Platelet activation at sites of vascular injury is essential for primary hemostasis, but also underlies arterial thrombosis leading to myocardial infarction or stroke 1,2 . Platelet activators such as adenosine diphosphate, thrombin or thromboxane A 2 (TXA 2 ) activate receptors that are coupled to heterotrimeric G proteins 1,3 . Activation of platelets through these receptors involves signaling through G q , G i and G z (refs. 4-6). However, the role and relative importance of G 12 and G 13 , which are activated by various platelet stimuli 7-9 , are unclear. Here we show that lack of Gα 13 , but not Gα 12 , severely reduced the potency of thrombin, TXA 2 and collagen to induce platelet shape changes and aggregation in vitro. These defects were accompanied by reduced activation of RhoA and inability to form stable platelet thrombi under high shear stress ex vivo. Gα 13 deficiency in platelets resulted in a severe defect in primary hemostasis and complete protection against arterial thrombosis in vivo. We conclude that G 13 -mediated signaling processes are required for normal hemostasis and thrombosis and may serve as a new target for antiplatelet drugs.

Platelets play a crucial role in the physiology of primary hemostasis and pathophysiologic processes such as arterial thrombosis. Accumulating evidence suggests a role of reactive oxygen species (ROSs) in platelet activation. Here we show that platelets activated with different agonists produced intracellular ROSs, which were reduced by reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase inhibitors and superoxide scavengers. In addition, we demonstrate that ROSs produced in platelets significantly affected ␣IIb␤3 integrin activation but not alpha and dense granule secretion and platelet shape change. Thrombin-induced integrin ␣IIb␤3 activation was significantly decreased after pretreatment of platelets with NAD (

Background: Clopidogrel is a potent drug for prevention of adverse effects during and after coronary intervention. Increasing experience indicates that a significant proportion of patients do not respond adequately to clopidogrel. Because failure of antiplatelet therapy can have severe consequences, there is need for a reliable assay to quantify the effectiveness of clopidogrel treatment. Methods: Of 24 healthy volunteers admitted to the study, 18 were treated for 1 week with clopidogrel (300-mg loading dose and 75-mg maintenance dose), and 6 with placebo. Platelet function was monitored by 2 assays, based on flow cytometry and enzyme immunoassay, that measure the phosphorylation status of vasodilator-stimulated phosphoprotein (VASP) and by aggregometry, flow cytometry of P-selectin, and the platelet function analyzer at baseline, on days 1-5, and on day 9 of treatment. Results: Aggregometry and VASP phosphorylation revealed a loss of platelet response to ADP within 12 h after clopidogrel intake. The phosphorylation status of VASP correlated with the inhibition of platelet aggregation. In contrast, neither P-selectin expression nor PFA-100 closure time was a clear indicator of clopidogrel effects on platelets. Conclusions: VASP phosphorylation assays are reliable for quantifying clopidogrel effects. Because the VASP assay directly measures the function of the clopidogrel target, the P2Y12 receptor, the assay is selective for

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 α 2 β 1 , 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 α 2 −/− or α 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 A 2 (TxA 2 ) receptor stimulation restored defective adhesion of anti–GP VI–treated wild-type but not α 2 −/− or α 2 +/− platelets to collagen. This process required the simultaneous activation of the G q and G 13 signaling pathways, as demonstrated by use of the respective knockout strains. Conversely, inhibition of TxA 2 production by aspirin severely compromised hemostasis in anti–GP VI–treated or GP VI/Fc receptor γ-chain–deficient but not control mice. Conclusions— Anti–GP VI therapy may result in defective hemostasis in patients with reduced α 2 β 1 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.

Glycoprotein (GP) VI is an essential collagen receptor on platelets and may serve as an attractive target for antithrombotic therapy. We have previously shown that a monoclonal antibody (mAb) against the major collagen-binding site on mouse GPVI (JAQ1) induces irreversible downregulation of the receptor and, consequently, long-term antithrombotic protection in vivo. To determine whether this unique in vivo effect of JAQ1 is based on its interaction with the ligand-binding site on GPVI, we generated new mAbs against different epitopes on GPVI (JAQ2, JAQ3) and tested their in vitro and in vivo activity. We show that none of the mAbs inhibited platelet activation by collagen or the collagen-related peptide in vitro. Unexpectedly, however, injection of either antibody induced depletion of GPVI with the same efficacy and kinetics as JAQ1. Importantly, this effect was also seen with IntroductionCoronary artery thrombosis is often initiated by platelet adhesion and aggregation on subendothelial collagens exposed on the surface of the ruptured atherosclerotic plaque. [1][2][3][4] Platelet-collagen interactions are complex and involve a large number of receptors that directly or indirectly interact with the matrix protein, most importantly GPIb-V-IX, 5 integrins ␣ 2 ␤ 1 6 and ␣ IIb ␤ 3 , 5 and glycoprotein (GP) VI. 7 Among these, GPVI plays a central role as it mediates the activation of integrins ␣ 2 ␤ 1 and ␣ IIb ␤ 3 , which is a prerequisite for firm platelet adhesion and thrombus growth. 8 GPVI (62 kDa) belongs to the immunoglobulin superfamily 9,10 and is noncovalently associated with the Fc receptor (FcR)␥ chain. 11,12 GPVI-deficient patients suffer from a mild bleeding diathesis and their platelets show severely impaired responses to collagen. 7,13,14 Similarly, platelets from FcR␥ chain-deficient mice, which lack GPVI, 15 are unresponsive to collagen 16 but no major bleeding has been observed in those mice, making GPVI an interesting target for safe antithrombotic therapy.The first direct proof for effective antithrombotic protection by anti-GPVI treatment came from studies in mice using the anti-GPVI monoclonal antibody (mAb), JAQ1. JAQ1 is directed against the major collagen-binding site on GPVI and inhibits platelet activation by collagen and the collagen-related peptide (CRP). 15,17 In vivo treatment of mice with JAQ1 or monovalent F(ab) fragments of the antibody induces the internalization and proteolytic degradation of GPVI in circulating platelets resulting in a prolonged GPVI-knock-out-like phenotype. 18 Similar mechanisms of GPVI down-regulation appear to exist in humans because one GPVI-deficient patient had developed highly specific antibodies against the apparently absent receptor suggesting that she suffered from an acquired GPVI deficiency based on antibodyinduced clearing of the receptor from her platelets. 13 F(ab) fragments of the antibodies isolated from this patient inhibited collageninduced activation of normal human platelets. 7,13 Together, these observations led to the hypothesis that targ...

Background and Purpose-Dipyridamole and in particular dipyridamole in combination with low-dose aspirin are very effective in preventing recurrent stroke. However, the mechanism(s) underlying this dipyridamole effect have not been elucidated. Since dipyridamole inhibits the cGMP-specific phosphodiesterase type V in vitro, we hypothesized and tested whether therapeutically relevant dipyridamole concentrations enhance NO/cGMP-mediated effects in intact human platelets studied ex vivo. Methods-Phosphorylation of vasodilator-stimulated phosphoprotein (VASP), an established marker of NO/cGMP effects in human platelets, was quantified by phosphorylation-specific antibodies and Western blots. Serotonin secretion and thromboxane synthase activity were determined by fluorometric quantification of derivatized serotonin and synthase products, respectively. Results-Endothelium-derived factors such as NO and prostaglandin I 2 are known to elevate both cGMP and cAMP levels with concomitant platelet inhibition and VASP phosphorylation. In our in vitro experiments, therapeutically relevant concentrations (3.5 mol/L) of dipyridamole amplified only cGMP-mediated VASP phosphorylation due to the NO donor sodium nitroprusside, but not cAMP-mediated effects. Furthermore, thromboxane synthase activity and serotonin secretion, events important for initial platelet activation, were inhibited by sodium nitroprusside, an effect also enhanced by dipyridamole, demonstrating the functional relevance of these observations. Finally, the ex vivo enhancement of NO/cGMP effects was also observed with platelets obtained from healthy volunteers treated with extended-release dipyridamole. Key Words: dipyridamole Ⅲ phosphodiesterase inhibitors Ⅲ platelet aggregation inhibitors Ⅲ stroke T he role of activated platelets in acute and chronic vascular diseases is well established. Accordingly, antiplatelet drugs significantly reduce the risk of severe events, such as ischemic stroke, myocardial infarction, and vascular death in atherosclerotic vascular disease. In numerous clinical studies, the efficacy of antiplatelet drugs in the treatment and secondary prevention of vascular diseases, particularly stroke, has been proven. [1][2][3] Platelet activation is inhibited by factors released from endothelial cells that constitute the inner cell layer of the vessel wall. The most important inhibitory endotheliumderived factors are NO and prostaglandin I 2 (PGI 2 ), which inhibit platelets by increasing the level of intracellular cyclic nucleotides. 4,5 Subsequent stimulation of cGMP-and cAMPdependent protein kinases leads to the phosphorylation of a variety of proteins. 6 In platelets, this inhibits agoniststimulated calcium signaling, 7,8 fibrinogen binding, 9 adhesion, 10 and aggregation 5 and stimulates phosphorylation of the cytoskeletal and focal adhesion protein vasodilatorstimulated phosphoprotein (VASP). 4,5,11 This phosphorylation correlates strongly with the inhibition of platelets. 9,11 Interestingly, antiplatelet treatment with clopidogrel or ticlo...

Summary.  Platelets play a central role in hemostasis and thrombosis but also in the initiation of atherosclerosis, making platelet receptors and their intracellular signaling pathways important molecular targets for antithrombotic and anti‐inflammatory therapy. Historically, much of the knowledge about hemostasis and thrombosis has been derived from patients suffering from bleeding and thrombotic disorders and the identification of the underlying molecular defects. In recent years, the availability of genetically modified mouse strains with defined defects in platelet function and the development of in vivo models to assess platelet‐related physiologic and pathophysiologic processes have opened new ways to identify the individual roles and the interplay of platelet proteins in adhesion, activation, aggregation, secretion, and procoagulant activity in vitro and in vivo. This review will summarize key findings made by these approaches and discuss them in the context of human disease.

Aspirin is effective in the therapy of cardiovascular diseases, because it causes acetylation of cyclooxygenase 1 (COX-1) leading to irreversible inhibition of platelets. Additional mechanisms can be suspected, because patients treated with other platelet COX inhibitors such as indomethacin do not display an increased bleeding tendency as observed for aspirin-treated patients. Recently, aspirin and other anti-inflammatory drugs were shown to induce shedding of L-selectin in neutrophils in a metalloproteinasedependent manner. Therefore, we investigated the effects of aspirin on the von Willebrand Factor receptor complex glycoprotein (GP) Ib-V-IX, whose lack or dysfunction causes bleeding in patients. As quantified by fluorescence-activated cell sorting analysis in whole blood, aspirin, but not its metabolite salicylic acid, induced dose-dependent shedding of human and murine GPIb␣ and GPV from the platelet surface, whereas other glycoproteins remained unaffected by this treatment. Biotinylated fragments of GPV were detected by immunoprecipitation in the supernatant of washed mouse platelets, and the expression level of GPIb␣ was decreased in these platelets as measured by Western blot analysis. Although shedding occurred normally in COX-1-deficient murine platelets, shedding was completely blocked by a broad-range metalloproteinase inhibitor and, more importantly, in mouse platelets expressing an inactive form of ADAM17. Shed fragments of GPIb␣ and GPV were elevated in the plasma of aspirin-injected mice compared with animals injected with control buffer. These data demonstrate that aspirin at high concentrationsinducessheddingofGPIb␣andGPVbyanADAM17-dependent mechanism and that this process can occur in vivo.The activation of platelets at sites of vascular injury, followed by controlled aggregate formation, is required for normal hemostasis, but can cause thrombosis in pathologically altered arteries leading to myocardial infarction or stroke (1). Under conditions of elevated shear, platelet adhesion is initiated by the von Willebrand Factor receptor glycoprotein (GP) 2 Ib-V-IX, a structurally unique receptor complex expressed in platelets and megakaryocytes. The interaction of GPIb␣ with von Willebrand factor bound to subendothelial collagen or on the surface of activated adherent platelets is essential for platelet adhesion and thrombus formation (1, 2). In patients, a reduced expression or malfunction of GPIb␣ determines the Bernard-Soulier syndrome, a congenital bleeding disorder, which is characterized by a macrothrombocytopenia, an inability of platelets to adhere to subendothelial matrices, and a dramatically prolonged bleeding time (3).The expression levels of GPIb and GPV are physiologically regulated by internalization processes and/or by proteolytic ectodomain cleavage ("shedding"), which might play a role in the clearance of aged platelets from the circulation (4). In contrast to the well known protease activity of the platelet activator thrombin that directly releases a 69-kDa fragment from GPV (GPV...