2.4. Thrombin generation assay

Thrombin generation was performed as previously described with slight variations.²³ Either 40 uL pooled normal citrated (50 ug mL⁻¹ CTI) or FV‐deficient plasma were mixed with different combinations of FV‐Short (before and after activation with 1.6 nmol L⁻¹ thrombin (0.5 units mL⁻¹) for 10 minutes and then inhibition with 1 unit/ml hirudin), anti‐bodies against protein S or TFPI (100 ug mL⁻¹ final concentration), TFPIα (40 uL) and 30 uL PL (10 umol L⁻¹; 10/20/70 of PS/PE/PC)/TF (1.4 pmol L⁻¹) mixture as indicated in each experiment. The concentrations given are final concentrations. Plasma was diluted 3.25 times in the assay and the contribution of intrinsic TFPIα (approximately 0.06‐0.08 nmol L⁻¹) was disregarded. After 1 minute incubation at 37°C, the reaction was initiated by addition of 20 uL fluorescent substrate (Z‐Gly‐Gly‐Arg‐AMC*HCl, 300 umol L⁻¹) containing Ca²⁺ (16.7 mmol L⁻¹). The reaction was monitored for 30 minutes in a Tecan Infinite 200 and the results analyzed with GraphPad Prism and expressed as ΔFU/min a response of 300 ΔFU/min corresponding to ≈135 nM thrombin.