The potency of selatogrel, a reversible antagonist of the P2Y12 receptor, is affected by calcium concentration
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Here, we report the in vitro characterization of the P2Y12 receptor antagonist selatogrel (ACT-246475). Binding studies with radiolabeled selatogrel demonstrated that selatogrel is a competitive antagonist of ADP binding to the P2Y12 receptor with a fast onset of action. Consequently, selatogrel was confirmed to be a potent inhibitor of P2Y12-mediated intra-platelet signaling and ADP-induced platelet activation. Characterization of selatogrel in platelet-rich plasma in vitro demonstrated that the mode of anti-coagulation affected the anti-platelet potency. Specifically, in platelet-rich plasma containing physiological calcium concentration (anticoagulated with a direct thrombin inhibitor), selatogrel achieved half-maximal inhibition of ADP-induced platelet aggregation at a 3-fold lower concentration than in conditions with low calcium concentration (anticoagulated with citrate). Furthermore, calcium-dependent reduction in selatogrel potency was observed in whole blood platelet aggregation using the VerifyNow™ system with a 3.7-fold potency loss in low calcium conditions. A comparable potency loss was also observed with the reversible P2Y12 receptor antagonists ticagrelor, cangrelor and elinogrel. Furthermore, receptor-binding experiments using radiolabeled selatogrel confirmed a 3-fold lowering of selatogrel binding affinity to the P2Y12 receptor in low calcium conditions. In conclusion, our data suggest that in low calcium conditions (i.e., citrate-anticoagulated blood), there is a risk of underestimating the potency of reversible P2Y12 receptor antagonists. To avoid overdosing, and a potential increase in bleeding risk, we propose that the ex vivo evaluation of reversible P2Y12 receptor antagonists should be performed with platelet assay systems containing physiological calcium concentration.White cell (WBC)-reduction filters that remove more than 99 percent of the WBCs from platelet concentrates are rapidly being introduced into routine use. Using activation-dependent monoclonal antibodies and flow cytometry, platelet activation was evaluated before and after WBC reduction in 10 platelet concentrates prepared manually from whole blood obtained from five male and five female regular volunteer blood donors. In general no significant increases were found in platelet activation markers after WBC reduction using filters. However, if platelets were activated during preparation, increased numbers of platelets were found expressing the activation marker CD62, and this correlated with the decrease in the platelet count after WBC reduction. These observations may explain increased platelet loss following WBC reduction in some platelet components.
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Platelets are transfused to patients to prevent bleeding. Since both preparation and storage can impact the hemostatic functions of platelets, we studied platelet concentrates (PCs) with different initial composition in regard to platelet fragmentation and its impact on storage-induced changes in activation potential. Ten whole blood derived PCs were assessed over 7 storage days. Using flow cytometry, platelet (CD41+) subpopulations were characterized for activation potential using activation markers (PAC-1, P-selectin, and LAMP-1), phosphatidylserine (Annexin V), and mitochondrial integrity (DiIC1(5)). Aggregation response, coagulation, and soluble activation markers (cytokines and sGPVI) were also measured. Of the CD41+ events, the PCs contained a median of 82% normal-sized platelets, 10% small platelets, and 8% fragments. The small platelets exhibited procoagulant hallmarks (increased P-selectin and Annexin V and reduced DiIC1(5)). Normal-sized platelets responded to activation, whereas activation potential was decreased for small and abolished for fragments. Five PCs contained a high proportion of small platelets and fragments (median of 28% of CD41+ events), which was significantly higher than the other five PCs (median of 9%). A high proportion of small platelets and fragments was associated with procoagulant hallmarks and decreased activation potential, but, although diminished, they still retained some activation potential throughout 7 days storage.
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Platelet activation occurs in response to vessel injury and is important for the arrest of bleeding. Platelet activation during disease states leads to vascular occlusion and ischemic damage. The P2Y12 receptor, activated by ADP, plays a central role in platelet activation and is the target of P2Y12 receptor antagonists that have proven therapeutic value.
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P-selectin
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Background and Objectives: Hyperconcentration of platelets may lead to platelet activation and loss of platelet function. Materials and Methods: Platelet activation following hyperconcentration was assessed using flow-cytometric detection of platelet P-selectin expression and platelet swirling. Results: Platelet hyperconcentration led to a minimal increase in P-selectin expression and no differnce in platelet swirling. Conclusion: Hyperconcentration was not associated with a clinically significant change in platelet activation and had no significant effect on platelet quality as detected by pH and platelet yield.
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Abstract Background and Objectives: Hyperconcentration of platelets may lead to platelet activation and loss of platelet function. Materials and Methods: Platelet activation following hyperconcentration was assessed using flow‐cytometric detection of platelet P‐selectin expression and platelet swirling. Results: Platelet hyperconcentration led to a minimal increase in P‐selectin expression and no differnce in platelet swirling. Conclusion: Hyperconcentration was not associated with a clinically significant change in platelet activation and had no significant effect on platelet quality as detected by pH and platelet yield.
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This article was originally published online on 23 April 2013 Animal models of allergic asthma indicate that intravascular platelet activation is essentail for the development of allergen induced chronic airway inflammation. P2Y12, the third CysLT receptor, is expressed on platelets and has been an important pathophysiological role in LTE4 mediated pulmonary inflammation. We investigated platelet activation status in asthmatic patients compared to controls. Fifty asthmatic patients and 20 healthy controls were enrolled from Ajou University Hospital, Suwon, Korea Surface expression of P-selectin and P2Y12 on platelets were determined by flowc ytometry. Plasma soluble P-selectin level was measured by ELISA. The asthmatic subjects were classified into two groups depending on high (>mean + 2 SD of controls) and low expression of P2Y12. The expressions of platelet P-selectin, P2Y12 and soluble p-selectin level were significantly higher in asthmatic patients than in controls, (p<0.001, p=0.001, p<0.001, respectively). No significant correlations were found between clinical parameters and platelet activation markers. Expressions of P2Y12 on platelet did not increase significantly after the treatment with LTE4 or aspirin. Higher expression group of P2Y12 had significantly higher peripheral eosinophil count (P=0.021). Platelet activation may play a role in asthma pathogenesis. A possible interaction between platelet and eosinophil via P2Y12 was suggested.
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Allergic Inflammation
Pathophysiology
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The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders.
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