Abstract
Platelets protect the vascular system during damage or inflammation, but platelet activation can result in pathological thrombosis. Activated platelets release a variety of extracellular vesicles (EVs). EVs shed from the plasma membrane often expose phosphatidylserine (PS). These EVs are pro-thrombotic and increased in number in many cardiovascular and metabolic diseases. The mechanisms by which PS-exposing EVs are shed from activated platelets are not well characterised. Cholesterol-rich lipid rafts provide a platform for coordinating signalling through receptors and Ca2+ channels in platelets. We show that cholesterol depletion with methyl-β-cyclodextrin or sequestration with filipin prevented the Ca2+-triggered release of PS-exposing EVs. Although calpain activity was required for release of PS-exposing, calpain-dependent cleavage of talin was not affected by cholesterol depletion. P2Y12 and TPα, receptors for ADP and thromboxane A2, respectively, have been reported to be in platelet lipid rafts. However, the P2Y12 antagonist, AR-C69931MX, or the cyclooxygenase inhibitor, aspirin, had no effect on A23187-induced release of PS-exposing EVs. Together, these data show that lipid rafts are required for release of PS-exposing EVs from platelets.
Highlights
Platelets protect the vascular system during damage or inflammation, but inappropriate or excessive platelet activation results in pathological thrombosis[1]
Washed platelets were stimulated with the Ca2+ ionophore, A23187, to trigger release PS-exposing extracellular vesicle (EVs)
Platelet-derived PS-exposing EVs were defined as CD41+/annexinV+ events that were smaller than 1 μm (Fig. 1)
Summary
Platelets protect the vascular system during damage or inflammation, but inappropriate or excessive platelet activation results in pathological thrombosis[1]. EVs shed from the plasma membrane often expose phosphatidylserine (PS) on their outer leaflet. An increase in intracellular Ca2+ concentration ([Ca2+]i) during platelet activation can trigger loss of plasma membrane asymmetry and exposure of PS in the outer leaflet[6]. PS-exposing EVs increase the rate and extent of thrombin generation, promoting thrombosis[10]. They regulate wound healing, inflammation and vascular integrity[11,12]. Cytoskeletal disruption by the Ca2+-dependent protease, calpain, is required[7,28,29] Beyond these events, little is known about how the process of shedding PS-exposing EVs from platelets is regulated
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