Intracellular cyclophilin A is an important Ca2+ regulator in platelets and critically involved in arterial thrombus formation

Abstract

Platelet adhesion and aggregation play a critical role in primary hemostasis. Uncontrolled platelet activation leads to pathologic thrombus formation and organ failure. The decisive central step for different processes of platelet activation is the increase in cytosolic Ca 2؉ activity ([Ca 2؉ ] i ). Activation-dependent depletion of intracellular Ca 2؉ stores triggers Ca 2؉ entry from the extracellular space. Stromal interaction molecule 1 (STIM1) has been identified as a Ca 2؉ sensor that regulates store-operated Ca 2؉ entry through activation of the pore-forming subunit Orai1, the major store-operated Ca 2؉ entry channel in platelets. In the present study, we show for the first time that the chaperone protein cyclophilin A (CyPA) acts as a Ca 2؉ modulator in platelets. CyPA deficiency strongly blunted activation-induced Ca 2؉ mobilization from intracellular stores and Ca 2؉ influx from the extracellular compartment and thus impaired platelet activation substantially. Furthermore, the phosphorylation of the Ca 2؉ sensor STIM1 was abrogated upon CyPA deficiency, as shown by immunoprecipitation studies. In a mouse model of arterial thrombosis, CyPAdeficient mice were protected against arterial thrombosis, whereas bleeding time was not affected. The results of the present study identified CyPA as an important Ca 2؉ regulator in platelets, a critical mechanism for arterial thrombosis. IntroductionPlatelet adhesion and aggregation are essential for hemostasis at sites of vascular injury to avoid excessive blood loss. 1,2 In contrast, uncontrolled platelet activation can induce acute vessel occlusion, leading to myocardial infarction or stroke at areas of atherosclerotic plaque rupture. 3,4 Platelet activation and thrombus formation is a multistage process that involves different signaling pathways to trigger platelet shape change, integrin activation, and degranulation. 4,5 The signaling pathways converge in the activation of phospholipase C (PLC), leading to the formation of inositol 1,4,5-triphosphate (IP 3 ) and diacylglycerol. 6 IP 3 is then able to bind to its receptor at the endoplasmic reticulum (ER) and mediates Ca 2ϩ efflux from the intracellular stores into the cytoplasm. 6,7 Compromised PLC activation impairs Ca 2ϩ mobilization, which is followed by defective thrombus formation under flow conditions. 8,9 Stromal interaction molecule 1 (STIM1) is a type I singletransmembrane protein with an N-terminal EF hand domain (helix-loop-helix structural domain) that binds Ca 2ϩ in the lumen of the ER. STIM1 binding to Ca 2ϩ is interrupted upon store release and induces redistribution of STIM1 to the plasma membrane to open store-operated Ca 2ϩ (SOC) channels, thereby stimulating store-operated Ca 2ϩ entry (SOCE). 10,11 Orai1 was identified as the major SOCE channel in platelets known to be regulated by the Ca 2ϩ sensor STIM1. 12,13 Immunophilins are endogenous cytosolic peptidyl-prolyl isomerases (PPIs) that interconvert between the cis and trans positions of target proteins. 14,15 According to their sensitivity t...

Phosphorylation of STIM1 at ERK1/2 Target sites regulates the interaction with the microtubule plus-end binding protein EB1

“…Thus, our results provide a new potential mediator, STIM1, in those signaling pathways where MAPK and Ca 2+ entry are involved. This would be the case for platelet activation (Elvers et al, 2012) and myoblast differentiation (Lee et al, 2012), although the hypothesis has not been fully confirmed in those cell types due to the lack of phosphospecific antibodies against those sites. Because our work has focused on the molecular mechanism underlying the regulation of STIM1 activity by phosphorylation, the involvement of STIM1 phosphorylation in signaling pathways and physiological events mediated by ERK1/2 will require further study in the future.…”

“…In addition to HEK293 cells, STIM1 phosphorylation, detected by an anti-phospho-MAPK/CDK substrate antibody, is enhanced by thapsigargin in neonatal rat ventricular myocytes (Zhu-Mauldin et al, 2012). Moreover, it has been reported that STIM1 phosphorylation at Ser575 promotes C2C12 myoblasts differentiation to myocytes (Lee et al, 2012), and that Ser to Ala mutation of residues 575, 608, and 621 impairs platelet adhesion to fibrinogen (Elvers et al, 2012), further supporting a role for STIM1 phosphorylation in cell physiology.…”

RhoA/Rho-Kinase in the Cardiovascular System

“…CyPA plays an important role as a Ca 2+ regulator in platelets. 150 Moreover, extracellular CyPA activates platelets via basigin (CD147)-mediated phosphoinositide-3-kinase/Akt signaling, leading to enhanced adhesion and thrombus formation. 151,152 Moreover, thrombin suppresses eNOS in EC via Rho-kinase pathway.…”

Platelets, inflammation and anti-inflammatory effects of antiplatelet drugs in ACS and CAD

“…Furthermore, CypA-PPIase facilitates ubiquitination of CXCR7 translocating it to the platelet surface (31). CypA is also involved in influencing thrombosis and haemostasis functions of platelets as elaborated in CypA -/-mice (37,38). CypA functions as a critical calcium sensor and regulator for store release and refill as mediated by Orai1 (38).…”

“…CypA is also involved in influencing thrombosis and haemostasis functions of platelets as elaborated in CypA -/-mice (37,38). CypA functions as a critical calcium sensor and regulator for store release and refill as mediated by Orai1 (38). CypA-dependent increase in ROS production leads to inside-out signalling causing increased GPIIb/IIIa activation and enhanced binding to fibrinogen.…”