Nucleotide receptor signaling in platelets

Upon injury to a vessel wall the exposure of subendothelial collagen results in the activation of platelets. Platelet activation culminates in shape change, aggregation, release of granule contents and generation of lipid mediators. These secreted and generated mediators trigger a positive feedback mechanism potentiating the platelet activation induced by physiological agonists such as collagen and thrombin. Adenine nucleotides, adenosine diphosphate (ADP) and adenosine triphosphate (ATP), released from damaged cells and that are secreted from platelet-dense granules, contribute to the positive feedback mechanism by acting through nucleotide receptors on the platelet surface. ADP acts through two G protein-coupled receptors, the Gq-coupled P2Y1 receptor, and the Gi-coupled P2Y12 receptor. ATP, on the other hand, acts through the ligand-gated channel P2X1. Stimulation of platelets by ADP leads to shape change, aggregation and thromboxane A2 generation. ADP-induced dense granule release depends on generated thromboxane A2. Furthermore, costimulation of both P2Y1 and P2Y12 receptors is required for ADP-induced platelet aggregation. ATP stimulation of P2X1 is involved in platelet shape change and helps to amplify platelet responses mediated by agonists such as collagen. Activation of each of these nucleotide receptors results in unique signal transduction pathways that are important in the regulation of thrombosis and hemostasis.     

Protease-activated receptor-induced Akt activation – regulation and possible function

BACKGROUND: Thrombin induces the activation of the platelet serine/threonine kinase Akt. Akt activation is dependent on its phosphorylation at Thr308 and Ser473. The mechanism by which thrombin induces Akt phosphorylation is controversial, as is the role of Akt in platelet function. OBJECTIVES: To investigate how protease-activated receptors (PARs) stimulate Akt and the role that Akt plays in human platelet function. METHODS: Platelets were stimulated through PAR1 or PAR4. Specific inhibitors were used to evaluate, by Western blotting, signaling pathways regulating Akt phosphorylation, and the role of activated Akt was evaluated by aggregometry and flow cytometry. RESULTS: Phospholipase C (PLC) controls Akt phosphorylation elicited by PARs. Stimulation of PAR1 or PAR4 resulted in rapid Akt phosphorylation, independently of secreted ADP and phosphatidylinositol-3-kinase (PI3K) activation. Akt phosphorylation approximately 60 s after PAR1 stimulation became entirely dependent on the purinergic receptor P2Y(12) and the activation of PI3K. In contrast, PAR4 partially sustained Akt phosphorylation independently of P2Y(12) and PI3K for up to 300 s. Pharmacologic inhibition of Akt reduced P-selectin expression and fibrinogen binding in platelets stimulated through PAR1, and delayed platelet aggregation in response to submaximal PAR1 or PAR4 stimulation, although aggregation at 300 s was unaffected. CONCLUSIONS: Platelet PAR stimulation causes rapid Akt phosphorylation downstream of PLC, whereas with continuous stimulation, ADP and PI3K are required for maintaining Akt phosphorylation. Activated Akt regulates platelet function by modulating secretion and alpha(IIb)beta(3) activation.     

Differences and similarities in tyrosine phosphorylation of proteins in platelets from human and pig species

Summary. Background: Pigs have been widely used as animal models to study hemostasis. However, there are significant differences when comparing the hemostatic behavior of pig and human platelets. Objective: To investigate signaling through tyrosine‐phosphorylation of proteins in pig platelets after activation in suspension or by adhesion under flow conditions, in comparison with human platelets. Methods: Activation of platelet suspensions was performed with thrombin (T; 0.1 and 1 U mL^?1) and type I collagen (Col‐I; 20 µg mL^?1), at two different time points (30 and 90 s). Activation by adhesion was carried out on Col‐I‐coated coverslips, using citrated whole blood samples perfused through a parallel‐plate chamber. Results and conclusions: Significant differences between pig and human platelets were detected before and after activation. Activation of pig platelets required higher concentrations of thrombin, as well as increased activation times, to achieve similar levels of tyrosine phosphorylation. Proteins p160, p140, p85 and pp62, present in human platelets, were not detected in profiles corresponding to activated pig platelets. A protein of 70 kDa appeared only in pig platelet profiles, p55 was highly phosphorylated, and the phosphorylation levels of some proteins were significantly different from those found in human platelet profiles. In profiles corresponding to adhered pig platelets, p85 and p62 were absent, and p115 appeared highly phosphorylated. As observed in suspension studies, p70 and p55 appeared specifically in adhered pig platelets. Our study shows that the phosphotyrosine proteins involved in the activation of pig platelets are significantly different from those observed in activated human platelets. These findings may help to explain the differing adhesive and cohesive properties of platelets from both species, which should be considered when extrapolating results.     

P2Y12 protects platelets from apoptosis via PI3k‐dependent Bak/Bax inactivation

Summary. Background: Platelet ADP receptor P2Y₁₂ is well studied and recognized as a key player in platelet activation, hemostasis and thrombosis. However, the role of P2Y₁₂ in platelet apoptosis remains unknown. Objectives: To evaluate the role of the P2Y₁₂ receptor in platelet apoptosis. Methods: We used flow cytometry and Western blotting to assess apoptotic events in platelets treated with ABT‐737 or ABT‐263, and stored at 37 °C, combined with P2Y₁₂ receptor antagonists or P2Y₁₂‐deficient mice. Results: P2Y₁₂ activation attenuated apoptosis induced by ABT‐737 in human and mouse platelets in vitro, evidenced by reduced phosphatidylserine (PS) exposure, diminished depolarization of mitochondrial inner transmembrane potential (ΔΨm) and decreased caspase‐3 activation. Through increasing the phosphorylation level of Akt and Bad, and changing the interaction between different Bcl‐2 family proteins, P2Y₁₂ activation inactivated Bak/Bax. This antiapoptotic effect could be abolished by P2Y₁₂ antagonism or PI3K inhibition. We also observed the antiapoptotic effect of P2Y₁₂ activation in platelets stored at 37 °C. P2Y₁₂ activation improved the impaired activation responses of apoptotic platelets stressed by ABT‐737. In platelets from mice dosed with ABT‐263 in vivo, clopidogrel or deficiency of P2Y₁₂ receptor enhanced apoptosis along with increased Bak/Bax activation. Conclusions: This study demonstrates that P2Y₁₂ activation protects platelets from apoptosis via PI3k‐dependent Bak/Bax inactivation, which may be physiologically important to counter the proapoptotic challenge. Our findings that P2Y₁₂ blockade exaggerates platelet apoptosis induced by ABT‐263 (Navitoclax) also imply a novel drug interaction of ABT‐263 and P2Y₁₂ antagonists.     

(N)-methanocarba-2MeSADP (MRS2365) is a subtype-specific agonist that induces rapid desensitization of the P2Y1 receptor of human platelets

Adenosine diphosphate (ADP) initiates and maintains sustained aggregation of platelets through simultaneous activation of both the Gq-coupled P2Y1 receptor and the Gi-coupled P2Y12 receptor. We recently described the synthesis and P2Y1 receptor-specific agonist activity of (N)-methanocarba-2MeSADP (MRS2365). Consequences of selective activation of the P2Y1 receptor by MRS2365 have been further examined in human platelets. Whereas MRS2365 alone only induced shape change, addition of MRS2365 following epinephrine treatment, which activates the Gi/z-linked, alpha2A-adrenergic receptor, resulted in sustained aggregation that was indistinguishable from that observed with ADP. Conversely, the platelet shape change promoted by ADP in the presence of the GPIIb/IIIa antagonist eptifibatide was similar to that promoted by MRS2365. Preaddition of the high affinity P2Y1 receptor antagonist MRS2500 inhibited the effect of MRS2365, whereas addition of MRS2500 subsequent to MRS2365 reversed the MRS2365-induced shape change. Preactivation of the P2Y1 receptor with MRS2365 for 2 min resulted in marked loss of capacity of ADP to induce aggregation as evidenced by a greater than 20-fold rightward shift in the concentration effect curve of ADP. This inhibitory effect of P2Y1 receptor activation was dependent on the concentration of MRS2365 (EC50 = 34 nm). The inhibitory effect of preincubation with MRS2365 was circumvented by activation of the Gq-coupled 5-HT2A receptor suggesting that MRS2365 induces loss of the ADP response as a consequence of desensitization of the Gq-coupled P2Y1 receptor. The time course of MRS2365-induced loss of aggregation response to epinephrine was similar to that observed with ADP. These results further demonstrate the P2Y1 receptor selectivity of MRS2365 and illustrate the occurrence of agonist-induced desensitization of the P2Y1 receptor of human platelets studied in the absence of P2Y12 receptor activation .     

Quantification of ADP and ATP receptor expression in human platelets

Summary.  The mechanism of ADP-mediated platelet activation has been difficult to unravel due to the large number of receptors for extracellular nucleotides (P2 receptors). mRNA levels in circulating platelets are very low, but have been shown to be translationally active. By optimizing mRNA extraction and using real time (RT)-PCR we were able to establish a protocol for highly sensitive platelet mRNA quantification in human regular blood samples. In platelets from healthy volunteers, only P2X₁, P2Y₁ and P2Y₁₂ were found in significant levels, with the following order of expression: P2Y₁₂ >> P2X₁ > P2Y₁. Other P2 receptors (P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₃, P2X₄, P2X₇) had very low expression. As a control measurement to exclude contamination, P2 receptors in buffy coat were quantified but had a completely different profile. Incubation in vitro revealed a more rapid degradation rate for P2X₁ receptor mRNA than for P2Y₁ and P2Y₁₂, indicating that the level of P2X₁ may be relatively higher in newly released platelets and in megacaryocytes. In conclusion, we have developed the first protocol for quantifying mRNA expression in human platelets limiting the P2 receptor drug development targets to P2Y₁₂, P2Y₁ and P2X₁. Furthermore, the method could be used to study platelet expression for any gene in human materials.     

Interaction of the active metabolite of prasugrel,R‐138727, with cysteine 97 and cysteine 175 of the human P2Y12 receptor

Summary. Background: The P2Y₁₂ receptor plays a crucial role in platelet aggregation and is the target of platelet aggregation inhibitors, including the thienopyridine compound prasugrel. Objective: The present study analyzed the effects of R‐138727 (2‐[1‐[2‐cyclopropyl‐1‐(2‐fluorophenyl)‐2‐oxoethyl]‐4‐mercapto‐3‐piperidinylidene]acetic acid), the active metabolite of prasugrel, on recombinant wild‐type and mutant human P2Y₁₂ receptors in order to identify the molecular site of action of R‐138727. Methods: The function of wild‐type and mutant P2Y₁₂ receptors stably expressed in Chinese hamster ovary cells was assessed by measuring the 2‐methylthio‐ADP‐mediated inhibition of forskolin‐stimulated cellular cAMP production. Results: In cells expressing wild‐type receptors, R‐138727 potently inhibited receptor function with a half‐maximal concentration below 1 μm . The mode of action was irreversible. The same effect of R‐138727 was observed in cells expressing Cys17Ala/Cys270Ala constructs. In contrast, in cells expressing either a Cys97Ala construct or a Cys175Ala construct, R‐138727 failed to inhibit the response to the agonist. When cells expressing wild‐type receptors were pretreated with the P2 receptor antagonists ATP or suramin, no effect of R‐138727 was observed. Similar experiments with N‐acetylcysteine 10 μm showed no interference of N‐acetylcysteine with R‐138727. Conclusions: The experiments demonstrate a potent and irreversible action of R‐138727 at the recombinant human P2Y₁₂ receptor. The data suggest that R‐138727 interacts with cysteine 97 (upper portion of the predicted third transmembrane region) and cysteine 175 (second extracellular loop) of the receptor, which are likely to form a disulfide bridge in native receptors. Moreover, the data also suggest that this site of action of R‐138727 is close to the ligand‐binding site of the receptor.     

P2Y12 receptor-mediated potentiation of thrombin-induced thromboxane A2 generation in platelets occurs through regulation of Erk1/2 activation

BACKGROUND: Thromboxane A2 (TXA2) is a positive feedback lipid mediator that is generated upon stimulation of platelets with various agonists. Aspirin works as an antithrombotic drug by blocking the generation of TXA2. The aim of this study was to evaluate the role of the purinergic P2Y receptors in thrombin-induced TXA2 generation. RESULTS: PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin, induced the activation of cytosolic phospholipase A2 (cPLA2), release of arachidonic acid (AA) from membrane-bound phospholipids, and subsequent TXA2 generation in human platelets. The actions of these agonists were significantly inhibited in the presence of the P2Y12 receptor antagonist, AR-C69931MX, but not the P2Y1 receptor antagonist, MRS2179. In addition, AYPGKF- and thrombin-induced TXA2 generation was significantly reduced in platelets from mice dosed with clopidogrel, confirming the results obtained with the human platelets. Also, Pearl mouse platelets that lack releasable nucleotides generated significantly less TXA2 when compared with the wild-type littermates in response to PAR stimulation. Inhibition of extracellular signal-regulated protein kinase 1/2 (Erk 1/2) activation using U0126, an inhibitor of MAP kinase kinase (MEK), suppressed PAR-mediated cPLA2 phosphorylation and TXA2 generation. Further, platelets that were pretreated with AR-C69931MX, as well as Pearl mouse platelets, displayed the reduced levels of Erk1/2 phosphorylation upon stimulation with the PAR agonists. CONCLUSIONS: Based on these findings, we conclude that thrombin-induced Erk1/2 activation is essential for PAR-mediated TXA2 generation, which is potentiated by the P2Y12 receptor-mediated signaling pathway but not the P2Y1 receptor-mediated signaling pathway. Finally, using selective inhibitors of Src kinases, we show that PAR-mediated Src activation precedes Erk1/2 activation.     

Release of sphingosine‐1‐phosphate from human platelets is dependent on thromboxane formation

Summary. Background: Platelets release the immune‐modulating lipid sphingosine‐1‐phosphate (S1P). However, the mechanisms of platelet S1P secretion are not fully understood. Objectives: The present study investigates the function of thromboxane (TX) for platelet S1P secretion during platelet activation and the consequences for monocyte chemotaxis. Methods: S1P was detected using thin‐layer chromatography in [³H]sphingosine‐labeled platelets and by mass spectrometry. Monocyte migration was measured in modified Boyden chamber chemotaxis assays. Results: Release of S1P from platelets was stimulated with protease‐activated receptor‐1‐activating peptide (PAR‐1‐AP, 100 μm ). Acetylsalicylic acid (ASA) and two structurally unrelated reversible cyclooxygenase inhibitors diclofenac and ibuprofen suppressed S1P release. Oral ASA (500‐mg single dose or 100 mg over 3 days) attenuated S1P release from platelets in healthy human volunteers ex vivo. This was paralleled by inhibition of TX formation. S1P release was increased by the TX receptor (TP) agonist U‐46619, and inhibited by the TP antagonist ramatroban and by inhibitors of ABC‐transport. Furthermore, thrombin‐induced release of S1P was attenuated in platelets from TP‐deficient mice. Supernatants from PAR‐1‐AP‐stimulated human platelets increased the chemotactic capacity of human peripheral monocytes in a S1P‐dependent manner via S1P receptors‐1 and ‐3. These effects were inhibited by ASA‐pretreatment of platelets. Conclusions: TX synthesis and TP activation mediate S1P release after thrombin receptor activation. Inhibition of this pathway may contribute to the anti‐inflammatory actions of ASA, for example by affecting activity of monocytes at sites of vascular injury.     

PKC inhibition markedly enhances Ca2+ signaling and phosphatidylserine exposure downstream of protease‐activated receptor‐1 but not protease‐activated receptor‐4 in human platelets

To cite this article: Harper MT, Poole AW. PKC inhibition markedly enhances Ca²⁺ signaling and phosphatidylserine exposure downstream of protease‐activated receptor‐1 but not protease‐activated receptor‐4 in human platelets. J Thromb Haemost 2011; 9 : 1599–607. Summary. Background: Cytosolic calcium concentration is a critical regulator of platelet activation, and so platelet Ca²⁺ signaling must be tightly controlled. Thrombin‐induced Ca²⁺ signaling is enhanced by inhibitors of protein kinase C (PKC), suggesting that PKC negatively regulates the Ca²⁺signal, although the mechanisms by which this occurs and its physiological relevance are still unclear. Objectives: To investigate the mechanisms by which PKC inhibitors enhance thrombin‐induced Ca²⁺ signaling, and to determine the importance of this pathway in platelet activation. Methods: Cytosolic Ca²⁺ signaling was monitored in fura‐2‐loaded human platelets. Phosphatidylserine (PS) exposure, a marker of platelet procoagulant activity, was measured by annexin V binding and flow cytometry. Results: PKC inhibition by bisindolylmaleimide‐I (BIM‐I) enhanced α‐thrombin‐induced Ca²⁺ signaling in a concentration‐dependent manner. PAR1 signaling, activated by SFLLRN, was enhanced much more strongly than PAR4, activated by AYPGKF or γ‐thrombin, which is a potent PAR4 agonist but a poor activator of PAR1. BIM‐I had little effect on α‐thrombin‐induced signaling following treatment with the PAR1 antagonist, SCH‐79797. BIM‐I enhanced Ca²⁺ release from intracellular stores and Ca²⁺ entry, as assessed by Mn²⁺ quench. However, the plasma membrane Ca²⁺ ATPase inhibitor, 5(6)‐carboxyeosin, did not prevent the effect of BIM‐I. PKC inhibition strongly enhanced α‐thrombin‐induced PS exposure, which was reversed by blockade of PAR1. Conclusions: Together, these data show that when PAR1 is stimulated, PKC negatively regulates Ca²⁺ release and Ca²⁺ entry, which leads to reduced platelet PS exposure.     

Clinical effects and outcomes with new P2Y12 inhibitors in ACS

Thienopyridines have become the cornerstone of treatment for percutaneous coronary intervention although no survival benefit has ever been shown with clopidogrel despite increasing loading doses. Newly developed P2Y12 inhibitors are more potent, more predictable, and have a faster onset of action than clopidogrel, characteristics that make them particularly attractive for high-risk percutaneous coronary intervention (PCI). Four new P2Y12 inhibitors have been tested each of them having particular individual properties. Prasugrel is an oral pro-drug leading to irreversible blockade of the P2Y12 receptor and is approved worldwide for ACS PCI. Ticagrelor is a direct-acting and reversible inhibitor of the P2Y12 receptor with potentially more pleiotropic effects. Cangrelor is an intravenous direct and reversible inhibitor of the P2Y12 receptor providing the highest level of inhibition, and elinogrel is an intravenous and oral P2Y12 antagonist with a direct and reversible action. Both prasugrel and ticagrelor, opposed to clopidogrel, have shown that stronger P2Y12 inhibition led respectively to significant 19 and 16% relative risk reduction of a similar primary end point combining cardiovascular death, non-fatal myocardial infarction, or non-fatal stroke. Both drugs showed a significant 0.6% absolute excess of TIMI major bleeding not related to CABG surgery. Because in clinical trials, patients perceived to be at higher risk of bleeding usually are excluded, the risk of major and even fatal bleeding might even be higher in a 'real-world' setting, i.e. in the elderly patient with comorbidities. On the other hand, these newly developed P2Y12 inhibitors decrease mortality after PCI compared with clopidogrel. The risk/benefit ratio is particularly favorable in PCI for patients with STEMI.     

Thrombostatin FM compounds: direct thrombin inhibitors – mechanism of action in vitro and in vivo

Summary.  Background:  Novel pentapeptides called Thrombostatin FM compounds consisting mostly of D-isomers and unusual amino acids were prepared based upon the stable angiotensin converting enzyme breakdown product of bradykinin – RPPGF. Methods and Results:  These peptides are direct thrombin inhibitors prolonging the thrombin clotting time, activated partial thromboplastin time, and prothrombin time at ≥0.78, 1.6, and 1.6 μ m , respectively. They competitively inhibit α-thrombin-induced cleavage of a chromogenic substrate at 4.4–8.2 μ m . They do not significantly inhibit plasma kallikrein, factor (F) XIIa, FXIa, FIXa, FVIIa-TF, FXa, plasmin or cathepsin G. One form, FM19 [rOicPaF( p -Me)], blocks α-thrombin-induced calcium flux in fibroblasts with an IC₅₀ of 6.9 ± 1.2 μ m . FM19 achieved 100% inhibition of threshold α- or γ-thrombin-induced platelet aggregation at 8.4 ± 4.7 μ m and 16 ± 4 μ m , respectively. The crystal structure of thrombin in complex with FM19 shows that the N-terminal D-Arg retrobinds into the S1 pocket, its second residue Oic interacts with His-57, Tyr-60a and Trp-60d, and its C-terminal p -methyl Phe engages thrombin's aryl binding site composed of Ile-174, Trp-215, and Leu-99. When administered intraperitoneal, intraduodenal, or orally to mice, FM19 prolongs thrombin clotting times and delays carotid artery thrombosis. Conclusion:  FM19, a low affinity reversible direct thrombin inhibitor, might be useful as an add-on agent to address an unmet need in platelet inhibition in acute coronary syndromes in diabetics and others who with all current antiplatelet therapy still have reactive platelets.     

Multiple electrode aggregometry predicts stent thrombosis better than the vasodilator‐stimulated phosphoprotein phosphorylation assay

Summary. Background and Aim: The prognostic value of the vasodilator‐stimulated phosphoprotein (VASP) phosphorylation assay and multiple electrode aggregometry (MEA) for thrombotic adverse events has been shown in independent studies. As no direct comparison between the two methods has been made so far, we investigated which laboratory approach has a better predictive value for stent thrombosis. Methods: The VASP phosphorylation assay and MEA were performed in 416 patients with coronary artery disease undergoing percutaneous coronary intervention. The rate of stent thrombosis was recorded during a 6‐month follow‐up. Results: Definite stent thrombosis occurred in three patients (0.7%) and probable stent thrombosis in four (1%). Receiver operating characteristic (ROC) analysis demonstrated that MEA distinguishes between patients with or without subsequent stent thrombosis better than the VASP phosphorylation assay: the area under the ROC curve was higher for MEA (0.92; P = 0.012) than for the VASP phosphorylation assay (0.60; P = 0.55). At equal levels of sensitivity (100%), the specificity was greater for MEA than for the VASP phosphorylation assay (86% vs. 37%). Stent thrombosis occurred in 9% of patients with platelet hyperreactivity in MEA, who were simultaneously clopidogrel non‐responders in the VASP phosphorylation assay. Interestingly, clopidogrel non‐responders in the VASP phosphorylation assay without platelet hyperreactivity in MEA did not suffer from stent thrombosis. Conclusions: Platelet hyperreactivity in MEA might be a better risk predictor for stent thrombosis than the assessment of the specific clopidogrel effect with the VASP phosphorylation assay.