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)

Tumor necrosis factor/cachectin (TNF) is a mediator of the septic state, which involves diffuse abnormalities of coagulation throughout the vasculature. Since previous studies have shown that endothelial cells can play an active role in coagulation, we wished to determine whether TNF could modulate endothelial cell hemostatic properties. Incubation of purified recombinant TNF with cultured endothelial cells resulted in a time- and dose-dependent acquisition of tissue factor procoagulant activity. Concomitant with enhanced procoagulant activity, TNF also suppressed endothelial cell cofactor activity for the anticoagulant protein C pathway; both thrombin-mediated protein C activation and formation of functional activated protein C-protein S complex on the cell surface were considerably attenuated. Comparable concentrations of TNF (half-maximal affect at approximately 50 pM) and incubation times (half-maximal affect by 4 h after addition to cultures) were required for each of these changes in endothelial cell coagulant properties. This unidirectional shift in cell surface hemostatic properties favoring promotion of clot formation indicates that, in addition to leukocyte procoagulants, endothelium can potentially be instrumental in the pathogenesis of the thrombotic state associated with inflammatory and malignant disorders.
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PMID:Modulation of endothelial cell hemostatic properties by tumor necrosis factor. 375 96

125I-labeled heparin cofactor II (HCII) was mixed with plasma and coagulation was initiated by addition of CaCl2, phospholipids, and kaolin or tissue factor. In the presence of 67 micrograms/ml of dermatan sulfate, radioactivity was detected in a band which corresponded to the thrombin-HCII complex (Mr = 96,000) upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No other complexes were observed. The thrombin-HCII complex was undetectable when 5 units/ml of heparin was present or when prothrombin-deficient plasma was used. In experiments with purified proteases, HCII did not significantly inhibit coagulation factors VIIa, IXa, Xa, XIa, XIIa, kallikrein, activated protein C, plasmin, urokinase, tissue plasminogen activator, leukocyte elastase, the gamma-subunit of nerve growth factor, and the epidermal growth factor-binding protein. HCII inhibited leukocyte cathepsin G slowly, with a rate constant of 8 X 10(4) M-1 min-1 in the presence of dermatan sulfate. These results indicate that the protease specificity of HCII is more restricted than that of other plasma protease inhibitors and suggest that the anticoagulant effect of dermatan sulfate is due solely to inhibition of thrombin by HCII.
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PMID:The protease specificity of heparin cofactor II. Inhibition of thrombin generated during coagulation. 383 15

HeLa cells have undetectable tissue factor (thromboplastin) activity when measured by a one-stage coagulation assay. In contrast, these cells accelerated the factor VII-catalyzed cleavage of factor X. The two assays gave similar results after either heating the samples to 100 degrees C for 2 min or exposure to thrombin. Neither of these treatments altered the tissue factor activity of human foreskin fibroblasts, a cell type with high tissue factor activity. HeLa cells contain an inhibitor(s) directed against factor Xa but not thrombin. The inhibitor(s) was inactivated by exposure to thrombin or by heat treatment. Inhibition of factor Xa-catalyzed cleavage of a synthetic peptide was blocked by ethyleneglycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) so the inhibition was apparently dependent on divalent cations. Inhibition was not accelerated by heparin. The inhibitor(s) was not protein C or other serine proteases since it was not inactivated by diisopropylfluorophosphate. The factor Xa inhibitor(s) has been isolated from HeLa cells with an approximate 500-fold increase in specific activity. After SDS-polyacrylamide gel electrophoresis factor Xa-inhibitory activity was recovered from a region corresponding to the major Coomassie-staining band at 43 kDa and in lesser amounts from regions corresponding to 26 and 17 kDa. Cellular inhibitors of coagulation may partially explain the low apparent tissue factor observed in some in vitro cells and may serve a regulatory role in limiting the expression of tissue factor.
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PMID:Alterations in the apparent tissue factor (thromboplastin) expression in HeLa cells by a cellular factor Xa inhibitor. 663 60

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

Two classes of antiphospholipid antibodies (APA) are associated with adverse pregnancy outcomes. Those APA identified by immunoassays using phospholipid-coated surfaces (e.g., anticardiolipin antibodies) seem to bind to the 57 kD anticoagulant protein, beta 2-glycoprotein-I, when complexed with anionic phospholipid bilayers. Such APA may or may not prolong phospholipid-dependent clotting assays. A second class of APA are identified by their interference with phospholipid-dependent clotting assays (i.e., lupus anticoagulants). The latter bind to phospholipids present in a unique hexagonal phase either alone or complexed with prothrombin or beta 2-glycoprotein-I. There is evidence that both classes of APA are directly responsible for adverse pregnancy outcomes including spontaneous abortions, stillbirths, fetal growth retardation, thrombosis, thrombocytopenia, and preeclampsia. Putative APA-mediated pathogenic mechanisms include intervillous thrombosis, intravillous infarctions and decidual vasculopathy. The thrombogenicity of APA may result from their interference with endothelial phospholipids required for antithrombin III and protein C and S anticoagulant activity and prostacyclin synthesis and/or increased endothelial expression of the procoagulants: tissue factor, von Willebrand factor, platelet-activating factor, and plasminogen activator inhibitor type-1. Other prothrombotic properties seem to include: increased platelet aggregation, and reduced beta 2-glycoprotein-1 and annexin V anticoagulant activity. Rigorous diagnostic criteria must be applied to the detection of both classes of APA because the prevention of adverse pregnancy outcomes requires potentially hazardous anticoagulant therapy.
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PMID:The immunobiology and obstetrical consequences of antiphospholipid antibodies. 752 11

Initiation of blood coagulation occurs when the plasma serine protease factor VIIa (fVIIa) binds to its cell-surface receptor/cofactor, tissue factor (TF). This binding interaction mediates a large enhancement in both the proteolytic activity and the amidolytic activity (hydrolysis of small peptidylamide substrates) of fVIIa. This necessitates local changes in the catalytic center of fVIIa of which little is understood. Studies with thrombin and activated protein C have demonstrated that residue E192 (chymotrypsinogen numbering system) near the active site of these proteases is an important determinant for substrate and inhibitor specificity. By homology, residue 192 in fVIIa is K, bringing into question the potential role of this residue in fVIIa. We have prepared two mutants of fVIIa in which K192 has been replaced by either Q (as in factors IX and X) or E (as in thrombin). Both mutants were found to be defective in clotting: fVIIK 192Q was 44% active, while fVIIK192E was completely ineffective. This defect was attributable to proportional decreases in specificity constants for activation of factor X. Although both mutant enzymes were catalytically competent with respect to amidolytic activity, the selectivity of fVIIaK192E was greatly altered. Inhibition of both mutants by the TF pathway inhibitor (TFPI) and bovine pancreatic trypsin inhibitor (BPTI) was also drastically altered. Neither mutant was inhibited by TFPI, while fVIIaK192Q was inhibited by BPTI better than wild-type fVIIa. In contrast, fVIIaK192E was poorly inhibited by BPTI and made more refractory to inhibition when bound to TF. These results suggest a potential role for K192 in governing the substrate and inhibitor specificities of fVIIa.
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PMID:Alteration of the substrate and inhibitor specificities of blood coagulation factor VIIa: importance of amino acid residue K192. 754 28

Phage displaying APPI Kunitz domain libraries have been used to design potent and selective active site inhibitors of human plasma kallikrein, a serine protease that plays an important role in both inflammation and coagulation. Selected clones from two Kunitz domain libraries randomized at or near the binding loop (positions 11-13, 15-19, and 34) were sequenced following five rounds of selection on immobilized plasma kallikrein. Invariant preferences for Arg at position 15 and His at position 18 were found, whereas His, Ala, Ala, and Pro were highly preferred residues at positions 13, 16, 17, and 19, respectively. At position 11 Pro, Asp, and Glu were favored, while hydrophobic residues were preferred at position 34. Selected variants, purified by trypsin affinity chromatography and reverse phase high performance liquid chromatography, potently inhibited plasma kallikrein, with apparent equilibrium dissociation constants (Ki*) ranging from approximately 75 to 300 pM. From sequence and activity data, consensus mutants were constructed by site directed mutagenesis. One such mutant, KALI-DY, which differed from APPI at 6 key residues (T11D, P13H, M17A, I18H, S19P, and F34Y), inhibited plasma kallikrein with a Ki* = 15 +/- 14 pM, representing an increase in binding affinity of more than 10,000-fold compared to APPI. Similar to APPI, the variants also inhibited Factor XIa with high affinity, with Ki* values ranging from approximately 0.3 to 15 nM; KALI-DY inhibited Factor XIa with a Ki* = 8.2 +/- 3.5 nM. KALI-DY did not inhibit plasmin, thrombin, Factor Xa, Factor XIIa, activated protein C, or tissue factor. Factor VIIa. Consistent with the protease specificity profile, KALI-DY did not prolong the clotting time in a prothrombin time assay, but did prolong the clotting time in an activated partial thromboplastin time assay > 3.5-fold at 1 microM.
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PMID:Potent and selective Kunitz domain inhibitors of plasma kallikrein designed by phage display. 759 8

All-trans-retinoic acid (ATRA) induces complete remission (CR) in up to 90% of acute promyelocytic leukemia (APL) patients with rapid amelioration of the bleeding syndrome. Previous studies indicate that ATRA treatment in vitro of the APL NB4 cell line can affect their procoagulant activity (PCA). To assess whether ATRA has this effect also in vivo, we prospectively studied the PCA of bone marrow blasts from APL patients on therapy with ATRA alone or associated with chemotherapy. Samples were obtained before, during, and after ATRA. To characterize the coagulopathy, we measured a series of plasma hemostatic variables before and during the first two weeks of therapy, as follows: (1) markers of hypercoagulability; (2) natural anticoagulants; (3) fibrinolysis proteins; and (4) elastase. The results by enzymatic and immunologic methods show that both total (tissue factor-like) and factor VII-independent (cancer procoagulant-like) blast cell PCAs, present before therapy, were reduced during (69% and 65% decrement, respectively) and virtually undetectable after ATRA. The plasma hemostatic assessment of patients before treatment was elevated hypercoagulability markers, low mean protein C, normal fibrinolysis proteins, and increased elastase. After starting ATRA, hypercoagulability markers were reduced within 4 to 8 days, protein C augmented, the overall fibrinolytic balance was unmodified, and elastase remained elevated. These results were not different either with or without chemotherapy and are consistent with the clinical findings of rapid improvement of the coagulopathy.
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PMID:Loss of blast cell procoagulant activity and improvement of hemostatic variables in patients with acute promyelocytic leukemia administered all-trans-retinoic acid. 762 Jan 61

A highly purified preparation of human plasma factor VIIa was submitted to chromogenic assays with S-2288 factors IXa, Xa, activated protein C and thrombin being absent. Factor VIIa alone or in the presence of calcium, kept its activity even in the presence of high concentrations of aprotinin, inhibition appeared only in the presence of a factor VIIa-tissue factor complex. A two-stage amidolytic assay using activation of purified factor X and hydrolysis of S-2765 chromogenic substrate by the generated Xa was used to show a competitive inhibition with a Ki value of 30 microM. Aprotinin had no effect on factor Xa amidolytic activity per se. The factor VIIa-tissue factor complex could be adsorbed to immobilized aprotinin and removed by a chaotropic ion like KSCN 3 M. The assays with the DFP inactivated VIIa-tissue factor complex proved that the interaction involved the active site of factor VIIa. The inhibition of the VIIa-tissue factor complex was demonstrated in a clotting assay using aprotinin enriched normal or factor VIII deficient plasma.
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PMID:Aprotinin is a competitive inhibitor of the factor VIIa-tissue factor complex. 769 18

The original activated partial thromboplastin time-based assay for activated protein C (APC)-resistant factor Va (FVa) requires carefully prepared fresh plasma and cannot be used in patients receiving warfarin or in patients with antiphospholipid antibodies. A new test is described here that circumvents these limitations and distinguishes without overlap heterozygotes for APC-resistant FVa from persons with normal FV. A diluted test plasma is incubated with an FV-deficient substrate plasma and tissue factor and then clotted with Ca2+ or Ca2+ plus APC. Test results are independent of the FV level or the dilution of the test plasma used. Of 39 controls, 37 gave normal results. Two controls (5%) gave results indicative of APC resistant FVa and on DNA analysis were found to be heterozygous for FV R506Q. Twenty of 21 randomly selected patients receiving warfarin gave normal results. In the single patient with abnormal results, heterozygous FV R506Q was confirmed by DNA analysis. Two of 15 patients with protein S deficiency and 5 of 29 patients with a lupus anticoagulant had abnormal results. APC resistance caused by FV R506Q was confirmed in the five of these seven patients available for DNA analysis. APC-resistant FVa was also detected in 10 of 21 (46%) stored plasma from unrelated patients with venous thrombosis and negative earlier evaluation for a lupus anticoagulant or a deficiency of protein C, protein S, or antithrombin, which confirms a high incidence of this defect among patients with venous thrombosis.
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PMID:Use of a generally applicable tissue factor--dependent factor V assay to detect activated protein C-resistant factor Va in patients receiving warfarin and in patients with a lupus anticoagulant. 770 80


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