ON THE MECHANISM OF THE ANTIFIBRINOLYTIC ACTIVITY OF PLASMA CARBOXYPEPTIDASE-B

The precursor of plasma carboxypeptidase B (pCPB) also known as thromb in-activable fibrinolysis inhibitor can be converted by thrombin to an active enzyme capable of eliminating C-terminal Lys- and Arg-residues from proteins. The activation is about 1000-fold more efficient in th e presence of thrombomodulin (TM), We investigated the antifibrinolyti c potency of maximally activated pCPB in plasma and explored the antif ibrinolytic mechanism of pCPB, During clotting of plasma in the presen ce of 3.3 NIH units/ml thrombin and 1 mu g/ml soluble TM, more than 80 % pro-pCPB was converted into the active form causing an increase of p lasma carboxypeptidase activity from 100 units/liter (constitutive act ivity ascribed to plasma carboxypeptidase N) to 430 units/liter as mea sured with furoylacroleyl-alanyl-arginine substrate, Under these condi tions, lysis of a plasma clot induced by a range of tissue-type plasmi nogen activator (t-PA) concentrations (0.2-2 mu g/ml) was retarded mor e than 4-fold. A considerable retardation of fibrinolysis was observed upon addition of as little as 12 ng/ml soluble TM, a concentration co mparable with physiological concentrations of soluble TM in human plas ma, The presence of Ca2+ appeared to be a critical requirement for eff ective activation of pro-pCPB by thrombin-TM in plasma, Plasminogen-bi nding sites (C-terminal lysines) on the surface of a plasmin-treated f ibrin clot were eliminated within 1-3 min by plasma with maximally act ivated pCPB, as studied in a recently described model involving fluore scence microscopy, Confocal fluorescence microscopy showed that in the absence of TM plasminogen strongly accumulated on fibrin fibers durin g t-PA-induced lysis of a plasma clot. In the presence of TM (and a co ncomitant pro-pCPB activation), lysis was slow and was not accompanied by accumulation of plasminogen on the fibers, In conclusion, generati on of active pCPB during clotting of plasma in the presence of Ca2+ an d TM leads to a retardation of plasma clot lysis in a wide range of t- PA concentrations, from low to therapeutic, and to a fast elimination of plasminogen-binding sites on partially degraded fibrin, This is a l ikely mechanism for the antifibrinolytic effect of active pCPB.