Although cGMP-dependent protein kinases (cGPKs) 1 have been recognized as important components of major signal transduction pathways (1-3), quantitative analysis of cGPK activation in intact cells has been very difficult (1-4). This is because of the relatively low expression of cGPK in most cell types compared with the relatively high expression of its closest functional homolog, the cAMP-dependent protein kinase (cAPK), and the scarcity of specific cGPK substrates. Unfortunately, the mediating role of cGPK for a given effect/function is often implied or excluded by the use of cGPK activators and/or inhibitors alone, which is clearly insufficient to establish or rule out functional roles of cGPKs (1-4). One of the few established cGPK substrates is the 46-kDa/50-kDa vasodilator-stimulated phosphoprotein (VASP), which was initially discovered and characterized as a substrate of both cAPK and cGPK in human platelets (5-8). VASP phosphorylation in response to cyclic nucleotide-regulating vasodilators (i.e. cAMP-elevating prostaglandins and cGMP-elevating nitric oxide donors) closely correlates with platelet inhibition and in particular with the inhibition of fibrinogen binding to the integrin ␣ IIb ␤ 3 of human platelets (9 -11). Molecular cloning of human, canine, and mouse VASP predicted highly homologous proteins and revealed a proline-rich protein that is organized into three structural segments of different sequence complexity (12,13). VASP is the founding member of a new family of proline-rich proteins, which includes Enabled (Ena), a dose-dependent suppressor of Drosophila Abl-and Disabled-dependent phenotypes, its mammalian homolog Mena, and the Ena-VASP-like protein Evl (14 -16). These proteins all share an overall domain organization consisting of highly homologous NH 2 -terminal and COOHterminal domains (Ena-VASP homology domains 1 and 2, EVH1 and EVH2), which are separated by a proline-rich central domain of low complexity (12-16). In platelets and many other cells including vascular smooth muscle cells, endothelial cells, and fibroblasts, VASP has been found to be associated with stress fibers, focal adhesions, cell-cell contacts, and highly dynamic membrane regions (16,17). VASP colocalizes with profilins and binds directly to their poly(L-proline) binding site (18), binds to and colocalizes with zyxin and vinculin (16,19), and also directly binds to Listeria monocytogenes surface protein ActA, which is essential for the actin polymerization-based intracellular motility of this pathogen (20). Functional evidence indicates that VASP is a crucial factor involved in the enhancement of spatially confined actin filament formation (16,20,21).Three distinct phosphorylation sites were biochemically identified in VASP (serine 157, serine 239, and threonine 278) which are used in vitro and in intact human platelets by both cAPK and cGPK and by the serine/threonine protein phosphatases 2A and 2B with overlapping selectivity (8,22). Phosphorylation of serine 157, the site preferred by the cAPK, leads to a marked...

Integrins and other adhesion receptors are essential components for outside-in and inside-out signaling through the cell membrane. The platelet glycoprotein IIb-IIIa (also known as fibrinogen receptor or integrin q&) is activated by platelet agonists, inhibited by cyclic-nucleotide-elevating agents, and is involved in the activation of protein tyrosine kinases including the 125-kDa focal adhesion kinase (~~1 2 5~~~) .However, the molecular details of glycoprotein IIb-IIIa regulation are not well understood. Here we report that in ADP-activated human platelets CAMP-and cGMPdependent protein-kinase-mediated phosphorylation of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) at Ser157 correlates well with glycoprotein IIb-IIIa inhibition. Human platelets contain similar concentrations of glycoprotein IIb-IIIa complexes (fibrinogen binding sites) and VASP. Using gel-filtered platelets, CAMP-elevating agents [e.g. prostaglandin E, and the forskolin analog 6-(3-dimethylaminopropionyl)forskolin (NKH 477)] caused VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa activation up to 70-100%. NO-generating, cGMP-elevating agents [e.g. 3-morpholinosydnonimine hydrochloride (SIN1) and sodium nitroprusside] stimulated VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa activation up to a maximal extent of 30-50%. The effects of CAMP-and cGMP-elevating agents on VASP phosphorylation and fibrinogen binding were reversible and could be mimicked by membrane-permeant selective activators of platelet CAMP-or cGMP-dependent protein kinase, respectively. Using threshold concentrations, the nitrovasodilator SIN 1 potentiated the effects of the forskolin analog NKH 477 with respect to inhibition of platelet aggregation, VASP phosphorylation and glycoprotein IIb-IIIa inhibition. It is proposed that the inhitition of glycoprotein IIb-IIIa induced by cyclic nucleotide involves CAMPand cGMP-dependent protein-kinase-mediated VASP phosphorylation at Ser157.Adhesion, activation and aggregation of platelets and platelet -vessel-wall interactions are tightly regulated under physiological conditions and often impaired in diseases including congenital hemorrhagic diseases, atherosclerosis, hypertension and diabetes 11, 21. Important components of this vascular cell -cell and cell -matrix interaction are integrins and other adhesion receptors [3 -81. The platelet glycoprotein IIb-IIIa ((xllbp3) is one of the best-studied integrins and represents the functional fibrinogen receptor which is absent or impaired in the hereditary disorder Glanzmann's thrombasthenia [l, 5-81. Activated platelets contain at their sur-

Abstract-Clopidogrel is an effective new antiplatelet agent useful for the treatment of ischemic cerebrovascular, cardiac, and peripheral arterial disease. However, the mechanism of clopidogrel action is not well understood, although it is known to inhibit ADP-evoked platelet aggregation. In the current study, the effect of clopidogrel on recently identified human platelet ADP receptors and their signaling pathways was investigated by using platelets from clopidogrel-treated subjects, 6 healthy volunteers (2 females and 4 males) who received 75 mg of clopidogrel daily for 7 days. Blood was taken and various platelet receptor signaling pathways were analyzed before treatment, after 7 days of medication, and 4 weeks after treatment had ceased. Platelet tests included the analysis of aggregation, rapid calcium influx, calcium mobilization from intracellular stores, adenylyl cyclase, and phosphorylation of vasodilator-stimulated phosphoprotein (VASP). The data indicate that clopidogrel does not affect those platelet ADP receptors coupled to cation influx (P2X1 ADP receptors) or calcium mobilization (P2Y1 ADP receptors). In contrast, clopidogrel treatment specifically impairs the ADP receptor coupled to G i /adenylyl cyclase (P2Y AC ADP receptors). Clopidogrel abolishes the inhibitory P2Y AC receptor-mediated ADP effects on prostaglandin E 1 -stimulated, cAMP-dependent phosphorylation of VASP without affecting epinephrine, thrombin, and thromboxane signaling. VASP phosphorylation is known to be closely correlated with the inhibition of platelet and fibrinogen receptor (glycoprotein IIb/IIIa) activation. Therefore, inhibition of the platelet P2Y AC ADP receptor and its intracellular signaling, including decreased VASP phosphorylation, is suggested as a molecular mechanism of clopidogrel action. Key Words: platelet inhibition Ⅲ purinergic receptors Ⅲ vasodilator-stimulated phosphoprotein I ncreased platelet activation and aggregation are central to the pathophysiology of acute and chronic arterial vascular diseases. This concept has gained broad acceptance since platelet inhibitors have been proven as effective agents for the treatment of both chronic and acute diseases of the arterial vessel wall. [1][2][3][4][5][6] Platelets are activated by numerous agents and conditions, but ADP is thought to play a key role in the development of arterial thrombosis. 7,8 Recently, long-term administration of clopidogrel to patients with atherosclerotic vascular disease was shown to be more effective than aspirin in reducing the combined risk of ischemic stroke, myocardial infarction, and vascular death. 9 Clopidogrel and the chemically related ticlopidine are thienopyridines that selectively and specifically interfere with ADP-mediated platelet activation. 5,10 In contrast to ticlopidine, which may cause neutropenia, clopidogrel appears to be a safe and well-tolerated drug. 9 Thienopyridines are inactive in-vitro, require in vivo metabolism, and cause an irreversible inhibition of platelet function. However, the mechanism of thieno...

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