Human neutrophil alpha-defensins induce formation of fibrinogen and thrombospondin-1 amyloid-like structures and activate platelets via glycoprotein IIb/IIIa.

BACKGROUND: Human neutrophil α-defensins (HNPs) are important constituents of the innate immune system. Beyond their antimicrobial properties, HNPs also have pro-inflammatory features. While HNPs in plasma from healthy individuals are barely detectable, their level is strongly elevated in septic plasma and plasma from patients with acute coronary syndromes. OBJECTIVES: As thrombosis and inflammation are intertwined processes and activation of human polymorphonuclear leukocytes (PMNL) and subsequent degranulation is associated with full activation of surrounding platelets, we studied the effect of HNPs on platelet function. METHODS: The effect of HNPs on platelet activation parameters and apoptosis was investigated via aggregometry, flow cytometry, confocal microscopy and the ELISA technique. RESULTS: It was found that HNPs activate platelets in pathophysiologically relevant doses, inducing fibrinogen and thrombospondin-1 binding, aggregation, granule secretion, sCD40L shedding, and procoagulant activity. HNPs bound directly to the platelet membrane, induced membrane pore formation, microparticle formation, mitochondrial membrane depolarization and caspase-3-activity. Confocal microscopy revealed the HNP-induced formation of polymeric fibrinogen and thrombospondin-1 amyloid-like structures, which bound microorganisms. Platelets adhered to these structures and formed aggregates. Blocking of glycoprotein IIb/IIIa (GPIIb/IIIa) markedly inhibited HNP-induced platelet activation. In addition, heparin, heparinoid, serpins and α(2)-macroglobulin, which all bind to HNPs, blocked HNP-1-induced platelet activation in contrast to direct thrombin inhibitors such as hirudin. CONCLUSIONS: HNPs activate platelets and induce platelet apoptosis by formation of amyloid-like proteins. As these structures entrapped bacteria and fungi, they might reflect an additional function of HNPs in host defense. The described mechanism links again thrombosis and infection.

Extracellular protein disulfide isomerase regulates feedback activation of platelet thrombin generation via modulation of coagulation factor binding.

BACKGROUND: Protein disulfide isomerase (PDI) controls platelet integrin function, tissue-factor (TF) activation, and concentrates at fibrin and thrombus formation sites of vascular injury. OBJECTIVE: To investigate the involvement of surface thiol isomerases and especially PDI, in thrombin-mediated thrombin amplification on human platelets. METHODS/RESULTS: Using a newly developed thrombin-dependent platelet thrombin generation assay, we observed that the feedback activation of thrombin generation on the platelet surface does not depend on TF, as anti-TF antibodies inhibiting TF-induced thrombin formation in platelet-depleted plasma had no effect compared with vehicle-treated controls. Feedback activation of thrombin generation in the presence of platelets was significantly diminished by membrane impermeant thiol blockers or by the thiol isomerase-inhibitors bacitracin and anti-PDI antibody RL90, respectively. Platelet thrombin formation depends on binding of coagulation factors to the platelet surface. Therefore, involvement of thiol isomerases in this binding was investigated. As shown by confocal microscopy and flow cytometry, thrombin-stimulated platelets exhibited increased surface-associated PDI as well as extracellular disulfide reductase activity compared with unstimulated platelets. Flow cytometric analysis revealed that membrane impermeant thiol blockers or PDI inhibitors, which had been added after platelet stimulation and after phosphatidylserine exposure to exclude their influence on primary platelet activation, significantly inhibited binding of all coagulation factors to thrombin-stimulated platelets. CONCLUSIONS: Thus, surface-associated PDI is an important regulator of coagulation factor ligation to thrombin-stimulated platelets and of subsequent feedback activation of platelet thrombin generation. Cell surface thiol isomerases might be therefore powerful targets to control hemostasis and thrombosis.