Abstract
Activated, procoagulant platelets shed phosphatidylserine (PS)-exposing extracellular vesicles (EVs) from their surface in a Ca2+- and calpain-dependent manner. These PS-exposing EVs are prothrombotic and proinflammatory and are found at elevated levels in many cardiovascular and metabolic diseases. How PS-exposing EVs are shed is not fully understood. A clearer understanding of this process may aid the development of drugs to selectively block their release. In this study we report that 2-aminoethoxydiphenylborate (2-APB) significantly inhibits the release of PS-exposing EVs from platelets stimulated with the Ca2+ ionophore, A23187, or the pore-forming toxin, streptolysin-O. Two analogues of 2-APB, diphenylboronic anhydride (DPBA) and 3-(diphenylphosphino)-1-propylamine (DP3A), inhibited PS-exposing EV release with similar potency. Although 2-APB and DPBA weakly inhibited platelet PS exposure and calpain activity, this was not seen with DP3A despite inhibiting PS-exposing EV release. These data suggest that there is a further target of 2-APB, independent of cytosolic Ca2+ signalling, PS exposure and calpain activity, that is required for PS-exposing EV release. DP3A is likely to inhibit the same target, without these other effects. Identifying the target of 2-APB, DPBA and DP3A may provide a new way to inhibit PS-exposing EV release from activated platelets and inhibit their contribution to thrombosis and inflammation.
Highlights
Platelets are the main cellular component of haemostasis
In this study, we investigated the mechanism of how 2-APB inhibits the release of annexin V (AnV)+ extracellular vesicles (EVs) from platelets
AnV+ EV release from permeabilised platelets was inhibited by 2-APB, diphenylboronic anhydride (DPBA) and DP3A, but not the other analogues (Fig. 5c, d)
Summary
Platelets are the main cellular component of haemostasis. They adhere, activate and aggregate at sites of vascular injury, forming a haemostatic plug that prevents blood loss. Procoagulant platelets release PS-exposing extracellular vesicles (EVs). Little is known of the mechanism beyond these events It is not clear whether any events other than PS exposure and calpain activity are required downstream of increased [Ca2+]cyt. In this study, we investigated the mechanism of how 2-APB inhibits the release of AnV+ EVs from platelets. We find that the effect of 2-APB does not depend on inhibition of Ca2+ signalling, PS exposure or calpain activity. These results indicate that events in addition to PS and calpain activation are required for release of PS-exposing EVs and suggest the existence of other potential targets to inhibit this important pathological process
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