Figure 3.

Interference with XPR1 increases platelet polyP and promotes activated platelet-driven coagulation via the polyP/FXII pathway. (A-B) Washed human platelets were preincubated with XVDL (A), PVDL (B), or XVDL (200 μg/ml) and increasing PVDL concentrations (0-600 μg/ml) (C) for 1, 4, 8, or 24 hours. PolyP was measured as monophosphate with a malachite green assay. PolyP content before treatment was set to 100%. Analysis of released soluble polyP in the supernatant (D-E) and insoluble platelet membrane–associated polyP in collagen-stimulated (10 μg/ml) platelets (F-G) after a 4-hour incubation with XVDL and PVDL, respectively. PolyP was measured as in panel C. (H-I) Real-time thrombin formation in collagen-stimulated human PRP that was preincubated for 4 hours with XVDL or PVDL (2-200 μg/ml). Results are representative of 5 experiments. Endogenous thrombin potential (ETP) (J) and maximum (peak) thrombin (K) triggered in collagen-stimulated human PRP preincubated for 4 hours with XVDL (200 μg/ml) in the presence of PPX (500 μg/ml), PPX_Δ12 (500 μg/ml), 3F7 (650 nM), or infestin-4 (500 μg/ml). Data are expressed as the mean ± standard error of the mean; n = 3; *P < .05; **P < .01; n.s. nonsignificant.