title: On the mechanism of the antifibrinolytic activity of plasma carboxypeptidase B.
abstract: The precursor of plasma carboxypeptidase B (pCPB) also known as thrombin-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 the presence of thrombomodulin (TM). We investigated the antifibrinolytic potency of maximally activated pCPB in plasma and explored the antifibrinolytic mechanism of pCPB. During clotting of plasma in the presence of 3.3 NIH units/ml thrombin and 1 microg/ml soluble TM, more than 80% pro-pCPB was converted into the active form causing an increase of plasma carboxypeptidase activity from 100 units/liter (constitutive activity ascribed to plasma carboxypeptidase N) to 430 units/liter as measured with furoylacroleyl-alanyl-arginine substrate. Under these conditions, lysis of a plasma clot induced by a range of tissue-type plasminogen activator (t-PA) concentrations (0.2-2 microg/ml) was retarded more than 4-fold. A considerable retardation of fibrinolysis was observed upon addition of as little as 12 ng/ml soluble TM, a concentration comparable with physiological concentrations of soluble TM in human plasma. The presence of Ca2+ appeared to be a critical requirement for effective activation of pro-pCPB by thrombin-TM in plasma. Plasminogen-binding sites (C-terminal lysines) on the surface of a plasmin-treated fibrin clot were eliminated within 1-3 min by plasma with maximally activated pCPB, as studied in a recently described model involving fluorescence microscopy. Confocal fluorescence microscopy showed that in the absence of TM plasminogen strongly accumulated on fibrin fibers during t-PA-induced lysis of a plasma clot. In the presence of TM (and a concomitant pro-pCPB activation), lysis was slow and was not accompanied by accumulation of plasminogen on the fibers. In conclusion, generation of active pCPB during clotting of plasma in the presence of Ca2+ and 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 likely mechanism for the antifibrinolytic effect of active pCPB.
authors.affiliation: Gaubius Laboratory, TNO Prevention and Health, 2301 CE Leiden, The Netherlands. dv.sakharov@pg.tno.nl
authors.forename: D V
authors.initials: DV
authors.lastname: Sakharov
authors.forename: E F
authors.initials: EF
authors.lastname: Plow
authors.forename: D C
authors.initials: DC
authors.lastname: Rijken
mesh_terms: D000933:Antifibrinolytic Agents / Q000378:metabolism / Q000494:pharmacology*; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000378:metabolism / Q000494:pharmacology*; D004789:Enzyme Activation; D005342:Fibrinolysis / Q000187:drug effects*; D006801:Humans; D018613:Microscopy, Confocal
chemical_list: D000933:Antifibrinolytic Agents; D002268:Carboxypeptidases; D043424:Carboxypeptidase B