Specific impairment of human platelet P2Y(AC) ADP receptor-mediated signaling by the antiplatelet drug clopidogrel.

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.

Analysis and regulation of vasodilator-stimulated phosphoprotein serine 239 phosphorylation in vitro and in intact cells using a phosphospecific monoclonal antibody.

The development and functional analysis of a monoclonal antibody (16C2) are reported; the antibody recognizes vasodilator-stimulated phosphoprotein (VASP; an established substrate of both cAMP- and cGMP-dependent protein kinase) only when serine 239 is phosphorylated. VASP serine 239 represents one of the best characterized cGMP-dependent protein kinase phosphorylation sites in vitro and in intact cells. Experiments with purified, recombinant human VASP and various VASP constructs with mutated phosphorylation sites (S157A, S239A, T278A) and experiments with intact cells (human/rat platelets and other cells) treated with cyclic nucleotide-elevating agents demonstrated the specificity of the monoclonal antibody 16C2. Quantitative analysis of the VASP shift from 46 to 50 kDa (indicating VASP serine 157 phosphorylation) and the appearance of VASP detected by the 16C2 monoclonal antibody (VASP serine 239 phosphorylation) in human platelets stimulated by selective protein kinase activators confirmed that serine 239 is the VASP phosphorylation site preferred by cGMP-dependent protein kinase in intact cells. Immunofluorescence experiments with human platelets treated with cGMP analogs showed that the 16C2 monoclonal antibody also detects VASP serine 239 phosphorylation in situ at established intracellular localization sites. Analysis of VASP serine 239 phosphorylation by the 16C2 antibody appears to be the best method presently available to measure cGMP-dependent protein kinase activation in intact cells. Also, the 16C2 antibody promises to be an excellent tool for the evaluation of VASP function in intact cells.

Ligand specificity and ticlopidine effects distinguish three human platelet ADP receptors.

Human platelets express adenosine 5'-diphosphate (ADP)-specific purinoceptors of the P2X and P2Y receptor superfamily, but their structure, diversity, and precise pharmacological profile is not well understood. Here, functional assays with intact platelets and well-characterized nucleotide derivatives were performed in order to characterize the ligand specificity of these platelet-specific purinoceptors. For the signalling pathways investigated (aggregation, rapid Ca2+-influx, desensitization of Ca2+-influx, Ca2+-mobilization, inhibition of adenylyl cyclase), significant differences in ligand specificity were demonstrated. ADP activated all purinoceptors of human platelets, while adenosine 5'-triphosphate (ATP) was a weak agonist for the P2X receptor and an antagonist for the P2Y receptors. The ADP-receptor pathway-antagonist ticlopidine inhibited ADP-evoked aggregation and adenylyl cyclase inhibition but did not affect platelet purinoceptors associated with Ca2+-influx and Ca2+-mobilization. These results indicate the presence of three distinct ADP-selective purinoceptors on human platelets.

Synergistic phosphorylation of the focal adhesion-associated vasodilator-stimulated phosphoprotein in intact human platelets in response to cGMP- and cAMP-elevating platelet inhibitors.

The mechanism underlying the synergistic inhibition of platelet activation by cGMP- and cAMP-elevating vasodilators was investigated using washed human platelets and platelet-rich plasma. With both types of human platelet preparations, low concentrations of sodium nitroprusside increased the cAMP-elevating potency of low concentrations of prostaglandin E1 (PG-E1). Using threshold concentrations of both sodium nitroprusside and PG-E1, the NO-donor potentiated the effect of PG-E1 with respect to the phosphorylation of the focal adhesion-associated vasodilator-stimulated phosphoprotein (VASP) at serine157. In contrast, threshold concentrations of cell-membrane permeant selective activators of the platelet cGMP-dependent protein kinase or the cAMP-dependent protein kinase had only additive effects on VASP serine157 phosphorylation in washed human platelets. The data demonstrate that low intracellular levels of cGMP effectively inhibit type III cGMP-inhibited phosphodiesterase in human platelets despite the high levels of cGMP-dependent protein kinase present in this cell type. This study provides the first evidence that the simultaneous activation of both cGMP- and cAMP-dependent protein kinase results in additive effects on VASP serine157 phosphorylation, whereas the supra-additive effects observed with the combination of sodium nitroprusside and PG-E1 are due to cGMP-mediated inhibition of type III phosphodiesterase. VASP phosphorylation at serine157 may be an important component underlying the synergistic inhibition of human platelets by cGMP-and cAMP-elevating agents.

Phosphorylation of focal adhesion vasodilator-stimulated phosphoprotein at Ser157 in intact human platelets correlates with fibrinogen receptor inhibition.

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 alpha IIb beta 3) 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 (pp125FAK). However, the molecular details of glycoprotein IIb-IIIa regulation are not well understood. Here we report that in ADP-activated human platelets cAMP- and cGMP-dependent 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 E1 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 inhibition of glycoprotein IIb-IIIa induced by cyclic nucleotide involves cAMP-and cGMP-dependent protein-kinase-mediated VASP phosphorylation at Ser157.

Defective nitrovasodilator-stimulated protein phosphorylation and calcium regulation in cGMP-dependent protein kinase-deficient human platelets of chronic myelocytic leukemia.

The presence and functional role of the cyclic nucleotide signal transduction system was investigated in platelets from patients with myeloproliferative disorders. Platelets from certain patients with chronic myelocytic leukemia showed decreased expression of cGMP-dependent protein kinase, and platelets from two such patients were studied in some detail. These platelets had very little if any cGMP-dependent protein kinase but a normal level of cAMP-dependent protein kinase. They also contained a normal level of VASP (vasodilator-stimulated phosphoprotein, a specific substrate of both cAMP- and cGMP-dependent protein kinase), as well as a functionally intact prostaglandin E1-stimulated cAMP-mediated VASP phosphorylation. In contrast, sodium nitroprusside-stimulated VASP phosphorylation was severely impaired in these cGMP-dependent protein kinase-deficient platelets, despite an exaggerated cGMP response to sodium nitroprusside. Furthermore, whereas selective activation of the cGMP-dependent protein kinase by 8-(4-chlorophenylthio)-cGMP strongly inhibited the ADP- or thrombin-evoked calcium mobilization from intracellular stores in normal platelets, this agonist-evoked calcium response was not inhibited by the cGMP analog in cGMP-dependent protein kinase-deficient platelets. The results demonstrate a defect in the nitrovasodilator-/cGMP-regulated signal transduction system in human platelets from some patients with myeloproliferative disorders, and underscore that a cGMP-dependent protein kinase regulatory system, distinct from that of cAMP-dependent protein kinase or other cGMP-dependent effectors is operative in normal human platelets.