SummaryProtein S, an important cofactor of activated protein C, and C4b-binding protein were purified from human plasma. Specific antibodies against the purified proteins were raised in rabbits and used for the development of immunologic assays for these proteins in plasma: an immunoradiometric assay for protein S (which measures both free protein S and protein S complexed with C4b-binding protein) and an electroimmunoassay for C4b- binding protein. Ranges for the concentrations of these proteins were established in healthy volunteers and patients using oral anticoagulant therapy. A slight decrease in protein S antigen was observed in patients with liver disease (0.78 ± 0.25 U/ml); no significant decrease in protein S was observed in patients with DIC (0.95 ± 0.25 U/ml).Criteria were developed for the laboratory diagnosis of an isolated protein S deficiency

SummaryProtein S, a vitamin K-dependent coagulation factor, is involved in the regulation of the anticoagulant activity of activated protein C.Using an immunoradiometric assay for total protein S in plasma we identified 14 patients (7 male and 7 female) in three unrelated Dutch families as fulfilling the criteria for an isolated protein S deficiency. In 9 patients who were not receiving oral anticoagulant treatment the mean total protein S antigen concentration was 0.50 ± 0.08 U/ml (± S.D.) and the calculated free protein S concentration was 0.15 ± 0.01 U/ml (± S.D.). In the five patients who were on oral anticoagulant treatment the mean total protein S antigen was 0.23 ± 0.05 U/ml (± S.D.).Seven of the 14 patients had a history of venous thromboembolism occurring at a mean age of 25 years and often without an apparent cause. Protein S deficiency is inherited as an autosomal dominant trait.

SummaryWe studied activation of human coagulation factors IX and X by factor VIIa in the presence of calcium ions, phospholipid (phosphatidylserine/phosphatidylcholine, 50/50, mol/mol) and purified tissue factor apoprotein. Activation of factor IX and factor X was found to occur without a measurable lag-phase and hence initial rates of factor IXa and factor Xa formation could be determined. Like previously observed for the activation of factor X, the activation of factor IX was saturable with respect to factor VIIa, tissue factor apoprotein and phospholipid. The results suggested that in the presence of a Ca2+ ions the same ternary complex of factor VIIa-tissue factor apoprolein-phospholipid is responsible for the activation of factor IX and factor X. Roth the apparent Km of 22 nM-factor IX and the apparent Kcat of 28 min−1 were about 3-fold lower than the coiicsponding parameters of factor X activation by this complex. Hence, the catalytic efficiency (Kcat/Km) of factor IX and factor X activation was about equal. However, the two substrates inhibited the activation of each other by competition for the same catalytic sites. The apparent Kinh of factor IX for inhibition of extrinsic factor X activation is 30 nM. The apparent Kinh of factor X for inhibition of extrinsic factor IX activation is 116 nM. From these kinetic data it was calculated that at plasma concentration of factors IX and X, the rate of extrinsic factor IX activation would be half the rate of factor X activation. These relative rates of extrinsic factor IX and factor X activation in combination with previously reported kinetic data on the activation of factor X by factor IXa in the presence of factor VIIIa provide support for the concept that at low levels of tissue factor, factor IXa formation might play an important role in the extiinsic pathway of coagulation in vivo.

In previous studies using purified human tissue factor (TF) apoprotein reconstituted with PS/PC membranes, no dramatic differences between the kinetic parameters for extrinsic activation of factor X (FX)and factor IX (FIX)could be observed. In vivo however, TF is an integral part of a complex biological membrane. Therefore we studied the kinetics of extrinsic FX and FIX activation on endothelial cells.TF expression was stimulated by incubation of cultured human endothelial cells with endotoxin (4 hr). The cells were washed and incubated with FVII(a), FX and/or FIX n the presence of CaCl2. FXa and FIXa formation were measured with a sensitive spectrophotometric assay (S2337) and an immunoradiometric assay (IRMA-IXa), respectively. Unstimulated cells did not induce significant activation of FX or IX. Stimulation of the cells and recombination of exposed TF withFVIIa resulted in rapidactivation of both substrates.Rates of FXa formation reached a maximum after a lag of about 1 min. Upon prolonged incubation the rate of FXa formation progressively decreased, probably by inactivation of FVIIa by product Xa. At the other hand, FIXa formation was linear in time for at least 30 min. When added together, FIX was found to be a weak, competitive inhibitor of FX activation, while FIX activation was severely inhibited by FX. Both FX and FIX activation were dependent on the presence of TF (sensitive to µ-TF antibodies) and FVIIa. The calculated Km of FIX (∼0.09µM) and of FX (∼0.07µM) for extrinsic activation at the endothelial surface were both higher than those observed in a cell free system using purified TF apoprotein and the ratio of Km-FIX/Km-FX was about 6-fold increased. The ratios of Vmax-FIX activation/Vmax-FX activation for both systems were similar (−0.3). These data indicate that the microenvironment of TF in the biological membrane does not introduce important alterations in the kinetic parameters of TF-FVIIa dependent activation reactions in favour of extrinsic FIX activation.