BFP analysis of integrin-dependent transduction of mechanical force leading to PS exposure and in vitro analysis of the role of integrin outside-in signaling in PPA. (A) Integrin ligand FN-conjugated BFP was allowed to repeatedly touch and pull the membrane surface of a resting or thrombin-stimulated platelet treated with or without mP6 or a scrambled control peptide (mP6Scr). Annexin V binding frequency to the same platelet was then assessed using a second BFP conjugated with annexin V (supplemental Figure 1). Data are shown as median (center bars) ± interquartile range (boxes) and maximal/minimal range (range bars). Note that annexin V binding to thrombin-stimulated platelets was significantly enhanced by frequent pulling applied through FN-coated BFP, which was inhibited by mP6 but not by mP6Scr. (B) The recalcification-induced clotting time (mean ± standard error of the mean [SEM]) of citrated human platelet-depleted plasma reconstituted with washed wild-type (WT) or β₃^−/− mouse platelets (WT, n = 6; β₃^−/−−, n = 8) under stirring conditions. (C-D) The recalcification-induced clotting time (mean ± SEM) of human citrated platelet-rich plasma treated with 40 µM of mP6 or a control peptide was monitored under stirring conditions in a turbidometric platelet aggregometer (C) (n = 9) or detected using a cone-and-plate rheometer under shear rate of 6000 s⁻¹ (D) (n = 5). *P < .05, **P < .01, and ****P < .0001. N.S., not significant.