Mitochondrially-Mediated Platelet Integrin AlphaIIbbeta3 Inactivation Limits Platelet Recruitment and Thrombus Growth,
Authors: Liu Fang , Gamez Graciela , Myers David R , Lam Wilbur , Jobe Shawn M 
Published: October 2019

Abstract
Abstract 3251
Platelet stimulation with strong agonist(s) results in the formation of a phenotypically unique platelet subpopulation known as procoagulant platelets. All platelets stimulated in strongly-activating conditions undergo granule release, activation of integrin αIIbβ3, and spreading. However, minutes later a subpopulation of these activated platelets undergo additional phenotypic changes including phosphatidylserine (PS) exposure, adoption of a vesiculated and balloon-like morphology, and an altered integrin αIIbβ3 conformation that has a decreased binding affinity for activation-dependent antibodies such as PAC-1 or JON/A. Deletion of the mitochondrial protein cyclophilin D (CypD), a critical regulator of mitochondrial permeability transition pore (mPTP) formation, results in marked abrogation of procoagulant platelet formation. We have previously demonstrated remarkable impairment of dual-agonist-initiated PS exposure and procoagulant activity in CypD−/− platelets (Jobe et al, Blood 2008). In this study the functional importance of the morphological and adhesive changes that occur in procoagulant platelets were examined using CypD−/− platelets and mice with platelet-specific deficiency of CypD. (CypDplt−/−).
The morphology and phenotype of fibrinogen-adherent CypD+/+ (WT) and CypD−/− platelets were examined following dual stimulation with thrombin and the GPVI agonist convulxin. In dual-stimulated WT platelets, platelets initially spread; then after three minutes, nearly half of the platelets demonstrated lamellopodial and filipodial retraction with platelet rounding and vesiculation, and these changes were associated with increased PS exposure and decreased binding of JON/A. These changes were delayed in CypD−/− platelets, but eventually started to be seen 30 minutes subsequent to stimulation. Adhesion and platelet recruitment to strongly-stimulated platelets were studied in flow conditions. Increased platelet accumulation was noted when unstimulated platelets were flowed over strongly-stimulated adherent CypD−/− platelets relative to WT platelets. Similarly, increased platelet accumulation of CypD−/− platelets was noted on a collagen surface compared to WT platelets. In platelet aggregation assays, dual stimulated WT platelets demonstrated a chaotic pattern of aggregation two to three minutes after activation consistent with aggregate disruption, a pattern that was not observed in CypD−/− platelets. Together these studies indicate that procoagulant platelet formation results in integrin inactivation. Since this process is impaired in the absence of CypD, CypD−/− platelets demonstrate increased platelet accumulation in flow assays and stable aggregate formation. Adhesive and cytoskeletal proteins were investigated in strongly-stimulated platelets. Western blot analysis demonstrated significant proteolytic cleavage of both talin and the cytoplasmic domain of integrin β3 in WT platelets stimulated with thrombin plus convulxin, and these events were only minimally observed in single-agonist stimulated platelets. Proteolytic cleavage of both talin and integrin β3 were markedly decreased in CypD−/− platelets and in WT platelets treated with calpain inhibitors.
CypD is a ubiquitously expressed protein; therefore, in vivo thrombosis and hemostasis were tested in CypDplt−/− mice. Following photochemically mediated mesenteric endothelial injury, time to occlusion was shortened in CypDplt−/− mice (723 ± 111 sec in CypDplt+/− vs. 371 ± 106 sec in CypDplt−/− mice), and complete arterial occlusion was increased in CypDplt−/− mice (55% in CypDplt+/− vs. 86% in CypDplt−/−) (p=0.02). Our in vitro and in in vivo results are consistent with the hypothesis that in strongly-stimulated platelets mitochondrially-mediated events initiate proteolytic cleavage of talin and integrin β3 accompanied by platelet integrin αIIbβ3 inactivation, and that this process limits platelet recruitment and thrombus growth. Since these events are limited to platelets stimulated by strong agonist(s), these results suggest a novel negative feedback mechanism initiated by accumulation of multiple or strong agonist(s) that limits thrombotic occlusion in vivo.

Disclosures:
No relevant conflicts of interest to declare.