A functional test for protein S activity in plasma.

The physiological role of coagulation cofactor Protein S (PrS) for activated Protein C (APC) has recently been appreciated by the description of patients with PrS-deficiency, suffering from thromboembolism. The present study introduces a one-stage clotting assay for the assessment of PrS functional activity in plasma samples. The assay procedure is based on a factor Xa-initiated clotting test utilizing a mixture of AL(OH)3-adsorbed substrate plasma and patient's plasma supplemented with purified prothrombin (0.15 microM) and APC (0.05 microM), with phospholipids and CaCl2. Owing to the varying concentration of PrS in the sample plasma, clotting times were prolonged up to 25 seconds in the presence of APC, whereas no prolongation occurred in its absence. The test procedure proved to be specific for PrS, since preincubation with monospecific antibodies against PrS abolished the prolongation of clotting time, while reconstitution of adsorbed plasma with purified PrS restored its cofactor activity completely. The functional assay showed an inter-assay and intra-assay variation in the normal range of 11.7% and 10.1%, respectively (n = 20). PrS activity in a group of unselected patients (n = 34), revealing no abnormalities in global coagulation tests, amounted to 95.8 +/- 16.5% (mean +/- S.D.) with a range from 67% to 136% when analyzed in comparison to a plasma pool constituted from healthy volunteers. Patients (n = 32) undergoing oral anticoagulant therapy presented 21.1 +/- 10.8% residual PrS-activity accompanied by a concomitant decrease in PrS-antigen levels to 69.9 +/- 21.2%. The assay described is sensitive, it can be performed on routine basis and allows the detection of patients with PrS-deficiency.