Inner Mitochondrial Membrane Disruption Links Apoptotic and Agonist-Initiated Phosphatidylserine Externalization in Platelets

Abstract

Objectives Phosphatidylserine (PSer) exposure mediates platelet procoagulant function and regulates platelet lifespan. Apoptotic, necrotic, and integrin-mediated mechanisms have been implicated as intracellular determinants of platelet PSer exposure. Here we investigate 1) the role of mitochondrial events in platelet PSer exposure initiated by these distinct stimuli and 2) the cellular interactions of the procoagulant platelet in vitro and in vivo. Approach and results Key mitochondrial events were examined, including cytochrome c release and inner mitochondrial membrane (IMM) disruption. In both ABT-737 (apoptotic) and agonist (necrotic)-treated platelets PSer externalization was temporally correlated with IMM disruption. Agonist stimulation resulted in rapid cyclophilin D - dependent IMM disruption that coincided with PSer exposure. ABT-737 treatment caused rapid cytochrome c release, eventually followed by caspase-dependent IMM disruption that again closely coincided with PSer exposure. A non-mitochondrial and integrin-mediated mechanism has been implicated in the formation of a novel PSer-externalizing platelet subpopulation. Using image cytometry, this subpopulation is demonstrated to be the result of the interaction of an aggregatory platelet and a procoagulant platelet rather than indicative of a novel intracellular mechanism regulating platelet PSer externalization. Using electron microscopy, similar interactions between aggregatory and procoagulant platelets are demonstrated in vitro and in vivo within a mesenteric vein hemostatic thrombus. Conclusions Platelet PSer externalization is closely associated with the mitochondrial event of IMM disruption identifying a common pathway in PSer externalizing platelets. The limited interaction of procoagulant platelets and integrin-active aggregatory platelets identifies a potential mechanism for procoagulant platelet retention within the hemostatic thrombus.

Procoagulant Phosphatidylserine-Exposing Platelets in vitro and in vivo

“…Heterogeneity within the PS-exposing platelet subpopulation has also been reported by Topalov et al (80): one subset with high Ca 2+ cyt , m loss, and inactive αIIbβ3; and another with low Ca 2+ cyt , intact m , and active αIIbβ3. Subsequently, this latter subset was described to be the result of the interaction between a procoagulant platelet and an aggregatory (non-PS-exposing) platelet (81).…”

“…It is also caspase dependent, as responses are abolished in the presence of a caspase inhibitor (52,78). However, it does not require increases in Ca 2+ cyt or calpain activity, which are necessary for agonist-induced PS exposure (52) (Agonist-Induced Phosphatidylserine Exposure) There is early mitochondrial outer membrane permeabilization (MOMP), followed by later IMM disruption concomitant with PS exposure (81): MPTP formation may not be involved, and m depolarization may occur (52,(103)(104)(105). PS-exposing platelets take on a rounded morphology but maintain cytoplasmic components (105,106); EV formation is not observed early on, but increases with time (103,104,107).…”

Comparison of putative BH3 mimetics AT-101, HA14-1, sabutoclax and TW-37 with ABT-737 in platelets

“…To investigate the effect of these putative BH3 mimetics on mitochondrial membrane potential, platelets were loaded with TMRM. This reflects late damage to the mitochondria rather than early release of cytochrome c [4,18]. The timecourse of loss of TMRM fluorescence matched that AnV binding.…”

Procoagulant Platelets

“…As noted, mitochondrial depolarization is essential for procoagulant platelet synthesis, although the details of how this occurs are poorly understood. Choo et al 15 have cleverly examined several mitochondrial changes and observed that disruption of the inner mitochondrial membrane, with either proapoptotic agents or dual agonist stimulation, is critical to the process of PS exposure. However, disruption of the outer mitochondrial membrane and release of cytochrome C, as observed in apoptosis, does not occur in procoagulant platelet formation.…”

“…The second observation by Choo et al 15 concerned a report that challenged the pentad of characteristics for procoagulant platelets. Topalov et al 16 observed that high levels of thrombin stimulation produced PS-positive platelets that retained polarized mitochondria and functional GP (glycoprotein) IIb/ IIIa receptors, labeling their new class of platelets as integrinregulated procoagulant platelets.…”