|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Prepublished online as a Blood First Edition Paper on June 21, 2002; DOI 10.1182/blood-2002-04-1234.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Departments of Internal Medicine and
Pediatrics, University of Iowa College of Medicine, and Veterans
Affairs Medical Center, Iowa City; Abramson Family Cancer Research
Institute, Department of Pathology and Laboratory Medicine, University
of Pennsylvania School of Medicine, Philadelphia.
The adapter protein SLP-76 is a critical mediator of signal
transduction via the platelet collagen receptor glycoprotein VI (GPVI)
and its coreceptor FcR Collagen is a major physiologic agonist for
platelet activation, mediating shape change, granule release,
aggregation, and expression of procoagulant activity. Several putative
collagen receptors have been identified on platelets,1 but
the major signal transducing collagen receptor is a member of the
immunoglobulin superfamily, glycoprotein VI (GPVI).2,3
Signal transduction through GPVI is dependent on coexpression of the Fc
receptor The hematopoietic cell adaptor protein SLP-76 is a critical component
of the GPVI/FcR Although collagen is known to be a potent agonist for the
platelet procoagulant response, the signal transduction pathways leading to expression of platelet procoagulant activity are poorly characterized.15 The procoagulant activity of activated
platelets is mediated by expression of anionic membrane phospholipids
that support the assembly and activity of procoagulant enzyme
complexes,16 and can be detected by functional
assays17 or annexin V binding.18 It is not
known whether SLP-76 is necessary for collagen-induced expression of
platelet procoagulant activity.19
In this study, we tested the hypothesis that SLP-76 is required for
collagen-induced expression of platelet procoagulant activity in murine
platelets. Platelets were isolated from SLP-76-deficient mice, and the
procoagulant response to collagen was measured by annexin V binding and
a prothrombinase assay. Our results demonstrate that collagen induces
procoagulant responses in murine platelets despite the absence of
SLP-76 and that the GPVI agonist convulxin20 fails to
produce detectable platelet procoagulant activity in murine platelets.
SLP-76 is required, however, for maximal procoagulant activity when
platelets are stimulated simultaneously with thrombin and collagen.
These findings demonstrate that the GPVI/FcR Reagents and mice
Platelet isolation and aggregation
Flow cytometry Washed platelets (1 × 106/reaction) were suspended in modified Tyrode buffer containing 2.0 mM CaCl2, and either left unstimulated ("control") or stimulated with convulxin (250 ng/mL), thrombin (0.5 U/mL), thrombin plus convulxin, or ionomycin (3 µM) for 15 minutes at 37°C without stirring. Platelets were then incubated for 10 minutes at 23°C with PE-conjugated rat antimouse CD49b and either FITC-conjugated rat antimouse CD62P, FITC-conjugated annexin V, or FITC-conjugated rat IgG1 (isotype control). Platelets were analyzed on a Becton Dickinson (San Diego, CA) FACScan flow cytometer as described previously.14 Platelets were gated by light scatter and expression of CD49b ( 2 integrin).
Platelet procoagulant activity Platelet procoagulant activity was measured in a prothrombinase assay that was modified from a method originally developed for use with human platelets.22 Washed murine platelets (6 × 106/reaction) were either left unstimulated ("control") or stimulated with convulxin (250 ng/mL), type I fibrillar collagen (40 µg/mL), thrombin (0.5 U/mL), thrombin plus collagen, or ionomycin (3 µM) at 37°C, without agitation but with slow mixing (100 rpm), in modified Tyrode buffer containing 2.9 mM CaCl2 and 0.05% (wt/vol) fatty acid-free bovine serum albumin (BSA). After 10 minutes, factor Va (6 nM) and factor Xa (3 nM) were added, followed 1 minute later by prothrombin (4 µM). The concentration of CaCl2 in the final reaction mixture was 2.0 mM. The rate of thrombin generation was measured by subsampling the reaction mixture into 0.05 M Tris (tris(hydroxymethyl)aminomethane)-HCl, 120 mM NaCl, 2.0 mM EDTA (ethylenediaminetetraacetic acid), pH 7.5, and measuring thrombin activity using the chromogenic substrate Chromozym TH (0.32 mM) in a Spectramax 190 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA).To verify that the rate of thrombin generation was dependent on the concentration of procoagulant phospholipid, control experiments were performed with murine platelets that had been subjected to 3 cycles of freezing and thawing to expose anionic membrane phospholipids. The initial rate of thrombin generation demonstrated a logarithmic relationship to the amount of added platelet phospholipid. Confocal microscopy Glass coverslips (12 mm) were coated with 1.0 mg/mL human fibrinogen (Sigma Chemicals) as described previously.14 After 2 rinses with phosphate-buffered saline (PBS), the coverslips were blocked with Tyrode buffer containing 2% BSA for 30 minutes at room temperature. Washed platelets (1 × 105/well) in modified Tyrode buffer containing 2.0 mM CaCl2 were added to the fibrinogen-coated coverslips, and then stimulated with thrombin (0.5 U/mL), collagen (50 µg/mL), or thrombin plus collagen for 30 minutes at 37°C in the presence of FITC anti-CD49b and PE-conjugated annexin V. Platelets were then fixed with 1% paraformaldehyde, washed with PBS, and analyzed by confocal microscopy (Bio-Rad 1024 confocal microscope; Hercules, CA).Statistical analysis The unpaired, 2-tailed Student t test was used to compare values in SLP-76+/ and SLP-76/
mice. The paired, 2-tailed Student t test was used to
compare values obtained with different platelet agonists. A value of
P < .05 was used to define statistical significance.
Values are reported as mean ± SE.
Platelet aggregation and granule release Platelets were isolated from SLP-76+/ or
SLP-76/ mice, and aggregation was measured in response
to equine fibrillar collagen, the GPVI agonist convulxin, or bovine
-thrombin. SLP-76+/ platelets aggregated normally in
response to all 3 agonists (Figure 1A).
SLP-76/ platelets aggregated normally in response to
thrombin, but failed to aggregate in response to collagen or convulxin
(Figure 1B).
To monitor granule release, SLP-76+/
Annexin V binding To measure surface exposure of anionic phospholipids, platelets from SLP-76+/ and SLP-76/ mice were
stimulated with thrombin, convulxin, or ionomycin, and annexin V
binding was measured by flow cytometry. Stimulation with thrombin
enhanced annexin V binding to both SLP-76+/ and
SLP-76/ platelets (Figure
3A-B). After stimulation with thrombin,
32% ± 4% of SLP-76+/ platelets and 39% ± 5% of
SLP-76/ platelets stained positively for annexin V
binding, compared with less than 25% of unstimulated control platelets
(P < .05; Figure 3C). In contrast, convulxin failed to
induce annexin V binding to either SLP-76+/ or
SLP-76/ platelets. Ionomycin induced a large increase
in annexin V binding to both SLP-76+/ and
SLP-76/ platelets, with more than 85% of platelets
staining positively for annexin V after stimulation with ionomycin
(Figure 3C).
Platelet procoagulant activity Platelet procoagulant activity was measured in a modified prothrombinase assay. After stimulation with various agonists, the ability of platelets to support the generation of thrombin from prothrombin was measured in the presence of exogenous factor Va and factor Xa. Convulxin failed to stimulate a detectable increase in prothrombinase activity with platelets from SLP-76+/ or
SLP-76/ mice, whereas collagen produced a significant
increase in prothrombinase activity with platelets from either
SLP-76+/ or SLP-76/ mice
(P < .05 versus unstimulated platelets; Figure
4A). These results suggested that the
GPVI/FcR /SLP-76 pathway is not absolutely necessary for
collagen-induced prothrombinase activity. Further support for this
finding was obtained with platelets from FcR/ mice,
in which prothrombinase activity was induced by collagen but not
convulxin (Figure 4A). Ionomycin produced a large (> 10-fold) increase in prothrombinase activity with SLP-76+/,
SLP-76/ , or FcR/ platelets
(P < .01).
To further define the role of SLP-76 in platelet procoagulant activity,
platelets from SLP-76+/ Annexin V binding after costimulation with thrombin and convulxin We next measured annexin V binding with platelets that were costimulated with thrombin and convulxin. After simultaneous activation with thrombin plus convulxin, SLP-76+/ and
SLP-76/ platelets exhibited equivalent surface
expression of P-selectin (Figure 5A-B),
but SLP-76/ platelets had markedly less annexin V
binding than SLP-76+/ platelets (Figure 5C,D). The flow
cytometric pattern of annexin V binding observed with
SLP-76/ platelets after costimulation with thrombin and
convulxin (Figure 5D) was very similar to that observed when
SLP-76/ platelets were stimulated with thrombin alone
(Figure 3B). This result was confirmed with platelets from several
additional pairs of SLP-76+/ and SLP-76/
mice (Figure 5E). These findings demonstrate that the GPVI/SLP-76 signal transduction pathway does contribute to anionic phospholipid exposure when platelets are stimulated simultaneously with thrombin and convulxin.
Annexin V binding after costimulation with thrombin and collagen Annexin V binding was examined with fibrinogen-adherent platelets by confocal microscopy (Figure 6). Following adherence to fibrinogen-coated coverslips, platelets were stimulated with thrombin, collagen, or thrombin plus collagen. Adherent platelets and platelet aggregates were identified by staining with FITC anti-CD49b (green fluorescence) and annexin V binding was detected by staining with PE-conjugated annexin V (red fluorescence). A low level of annexin V staining was detected with both SLP-76+/ and
SLP-76/ platelets after stimulation with thrombin
(Figure 6A,D). Moderate staining for annexin V was observed with
SLP-76+/ platelets after stimulation with collagen
(Figure 6B) and strong staining for annexin V was seen with
SLP-76+/ platelets after costimulation with thrombin and
collagen (Figure 6C). With SLP-76/ platelets, however,
stimulation with collagen (Figure 6E) or costimulation with thrombin
and collagen (Figure 6F) did not produce an evident increase in annexin
V staining over that observed after stimulation with thrombin
alone (Figure 6D). These findings indicate that SLP-76 is necessary for
maximal anionic phospholipid exposure when fibrinogen-adherent
platelets are stimulated with collagen or costimulated with thrombin
and collagen.
The adapter protein SLP-76 is essential for platelet activation
responses that are downstream of the major signal transducing collagen
receptor GPVI.7 Platelets that lack SLP-76 exhibit marked
impairment of several activation responses to collagen, including shape
change, aggregation, and granule release.8,9 We
hypothesized, therefore, that deficiency of SLP-76 also may impair
platelet procoagulant responses to collagen. Contrary to our
expectations, we found that SLP-76 was not necessary for platelet procoagulant activity when platelets in suspension were stimulated by
collagen as a single agonist. Consistent with this finding, we also
found that deficiency of FcR Our finding that stimulation of murine platelets with convulxin as a
single agonist did not induce detectable annexin V binding or
expression of procoagulant activity is discordant with the observations
of Furihata et al, who found an association between GPVI content and
convulxin-induced procoagulant activity in human platelets.23 This discordancy may be related to
differences between murine and human platelets or to differences in the
sensitivities of the procoagulant activity assays to platelet-derived
factor V. Because the prothrombinase assay used by Furihata and
coworkers relied on activated platelets as a source of factor V, it is
very likely that a component of the platelet procoagulant activity detected in their study was due to convulxin-induced release of factor
V from There is considerable evidence that GPVI is a major signal transducing
collagen receptor on platelets, but it is likely that other surface
receptors also contribute to platelet-collagen
interactions.6 The initial tethering of platelets to
collagen surfaces under conditions of high shear stress is dependent on
an interaction between collagen-bound von Willebrand factor (VWF) and
the glycoprotein Ib/V/IX complex.25 VWF-mediated tethering
facilitates subsequent interactions between collagen and GPVI and other
platelet surface receptors such as Although our results clearly demonstrate that collagen can induce
platelet procoagulant activity in the absence of SLP-76, it is evident
that the GPVI/FcR In agreement with previous results with human platelets,24,31 we found that the calcium ionophore ionomycin is a potent inducer of annexin V binding and prothrombinase activity. The annexin V binding response to ionomycin was similar to that observed after costimulation with thrombin and collagen (Figure 5), which suggests that a very potent calcium mobilization signal is sufficient to generate maximum platelet procoagulant activity. Alternatively, ionomycin may produce membrane effects that result in exposure of anionic phospholipids independently of its effects on calcium influx. In summary, we have found that stimulation of murine platelets with the GPVI agonist convulxin produces aggregation and granule release, but little procoagulant activity. In contrast, stimulation with fibrillar collagen produces substantial platelet procoagulant activity in addition to aggregation and granule release. SLP-76 is necessary for aggregation and granule release, which is consistent with a major role for GPVI in these activation responses to collagen. SLP-76 is dispensable for procoagulant activity when platelets in suspension are stimulated by collagen as a single agonist, but required for maximal procoagulant activity induced by costimulation with thrombin and collagen. We conclude that both GPVI-independent (activated by collagen alone) and GPVI-dependent (activated by collagen plus thrombin) signaling events contribute to platelet procoagulant activity.
We thank Todd Goldman for technical assistance and Justin Fishbaugh, Technical Director of the University of Iowa Holden Comprehensive Cancer Center Flow Cytometry Facility, for expert advice. Confocal microscopy was performed in the University of Iowa Central Microscopy Research Facility.
Submitted April 24, 2002; accepted June 7, 2002.
Prepublished online as Blood First Edition Paper, June 21, 2002; DOI 10.1182/blood-2002-04-1234.
Supported by National Institutes of Health grants HL063017, DK025295, HL004460, HD027748, and HL063943, and the Office of Research and Development, Department of Veterans Affairs.
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.
Reprints: Steven R. Lentz, Department of Internal Medicine, C303 GH, University of Iowa, Iowa City, IA 52242; e-mail: steven-lentz{at}uiowa.edu.
1. Clemetson KJ, Clemetson JM. Platelet collagen receptors. Thromb Haemost. 2001;86:189-197[Medline] [Order article via Infotrieve].
2.
Clemetson JM, Polgar J, Magnenat E, Wells TN, Clemetson KJ.
The platelet collagen receptor glycoprotein VI is a member of the immunoglobulin superfamily closely related to Fc
3.
Jandrot-Perrus M, Busfield S, Lagrue AH, et al.
Cloning, characterization, and functional studies of human and mouse glycoprotein VI: a platelet-specific collagen receptor from the immunoglobulin superfamily.
Blood.
2000;96:1798-1807 4. Poole A, Gibbins JM, Turner M, et al. The Fc receptor gamma-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen. EMBO J. 1997;16:2333-2341[CrossRef][Medline] [Order article via Infotrieve].
5.
Nieswandt B, Bergmeier W, Schulte V, Rackebrandt K, Gessner JE, Zirngibl H.
Expression and function of the mouse collagen receptor glycoprotein VI is strictly dependent on its association with the FcRgamma chain.
J Biol Chem.
2000;275:23998-24002 6. Watson SP, Asazuma N, Atkinson B, et al. The role of ITAM- and ITIM-coupled receptors in platelet activation by collagen. Thromb Haemost. 2001;86:276-288[Medline] [Order article via Infotrieve]. 7. Judd BA, Koretzky GA. The role of the adapter molecule SLP-76 in platelet function. Oncogene. 2001;20:6291-6299[CrossRef][Medline] [Order article via Infotrieve]. 8. Clements JL, Lee JRGB, Yang B, et al. Fetal hemorrhage and platelet dysfunction in SLP-76-deficient mice. J Clin Invest. 1999;103:19-25[Medline] [Order article via Infotrieve].
9.
Gross BS, Lee JR, Clements JL, et al.
Tyrosine phosphorylation of SLP-76 is downstream of Syk following stimulation of the collagen receptor in platelets.
J Biol Chem.
1999;274:5963-5971
10.
Judd BA, Myung PS, Obergfell A, et al.
Differential requirement for LAT and SLP-76 in GPVI versus T cell receptor signaling.
J Exp Med.
2002;195:705-717
11.
Obergfell A, Judd BA, del P, Schwartz MA, Koretzky GA, Shattil SJ.
The molecular adapter SLP-76 relays signals from platelet integrin
12.
Clements JL, Yang B, Ross-Barta SE, et al.
Requirement for the leukocyte-specific adapter protein SLP-76 for normal T cell development.
Science.
1998;281:416-419 13. Pivniouk V, Tsitsikov E, Swinton P, Rathbun G, Alt FW, Geha RS. Impaired viability and profound block in thymocyte development in mice lacking the adaptor protein SLP-76. Cell. 1998;94:229-238[CrossRef][Medline] [Order article via Infotrieve].
14.
Judd BA, Myung PS, Leng L, et al.
Hematopoietic reconstitution of SLP-76 corrects hemostasis and platelet signaling through alpha IIb beta 3 and collagen receptors.
Proc Natl Acad Sci U S A.
2000;97:12056-12061
15.
Zwaal RF, Schroit AJ.
Pathophysiologic implications of membrane phospholipid asymmetry in blood cells.
Blood.
1997;89:1121-1132
16.
Rosing J, van Rijn JL, Bevers EM, van Dieijen G, Comfurius P, Zwaal RF.
The role of activated human platelets in prothrombin and factor X activation.
Blood.
1985;65:319-332 17. Bevers EM, Comfurius P, Zwaal RF. Platelet procoagulant activity: physiological significance and mechanisms of exposure. Blood Rev. 1991;5:146-154[CrossRef][Medline] [Order article via Infotrieve].
18.
Dachary-Prigent J, Freyssinet JM, Pasquet JM, Carron JC, Nurden AT.
Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study showing a role for free sulfhydryl groups.
Blood.
1993;81:2554-2565 19. Sims PJ, Wiedmer T. Unraveling the mysteries of phospholipid scrambling. Thromb Haemost. 2001;86:266-275[Medline] [Order article via Infotrieve].
20.
Polgar J, Clemetson JM, Kehrel BE, et al.
Platelet activation and signal transduction by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor.
J Biol Chem.
1997;272:13576-13583 21. Takai T, Li M, Sylvestre D, Clynes R, Ravetch JV. FcR gamma chain deletion results in pleiotrophic effector cell defects. Cell. 1994;76:519-529[CrossRef][Medline] [Order article via Infotrieve]. 22. Bevers EM, Comfurius P, Reutelingsperger CPM, Zwaal RF. Platelet procoagulant activity and its measurement. In: Watson SP,Authi KS, eds. Platelets: a Practical Approach. Oxford: Oxford University Press; 1996:319-340.
23.
Furihata K, Clemetson KJ, Deguchi H, Kunicki TJ.
Variation in human platelet glycoprotein VI content modulates glycoprotein VI-specific prothrombinase activity.
Arterioscl Thromb Vasc Biol.
2001;21:1857-1863
24.
Siljander P, Farndale RW, Feijge MA, et al.
Platelet adhesion enhances the glycoprotein VI-dependent procoagulant response: involvement of p38 MAP kinase and calpain.
Arterioscl Thromb Vasc Biol.
2001;21:618-627 25. Savage B, Cattaneo M, Ruggeri ZM. Mechanisms of platelet aggregation. Curr Opin Hematol. 2001;8:270-276[CrossRef][Medline] [Order article via Infotrieve].
26.
Chen J, Diacovo TG, Grenache DG, Santoro SA, Zutter MM.
The 27. Nieswandt B, Brakebusch C, Bergmeier W, et al. Glycoprotein VI but not alpha 2 beta 1 integrin is essential for platelet interaction with collagen. EMBO J. 2001;20:2120-2130[CrossRef][Medline] [Order article via Infotrieve].
28.
Diaz-Ricart M, Tandon NN, Carretero M, Ordinas A, Bastida E, Jamieson GA.
Platelets lacking functional CD36 (glycoprotein IV) show reduced adhesion to collagen in flowing whole blood.
Blood.
1993;82:491-496
29.
Moog S, Mangin P, Lenain N, et al.
Platelet glycoprotein V binds to collagen and participates in platelet adhesion and aggregation.
Blood.
2001;98:1038-1046
30.
Zheng YM, Liu CD, Chen H, Locke D, Ryan JC, Kahn ML.
Expression of the platelet receptor GPVI confers signaling via the Fc receptor gamma-chain in response to the snake venom convulxin but not to collagen.
J Biol Chem.
2001;276:12999-13006
31.
Alberio L, Safa O, Clemetson KJ, Esmon CT, Dale GL.
Surface expression and functional characterization of alpha-granule factor V in human platelets: effects of ionophore A23187, thrombin, collagen, and convulxin.
Blood.
2000;95:1694-1702 32. Dale GL, Friese P, Batar P, et al. Stimulated platelets use serotonin to enhance their retention of procoagulant proteins on the cell surface. Nature. 2001;415:175-179.
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  S. M. Jobe, K. M. Wilson, L. Leo, A. Raimondi, J. D. Molkentin, S. R. Lentz, and J. Di Paola Critical role for the mitochondrial permeability transition pore and cyclophilin D in platelet activation and thrombosis Blood, February 1, 2008; 111(3): 1257 - 1265. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Dayal, K. M. Wilson, L. Leo, E. Arning, T. Bottiglieri, and S. R. Lentz Enhanced susceptibility to arterial thrombosis in a murine model of hyperhomocysteinemia Blood, October 1, 2006; 108(7): 2237 - 2243. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Jobe, L. Leo, J. S. Eastvold, G. Dickneite, T. L. Ratliff, S. R. Lentz, and J. Di Paola Role of FcR{gamma} and factor XIIIA in coated platelet formation Blood, December 15, 2005; 106(13): 4146 - 4151. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. F.W. Keuren, S. J.H. Wielders, H. Ulrichts, T. Hackeng, J. W.M. Heemskerk, H. Deckmyn, E. M. Bevers, and T. Lindhout Synergistic Effect of Thrombin on Collagen-Induced Platelet Procoagulant Activity Is Mediated Through Protease-Activated Receptor-1 Arterioscler. Thromb. Vasc. Biol., July 1, 2005; 25(7): 1499 - 1505. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. Kempton, M. Hoffman, H. R. Roberts, and D. M. Monroe Platelet Heterogeneity: Variation in Coagulation Complexes on Platelet Subpopulations Arterioscler. Thromb. Vasc. Biol., April 1, 2005; 25(4): 861 - 866. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Gibbins Platelet adhesion signalling and the regulation of thrombus formation J. Cell Sci., July 15, 2004; 117(16): 3415 - 3425. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
3 to the
platelet cytoskeleton.
P < .05 versus
SLP-76+/
