Clot retraction assays

Whole murine blood was anti-coagulated with sodium citrate and PRP prepared as above. The platelet count was adjusted to 3 × 10⁸ /ml with HEPES-Tyrodes containing CaCl₂ (2 mM) and fibrinogen (2 mg/ml). 400 μl of this mix was placed into an aggregometer tube and incubated at 37°C for 5 min. 2 μl of mouse erythrocytes were added for colour contrast. Thrombin (10 U/ml) was added and mixed with a paper-clip and clot retraction was allowed to proceed at 37°C for 1 hour with the paper-clip present. At appropriate time points, the clot was pulled out with the paper-clip and the remaining serum volume measured. These experiments were performed blind.