Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A low molecular weight platelet inhibitor of factor XIa (PIXI) has been purified 250-fold from releasates of washed and stimulated human platelets. Molecular weight estimates of 8400 and 8500 were determined by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, although a second band of Mr 5000 was present upon electrophoresis. The inhibitor does not appear to be one of the platelet-specific, heparin-binding proteins, since it neither bound to nor was affected by heparin. An amount of PIXI which inhibited by 50% factor XIa cleavage of the chromogenic substrate S2366 (Pyr-Glu-Pro-Arg-pNA-2H2O) only slightly inhibited (5-9%) factor XIIa, plasma kallikrein, plasmin, and activated protein C and did not inhibit factor Xa, thrombin, tPA, or trypsin, suggesting specificity for factor XIa. Kinetic analyses of the effect of PIXI on factor XIa activity demonstrated mixed-type, noncompetitive inhibition of S2366 cleavage and of factor IX activation with Ki's of 7 x 10(-8) and 3.8 x 10(-9) M, respectively. Immunoblot analysis showed that PIXI is not the inhibitory domain of protease nexin II, a potent inhibitor of factor XIa also secreted from platelets. Amino acid analysis showed that PIXI has no cysteine residues and, therefore, is not a Kunitz-type inhibitor. PIXI can prevent stable complex formation between alpha 1-protease inhibitor and factor XIa light chain as demonstrated by SDS-polyacrylamide gel electrophoresis. The inhibition by PIXI of factor XIa-catalyzed activation of factor IX and its capacity to prevent factor XIa inactivation by alpha 1-protease inhibitor, combined with the specificity of PIXI for factor XIa among serine proteases found in blood, suggest a role for PIXI in the regulation of intrinsic coagulation.
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PMID:A low molecular weight platelet inhibitor of factor XIa: purification, characterization, and possible role in blood coagulation. 173 24

In this communication some of the important regulatory mechanisms involving endothelial cell surface associated anticoagulant reactions as well as endothelial cell surface expressed receptors which directly contribute to the inhibition of coagulation are reviewed. In particular, the mechanism of action of protease inhibitors such as antithrombin III, heparin cofactor II, or protease nexin I and their possible interaction with glycosaminoglycan components of the endothelial cells is critically summarized. Thrombin binding to endothelial cells, in particular to thrombomodulin, is believed to be a major event in the induction of anticoagulatory mechanisms such as the protein C/protein S system which warrant a balanced hemostatic system. Additional components such as vascular anticoagulant or extrinsic pathway inhibitor may also contribute to the anticoagulant potential of the vessel wall. Furthermore, the modulation of these membrane-associated anticoagulant reactions by other components such as heparin-binding proteins is discussed.
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PMID:Anticoagulant potential of endothelial cell membrane components. 306 43

Protein C inhibitor (PCI) is a heparin-binding serine proteinase inhibitor (serpin) which is thought to be a physiological regulator of activated protein C (APC). The residues F353-R354-S355 (P2-P1-P1') constitute part of the reactive site loop of PCI with the R-S peptide bond being cleaved by the proteinase. Changing the reactive site P1 and P2 residues to those of either proteinase nexin-1, alpha 1-proteinase inhibitor or heparin cofactor II resulted in a decrease in inhibitory activity towards thrombin and APC. Changing the P2 residue F353-->P generated a rPCI which was a better thrombin inhibitor, but was 10-fold less active with APC. While these results support the concept that the P1 and P2 residues are important in the specificity of PCI, they suggest that the reactive site residues are not the only determinant of serpin specificity. Kinetic analysis of the rPCI variants was consistent with PCI operating by a mechanism similar to that proposed for other serpins. In this model an intermediary complex forms between inhibitor and proteinase that can proceed to either cleavage of the inhibitor as substrate or formation of an inactive complex.
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PMID:Reactive site mutants of recombinant protein C inhibitor. 781 27

The inhibition of activated protein C by six different serine protease inhibitors (serpins) that have arginine residues in the P1 position has been investigated. Micromolar concentrations of C1-inhibitor failed to inhibit the enzyme, and it was inhibited only slowly by antithrombin III with an association rate constant (kass.) of 0.15 M-1.s-1. The kass. values for the other serpins tested (protease nexin I, protein C inhibitor, and mutants of alpha 1-antichymotrypsin and alpha 1-antitrypsin with P1 arginine residues) were at least 1000-fold higher, with P1-Arg-alpha 1-antitrypsin (kass. = 7 x 10(4) M-1.s-1) being the most effective inhibitor. The inhibition with these four serpins appeared to be reversible, with inhibition constants in the nanomolar range. The relatively high value of kass. for protease nexin I (5 x 10(3) M-1.s-1) suggested that it may be involved in the control of activated protein C on the surface of platelets where protein nexin I is present at relatively high concentrations. The value of kass. for protease nexin I, protein C inhibitor and antithrombin III showed a bell-shaped dependence on heparin concentration. At optimal concentrations, heparin accelerated the rate of inhibition by protease nexin I, protein C inhibitor and antithrombin III by 44-, 18- and 13-fold respectively. The kinetic constants for the inhibition of thrombin were also determined, and in all cases the serpins were more effective inhibitors of thrombin. Comparison of the sequences of the active-site regions of activated protein C and thrombin suggested that the more hydrophobic active site of thrombin may be more favourable for interactions with serpins.
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PMID:Interaction of activated protein C with serpins. 821 24

Thrombin's potent effects on astrocytes are mediated by a specific receptor and inhibited by a serpin, protease nexin I (PNI). Thrombomodulin (TM), a membrane protein that forms complexes with thrombin, changing its enzymatic specificity, has not been studied in astrocytes. In primary astrocyte cultures, using Western blotting and immunocytochemistry, we found a 70 kDa TM band and TM localized to the surface with an anti-mouse TM monoclonal antibody. By reverse transcriptase coupled with polymerase chain reaction (RT-PCR), we found the correct sequence for mouse TM mRNA in astrocytes. Finally, we documented calcium-dependent activation of protein C by a thrombin:TM complex with thrombin added to the astrocytes. These results indicate the presence of functionally active TM at the astrocyte surface and add support to a role for thrombin signaling in the nervous system.
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PMID:Novel expression and localization of active thrombomodulin on the surface of mouse brain astrocytes. 906 32

Members of the serpin (serine protease inhibitor) family share a similar backbone structure but expose a variable reactive-site loop, which binds to the catalytic groove of the target protease. Specificity originates in part from the sequence of this loop and also from secondary binding sites that contribute to the inhibitor function. To clarify the intrinsic contribution of the reactive-site loop, alpha1-antichymotrypsin has been utilized as a scaffold to construct chimeras carrying the loop of antithrombin III, protease nexin 1, or alpha1-antitrypsin. Reactive-site loops not only vary in sequence but also in length; therefore, the length of the reactive-site loop was also varied in the chimeras. The efficacy of the specificity transfer was evaluated by measuring the stoichiometry of the reaction, the ability to form an SDS-stable complex, and the association rate constant with a number of potential targets (chymotrypsin, neutrophil elastase, trypsin, thrombin, factor Xa, activated protein C, and urokinase). Overall, substitution of a reactive-site loop was not sufficient to transfer the specificity of a given serpin to alpha1-antichymotrypsin. Specificity of the chimera partly matched that of the loop donor and partly that of the acceptor, whereas the behavior as an inhibitor or a substrate depended upon the targeted protease. Results suggest that, aside from the contributions of the loop sequence and the framework-specific secondary binding sites, an intramolecular control may be essential for productive interaction.
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PMID:Intrinsic specificity of the reactive site loop of alpha1-antitrypsin, alpha1-antichymotrypsin, antithrombin III, and protease nexin I. 919 29

Protein C inhibitor (PCI) is a heparin-binding serine protease inhibitor (serpin) that regulates hemostatic proteases such as activated protein C (APC) and thrombin. The work described here provides further evidence that the PCI H helix, but not the D helix, has a major role in heparin-accelerated inhibition of APC and thrombin. We previously identified Arg-269 and Lys-270 of the H helix [R269A/K270A "H1" recombinant PCI (rPCI)] as important residues both for heparin-accelerated inhibition of thrombin and APC and for heparin-Sepharose binding (Shirk, R. A., Elisen, M. G. L. M., Meijers, J. C. M., and Church, F. C. (1994) J. Biol. Chem. 269, 28690-28695). H1 rPCI was used as a template for Ala-scanning mutagenesis of other H helix basic residues (H1-K266A, H1-K273A, and H1-K266A/K273A) and of the D helix basic residues (H1-K82A, H1-K86A, H1-R90A, and H1-K82A/K86A/R90A). Compared to wild-type rPCI/heparin (k2 = 2.2 x 10(7) M-1 min-1 for thrombin), heparin-accelerated thrombin inhibition was decreased 2.4-fold by H1 rPCI, 4.4-fold by H1-K266A rPCI, and 8-fold by H1-K273A rPCI. H1-K266A/K273A rPCI thrombin inhibition was essentially not accelerated by heparin. A similar trend was found for APC-heparin inhibition using these H helix rPCI mutants. In contrast, the D helix rPCI mutants did not have further reduced heparin-stimulated thrombin or APC inhibition compared to H1 rPCI. Interestingly, all of the H and D helix rPCI mutants had reduced heparin-Sepharose binding activity (ranging from 180 to 360 mM NaCl) compared to wild-type rPCI and H1 rPCI, which eluted at 650 and 430 mM NaCl, respectively. These data suggest that all four basic residues (Lys-266, Arg-269, Lys-270, Lys-273) in the H helix of PCI form a heparin binding site. Our results also imply that while the D helix basic residues (Lys-80, Lys-86, and Arg-90) contribute to overall heparin binding, they are not necessary for heparin-accelerated activity. We conclude that the primary heparin binding site of PCI is the H helix and not the D helix as found in other homologous heparin-binding serpins such as antithrombin III, heparin cofactor II, and protease nexin 1.
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PMID:Contribution of basic residues of the D and H helices in heparin binding to protein C inhibitor. 964 72

Inhibition of factor XIa by protease nexin II (K(i) approximately 450 pM) is potentiated by heparin (K(I) approximately 30 pM). The inhibition of the isolated catalytic domain of factor XIa demonstrates a similar potentiation by heparin (K(i) decreasing from 436 +/- 62 to 88 +/- 10 pM) and also binds to heparin on surface plasmon resonance (K(d) 11.2 +/- 3.2 nM vs K(d) 8.63 +/- 1.06 nM for factor XIa). The factor XIa catalytic domain contains a cysteine-constrained alpha-helix-containing loop: (527)CQKRYRGHKITHKMIC(542), identified as a heparin-binding region in other coagulation proteins. Heparin-binding studies of coagulation proteases allowed a grouping of these proteins into three categories: group A (binding within a cysteine-constrained loop or a C-terminal heparin-binding region), factors XIa, IXa, Xa, and thrombin; group B (binding by a different mechanism), factor XIIa and activated protein C; and group C (no binding), factor VIIa and kallikrein. Synthesized peptides representative of the factor XIa catalytic domain loop were used as competitors in factor XIa binding and inhibition studies. A native sequence peptide binds to heparin with a K(d) = 86 +/- 15 nM and competes with factor XIa in binding to heparin, K(i) = 241 +/- 37 nM. A peptide with alanine substitutions at (534)H, (535)K, (538)H, and (539)K binds and competes with factor XIa for heparin-binding in a manner nearly identical to that of the native peptide, whereas a scrambled peptide is approximately 10-fold less effective, and alanine substitutions at residues (529)K, (530)R, and (532)R result in loss of virtually all activity. We conclude that residues (529)K, (530)R, and (532)R comprise a high-affinity heparin-binding site in the factor XIa catalytic domain.
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PMID:Localization of a heparin binding site in the catalytic domain of factor XIa. 1141 11

The serine proteases, cofactors and cell-receptor molecules that comprise the haemostatic mechanism are highly conserved modular proteins that have evolved to participate in biochemical reactions in blood coagulation, anticoagulation and fibrinolysis. Blood coagulation is initiated by exposure of tissue factor, which forms a complex with factor VIIa and factor X, which results in the generation of small quantities of thrombin and is rapidly shutdown by the tissue factor pathway inhibitor. The generation of these small quantities of thrombin then activates factor XI, resulting in a sequence of events that lead to the activation of factor IX, factor X and prothrombin. Sufficient thrombin is generated to effect normal haemostasis by converting fibrinogen into fibrin. The anticoagulant pathways that regulate blood coagulation include the protein C anticoagulant mechanism, the serine protease inhibitors in plasma, and the Kunitz-like inhibitors, tissue factor pathway inhibitor and protease nexin 2. Finally, the fibrinolytic mechanism that comprises the activation of plasminogen into plasmin prevents excessive fibrin accumulation by promoting local dissolution of thrombi and promoting wound healing by reestablishment of blood flow.
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PMID:Proteases in blood clotting. 1246 64

A large variety of biological processes is mediated by stimulation of the receptor tyrosine kinase MET. Screening a mouse embryo cDNA library, we were able to identify several novel, putative intracellular TPR/MET-substrates: SNAPIN, DCOHM, VAV-1, Sorting nexin 2, Death associated protein kinase 3, SMC-1, Centromeric protein C, and hTID-1. Interactions as identified by yeast two-hybrid analysis were validated in vitro and in vivo by mammalian two-hybrid studies, a far-western assay and coimmunoprecipitation. Participation in apoptosis-regulating mechanisms through interaction with DAPK-3 and cell cycle control via binding to nuclear proteins such as CENPC and SMC-1 are possible new aspects of intracellular MET signaling.
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PMID:Novel interaction partners of the TPR/MET tyrosine kinase. 1554 61


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