Figure 4.

Coagulation assays for the direct anticoagulant activity of murine plasma protein S. In all cases, plasma was treated with neutralizing antibody (TVM-1) against murine APC to exclude any contribution of protein S co-factor activity for APC. (A) Thrombin generation profile for mouse plasma. Following addition of calcium and phospholipids to mouse plasma, thrombin generation was monitored over time in a modified ETP assay as described in the Design and Methods section. Normal mouse plasma, solid line; mouse plasma with addition of partially purified recombinant murine protein S (0.1 μg), dashed line. (B) Thrombin generation in normal mouse plasma with and without addition of 2 mg/mL neutralizing rabbit anti-human protein S. (C) Thrombin generation in mixtures of normal mouse plasma and rat PSdP. See the legend to Figure 3 for mixtures. (D) Variation in ETP parameters with increasing dose of mouse normal plasma. In mixtures of pooled normal mouse plasma with rat PSdP, the area under the curve and peak thrombin generation in the ETP assay decreased in a linear manner with increasing doses of murine plasma containing protein S. The quantity of plasma used was 12 μL in the experiment represented in (B) and 15 μL in the experiments represented in the other panels.