THE EFFECT OF ACTIVATED PROTEIN-C ON FIBRINOLYSIS IN CELL-FREE PLASMACAN BE ATTRIBUTED SPECIFICALLY TO ATTENUATION OF PROTHROMBIN ACTIVATION

The effect of human activated protein C (APC) on tissue plasminogen ac tivator (tPA)-induced fibrinolysis was studied in cell free plasma and in a system of purified components. Clots were produced by adding pla sma or a solution of fibrinogen and plasminogen to the wells of a micr otiter plate containing small separated aliquots of Ca2+, thrombin, an d tPA, plus and minus APC. Initial clotting and subsequent fibrinolysi s were monitored continuously by turbidity. The lysis time of dialyzed normal human plasma (NHP) was longer than that of dialyzed barium cit rate-adsorbed plasma (BAP). APC had no effect on the lysis time of BAP but shortened the lysis time of NHP to that of BAP. Two fractions wer e produced from material eluted from the barium citrate pellet by prec ipitation of selective components with polyethylene glycol 8000 (PEG). One fraction comprised materials which precipitated at 5% PEG (5% PF) and the other materials which precipitated between 5 and 40% PEG (5-4 0% PF). Both fractions together, but neither alone, prolonged the lysi s time of BAP, an effect which could be reversed by APC. Fractionation of the 5% PF showed that the component with the required activity has properties of the procoagulant surface and can be replaced with vesic les of phosphatidylcholine/phosphatidylserine (PCPS). In addition, the 5-40% PF can be replaced with either the combination of purified coag ulation Factors II, IX, and X or Factor II plus the prothrombin activa tor Factor Xa. When Factor Xa was used as the activator in BAP plus PS PC vesicles, a dose-dependent saturable increase in lysis time was obs erved with a half-maximal increase occurring at 32 pM Factor Xa. This effect was eliminated by APC. In a system of purified components compr ising PCPS vesicles, Factors V and 11, protein S, plasminogen and fibr inogen; the prothrombin activators Factor Xa and ecarin both induced a prolongation of the lysis time. APC prevented prolongation by Factor Xa but not by ecarin. The time courses of the generation of thrombin a nd plasmin during fibrinolysis of clots produced from systems of purif ied components in the presence and absence of APC, and with Factor Xa as the prothrombin activator, were determined by standardized activity assays using chromogenic substrates. In the absence of APC the lysis time was 145 min, and prothrombin was quantitatively converted to thro mbin. In the presence of APC, however, the lysis time was reduced to 1 00 min with no evidence for the activation of prothrombin. The time co urse of the generation of plasmin during lysis was retarded in the abs ence of APC. The final concentrations of plasmin produced in both cond itions were indistinguishable, however. These data indicate that activ ation of prothrombin generates an as yet unidentified antifibrinolytic component(s), and the profibrinolytic effect of APC can be rationaliz ed by its ability to inhibit the activation of prothrombin.