New functional test for the TFPI α cofactor activity of Protein S working in synergy with FV ‐Short

Essentials Protein S and FV‐Short are synergistic cofactors to Tissue Factor Pathway Inhibitor α (TFPI α ). An assay for the TFPI α synergistic cofactor activity of protein S with FV‐Short was developed. The assay was specific for the synergistic TFPI α ‐cofactor activity of free protein S. Protein S deficient individuals with known mutations were correctly distinguished from controls.Summary Background Protein S is an anticoagulant cofactor to both activated protein C and tissue factor pathway inhibitor ( TFPI α ). The TFPI α ‐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 α ‐cofactor activity between protein S and FV ‐Short to develop a functional test for plasma protein S. Patients/Methods TFPI α ‐mediated inhibition of FX a in the presence of FV ‐Short, protein S and negatively charged phospholipid vesicles was monitored in time by synthetic substrate S2765. TFPI α , FX a 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 C4 BP β +‐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 α ‐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 α ‐cofactor activity.