Antithrombotic Potential of Blockers of Store-Operated Calcium Channels in Platelets

Abstract

Objective— Platelet Orai1 channels mediate store-operated Ca 2+ entry (SOCE), which is required for procoagulant activity and arterial thrombus formation. Pharmacological blockage of these channels may provide a novel way of antithrombotic therapy. Therefore, the thromboprotective effect of SOCE blockers directed against platelet Orai1 is determined. Methods and Results— Candidate inhibitors were screened for their effects on SOCE in washed human platelets. Tested antagonists included the known compounds, SKF96365, 2-aminoethyl diphenylborate, and MRS1845 and the novel compounds, Synta66 and GSK-7975A. The potency of SOCE inhibition was in the order of Synta66>2-aminoethyl diphenylborate>GSK-7975A>SKF96365>MRS1845. The specificity of the first 3 compounds was verified with platelets from Orai1-deficient mice. Inhibitory activity on procoagulant activity and high-shear thrombus formation was assessed in plasma and whole blood. In the presence of plasma, all 3 compounds suppressed platelet responses and restrained thrombus formation under flow. Using a murine stroke model, arterial thrombus formation was provoked in vivo by transient middle cerebral artery occlusion. Postoperative administration of 2-aminoethyl diphenylborate markedly diminished brain infarct size. Conclusion— Plasma-soluble SOCE blockers such as 2-aminoethyl diphenylborate suppress platelet-dependent coagulation and thrombus formation. The platelet Orai1 channel is a novel target for preventing thrombotic events causing brain infarction.

Survival protein anoctamin‐6 controls multiple platelet responses including phospholipid scrambling, swelling, and protein cleavage

“…On the other hand, we could not detect Ano6 protein expression and hence found no evidence for a role of these splice variants, in the residual PS exposure in human Scott syndrome or mouse Ano6 Avor–/– platelets. Not unlikely, other Ca 2+ ‐dependent Ano isoforms (23), enforced by Orai1 and TRPC Ca 2+ entry channels (43, 44), may account for the low PS exposure in the absence of Ano6 channels.…”

“…The swelling seems to be a consequence of multiple ion influxes, as we found that specific depletion of Cl 2 or Na + ions was without effect (unpublished results). On the other hand, it should be noted that also other Ca 2+ channels, in particular Orai1 and TRPC isoforms, can contribute to PS exposure in platelets (43,44). Scott syndrome has been described as a moderate bleeding disorder, with hemorrhagic episodes only after trauma or childbirth (2,3).…”

Selective ORAI1 Inhibition Ameliorates Autoimmune Central Nervous System Inflammation by Suppressing Effector but Not Regulatory T Cell Function

“…AMG1 (N-(5-(2-chloro-5-(trifluoromethyl)phenyl)-2-pyrazinyl)-2,6-difluorobenzamid) is a selective inhibitor of the CRAC channel synthesized by Amgen that is in the same chemical series as Synta 66 (32, 33) and is described in patents WO2006081391 A2 and US2006/002874. For in vivo administration, active EAE was induced in WT mice by immunization with MOG 35-55 peptide in CFA.…”

The unique contribution of ion channels to platelet and megakaryocyte function

“…Thus, Orai1 blockers are a good candidate for anti-thrombotic therapy. Indeed, a recent study demonstrates how Orai1 antagonists inhibit platelet responses and can be used to reduce brain infarction in a murine model of stroke [124]. A key question is the identity of the Ca 2+ influx pathway(s) that compensate for the absence of Orai1 to support relatively normal levels of procoagulant activity (due to phosphatidylserine exposure) when platelets are co-stimulated by thrombin and a GPVI stimulus [125].…”