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)

Murine monoclonal antibodies, developed following immunization with human protein C, were characterized for their ability to bind antigen in the presence of either CaCl2 or excess EDTA. Three stable clones were obtained which produced antibodies that bound to protein C only in the presence of EDTA. All three antibodies bound to the light chain of protein C on immunoblots and also bound to the homologous proteins factor X and prothrombin in solid-phase radioimmunoassays. One antibody, 7D7B10 was purified and studied further. The binding of 7D7B10 to human protein C was characterized by a KD of 1.4 nM. In competition studies, it was found that the relative affinity of the antibody for protein C was 20-40-fold higher than for prothrombin, fragment 1 of prothrombin, or factor X. In contrast, 7D7B10 was unable to bind to factor IX or bovine protein C. The effect of varying Ca2+ concentration on the interaction of the antibody with protein C was complex. Low concentrations of Ca2+ enhanced the formation of the protein C-antibody complex with half-maximal effect occurring at approximately 60 microM metal ion. However, higher concentrations of Ca2+ completely inhibited 7D7B10 binding to protein C with a K0.5 of 1.1 mM. Furthermore, millimolar concentrations of Mn2+, Ba2+, or Mg2+ also completely abolished antibody binding to protein C. The location of the epitope was delineated by immunoblotting and peptide studies and found to be present in the NH2-terminal 15 residues of protein C. Although residues corresponding to positions 10-13 of human protein C were necessary for maximal binding of the antibody, they were not sufficient. No evidence could be found for involvement of the epitope in metal binding per se. Therefore, the effect of Ca2+ on antibody binding is thought to be due to metal-dependent conformational changes in protein C. It seems likely that Ca2+ occupation of a high affinity site, shown by others to be located in the epidermal growth factor-like domain, causes a conformational change in the NH2-terminal region of protein C which is favorable for antibody interaction, whereas Ca2+ binding to the low affinity site(s), known to be present in the gamma-carboxyglutamic acid domain, causes an unfavorable conformational change.
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PMID:Conformational changes in an epitope localized to the NH2-terminal region of protein C. Evidence for interaction of protein C domains. 247 52

A patient with microvascular thrombosis and thrombocytopenia was found to have a high-titre lupus anticoagulant. The biological effects of the patient's lupus anticoagulant were studied using whole patient serum and plasma. Staph Protein A eluate, and affinity-purified lupus anticoagulant. The latter was isolated by immunoadsorption of serum onto cardiolipin/phosphatidylserine/cholesterol liposomes. Each source of lupus anticoagulant demonstrated 'anticoagulant' activity, defined as prolongation of a modified kaolin clotting time, and contained antibody which bound to endothelial monolayers. Each interfered with thrombin-mediated prostacyclin release from endothelial cells, but had no effect on arachidonate-induced prostacyclin release. In addition, the lupus anticoagulant selectively blocked platelet aggregation in response to thrombin, but not in response to arachidonate, ADP or epinephrine. Lupus anticoagulant also reduced thrombin-stimulated shifts in cytosolic calcium. Thrombin-mediated membrane inositol metabolism and total thrombin binding to endothelium were unaffected by lupus anticoagulant, and another endothelial anticoagulant function related thrombin binding. Protein C activation by thrombomodulin, was not altered. We conclude that the binding of lupus anticoagulant to endothelial cells and platelets does not prevent all thrombin signalling events, but does interrupt prostacyclin production.
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PMID:Lupus anticoagulant induces a selective defect in thrombin-mediated endothelial prostacyclin release and platelet aggregation. 249 19

Plasminogen activator inhibitor (PAI) was purified in active form from porcine platelets under nondenaturing conditions. The purified inhibitor (Mr 47,000) reacts with tissue-type plasminogen activator (t-PA), urokinase (UK), and activated protein C (APC) to yield both SDS-stable complexes and a modified PAI of slightly reduced molecular weight. The second-order rate constants for the inhibition of t-PA and UK by PAI are 3.5 X 10(7) and 3.4 X 10(7) M-1 s-1, respectively. Activated protein C reacts with PAI with a second-order rate constant of 1.1 X 10(4) M-1 s-1. This rate is not accelerated by protein S, phospholipid, and calcium, or heparin. It is concluded that (1) PAI can function as both inhibitor and substrate of its target proteases, (2) if APC promotes fibrinolysis via inactivation of PAI, then APC must be present in concentrations several orders of magnitude greater than t-PA, or the interaction of APC and PAI must be accelerated by presently unknown mechanisms, and (3) in the absence of heparin, platelet PAI is the most rapid inhibitor of APC yet described.
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PMID:Platelet plasminogen activator inhibitor: purification and characterization of interaction with plasminogen activators and activated protein C. 250 42

Presentation of Ag to type I CD4+ T cell clones by chemically fixed APC results in the induction of a long-lasting state of proliferative unresponsiveness in the T cell. Ag-specific TCR interactions do occur during this stimulation, as Ag- and Ia molecule-dependent increases in intracellular calcium free ion concentration can be demonstrated, yet free inositol phosphate generation is low and neither IL-2 synthesis nor proliferation occur. The addition of normal allogeneic accessory cells during this stimulation can restore the T cell proliferative response, as well as prevent the induction of unresponsiveness, thus defining an accessory cell-dependent costimulatory activity necessary for proliferation. We have now examined the biochemical effects of this costimulatory activity on early T cell activation. Normal accessory cell costimulatory activity was found to be incapable of augmenting the generation of free inositol phosphate in response to either fixed APC plus Ag or Con A alone. Furthermore, protein kinase C-dependent CD3 gamma-chain phosphorylation occurred in response to either fixed APC plus Ag or Con A alone, and the addition of normal accessory cells had no effect on the level of this phosphorylation. Finally, minimal CD3 zeta-chain tyrosine phosphorylation occurred during the induction of unresponsiveness with Ag and fixed APC alone and this also was not affected by the costimulatory activity. Our results demonstrate that T cell Ag receptor-mediated increases in intracellular calcium free ion concentration and protein kinase C activation occur independently of an accessory cell-derived costimulatory signal. In the absence of this costimulatory signal, these two intracellular second messengers are insufficient to induce a maximal proliferative response and in fact lead to a state of unresponsiveness.
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PMID:An accessory cell-derived costimulatory signal acts independently of protein kinase C activation to allow T cell proliferation and prevent the induction of unresponsiveness. 252 63

Total and free protein S antigen and C4b-binding protein (C4bp) were determined by rocket immuno-electrophoresis, and functional protein S was assayed by a coagulation method, throughout pregnancy and normal puerperium and in a group of normal full-term newborns (FTN). The functional protein S assay is based on a modification of the APTT, using a mixture of test sample, protein S deficient plasma, activated protein C, phospholipids and calcium. This protein S functional assay is specific for protein S since the APTT prolongation by normal plasma was abolished by incubation of plasma with monospecific, rabbit anti-protein S IgG. The ratios of functional protein S/free protein S antigen in healthy men (n = 13) and women (n = 14) were 1.0 +/- 0.13 (mean +/- SD) and 1.03 +/- 0.20, respectively. During pregnancy there is a decrease in functional protein S and a progressive decrease in total and free protein S antigen, with a functional/free protein S ratio of 0.75 +/- 0.28 in the third trimester of pregnancy (n = 16). In early puerperium the functional protein S level was lower than the free protein S antigen level (ratio about 0.5). In the FTN group, the free protein S level was 39% and protein S activity was about 70% that of adults, with a functional/free protein S ratio of 1.84 +/- 0.31. C4bp values were 23.5 +/- 10.3% in the FTN group, and crossed immunoelectrophoresis showed that in this group the major protein S peak corresponded to free protein S.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Functional and immunologic protein S in normal pregnant women and in full-term newborns. 252 62

Four regulatory phenomena appear to regulate differentially the activation of TH1, TH2, and CTL clones. First, IFN-gamma selectively inhibits proliferation of TH2 but not TH1 cells; lymphokine production by TH2 cells is not affected by IFN-gamma. In addition, when fresh OVA-specific HTL clones are derived in the presence of rIL-2 TH2 cells are preferentially obtained, whereas TH1 cells predominate if cloning is performed in rIL-2 plus rIFN-gamma. These results suggest that the presence of IFN-gamma during the course of an immune response would result in the preferential expansion of HTL of the TH1 phenotype. Proliferation of CTL clones is not influenced by IFN-gamma. Second, different APC populations appear to differentially activate TH1 and TH2 clones. Purified splenic B cells stimulate optimal proliferation of TH2 but not TH1 cells, whereas macrophage/dendritic cells appear to stimulate optimal proliferation of TH1 but not TH2 cells. Since both APC types stimulate lymphokine production by each of the HTL subsets, these results suggest the existence of TH1- and TH2-specific cofactors for growth. Third, high doses of immobilized anti-CD3 mAb inhibit IL-2-dependent proliferation of TH1 but not TH2 clones. Since this effect appears to require calcium, this observation suggests that TCR-mediated signalling events might differ between the two HTL subsets. Indeed, little or no increase in [Ca++]i can be detected in TH2 clones stimulated with Con-A, while such an increase is easily discernible in TH1 cells. Although high concentrations of immobilized anti-CD3 mAb inhibit IL-2-dependent proliferation of CTL clones, proliferation of these cells in response to immobilized anti-CD3 alone reaches a plateau. Since activation with anti-CD3 is thought to mimic antigenic stimulation, these results suggest that antigen concentration may play a role in determining which predominant T-cell types proliferate in a particular immunological situation. Fourth, pretreatment of TH1 cells, but not TH2 cells or CTL, with IL-2 results in decreased lymphokine production and proliferation in response to subsequent stimulation via the TCR. This antigen-responsive state appears to involve a defect in calcium-dependent signalling, providing additional evidence for different signalling mechanisms in TH1 and TH2 clones.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Regulation of T-cell activation: differences among T-cell subsets. 253 16

Four monoclonal antibodies to human thrombomodulin were characterized. Binding of two of these antibodies was dependent on the presence of calcium ions, and approximately 5 mM calcium was required for their maximum binding. These two antibodies inhibited the binding of thrombin to thrombomodulin, thereby inhibiting activation of protein C catalyzed by thrombin-thrombomodulin complex. These two antibodies bind to a major active fragment formed by limited proteolytic digestions of thrombomodulin with elastase and trypsin, suggesting that the antibodies bind to the thrombin-binding site (or its vicinity) located in the epidermal growth factor (EGF)-homology domain. One of the other calcium-independent antibodies also inhibited the binding of thrombin and the activation of protein C, but the inhibition was very weak and was observed only when the antibody was present in a molar excess over thrombomodulin. This antibody did not bind to the protease digests of thrombomodulin. Another calcium-independent antibody did not inhibit either thrombin binding or protein C activation, but bound to the active fragment of protease digests, suggesting that the antibody binds to a region other than the thrombin-binding site in the EGF-homology domain. These observations suggest that thrombomodulin undergoes a calcium-dependent conformational change which may occur in proximity to a thrombin-binding site located in the EGF-homology domain.
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PMID:Monoclonal antibodies to human thrombomodulin whose binding is calcium dependent. 254 63

The location of the active site of the membrane-bound anticoagulant complex of thrombin and thrombomodulin has been determined relative to the membrane surface using fluorescence energy transfer. Thrombin was reacted with 5-(dimethylamino)-1-naphthalenesulfonylglutamylglycylarginyl chloromethyl ketone (DEGR-CK) to yield DEGR-thrombin, an analogue of thrombin with a fluorescent dye covalently attached to its active site. When DEGR-thrombin was titrated with thrombomodulin that had been reconstituted into phospholipid vesicles containing octadecylrhodamine, singlet-singlet energy transfer was observed between the donor dyes, each in an active site of a DEGR-thrombin bound to thrombomodulin, and the acceptor dyes at the outer surface of the phospholipid bilayer. The extent of energy transfer reached a maximum when DEGR-thrombin and thrombomodulin were equimolar in the sample, as expected for the formation of a 1:1 complex between thrombin and thrombomodulin. This energy transfer was dependent upon the binding of DEGR-thrombin to thrombomodulin because no energy transfer was observed with vesicles that lacked thrombomodulin, and the extent of energy transfer was reduced greatly by the addition of excess unmodified nonfluorescent thrombin to compete with DEGR-thrombin for binding to the thrombomodulin. From the dependence of the energy transfer upon the acceptor density and assuming kappa 2 = 2/3, the distance of closest approach between a dye in the active site of the thrombin-thrombomodulin complex and a dye at the membrane surface was determined to average 66 A (65 +/- 3 A for phosphatidylcholine vesicles without and 67 +/- 5 A for those with 20% phosphatidylserine). This distance was also insensitive to the presence or absence of Ca2+. These direct measurements indicate that the active site of the membrane-bound thrombin-thrombomodulin complex is located far above the phospholipid surface, that the peptide bond cleaved during the activation of protein C is situated about 66 A above the membrane, that the thrombin binding site on thrombomodulin is positioned more than 45 A above the membrane, ant that thrombin, with a diameter near 40 A, is not positioned alongside thrombomodulin near the membrane to form the thrombin-thrombomodulin complex but is instead bound "on top" of thrombomodulin.
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PMID:The active site of the thrombin-thrombomodulin complex. A fluorescence energy transfer measurement of its distance above the membrane surface. 254 42

A complex composed of factor Xa and phospholipid vesicles assembled in the presence of calcium ions catalyzes a discrete cleavage of the heavy chain of bovine protein C that is indistinguishable from that produced by thrombin as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This cleavage generates an active site capable of hydrolyzing small substrates and inactivating factor Va function in the prothrombinase complex. Activation of protein C by factor Xa requires both calcium ions and phospholipid vesicles and proceeds at a rate an order of magnitude greater than that observed for alpha-thrombin in solution. gamma-Carboxyglutamic acid-domainless protein C is not activated by factor Xa, consistent with the requirement for phospholipid and distinguishing this reaction from protein C activation by thrombin. Thrombomodulin serves as a cofactor for the factor Xa-catalyzed reaction, forming a 1:1 complex with factor Xa (apparent Kd = 5.7 X 10(-10) M) and stimulating the saturated rate of protein C activation by factor Xa (kcat = 149 min-1) to levels comparable with the thrombin-thrombomodulin complex. Protein C activation by factor Xa is not inhibited by the specific thrombin inhibitor dansyl-N-(3-ethyl-1,5-pentanediyl)amide but is inhibited by antithrombin III, tripeptide-chloromethyl ketones, and the monoclonal antibody alpha-BFX-2b that is highly specific for factor Xa. These data indicate that thrombomodulin is promiscuous in its role as a cofactor and suggest the existence of an alternative pathway for protein C activation in vivo.
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PMID:The activation of bovine protein C by factor Xa. 255 Apr 35

Plasma protein C inhibitor (PCI) was purified to homogeneity (greater than 95%) with good recovery (greater than 25%) and reproducibility, and the inhibition of a number of blood clotting and fibrinolytic enzymes by purified PCI was studied. PCI inhibited activated protein C (APC), two-chain urokinase (2c-uPA), two-chain tissue plasminogen activator (2c-tPA), thrombin, factor Xa, plasma kallikrein and factor XIa, and this inhibition was accelerated by heparin. The inhibition of each enzyme was accompanied by formation of enzyme inhibitor complexes and by degradation of the inhibitor to lower molecular weight derivatives. Plasma kallikrein and factor XIa cleaved PCI of native Mr = 57,000 into two products with Mr = 54,000 and 52,000 whereas the other enzymes converted the PCI to a product with Mr = 54,000. PCI did not detectably inhibit alpha-factor XIIa or plasmin. Kinetic studies using PCI yielded the following second-order rate constants for inhibition of human APC, 2c-uPA, 2c-tPA, thrombin, factor Xa, kallikrein and factor XIa respectively: 0.65 x 10(4), 0.22 x 10(4), 0.08 x 10(4), 0.61 x 10(4), 2.01 x 10(4), 6.50 x 10(4), and 9.03 x 10(4) M-1s-1 in the absence of heparin and 1.58 x 10(6), 0.43 x 10(6), 0.03 x 10(6), 0.52 x 10(6), 0.09 x 10(6), 0.18 x 10(6) and 0.74 x 10(6) M-1s-1 in the presence of optimal concentrations of heparin. The rate constants for the inhibition of factor XIa and 2c-uPA by PCI suggest a possible role of PCI in the physiologic regulation of these enzymes. The second order rate constants for inhibition of bovine APC and Gla-domainless bovine APC by human PCI were 0.61 x 10(4) and 0.26 x 10(4) M-1s-1 in the absence of heparin and 0.54 x 10(6) and 0.71 x 10(6) M-1s-1 in the presence of heparin, respectively. Calcium ions (0.05 to 4 mM) did not affect these rate constants. The results obtained with normal and Gla-domainless APC indicate that the Gla domain of APC is not required for inactivation by PGI and is not essential for the heparin stimulation of this reaction.
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PMID:Purification and characterization of plasma protein C inhibitor. 255 Oct 64


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