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2-Aminoethoxydiphenylborate (2-APB) inhibits release of phosphatidylserine-exposing extracellular vesicles from platelets.

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.

SUBMITTER: Wei H

PROVIDER: S-EPMC7051957 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundUpon platelet activation, a subpopulation of procoagulant platelets is formed, characterized by the exposure of the anionic aminophospholipid phosphatidylserine (PS) on the surface membrane.ObjectiveTo evaluate procoagulant PS-exposing platelets by imaging flow cytometry.MethodsPlatelet ultrastructure was examined by transmission electron microscopy, and a comprehensive analysis of procoagulant platelets was performed using imaging flow cytometry; platelets were fluorescently labeled for the markers glycoprotein (GP)IX, activated integrin αIIbβ3, CD62P, and PS exposure.ResultsA subpopulation of platelets stimulated in suspension by the physiological agonists thrombin+collagen, and all platelets stimulated by the calcium ionophore A23187, had a distinct round morphology. These platelets were PS-exposing, larger in size, had an increased circularity index, and had reduced internal complexity compared with non-PS-exposing platelets. They expressed CD62P and αIIbβ3 in an inactive conformation on the surface, and demonstrated depolarized inner mitochondrial membranes. For the first time, using imaging flow cytometry, a large proportion of PS-exposing platelets possessing platelet-associated extracellular vesicles (EVs) was observed, which demonstrated heterogeneous platelet marker expression that was different from free released EVs.ConclusionsInnovative imaging flow cytometry allowed detailed fluorescence-based, quantitative morphometric analysis of PS-exposing platelets; in becoming procoagulant, platelets undergo remarkable morphological changes, transforming into spherical "balloons," almost devoid of their normal internal architecture. Almost all PS-exposing platelets have associated EVs that are not detectable by traditional flow cytometry. While their functions have yet to be fully elucidated, the heterogeneity of platelet-associated and released EVs suggests that they may contribute to different aspects of hemostasis and of thrombosis.

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2-Aminoethoxydiphenylborate (2-APB) inhibits release of phosphatidylserine-exposing extracellular vesicles from platelets.

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