Protein S levels modulate the activated protein C resistance phenotype induced by elevated prothrombin levels

Abstract

Elevated plasma prothrombin levels, due to the prothrombin 20210 G/A mutation or to acquired causes, are a risk factor for venous thrombosis, partly because of prothrombin-mediated inhibition of the protein C anticoagulant pathway and consequent activated protein C (APC) resistance. We determined the effect of plasma prothrombin concentration on the APC resistance phenotype and evaluated the role of protein S levels as a modulating variable. The effect of prothrombin and protein S levels on APC resistance was investigated in reconstituted plasma systems and in a population of healthy individuals using both the aPTT-based and the thrombin generation-based APC resistance tests. In reconstituted plasma, APC resistance increased at increasing prothrombin concentration in both assays. Enhanced APC resistance was caused by the effect of prothrombin on the clotting time in the absence of APC in the aPTT-based test, and on thrombin formation in the presence of APC in the thrombin generation-based test. In plasma from healthy individuals prothrombin levels were highly correlated to protein S levels. Since prothrombin and protein S had opposite effects on the APC resistance phenotype, the prothrombin/protein S ratio was a better predictor of APC resistance than the levels of either protein alone. Prothrombin titrations in plasmas containing different amounts of protein S confirmed that protein S levels modulate the ability of prothrombin to induce APC resistance. These findings suggest that carriers of the prothrombin 20210 G/A mutation, who have a high prothrombin/protein S ratio, may experience a higher thrombosis risk than non-carriers with comparable prothrombin levels.

Thrombin generation and activated protein C resistance in patients with essential thrombocythemia and polycythemia vera

“…This is plausible as it has been demonstrated that small variations in the plasma concentration of free PS can cause significant variation in the ETP values determined in the presence of added APC. 34 Reductions in the plasma levels of natural anticoagulants, particularly PS, were previously reported in patients with ET and PV. [37][38][39]44 In our study, we not only confirmed these previous observations, but we could also demonstrate that the alterations in the coagulation protein levels induce an APC-resistant phenotype.…”

“…The assay was performed in the absence and presence of APC, essentially as described. 34 Briefly, 80 L plasma was pipetted into the well of a microtiter plate together with 15 L of a mixture of TF (Innovin; Dade Behring, Deerfield, IL) and synthetic phospholipid vesicles (DOPS/DOPC/ DOPE, 20/60/20 mol/mol/mol). After 5 minutes of prewarming at 37°C, 10 L buffer or purified APC (Innovative Research, Southfield, MI) was added to the respective wells, and the reaction was started with 20 L of a fluorogenic substrate (Z-Gly-Gly-Arg-AMC; Bachem, Bubendorf, Switzerland)/CaCl 2 mixture.…”

“…FII levels were determined by a chromogenic assay after complete activation of FII with Ecarin in 1:500-diluted plasma. 34 FV levels were measured by a prothrombinase-based assay after complete activation of FV with thrombin in 1:1000-diluted plasma. 34 Total PS antigen levels were quantified by homemade enzyme-linked immunosorbent assay, as previously described.…”

“…34 FV levels were measured by a prothrombinase-based assay after complete activation of FV with thrombin in 1:1000-diluted plasma. 34 Total PS antigen levels were quantified by homemade enzyme-linked immunosorbent assay, as previously described. 35 Free-PS and free-TFPI antigen levels were quantified with commercial enzyme-linked immunosorbent assay kits (Asserachrom; Diagnostica Stago, Asnières sur Seine, France) according to the manufacturer's instructions.…”

“…29,32 In particular, the aPTT test is not sensitive to PS levels in the 50% to 100% range. 34 Because APC resistance, in the absence of FV Leiden, is also associated with an increased risk of thrombosis in other conditions, including pregnancy, oral contraceptive use, hormone replacement therapy, and cancer, 31,35,[40][41][42] acquired APC resistance in ET and PV patients may account for the thrombotic risk associated with these disorders.…”