Jinmi Zou,Pengyu Zhang,Fiorella A Solari,Claudia Schönichen,Isabella Provenzale,Nadine J A Mattheij,Marijke J E Kuijpers,Julia S Rauch,Frauke Swieringa,Albert Sickmann,Barbara Zieger,Kerstin Jurk,Johan W M Heemskerk
{"title":"Suppressed ORAI1-STIM1-dependent Ca2+ entry by protein kinase C isoforms regulating platelet procoagulant activity.","authors":"Jinmi Zou,Pengyu Zhang,Fiorella A Solari,Claudia Schönichen,Isabella Provenzale,Nadine J A Mattheij,Marijke J E Kuijpers,Julia S Rauch,Frauke Swieringa,Albert Sickmann,Barbara Zieger,Kerstin Jurk,Johan W M Heemskerk","doi":"10.1016/j.jbc.2024.107899","DOIUrl":null,"url":null,"abstract":"Agonist-induced rises in cytosolic Ca2+ control most platelet responses in thrombosis and hemostasis. In human platelets we earlier demonstrated that the ORAI1-STIM1 pathway is a major component of extracellular Ca2+ entry, in particular when induced via the ITAM-linked collagen receptor, glycoprotein VI (GPVI). In the present paper, using functionally defective platelets from patients with a loss-of-function mutation in ORAI1 or STIM1, we show that Ca2+ entry induced by the endoplasmic reticulum ATPase inhibitor, thapsigargin, fully relies on this pathway. We demonstrate that both the GPVI-induced and thapsigargin-induced Ca2+ entry is strongly suppressed by protein kinase C (PKC) activation, while leaving intracellular Ca2+ mobilization unchanged. Comparing effects of a PKC inhibitory panel pointed to redundant roles of beta and theta PKC isoforms in Ca2+-entry suppression. In contrast, tyrosine kinases positively regulated GPVI-induced Ca2+ entry and mobilization. Label-free and stable isotope phosphoproteome analysis of GPVI-stimulated platelets suggested a regulatory role of bridging integrator-2 (BIN2), known as important mediator of the ORAI1-STIM1 pathway in mouse platelets. Identified were 25-45 regulated phospho- sites in BIN2 and 16-18 in STIM1. Five of these were characterized as direct substrates of the expressed PKC isoforms alpha, beta delta and theta. Functional platelet testing indicated that the downregulation of Ca2+ entry by PKC resulted in suppressed phosphatidylserine exposure and plasmatic thrombin generation. Conclusively, our results indicate that in platelets multiple PKC isoforms constrain the store-regulated Ca2+ entry via ORAI1-BIN2-STIM1, and hence downregulate platelet-dependent coagulation.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":null,"pages":null},"PeriodicalIF":4.0000,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Biological Chemistry","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1016/j.jbc.2024.107899","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Agonist-induced rises in cytosolic Ca2+ control most platelet responses in thrombosis and hemostasis. In human platelets we earlier demonstrated that the ORAI1-STIM1 pathway is a major component of extracellular Ca2+ entry, in particular when induced via the ITAM-linked collagen receptor, glycoprotein VI (GPVI). In the present paper, using functionally defective platelets from patients with a loss-of-function mutation in ORAI1 or STIM1, we show that Ca2+ entry induced by the endoplasmic reticulum ATPase inhibitor, thapsigargin, fully relies on this pathway. We demonstrate that both the GPVI-induced and thapsigargin-induced Ca2+ entry is strongly suppressed by protein kinase C (PKC) activation, while leaving intracellular Ca2+ mobilization unchanged. Comparing effects of a PKC inhibitory panel pointed to redundant roles of beta and theta PKC isoforms in Ca2+-entry suppression. In contrast, tyrosine kinases positively regulated GPVI-induced Ca2+ entry and mobilization. Label-free and stable isotope phosphoproteome analysis of GPVI-stimulated platelets suggested a regulatory role of bridging integrator-2 (BIN2), known as important mediator of the ORAI1-STIM1 pathway in mouse platelets. Identified were 25-45 regulated phospho- sites in BIN2 and 16-18 in STIM1. Five of these were characterized as direct substrates of the expressed PKC isoforms alpha, beta delta and theta. Functional platelet testing indicated that the downregulation of Ca2+ entry by PKC resulted in suppressed phosphatidylserine exposure and plasmatic thrombin generation. Conclusively, our results indicate that in platelets multiple PKC isoforms constrain the store-regulated Ca2+ entry via ORAI1-BIN2-STIM1, and hence downregulate platelet-dependent coagulation.
期刊介绍:
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