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)

To estimate the minimal structural requirements for cross-reaction of idiotypic determinants, we determined the capacity of monoclonal antibodies specific for the idiotype of the phosphorylcholine (PC)-binding myeloma protein TEPC-15 for cross-reactivities with the PC-binding, acute-phase protein C-reactive protein (CRP) and the hemagglutinin from the horseshoe crab Limulus polyphemus (limulin), which binds sialic acid and PC. Certain monoclonal antibodies (MAb) to the TEPC-15 idiotype showed strong cross-reactions with CRP and limulin when tested by enzyme-linked immunoadsorbent assays. The specificity of the cross-reactivities was confirmed by testing the binding of the reactive anti-TEPC-15 MAb to both CRP and limulin in the presence of p-nitrophenylphosphorylcholine (pNPPC), N-acetylneuraminic acid, and bovine submaxillary mucin. The binding of the MAb to both CRP and limulin was strongly decreased by pNPPC, partially decreased by free PC, and not affected by N-acetylneuraminic acid or bovine submaxillary mucin. Neither CRP nor limulin showed significant overall sequence homology to vertebrate immunoglobulins. However, CRP, limulin, and TEPC-15 variable region heavy chain (VH) shared short stretches of homology (8-10 amino acids) that mapped to a stretch comprised of the second complementarity determining region and third framework region of the TEPC-15 VH. These results might reflect either evolutionary convergence forced upon molecules of diverse evolutionary histories because of steric requirements of binding the same ligand, or a conservation of primitive combining site gene segments in evolution.
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PMID:Invertebrate recognition protein cross-reacts with an immunoglobulin idiotype. 620 May 68

Purified human factor VIII procoagulant protein (VIII:C) was treated with purified human activated protein C (APC) and the loss of VIII:C activity correlated with proteolysis of the VIII:C polypeptides. APC proteolyzed all VIII:C polypeptides with mol wt = 92,000 or greater, but not the doublet at mol wt = 79-80,000. These results and our previous thrombin activation studies of purified VIII:C, are analogous with similar studies of factor V and form the basis for the following hypothesis: activated VIII:C consists of heavy and light chain polypeptides [mol wt = 92,000 and mol wt = 79-80,000 (or 71-72,000), respectively] which are similar in Mr to the heavy and light chains of activated factor V. Thrombin activates VIII:C and V by generating these polypeptide chains from larger precursors and APC inactivates both molecules by cleavage at a site located in the heavy chain region of activated VIII:C and V.
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PMID:Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V. 641 99

Activated factor V (factor Va) is composed of two nonidentical subunits which can be dissociated on chelation of the bound Ca2+ with EDTA. The isolated subunits can be recombined in the presence of Ca2+ to form factor Va. The factor Va heavy chain (Mr = 94,000) binds to prothrombin in a specific and Ca2+-independent fashion. Following inactivation of either factor Va or the factor Va heavy chain by limited proteolysis with activated protein C, factor Va no longer binds to the immobilized prothrombin. Factor Va also binds specifically to (p-amidinophenyl)-methanesulfonyl-factor Xa-Affi-Gel 15. However, neither isolated subunit binds to this column. Factor Va inactivated by activated protein C is no longer retained by the factor Xa column. This data suggests that both subunits are required for optimal factor Va-factor Xa interaction and that inactivation of factor Va with activated protein C reduces the affinity of factor Va for both prothrombin and factor Xa.
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PMID:Loss of prothrombin and of factor Xa-factor Va interactions upon inactivation of factor Va by activated protein C. 643 88

The two-subunit structure of the factor Va molecule is essential to its function in the prothrombinase complex. In the presence of phospholipids, the cleavage of the light chain of bovine factor Va by activated protein C proceeded at the same rate as the cleavage of the heavy chain. The limited proteolysis of factor Va is accompanied by a parallel loss of factor Va activity. Evidence that loss of activity was solely the result of the cleavage of the heavy chain, was obtained from reconstitution experiments utilizing cleaved and intact chains. The pseudo first-order rate constant of factor Va inactivation by activated protein C was found to be dependent on the amount of phospholipid-bound activated protein C and not on the amount of phospholipid-bound factor Va. However, phospholipids enhance the rate of proteolysis of the phospholipid-binding subunit, i.e. the light chain, and not the cleavage of the heavy chain. Cleavage of the heavy chain and as a consequence the inactivation of factor Va by activated protein C is mediated by phospholipid-bound light chain. After cleavage of the light chain, the 'two-subunit' structure, as well as the phospholipid-binding properties of factor Va were found to be conserved.
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PMID:Functional properties of factor Va subunits after proteolytic alterations by activated protein C. 654 61

Protein C is a precursor to a serine protease that is present in mammalian plasma. In its activated form, it readily inactivates factor Va and factor VIIIa, two proteins that participate as cofactors in the blood coagulation cascade. In the present studies, a lambda gt11 library containing cDNA inserts prepared from human liver mRNA has been screened with an antibody to human protein C. Seven positive clones were isolated from 2 X 10(6) phage and were plaque-purified. The cDNA inserts of two of these phage were sequenced and shown to code for human protein C. Each cDNA insert coded for a portion of the light chain of the molecule, a connecting region, the heavy chain, a stop codon, a 3'-noncoding region, and a poly(A) tail. The length of the noncoding sequence on the 3' end differed in the two clones, but each contained a processing or polyadenylylation signal followed by a poly(A) tail. The amino acid sequence as determined from the cDNA indicates that protein C is synthesized as a single-chain polypeptide containing the light chain and the heavy chain connected by a dipeptide of Lys-Arg. The single-chain molecule is then converted to the light and heavy chains by cleavage of two or more internal peptide bonds. In plasma, the heavy and light chains of protein C are linked together by a disulfide bond. The amino acid sequence of human protein C shows a high degree of homology with that of the bovine molecule. The DNA sequence coding for the catalytic region near the active site serine in human protein C also showed a high degree of DNA and amino acid sequence identity with prothrombin, factor IX, and factor X, three of the other vitamin K-dependent serine proteases that are present in plasma.
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PMID:Characterization of a cDNA coding for human protein C. 658 23

Thrombin-activated vitamin K-dependent protein C purified from human plasma has a potent anticoagulant effect on human plasma, whereas its bovine counterpart has a very weak anticoagulant effect on human plasma. This species difference was found to be partly due to a more rapid degradation of human factor Va by human than by bovine activated protein C. In the presence of phospholipid, activated human protein C cleaves several peptide bonds in fragment D (heavy chain of factor Va), whereas in fragment F1F2 (light chain of factor Va) there appears to be only one peptide bond that is slowly cleaved. The degradation of fragment D is accompanied by a parallel loss of factor V activity. With the blood coagulation factor Xa bound to factor Va, fragment D is protected from degradation by activated protein C, and factor Va remains active. Fragment D isolated from factor Va was exposed to activated protein C in the presence of phospholipid and was found not to be degraded. This observation suggests that fragment D of factor V is bound to phospholipid via fragment F1F2.
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PMID:Inactivation of human coagulation factor V by activated protein C. 668 87

The amino acid sequence of the heavy chain of protein C from bovine plasma is reported together with supporting data. The heavy chain contains 260 amino acid residues and it has three carbohydrate side chains. Its structure was determined by sequenator degradation of fragments obtained by enzymatic and chemical cleavage. The sequence is homologous with those of the other serine proteases. On activation, an NH2-terminal tetradecapeptide is removed.
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PMID:Amino acid sequence of the heavy chain of bovine protein C. 689 77

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 (PC) is a vitamin K-dependent zymogen that inactivates factors Va and VIIIa after its activation by thrombin complexed to thrombomodulin. We characterized a monoclonal antibody (mAb) against PC, whose only influence on PC functions was to inhibit PC activation by the thrombin-thrombomodulin complex. It recognized an epitope in the PC heavy chain, the conformation of which is calcium-dependent. The mAb did not recognize a natural PC variant that was not activated by the thrombin-thrombomodulin complex (mutation R229Q) and did recognize a synthetic peptide corresponding to PC amino acids 225-235 in an Elisa assay. The peptide inhibited PC activation by the thrombin-thrombomodulin complex. These data confirm that the calcium-binding loop of the serine-protease domain is involved in the interaction of PC with the thrombin-thrombomodulin complex.
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PMID:Amino acids 225-235** of the protein C serine-protease domain are important for the interaction with the thrombin-thrombomodulin complex. 754 Sep 90

The inactivation of human platelet factor Va by activated protein C (APC) was analyzed by functional assessment of cofactor activity and Western blotting analysis to visualize the factor Va fragments accompanying proteolysis. Platelets were treated with thrombin to facilitate both their activation as well as the release and further activation of platelet factor Va, followed by APC addition. The rates of inactivation were donor-dependent such that 15-60% of the initial cofactor activity was lost within 5 min of APC addition with as much as 10-20% of the activity still remaining after 2 h of incubation. Western blot analysis using a monoclonal antibody that recognizes an epitope between amino acid residues 307 and 506 of the factor V molecule suggested that the factor Va activity resistant to APC inactivation was due to residual heavy chain. Furthermore, in contrast to studies with normal plasma-derived factor Va, two possible cleavage mechanisms could explain the platelet factor Va fragments observed. APC can cleave platelet factor Va initially at Arg506, with subsequent cleavages occurring at Arg306 and Arg679. Alternatively, APC can cleave at Arg306 initially, with further cleavage at Arg679 then at Arg506 or at Arg506 followed by cleavage at Arg679. Similar results were obtained if platelets were removed from the inactivation mixtures and phospholipid vesicles were used to supply the membrane surface required for inactivation, suggesting that the order of platelet factor Va peptide bond cleavage or the amount of cofactor activity remaining was not altered by either of these surfaces. Thus, APC is unable to effect the complete inactivation of platelet factor Va, even though it would appear that the same cleavages which render the plasma cofactor inactive are occurring in the platelet cofactor. Analogous protocols were used to study an individual heterozygous for the Arg506-->Gln506 mutation (Factor V Leiden, Factor VR506Q). With respect to the mutant platelet factor Va in the presence of APC, > 70% of the initial cofactor activity remained after 1 min, with 30% activity still remaining after 2 h. As seen in studies of the APC-catalyzed inactivation of plasma factor VaR506Q, proteolysis of the mutant platelet factor Va confirms that even though cleavage at Arg306 will occur in the absence of cleavage at Arg506, the rate of inactivation is slower. Collectively these data suggest that when compared to normal plasma factor Va, differences in normal platelet factor Va which define: 1) whether the heavy chain is susceptible to cleavage at Arg306 or Arg506 and 2) the extent to which it is cleaved initially at Arg306, in contrast to cleavage of Arg506, will define both the extent and rate of inactivation.
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PMID:The mechanism of inactivation of human platelet factor Va from normal and activated protein C-resistant individuals. 765 63


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