[Show abstract][Hide abstract] ABSTRACT: Thrombomodulin (TM) increases the catalytic efficiency of thrombin (IIa)-mediated activation of thrombin-activable fibrinolysis inhibitor (TAFI) 1250-fold. Negatively charged residues of the C-loop of TM-EGF-like domain 3 are required for TAFI activation. Molecular models suggested several positively charged residues of TAFI with which the C-loop residues could interact. Seven TAFI mutants were constructed to determine if these residues are required for efficient TAFI activation. TAFI wild-type or mutants were activated in the presence or absence of TM and the kinetic parameters of TAFI activation were determined. When the three consecutive lysine residues in the activation peptide of TAFI were substituted with alanine (K42/43/44A), the catalytic efficiencies for TAFI activation with TM decreased 8-fold. When other positively charged surface residues of TAFI (Lys-133, Lys-211, Lys-212, Arg-220, Lys-240, or Arg-275) were mutated to alanine, the catalytic efficiencies for TAFI activation with TM decreased by 1.7-2.7-fold. All decreases were highly statistically significant. In the absence of TM, catalytic efficiencies ranged from 2.8-fold lower to 1.24-fold higher than wild-type. None of these, except the 2.8-fold lower value, was statistically significant. The average half-life of the TAFIa mutants was 8.1+/-0.6 min, and that of wild type was 8.4+/-0.3 min at 37 degrees C. Our data show that these residues are important in the activation of TAFI by IIa, especially in the presence of TM. Whether the mutated residues promote a TAFI-TM or TAFI-IIa interaction remains to be determined. In addition, these residues do not influence spontaneous inactivation of TAFIa.
Journal of Biological Chemistry 01/2009; 284(11):7059-67. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Studies have shown that inhibition of TAFI by small peptides enhances pharmacological effects of tPA in animal models of thrombosis, suggesting that TAFI modulates the fibrinolytic system. In this study, we investigated the effect of activated human TAFI (TAFIa) on endogenous fibrinolysis in a rat model of intravascular fibrin deposition. (125)I-labeled fibrinogen was injected intravenously followed by a bolus injection of batroxobin, a thrombin-like enzyme. Batroxobin cleaved fibrinogen to form insoluble fibrin that was deposited in tissues, including the lungs. This was shown by a decrease of radioactivity in the blood as a result of consumption of (125)I-labeled fibrinogen and an elevation of radioactivity in the lungs 5 min following batrox-obin administration. Endogenous fibrinolysis was detected by a gradual increase in radioactivity in the blood and a decrease in radioactivity in the lungs at 30 min, an indication of radiolabeled fibrin degradation products (FDPs) being released into the circulation from the tissues. Intravenous administration of human TAFIa dose-dependently attenuated the later phase reduction of radioactivity in the lungs. When the dose of TAFIa was 218 micro g/kg, giving a peak plasma level of TAFIa 0.9 +/- 0.05 micro g/ml, the spontaneous fibrinolysis was completely prevented. These results provide direct evidence that an increase in circulating TAFIa impairs endogenous clot lysis in a rat model of fibrin deposition.
Thrombosis and Haemostasis 10/2003; 90(3):414-21. · 5.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family that is involved in a variety of homeostatic processes. Here we characterize the processing essential for the conversion of the precursor, human pro-CNP, to the biologically active hormone. In human embryonic kidney 293 and chondrosarcoma SW 1353 cells, recombinant pro-CNP was converted into a mature peptide intracellularly as detected by Western analysis. Expression of recombinant human corin, a proatrial natriuretic peptide convertase, did not enhance the processing of pro-CNP in these cells. The processing of pro-CNP was inhibited in the presence of an inhibitor of the endoprotease furin but was not affected by inhibitors of matrix metalloproteinases and tumor necrosis factor-alpha convertase. In furin-deficient human colon adenocarcinoma LoVo cells, no conversion of recombinant pro-CNP to CNP was detected. Expression of recombinant human furin in LoVo cells restored the ability of these cells to process pro-CNP. Furthermore, incubation of purified recombinant human furin with LoVo cell lysate containing pro-CNP led to the conversion of the precursor to a mature peptide. The furin-processed CNP was shown to be biologically active in a cell-based cGMP assay. These results demonstrate that furin is a critical enzyme for the processing of human pro-CNP.
Journal of Biological Chemistry 08/2003; 278(28):25847-52. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, we investigated if elevation of endogenous plasminogen activator inhibitor type 1 (PAI-1) by lipopolysaccharide (LPS) can retard thrombolysis in both a rat model of lung vasculature fibrin deposition and a platelet-rich thrombus model induced by endothelial injury. By 3 h following an intravenous bolus injection of 0.5 mg/kg LPS, the plasma PAI-1 level had increased to approximately 8 ng/ml. 125I-labeled fibrinogen was injected intravenously followed by an injection of batroxobin. Batroxobin converts fibrinogen into insoluble fibrin, which was then deposited in the lungs within 5 min, followed by spontaneous fibrinolysis that completely cleared fibrin deposition in the lungs by 30 min. In rats pre-treated with LPS, spontaneous fibrinolysis was significantly retarded. In the endothelial injury model, topical application of FeCl2 on the carotid artery induced an occlusive platelet-rich thrombus, which was not sensitive to endogenous thrombolysis. Exogenous tissue-type plasminogen activator (tPA) was required to recanalize the occlusive thrombus in a dose-dependent manner. Pre-treatment with LPS did not alter the dose-response curve of exogenous tPA-induced thrombolysis. These data indicate that batroxobin-induced lung vasculature fibrin deposition in rats, unlike the FeCl2 model, is sensitive to the impact of endogenous PAI-1 on fibrinolysis.
Thrombosis Research 02/2003; 111(6):381-7. · 2.43 Impact Factor