Functional responses and molecular mechanisms involved in platelet activation triggered by histones

CARESTIA, A; RIVADENEYRA, L; ROMANIUK, MA; FONDEVILA, C; SCHATTNER, M

Amsterdam

Congreso; XXIV Congress of International Society on Thrombosis and Haemostasis; 2013

Society on Thrombosis and Haemostasis

Background: Histones are highly alkaline proteins found in cell nuclei that can be released by dying or inflammatory cells. The recent observation that histones are major components of neutrophil extracellular DNA traps and promote platelet aggregation and platelet-dependent thrombin generation has highlighted these proteins as potent prothrombotic molecules. Aim: Since the mechanism(s) of platelet activation by histones is not completely elucidated, we explored the ability of individual human recombinant H1, H2A, H3 and H4 to induce platelet activation as well as the molecular mechanisms involved. Methods: Platelet adhesion and spreading were determined by confocal microscopy. Binding of fibrinogen, expression of P-selectin and formation of platelet-leukocytes aggregates were evaluated by flow cytometry. The release of von Willebrand factor (vWF) was analyzed by ELISA. Phosphorylation of platelet ERK, AKT, P38 and NFκB was studied by Western blot. Results: All histones (10-50 μg /ml) were substrates for platelet adhesion and spreading (C:1±1; H1: 1±2; H2A: 2±0.7; H3: 16±3; H4:43±6; number of cells per field, 10 μg/ml, n=3), and triggered fibrinogen binding (C: 12±6; H1: 74±24; H2A: 150±35; H3: 332±50; H4: 510±57, mean fluorescence intensity (MFI), n=5), von Willebrand factor release (C: 17±6; H1: 28.7±1; H2A: 118±33; H3: 144±35; H4: 180±22, ng/ml n=3), P-selectin exposure (C: 9±3; H1: 18±6; H2A: 26±10; H3: 76±24; H4: 123±46 MFI, n=5) and formation of platelet-leukocyte aggregates (C; 17±3; H1: 22±10; H2A: 37±13; H3: 135±69; H4: 215±81, MFI n=5). Histones synergize with thrombin in triggering fibrinogen binding and P-selectin expression. Western blot assays showed that histones induce the phosphorylation of platelet ERK, AKT, P38 and NFκB. Accordingly, platelet activation induced by histones was significantly impaired by pretreatment of platelets with two non-structurally related NFκB inhibitors (BAY 11-7082 and Ro 106-9920), ERK (U0126), PI3K (LY 294002) or P38 (SB 203580) blockers. Pre-incubation of platelets with aspirin or dexamethasone markedly decreased fibrinogen binding and adhesion mediated by histones without modification of P-selectin exposure. Functional platelet responses induced by H3 and H4, but not H1 and H2A, were partially mediated through interaction with Toll like receptors 2 and 4. Conclusion: Our data identify histones as important triggers of hemostatic and inflammatory-mediated platelet responses, which are partially inhibited by anti-inflammatory drugs.