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

Recent studies on the mechanism of initiation and regulation of blood coagulation are reviewed. In the intrinsic blood coagulation pathway, factor XII, prekallikrein (or factor XI) and high molecular weight kininogen from a complex on an anionic surface, such as exposed subendothelium at the site of vascular trauma. In complex, zymogen factor XII activates prekallikrein (or factor XI) by limited proteolysis to initiate the coagulation cascade. A similar initiating mechanism may be operative in the extrinsic pathway, where zymogen factor VII, complexed with a lipoprotein (tissue factor) and calcium ions, converts factor X to factor Xa. Factor Xa converts prothrombin to thrombin which converts soluble fibrinogen to an insoluble fibrin network which physically arrests the flow of blood from the damaged vasculature. In addition, thrombin converts protein C to activated protein C. Activated protein C functions as a negative regulator in the coagulation process by degrading factor VIIIa and factor Va.
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PMID:Enzymological aspects of blood coagulation. 668 3

Human factor VIII was purified 350,000-fold (relative to plasma) from a commercial factor VIII concentrate. The procedure used standard protein separation techniques and was performed in the absence of protease inhibitors. The product has a specific activity of 4,900 units/mg, an activity-to-antigen ratio of 75:1 (unit/unit) and no more than 0.1% von Willebrand protein. Electrophoresis of the reduced protein in a denaturing polyacrylamide gel showed a single major band of Mr 100,000. Procoagulant activity was eluted from a nondenaturing gel after electrophoresis in the region of the single major band. Thrombin converted the Mr 100,000 polypeptide to a polypeptide of Mr 75,000. The procoagulant activity was increased 10-fold by thrombin or factor Xa and was completely inhibited by activated protein C or factor VIII inhibitor plasma. This factor VIII preparation consists of a single high molecular weight polypeptide chain and has the highest specific activity thus far reported for human factor VIII.
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PMID:Purification and characterization of a highly purified human factor VIII consisting of a single type of polypeptide chain. 681 42

The coagulation protein Factor Va forms the receptor for the serine protease Factor Xa at the platelet surface. This membrane-bound complex of Factor Va and Factor Xa plus calcium constitutes the enzymatic complex prothrombinase, which effects the conversion of prothrombin to the clotting enzyme, thrombin. Studies were undertaken to investigate the proteolytic events accompanying the inactivation of platelet-bound Factor Va by activated protein C as well as the ability of Factor Xa to protect Factor Va from activated protein C inactivation. During the course of these studies, observations were made which indicated that Factor Va was also cleaved by both a platelet-associated protease, as well as Factor Xa. When Factor Va was incubated with washed platelets, electrophoresis and autoradiography of solubilized platelet pellets indicated that three Factor Va peptides were associated with the platelet: component D (Mr = 94,000), component E (Mr = 74,000), and a 90,000-dalton peptide (component D') which appeared with time as the result of a platelet-associated protease cleavage of component D. The Factor Va peptides bound to platelets were proteolytically inactivated by activated protein C, resulting in five peptide products, all of which remained associated with the platelet-membrane surface. Factor Va was protected from activated protein C proteolysis by complex formation with Factor Xa or active site-blocked Factor Xa. However, active Factor Xa cleaved platelet-bound Factor Va to peptide products which also remained associated with the platelet. Whereas activated protein C rapidly cleaved components D and D' with secondary cleavages occurring in component E, Factor Xa rapidly cleaved component E with secondary cleavages occurring in components D and D'. The Factor Xa-cleaved Factor Va is catalytically functional. To determine whether cleavage was necessary for function, prothrombin conversion reaction mixtures were monitored for thrombin formation and Factor Va cleavage with time in a defined phospholipid vesicle model system. The results indicated that Factor Xa cleavage of Factor Va is not essential for Factor Va activity but may promote its ability to function in the prothrombinase complex.
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PMID:Proteolytic alterations of factor Va bound to platelets. 684 22

Factor Xa binds to a receptor available on the platelet surface after the release reaction. The receptor consists of phospholipid and factor V. Factor Xa bound to the receptor catalyses the activation of prothrombin effectively. The effects of bovine protein C, a vitamin-K-dependent zymogen of a serine protease, on prothrombin activation by platelet-bound bovine factor Xa has been studied. Protein C was found to be activated (protein Ca) by thrombin formed in the prothrombin-platelet-factor Xa incubation. Protein Ca in contrast to the zymogen, protein C, or protein Ca inactivated with diisopropylphosphofluoridate inhibited prothrombin activation by factor Xa in the presence of platelets. protein Ca was found to destroy the receptor by proteolysis whereas direct binding of protein Ca to the receptor could not be demonstrated. The inhibition by protein Ca could be monitored as a parallel decrease in factor Xa binding and prothrombin activation. The receptor was protected by factor Xa from proteolysis by protein Ca. Protein Ca was also found to inhibit the interaction between prothrombin and the factor Xa platelet receptor. These results indicate that protein C after activation may have a role as a regulator of prothrombin activation in vivo.
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PMID:Inhibitory effect of activated protein C on activation of prothrombin by platelet-bound factor Xa. 689 81

Factor VIII has been purified approximately 300000-fold from bovine plasma by ammonium sulfate fractionation, glycine precipitation, DEAE-Sephadex column chromatography, sulfate--Sepharose column chromatography, Sephadex G-200 gel filtration, and factor X--Sepharose column chromatography. The highly purified preparation migrated as a triplet on sodium dodecyl sulfate/urea--polyacrylamide gel electrophoresis with apparent molecular weights of 93000, 88000, and 85000. The coagulant activity of the purified preparations was inhibited by antibodies raised in rabbits against either the purified factor VIII protein or a preparation of factor VIII/von Willebrand factor. Antibodies to the purified protein also inhibited the coagulant activity of factor VIII/von Willebrand factor preparations. The purified factor VIII contained no platelet-aggregating activity, as measured in human platelet-rich plasma. The purified preparation of factor VIII was required for the activation of factor X in the presence of factor IXa, calcium, and phospholipid. It was activated about 30-fold by thrombin or factor Xa plus calcium and phospholipid, and each of these reactions was accompanied by a change in the sodium dodecyl sulfate/urea--polyacrylamide gel electrophoresis pattern of the protein. Factor VIII was rapidly inactivated by bovine-activated protein C in a reaction requiring calcium and phospholipid. This reaction was also associated with a change in the sodium dodecyl sulfate/urea--polyacrylamide gel electrophoresis pattern of the highly purified protein. These experiments involving three highly specific serine proteases support the conclusion that the triplet observed on polyacrylamide gels is factor VIII.
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PMID:Preparation and properties of bovine factor VIII (antihemophilic factor). 735 33

Inactivation of membrane-bound factor Va by activated protein C (APC) proceeds via a biphasic reaction that consists of a rapid and a slow phase, which are associated with cleavages at Arg506 and Arg306 of the heavy chain of factor Va, respectively. We have investigated the effects of protein S and factor Xa on APC-catalyzed factor Va inactivation. Protein S accelerates factor Va inactivation by selectively promoting the slow cleavage at Arg306 (20-fold). Factor Xa protects factor Va from inactivation by APC by selectively blocking cleavage at Arg506. Inactivation of factor VaR506Q, which was isolated from the plasma of a homozygous APC-resistant patient and which lacks the Arg506 cleavage site, was also stimulated by protein S but was not affected by factor Xa. This confirms that the target sites of protein S and factor Xa involve Arg306 and Arg506, respectively. Factor Xa completely blocked APC-catalyzed cleavage at Arg506 in normal factor Va (1 nM) with a half-maximal effect (K1/2Xa) at 1.9 nM factor Xa. Expression of cofactor activity of factor Va in prothrombin activation required much lower factor Xa concentrations (K1/2Xa = 0.08 nM). When the ability of factor Xa to protect factor Va from inactivation by APC was determined at low factor Va concentrations during prothrombin activation much lower amounts of factor Xa were required (K1/2Xa = 0.03 nM). This indicates 1) that factor Va is optimally protected from inactivation by APC by incorporation into the prothrombinase complex during ongoing prothrombin activation, and 2) that the formation of a catalytically active prothrombinase complex and protection of factor Va from inactivation by APC likely involves the same interaction of factor Xa with factor Va. In accordance with the proposed mechanisms of action of protein S and factor Xa, we observed that the large differences between the rates of APC-catalyzed inactivation of normal factor Va and factor VaR506Q were almost annihilated in the presence of factor Xa and protein S. This observation may explain why, in the absence of other risk factors, APC resistance only results in a weak prothrombotic condition.
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PMID:Effects of protein S and factor Xa on peptide bond cleavages during inactivation of factor Va and factor VaR506Q by activated protein C. 749 57

Protein C inhibitor (PCI) inhibits multiple plasma serine proteases. To determine which residues contribute to its specificity of inhibition, 19 mutations in the reactive site loop of PCI (from Thr352 to Arg357) were generated and assayed with thrombin, activated protein C (APC), and factor Xa. To identify the intermolecular interactions responsible for these kinetics, a molecular model of PCI was generated using alpha 1-protease inhibitor and ovalbumin as templates. This model of PCI was docked with thrombin, followed by extensive energy minimization, to determine a lowest energy complex. The resulting docked complex was used as a template to form molecular models of PCI-APC and PCI-factor Xa complexes. The best inhibitors of thrombin contained Pro or Gly at the P2 position in place of Phe353, with 2- and 7-fold increases in activity, respectively. These substitutions reduced steric interactions with the 60-insertion loop unique to thrombin. The best inhibitors of APC and factor Xa contained Arg at the P3 position in place of Thr352, with 2- and 5-fold increases in inhibition rates, respectively. The molecular model predicts that Arg in this position could form a salt bridge with Glu217 of each protease. Changing Arg357 at the P3' position had little effect on protease inhibition, consistent with the observation in the model that this residue points toward the body of PCI, forming a salt bridge with Glu220. Given its broad specificity of inhibition, PCI has proven very useful in understanding the nature of serpin-protease interactions using multiple mutations in a serpin assayed with multiple proteases.
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PMID:Intermolecular interactions between protein C inhibitor and coagulation proteases. 754 57

The human intracellular serine proteinase inhibitor, proteinase inhibitor 6 (PI-6), was expressed in the methylotropic yeast Pichia pastoris. The PI-6 cDNA was modified to encode six histidine residues immediately after the initiation codon, and was placed under the control of the P. pastoris alcohol oxidase promoter in the vector pHIL-D2. On the methanol induction, active recombinant PI-6 was produced within the yeast cells, and following cell lysis, was separated from yeast proteins by affinity chromatography using nickel nitrilo-tri-acetic acid (NTA) resin. The interaction of recombinant PI-6 with a range of serine proteinases was studied. Second order association rate constants (ka) were derived for the interaction with trypsin (1.8 x 10(6) M-1 s-1), thrombin (1.2 x 10(5) M-1 s-1), urokinase plasminogen activator (4.0 x 10(4) M-1 s-1), plasmin (1.3 x 10(6) M-1 s-1), and activated protein C (7.5 x 10(3) M-1 s-1). By monitoring complex formation, recombinant PI-6 was also shown to interact with factor Xa. No complex formation was observed with chymotrypsin, human leukocyte elastase, cathepsin G and tissue plasminogen activator, although PI-6 is apparently a substrate for chymotrypsin, leukocyte elastase and cathepsin G.
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PMID:Production and characterization of recombinant human proteinase inhibitor 6 expressed in Pichia pastoris. 754 63

Protein C inhibitor (PCI), a plasma serine protease inhibitor, inhibits several proteases including the anticoagulant enzyme, activated protein C (APC), and the coagulation enzymes, thrombin and factor Xa. Previous studies have shown that thrombin and APC are inhibited at similar rates by PCI and that heparin accelerates PCI inhibition of both enzymes more than 20-fold. We now demonstrate that the thrombin-binding proteoglycan, rabbit thrombomodulin, accelerates inhibition of thrombin by PCI approximately equal to 140-fold (k2 = 2.4 x 10(6) in the presence of TM compared to 1.7 x 10(4) M-1 S-1 in the absence of TM). Most of this effect is mediated by protein-protein interactions since the active fragment of TM composed of epidermal growth factor-like domains 4-6 (TM 4-6) accelerates inhibition by PCI approximately equal to 59-fold (k2 = 1.0 x 10(6) M-1 S-1). The mechanism by which TM alters reactivity with PCI appears to reside in part in an alteration of the S2 specificity pocket. Replacing Phe353 with Pro at the P2 position in the reactive loop of PCI yields a mutant that inhibits thrombin better in the absence of TM (k2 = 6.3 x 10(5) M-1 S-1), but TM 4-6 enhances inhibition by this mutant approximately equal to 9-fold (k2 = 5.8 x 10(6) M-1 S-1) indicating that TM alleviates the inhibitory effect of the less favored Phe residue. These results indicate that PCI is a potent inhibitor of the protein C anticoagulant pathway at the levels of both zymogen activation and enzyme inhibition.
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PMID:Protein C inhibitor is a potent inhibitor of the thrombin-thrombomodulin complex. 759 94

Mutation of residue 192 (chymotrypsin numbering) from Glu to Gln in thrombin and activated protein C has been shown to dramatically alter substrate and inhibitor specificity, in large part by allowing these enzymes to accept substrates with acidic residues in the P3 and/or P3' positions. In factor Xa, residue 192 is already a Gln. We now compare the properties of a Q192E mutant of Gla-domainless factor X (GDFX). Kinetic analysis of prothrombin activation indicates similar affinity of factor Va for GDFXa and GDFXa Q192E (Kd(app) = 3.6 and 3.7 microM, respectively). Prothrombin activation rates are similar for both enzymes with factor Va, but are approximately 10-fold slower for the Q192E mutant without factor Va. This defect is in the activation of prethrombin 2 and is corrected by factor Va only in the presence of fragment 2. Without factor Va, fragment 2 has no influence on bovine prethrombin 2 activation by GDFXa, but fragment 2 enhances prethrombin 2 activation by the Q192E mutant at least 10-fold. These results indicate that the fragment 2 domain of prothrombin probably alters the conformation of the prethrombin 2 domain, selectively improving its presentation to GDFXa Q192E. With respect to inhibition, tissue factor pathway inhibitor and bovine pancreatic trypsin inhibitor are > or = 30 times poorer inhibitors of GDFXa Q192E than of GDFXa, but these enzymes are inhibited at comparable rates by antithrombin. These results indicate that Gln-192 in factor Xa is a key determinant of substrate/inhibitor specificity.
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PMID:Contribution of residue 192 in factor Xa to enzyme specificity and function. 760 83


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