Platelets are activated by IAV H1N1 through FcγRIIA signaling and thrombin activation. (A) Human platelets (100 × 106/mL) preincubated with blocking IV.3 in 2% HS, 2% heat-inactivated serum, or 2% HS depleted of selective complement proteins were incubated with HBSS (as control) or stimulated with IAV H1N1 for 30 minutes. The induction of PAC-1 binding and CD62P on platelets, expressed as mean percentage ± SEM, was determined by flow cytometry. (B-D) Human platelets (100 × 106/mL) in 2% HS were preincubated or not with IV.3 mAb and ± PPACK thrombin inhibitor followed by vehicle or IAV H1N1 treatments. The production of 12-HETE (B), formation of MPs (C), and induction of PAC-1 binding/CD62P (D) were next determined. (E) Human platelets (100 × 106/mL) in 2% IAV-naïve mouse serum were preincubated or not with IV.3 mAb and ± PPACK thrombin inhibitor followed by vehicle or IAV H1N1 treatments for 20 minutes. The induction of PAC-1 binding/CD62P was assessed by flow cytometry. N = 5; data are mean + SEM. *P < .05; **P < .001; ***P < .0001; NS, nonsignificant.
