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 g/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 g/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 Ca 2؉ 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 Ca 2؉ 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 plasminogenbinding sites on partially degraded fibrin. This is a likely mechanism for the antifibrinolytic effect of active pCPB.

A Kinetic Analysis of the Tissue Plasminogen Activator and DSPAα1 Cofactor Activities of Untreated and TAFIa-treated Soluble Fibrin Degradation Products of Varying Size

“…We, however, recently demonstrated that FDPs produced in a perfused, cross-linked clot vary in molecular weight from 250 (the mass of DD/E) to 10,000 kDa, with the majority of the mass contained in fragments larger than DD/E (9). This suggests that FDPs much larger than DD/E are released into circulation during fibrinolysis, and indeed FDP complexes of at least 2.0 ϫ 10 6 Da have been observed in the plasma of patients with disseminated intravascular coagulation (13,14) and chronic subdural hematoma (15). Whereas a large difference in activity between t-PA and DSPA␣1 would be expected with DD/E, this difference disappears with larger FDPs.…”

Deficiency of thrombin activatable fibrinolysis inhibitor in cirrhosis is associated with increased plasma fibrinolysis

“…10,11 The shortening of the lysis time caused by PTI is a measure of the antifibrinolytic activity of TAFIa and is expressed as PTI sensitivity ratio (hereafter referred to as PTI ratio), i.e., the ratio between lysis time in the absence of PTI and lysis time in the presence of PTI.…”

“…To that purpose, each plasma dilution was incubated for 5 minutes with thrombin (3 U/mL), solulin (1 g/mL), and CaCl 2 (20 mmol/L) to convert all TAFI to TAFIa. 10 Afterwards, the samples were processed as reported above for the determination of PTI-sensitive Cp activity. The amount of TAFIa generated during the clot lysis experiments is expressed as percentage of total TAFI present in normal plasma.…”

“…[7][8][9] Upon activation by thrombin or plasmin, it is converted to an enzyme (TAFIa) with carboxypeptidase B-like activity that inhibits fibrinolysis through the removal of C-terminal lysines from partially degraded fibrin. 10,11 In so doing, TAFIa reduces the cofactor function of fibrin in the plasminogen activation catalyzed by t-PA, thereby decreasing plasmin generation. The most likely physiologic activator of TAFI is thrombin, 12,13 which can work either alone, provided it is present at high concentrations, or in complex with thrombomodulin, in which case its efficiency is increased by more than 1,000-fold.…”

“…13 Under the latter condition, antifibrinolytic amounts of TAFIa are generated only if high amounts of thrombin are formed, likely via a FXI-dependent feedback mechanism, 15 and any impairment of TAFIa formation will translate in highly accelerated fibrinolysis because plasmin generated within a fibrin clot has a much higher impact on fibrinolysis than plasmin generated at the clot surface. 16,17 The potential physiologic role of TAFI in the fibrinolytic process is underscored by several in vitro and in vivo data, 7,10,[18][19][20][21] and evidence is accumulating suggesting that alterations in the TAFI pathway, either because of derangements in the TAFI activating capacity or because of changes in TAFI levels, may contribute to bleeding or thrombosis. [22][23][24] In cirrhosis, the plasma levels of TAFI antigen are markedly reduced most likely because of impaired synthesis.…”