Effect of RO318220 on thrombin generation in the presence of limiting (2 nM) or excess (20 nM) exogenous factor Va. Thrombin generation assays were performed with aspirin-treated, washed, and gel-filtered platelets treated with vehicle or inhibitor and activated with (A) α thrombin (0.2 U/mL; closed symbols) or (B) SFLLRN (50 μM; open symbols). Thrombin was generated in the presence of limiting (2 nM; A-B) or excess (20 nM) factor Va (C), then was assayed by S-2238 hydrolysis as described in “Materials and methods.” Squares indicate DMSO controls; crossed squares (B), matched controls for 50 μM RO318220; circles, 20 μM RO318220; and triangles, 50 μM RO318220. The insets to panel C show factor Va titrations with 1 nM factor Xa using (i) RO318220-treated (20 μM) platelets or (ii) control platelets with the abscissa adjusted to reflect added factor Va plus 1.6 nM released platelet factor Va. Line arrow in inset i indicates the thrombin formed using control platelets with no added factor Va. Means and standard errors are derived from 3 (RO318220 20 μM) or 6 (RO318220 50 μM) experiments performed in duplicate.