Figure 4. PPX and PPX_Δ12 interfere with polyP-induced coagulation.

(a,c) PPX and (b,d) PPX_Δ12 inhibit polyP-initiated thrombin formation. Real-time thrombin generation in the absence or presence of increasing concentrations of PPX or PPX_Δ12 in PPP stimulated with long-chain (LC; 1 μg ml⁻¹) polyP or short-chain (SC; 10 μg ml⁻¹) polyP. Representative thrombin generation curve of n=6 is shown. (e) FXIIa formation in human plasma was stimulated with buffer, LC (1 μg ml⁻¹), SC (10 μg ml⁻¹) or LC and SC polyP preincubated with PPX or PPX_Δ12 (100 μg ml⁻¹ each). FXIIa was measured by conversion of the chromogenic substrate D–Pro–Phe–Arg–p nitroanilide (S-2302) at λ=405 nm and t=60 min in the presence of inhibitors specified in the methods. Mean±s.e.m., n=6, ***P<0.001 by one-way analysis of variance (ANOVA). (f–h) Targeting polyP interferes with activated platelet-driven coagulation. (f) Real-time thrombin generation in collagen- (3.3 μg ml⁻¹) stimulated PRP in the absence or presence of PPX or PPX_Δ12 (500 μg ml⁻¹ each). (g) Recalcification clotting times in Trap6- (30 μM) or collagen- (33 μg ml⁻¹) stimulated human PRP dependent on addition of anti-FXIIa antibody (3F7; 375 nM), PPX, PPX_Δ12 or polyP pre-bound-PPX_Δ12 (500 μg ml⁻¹ each). Mean±s.e.m., n=4, ***P<0.001 versus buffer by one-way ANOVA. (h) Recalcification clotting times triggered by Ca²⁺ ionophore (A23187, 5 μM) or collagen (33 μg ml⁻¹) in PRP of WT or F12^−/− mice in the presence (+) or absence (−) of PPX or PPX_Δ12 (500 μg ml⁻¹ each). Clotting time reduction is given relative to untreated plasma. Mean±s.e.m., n=4. Insert: collagen failed to initiate zymogen FXII activation in PPP, while dextran sulfate (DXS; 100 μg ml⁻¹) activated all plasma FXII. (i) Role of polyP in FXI activation in the plasma. F12^−/− mouse plasma was incubated with α-thrombin (5 nM) in the absence or presence of polyP (LC or SC, 10 μg ml⁻¹ each) and FXII (30 μg ml⁻¹). Formed FXIa was measured by conversion of the chromogenic substrate S-2366 at λ=405 nm in the presence of inhibitors specified in the methods. Mean±s.e.m., n=4, ***P<0.001 versus buffer by one-way ANOVA.