Not dead yet

In this issue of Blood, Hua et al use a novel marker of platelet activity to demonstrate that the necrotic platelet, a highly procoagulant subpopulation of activated platelet, uniquely contributes to fibrin formation and platelet accumulation within the forming thrombus.¹ 

Necrotic platelets are a subpopulation of activated platelets that are formed in response to a strong in vitro stimulus.²  A jambalaya of descriptive names and acronyms has been ascribed to platelets with similar characteristics, among these procoagulant,³  balloon(ing),⁴  coated,⁵  SCiP (sustained collagen-induced platelets),⁶  and CoaT (collagen and thrombin) platelets.⁷  Distinguishing in vitro characteristics and functions of necrotic platelets includes high levels of platelet phosphatidylserine externalization facilitating the activity of the tenase and prothrombinase complex⁷ ; increased calpain activation and degradation of intracellular proteins, including the cytoplasmic domain of integrin α_IIbβ₃³; decreased adhesiveness⁸ ; and cellular rounding and microparticle formation.⁴ 

Although many features and functions have been described in vitro, the necrotic platelet’s role in hemostasis and thrombosis continues to be debated. Depending on the model, either hemostatic or thrombotic functions have been attributed. And in some settings, minimal to no contribution of the necrotic platelet has been suggested. Agonist-initiated platelet phosphatidylserine externalization, a key feature of the necrotic platelet, requires the channel protein TMEM16F, also known as anoctamin-6. TMEM16F deficiency results in a hemorrhagic diathesis, suggesting an important role for the procoagulant function of this subpopulation in hemostasis.⁹  Conversely, using a photochemically induced model of thrombosis, impaired necrotic platelet formation, occurring as a result of cyclophilin D deficiency, resulted in accelerated thrombotic occlusion.⁵  Finally, recent studies have even questioned the physiologic role of platelets in supporting local thrombin generation. Fibrin formation and prothrombinase activity are minimally associated with platelets in a laser-induced nonocclusive thrombus.¹⁰ 

In this study, Hua et al use a novel marker of necrotic platelet formation to investigate the localization and function of the necrotic platelet in vivo.¹  4-[N-(S-glutationylacetyl)amino] phenylarsonous acid (GSAO) is an arsenical with a high affinity for intracellular protein thiols. Labeled GSAO had been demonstrated to identify late apoptotic and necrotic-nucleated cells. Using this marker, the authors identify a necrotic-activated platelet subpopulation characterized by GSAO and surface P-selectin positivity. Validating the utility of GSAO as a marker of necrotic platelets, formation of this GSAO-positive subpopulation was regulated by the peptidylprolyl isomerase cyclophilin D, a mitochondrial protein that functions to regulate necrotic cell death in platelets.⁵ 

GSAO labeling of forming thrombi in murine arterioles demonstrated the spatial association of necrotic platelet formation with fibrin formation in an occlusive thrombus initiated by extramural ferric chloride application. That the necrotic platelet drove both fibrin formation and platelet accumulation in this model was clearly demonstrated by abrogation of GSAO labeling, fibrin formation, and platelet accumulation in mice with cyclophilin D null platelets. Platelet heterogeneity was observed both within the occlusive thrombus, as discrete foci of necrotic platelet formation, and between thrombi, depending on the mechanism of thrombus initiation. Distinct from the occlusive thrombus, few necrotic platelets, if any, were observed in a laser-induced nonocclusive thrombus injury model. This minimal role is consistent with the lack of association of fibrin formation with the platelet mass in this model.

As demonstrated here¹  and by others,⁴  necrotic platelet formation can be targeted independent of more traditional platelet activation pathways. This suggests that pathways regulating necrotic platelet formation might provide an alternative target for modulation of thrombotic and other platelet-dependent pathologies. Studies using the GSAO reagent described here in other models of platelet-facilitated thrombotic and inflammatory conditions can be expected to provide novel insights into the mechanism and function of necrotic platelets.