Platelet factor 4-containing immune complexes induce platelet activation followed by calpain-dependent platelet death

Abstract

Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy sometimes associated with thrombosis. The hallmark of HIT is antibodies to the heparin/platelet factor 4 (PF4) complex that cause thrombocytopenia and thrombosis through platelet activation. Despite the clinical importance, the molecular mechanisms and late consequences of immune platelet activation are not fully understood. Here, we studied immediate and delayed effects of the complexes formed by human PF4 and HIT-like monoclonal mouse anti-human-PF4/heparin IgG antibodies (named KKO) on isolated human platelets in vitro. Direct platelet-activating effect of the KKO/PF4 complexes was corroborated by the overexpression of phosphatidylserine (PS) and P-selectin on the platelet surface. The immune platelet activation was accompanied by a decrease of the mitochondrial transmembrane potential (ΔΨm), concurrent with a significant gradual reduction of the ATP content in platelets, indicating disruption of energy metabolism. A combination of PS expression and mitochondrial depolarization induced by the PF4-containing immune complexes observed in a substantial fraction of platelets was considered as a sign of ongoing platelet death, as opposed to a subpopulation of activated live platelets with PS on the plasma membrane but normal ΔΨm. Both activated and dying platelets treated with KKO/PF4 formed procoagulant extracellular microvesicles bearing PS on their surface. Scanning and transmission electron microscopy revealed dramatic morphological changes of KKO/PF4-treated platelets, including their fragmentation, another indicator of cell death. Most of the effects of KKO/PF4 were prevented by an anti-FcγRII monoclonal antibody IV.3. The adverse functional and structural changes in platelets induced by the KKO/PF4 complexes were associated with strong time-dependent activation of calpain, but only trace cleavage of caspase 3. The results indicate that the pathogenic PF4-containing HIT-like immune complexes induce direct prothrombotic platelet activation via FcγRIIA receptors followed by non-apoptotic calpain-dependent death of platelets, which can be an important mechanism of thrombocytopenia during HIT development.

Heparin-induced thrombocytopenia (HIT): Review of incidence, diagnosis, and management

“…Although thrombocytopenia is the hallmark of HIT, its underlying mechanisms are not completely understood. 21,22 A positive feedback loop propagates the prothrombotic loop. The roles of additional cell types in the pathogenesis of HIT are currently being investigated, including B and T-cells.…”

Platelet Activation in Heparin-Induced Thrombocytopenia is Followed by Platelet Death via Complex Apoptotic and Non-Apoptotic Pathways

“…In addition to the previously published paper [27], the present manuscript contains new evidence for the existence of high calpain activity associated with the apoptotic death pathway of platelets treated with KKO/PF4. Therefore, we conclude that HIT-like immune complexes cause platelet death through complex apoptotic and non-apoptotic pathways.…”

Controlling human platelet activation with calcium-binding nanoparticles

“…This will be determined both by the number and size of the pores, as well as the negative surface charge of the platelet glycocalyx. Upon platelet activation, the pores of the OCS have been shown to widen upon stimulation with thrombin or Ca 2+ ionophore [ 20 , 21 ]. Additionally the number of pores connecting to the platelet surface has been shown to increase [ 22 ].…”