.0.
*
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ODRLJIN ET AL
2052
ing to immobilized fibrin(ogen) was performed using
12.5 pglmL
heparin-BSA-biotin in the presence of increasing doses of heparin.
Heparin-agarose uffiniry chromatography. Heparin-agarosecol(IO
umns (2.5 mL bed volume) were washed with column buffer
mmolL Tris-HCI, pH 7.4). Fibrin(ogen) ( I mL at 300 pglmL) was
5 m m , washedwith 12.5 mL column
appliedtothecolumnat
buffer, and eluted with a I O mL linear gradient of 0 to 0.5 moln
NaCl in column buffer at 20 mm.'The conductivity of every other
fraction was measured in milliosmols and then converted to millimoles per liter of NaCI. Synthetic peptides (net charge), ie, BPI-42
( + l ) , 015-42 (+3), P24-42 (O), P18-31 ( + l ) , and P18-27 (0).were
chosen based on the charge density of basic (B) residues to other
(X) residues of XBBBXXBX, as described for an HBD consensus
sequence? The primary structure of BPI-42 is QGVNDNEEGFFSARGHRPLDKKREEAPSLRPAPPPISGGGYR. TheHBDpeptide
of the B1 chain of laminin (RwlYVVLPRPVCFEKGMNTVRWfl)'
was chosen as a positive control HBD peptide. Peptides (5 mglmL)
were bound to heparin-agarose and eluted with a 0 to 1.0 molL
linear gradient of NaCI.
Solid-phase binding ofjibrin(ogen)to hepurin. Heparin, heparan
sulfate, and desulfated heparin (20 pglmL) were bound to polystyrene microtiter plates. After washing and blocking, FBG, NDSK, or
Ha-NDSK (200 pglmL) was added to appropriate wells (n = 3) and
RT. Heparin bound protein was detected
incubated for 2 hours at
with IO pglmL PoAb anti-FBG"'N".35
Aa,a
-
[
BP-
Y-
WmB
Fibrinogen
-BB
- / p (- 1-42)
Fibrinogen
325
1
2
3
4
-
KD
RESULTS
SDS-PAGE and ELISA. Each preparation of fibrin(ogen)
used in this study was tested to assess the degree of enzymatic (Ha, plasmin, and protease 111) or chemical (CNBr)
cleavage. Both FBG and FBG-325 were analyzed, reduced,
by SDS-PAGE (Fig 1A) and by ELISA (Table 2). The FBG
starting material was free of contaminating FN, and minimal
y - y dimer formation was observed. Limited degradation of
the Aa chain was observed, as is normally the case in pooled
plasma. The BP chain was intact in the FBG starting material
(Fig 1A and Table 2). The majority of BPI-42 was cleaved
by protease 111 to produce the lower molecular weight (325
kD) FBG-325. However, about 10% of the BP chain was
not cleaved, leaving fibrinopeptide B and BPI-42 sequences
intact (Fig 1A and Table 2). One major band was observed
for bothNDSKand
h-NDSK by SDS-PAGE (Fig IB);
however, some heterogeneity wasnoted.Most likely, the
heterogeneity of NDSKs was due to variability in CNBr
cleavage at the C-terminal boundary of the central domain,
which does not alter the primary structure of the BP1-42 or
P15-42 domains. The heterogeneity of Ila-NDSK resulted
additionally from incomplete IIa-cleavage in that trace
amounts of fibrinopeptide B were detected with MoAb I8C6
(Table 2). Plasmin-cleaved FBG fragment D was composed
of fragments D,, D*, and D3 (Fig 1B and Table l), and
ELISA of FBG E fragments with MoAbs T2G1 and 18C6
showed that the preparations were composed mostly of the
late cleavage fragment E3, which is missing residues BPI53 (Table 2).3s
Heparin-jhrin(ogen) complex formation. To determine
whether fibrin(ogen) formed complexes with heparin, ClEP
was performed. FBG showed an anodal migratory shift in
the presence of heparin, whereas FBG-325 incubated with
heparin showed no migratory shift (Fig 2A and Table 2).
Three precitipin lines reflecting points of equivalence were
97
-
45
29
9-NDSK
Ile-NDSK
D
E
Fig 1. SDS-PAGE of fibrin(ogen1. (A) FBG and FBG-325 were resolved on a 7%-polyacrylamide Weber Osborn gel after reduction.
The positions of migration of the Am, m, Bp, p, and y chains are
noted. FBG-325 demonstrates two Bp chain bands, one faint band
migrating at theposition of the intact Bp chain and the otherin the
position of the protease Ill-cleaved p chain (missing residues Bp 142). (B)NDSK, Ila-NDSK, D, and E fragments resolved on a
1046polyacrylamide Laemmli gel under nonreducing conditions.
obtained with FBG-325, with a principal precipitin band of
dimerically cleaved BPI-42 andminor
bands of
both
cleaved uncleaved BP 1-42 and dimerically uncleaved BP 1 42. Plasmin fragments D and El showed no anodal migratory
shift in the presence of heparin, indicating no complex formation (Fig 2B); this was most likely because fragments D
and E3 lack BPI-42 (Table 2). Again, the multiple precipitin
lines obtained with D and E are consistent with heterogeneity
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CRYPTIC HEPARIN
FIBRINBINDING DOMAIN OF
2053
+
.
-. .
.
+
“1
0
t
.
0
Dimension
First
Dimension
First
B
FibrinogenFragment“D”FibrinogenFragment
“E:
+
t
t
Fig 2. CIEP of different preparations of
fibrin(ogen1
inthe
presence (+) orabsence (-1 of
heparin. (A) FBG and FBG-325
(15 pg/welll were incubated
without (-1 or with (+l 45 p g of
heparin before electrophoresis
in the first dimension. (B) Frag
ments D and E were used at 3
and 1.6 pglwell with9 and 5 pg/
well
heparin,
of
respectively.
0
+-
FirstDimension
of the plasmin-cleaved FBG preparations. In the presence of
heparin, Ha-NDSK showed a greater anodal migratory shift
than did NDSK, suggesting that Ila-cleavage enhances the
availability of a specific HBD (Fig 2C). The two peaks of
precipitin line obtained in the second dimension with IIaNDSK in the presence of heparin, coupled with the degree
of shift in anodal migration, suggest a population of NDSK
heterodimerically cleaved with Ita, which would leave one
First dimension
4
intact fibrinopeptide B per molecule (Table 2). Comparison
of the degree of shift in anodal migration due to protein
complexing withheparin indicates that fragments lacking
BPI-42 (D, E, and FBG-325) showed no complexing with
heparin (CIEP shift factor of 1.0). Fibrin(ogen) with at least
one intact fibrinopeptide B showed an intermediary shift
(FBG, 2.0; NDSK, 1.15); whereas homodimerically cleaved
Ila-NDSK showed dramatic complexing with heparin (3.59;
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ODRLJIN ET AL
2054
C
Fibrinogen NDSK
Fibrin NDSK
+
A-.
0
.L.'
-g +
C
0
O Q
Q
_.
.
"
Dimension
FirstDimension
First
Table 2). The results indicate that BPI-42 is required for
complex formation with heparin andthat IIa-cleavage enhances binding of heparin withthe fibrin P15-42 N-terminus.
Saturation andinhibition of binding of jbrin(ogen) to
heparin-agarose. Scatchard analysis of the binding data
was analyzed by the LIGAND program, which suggested a
single class of binding sites (Fig 3A through C) with an
average kd of 0.8 pmol/L for NDSK, 18 pmol/L for FBG,
and 0.3 pnol/L for IIa-NDSK. These results indicate that
Ila-NDSK binds heparin with 2.7-fold higher affinity than
does NDSK, but that both bind with much
higher affinity
than intact FBG. The specificity of FBG binding was confirmed by the inhibition of its binding with '*'I-FBG (data
not shown) and with heparin (IC5oof 42 mmol/L; Fig 3D).
Saturation and inhibitionof binding of heparin to immobilizedjhrin(ogen). Because intact fibrin is insoluble, kd determinations as described above could notbe performed.
Therefore, a reciprocal binding assay was developed to measure the binding of biotin-tagged heparin to fibrin and FBG
immobilized on a solid surface. Heparin-BSA-biotin bound
toboth immobilized FBGandfibrin
in a dose-dependent
and saturable manner (Fig 4A); however, maximum binding
of heparin at saturation was twofold higher to fibrin than to
FBG. These results indicate that IIa-cleavage of FBG resulted in exposure of twice the number of heparin binding
sites. Competitive inhibition of heparin binding showed that
the amount of heparin that caused a 50% inhibition in the
binding of the biotin-BSA-heparin (IC'o) to immobilized
fibrin was 5.5-fold greater (ICs0 = 6.6 pmol/L) compared
with the amount required to inhibit binding to FBG (IC50 =
1.2 pmol/L; Fig 4B), suggesting higher affinity of fibrin than
FBG for heparin.
v)
*-&m
Fig 2 (cont'd). (C) Ila-NDSK
was used at 2.6 pglwell with7.8
pglwell
heparin,
of
whereas
NDSK was used at 10 pglwell
and 30 pglwell of heparin. The
( + l and (-1 symbols in the righthand margins denote protein
preincubated with or without
heparin,
respectively.
The I + )
symbols at the ends of the
arrows denote the direction of
electrophoresis towards the
anode.
Binding of jbrin(ogen) to heparin-agarose. Fibrin(ogen) was eluted from heparin-agarose with a linear gradient
of 0 to 0.5 mol/L NaCI. Whereas 96.3%of Kabi FBG bound
to heparin-agarose, only 40.3% of FBG-325 loaded was retained (Table 3). The differences in binding support the hypothesis that BPI-42 of FBG contributes to an HBD. FBG
fragment D bound (56.8%) to heparin-agarose, as expected
based on the localization of HBDs to both thep and y chains
in the D domain.' In contrast, fragment E:, did not bind to
heparin-agarose, because greater than 95% flowed through
(Table 3). suggesting a requirement for the presence of BPI42 to support FBG binding to heparin. Further support was
obtained using the NDSKs ofFBGandIIa-fibrin;
greater
than 95% of both NDSK and IIa-NDSK bound to heparinagarose (Fig 5A and Table 3). Comparison of the molarity
of salt required to elute the peak fractions of each preparation
showed that both FBG-325 and D fragments eluted below
physiologic salt concentrations. Intact FBG eluted at below
physiologic salt ( 1 20 mmol/L) as well, suggesting that FBG
binding to heparin is not physiologically relevant. The reduced binding to heparin-agarose and elution of fragments
D and FBG-325 with low salt indicated weak complexing
of these fragments with heparin, which was borne out by
the absence of detectable complex formation by CIEP (Fig
2). NDSKboundwith
higher affinity (200 mmol/L) than
intactFBG; however, IIa-NDSK (350 mmoVL) bound to
heparin-agarose with the highest affinity (Fig 5B).
Mapping theprimary structure of thejbrin(ogen)p chain
HBD. Laminin F9 heparin binding peptide was usedto
show the efficacy of the heparin-agarose column for binding
peptides. Fibrin peptide P15-42 bound (96%) to heparinagarose to a similar degree as laminin F9 peptide (96.9%),
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2055
CRYPTIC HEPARIN BINDING DOMAIN OF FIBRIN
OJ
0
i
81.
2
3
4
l4
5
B
zol
A
6
D
FlmlrM)
W-
Fig 3. Saturation binding of I’mllfibrin(ogen) to heparin. The binding of increasing concentrationsof iodinated NDSK (A), FBG (B), and IlaNDSK (C) to heparin-agarose was measured. Nonspecific binding measured in the presence of 60 mg/mL heparin and 20 mg/mL FBG was
always l e s s than 5%. Data are represented as a saturation curve ofbound versus free. Insets in (A through (C): Scatchard plot of bound (B)/
free (F) versus B, in which the best fit of the data was determined by analysis with the LIGAND computer program. Binding of ‘=l-FBG to
heparin-agarosewas inhibited in a dose-dependent mannerwith increasing concentrationsof soluble heparin (D). Bars represent
f 1 standerd
deviation (n = 6 to 12 per data point; data were collected usingtwo different preparations of radiolabeled NDSK and Ila-NDSK).
with each peptide contributing a net +3 charge (Fig 6). FBG
peptide Bpl-42 with a lower net positive charge (+ 1) also
bound to heparin-agarose, buttoa lesser extent (67.2%).
The unbound fraction of PI-42 was reapplied and none of
the pl-42 bound, indicating the fraction of peptide is nonbinding and that the heparin-agarose column was not overloaded. Very little ,618-31 bound (8.5%) to heparin-agarose,
even though this peptide has the same net charge as BPI42 of + l. Furthermore, neither neutral peptide 018-27 nor
p24-42 bound to heparin-agarose ( 4 % ; Fig 6), indicating
that charge is not the sole determining factor for the binding
of BP chain N-terminal sequences to heparin. Significant
binding was obtained with peptides having net charges of
+3 and + 1, whereas peptides with + 1 net charge and neutral
peptides containing basically charged Arg and Lys (p18-31
and p18-27, respectively) did notbind.We
propose that
residues Bpl-42 constitute, at least in part, an FBG HBD;
similarly, P15-42 constitute a fibrin HBD. Peptides p18-27,
p18-31, and P24-42 did not bind to heparin-agarose (Fig 6),
suggesting further that residues within p15-23, and perhaps
p32-42, are essential for conferring the specificity and proper
conformation on the p chain N-terminus to mediate fibrin
(ogen) binding to heparin (Table 4).
SpecQicity offibrin(ogen) binding to proteoglycans and glycosaminoglycans. Comparison of fibrin(ogen) binding to heparin, chondroitin sulfate, d e m t a n sulfate, heparan sulfate, and
keratan sulfate indicated that fibrin(ogen) bound with the highest
affinity to heparin(datanot shown). When the bindingof FBG,
NDSK, andplasminfragment D toheparin,heparansulfate,
and desulfated heparin fixed to a solid surface was compared,
little difference in binding to the various heparin derivatives
wasobserved(Fig 7). In contrast,whenthebindingofHaNDSK to all three heparin derivatives was compared, the results
indicated maximal binding to heparin followed by desulfated
heparin and heparan sulfate, which is sulfated to a lesser degree
than heparin(Fig 7). IIa-NDSKshowedfourfoldtosixfold
higher binding toall threeheparin derivatives than was observed
for FBG and for NDSK (Fig 7). Very little binding to any of
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2056
ODRLJIN ET AL
A
0.4
-E
0.3
4
0
a,
(U
v
a,
0
C
0.2
m
e
9
0.1
0.0
10
0
B
20
30
.
00
10
20
F M
the heparin derivatives was observed with FBG fragment D in
the ELISA format. Together, these results suggest that fibrin
binding is specific for heparin saccharide sequences and is not
solely dependent on a charge interactionof p15-42 (net charge
+3) with the highly negatively charged glycosaminoglycans.
DISCUSSION
The data presented in this report clearly show that Ilacleavage within the FBG Bpl-42 enhances exposure of an
30
Fig 4. Saturation andcompetitive inhibition of
heparin
binding to solid-phaae fibrinogencompared wkh fibrin. (A)
The binding d increasing concentrations of heparin-BSA-biotop curve) orFBG
tin tofibrin (0,
(0,bottom curve) bound to the
surface ofpolystyrenemicrotiter
wells was meesured. (B) Inereasing concentrationsofheparin
were wed to inhibit competitively the binding ofheparinBSA-biotin to fibrin (0.top
curve)
and
FBG IO, bottom
curve) immobilized to a solid
surface. Bars represent2 1 standard deviation.
HBD at fibrin p15-42. Previous s t ~ d i e s ' ~have
, ~ ~ .not
~~
mapped the fine point structure o f the fibrin(ogen) HBD;
therefore, we performed a detailed study of the minimally
relevant structure required to support binding of fibrin(ogen)
to heparin. CIEP of fibrin(ogen) in the presence of heparin
showed protein-glycosaminoglycan complex formation. By
comparing the binding of various synthetic peptides and purified proteolytic and chemical cleavage products of fibrin
(ogen) to heparin, we conclude that regions Bpl-42 of FBG
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2057
CRYPTIC HEPARIN BINDING DOMAIN OF FIBRIN
Table 3. Comparison of the Amount of Fibrin(ogen1That Bound to
the Heparin-Agarose Column to theAmount That Flowed Through
the Column Based on Total Amount of Protein Recovered
FBG
Preparation
% Bound
% Not Bound
FBG
FBG-325
NDSK
Ila-NDSK
Dl.3
96.3
40.3
95.6
96.7
56.8
4.4
3.7
59.7
4.4
3.3
43.2
96.6
E3
and p15-42 of fibrin constitute, at least in part, the HBDs
of FBG and fibrin, respectively. Mohri et aIx showed that
HBDs are localized to regions of the Bp and y chains in
the C-terminal domain of FBG that are generated by V8
protease cleavage. Our data confirm the presence of these
HBDs in that D fragments bound to heparin-agarose, albeit
with much lower affinity, than did the NDSKs of both FBG
andfibrin. The FBG Ply HBDs differ considerably from
HBDs described for other adhesive glycoproteins. The capacity of the FBG Ply HBD to bindto heparin was not
eliminated by reductionand alkylation: as would be expected.' In addition, the synthetic peptide with neutralcharge
that corresponds to the platelet recognition domain 739841 1, yGQQHHLGGAKQAGDV, inhibited FBG binding to
heparin-Sepharose..' This peptide neither bears resemblance
to the structures of other known HBD peptides nor does
it share similarity to the consensus sequence described for
A
5
0
m
a
V
em
2
n
a
H B D s . ~In contrast, the structure of P15-42 of fibrin bears
considerable likeness to the HBD consensus sequence (Table
4 and Fig 8).'
The strength of the complex formed between heparin and
fibrin(ogen) fragments was shown to vary. Ila-NDSK bound
with higher affinity than did NDSK to heparin-agarose, as
shown by their elution profiles and dissociation constants.
FBG E3 fragments lacking 01-53.'' did not bind to heparinagarose. FBG-325 lacking BPI-42, FBG D fragments lacking BPI-133, and intact FBG exhibited weaker binding to
heparin-agarose because all eluted at less than 120 mmol/L
NaCI. Compared with binding of FBG, tighter binding of
NDSK ( I .6-fold) and IIa-NDSK (2.9-fold) to heparin-agarose was observed. A comparison of the kd shows that IIaNDSK bound toheparin-agarose with 2.7-fold higher affinity
than did NDSK, with both binding with much higher affinity
than intact FBG. These data are compatible with one class
of binding site located on the NDSK within BPI-42. Also,
the kd are in the same range determined by others for soluble
FBG andfibrinmonomer.Fibrinmonomer
(5.7 pmol/L)
exhibited a 3.5-fold higher affinity than FBG (20 pmol/L)'y
for heparin, consistent with our data showing that Ila-cleavage of FBG exposes a higher affinity HBD in fibrin. In
addition, the low affinity of intactFBG for heparin (1 8 prnoll
L this study; 20 pmol/L per Hogg and Jackson") suggests
that the heparin binding site in the D domainx," is unlikely
to be physiologically relevant compared withthe HBD at
the fibrin(ogen) BP chain N-terminus.
However, it is unlikely that the 30- to 60-fold enhancement of binding of fibrin(ogen) NDSKs to heparin compared
r
0.5
A
0.4
0.3
0.2
0.1
0
x)
20
30
Fractlon Number
Fig 5. Heparin-agarose affinity chromatography.
(A) Elution profile (Am, left ordinate) of NDSK (broken line) and Ila-NDSK (solid line) from heparin-agarose. The downward arrow indicates the point at
which the 0 to 0.5 mollL NaCl gradient (right ordinate) was appliedto the column. The gradient profile
is represented by the straight line. (B) Comparison
of the molarity of NaCl required to elute the various
fibrin(ogen1 peak fractionsfrom heparin-agarose.
40
50
60
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ODRLJIN ET AL
2058
l-
4
p 18-31
15-42
FRACTION
Fig 6. Heparin-agarose affinity chromatography
of Bp-chain peptides. Data represent elution profile
of the relative Am plotted against fraction number.
Only the NaCl gradient from 0 to 0.4 mol/L NaCl is
plotted, as represented by the straight line.
NUMBER
with intact FBG reflects the in vivo situation. We hypothesize that, in vivo, the p15-42 domain of FBG is probably
inaccessible to heparinheparan sulfate proteoglycans. Subsequent IIa-cleavage and resulting conformational changes
occumng during fibrin f ~ r m a t i o n ’ ~ . ’are
~.~
most
” ~ ~likely required to make the p15-42 site available for fibrin-cell interactions involved in hemostasis and wound repair.””’ Unfortunately, it is extremely difficult to compare the physiologic
soluble FBG to the physiologic insoluble fibrin in binding
assays without experimentally manipulating the ligands so
that they are either both soluble or both immobilized to a
solid surface. It is known that disruption of the rigid coiledcoil region of fibrin(ogen), as would occur by CNBr cleavage, experimentally alters the native conformation by
allowing more flexibility in the N-terminus.4”Consequently,
CNBr cleavage may partially expose the 015-42 HBDin
fibrin(ogen) NDSKs. Similarly, the HBD of VN is exposed
by either proteolytic ~leavage’~.’’
or chemical denat~ration.~’
Immobilization of FBG ona surface also alters native conformation by exposing neo-epitopes not accessible on FBG in
s~lution.~’
Further, urea treatment of FBG results in partial
exposure of the conformational fibrin-p15-42 epitope recognized by T2Gl,“.’* which is in close proximity to the fibrin
HBD.
Fibrin monomer prepared inthe presence of GPRP to
inhibit polymerization was considered asan alternative li-
gand to Ila-NDSK for Scatchard analysis. However, the
efficiency of fibrinopeptide B cleavage is acceleratedas polymerization of fibrin proceeds, suggesting that polymerization-induced conformational changes facilitate IIa-release of
fibrinopeptide
Based on this interpretation, we chose
not to inhibit fibrin polymerization with GPRP in case this
prevented efficient release of fibrinopeptide B, a consideration crucial to our experimental objectives. In addition,
when fibrinopeptide B is fully released, fibrin aggregation
occurs, even in the presence of GPRP (J.R.S., unpublished
data). Therefore, at the higher concentrations of protein required for the Scatchard analysis as performed in this report,
the potential for fibrin monomer to become insoluble aggregates would have clouded interpretation of the data. The use
of NDSK and IIa-NDSK provided soluble fragments that
could never polymerize due to the absence of the complementary “a” and “b” polymerization sites foundon y
chains of the D fragment.
Therefore, because it is not yet experimentally feasible to
compare directly the binding affinities of the physiologic
relevant forms of FBG and fibrin and mostof the experiments conducted in this and other studies were performed
with soluble fragments of fibrin, we developed two additional assays to measure (1) the binding of heparin to intact
FBG and to fibrin
immobilized to a solid surface and ( 2 )
the binding of fibrin(ogen) fragments to a panel of various
Table 4. Alignment of Fibrin p15-42 Sequence With the Heparin Binding Domain Consensus Sequence
+
G
H
R
P
L
X
B
B
-
+
+
D
K
B
K
X
+
R
X
-
B
X
*
+
-
E
* The heparin binding domain consensus sequence is
represents basic amino acid residues.
E
A
P
S
L
R
+
P
A
as defined by Cardin and Weintraub?
P
P
I
S
G
G
G
Y
X represents any amino acid residue and
R
B
PARIN
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CRYPTIC
FIBRIN
DOMAIN OF
2059
B Heparin
I
o0.357
0.30
1
0.25
0
Heparansulfate
El Desulfated
heparin
-
0
m
d
0.20-
c3
0
0.15I
,.IO{
0.05
nnn
FBS
FBG-NDSK
Ila-NDSK
D
Fig 7. Analysis of the degree of heparin sulfation on fibrin(ogen1
binding. The binding of fibrin(ogen1to heparin, heparan sulfate (HS),
and desulfated heparin (DSH) was measured in an ELSA format in
which the heparin derivative was coated on the surface ofthe microtiter plate, and the fibrin(ogen) fragment bound to the heparin derivative was detected with PoAb antihuman FBGIFN". Bars represent 2
l standard deviation.
glycosaminoglycans and proteoglycans immobilized to a
surface. Heparin bound toimmobilized FBG, but the binding
of heparin to immobilized fibrinwas enhanced twofold, consistent with release of fibrinopeptide B and exposure of two
additional 015-42 HBDs per molecule. Thus, IIa-cleavage
of FBGdoubles the number of heparin binding sites in fibrin.
To substantiate the hypothesis that the newly exposed HBDs
on fibrinhave higher affinity for heparin thanthe same region
in FBG, competitive inhibition of heparin binding to immobilized FBG and fibrin wasperformed. The 5.5-fold stronger
binding of heparin to immobilized fibrin is most likely due
to a combination of the doubling of the actual number of
binding sites and the higher binding affinity of fibrin 015-
42 for heparin. Further support for this hypothesis was obtained by kd determinations with the soluble forms of NDSK
and Ila-NDSK, which indicate a 2.7-fold increase in affinity
after IIa-cleavage. Further, IIa-NDSK bound to desulfated
heparin and heparan sulfate to a fourfold to sixfold higher
degree thandidFBG
fragments. These data suggest that,
in addition to the charge interaction and primary structure
specificity of 015-42, there may be a defined specificity of
saccharide structures for heparin binding to 015-42, as is
the case for antithrombin Ill binding to a pentasaccharide
sequence of heparin?
The ability of heparin to modulate secondary conformationatheparin
binding domains of glycoproteins iswell
described." Heparin binding induces amphiphatic a-helices,
segregating basic residues primarily to one side of the helical
face, which may serve to lock or stablilize the conformation
of the HBD.9Secondary structure determinations predict that
the 019-26 region of fibrin(ogen) forms an a-helix, which
after IIa-cleavage may mediate conformational changes that
induce the proper folding of the fibrin 0 chain N-terminu~.~~
Basic residues of 015-32 segregate primarily to one side of
the helical face in a helical wheel diagram (Fig 8), forming
a region of high positive charge density that compares to
authentic HBDs.'We propose that conformational changes
in the 0 chain N-terminus, caused by 1Ia-release of fibrinopeptide B (BflI-14),14.37enhance the exposure of the HBD
in fibrin, thus allowing more avidbinding of heparin to fibrin
thantoFBG. An analogous situation isfound for VN, in
which y-thrombin cleavage alters its conformation to expose
a cryptic HBD."."
Although the significance of heparin binding to domains
of fibrin(ogen) has not been documented in vivo, there is a
considerable body of in vitro evidence showing that heparin
binding to Ila-fibrin monomer in a ternary complex could
profoundly affect the regulatory balance betweenhemostatic
and thrombotic events. The effects of heparinonternary
complex formation and IIa-binding to fibrin have been studied in detail.18'?fl.22-2sHBDs of adhesive glycoproteins participate in many different cell-matrix andcell-cellinteracIndeed, the HBD of VN mediates binding of
tions.l.7.16.17.26.27
Antithrombin-Ill
ECGF/aFGF
125-142
101-118
Fibrin(ogen)
p1 5-32
R
Fig 8. Helicalwheel diagram of fibrinlogen) HBD. Ahelicalwheel diagram was generatedwith Bp15-32 chain sequences according
to Cardin
and Weintraub? Basic residues of HBDs of antithrombin 111 and EC growth factorlacidic fibroblast growth factor segregate to one side of the
helical face.
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2060
ODRLJIN ET AL
the ternary complex VN-IIa-antithrombin 111 to proteoglycans on the apical surface of EC.4h We have data indicating
that fibrin fragments, but not FBG, bind to EC by a heparindependent mechanism involving residues 01 S-42 (manuscript submitted), which is compatible with the knowledge
that fibrin p1S-42 sequences interact directly with the EC
cell surface." Also consistent with this is the fact that IIacleavage of fibrinopeptide B is required
for fibrin-induced
EC capillary tube formation in vitro with fibrin on the apical
surface of the EC.47 Moreover, a C-terminal HBD of FN
functions in focal adhesion assembly' by interacting with a
cellsurfaceheparansulfate
proteoglycan(syndecan 4) in
addition to the primary receptor:ligand interaction, which is
mediated through either 01- or 03-integrins with the RGDcell binding domain of
From these
observations,
we
propose that fibrin(ogen) functions similarly to other heparin
binding adhesive glycoproteins in cel1:matrix interactions.
Dual-receptor binding may promote formationof a multimolecular complex, converting an initially low-affinity interaction into ahigh-aviditycomplex.
We postulatethatlowaffinity
cell
surface
heparan
sulfate
proteoglycan
may
interact with the 015-42 HBD of fibrin while the integrin
receptor a v P 3 interacts with the fibrin(ogen) Aa-chain RGD
domain. Thus, the interaction of the integrincellreceptor
withamatrixproteinmaylead
to adhesionlackingfocal
contacts that, upon ligation of cell surface heparan sulfate
proteoglycan with the HBD, could promote stable focal adhesions and stress fibers,'.' which are indicative of cell proliferation." Alternatively, binding of fibrinP1S-42 to theapical
surface of EC (manuscript submitted), presumably via heparan sulfate proteoglycan coreceptors, may induce a migratory phenotype to promote capillary tube formation" during
angiogenesis.Together, theseobservationsunderscore
the
importance of elucidating thephysiologic mechanism(s) that
is involved in mediating fibrin-EC interactions dependent on
Ila-induced exposure of the ,B1 S-42 domain."'~''~47
ACKNOWLEDGMENT
The technical assistance of Jann Whiteis greatly appreciated. We
thank Drs L.A. Bunce, L.A. Sporn, P.J. Fay, V.J. Marder, and C.W.
Francis for their enthusiastic support and helpful discussions regarding the work. The authors gratefully acknowledge the editorial contributions of Oliver Medzihradsky to the manuscript. We thank Dr
T. Maciag (American Red Cross, Rockville, MD) for suggestions
thateventually led to our investigation of heparin-fibrin binding
interactions. and for his comments on the manuscript.
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1996 88: 2050-2061
Thrombin cleavage enhances exposure of a heparin binding domain
in the N-terminus of the fibrin beta chain
TM Odrljin, JR Shainoff, SO Lawrence and PJ Simpson-Haidaris
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