Published online before print October 25, 2004, doi: 10.1161/01.CIR.0000146341.63677.3C
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Circulation. 2004;110:2946-2951.)
© 2004 American Heart Association, Inc.
Vascular Medicine |
Anti–Glycoprotein VI Treatment Severely Compromises Hemostasis in Mice With Reduced 
2ß1 Levels or Concomitant Aspirin Therapy
From the Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, University of Würzburg, Würzburg (S.G., M.P., B.A., V.S., B.N.); the Institute of Pharmacology, University of Heidelberg, Heidelberg (A.M., S.O.); and the Department of Dermatology, University of Cologne, Cologne (T.K., B.E.), Germany.
Correspondence to Bernhard Nieswandt, PhD, Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Versbacher Straße 9, 97078 Würzburg, Germany. E-mail bernhard.nieswandt{at}virchow.uni-wuerzburg.de
Received March 5, 2004; revision received May 13, 2004; accepted May 21, 2004.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background— Platelet inhibition is a major strategy to prevent arterial thrombosis, but it is frequently associated with increased bleeding because of impaired primary hemostasis. The activating platelet collagen receptor, glycoprotein VI (GP VI), may serve as a powerful antithrombotic target because its inhibition or absence results in profound protection against arterial thrombosis but no major bleeding in mice.
Methods and Results— Mice lacking (–/–) or expressing half-levels (+/–) of the other major platelet collagen receptor, integrin 
2ß1, were injected with the anti–GP VI antibody JAQ1 and analyzed on day 5. Anti–GP VI treatment resulted in a marked hemostatic defect in 2–/– or 2+/– mice, as shown by dramatically prolonged tail bleeding times. Platelet adhesion to collagen was studied in an ex vivo whole-blood perfusion system under high shear conditions. Weak integrin activation by thromboxane A2 (TxA2) receptor stimulation restored defective adhesion of anti–GP VI–treated wild-type but not 2–/– or 2+/– platelets to collagen. This process required the simultaneous activation of the Gq and G13 signaling pathways, as demonstrated by use of the respective knockout strains. Conversely, inhibition of TxA2 production by aspirin severely compromised hemostasis in anti–GP VI–treated or GP VI/Fc receptor 
-chain–deficient but not control mice.
Conclusions— Anti–GP VI therapy may result in defective hemostasis in patients with reduced 2ß1 levels or concomitant aspirin therapy. These observations may have important implications for a potential use of anti–GP VI–based therapeutics in the prevention of cardiovascular disease.
Key Words: collagen • glycoproteins • receptors • platelets • thrombosis
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Platelet adhesion and aggregation on the extracellular matrix is a key mechanism for normal hemostasis limiting blood loss after tissue trauma but may also lead to occlusion and irreversible tissue damage or infarction in diseased vessels.1,2 Therefore, platelet inhibition has become an important strategy for the control of cardiovascular diseases.1,2 Collagen is one of the major constituents of the subendothelial extracellular matrix and initiates adhesion and aggregation of platelets. In addition to glycoprotein (GP) Ib-V-IX and
IIbß3 integrin, which indirectly interact with collagen via von Willebrand factor,3 several direct collagen receptors have been identified on platelets, most importantly 2ß1 integrin4 and GP VI,5,6 which noncovalently associates with the signal transducing Fc receptor (FcR) -chain in the platelet membrane.7 In vitro, GP VI is essential for platelet-collagen interaction because it mediates the activation of ß1- and ß3-integrins, thereby allowing firm adhesion and thrombus growth.8 In vivo, GP VI/FcR deficiency results in profound protection in different mouse models of arterial thrombosis,9,10 but GP VI–deficient humans5,11 and mice12,13 display no major bleeding defect, suggesting that other receptors can substitute for GP VI during normal hemostasis. Thus, GP VI may serve as an attractive antithrombotic target, particularly because the receptor can be irreversibly depleted from circulating platelets in vivo, resulting in a GP VI–knockout-like phenotype and prolonged antithrombotic protection.12,14
The role of 2ß1 in platelet-collagen interactions has been controversial. Initially, 2ß1 was thought to be essential for shear-resistant adhesion on collagen but not to be involved in the activation process.15 However, it is now recognized that the integrin plays a significant but not essential role for the adhesion process8 and that it contributes to signaling directly16 and indirectly by reinforcing GP VI–collagen interactions.17 Despite this clear function of 2ß1 in vitro, 2-deficient mice display no hemostatic defect,18,19 and they form occlusive thrombi in the injured carotid artery,20 although in one study, this process was found to be delayed.21 In healthy humans, 2ß1 expression levels on platelets vary up to 10-fold,22 demonstrating that even individuals with very low platelet 2ß1 levels suffer no bleeding problem.
Although 2ß1 plays a supportive rather than an essential role in normal platelets, recent in vitro studies have revealed a more prominent function of the integrin when GP VI levels are reduced or the function of the receptor is inhibited.8,23 Signaling through 2ß1 is similar to that induced by GP VI, because both receptors regulate a similar set of intracellular signaling molecules, including Syk, SLP-76, and phospholipase C2.16 Thus, 2ß1 and GP VI appear to have partially redundant functions, but the in vivo significance of this is unclear.
We report here that the induction of a GP VI deficiency in mice lacking or expressing half-levels of 2ß1 results in a severe hemostatic defect, as shown by markedly increased bleeding times. Thromboxane (Tx) A2 restored defective adhesion of GP VI–depleted wild-type but not 2–/– or 2+/– platelets to collagen in vitro and was necessary to arrest bleeding in anti–GP VI–treated or FcR chain–deficient but not wild-type mice. These results provide the first evidence that platelet collagen receptors are essential for normal hemostasis and strongly indicate that anti–GP VI therapy may induce a bleeding defect in patients with reduced 2ß1 levels or concomitant aspirin therapy.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animals
All animal experiments and care were approved by the local Animal Care and Use Committee. Classic mouse mutants deficient in the
2 integrin- (2–/–) and Gq-subunits (Gq–/–) as well as the conditional G13-deficient mouse line (G13–/–) were produced as described.18,24,25 Both mutant and wild-type control animals were of 129/Sv x C57BL/6 genetic background and were used at the age of 10 to 16 weeks. To induce GP VI depletion, mice were injected with 100 µg JAQ1 intraperitoneally, and platelets were used for analysis on day 5.
Antibodies and Chemicals
JON/A-PE, which preferentially binds to activated integrin IIbß3,26 and anti–GP Ib-FITC were from Emfret Analytics. The anti–GP VI monoclonal antibody JAQ214 and all other antibodies and chemicals were from previously described sources.12
Platelet Preparation
Platelet preparation was performed as described previously.12 Briefly, whole blood was drawn from the retro-orbital plexus of anesthetized mice and collected in TBS containing 20 U/mL heparin. After washing the platelets twice with HEPES-Tyrode’s buffer (137 mmol/L NaCl, 2 mmol/L KCl, 12 mmol/L NaHCO3, 0.3 mmol/L NaH2PO4, 5.5 mmol/L glucose, 5 mmol/L HEPES, 0.35% BSA), platelets were resuspended in Tyrode’s buffer containing 1 mmol/L CaCl2 and 1 mmol/L MgCl2 to 2x106 platelets/µL.
Flow Cytometry
Platelets (2x106) were left untreated or stimulated with ADP (5 µmol/L), U46619 (1 µmol/L), or both and then stained with fluorophore-conjugated monoclonal antibodies at saturating concentrations for 15 minutes at room temperature and analyzed directly on a FACScalibur (Becton Dickinson). Platelets were identified by FSC/SSC characteristics.
Aggregometry
To determine platelet aggregation, light transmission was recorded by use of platelet-rich plasma (200 µL with 0.5x106 platelets/µL) in a Fibrintimer 4 channel aggregometer (APACT Laborgeräte und Analysensysteme).
Adhesion Under Flow Conditions
Mouse blood (1 vol) was collected into 0.5 vol of HEPES buffer, pH 7.45, containing 137 mmol/L NaCl, 5.6 mmol/L glucose, 5 mmol/L HEPES, 2.7 mmol/L KCl, 2 mmol/L MgCl2, 0.42 mmol/L NaH2PO4, and 0.1% BSA, 120 µmol/L PPACK, and 15 U/mL heparin. Rectangular coverslips (24x60 mm) were coated with 0.25 mg/mL "Horm type" collagen (Nycomed) for 1 hour at 37°C and blocked with 1% BSA. Perfusion of whole blood was performed as described.8 Briefly, transparent flow chambers with a slit depth of 50 µm, equipped with the collagen-coated coverslips, were rinsed with HEPES buffer supplemented with 2 mmol/L CaCl2 and 1 U/mL heparin and connected to a syringe filled with the anticoagulated blood. Perfusion was performed at room temperature with a pulse-free pump at high shear stress (4 minutes, flow rate of 7.53 mL/h, equivalent to a wall shear rate of 1000 s–1). When indicated, ADP (5 µmol/L), U46619 (1 µmol/L), or a combination of the 2 agonists was coinfused to the blood directly before it entered the flow chamber. During perfusion, microscopic phase-contrast images were recorded in real time. Thereafter, the chambers were rinsed by a 10-minute perfusion with HEPES buffer, pH 7.45 (2 mmol/L CaCl2 and 1 U/mL heparin), at the same shear stress. Phase-contrast images were recorded from at least 5 different microscopic fields (x63 objectives). Image analysis was performed offline with Metamorph software (Visitron). The platelet adhesion results are expressed as the mean percentage of total area covered or single platelets per microscopic field.
Bleeding-Time Experiments
Mice were anesthetized by intraperitoneal injection of ketamine/xylazine (ketamine 100 mg/kg, Parke-Davis; xylazine 5 mg/kg, Bayer AG), and a 3-mm segment of the tail tip was cut off with a scalpel. Tail bleeding was monitored by gently absorbing the bead of blood with a filter paper without contacting the wound site. When no blood was observed on the paper after 15-second intervals, bleeding was determined to have ceased.27 The experiment was stopped after 20 minutes. In some experiments, mice were treated with 100 mg/kg acetylsalicylic acid (Aspisol, Bayer AG) in sterile PBS intravenously 3 hours before the experiments.
Statistical Evaluation
Statistical analysis was performed by use of ANOVA (Scheffé test).
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
To better define the function of GP VI and
2ß1 in hemostasis, we depleted GP VI in 2–/– mice by injection of the anti–GP VI antibody JAQ1. In agreement with previous results,12 on day 5, JAQ1-treated wild-type and 2–/– mice had normal platelet counts (Figure 1a) but were GP VI–deficient, as shown by analysis with the anti–GP VI monoclonal antibody JAQ214 by flow cytometry (Figure 1b) and Western blot analysis (data not shown). In contrast, all other receptors examined, including GP Ib and IIbß3, were not affected by the treatment. Because of the absence of GP VI, both wild-type and 2–/– platelets did not aggregate in response to high doses of collagen (20 µg/mL), whereas they normally responded to other agonists such as ADP (5 µmol/L) or the stable TxA2 analog U46619 (1 µmol/L) (Figure 1c). To test whether the 2/GP VI double deficiency would have any effect under in vivo conditions, we determined tail bleeding times as a measure of primary hemostasis. As reported previously,12 JAQ1-treated wild-type mice displayed only slightly increased bleeding times. In marked contrast, however, in 2–/– mice, the same treatment induced a severe hemostatic defect, with 20 of 33 mice (60.6%) bleeding longer than 20 minutes (Figure 1d). Furthermore, the mean bleeding time of the other 13 mice was 2.6 times increased compared with JAQ1-treated wild-type mice. This finding demonstrated that the presence of either functional GP VI or 2ß1 is absolutely required for normal primary hemostasis.
|
It is well documented that 2ß1 expression levels in platelets vary greatly (up to 10-fold) among the normal population because of several linked polymorphisms within the 2 gene.22 Although there is growing evidence that high 2ß1 levels may be associated with an increased risk of cardiovascular disease, low levels of the integrin do not seem to affect hemostasis in otherwise hemostatically normal individuals.28 However, low 2ß1 levels may become clinically significant in a setting in which the overall platelet-collagen interaction is already partially compromised, as in the case of mild type I von Willebrand disease.29 To test whether reduced 2ß1 levels would interfere with the safety of anti–GP VI treatment, we examined 2+/– mice, which express 50% of normal 2ß1 on their platelets18 (Figure 2a). Five days after injection of 100 µg JAQ1, these mice had normal platelet counts and were GP VI–deficient (not shown). Very unexpectedly, the anti–GP VI treatment resulted in a marked bleeding time prolongation in the heterozygous mice that was similar to that observed in the homozygous mutant mice, with 8 of 14 animals (57.1%) bleeding longer than 20 minutes (Figure 2b). Thus, a 50% reduction in the 2ß1 level is sufficient to induce a severe hemostatic defect in anti–GP VI–treated mice. Although the data obtained in mice cannot be directly extrapolated to the situation in humans, these results strongly suggest that anti–GP VI treatment would severely impair primary hemostasis in humans with low 2ß1 levels, thereby potentially increasing the risk of uncontrolled bleeding.
|
To examine the molecular determinants of hemostasis, and in particular the role of 2ß1, in the absence of functional GP VI, we assessed platelet adhesion in ex vivo whole-blood perfusion studies. GP VI–deficient or –blocked platelets fail to adhere to collagen because of defective activation of integrins 2ß1 and IIbß3.8 To test whether adhesion of GP VI–deficient platelets can be restored by integrin activation through other signaling pathways, we coinfused ADP, U46619, or a combination of the 2 agonists into the flowing blood (1000 s–1) directly before it entered the flow chamber.8 In the case of ADP (5 µmol/L), which induces reversible integrin activation at intermediate levels (Figure 3a), both GP VI–depleted wild-type and 2–/– platelets formed large thrombi on the collagen surface within 2 minutes. These thrombi were, however, very unstable and completely washed out when the flow chamber was rinsed (data not shown). In contrast, coinfusion of U46619 (1 µmol/L), which on its own induces weak but sustained integrin activation30 (Figure 3a), consistently resulted in firm adhesion of single GP VI–depleted wild-type but not 2–/– or 2+/– platelets (Figure 3b). When strong and sustained integrin activation was induced by the coinfusion of ADP and U46619 in combination30 (Figure 3a), GP VI–depleted wild-type, 2–/–, and 2+/– platelets formed large thrombi on the collagen, the size and stability of which was comparable in all groups (Figure 3b). This 2ß1-independent adhesion is mediated by IIbß3–von Willebrand factor interaction, because it was completely inhibited in the presence of Fab' fragments of the anti-IIbß3 antibody JON/A (30 µg/mL, Figure 3c). These findings suggest that 2ß1 plays a major role for shear-resistant platelet adhesion on collagen under conditions of weak but not strong integrin activation, because under the latter conditions, IIbß3 is sufficient to arrest the cells. This is in line with the well-documented requirement for 2ß1 for adhesion to monomeric or degraded collagen, which is a weak agonist at GP VI and therefore induces only low-level integrin activation.31
|
On fibrillar collagens as found in the extracellular matrix, GP VI mediates strong and sustained integrin activation,31 which may explain why 2ß1 is not required for hemostasis under normal conditions.18,19 In anti–GP VI–treated mice, however, 2ß1 is strictly required to arrest bleeding, suggesting that the stimuli leading to integrin activation in the absence of functional GP VI must be rather weak, ie, insufficient to induce IIbß3-dependent adhesion. We suspected TxA2 of playing a role in this process, because in vitro, this activation pathway induces 2ß1-dependent adhesion of GP VI–deficient platelets to collagen (Figure 3b). To test this hypothesis directly, we inhibited TxA2 synthesis in control and JAQ1-treated wild-type mice (day 5) with aspirin (100 mg/kg) and determined the tail bleeding times 3 hours after aspirin treatment. Strikingly, although aspirin had only a minor effect on bleeding times in control mice, it severely compromised hemostasis in anti–GP VI–treated mice, with 10 of 18 animals (55.5%) bleeding longer than 20 minutes (Figure 4a). To further substantiate this surprising finding, we used FcR-chain–deficient mice, which fail to express GP VI on their platelets.31 These mice are largely protected from arterial thrombus formation and subsequent neointimal hyperplasia9 as well as collagen-induced thromboembolism,21 but they display no increased bleeding tendency.31 Treatment of these mice with aspirin, however, almost completely blocked hemostasis, as shown by bleeding times >20 minutes in 7 of 10 mice (Figure 4a). These results demonstrate that the TxA2-mediated activation pathway becomes crucial for primary hemostasis in the absence of functional GP VI, suggesting that the safety of anti–GP VI treatment might be blunted in patients concomitantly taking aspirin at antithrombotic, analgetic, or antiphlogistic dosages.
|
In platelets, the TxA2 receptor couples to the Gq and G12/G13 signaling pathways, both of which are crucial for the formation of stable thrombi under flow conditions in vitro and in vivo.24,25 To determine which of these signaling pathways is required for U46619-induced adhesion of GP VI–deficient platelets, we injected wild-type, Gq-, and G13-deficient mice with JAQ1 and tested the adhesion of their platelets to collagen under flow conditions ex vivo on day 5. Interestingly, U46619 was unable to induce adhesion of Gq–/– or G13–/– platelets, demonstrating that both signaling pathways are essential for this process to occur (Figure 4b).
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Thus, platelet adhesion in the absence of functional GP VI requires the concerted action of different G-protein–mediated signaling pathways. This indicates that a reduced activity of one of these pathways, which by itself may not be sufficient to affect hemostasis under normal conditions, could cause a severe bleeding defect in individuals receiving anti–GP VI treatment. Although there is only a weak correlation between the bleeding time and bleeding risk, it is tempting to speculate that the combination of anti–GP VI agents with other antiplatelet compounds to improve the antithrombotic effectiveness of the therapy, as currently discussed for clopidogrel and aspirin,32 would have to be carefully evaluated to avoid uncontrolled bleeding in humans.
GP VI mediates platelet activation at sites of vascular injury and plays a central role in pathological thrombus formation.9,10 Therefore, this receptor has been recognized as a promising antithrombotic target particularly because its inhibition or absence does not cause major bleeding in humans or mice. Identifying the mechanisms underlying primary hemostasis in GP VI–compromised individuals is important to develop effective anti–GP VI agents and to predict their safety. Our data clearly indicate that the loss of GP VI signaling is compensated for by a very sensitive network of different G-protein–mediated signaling pathways that converge in the activation of 2ß1, allowing it to arrest the cells and to reinforce activation through "outside-in" signals.16 Any impairment of this alternative pathway, which may not be of any significance under normal conditions, might lead to an intolerable bleeding risk under anti–GP VI treatment. These observations may have important implications for the development and potential use of anti–GP VI–based therapeutics for the prevention of ischemic cardiovascular disease.
|
Acknowledgments |
|---|
This work was supported by grant Ni556/4-1 (to Dr Nieswandt) and SFB 589 (to Dr Eckes and Dr Krieg) from the Deutsche Forschungsgemeinschaft (DFG). We thank M. Koch and S. Hartmann for excellent technical assistance.
|
Footnotes |
|---|
*The first 2 authors contributed equally to this work.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Ruggeri ZM. Platelets in atherothrombosis. Nat Med. 2002; 8: 1227–1234.[CrossRef][Medline] [Order article via Infotrieve]
2. Bhatt DL, Topol EJ. Scientific and therapeutic advances in antiplatelet therapy. Nat Rev Drug Discov. 2003; 2: 15–28.[CrossRef][Medline] [Order article via Infotrieve]
3. Savage B, Almus-Jacobs F, Ruggeri ZM. Specific synergy of multiple substrate-receptor interactions in platelet thrombus formation under flow. Cell. 1998; 94: 657–666.[CrossRef][Medline] [Order article via Infotrieve]
4. Santoro SA. Identification of a 160,000 dalton platelet membrane protein that mediates the initial divalent cation-dependent adhesion of platelets to collagen. Cell. 1986; 46: 913–920.[CrossRef][Medline] [Order article via Infotrieve]
5. Moroi M, Jung SM, Okuma M, et al. A patient with platelets deficient in glycoprotein VI that lack both collagen-induced aggregation and adhesion. J Clin Invest. 1989; 84: 1440–1445.[Medline] [Order article via Infotrieve]
6. Clemetson JM, Polgar J, Magnenat E, et al. The platelet collagen receptor glycoprotein VI is a member of the immunoglobulin superfamily closely related to FcalphaR and the natural killer receptors. J Biol Chem. 1999; 274: 29019–29024.
7. Gibbins JM, Okuma M, Farndale R, et al. Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor gamma-chain. FEBS Lett. 1997; 413: 255–259.[CrossRef][Medline] [Order article via Infotrieve]
8. Nieswandt B, Brakebusch C, Bergmeier W, et al. Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen. EMBO J. 2001; 20: 2120–2130.[CrossRef][Medline] [Order article via Infotrieve]
9. Konishi H, Katoh Y, Takaya N, et al. Platelets activated by collagen through immunoreceptor tyrosine-based activation motif play a pivotal role in initiation and generation of neointimal hyperplasia after vascular injury. Circulation. 2002; 105: 912–916.
10. Massberg S, Gawaz M, Gruner S, et al. A crucial role of glycoprotein VI for platelet recruitment to the injured arterial wall in vivo. J Exp Med. 2003; 197: 41–49.
11. Sugiyama T, Okuma M, Ushikubi F, et al. A novel platelet aggregating factor found in a patient with defective collagen-induced platelet aggregation and autoimmune thrombocytopenia. Blood. 1987; 69: 1712–1720.
12. Nieswandt B, Schulte V, Bergmeier W, et al. Long-term antithrombotic protection by in vivo depletion of platelet glycoprotein VI in mice. J Exp Med. 2001; 193: 459–470.
13. Kato K, Kanaji T, Russell S, et al. The contribution of glycoprotein VI to stable platelet adhesion and thrombus formation illustrated by targeted gene deletion. Blood. 2003; 102: 1701–1707.
14. Schulte V, Rabie T, Prostredna M, et al. Targeting of the collagen-binding site on glycoprotein VI is not essential for in vivo depletion of the receptor. Blood. 2003; 101: 3948–3952.
15. Santoro SA, Walsh JJ, Staatz WD, et al. Distinct determinants on collagen support alpha 2 beta 1 integrin-mediated platelet adhesion and platelet activation. Cell Regul. 1991; 2: 905–913.[Medline] [Order article via Infotrieve]
16. Inoue O, Suzuki-Inoue K, Dean WL, et al. Integrin alpha2beta1 mediates outside-in regulation of platelet spreading on collagen through activation of Src kinases and PLCgamma2. J Cell Biol. 2003; 60: 769–780.
17. Kuijpers MJ, Schulte V, Bergmeier W, et al. Complementary roles of platelet glycoprotein VI and integrin alpha2beta1 in collagen-induced thrombus formation in flowing whole blood ex vivo. FASEB J. 2003; 17: 685–687.
18. Holtkotter O, Nieswandt B, Smyth N, et al. Integrin alpha 2-deficient mice develop normally, are fertile, but display partially defective platelet interaction with collagen. J Biol Chem. 2002; 277: 10789–10794.
19. Chen J, Diacovo TG, Grenache DG, et al. The alpha(2) integrin subunit-deficient mouse: a multifaceted phenotype including defects of branching morphogenesis and hemostasis. Am J Pathol. 2002; 161: 337–344.
20. Gruner S, Prostredna M, Schulte V, et al. Multiple integrin-ligand interactions synergize in shear-resistant platelet adhesion at sites of arterial injury in vivo. Blood. 2003; 102: 4021–4027.
21. He L, Pappan LK, Grenache DG, et al. The contributions of the alpha2beta1 integrin to vascular thrombosis in vivo. Blood. 2003; 102: 3652–3657.
22. Kunicki TJ, Kritzik M, Annis DS, et al. Hereditary variation in platelet integrin alpha 2 beta 1 density is associated with two silent polymorphisms in the alpha 2 gene coding sequence. Blood. 1997; 89: 1939–1943.
23. Chen H, Kahn ML. Reciprocal signaling by integrin and nonintegrin receptors during collagen activation of platelets. Mol Cell Biol. 2003; 23: 4764–4777.
24. Offermanns S, Toombs CF, Hu YH, et al. Defective platelet activation in G alpha(q)-deficient mice. Nature. 1997; 389: 183–186.[CrossRef][Medline] [Order article via Infotrieve]
25. Moers A, Nieswandt B, Massberg S, et al. G(13) is an essential mediator of platelet activation in hemostasis and thrombosis. Nat Med. 2003; 9: 1418–1422.[CrossRef][Medline] [Order article via Infotrieve]
26. Bergmeier W, Schulte V, Brockhoff G, et al. Flow cytometric detection of activated mouse integrin alphaIIbbeta3 with a novel monoclonal antibody. Cytometry. 2002; 48: 80–86.[CrossRef][Medline] [Order article via Infotrieve]
27. Carmeliet P, Stassen JM, Schoonjans L, et al. Plasminogen activator inhibitor-1 gene-deficient mice, II: effects on hemostasis, thrombosis, and thrombolysis. J Clin Invest. 1993; 92: 2756–2760.[Medline] [Order article via Infotrieve]
28. Santoro SA. Platelet surface collagen receptor polymorphisms: variable receptor expression and thrombotic/hemorrhagic risk. Blood. 1999; 93: 3575–3577.
29. Di Paola J, Federici AB, Mannucci PM, et al. Low platelet alpha2beta1 levels in type I von Willebrand disease correlate with impaired platelet function in a high shear stress system. Blood. 1999; 93: 3578–3582.
30. Nieswandt B, Schulte V, Zywietz A, et al. Costimulation of Gi- and G12/G13-mediated signaling pathways induces integrin alpha IIbbeta 3 activation in platelets. J Biol Chem. 2002; 277: 39493–39498.
31. Nieswandt B, Watson SP. Platelet-collagen interaction: is GP VI the central receptor? Blood. 2003; 102: 449–461.
32. Peters RJ, Mehta SR, Fox KA, et al. Effects of aspirin dose when used alone or in combination with clopidogrel in patients with acute coronary syndromes: observations from the Clopidogrel in Unstable angina to prevent Recurrent Events (CURE) study. Circulation. 2003; 108: 1682–1687.
This article has been cited by other articles:
 |
|
 |
|
  B. Dumont, D. Lasne, C. Rothschild, M. Bouabdelli, V. Ollivier, C. Oudin, N. Ajzenberg, B. Grandchamp, and M. Jandrot-Perrus Absence of collagen-induced platelet activation caused by compound heterozygous GPVI mutations Blood, August 27, 2009; 114(9): 1900 - 1903. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. J.H. Sachs and B. Nieswandt In Vivo Thrombus Formation in Murine Models Circ. Res., April 13, 2007; 100(7): 979 - 991. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. F. Samaha and M. L. Kahn Novel Platelet and Vascular Roles for Immunoreceptor Signaling Arterioscler Thromb Vasc Biol, December 1, 2006; 26(12): 2588 - 2593. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Schulte, H. P. Reusch, M. Pozgajova, D. Varga-Szabo, C. Gachet, and B. Nieswandt Two-Phase Antithrombotic Protection After Anti-Glycoprotein VI Treatment in Mice Arterioscler Thromb Vasc Biol, July 1, 2006; 26(7): 1640 - 1647. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z.-G. Zhang, I. Bothe, F. Hirche, M. Zweers, D. Gullberg, G. Pfitzer, T. Krieg, B. Eckes, and M. Aumailley Interactions of primary fibroblasts and keratinocytes with extracellular matrix proteins: contribution of {alpha}2{beta}1 integrin. J. Cell Sci., May 1, 2006; 119(Pt 9): 1886 - 1895. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. Freedman Molecular Regulation of Platelet-Dependent Thrombosis Circulation, October 25, 2005; 112(17): 2725 - 2734. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. L. Sarratt, H. Chen, M. M. Zutter, S. A. Santoro, D. A. Hammer, and M. L. Kahn GPVI and {alpha}2{beta}1 play independent critical roles during platelet adhesion and aggregate formation to collagen under flow Blood, August 15, 2005; 106(4): 1268 - 1277. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||