Solulin increases clot stability in whole blood from humans and dogs with hemophilia.

Solulin is a soluble form of thrombomodulin that is resistant to proteolysis and oxidation. It has been shown to increase the clot lysis time in factor VIII (fVIII)-deficient plasma by an activated thrombin-activatable fibrinolysis inhibitor (TAFIa)-dependent mechanism. In the present study, blood was drawn from humans and dogs with hemophilia, and thromboelastography was used to measure tissue factor-initiated fibrin formation and tissue-plasminogen activator-induced fibrinolysis. The kinetics of TAFI and protein C activation by the thrombin-Solulin complex were determined to describe the relative extent of anticoagulation and antifibrinolysis. In severe hemophilia A, clot stability increased by > 4-fold in the presence of Solulin while minimally affecting clot lysis time. Patients receiving fVIII/fIX prophylaxis showed a similar trend of increased clot stability in the presence of Solulin. The catalytic efficiencies of TAFI and protein C activation by the thrombin-Solulin complex were determined to be 1.53 and 0.02/µM/s, respectively, explaining its preference for antifibrinolysis over anticoagulation at low concentrations. Finally, hemophilic dogs given Solulin had improved clot strength in thromboelastography assays. In conclusion, the antifibrinolytic properties of Solulin are exhibited in hemophilic human (in vitro) and dog (in vivo/ex vivo) blood at low concentrations. Our findings suggest the therapeutic utility of Solulin at a range of very low doses.

Kinetics of activated thrombin-activatable fibrinolysis inhibitor (TAFIa)-catalyzed cleavage of C-terminal lysine residues of fibrin degradation products and removal of plasminogen-binding sites.

Partial digestion of fibrin by plasmin exposes C-terminal lysine residues, which comprise new binding sites for both plasminogen and tissue-type plasminogen activator (tPA). This binding increases the catalytic efficiency of plasminogen activation by 3000-fold compared with tPA alone. The activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis by removing these residues, which causes a 97% reduction in tPA catalytic efficiency. The aim of this study was to determine the kinetics of TAFIa-catalyzed lysine cleavage from fibrin degradation products and the kinetics of loss of plasminogen-binding sites. We show that the k(cat) and K(m) of Glu(1)-plasminogen (Glu-Pg)-binding site removal are 2.34 s(-1) and 142.6 nm, respectively, implying a catalytic efficiency of 16.21 µm(-1) s(-1). The corresponding values of Lys(77)/Lys(78)-plasminogen (Lys-Pg)-binding site removal are 0.89 s(-1) and 96 nm implying a catalytic efficiency of 9.23 µm(-1) s(-1). These catalytic efficiencies of plasminogen-binding site removal by TAFIa are the highest of any TAFIa-catalyzed reaction with a biological substrate reported to date and suggest that plasmin-modified fibrin is a primary physiological substrate for TAFIa. We also show that the catalytic efficiency of cleavage of all C-terminal lysine residues, whether they are involved in plasminogen binding or not, is 1.10 µm(-1) s(-1). Interestingly, this value increases to 3.85 µm(-1) s(-1) in the presence of Glu-Pg. These changes are due to a decrease in K(m). This suggests that an interaction between TAFIa and plasminogen comprises a component of the reaction mechanism, the plausibility of which was established by showing that TAFIa binds both Glu-Pg and Lys-Pg.

Thrombin-activatable fibrinolysis inhibitor is activated in vivo in a baboon model of Escherichia coli induced sepsis.

Activated thrombin-activatable fibrinolysis inhibitor (TAFIa or CPU) is a carboxypeptidase that is able to attenuate fibrinolysis. Although its role in fibrinolysis and inflammation has been studied extensively in vitro, its levels and subsequent effect in vivo has not been studied to the same extent. Using our recently developed assay that is specific for TAFIa, we were able to quantify its levels in plasma samples obtained from an Escherichia coli (E. coli) challenged baboon sepsis model. TAFIa levels accumulated appeared to be E. coli dose dependent, where the lethal dose of 10(10) CFU/kg generated a peak TAFIa level of 24 nM by 2 h, which represents almost 32% of total plasma level of its precursor, thrombin-activatable fibrinolysis inhibitor (TAFI or proCPU). Furthermore, our data suggest that there is continual TAFI activation under lethal level of E. coli as the apparent half-life of TAFIa is increased from 8 min to 2.2 h. Two sublethal doses of 10(8) and 10(6) CFU/kg generated peak TAFIa levels of 1.1 and 0.4 nM, respectively, both by 6 h. Taken together, our data show that TAFIa is generated at systemic levels, in a dose-dependent manner, that can substantially affect both fibrinolysis and inflammatory response in the E. coli challenged baboon sepsis model.

Differential signaling of cysteinyl leukotrienes and a novel cysteinyl leukotriene receptor 2 (CysLT2) agonist, N-methyl-leukotriene C4, in calcium reporter and ß arrestin assays.

The cysteinyl leukotrienes (cysLTs) LTC4, LTD4, and LTE4 are lipid mediators with physiological and pathophysiological functions. They exert their effects through G protein-coupled receptors (GPCRs), most notably via CysLT1 and CysLT2 receptor. The roles of the CysLT2 receptor are beginning to emerge. Both LTC4 and LTD4 are potent agonists for the CysLT2 receptor; however, LTC4 is rapidly converted to LTD4, which is also the main endogenous ligand for the CysLT1 receptor. A selective and potent agonist at the CysLT2 receptor would facilitate studies to discern between receptor subtypes. We show here that N-methyl LTC4 (NMLTC4), a metabolically stable LTC4 mimetic, is a potent and selective CysLT2 receptor agonist. Two expression systems were used to evaluate the functional activity of NMLTC4 at human and/or mouse CysLT1 and CysLT2 receptors. Through the aequorin cell-based assay for calcium-coupled GPCRs, NMLTC4 was almost equipotent to LTC4 at CysLT2 receptors but was the least efficacious at CysLT2 receptors. In a ß-galactosidase-ß-arrestin complementation assay, the human (h) CysLT2 receptor can couple with ß-arrestin-2, and NMLTC4 is slightly more potent for eliciting ß-arrestin-2 binding compared with cysLTs. Furthermore, LTE4 is nearly inactive in this assay compared with its weak partial agonist activity in the aequorin system. In a vascular leakage assay, NMLTC4 is potent and active in mice overexpressing hCysLT2 receptor in endothelium, whereas the response is abrogated in CysLT2 receptor knockout mice. Therefore, NMLTC4 is a potent subtype selective agonist for the CysLT2 receptor in vitro and in vivo, and it will be useful to elucidate its biological roles.

Differences in prethrombin-1 activation with human or bovine factor Va can be attributed to the heavy chain.

Human and bovine factor Va (FVa) function similarly in the activation of prothrombin but differently in the activation of prethrombin-1 (Pre-1). Pre-1 activation with human FVa proceeds at about 22 percent of the rate with bovine FVa. The dependencies of initial rates on the FVa and Pre-1 concentrations indicate that the differential activity is expressed in kcat differences, rather than differences in the assembly of prothrombinase or the K(m) value of the substrate. The heavy and light chains of both species of FVa were separated and interspecies hybrids were constructed in the presence of Ca(++). Studies of the activation of Pre-1 with these hybrids indicate that the species difference can be attributed specifically to the heavy chain of FVa. Analyses of the reactions by SDS-PAGE indicated that cleavage at Arg271 occurs at about the same rate with both species of FVa, but cleavage at Arg320 with human FVa is specifically retarded. A major difference in primary structure between the human and bovine FVa heavy chains comprises 10 residues at COOH-terminus, adjacent to the negatively charged hirudin-like DYDYQ sequence. These residues have pI values of 12.5 and 4.26 in human and bovine FVa, respectively. The lower value would complement the negatively charged DYDYQ sequence but the higher value would counteract it. Thus, we suggest that the differences in the COOH-terminus of the heavy chain are responsible for the differences in Pre-1 activation, and that it specifically influences cleavage at Arg320 in Pre-1.

The interaction of fragment 1 of prothrombin with the membrane surface is a prerequisite for optimum expression of factor Va cofactor activity within prothrombinase.

Incorporation of factor (F) Va into prothrombinase directs prothrombin activation by FXa through the meizothrombin pathway, characterized by initial cleavage at Arg(320). We have shown that a pentapeptide with the sequence DYDYQ specifically inhibits this pathway. It has been also established that Hir(54-65)(SO(3)(-)) is a specific inhibitor of prothrombinase. To understand the role of FVa within prothrombinase at the molecular level, we have studied thrombin formation by prothrombinase in the presence of various prothrombin-derived fragments alone or in combination. Activation of prethrombin 1 is slow with cleavages at Arg(320) and Arg(271) occurring with similar rates. Addition of purified fragment 1 to prethrombin 1 accelerates both the rate of cleavage at Arg(320) and thrombin formation. Both reactions were inhibited by Hir(54-65)(SO(3)(-)) while DYDYQ had no significant inhibitory effect on prethrombin 1 cleavage in the absence or presence of fragment 1. Similarly, activation of prethrombin 2 by prothrombinase, is inhibited by Hir(54-65)(SO(3)(-)), but is not affected by DYDYQ. Addition of purified fragment 1*2 to prethrombin 2 accelerates the rate of cleavage at Arg(320) by prothrombinase. This addition also results in a significant inhibition of thrombin formation by DYDYQ and is concurrent with the elimination of the inhibitory effect of Hir(54-65)(SO(3)(-)) on the same reaction. Finally, a membrane-bound ternary complex composed of prethrombin 2/fragment 1*2/Hir(54-65)(SO(3)(-)) is inhibited by DYDYQ. Altogether, the data demonstrate that membrane-bound fragment 1 is required to promote optimum Fva cofactor activity which in turn is translated by efficient initial cleavage of prothrombin by prothrombinase at Arg(320).

Isolation and characterization of cotiaractivase, a novel low molecular weight prothrombin activator from the venom of Bothrops cotiara.

In this study, we isolated a novel prothrombin activator from the venom of Bothrops cotiara, a Brazilian lance-headed pit viper (Cotiara, Jararaca preta, Biocotiara), which we have designated "cotiaractivase" (prefix: cotiar- from B. cotiara; suffix: -activase, from prothrombin activating activity). Cotiaractivase was purified using a phenyl-Superose hydrophobic interaction column followed by a Mono-Q anion exchange column. It is a single-chain polypeptide with a molecular weight of 22,931 Da as measured by mass spectroscopy. Cotiaractivase generated active alpha-thrombin from purified human prothrombin in a Ca2+-dependent manner as assessed by S2238 chromogenic substrate assay and SDS-PAGE. Cotiaractivase cleaved prothrombin at positions Arg271-Thr272 and Arg320-Ile321, which are also cleaved by factor Xa. However, the rate of thrombin generation by cotiaractivase was approximately 60-fold less than factor Xa alone and 17 x 10(6)-fold less than the prothrombinase complex. The enzymatic activity of cotiaractivase was inhibited by the chelating agent EDTA, whereas the serine protease inhibitor PMSF had no effect on its activity, suggesting that it is a metalloproteinase. Interestingly, S2238 inhibited cotiaractivase activity non-competitively, suggesting that this toxin contains an exosite that allows it to bind prothrombin independently of its active site. Tandem mass spectrometry and N-terminal sequencing of purified cotiaractivase identified peptides that were identical to regions of the cysteine-rich and disintegrin-like domains of known snake venom metalloproteinases. Cotiaractivase is a unique low molecular weight snake venom prothrombin activator that likely belongs to the metalloproteinase family of proteins.

Lipoprotein-complexed C-reactive protein and the biphasic transmittance waveform in critically ill patients.

The 'biphasic transmittance waveform' (BTW) refers to a decrease in light transmittance that often occurs prior to clotting in coagulation assays of critically ill patient plasmas. It correlates with disseminated intravascular coagulation and mortality. The present work shows that the BTW is due to the rapid formation of a precipitate and a coincident change in turbidity in re-calcified plasma. The precipitate was isolated from patient plasma and contained lipids typical of very low density lipoprotein (VLDL), plus the proteins apolipoprotein B-100 and C-reactive protein (CRP). Precipitation also occurred in normal plasma supplemented with CRP. In addition, CRP precipitated with VLDL and intermediate density lipoprotein, but not low density lipoprotein or high density lipoprotein. The Kd value for the CRP/VLDL interaction is 340 nM. The IC50 value of Ca2+ for complex formation is 5.0 mM, and epsilon-aminocaproic acid inhibits the process. In 15 plasmas with the BTW from critically ill patients, CRP was highly elevated (77-398 microg/mL) and VLDL cholesterol ranged from 0.082 to 1.32 mM. The magnitude of the turbidity change on re-calcification correlated well with the calculated level of the CRP/VLDL complex. Thus, the Ca2+-dependent formation of a complex between CRP and VLDL accounts for the BTW.

Prothrombinase enhancement through quantitative and qualitative changes affecting very low density lipoprotein in complex with C-reactive protein.

The biphasic waveform that can predict for disseminated intravascular coagulation (DIC) is due to the formation of a calcium-dependent complex between C reactive protein (CRP) and very low density lipoprotein (VLDL). As thrombin generation is pivotal to DIC, this aspect has been specifically investigated and the VLDL component has been found to increase prothrombinase activity via both quantitative and qualitative changes. The specific prothrombinase activity of VLDL from patients manifesting the biphasic waveform was 2.5 times that of normal individuals or critically ill patients without the biphasic waveform. This activity was due to an increase in anionic phospholipid surfaces that could be inhibited with excess annexin V and which was dependent on structurally intact apolipoprotein B. The qualitative change appeared to be due to a deficiency of phosphatidylethanolamine in VLDL from patients with the biphasic waveform. The functional consequence of this enhanced prothrombinase activity was an increased procoagulant effect in plasma. Using a modified activated partial thromboplastin time assay, the mean normal clot time decreased significantly when VLDL from patients with biphasic waveforms was substituted. These results indicate that VLDL derived from patients with the biphasic waveform can enhance thrombin procoagulant activity. As the CRP-VLDL complex exists in vivo, it could have a pathogenic role in disseminating the process of intravascular coagulation.

Thrombin and fibrinolysis.

When the activities of the coagulation and fibrinolytic cascades are properly regulated, so that fibrin (FN) deposition and removal are properly balanced, the vascular system is protected from catastrophic blood loss at the site of an injury, while its fluidity is ensured elsewhere. When these activities are not properly regulated, however, the organism is subjected to either excessive bleeding or thrombosis. Thrombomodulin on the endothelial cell is very important in this regulation because it converts thrombin to an anticoagulant enzyme by directing it toward the activation of protein C. It also converts thrombin to an antifibrinolytic enzyme by directing it toward the activation of thrombin-activatable fibrinolysis inhibitor (TAFI). By doing so, it creates a direct molecular connection between the coagulation and fibrinolytic cascades, such that activation of the former suppresses the activity of the latter. Recent studies indicate that the TAFI pathway functions in vivo and is likely relevant in maintaining the proper balance between FN deposition and removal. Whether it will be a target for pharmaceutical manipulation of this balance remains to be determined.

Activated thrombin activatable fibrinolysis inhibitor (TAFIa) is associated with inflammatory markers in inflammatory bowel diseases TAFIa level in patients with IBD.

BACKGROUND: Thrombin activatable fibrinolysis inhibitor (TAFI) has been reported to be involved in the pathogenesis and progression of inflammatory bowel disease (IBD). Activated TAFI (TAFIa) attenuates fibrinolysis by cleaving C-terminal lysine residues thus down-regulating plasminogen activation. To date, no reports on TAFIa in IBD have been published. METHODS: Plasma levels of TAFIa were measured using a functional assay in 55 consecutive patients with ulcerative colitis (UC) and 50 with Crohn's disease (CD). Associations of TAFIa with disease activity, hemostatic variables and inflammatory markers were assessed. RESULTS: Plasma TAFIa was higher in CD patients than in those with UC. The disease activity correlated positively with TAFIa levels in the UC group, but not in the CD group. In UC patients, there were positive correlations of TAFIa with white blood cells, C-reactive protein and fibrinogen and an inverse correlation with albumin. In the CD group, a positive correlation was shown for C-reactive protein, fibrinogen and platelet count, while a negative correlation was noted for albumin. CONCLUSIONS: This study is the first to show that TAFIa is increased in CD patients compared with UC and its levels are associated with inflammatory markers in both forms of IBD. These findings fit in the hypothesis that TAFIa may be a marker of active IBD, and in particular of active UC.

Plasma carboxypeptidase B downregulates inflammatory responses in autoimmune arthritis.

The immune and coagulation systems are both implicated in the pathogenesis of rheumatoid arthritis (RA). Plasma carboxypeptidase B (CPB), which is activated by the thrombin/thrombomodulin complex, plays a procoagulant role during fibrin clot formation. However, an antiinflammatory role for CPB is suggested by the recent observation that CPB can cleave proinflammatory mediators, such as C5a, bradykinin, and osteopontin. Here, we show that CPB plays a central role in downregulating C5a-mediated inflammatory responses in autoimmune arthritis. CPB deficiency exacerbated inflammatory arthritis in a mouse model of RA, and cleavage of C5a by CPB suppressed the ability of C5a to recruit immune cells in vivo. In human patients with RA, genotyping of nonsynonymous SNPs in the CPB-encoding gene revealed that the allele encoding a CPB variant with longer half-life was associated with a lower risk of developing radiographically severe RA. Functionally, this CPB variant was more effective at abrogating the proinflammatory properties of C5a. Additionally, expression of both CPB and C5a in synovial fluid was higher in patients with RA than in those with osteoarthritis. These findings suggest that CPB plays a critical role in dampening local, C5a-mediated inflammation and represents a molecular link between inflammation and coagulation in autoimmune arthritis.

Down regulation of prothrombinase by activated protein C during prothrombin activation.

Activated protein C (aPC) proteolytically inactivates factor Va (FVa) and thereby downregulates prothrombinase. Although FVa inactivation by aPC has been studied extensively, the inactivation of prothrombinase during prothrombin activation has not. Therefore, prothrombin activation initiated both without and with aPC (5.0, 7.5 or 10.0 nM) was monitored over time by fluorescence. The experiments were performed with 0.075 nM FVa and 1.0 nM FXa, and with these concentrations reversed. The time courses of the residual prothrombinase activity with aPC, determined from the slopes of fluorescence over time, were pseudo first order with both limiting and excess FVa. With FVa limiting or in excess, the second rate constants for inactivation of prothrombinase were 1.98 +/- 0.09 x 10(5) M(-1)s(-1) and 2.54 +/- 0.13 x 10(5) M(-1)s(-1), respectively. The former value is 101-fold smaller than that for FVa inactivation by aPC alone. Since with limiting FVa the second order rate constants for prothrombinase inactivation and FVa inactivation are equal, FVa is protected 101-fold, presumably by both FXa and prothrombin. In contrast, with excess FVa, the calculated rate constant for FVa inactivation exceeds that for prothrombinase inactivation 17.3-fold, which reflects a loss of protection by FXa. Since the protective effects of the two proteins are theoretically multiplicative, FXa protected 17.3-fold and prothrombin protected 5.8-fold. With 150 nM protein S and limiting FVa, prothrombinase inactivation was two-fold faster, yet it was still protected 91-fold. These studies show that FVa is down-regulated by aPC during prothrombin activation, but both FXa and prothrombin protect FVa in a multiplicative way, with or without protein S.

The roles of selected arginine and lysine residues of TAFI (Pro-CPU) in its activation to TAFIa by the thrombin-thrombomodulin complex.

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.

Thrombin-activable fibrinolysis inhibitor zymogen does not play a significant role in the attenuation of fibrinolysis.

Activated thrombin-activable fibrinolysis inhibitor (TAFIa) plays a significant role in the prolongation of fibrinolysis. During fibrinolysis, plasminogen is activated to plasmin, which lyses a clot by cleaving fibrin after selected arginine and lysine residues. TAFIa attenuates fibrinolysis by removing the exposed C-terminal lysine residues. It was recently reported that TAFI zymogen possesses sufficient carboxypeptidase activity to attenuate fibrinolysis through a mechanism similar to TAFIa. Here, we show with a recently developed TAFIa assay that when thrombin is used to clot TAFI-deficient plasma supplemented with TAFI, there is some TAFI activation. The extent of activation was dependent upon the concentration of zymogen present in the plasma, and lysis times were prolonged by TAFIa in a concentration-dependent manner. Potato tuber carboxypeptidase inhibitor, an inhibitor of TAFIa but not TAFI, abolished the prolongation of lysis in TAFI-deficient plasma supplemented with TAFI zymogen. In addition, TAFIa but not TAFI catalyzed release of plasminogen bound to soluble fibrin degradation products. The data presented confirm that TAFI zymogen is effective in cleaving a small substrate but does not play a role in the attenuation of fibrinolysis because of its inability to cleave plasmin-modified fibrin degradation products.

An assay for measuring functional activated thrombin-activatable fibrinolysis inhibitor in plasma.

Thrombin-activatable fibrinolysis inhibitor (TAFI), also called procarboxypeptidase U (proCPU), is a plasma zymogen that can be activated by thrombin, the thrombin-thrombomodulin complex, or plasmin. The activated form of TAFI (TAFIa, CPU) removes C-terminal lysine residues of plasmin-modified fibrin (FN') that mediates a positive feedback mechanism in plasminogen (Pg) activation, thereby attenuating fibrinolysis. The plasma concentration of TAFI is approximately 75 nM. Because the half-maximal effect of TAFIa occurs at 1 nM, only approximately 1.3% of TAFI needs to be activated to exert an effect on clot lysis. The assay is performed by mixing soluble FN' covalently attached to a quencher and fluorescein-labeled Pg. The sample containing TAFIa is then added, and the rate of fluorescence increase due to removal of C-terminal lysine from FN' and loss of Pg binding is measured with a fluorescence plate reader. The assay was shown to be sensitive for TAFIa at a concentration as low as 12 pM. The intraassay variability and interassay variability of the assay were 6.3 and 8.3%, respectively. This assay was not confounded by the naturally occurring TAFI Thr325Leu polymorphism that affects the thermal stability of TAFIa or endogenous plasminogen in plasma.

Effect of single nucleotide polymorphisms on expression of the gene encoding thrombin-activatable fibrinolysis inhibitor: a functional analysis.

Thrombin-activable fibrinolysis inhibitor (TAFI) is a plasma zymogen that acts as a molecular link between coagulation and fibrinolysis. Numerous single nucleotide polymorphisms (SNPs) have been identified in CPB2, the gene encoding TAFI, and are located in the 5'-flanking region, in the coding sequences, and in the 3'-untranslated region (UTR) of the CPB2 mRNA transcript. Associations between CPB2 SNPs and variation in plasma TAFI antigen concentrations have been described, but the identity of SNPs that are causally linked to this variation is not known. In the current study, we investigated the effect of the SNPs in the 5'-flanking region on CPB2 promoter activity and SNPs in the 3'-UTR on CPB2 mRNA stability. Whereas the 5'-flanking region SNPs (with 2 exceptions) did not have a significant effect on promoter activity, either alone or in haplotypic combinations seen in the human population, all of the 3'-UTR SNPs substantially affected mRNA stability. We speculate that these SNPs, in part, contribute to variation in plasma TAFI concentrations via modulation of CPB2 gene expression through an effect on mRNA stability.

Further evidence for two functional forms of prothrombinase each specific for either of the two prothrombin activation cleavages.

Previous work showed that prothrombin derivatives cleavable only at Arg-320 (rMZ) or Arg-271 (rP2) are partial, rather than competitive, inhibitors of prothrombin activation by prothrombinase. A "ping-pong"-like model, which posits two equilibrating forms of prothrombinase, explained the inhibition pattern. The present studies were undertaken to further investigate this putative mechanism. Two models were developed, one allowing for one form of the enzyme and the other allowing for two forms. Both models also allowed channeling and ratcheting. The models were fit to full time courses of prothrombin, meizothrombin, prethrombin-2, and the B-chain. In the absence of ratcheting and channeling, neither model fits the data. In their presence, however, both models fit very well, and thus they could not be distinguished. Therefore, inhibition of rMZ activation by rP2 was studied. Inhibition was partial and the two-form model fit the data with randomly distributed residuals, whereas the one-form model did not. Initial rates of fluorescein-labeled prothrombin cleavage in the presence of various prothrombin derivatives reported by Brufatto and Nesheim (Brufatto, N., and Nesheim, M. E. (2003) J. Biol. Chem. 278, 6755-6764) were also analyzed using the two models. The two-form model fit the partial inhibition data well, whereas the one-form model did not. In addition, prothrombin at varying concentrations was activated, and subsequently, the initial rates were plotted with respect to the initial prothrombin concentration. When compared with the expected initial rates as determined by the simulation of the models, the two-form model fit the observed rates better than the one-form model. The results obtained here further support the existence of two functional forms of prothrombinase.

Molecular analysis of the human thrombin-activatable fibrinolysis inhibitor gene promoter.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like pro-enzyme that, once activated, attenuates fibrinolysis. Little is presently known of the factors that regulate expression of CPB2, the gene encoding TAFI. This study identified 10 potential transcription factor binding sites (denoted A-J) within the proximal promoter region of CPB2, spanning nucleotides -425 to +21; two of these represent previously-described binding sites for CCAAT/enhancer binding protein and glucocorticoid receptor. We identified additional transcription factors that bind within the proximal CPB2 promoter, namely, nuclear factor-Y (NF-Y) and hepatocyte nuclear factor-1alpha (HNF-1alpha). Binding of NF-Y to the region between nucleotides -76 to -59 (Site B) is important for basal CPB2 promoter activity; NF-Y may be a key factor for the recruitment of the transcriptional machinery to the TAFI gene promoter. HNF-1alpha binds at the interface between Sites C and B. Transient transfections of CPB2 promoter-reporter constructs showed that HNF-1alpha binding is essential for the activity of this promoter in HepG2 cells, indicating that HNF-1alpha is involved in the liver-specific expression of CPB2.