Barsom Aktas

G13 is an essential mediator of platelet activation in hemostasis and thrombosis

Platelet activation at sites of vascular injury is essential for primary hemostasis, but also underlies arterial thrombosis leading to myocardial infarction or stroke. Platelet activators such as adenosine diphosphate, thrombin or thromboxane A2 (TXA2) activate receptors that are coupled to heterotrimeric G proteins. Activation of platelets through these receptors involves signaling through Gq, Gi and Gz (refs. 4–6). However, the role and relative importance of G12 and G13, which are activated by various platelet stimuli, are unclear. Here we show that lack of Gα13, but not Gα12, severely reduced the potency of thrombin, TXA2 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. Gα13 deficiency in platelets resulted in a severe defect in primary hemostasis and complete protection against arterial thrombosis in vivo. We conclude that G13-mediated signaling processes are required for normal hemostasis and thrombosis and may serve as a new target for antiplatelet drugs.

Anti–Glycoprotein VI Treatment Severely Compromises Hemostasis in Mice With Reduced α2β1 Levels or Concomitant Aspirin Therapy

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 &agr;2&bgr;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 &agr;2−/− or &agr;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 &agr;2−/− or &agr;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 &ggr;-chain–deficient but not control mice. Conclusions—Anti–GP VI therapy may result in defective hemostasis in patients with reduced &agr;2&bgr;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.

Dipyridamole Enhances NO/cGMP-Mediated Vasodilator-Stimulated Phosphoprotein Phosphorylation and Signaling in Human Platelets In Vitro and In Vivo/Ex Vivo Studies

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 I2 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 &mgr;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. Conclusions— Under therapeutically relevant conditions, dipyridamole enhances platelet inhibition by amplifying the signaling of the NO donor sodium nitroprusside. These data support the concept that enhancement of endothelium-dependent NO/cGMP-mediated signaling may be an important in vivo component of dipyridamole action.

Platelets in atherothrombosis: lessons from mouse models

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 induces platelet receptor shedding via ADAM17 (TACE).

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 metalloproteinase-dependent 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 GPIbα 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 GPIbα 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 GPIbα 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 concentrations induces shedding of GPIbα and GPV by an ADAM17-dependent mechanism and that this process can occur in vivo.

The C-terminal peptide of thrombospondin-1 stimulates distinct signaling pathways but induces an activation-independent agglutination of platelets and other cells.

A peptide from the C‐terminal domain of thrombospondin‐1 (4N1‐1) has been proposed to stimulate platelet aggregation by a novel mechanism involving both an activation‐independent agglutination and an activation‐dependent, glycoprotein (GP) IIb/IIIa‐mediated aggregation which involves GPVI signaling but does not involve CD47. The present study demonstrates that 4N1‐1 stimulated a different pattern of signal transduction pathways than the GPVI agonist convulxin. Furthermore, 4N1‐1‐induced platelet aggregation was activation‐independent and not dependent on GPVI or GPIIb/IIIa. Interestingly, 4N1‐1 also stimulated activation‐independent agglutination of different megakaryocytic and non‐megakaryocytic cells. 4N1‐1‐induced cell agglutination but not platelet signaling was inhibited by anti‐CD47 antibodies.

Inhibition of platelet P2Y12 and α2A receptor signaling by cGMP-dependent protein kinase

The important role of cGMP and cGMP-dependent protein kinase (cGPK) for the inhibition of platelet activation and aggregation is well established and due to the inhibition of fundamental platelet responses such as agonist-stimulated calcium increase, exposure of adhesion receptors and actin polymerization. The diversity of cGMP binding proteins and their synergistic interaction with cAMP signaling in inhibiting platelets indicates that a variety of cGMP targets contribute to its antiplatelet action. Since stimulation of G(i)-proteins was recently shown to be essential for complete platelet activation/aggregation, the possibility that G(i)-signaling events are cGMP/cGPK targets was investigated. Thus, the effect of elevated cGMP levels and selective cGPK activation on purinergic and adrenergic receptor-evoked decrease of platelet cAMP content was closely examined. Experiments with a selective activator of cGPK demonstrate for the first time a cGMP-caused G(i)-protein inhibition and our data suggest that this effect is mediated by cGPK. Considering the essential role of G(i)-signaling for platelet activation, we propose that inhibition of G(i)-mediated signaling by cGMP/cGPK is an important mechanism of action underlying the platelet inhibition by cGMP-elevating endothelium derived factors and drugs.

Abstracts of papers presented at the XIXth European Platelet Meeting, 14th–16th October 2004, Dorint Resort & Spa, Bad Brückenau, Germany

Abstracts of papers presented at the XIXth European Platelet Meeting, 14th–16th October 2004, Dorint Resort & Spa, Bad Brückenau, Germanys of papers presented at the XIXth European Platelet Meeting, 14th–16th October 2004, Dorint Resort & Spa, Bad Brückenau, Germany Organising Committee: ULRICHWALTER, BERNHARDNIESWANDT, SABINE GRÜNER, & BARSOMAKTAS Institute for Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Germany, and Rudolf Virchow Center, Vascular Biology, University of Würzburg, Germany The organisers thank the following for sponsoring the conference: Main Sponsors Baxter, Biotest Pharma GmbH, DFG/SFB 355, IZKF des Universitätsklinikums Würzburg Sponsors Aventis, Bristol-Myers Squibb, Roche Diagnostics, Sanofi Synthelabo GmbH, Biocytex, ZLB Behring Platelets, May/June 2005; 16(3/4): 229–248 ISSN 0953–7104 print/ISSN 1369–1635 online 2005 Taylor & Francis Group Ltd DOI: 10.1080/09537100500147690 HRFVIIai (ASIS ) efficacy assayed in vitro in combination with ARC-69931MX and aspirin in volunteers with and without oral contraceptives (OC) A. Abu-Taleb 1 , W. Wegert 1,2 , S. Bassus 2 , S. Harder 1 , & C. M. Kirchmaier Pharmazentrum Frankfurt, Universitätsklinikum Frankfurt/M., Institut für klinische Pharmakologie, Frankfurt am Main, and GFF der Stiftung Deutsche Klinik für Diagnostik, FB Haemostaseologie und Angiologie,