Surface expression and functional characterization of α-granule factor V in human platelets: effects of ionophore A23187, thrombin, collagen, and convulxin

Blood ◽  
2000 ◽  
Vol 95 (5) ◽  
pp. 1694-1702 ◽  
Author(s):  
L. Alberio ◽  
O. Safa ◽  
K. J. Clemetson ◽  
C. T. Esmon ◽  
G. L. Dale
Keyword(s):  
Functional Characterization ◽  
Factor Xa ◽  
Annexin V ◽  
Surface Expression ◽  
Human Platelets ◽  
Ionophore A23187 ◽  
Factor V ◽  
Glycoprotein Vi ◽  
Activated Platelets ◽  
Simultaneous Activation

Factor V (FV) present in platelet -granules has a significant but incompletely understood role in hemostasis. This report demonstrates that a fraction of platelets express very high levels of surface-bound, -granule FV on simultaneous activation with 2 agonists, thrombin and convulxin, an activator of the collagen receptor glycoprotein VI. This subpopulation of activated platelets represents 30.7% ± 4.7% of the total population and is referred to as convulxin and thrombin–induced-FV (COAT-FV) platelets. COAT-FV platelets are also observed on activation with thrombin plus collagen types I, V, or VI, but not with type III. No single agonist examined was able to produce COAT-FV platelets, although ionophore A23187 in conjunction with either thrombin or convulxin did generate this population. COAT-FV platelets bound annexin-V, indicating exposure of aminophospholipids and were enriched in young platelets as identified by the binding of thiazole orange. The functional significance of COAT-FV platelets was investigated by demonstrating that factor Xa preferentially bound to COAT-FV platelets, that COAT-FV platelets had more FV activity than either thrombin or A23187–activated platelets, and that COAT-FV platelets were capable of generating more prothrombinase activity than any other physiologic agonist examined. Microparticle production by dual stimulation with thrombin and convulxin was less than that observed with A23187, indicating that microparticles were not responsible for all the activities observed. These data demonstrate a new procoagulant component produced from dual stimulation of platelets with thrombin and collagen. COAT-FV platelets may explain the unique role of -granule FV and the hemostatic effectiveness of young platelets.

Platelet Procoagulant Properties Studied with Snake Venom Prothrombin Activators

1987 ◽  
Vol 57 (03) ◽  
pp. 349-355 ◽  
Author(s):  
Han Speijer ◽  
José W P Govers-Riemslag ◽  
Robert F A Zwaal ◽  
Jan Rosing
Keyword(s):  
Snake Venom ◽  
Time Course ◽  
Considerable Increase ◽  
Factor Xa ◽  
Human Platelets ◽  
Ionophore A23187 ◽  
Activated Platelets ◽  
Essential Components ◽  
Echis Carinatus ◽  
Prothrombin Activation

SummaryPurified snake venom prothrombin activators were used to probe the procoagulant properties of platelet membranes. Human platelets were able to stimulate prothrombin activation by the venom activators from Oxyuranus scutellatus and Notechis scutatus, while the prothrombin activator from Echis carinatus was not affected by the presence of platelets. The prothrombinconverting activity of platelets was further studied with the venom activator from Oxyuranus scutellatus and with the factor Xa-Va complex as prothrombin activating enzymes. Stimulation of platelets with collagen, collagen plus thrombin or with the Ca-ionophore A23187 resulted in a considerable increase of platelet prothrombin converting activity probed with the factor Xa-Va complex as well as with the prothrombin activator from Oxyuranus scutellatus. The stimulatory effect of activated platelets on the rates of prothrombin activation by Oxyuranus scutellatus was similar to that determined for factor Xa-Va-catalyzed prothrombin activation. Compared to non-stimulated platelets, platelets stimulated with thrombin plus collagen exposed 20-times more procoagulant sites for as well the factor Xa-Va complex, as for the venom activator from Oxyuranus scutellatus. The actual number of procoagulant sites per platelet determined with the factor Xa-Va complex was in close agreement with the number of sites determined with the venom activator. Also the time course of appearance of procoagulant activity during platelet stimulation by collagen plus thrombin was comparable for both activator complexes. Phospholipase A2 treatment of stimulated platelets resulted in an almost complete loss of their ability to stimulate prothrombin activation by the enzyme from Oxyuranus scutellatus or by factor Xa-Va complex. The findings presented in this paper suggest: a) that the factor Xa-Va complex and the prothrombin activator from Oxyuranus scutellatus recognize the same procoagulant sites on both stimulated and unstimulated platelets and b) that negatively-charged phospholipids are essential components of these procoagulant sites.

scholarly journals Polyphosphate accelerates factor V activation by factor XIa

2015 ◽  
Vol 113 (03) ◽  
pp. 599-604 ◽  
Author(s):  
Sharon Choi ◽  
Stephanie Smith ◽  
James Morrissey
Keyword(s):  
Thrombin Generation ◽  
Factor Xa ◽  
Human Platelets ◽  
Critical Event ◽  
Factor V ◽  
Factor Va ◽  
Activated Platelets ◽  
Factor Xia ◽  
High Concentrations ◽  
Prothrombin Activation

SummaryFactor Va enhances the rate of prothrombin activation by factor Xa by four to five orders of magnitude. Production of initiating levels of factor Va from its precursor, factor V, is a critical event early in haemostasis, as factor V exhibits negligible cofactor activity. While thrombin is the most potent physiological back-activator of factor V, the first prothrombinase complexes require a source of factor Va prior to thrombin generation. A recent study by Whelihan et al. (J Thromb Haemost 2010; 8:1532–1539) identified factor XIa as a candidate for the initial thrombin-independent activation of factor V, although this reaction was slow and required relatively high concentrations of factors V and XIa. Activated platelets secrete polyphosphate, which we previously showed to be potently procoagulant. We now report that polyphosphate greatly accelerates factor V activation by factor XIa, and that this is supported by polyphosphate polymers of the size secreted by activated human platelets. This finding provides additional evidence that factor XIa-mediated generation of factor Va may contribute to the initiation of haemostasis.

Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

1998 ◽  
Vol 79 (01) ◽  
pp. 177-185 ◽  
Author(s):  
Ashia Siddiqua ◽  
Michael Wilkinson ◽  
Vijay Kakkar ◽  
Yatin Patel ◽  
Salman Rahman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Functional Characterization ◽  
Human Platelets ◽  
Western Blots ◽  
Activated Platelets ◽  
Reducing Conditions ◽  
Fibronectin Binding

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

scholarly journals The effect of platelets upon factor Xa-catalyzed activation of factor VII in vitro

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 396-401 ◽  
Author(s):  
LV Rao ◽  
SI Rapaport
Keyword(s):  
Surface Membrane ◽  
Calcium Ionophore ◽  
Factor Xa ◽  
Factor Vii ◽  
Ionophore A23187 ◽  
Exogenous Factor ◽  
Platelet Factor ◽  
Anionic Phospholipids ◽  
Factor Va ◽  
Activated Platelets

Abstract The authors have investigated the ability of platelets to enhance factor Xa-catalyzed activation of factor VII. Unstimulated platelets were without effect, whereas freeze/thawed platelets substantially enhanced activation. Antifactor V antibodies did not diminish the enhancement. Platelets activated by thrombin, collagen, or calcium ionophore A23187 also enhanced factor Xa-catalyzed activation of factor VII. In contrast to their lack of effect upon freeze/thawed platelets, antifactor V antibodies abolished augmented factor VII activation induced by activated platelets. Adding exogenous factor Va to unstimulated platelets failed to enhance factor Xa-catalyzed activation of factor VII, nor did adding exogenous factor Va to activated platelets augment activation beyond that observed with activated platelets alone. These observations can be interpreted as follows: (1) factor Va does not function as a cofactor for factor Xa-catalyzed activation of factor VII; (2) anionic phospholipids are a known cofactor for factor Xa-catalyzed activation of factor VII, and freeze/thawed platelets probably enhance activation by making anionic phospholipids on disrupted platelet membranes available to function as a cofactor; (3) the presumed binding of factor Xa to exogenous factor Va on unstimulated platelets is insufficient in itself to augment factor Xa-catalyzed activation of factor VII; (4) activated platelets augment factor Xa-catalyzed factor VII activation because activation allows both factor Xa to bind to released platelet factor V(a) and makes available a surface membrane component, probably anionic phospholipids, with which the bound factor Xa interacts.

scholarly journals The effect of platelets upon factor Xa-catalyzed activation of factor VII in vitro

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 396-401
Author(s):  
LV Rao ◽  
SI Rapaport
Keyword(s):  
Surface Membrane ◽  
Calcium Ionophore ◽  
Factor Xa ◽  
Factor Vii ◽  
Ionophore A23187 ◽  
Exogenous Factor ◽  
Platelet Factor ◽  
Anionic Phospholipids ◽  
Factor Va ◽  
Activated Platelets

The authors have investigated the ability of platelets to enhance factor Xa-catalyzed activation of factor VII. Unstimulated platelets were without effect, whereas freeze/thawed platelets substantially enhanced activation. Antifactor V antibodies did not diminish the enhancement. Platelets activated by thrombin, collagen, or calcium ionophore A23187 also enhanced factor Xa-catalyzed activation of factor VII. In contrast to their lack of effect upon freeze/thawed platelets, antifactor V antibodies abolished augmented factor VII activation induced by activated platelets. Adding exogenous factor Va to unstimulated platelets failed to enhance factor Xa-catalyzed activation of factor VII, nor did adding exogenous factor Va to activated platelets augment activation beyond that observed with activated platelets alone. These observations can be interpreted as follows: (1) factor Va does not function as a cofactor for factor Xa-catalyzed activation of factor VII; (2) anionic phospholipids are a known cofactor for factor Xa-catalyzed activation of factor VII, and freeze/thawed platelets probably enhance activation by making anionic phospholipids on disrupted platelet membranes available to function as a cofactor; (3) the presumed binding of factor Xa to exogenous factor Va on unstimulated platelets is insufficient in itself to augment factor Xa-catalyzed activation of factor VII; (4) activated platelets augment factor Xa-catalyzed factor VII activation because activation allows both factor Xa to bind to released platelet factor V(a) and makes available a surface membrane component, probably anionic phospholipids, with which the bound factor Xa interacts.

Differential proteome analysis of TRAP-activated platelets: involvement of DOK-2 and phosphorylation of RGS proteins

Blood ◽  
2004 ◽  
Vol 103 (6) ◽  
pp. 2088-2095 ◽  
Author(s):  
Angel García ◽  
Sripadi Prabhakar ◽  
Sascha Hughan ◽  
Tom W. Anderson ◽  
Chris J. Brock ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Proteome Analysis ◽  
Human Platelets ◽  
Rgs Proteins ◽  
Protein Signaling ◽  
Glycoprotein Vi ◽  
Activated Platelets ◽  
Tyrosine Kinase 2 ◽  
Proteomics Approach ◽  
Thrombotic Diseases

Abstract We have applied a proteomics approach to analyze signaling cascades in human platelets stimulated by thrombin receptor activating peptide (TRAP). By analyzing basal and TRAP-activated platelets using 2-dimensional gel electrophoresis (2-DE), we detected 62 differentially regulated protein features. From these, 41 could be identified by liquid chromatography–coupled tandem mass spectrometry (LC-MS/MS) and were found to derive from 31 different genes, 8 of which had not previously been reported in platelets, including the adapter downstream of tyrosine kinase 2 (Dok-2). Further studies revealed that the change in mobility of Dok-2 was brought about by tyrosine phosphorylation. Dok-2 tyrosine phosphorylation was also found to be involved in collagen receptor, glycoprotein VI (GPVI), signaling as well as in outside-in signaling through the major platelet integrin, αIIbβ3. These studies also provided the first demonstration of posttranslational modification of 2 regulator of G protein signaling (RGS) proteins, RGS10 and 18. Phosphorylation of RGS18 was mapped to Ser49 by MS/MS analysis. This study provides a new approach for the identification of novel signaling molecules in activated platelets, providing new insights into the mechanisms of platelet activation and building the basis for the development of therapeutic agents for thrombotic diseases.

Murine Platelets Retain High Amounts of Alpha Granule Proteins on Their Surface upon Stimulation with Multiple Agonists.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3897-3897 ◽  
Author(s):  
Shawn M. Jobe ◽  
Lorie Leo ◽  
Gerhard Dicknite ◽  
Steven R. Lentz ◽  
Jorge Di Paola
Keyword(s):  
Annexin V ◽  
Human Platelets ◽  
Factor Xiiia ◽  
Null Mouse ◽  
Specific Marker ◽  
Factor V ◽  
Phosphatidylserine Exposure ◽  
Activated State ◽  
Granule Proteins ◽  
Platelet Formation

Abstract Human platelets exposed to two strong agonists have recently been noted to form a unique subpopulation characterized by increased amounts of bound alpha granule constituents, including fibrinogen and factor V. We have identified and characterized a similar population of platelets in the mouse utilizing thrombin and the GPVI agonist, convulxin, as dual stimulants. We demonstrate that these platelets are analogous to those previously characterized in humans and examine their formation in both Fc Receptor γ-chain (FcRγ) and Factor XIIIa null mice. C57/Bl6 mouse platelets were exposed to convulxin, thrombin, and ionomycin singly or in combination at concentrations previously demonstrated to give maximal aggregation. Platelets were then examined by flow cytometry. Upon stimulation by any of the agonists, all platelets exhibited moderate amounts of fibrinogen binding and equivalent P-selectin exposure. Approximately 70% of convulxin and thrombin stimulated platelets (CoaT platelets) and 10% of thrombin stimulated platelets retained high amounts of alpha granule proteins, including fibrinogen and vWF. Interestingly, despite their high amount of bound fibrinogen, CoaT platelets demonstrated decreased binding of the GPIIb/IIIa activated state-specific antibody, JON/A, relative to single agonist stimulated platelets. Further characterization identified this platelet population as having high phosphatidylserine exposure (increased annexin V binding) and increased ability to bind the actin specific marker, phalloidin. All of these characteristics were present within two minutes of agonist stimulation. CoaT platelets could not be generated in the FcRγ-chain knockout mouse demonstrating the importance of this signaling pathway in their formation. Since human CoaT platelet formation is inhibited both by transglutaminase inhibitors and a monoclonal antibody to Factor XIIIa, CoaT platelet formation was examined in the FXIIIa null mouse. The absence of FXIIIa did not affect CoaT platelet formation, aggregation, or phosphatidylserine exposure. These findings demonstrate the presence of a unique subpopulation of platelets in the mouse formed on exposure to dual agonists analogous to the previously described CoaT platelets. This population is characterized by high amounts of bound alpha granule proteins, decreased binding of a GPIIb/IIIa activation specific mAb, and increased phosphatidylserine exposure; and in the mouse is not dependent on FXIIIa.

The Role of Ca2+I in Procoagulant Surface Expression and Apoptosis in Human Platelets.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3569-3569
Author(s):  
Adam M. Gwozdz ◽  
Hong Wang ◽  
K.W. Annie Bang ◽  
Marian A. Packham ◽  
John Freedman ◽  
...  
Keyword(s):  
In Vitro Study ◽  
Fold Increase ◽  
Surface Expression ◽  
Human Platelets ◽  
Apoptotic Pathway ◽  
Outer Leaflet ◽  
Activated Platelets

Abstract Asymmetry of phospholipids across the plasma membrane bilayer is a feature of all eukaryotic cells. When platelets are stimulated with certain agonsists, phospholipids are randomized by the action of a Ca2+-dependent scramblase enzyme, resulting in exposure of the anionic aminophospholipid phosphatidylserine (PS) on the outer leaflet that provides a procoagulant surface, catalyzing thrombin formation. We have previously demonstrated that the procoagulant surface of activated platelets persists in vitro for at least 4 hrs (Blood100:63b, 2002). Such persistence may propagate thrombosis in vivo when activated procoagulant platelets re-enter the circulation after fibrinolysis. There is currently little information concerning the mechanisms by which the procoagulant surface persists on activated platelets. In this in vitro study, the Ca2+-chelator BAPTA (0.1 μmol/109 platelets) was used to investigate the role of intracellular Ca2+ (Ca2+i) in procoagulant surface expression and persistence; PS expression was determined flow cytometrically by the binding of annexin A5-FITC. Unexpectedly, chelation of Ca2+i resulted in a 2–2.5x-fold increase in PS expression on the surface of platelets 5 min after activation with thrombin or thrombin+collagen (T+C), and this persisted for up to 4 hrs (last time point tested). Since PS expression is a hallmark of apoptosis in nucleated cells, we also examined another platelet apoptosis marker, the collapse of the mitochondrial inner membrane potential (ΔΨm), by flow cytometry using the potential-sensitive dye TMRM; PS expression was measured concurrently. This allowed us to distinguish between activated platelets expressing PS with an intact ΔΨm and apoptotic platelets expressing PS with a dissipated ΔΨm. 70–85% of the thrombin- or T+C-activated platelets expressing PS had an intact ΔΨm, which persisted for up to 4 hrs after activation. Thus, PS expression can occur independently of ΔΨm loss. However, chelation of Ca2+i with BAPTA resulted in 60–70% of the thrombin- or T+C-activated platelets persistently expressing PS to also have a collapsed ΔΨm, indicating that apoptotic pathways similar to those found in nucleated cells may modulate PS expression in platelets and may depend on Ca2+i concentrations. Caspases and calpain are centrally involved in apoptotic signaling and execution in nucleated cells. Caspases-9 and -3 have been identified in human platelets and may be responsible for downstream activation of calpain. We examined the effects of Ca2+i chelation in thrombin- and T+C- activated platelets on the activation of procaspases and calpain by Western blotting. In keeping with our observations of increased PS expression with concurrent ΔΨm loss in activated platelets with Ca2+i chelation, we observed cleavage of both procaspase-9, procaspase-3 and calpain, which did not occur in activated platelets without Ca2+i chelation. Taken together, our results indicate that Ca2+i levels in activated platelets may serve as a decisional checkpoint for the apoptotic pathway in human platelets, where procaspase-9 and procaspase-3 along with downstream calpain may function in a Ca2+-sensitive manner to protect platelets against PS exposure and ΔΨm collapse.

scholarly journals Functional characterization of thrombin Salakta: an abnormal thrombin derived from a human prothrombin variant

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 556-561 ◽  
Author(s):  
A Bezeaud ◽  
J Elion ◽  
MC Guillin
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Functional Characterization ◽  
Sodium Dodecyl ◽  
Factor Xa ◽  
Specific Pattern ◽  
Factor V ◽  
Functional Abnormality ◽  
V Protein ◽  
Fibrinogen Binding

Abstract The genetic variant prothrombin Salakta has been described in a patient presenting with a normal level of prothrombin antigen but reduced prothrombin activity. Initial studies indicated that factor Xa- catalyzed cleavages proceed normally but lead to the production of a thrombin molecule with an altered enzymatic activity. To characterize the functional abnormality of thrombin Salakta more precisely, it was purified by chromatography on heparin-Sepharose and diethylaminoethyl- Sephadex. The purified variant does not differ from normal thrombin by size, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and is 93.1% +/- 7.6% active by titration with p- nitrophenyl-p'-guanidinobenzoate. Its activity, however, is altered to various extents toward the following substrates: H-D-phenylalanyl-L- pipecolyl-L-arginine paranitroanilide (S 2238), fibrinogen, factor V, protein C, and antithrombin III. The Michaelis constant (Km) of thrombin Salakta for S 2238 is higher (12.2 +/- 3.3 mumol/L) than normal (2.8 +/- 0.7 mumol/L), whereas the turnover number (Kcat) is normal (84.4 +/- 6.6 s-1 v 85.9 +/- 14.0 s-1 for normal thrombin). The interaction of thrombin Salakta with benzamidine is also altered as evidenced by an increased inhibition constant (Ki = 3.5 mmol/L v 0.28 mmol/L for normal thrombin). The inability of fibrinogen to act as a competitor in the inactivation of thrombin Salakta by diisopropylfluorophosphate clearly indicates that fibrinogen binding to the fibrinopeptide groove is drastically impaired. In contrast, interactions involving sites remote from the active site such as those with fibrin and thrombomodulin are only slightly impaired. These results indicate that thrombin Salakta exhibits a specific pattern of functional alterations different from those reported for other variants. The structural defect seems to affect essentially the primary substrate binding site and to a lesser extent recognition site(s) remote from the catalytic site such as those for fibrin and thrombomodulin.

Role of the adapter protein SLP-76 in GPVI-dependent platelet procoagulant responses to collagen

Blood ◽  
2002 ◽  
Vol 100 (8) ◽  
pp. 2839-2844 ◽  
Author(s):  
Lorie Leo ◽  
Jorge Di Paola ◽  
Barbi A. Judd ◽  
Gary A. Koretzky ◽  
Steven R. Lentz
Keyword(s):  
Signal Transduction ◽  
Signal Transduction Pathway ◽  
Annexin V ◽  
Fold Increase ◽  
Surface Expression ◽  
Procoagulant Activity ◽  
Adapter Protein ◽  
Glycoprotein Vi ◽  
Granule Release

The adapter protein SLP-76 is a critical mediator of signal transduction via the platelet collagen receptor glycoprotein VI (GPVI) and its coreceptor FcRγ. We tested the hypothesis that SLP-76 is required for collagen-induced procoagulant responses in murine platelets. Platelets from SLP-76 null (SLP-76−/−) or heterozygous (SLP-76+/−) mice were activated with the GPVI agonist convulxin, and surface expression of P-selectin (a marker of granule release) and annexin V binding (a marker of procoagulant phospholipid) were determined by flow cytometry. Convulxin induced surface expression of P-selectin in SLP-76+/− platelets, but not SLP-76−/− platelets (P < .01), and failed to stimulate annexin V binding to either SLP-76+/−or SLP-76−/− platelets. Platelet procoagulant activity was measured in a prothrombinase assay. Convulxin did not stimulate procoagulant activity in either SLP-76+/− or SLP-76−/− platelets, but fibrillar collagen produced a 1.9-fold increase in procoagulant activity in both SLP-76+/− and SLP-76−/− platelets (P < .001 versus unstimulated platelets). Similar results were obtained with platelets from FcRγ null mice, for which collagen, but not convulxin, induced procoagulant activity (P < .01). Costimulation with thrombin and collagen produced a further (2.3-fold) increase in procoagulant activity in SLP-76+/− platelets (P < .05), but not in SLP-76−/− platelets. SLP-76−/− platelets also exhibited less annexin V binding than SLP-76+/−platelets after costimulation with thrombin and convulxin (P < .05). These findings demonstrate that an intact GPVI/FcRγ/SLP-76 signal transduction pathway is not essential for platelet procoagulant activity induced by collagen but is necessary for maximal procoagulant response to costimulation with thrombin plus collagen. Thus, both GPVI-dependent and GPVI-independent pathways contribute to collagen-induced platelet procoagulant activity.

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