Each experiment was performed at least in triplicate. Flow cytometry of platelets and platelet-derived microvesicles In flow cytometry experiments, platelets and platelet-derived microvesicles were identified by labeling them with PE-conjugated monoclonal antibodies to CD41 (platelet integrins subunit IIb). by a decrease of the mitochondrial transmembrane potential (m), 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 m. 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, GSK690693 another indicator of cell death. Most of the effects of KKO/PF4 were prevented by an anti-FcRII 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 FcRIIA receptors followed by non-apoptotic calpain-dependent death of platelets, which can be an important mechanism of thrombocytopenia during HIT development. is the number of experiments with platelets from independent donors. *is the number of experiments with platelets isolated from three independent donors for each experimental condition. *for 10?min to obtain platelet-rich plasma (PRP). Platelets from PRP were isolated by gel filtration at room temperature on Sepharose 2B equilibrated with Tyrodes buffer (4?mM HEPES, 135?mM NaCl, 2.7?mM KCl, 2.4?mM MgCl2, 5.6?mM D-glucose, 3.3?mM NaH2PO4, 0.35?mg/ml bovine serum albumin, and pH 7.4). Platelets were counted in a hemocytometer and used within 3?h after blood collection. Cell viability was about 93C98% based on maintenance of the m as determined by flow cytometry using a m-sensitive fluorescent dye MitoTracker DeepRed FM. Incubation of platelets with various activators A total of 200,000 isolated platelets in 150?l Tyrodes buffer were incubated at 37?C for various periods of time with PF4 (final concentration 10?g/ml), HIT-like pathogenic mouse monoclonal antibodies (KKO) (final concentration 50?g/ml), HIT-like non-pathogenic mouse monoclonal antibodies (RTO) (final concentration 50?g/ml), and KKO/PF4 or RTO/PF4 immune complexes preformed by mixing 50? g/ml KKO or RTO and 10?g/ml PF4 (final concentrations). The applied concentrations of PF4 and pathogenic antibodies have been earlier shown to affect platelet functionality49C51. Untreated platelets were used as a negative control and platelets incubated with 10?M calcium ionophore A23187 were used GSK690693 as a positive control. 4C18 independently GSK690693 isolated platelet preparations were studied under each experimental condition. Each experiment was performed at least in triplicate. Flow cytometry of platelets and platelet-derived microvesicles In flow cytometry experiments, platelets and platelet-derived microvesicles were identified by labeling them with PE-conjugated monoclonal antibodies to CD41 (platelet integrins subunit IIb). After incubation under various experimental conditions, platelets (150?l) were mixed with 300?l of a Ca2+-containing buffer (10?mM HEPES, 140?mM NaCl, 2.5?mM CaCl2, and pH 7.4) to ensure the binding of EPLG6 Annexin V-FITC that was used as a marker of phosphatidylserine expressed on the surface of platelets and microvesicles. To measure the m platelets were labeled with a m-sensitive fluorescent dye MitoTracker DeepRed FM. To assess platelet activation, the surface expression of P-selectin was measured using PE-conjugated anti-human CD62P antibodies. To evaluate the activity of caspases 3 and 7, platelets were incubated with CellEventTM Caspase-3/7 Green Detection Reagent GSK690693 (500?nM final concentration) for 30?min following incubation and 1?M SYTOX? AADvanced? dead cell stain for 5?min before the end of the incubation (total time of incubation 90?min) under various experimental conditions and analyzed using flow cytometry. Platelets were gated by their FSC/SSC characteristics after size-based calibration with 1?m, 2?m, and 4?m polystyrene beads and by their binding of anti-CD41-PE-labeled antibodies (Figs. S1A, S2). Platelet-derived microvesicles were identified and quantified as the events that reflected a platelet-specific marker CD41 (platelet integrins subunit IIb) and were characterized by forward light scatter (FSC) smaller than 1?m (Fig. S2). Unlabeled platelets and microvesicles.