Antiplatelet Agents Inhibit the Generation of Platelet-Derived Microparticles.

Alice Giacomazzi,Pietro Minuz,Maurizio Degan,Stefano Calabria,Alessandra Meneguzzi

doi:10.3389/fphar.2016.00314

Abstract

Platelet microparticles (PMPs) contribute to thrombogenesis but the effects of antiplatelet drugs on PMPs generation is undefined. The present study investigated the cellular events regulating PMPs shedding, testing in vitro platelet agonists and inhibitors. Platelet-rich plasma from healthy subjects was stimulated with arachidonic acid (AA), U46619, collagen type-I (10 and 1.5 μg/mL), epinephrine, ADP or TRAP-6 and pre-incubated with acetylsalicylic acid (ASA, 100 and 10 μmol/L), SQ-29,548, apyrase, PSB-0739, or eptifibatide. PMPs were detected by flow-cytometry using CD61 and annexin-V as fluorescent markers. Platelet agonists induced annexin V-positive PMPs shedding. The strongest response was to high concentration collagen. ADP-triggered PMPs shedding was dose-independent. ASA reduced PMPs induced by AA- (645, 347–2946 vs. 3061, 446–4901 PMPs/μL; median ad range, n = 9, P < 0.001), collagen 10 μg/mL (5317, 2027–15935 vs. 10252, 4187–46316 PMPs/μL; n = 13, P < 0.001), collagen 1.5 μg/mL (1078, 528–2820 vs. 1465, 582–5948 PMPs/μL; n = 21, P < 0.001) and TRAP-6 (2008, 1621–2495 vs. 2840, 2404–3031 PMPs/μL; n = 3, P < 0.01) but did not affect the response to epinephrine or ADP. The ADP scavenger apyrase reduced PMPs induced by U46619 (1256, 395–2908 vs. 3045, 1119–5494 PMPs/μL, n = 6, P < 0.05), collagen 1.5 μg/mL (1006, 780–1309 vs. 2422, 1839–3494 PMPs/μL, n = 3, P < 0.01) and TRAP-6 (904, 761–1224 vs. 2840, 2404–3031 PMPs/μL, n = 3, P < 0.01). The TP receptor antagonist SQ-29,548 and the P2Y12 receptor antagonist PSB-0739 markedly inhibited PMPs induced by low doses of collagen. Except for high-dose collagen, eptifibatide abolished agonist-induced PMPs release. Both TXA2 generation and ADP secretion are required as amplifiers of PMP shedding. The crucial role of the fibrinogen receptor and the collagen receptor in PMPs generation, independently of platelet aggregation, was identified.

Highlights

Activated platelets vesiculate to produce platelet microparticles (PMPs), a heterogeneous population of small membrane-coated vesicles, ranging from 0.1 to 1.0 μm in diameter (Shai et al, 2012; Hsu et al, 2013)
Previous studies showed a reduction in PMPs generation in hyperlipidemic patients with type II diabetes after treatment with statins and eicosapentaenoic acid (Nomura et al, 2009) and in patients with acute coronary syndromes treated with aspirin and P2Y12 receptor antagonists (Behan et al, 2005; Bulut et al, 2011)
In our experiments PMPs were detected in the particulate fraction shed from in vitro activated platelets and identified by flow cytometry (FCM) as small-size scatter events staining positive for integrin β3 (CD61), or integrin αIIb (CD41)

Summary

Introduction

Activated platelets vesiculate to produce platelet microparticles (PMPs), a heterogeneous population of small membrane-coated vesicles, ranging from 0.1 to 1.0 μm in diameter (Shai et al, 2012; Hsu et al, 2013). PMPs represent approximately 70–90% of circulating microparticles in the blood of healthy individuals (Horstman, 1999; Diamant et al, 2004) and elevations of their levels have been detected in a number of disorders including cancer, atherosclerosis, sepsis, diabetes, acute coronary syndromes (Shantsila et al, 2010; Varon and Shai, 2015). Previous studies showed a reduction in PMPs generation in hyperlipidemic patients with type II diabetes after treatment with statins and eicosapentaenoic acid (Nomura et al, 2009) and in patients with acute coronary syndromes treated with aspirin and P2Y12 receptor antagonists (Behan et al, 2005; Bulut et al, 2011). Microvesiculation by apoptotic platelets results from a disruption of the balance between Bcl survival and Bak apoptotic signals (Mason et al, 2007; Zhang et al, 2007; Schoenwaelder et al, 2009), independently of platelet activation (Zhang et al, 2013)

Methods

Results

Conclusion