New functional test for the TFPIα cofactor activity of Protein S working in synergy with FV-Short

Background Protein S is an anticoagulant cofactor to both activated protein C and tissue factor pathway inhibitor (TFPI alpha). The TFPI alpha-cofactor activity of protein S is stimulated by a short isoform of factor V (FV-Short), the two proteins functioning in synergy. Objective Using the synergistic TFPI alpha-cofactor activity between protein S and FV-Short to develop a functional test for plasma protein S. Patients/Methods TFPI alpha-mediated inhibition of FXa in the presence of FV-Short, protein S and negatively charged phospholipid vesicles was monitored in time by synthetic substrate S2765. TFPI alpha, FXa and FV-Short were purified proteins, whereas diluted plasma from protein S deficient patients or controls were used as source for protein S. Results The assay was specific for free protein S demonstrating good correlation to free protein S plasma levels (r = 0.92) with a Y-axis intercept of -5%. Correlation to concentrations of total protein S (free and C4BP beta+-bound) was lower (r = 0.88) and the Y-axis intercept was +46%, which is consistent with the specificity for free protein S. The test distinguished protein S-deficient individuals from 6 families with known ProS1 mutations from family members having no mutation. Protein S levels of warfarin-treated protein S deficient cases were lower than protein S in cases treated with warfarin for other causes. Conclusions We describe a new assay measuring the TFPI alpha-cofactor activity of plasma protein S. The test identifies type I/III protein S deficiencies and will be a useful tool to detect type II protein S deficiency having defective TFPI alpha-cofactor activity.