Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

TF mediated initiation of coagulation appears to play a critical role in normal hemostasis and probably pathologic thrombosis as well. Although teleological considerations would seem to suggest that a specific regulator of this process should exist, and although the presence in plasma of such an inhibitor was documented many years ago, it was not until the past five years that the inhibitor was characterized and its mechanism of action defined. LACI produces factor Xa-dependent feedback initiation of the VIIa/TF catalytic complex. The mechanism of this feedback inhibition is novel. First, LACI, a multi-headed protease inhibitor, binds factor Xa, a product of VIIa/TF catalysis, at one of its inhibitory domains. The Xa-LACI complex, possibly acting as a pseudosubstrate, then is able to bind to VIIa/TF in an appropriate conformation such that a second inhibitory domain of LACI is positioned to interact with factor VIIa in the VIIa/TF complex. Whether such a unique means of eliciting feedback inhibition in a protease cascade is repeated in nature is unknown. The existence of LACI appears to help explain the clinical need for both "extrinsic" and "intrinsic" coagulation pathways. In addition, data to the present are consistent with the notion that, in normal hemostasis at least, TF is responsible for an initial burst of factor Xa generation which provides sufficient thrombin to induce the aggregation of platelets and the activation of the critical coagulation cofactors factor V and factor VIII. Ultimate and persistent hemostasis, however, appears to require the continued production of additional factor Xa through the action of factor IXa and factor VIII. The fact that patients with factor XI deficiency suffers a variable but usually mild bleeding diathesis suggests that under certain conditions the initial burst of factor IXa formed through the action of VIIa/TF is insufficient and supplemental factor IXa generated by factor XIa is needed for normal hemostasis. The mechanism by which this factor XIa is generated in vivo, however, has not been determined. We stress that the predicted in vivo role of LACI is simply that--a prediction based on its known in vitro properties. Documentation of its physiologic importance remains to be provided and is an area of active research. Further, although significant progress has been made over the past few years in the characterization of LACI, many questions remain unanswered. For example: What is the mechanism for LACI's association with lipoproteins in plasma? What function, if any, does the third Kunitz-type protease inhibitor domain in LACI serve? (ABSTRACT TRUNCATED AT 400 WORDS)
 ...
PMID:The lipoprotein-associated coagulation inhibitor. 200 33

Protease nexin-2/amyloid beta-protein precursor (PN-2/A beta PP) is a Kunitz-type protease inhibitor which has been shown to be a tight-binding inhibitor of enzymes, factors XIa and IXa (FIXa), suggesting a role for this protein in hemostasis. Since coagulant reactions are modulated on biologic surfaces, we investigated how 25:75 (mol/mol) phosphatidylserine/phosphatidylcholine vesicles (PSPC), thrombin-activated platelets, or umbilical vein endothelial cells influence inactivation of FIXa by PN-2/A beta PP. The Km of human or porcine FIXa activation of human factor X in the presence of PSPC, activated platelets, or endothelial cells in the absence or presence of thrombin-activated factor VIII (FVIIIa) was similar, (0.05-0.39 microM). The presence of FVIIIa increased the catalytic efficiency (kcat/Km ratio) of human and porcine factor IXa's activation of factor X 4952-406-fold, respectively. In the presence of PSPC, the Ki of human and porcine FIXa inhibition by PN-2/A beta PP was Ki = 1.9 x 10(-9) M and 5.8 x 10(-9) M, respectively. After the addition of FVIIIa to the reaction, the Ki for both human and porcine FIXa inhibition by PN-2/A beta PP on PSPC increased 13- and 4-fold to Ki = 2.5 x 10(-8) M and 2.4 x 10(-8) M, respectively. These Ki for inhibition of human FIXa on phospholipid vesicles by PN-2/A beta PP were similar when factor X activation was measured by chromogenic or activation peptide release assays. FVIIIa reduced the inhibition of FIXa by PN-2/A beta PP only in the presence of PSPC.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:Factor IXa inhibition by protease nexin-2/amyloid beta-protein precursor on phospholipid vesicles and cell membranes. 782 67

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor with three tandem inhibitory domains (K1, K2, and K3) that regulates the initial reactions of the extrinsic blood coagulation pathway through K1 and K2. In the present study, the effect of thrombin on TFPI in a purified system was first examined using recombinant TFPI from Chinese hamster ovary (CHO) cells. TFPI was inactivated by thrombin with cleavage of three peptide bonds, Lys 254-Thr 255 in the C-terminal basic region, Arg 107-Gly 108 (reactive site toward factor Xa in K2), and Lys 86-Thr 87 between K1 and K2. Then, degradation of radiolabeled TFPI by thrombin was examined in two systems: (1) mixed with plasma and then tissue factor (TF) and calcium ion, and (2) mixed with fibrinogen and then thrombin. TFPI degradation was detected in serum from normal plasma and more extensively from anti-thrombin (AT)-depleted plasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Significant radioactivity was found in the clot after coagulation of the plasma, which decreased after 20 hours' incubation. These changes were more prominent in AT-depleted plasma than in normal plasma. When TFPI lacking the C-terminal basic region was used instead of full-length TFPI, most of the radioactivity was found in serum rather than in fibrin clots. Incorporation of TFPI into the fibrin clot was prevented by a synthetic C-terminal peptide of TFPI. Similar results were obtained after mixing radiolabeled TFPI with fibrinogen and then thrombin in the presence of calcium ion or EDTA. These results demonstrate a novel degradation pathway of TFPI, ie, incorporation into fibrin via the C-terminal basic region and degradation by thrombin (possibly fibrin-bound thrombin).
 ...
PMID:A novel degradation pathway of tissue factor pathway inhibitor: incorporation into fibrin clot and degradation by thrombin. 929 21

Tissue factor pathway inhibitor (TFPI), a Kunitz-type protease inhibitor with three tandem inhibitory domains (K1, K2 and K3), inhibits the initial reactions of the extrinsic blood coagulation pathway through its K1 and K2 domains. We prepared and characterized a monoclonal antibody (Mab8-1) against TFPI-factor Xa (TFPI-Xa) complex. The reactivities of Mab8-1 toward TFPI-Xa complex, TFPI without C-terminal (TFPI-C)-Xa complex, K1K2-Xa complex and K2K3-Xa complex were examined using a surface plasmon resonance analysis (Biacore). The Biacore system allowed a quantitative analysis of antibody-antigen interaction, in real time, from which the association and dissociation rate constants could readily be obtained. The bindings of Mab8-1 to TFPI-Xa complex, TFPI-C-Xa complex and K2K3-Xa complex were each concentration-dependent. However, no binding of Mab8-1 to the K1K2-Xa complex was observed. The binding of Mab8-1 to TFPI or Xa was also not observed. These results suggested that the epitope for Mab8-1 was exposed in the K3 domain of TFPI, which was generated by the conformational change after the formation of TFPI-Xa complex. We then developed an enzyme-linked immunosorbent assay method specific for TFPI-Xa complex using Mab8-1, and we used this assay to measure plasma levels of TFPI-Xa. The normal range assessed from analyses of plasma from 30 normal healthy volunteers was 17.7-66.7 with a mean of 35.5 +/- 11.7 pmol/l. In order to asses the clinical implication of TFPI-Xa complex in the plasma of patients with thrombotic disorders, plasma concentrations were measured in 37 patients with disseminated intravascular coagulation (DIC) caused by a variety of underlying diseases. The TFPI-Xa antigen levels were significantly higher in the patients with DIC (51.9 +/- 21.6 pmol/l) and the 36 patients with pre-DIC (55.1 +/- 20.2 pmol/l) than in the 137 non-DIC patients (37.9 +/- 13.1 pmol/l). In the patients with DIC or pre-DIC, there was no significant correlation between TFPI-Xa complex and the elevated levels of thrombin-antithrombin complex, plasmin-alpha2 plasmin inhibitor complex, D-dimer, soluble fibrin monomer, soluble thrombomodulin or tissue factor. These data indicate that the plasma level of TFPI-Xa seems to be a novel independent molecular marker of DIC and pre-DIC.
 ...
PMID:Monoclonal antibody specific for tissue factor pathway inhibitor-factor Xa complex: its characterization and application to plasmas from patients with disseminated intravascular coagulation and pre-disseminated intravascular coagulation. 1049 12

Recently we have described a novel secreted protein (the WFIKKN protein) that consists of multiple types of protease inhibitory modules, including two tandem Kunitz-type protease inhibitor-domains. On the basis of its homologies we have suggested that the WFIKKN protein is a multivalent protease inhibitor that may control the action of different proteases. In the present work we have expressed the second Kunitz-type protease inhibitor domain of the human protein WFIKKN in Escherichia coli, purified it by affinity chromatography on trypsin-Sepharose and its structure was characterized by CD spectroscopy. The recombinant protein was found to inhibit trypsin (Ki = 9.6 nm), but chymotrypsin, elastase, plasmin, pancreatic kallikrein, lung tryptase, plasma kallikrein, thrombin, urokinase or tissue plasminogen activator were not inhibited by the recombinant protein even at 1 microm concentration. In view of the marked trypsin-specificity of the inhibitor it is suggested that its physiological target may be trypsin.
 ...
PMID:Expression, purification and characterization of the second Kunitz-type protease inhibitor domain of the human WFIKKN protein. 1270 70

Bleeds in hemophilia are treated either on demand or prophylactically by intravenous replacement therapy with FVIII or FIX. However, there is a call for subcutaneous and less frequent drug administration, and this need may be met by administration of a suitable antibody. Pioneering studies in vitro] and in a rabbit hemophilia model suggest that blockage of tissue factor pathway inhibitor (TFPI) provides a potential alternative approach to current therapy of hemophilia patients. TFPI down-regulates the initiation of coagulation by inhibition of FVIIa/TF/FXa and blockage of TFPI enhances FXa and thrombin generation. In line with these discoveries, TFPI targeting reagents with different potential benefits are currently evaluated as possible novel therapeutic agents. The development and testing of these agents in in vitro and in vivo hemophilia models provide new information about the mode of action of TFPI and its role in hemostasis. Blockage of TFPI with various antagonists has been shown to effectively enhance FX activation by TF/FVIIa and improve clot formation in hemophilia blood and plasma. The monoclonal antibody, mAb 2021, is one such antagonist directed towards the Kunitz-type protease inhibitor (KPI) 2 domain of TFPI which is now being tested in preclinical and clinical trials. Using mAb 2021, we have confirmed the original findings, and further characterized the pro-haemostatic effect of this specific anti-KPI-2 mAb in preclinical studies.
 ...
PMID:Hemostatic properties of a TFPI antibody. 2240 86

Hemophilia is treated by IV replacement therapy with Factor VIII (FVIII) or Factor IX (FIX), either on demand to resolve bleeding, or as prophylaxis. Improved treatment may be provided by drugs designed for subcutaneous and less frequent administration with a reduced risk of inhibitor formation. Tissue factor pathway inhibitor (TFPI) down-regulates the initiation of coagulation by inhibition of Factor VIIa (FVIIa)/tissue factor/Factor Xa (FVIIa/TF/FXa). Blockage of TFPI inhibition may facilitate thrombin generation in a hemophilic setting. A high-affinity (K(D) = 25pM) mAb, mAb 2021, against TFPI was investigated. Binding of mAb 2021 to TFPI effectively prevented inhibition of FVIIa/TF/FXa and improved clot formation in hemophilia blood and plasma. The binding epitope on the Kunitz-type protease inhibitor domain 2 of TFPI was mapped by crystallography, and showed an extensive overlap with the FXa contact region highlighting a structural basis for its mechanism of action. In a rabbit hemophilia model, an intravenous or subcutaneous dose significantly reduced cuticle bleeding. mAb 2021 showed an effect comparable with that of rFVIIa. Cuticle bleeding in the model was reduced for at least 7 days by a single intravenous dose of mAb 2021. This study suggests that neutralization of TFPI by mAb 2021 may constitute a novel treatment option in hemophilia.
 ...
PMID:Hemostatic effect of a monoclonal antibody mAb 2021 blocking the interaction between FXa and TFPI in a rabbit hemophilia model. 2256 84