Roles of Platelet STIM1 and Orai1 in Glycoprotein VI- and Thrombin-dependent Procoagulant Activity and Thrombus Formation

Karen Gilio,Roger Van Kruchten,Attila Braun,Alejandro Berna-Erro,Marion A.H Feijge,David Stegner,Paola E.J Van Der Meijden,Marijke J.E Kuijpers,David Varga-Szabo,Johan W.M Heemskerk,Bernhard Nieswandt

doi:10.1074/jbc.m110.108696

Karen Gilio, Roger Van Kruchten + Show 9 more

Open Access

https://doi.org/10.1074/jbc.m110.108696

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Abstract

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca(2+) entry (SOCE) with Orai1 as principal Ca(2+) entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca(2+) entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1(-/-) and Orai1(-/-) platelets had greatly impaired glycoprotein (GP) VI-dependent Ca(2+) signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2(-/-) platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca(2+) signals of Stim1(-/-) and Orai1(-/-) platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1(-/-) and Orai1(-/-) platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca(2+) entry, inhibited Ca(2+) and procoagulant responses even in Stim1(-/-) and Orai1(-/-) platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca(2+) entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca(2+) entry and PS exposure, only one relying on STIM1-Orai1 interaction.

Highlights

STIM1 and Orai1 jointly contribute to glycoprotein VI (GPVI)-induced store-operated Ca2ϩ entry (SOCE), procoagulant activity, and thrombus formation; (ii) a compensating Ca2؉ entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca2؉
These results show that, in the presence of thrombin, GPVI-dependent PS exposure and procoagulant activity are only partly affected by deficiency in platelet STIM1 or Orai1
This study examines the contribution of recently discovered Ca2ϩ entry pathways to the regulation of platelet procoagulant activity and thrombus formation

Summary

EXPERIMENTAL PROCEDURES

Mouse Strains—Animal studies were approved by the local animal care and use committees. PPACK/heparin-anticoagulated blood of C57BL/6 mice transplanted with bone marrow from wild type, Stim1Ϫ/Ϫ or Orai1Ϫ/Ϫ animals was flowed over collagen at a shear rate of 1000 sϪ1. PPACK/heparin-anticoagulated blood of wild type or chimeric Stim1Ϫ/Ϫ or Orai1Ϫ/Ϫ mice was supplemented with 10% Fluo-4-loaded platelets of the same genotype. Blood samples were flowed over collagen at 1000 sϪ1, and fluorescence images from the collagen surface were recorded at 5 Hz. A, single-cell rises in Ca2ϩ of two representative platelets per genotype. Irradiated wild type animals with Stim1Ϫ/Ϫ, Orai1Ϫ/Ϫ, or wild type bone marrow cells Platelets from these mice, isolated at least 4 weeks after transplantation, were checked for the absence of STIM1 protein or Orai transcript (supplemental Fig. 1, A and B). Stim1Ϫ/Ϫ platelets showed reduced Ca2ϩ rises in the absence of CaCl2 and Ca2ϩ entry, supporting the concept that STIM1 controls the filling state of Ca2ϩ stores [24]

Blood from these chimeric mice was anticoagulated with

Findings

DISCUSSION