From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
The von Willebrand factor–reducing activity of thrombospondin-1 is located in
the calcium-binding/C-terminal sequence and requires a free thiol at position 974
John E. Pimanda, Douglas S. Annis, Mark Raftery, Deane F. Mosher, Colin N. Chesterman, and Philip J. Hogg
Plasma von Willebrand factor (VWF) is a
multimeric protein that mediates adhesion of platelets to sites of vascular injury; however, only the very large VWF
multimers are effective in promoting platelet adhesion in flowing blood. The multimeric size of VWF can be controlled by
the glycoprotein, thrombospondin-1 (TSP1), which facilitates reduction of the disulfide bonds that hold VWF multimers together. The TSP family of extracellular
glycoproteins consists of 5 members in
vertebrates, TSP-1 through TSP-4 and
TSP-5/COMP. TSP-1 and TSP-2 are structurally similar trimeric proteins composed of disulfide-linked 150-kDa monomers. Recombinant pieces of TSP-1 and
TSP-2 incorporating combinations of domains that span the entire subunit were
produced in insect cells and examined for
VWF reductase activity. VWF reductase
activity was present in the Caⴙⴙ-binding
repeats and C-terminal sequence of
TSP-1, but not of TSP-2. Alkylation of
Cys974 in the C-terminal TSP-1 construct,
which is a serine in TSP-2, ablated VWF
reductase activity. These results imply
that the reductase function of TSP-1 centers around Cys974 in the C-terminal sequence. (Blood. 2002;100:2832-2838)
© 2002 by The American Society of Hematology
Introduction
Platelet adhesion to von Willebrand factor (VWF) in the subendothelium of a damaged blood vessel is the initial step in formation of
a hemostatic plug at high shear rates (for a review, see Sadler1).
VWF also acts in synergy with fibrinogen in the formation of
interplatelet adhesive links to form a stable thrombus at arterial
shear rates.2,3 As a carrier for procoagulant factor VIII, VWF
prolongs its survival in the circulation by protecting it from
inactivation by activated protein C and factor Xa. VWF is
synthesized by vascular endothelial cells and megakaryocytes and
circulates in blood as a series of multimers containing a variable
number of about 500-kDa homodimers.4 The largest VWF multimers have a molecular mass of approximately 20 000 kDa,
comparable in length to the diameter of a medium platelet (2 ␮M),
and are released from endothelial cells following stimulation.
The assembly of VWF multimers follows a stepwise process.
Pro-VWF dimers are assembled in the endoplasmic reticulum via
disulfide bridges between cysteine residues located in the cysteine
knotlike domains at the C-terminal ends of the pro-VWF subunits.
Intersubunit disulfide bonds involve 1 or 3 of the cysteine residues
at positions 2008, 2010, and 2048.5 These tail-to-tail linked
pro-VWF dimers are subsequently multimerized within the Golgi
apparatus by head-to-head linkage by disulfide bonds near the
N-terminal domains.6 Interdimeric disulfide bonds involve Cys379
and one or more of the cysteine residues at positions 459, 462, and
464.7 After multimerization, the VWF propeptides are removed by
proteolysis.6
Only large multimeric forms of VWF are hemostatically
active.8 The unusually large VWF multimers secreted by
endothelial cells have been shown to be more effective than the
largest plasma forms in inducing platelet aggregation under
conditions of high fluid shear.9 This functional importance of
multimer size relates to the affinity of VWF for its ligands.
Large VWF multimers bind with an approximate 100-fold
greater affinity to both collagen and platelets than monomeric
VWF.8 Some thrombotic disorders are characterized by altered
VWF multimer size. Thrombotic thrombocytopenic purpura
(TTP) is often associated with unusually large VWF multimers
in the blood, which are thought to precipitate intravascular
platelet clumping.10,11 Conversely, lower than average multimer
size characterizes the bleeding diathesis of type IIA von
Willebrand disease. Modulation of VWF multimer size is,
therefore, critical to the control of its hemostatic activity.
We recently reported that the homotrimeric glycoprotein, thrombospondin-1 (TSP-1; for a review, see Lawler12), reduces the
average multimer size of plasma or purified VWF both in vitro and
in vivo.13,14 Incubation of TSP-1 with VWF results in formation of
thiol-dependent complexes of TSP-1 and VWF, generation of new
thiols in VWF, and reduction in the average multimer size of VWF.
Moreover, the ratio of the concentrations of TSP-1 and VWF in
plasma reflect the average multimer size of VWF. The higher the
plasma TSP-1/VWF molar ratio the smaller the average VWF
multimer size. These results indicate that TSP-1 regulates the
multimeric size and therefore hemostatic activity of VWF. We
show herein that the VWF reductase activity of TSP-1 resides in the
Ca⫹⫹-binding and C-terminal sequences and requires a free thiol
at Cys974.
From the Centre for Thrombosis and Vascular Research and Cytokine
Research Unit, School of Medical Sciences, University of New South Wales
and Department of Haematology, Prince of Wales Hospital, Sydney, Australia;
and Section of Hematology, Department of Medicine, University of Wisconsin,
Madison.
Department, and National Institutes of Health grant HL54462.
Submitted March 14, 2002; accepted June 7, 2002. Prepublished online as
Blood First Edition Paper, June 21, 2002; DOI 10.1182/blood-2002-03-0770.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by the National Health and Medical Research Council of Australia,
the National Heart Foundation of Australia, the New South Wales Health
2832
Reprints: Philip Hogg, Centre for Thrombosis and Vascular Research, School
of Medical Sciences, University of New South Wales, Sydney NSW 2052
Australia; e-mail: [email protected].
© 2002 by The American Society of Hematology
BLOOD, 15 OCTOBER 2002 䡠 VOLUME 100, NUMBER 8
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 15 OCTOBER 2002 䡠 VOLUME 100, NUMBER 8
Materials and methods
Proteins and reagents
2-Nitro-5-thiocyanobenzoic acid (NTCB), 5,5⬘-dithiobis(2-nitrobenzoic
acid) (DTNB), N-ethylmaleimide (NEM), reduced glutathione (GSH),
and EDTA (ethylenediaminetetraacetic acid) were from Sigma (St
Louis, MO). 3-(N-maleimidylpropionyl)biocytin (MPB) was from Molecular Probes (Eugene, OR). Purified human plasma VWF and recombinant human VWF was used in this study. Plasma VWF was a gift from
Dr Michael Berndt; recombinant VWF produced in baby hamster kidney
cells and purified by immunoaffinity chromatography was a gift from Dr
Eric Huizinga. VWF concentration was measured by BCA protein assay
(Pierce, Rockford, IL). TSP-1 was purified from human platelet
concentrates as described previously15 with some modifications.16
Buffers containing 0.1 mM CaCl2 were used throughout the chromatographic purification of TSP-1. TSP-1 concentration was calculated using
an absorption coefficient of 10.9 for a 1% solution at 280 nm. All other
reagents were of analytical grade.
TSP-1 and TSP-2 fragments
Modular pieces of TSP-1 and TSP-2 were expressed using a baculovirus
expression system and purified by nickel chelate chromatography as
described.17-19 Briefly, DNA encoding for TSP-1 proteins NoC-1 (residues
1-356), CP123-1 (294-529), P3E123-1 (473-673), E3CaG-1 (630-1152),
and delNo-1 (294-1152) and TSP-2 proteins delNo-2 (290-1154) and
E3CaG-2 (632-1154) were cloned into the baculovirus transfer vector
pAcGP67.coco. The Baculogold transfection module (BD Biosciences
Pharmingen, San Diego, CA) was cotransfected with a pAcGP67.coco
clone into SF9 cells. Individual viral clones were isolated, and high-titer
viral stocks (⬎ 108 pfu/mL) were obtained. The TSP-derived proteins were
expressed by infection of High 5 cells in SF900II serum-free media at a
multiplicity of infection (MOI) of about 5 followed by growth at 27°C for
65 to 72 hours. The His-tagged proteins were purified from the conditioned
media by nickel chelate chromatography.
Assays for VWF multimer size
The VWF (8 nM) was incubated with TSP-1 or TSP-1/TSP-2 fragments
(0.8, 8, or 80 nM) for 1 hour at 37°C. All dilutions were made with 50
mM HEPES (N-2-hydroxyethylpiperazine-N⬘2-ethanesulfonic acid),
0.125 M NaCl, pH 7.4 buffer (HEPES-buffered saline) containing 0.1
mM CaCl2. Aliquots (30 ␮L) of the reactions were diluted 20-fold in 20
mM imidazole, 5 mM citric acid, 0.12 M NaCl, pH 7.3 buffer containing
5% bovine serum albumin and assayed for collagen-binding affinity and
VWF antigen as described by Favaloro et al20 and Xie et al.13 Reactions
were assayed in triplicate for collagen-binding affinity and VWF antigen
and the ratio of the 2 measurements was reported. The overall error was
calculated by adding the relative errors (1 SD) of each measurement.
The data groups were compared using a one-way ANOVA and a Tukey
post-hoc test was applied to compute significance between groups.
Citrated normal plasma was incubated with an equal volume of
purified platelet TSP-1, E3CaG-1, or E3CaG-2 in HEPES-buffered
saline containing 50 mM CaCl2 and 10 ␮M D-Phe-Pro-Arg-chloromethylketone (Calbiochem-Novabiochem, Bad Soden, Germany) for 1 hour
at 37°C. The final concentrations of TSP-1, E3CaG-1, and E3CaG-2
were 40 nM, 400 nM, and 400 nM, respectively. Aliquots of the
reactions (10 ␮L) were resolved on 1% agarose gel electrophoresis,21
transferred to polyvinylidene difluoride (PVDF) membrane (DuPont
NEN, Boston, MA), blotted with 2 ␮g/mL peroxidase-conjugated
anti-VWF polyclonal antibodies (Dako, Carpinteria, CA) and visualized
using chemiluminescence (DuPont NEN).
Assay for formation of new thiols in VWF
The biotin-linked maleimide, MPB, was used to measure reduction of
VWF disulfide bond(s) by TSP-1 or TSP-1/TSP-2 fragments. The
CONTROL OF VWF MULTIMER SIZE BY TSP-1
2833
protocol was essentially as described by Xie et al.13 Briefly, aliquots
(250 ␮L) of the incubation mixtures used to measure VWF multimer
size were labeled with MPB (100 ␮M) for 10 minutes at 37°C and
the unreacted MPB was quenched with GSH (200 ␮M) for 10 minutes
at 37°C. The MPB-labeled VWF was incubated in microtiter plate
wells coated with antihuman VWF polyclonal antibodies (Dako),
and the biotin label was detected using StreptABComplex/HRP (Dako).
The reactions were assayed in triplicate and the mean and 1 SD
is reported.
Quantitation of thiols in E3CaG-1
The number of thiols in E3CaG-1 was measured using DTNB. E3CaG-1
(⬃10 ␮M) was incubated with DTNB (⬃1 mM) in 0.1 M HEPES, 0.3 M
NaCl, 10 mM EDTA, pH 7.0 buffer for 10 minutes at room temperature
and the TNB was measured from the absorbance at 412 nm using a
Molecular Devices Thermomax Plus (Palo Alto, CA) microplate reader.
The extinction coefficient for the TNB dianion at pH 7.0 is 14 150
M⫺1cm⫺1 at 412 nm.22
Alkylation of E3CaG-1 with maleimides
E3CaG-1 (0.5 mg/mL) was incubated with 10 mM NEM for 20 hours in
HEPES-buffered saline containing 0.1 mM CaCl2, and the excess NEM was
removed by dialysis against the same HEPES buffer. The unreacted and
alkylated E3CaG-1 (10 ␮g/mL) was labeled with MPB (100 ␮M) for 30
minutes at room temperature in HEPES-buffered saline containing 10 mM
EDTA. Samples of the labeled proteins (0.2 ␮g) were resolved on 8% to
16% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) under nonreducing conditions, transferred to PVDF membrane,
blotted with a 1:2000 dilution of streptavidin-peroxidase (Dako), and
visualized using chemiluminescence.
Mapping the free thiol in E3CaG-1
NTCB specifically S-cyanylates cysteine thiols and the peptide bond on
the N-terminal side of the cyanylated cysteine is then cleaved under
mildly alkaline conditions.23,24 E3CaG-1 (0.2 mg/mL) in 0.1 M Tris
(tris(hydroxymethyl)aminomethane), pH 8.0 buffer containing either
0.1 mM or 2 mM Ca⫹⫹ was incubated with NTCB (20 mM) for 60
minutes at 37°C to cyanylate the cysteine thiols. The pH of the reaction
was adjusted to 9.0 using 3.0 M Tris base and incubated at 37°C for a
further 60 minutes. The resulting fragments were reduced with dithiothreitol, alkylated with iodoacetamide, and resolved by 8% to 16%
SDS-PAGE. The Coomassie-stained fragments were cut from the gel
and analyzed by mass spectrometry.
Peptide mass fingerprinting
The SDS-PAGE gel pieces were completely destained in 1:1 acetonitrile
and 25 mM NH4HCO3 (4 ⫻ 200 ␮L, 30 minutes), then acetonitrile (100
␮L, 10 minutes) and dried in a vacuum centrifuge. The gel pieces were
rehydrated in 20 ␮L 10 ␮M NH4HCO3 containing about 10 ␮g/mL trypsin
and incubated at 37°C overnight. Aliquots (0.5 ␮L) of each sample were
added to a matrix (1 ␮L 10 mg/mL 2,5-dihydroxybenzoic acid) and spotted
onto a 100-well sample plate and analyzed by matrix-assisted laser
desorption/ionization reflectron time of flight mass spectrometry (MALDIrTOFMS) as described.25 The molecular mass profile of the trypsindigested fragments was then compared with the theoretical tryptic digestion
of E3CaG-1.
Results
TSP-1 and TSP-2 fragments
The domain structures of TSP-1 and TSP-2 and the recombinant
pieces used in our experiments are shown in Figure 1A. Overlapping TSP-1 constructs span the entire subunit. The TSP-2 constructs focused on the region that was active in the TSP-1
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2834
PIMANDA et al
BLOOD, 15 OCTOBER 2002 䡠 VOLUME 100, NUMBER 8
Figure 2. The VWF-reducing activity of TSP-1 is contained in the E3CaG-1
fragment. VWF (8 nM) was incubated with 0.8, 8, or 80 nM TSP-1, delNo-1, NoC-1,
CP123-1, P3E123-1, or E3CaG-1. Aliquots of the reactions were analyzed for the
average VWF multimer size and for the generation of new thiols in VWF. The average
VWF multimer size was estimated from the ratio of the collagen-binding activity and
VWF antigen level (䡺). Generation of new free thiols in VWF was assessed from
incorporation of the biotin-linked maleimide, MPB (f). The dotted lines represent no
change in VWF multimer size (top line) or thiols in VWF (bottom line). The asterisks
indicate significant difference compared to control (P ⬍ .05).
Figure 1. TSP-1 and TSP-2 fragments. (A) The subgroup A thrombospondins,
TSP-1 and TSP-2, share a similar molecular architecture comprising a unique
heparin-binding domain at the N-terminus followed by a connecting region that links 3
subunits via interchain disulfide bonds, a procollagenlike module, 3 properdinlike or
type 1 modules, 3 EGF-like or type 2 modules, 7 type-3 repeats (12 unique
calcium-binding loops), and a unique C-terminal sequence. The NoC-1 fragment
contains the connecting region and is trimeric. The residue numbers are for the
mature proteins. TSP-1 begins at Asn1, while TSP-2 begins at Gly1. (B) SDS-PAGE
profile of the TSP-1 and TSP-2 fragments. Samples (4 ␮g) of TSP-1 (lane 1) and the
TSP-1 (lanes 2-6) and TSP-2 (lanes 7 and 8) fragments were resolved on 8% to 16%
SDS-PAGE under reducing conditions and stained with Coomassie blue. The
positions of Mr markers are shown at left.
constructs. The SDS-PAGE profile of the reduced pieces are shown
in Figure 1B. Without reduction, all migrated similarly except for
NoC-1, which was trimeric.
The VWF-reducing activity of TSP-1 was contained in the
E3CaG-1 fragment
Increasing concentrations of TSP-1 and TSP-1 constructs were
incubated with VWF for 1 hour and VWF reductase activity was
identified by the concurrent reduction of VWF multimer size and
generation of new thiols in VWF. The ratio of collagen-binding
affinity to VWF antigen (CBA/VWF:Ag ratio) was used as a
surrogate measure of the average VWF multimer size. Reduction of
disulfide bonds in VWF was measured from incorporation of the
biotin-linked maleimide, MPB.
The VWF reductase activity was limited to 2 TSP-1 constructs,
delNo-1 and E3CaG-1, which both contain the third type 2 repeat,
the 7 type 3 domains, and the C-terminal sequence (Figure 2).
CP123-1 and P3E123-1 were devoid of VWF reductase activity
Figure 3. The effect of NoC-1 on the collagen binding of VWF is independent of
calcium ions. (A) VWF (8 nM) was incubated with 0.8 to 80 nM E3CaG-1 or 0.008 to
800 nM NoC-1. Aliquots of the reactions were analyzed for the average VWF
multimer size (䡺) and for the generation of new thiols in VWF (f). The dotted lines
represent no change in VWF multimer size (top line) or thiols in VWF (bottom line).
The asterisks indicate significant difference compared to control (P ⬍ .05). The small
increase in MPB labeling (f) at high concentrations of NoC-1 was not significantly
different compared to control (P ⬎ .05). (B) VWF (8 nM) was incubated with TSP-1 or
the NoC-1 fragment (80 nM) in the absence or presence of EDTA (10 mM). Aliquots of
the reactions were analyzed for the collagen-binding affinity and VWF antigen level.
The asterisks indicate significant difference compared to control (P ⬍ .05).
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 15 OCTOBER 2002 䡠 VOLUME 100, NUMBER 8
CONTROL OF VWF MULTIMER SIZE BY TSP-1
2835
Figure 4. TSP-2 fragments do not contain VWFreducing activity. (A) VWF (8 nM) was incubated with
0.8, 8, or 80 nM TSP-1, delNo-2, or E3CaG-2. Aliquots of
the reactions were analyzed for the average VWF multimer size (䡺) and for the generation of new thiols in VWF
(f). The dotted lines represent no change in VWF
multimer size (top line) or thiols in VWF (bottom line). The
asterisks indicate significant difference compared to control (P ⬍ .05). (B) Citrated normal plasma (NP) was
incubated with 40 nM TSP-1 (lane 2), 400 nM E3CaG-1
(lane 3), or 400 nM E3CaG-2 (lane 4), and aliquots of the
reactions were resolved on 1% agarose gel electrophoresis. The VWF was analyzed by Western blot using
peroxidase conjugated anti-VWF polyclonal antibodies.
over the same concentration range used for intact TSP-1, delNo-1,
or E3CaG-1.
The NoC-1 fragment was unusual in that it reduced the
CBA/VWF:Ag ratio but did not result in the formation of new
thiols in VWF (Figure 2). This result was confirmed over a 5-log
concentration range of NoC-1 (Figure 3A). Calcium ions were not
required for the effect of the NoC-1 fragment on the CBA/VWF:Ag
ratio, which is in contrast to the requirement for calcium ions for
the VWF reductase activity of TSP-1 (Figure 3B).13,14 In addition,
incubation of VWF with NoC1 did not change VWF multimer size
measured by agarose gel electrophoresis (not shown). These results
suggest a mechanism for the effect of NoC-1 other than the
reduction of VWF multimer size. The simplest explanation is that
NoC-1 competed with VWF for binding to collagen, although this
would imply that the affinity of NoC-1 for collagen is significantly
higher than the affinity of intact TSP-1 for collagen.
TSP-2 fragments did not contain VWF-reducing activity
Increasing concentrations of recombinant TSP-1 or TSP-2 constructs were incubated with VWF for 1 hour and VWF reductase
activity was identified by the concurrent reduction of VWF
multimer size and generation of new thiols in VWF. Neither the
delNo-2 nor E3CaG-2 fragments expressed VWF reductase activity (Figure 4A).
The reduction of the average VWF multimer size by E3CaG-1
but not E3CaG-2 was confirmed by resolving aliquots of the
reaction mixtures on 1% agarose gel electrophoresis and Western
blotting for VWF (Figure 4B).
Alkylation of the cysteine thiol in E3CaG-1 ablated the
VWF-reducing activity
Each TSP-1 subunit contains a single free thiol at Cys974 when
TSP-1 is purified in buffers containing 0.1 mM Ca⫹⫹.26,27 The
homologous residue in TSP-2 is serine and the cysteine in
E3CaG-2 are all in disulfides.17 The number of thiols in E3CaG-1
was quantitated using DTNB and found to be 0.94 mol thiol per
mol E3CaG-1. E3CaG-1, therefore, contains a single free thiol, like
the intact TSP-1 subunit.
The thiol in E3CaG-1 in buffer containing 0.1 mM Ca⫹⫹ was
alkylated with NEM, and the fragment was tested for VWF
reductase activity. Extent of alkylation of E3CaG-1 was assessed by labeling with the biotin-linked maleimide, MPB, and
detecting incorporation of the label by blotting with streptavidinperoxidase. MPB labeled E3CaG-1 but not the alkylated protein
(Figure 5A), which indicated that the majority of the thiols in the
E3CaG-1 preparation were blocked by NEM. TSP-1 or unreacted or alkylated E3CaG-1 was incubated with VWF for 1 hour,
and VWF reductase activity was identified by the concurrent
reduction of VWF multimer size and generation of new thiols in
VWF. Alkylation of E3CaG-1 ablated VWF reductase activity
(Figure 5B).
Figure 5. Alkylation of the cysteine thiol in E3CaG-1
ablates the VWF-reducing activity. (A) E3CaG-1 or
NEM-alkylated E3CaG-1 (NEM-E3CaG-1) was incubated with MPB, resolved on SDS-PAGE, and blotted
with streptavidin-peroxidase to detect the biotin label.
The positions of Mr markers are shown at left. (B) VWF (8
nM) was incubated with 80 nM TSP-1, 8 or 80 nM
E3CaG-1 or 80 nM NEM-E3CaG-1. Aliquots of the
reactions were analyzed for the average VWF multimer
size (䡺) and for the generation of new thiols in VWF (f).
The dotted lines represent no change in VWF multimer
size (top line) or thiols in VWF (bottom line). The asterisks
indicate significant difference compared to control
(P ⬍ .05).
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2836
BLOOD, 15 OCTOBER 2002 䡠 VOLUME 100, NUMBER 8
PIMANDA et al
The cysteine thiol in E3CaG-1 was at position 974
Specific chemical cleavage and mass spectrometry was used to
establish the position of the free thiol in the E3CaG-1 fragment.23,24
NTCB specifically S-cyanylates unpaired cysteine residues at pH 8.
Cleavage of the peptide bond N-terminal to the cyanylated cysteine
is achieved by transfer/migration of the cysteine residue’s nitrogen
to the cyano group at pH 9, forming a 2-iminothiazolidine-4carboxylyl (ITC) peptide. This cleavage will occur at every
cyanylated cysteine, and, therefore, the number of ITC peptides
corresponds to the number of unpaired cysteines in the protein.
E3CaG-1 in pH 8.0 buffer containing either 0.1 mM or 2 mM
Ca⫹⫹ was reacted with NTCB and the peptide bond N-terminal of
the cyanylated cysteine was then cleaved at pH 9. The protein was
reduced and alkylated and resolved by SDS-PAGE. The E3CaG-1
fragment migrated at about 75 kDa, and NTCB cleavage in buffer
containing either 0.1 mM or 2 mM Ca⫹⫹ yielded peptides of about
60 and about 20 kDa (Figure 6A). The expected molecular mass of
the E3CaG-1 fragment was 58 849 Da and NTCB cleavage at
Cys974 should yield an N-terminal fragment of 38 278 Da and an
ITC peptide of 20 726 Da. There is agreement between the
expected and observed mass of the ITC peptide. The discrepancy in
size on SDS-PAGE of the parent molecule and the N-terminal
peptide is probably due to the high aspartate content (17%) of these
fragments.
The 3 fragments were cut from the gel and digested with trypsin
and the resulting peptides analyzed by MALDI-rTOFMS (Figure
6). Residues Asp914-Arg962 mapped to the approximate 60-kDa
fragment. This is consistent with NTCB cleavage of E3CaG-1 at
Cys974. NTCB cleavage at Cys892 or Cys928, for example, would
have resulted in part or all of these amino acids being located
within the ITC fragment. Furthermore, NTCB cleavage at Cys892
or Cys928 would have resulted in N-terminal/ITC peptides of
29 011/29 838 Da and 32 939/25 910 Da, respectively, which is
contrary to the peptide masses resolved by SDS-PAGE. These
results indicate that the free thiol in E3CaG-1 was at Cys974, which
is the same position of the free thiol in intact TSP-1.27
Discussion
The TSPs are a family of extracellular glycoproteins that participate in cell-cell and cell-matrix communication. They approximate
Figure 6. The cysteine thiol in E3CaG-1 is at position 974. (A) E3CaG-1 in pH
8.0 buffer containing either 0.1 mM (lanes 2 and 3) or 2 mM Ca⫹⫹ (lanes 4 and 5)
was untreated (lanes 2 and 4) or reacted with NTCB (lanes 3 and 5). The peptide
bond N-terminal of the cyanylated cysteine was then cleaved at pH 9. The protein
was reduced and alkylated and resolved by SDS-PAGE. The Mr markers are
shown in lane 1. (B-D) The 3 Coomassie-stained fragments were cut from the gel,
digested with trypsin, and the resulting peptides analyzed by MALDI-rTOFMS. (B)
Profile of band i in panel A, lane 5; (C), profile of band ii in panel A, lane 5; (D),
profile of band iii in panel A, lane 5. Band i is undigested E3CaG-1, and bands ii
and iii are the result of a single cleavage of band i. The Mr’s of the major peptides
were matched to the E3CaG-1 amino acid sequence. The amino acid residues of
selected peptides are shown in the tables at the right of the profiles.
Figure 7. Model of the Caⴙⴙ-binding loops and C-terminal sequence of TSP-1
and positions of reactive cysteine. Small circles represent the relative positions of
the 19 cysteines in the Ca⫹⫹-binding loops and C-terminal sequence. Open circles,
shaded circles, and solid circles represent cysteines not involved in disulfide
exchange, moderately involved in disulfide exchange, and heavily involved in
disulfide exchange, respectively, in Ca⫹⫹-depleted TSP-1.26 In TSP-1 purified in
buffers containing 0.1 mM Ca⫹⫹,27 as well as in E3CaG-1, the free thiol is at Cys974.
The dotted lines represent the likely disulfide bonding based on the disulfide
connectivity of E3CaG-2.17 Cys974 may exchange with Cys928 or Cys892 (arrows).
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 15 OCTOBER 2002 䡠 VOLUME 100, NUMBER 8
and regulate cytokines at cell surfaces and play a role in the growth
and differentiation of tissues. The TSP family consists of 5
members in vertebrates, TSP-1 through TSP-428-32 and TSP-5 (also
known as cartilage oligomeric matrix protein).33 A single member,
dTSP, has also been identified in Drosophila.34,35 Based on their
molecular architecture, the TSP gene family can be divided into 2
groups. TSP-1 and TSP-2 (subgroup A) are structurally similar
trimeric proteins, composed of identical disulfide-linked 150-kDa
monomers (Figure 1). The members of subgroup B, TSP-3, TSP-4,
and TSP-5/COMP, are pentameric and differ from subgroup A in
that they lack the procollagen and properdin modules and contain
an extra epidermal growth factor (EGF)–like module (for a review,
see Adams36). The aspartate-rich, Ca⫹⫹-binding repeats and Cterminal sequence are common to all TSPs and have been
extraordinarily well conserved. Using baculovirus-expressed recombinant overlapping constructs of TSP-1 that span the entire subunit
and parallel TSP-2 constructs, we have shown that VWF reductase
activity resides in the Ca⫹⫹-binding repeats and C-terminal sequence of TSP-1 (E3CaG-1), but not in the parallel sequence of
TSP-2 (E3CaG-2). Alkylation of the free thiol at Cys974 in the
C-terminal TSP-1 fragment ablated VWF reductase activity.
Each subunit of TSP-1 contains a free thiol.26 TSP-2, in
contrast, does not contain unpaired cysteine.29,30 The TSP-1 thiol is
remarkably fluid and can reside on any one of 12 different cysteines
in the Ca⫹⫹-binding repeats and C-terminal sequence of TSP-1 on
release from platelets and chelation of Ca⫹⫹ with EDTA.26 In
contrast, TSP-1 purified in buffers containing 0.1 mM Ca⫹⫹ is a
homogeneous TSP-1 population in that the free thiol is at Cys974.27
A model of the cysteine in the Ca⫹⫹-binding repeats and Cterminal sequence of TSP-1 is shown in Figure 7.
We have proposed that nucleophilic attack by a TSP-1 thiol on a
VWF intersubunit disulfide bond results in reduction of the
disulfide bond with formation of an intermediate disulfide-linked
CONTROL OF VWF MULTIMER SIZE BY TSP-1
2837
complex between TSP-1 and VWF. Attack by a second TSP-1 thiol
results in release of VWF and formation of an intramolecular
disulfide bond in TSP-1. We suggest that Cys974 is the TSP-1 thiol
that mediates these events. This is supported by the demonstration
that the free thiol in the E3CaG-1 fragment resides predominantly,
if not exclusively, at Cys974 and that alkylation of this thiol ablates
VWF reductase activity. It remains to be determined whether
Cys974 operates in isolation or that exchange of Cys974 with
Cys928 or Cys892 (Figure 7), or other TSP-1 cysteines, is
important for VWF reductase activity.
Misenheimer et al17 have determined the disulfide connectivity
of E3CaG-2. The disulfide pairing of the 18 cyteines in the
Ca⫹⫹-binding repeats and C-terminal sequence is sequential, that
is, a 1-2, 3-4, 5-6, and so forth pattern. The corresponding disulfide
connectivity in the Ca⫹⫹-binding repeats and C-terminal sequence
of TSP-1 is shown in Figure 7. It will be informative if the unpaired
Cys974 in TSP-1 results in different disulfide connectivity.
TSP-3,31 TSP-4,32 TSP-5/COMP,33 and dTSP34,35 each contain
an unpaired cysteine, although it is not at a position equivalent to
Cys974 in TSP-1 but is very close to the C-terminus. It may be that
these other TSP family members also have VWF reductase activity,
although their tissue distribution (cartilage, bone, ligaments, lung,
and brain) does not support this function in vivo.37-39 In contrast,
TSP-1 is readily demonstrable in and around blood vessels, which
is where VWF acts and the regulation of its multimer size has
functional relevance.
Acknowledgments
The authors thank Dr Michael Berndt and Dr Eric Huizinga for the
von Willebrand factor.
References
1. Sadler JE. Biochemistry and genetics of von Willebrand factor. Annu Rev Biochem. 1998;67:395424.
2. Ruggeri ZM, Dent JA, Saldivar E. Contribution of
distinct adhesive interactions to platelet aggregation in flowing blood. Blood. 1999;94:172-178.
3. Savage B, Sixma JJ, Ruggeri ZM. Functional
self-association of von Willebrand factor during
platelet adhesion under flow. Proc Natl Acad Sci
U S A. 2002;99:425-430.
4. Counts RB, Paskell SL, Elgee SK. Disulfide
bonds and the quaternary structure of factor VIII/
von Willebrand factor. J Clin Invest. 1978;62:702709.
5. Katsumi A, Tuley EA, Bodó I, Sadler JE. Localization of disulfide bonds in the cysteine knot domain
of human von Willebrand factor. J Biol Chem.
2000;275:25585-25594.
6. Wagner DD, Lawrence SO, Ohlsson-Wilhelm BM,
Fay PJ, Marder VJ. Topology and order of formation of interchain disulfide bonds in von Willebrand factor. Blood. 1987;69:27-32.
7. Dong Z, Thoma RS, Crimmins DL, McCourt DW,
Tuley EA, Sadler JE. Disulfide bonds required to
assemble functional von Willebrand factor multimers. J Biol Chem. 1994;269:6753-6758.
8. Furlan M. von Willebrand factor: molecular size
and functional activity. Ann Hematol. 1996;72:
341-348.
9. Moake JL, Turner NA, Stathopoulos NA, Nolasco
LH, Hellums JD. Involvement of large plasma von
Willebrand factor (vWF) multimers and unusually
large vWF forms derived from endothelial cells in
shear stress-induced platelet aggregation. J Clin
Invest. 1989;78:1456-1461.
10. Moake JL, Rudy CK, Troll JH, et al. Unusually
large plasma factor VIII:von Willebrand factor
multimers in chronic relapsing thrombotic thrombocytopenic purpura. N Engl J Med. 1982;307:
1432-1435.
19. Mosher DF, Huwiler KG, Misenheimer TM, Annis
DS. Expression of recombinant matrix components using baculoviruses. In: Adams JC, ed.
Methods in Cell-Matrix Adhesion. San Diego, CA:
Academic Press; 2002:69-81.
11. Moake JL. Studies on the pathophysiology of
thrombotic thrombocytopenic purpura. Semin Hematol. 1997;34:83-89.
20. Favaloro EJ, Grispo L, Exner T, Koutts J. Development of a simple collagen based ELISA assay
aids in the diagnosis of, and permits sensitive discrimination between type I and type II, von Willebrand’s disease. Blood Coagul Fibrinolysis.
1991;2:285-291.
12. Lawler, J. The functions of thrombospondin-1 and
-2. Curr Opin Cell Biol. 2000;12:634-640.
13. Xie L, Chesterman CN, Hogg PJ. Reduction of
von Willebrand factor by endothelial cells.
Thromb Haemost. 2000;84:506-513.
14. Xie L, Chesterman CN, Hogg PJ. Control of von
Willebrand factor multimer size by thrombospondin-1. J Exp Med. 2001;193:1341-1349.
15. Murphy-Ullrich JE, Mosher DF. Localization of
thrombospondin in clots formed in situ. Blood.
1985;66:1098-1104.
16. Hogg PJ, Hotchkiss KA, Jiménez BM, Stathakis
P, Chesterman CN. Interaction of platelet-derived
growth factor with thrombospondin 1: dependence on the disulfide-bond arrangement in
thrombospondin 1. Biochem J. 1997;326:709716.
17. Misenheimer TM, Hahr AJ, Harms AC, Annis DS,
Mosher DF. Disulfide connectivity of recombinant
C-terminal region of human thrombospondin 2.
J Biol Chem. 2001;276:45882-45887.
18. Misenheimer TM, Huwiler KG, Annis DS, Mosher
DF. Physical characterization of the procollagen
module of human thrombospondin 1 expressed in
insect cells. J Biol Chem. 2000;275:4093840945.
21. Ruggeri ZM, Zimmerman TS. Variant von Willebrand’s disease: characterization of two subtypes by analysis of multimer composition of factor VIII/von Willebrand factor in plasma and
platelets. J Clin Invest. 1980;65:1318-1325.
22. Riddles PW, Blakeley RL, Zerner B. Reassessment of Ellman’s reagent. Methods Enzymol.
1983;91:49-60.
23. Jacobson GR, Schaffer MH, Stark GR, Vanaman
TC. Specific chemical cleavage in high yield at
the amino peptide bonds of cysteine and cystine
residues. J Biol Chem. 1973;248:6583-6591.
24. Wu J, Gage DA, Watson JT. A strategy to locate
cysteine residues in proteins by specific chemical
cleavage followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal Biochem. 1996;235:161-174.
25. Raftery MJ, Yang Z, Valenzuela SM, Geczy CL.
Novel intra- and inter-molecular sulfonamide
bonds in S100A8 produced by hypochlorite oxidation. J Biol Chem. 2001;276:33393-33401.
26. Speziale MV, Detwiler TC. Free thiols of platelet
thrombospondin. J Biol Chem. 1990;265:1785917867.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2838
PIMANDA et al
27. Sun X, Skorstengaard K, Mosher DF. Disulfides
modulate RGD-inhibitable cell adhesive activity of
thrombospondin. J Cell Biol. 1992;118:693-701.
28. Lawler J, Hynes RO. The structure of human
thrombospondin, an adhesive glycoprotein with
multiple calcium-binding sites and homologies
with several different proteins. J Cell Biol. 1986;
103:1635-1648.
29. Bornstein P, O’Rourke K, Wikstrom K, et al. A second, expressed thrombospondin gene (Thbs2)
exists in the mouse genome. J Biol Chem. 1991;
266:12821-12824.
30. LaBell TL, Milewicz DJ, Disteche CM, Byers PH.
Thrombospondin II: partial cDNA sequence, chromosome location, and expression of a second
member of the thrombospondin gene family in
humans. Genomics. 1992;12:421-429.
BLOOD, 15 OCTOBER 2002 䡠 VOLUME 100, NUMBER 8
31. Vos HL, Devarayalu S, de Vries Y, Bornstein P.
Thrombospondin 3 (Thbs3), a new member of the
thrombospondin gene family. J Biol Chem. 1992;
267:12192-12196.
32. Lawler J, Duquette M, Whittaker CA, Adams JC,
McHenry K, DeSimone DW. Identification and
characterization of thrombospondin-4, a new
member of the thrombospondin gene family.
J Cell Biol. 1993;120:1059-1067.
33. Oldberg A, Antonsson P, Lindblom K, Heinegard
D. COMP (cartilage oligomeric matrix protein) is
structurally related to the thrombospondins. J Biol
Chem. 1992;267:22346-22350.
34. Adams MD, Celniker SE, Holt RA, et al. The genome sequence of Drosophila melanogaster. Science. 2000;287:2185-2195.
35. Adolph KW. A thrombospondin homologue in Dro-
sophila melanogaster: cDNA and protein structure. Gene. 2001;269:177-184.
36. Adams JC. Thrombospondins: multifunctional
regulators of cell interactions. Annu Rev Cell Dev
Biol. 2001;17:25-51.
37. Tooney PA, Sakai T, Sakai K, Aeschlimann D,
Mosher DF. Restricted localization of thrombospondin-2 protein during mouse embryogenesis: a comparison to thrombospondin-1. Matrix
Biol. 1998;17:131-143.
38. Iruela-Arispe ML, Liska DJ, Sage EH, Bornstein
P. Differential expression of thrombospondin 1, 2,
and 3 during murine development. Dev Dyn.
1993;197:40-56.
39. Tucker RP, Adams JC, Lawler J. Thrombospondin-4 is expressed by early osteogenic
tissues in the chick embryo. Dev Dyn. 1995;203:
477-490.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2002 100: 2832-2838
doi:10.1182/blood-2002-03-0770 originally published online June
21, 2002
The von Willebrand factor−reducing activity of thrombospondin-1 is located
in the calcium-binding/C-terminal sequence and requires a free thiol at
position 974
John E. Pimanda, Douglas S. Annis, Mark Raftery, Deane F. Mosher, Colin N. Chesterman and Philip J.
Hogg
Updated information and services can be found at:
http://www.bloodjournal.org/content/100/8/2832.full.html
Articles on similar topics can be found in the following Blood collections
Hemostasis, Thrombosis, and Vascular Biology (2485 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.