Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.37 (neutrophil elastase)
 4,078 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The degradation of the coagulation cofactors Va and VIIIa by activated protein C (APC), is dependent on calcium, a suitable surface and protein S. The latter is a vitamin K-dependent protein which functions as a cofactor in the reaction. In this study we investigated the role of neutrophils in regulating the activity of protein S. Protein S specifically bound to neutrophils pretreated with di-isopropyl fluorophosphate (DFP) in a time-dependent and saturable reaction. Double reciprocal plot analysis indicated 5 x 10(6) protein S binding sites per neutrophil with half saturable binding occurring at a protein S concentration of 18 nM. Binding was unaffected by the presence of APC, factor V or factor Va. Failure to preincubate the cells with DFP allowed a surface protease to rapidly cleave protein S resulting in its dissociation from its binding site. The cleavage was associated with loss of protein S cofactor activity. The major neutrophil surface associated enzyme that caused this cleavage was identified as elastase. Exposure of neutrophils to ionophore A23187, soluble heat aggregated IgG and serum opsonized zymosan caused the release of a protease/s which cleaved and inactivated protein S. Purification of the protease revealed that it was elastase. This was verified by incubating the neutrophil releasate with anti-neutrophil elastase antibody which inhibited protein S cleavage. Purified neutrophil elastase in concentrations observed under physiological conditions resulted in rapid cleavage and inactivation of protein S. We propose that neutrophils by binding and inactivating protein S serve as an important control for the activity of this natural anticoagulant.
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PMID:The binding and regulation of protein S by neutrophils. 183 84

Protein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC. In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor Xa was inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor Xa mediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S. These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.
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PMID:The anticoagulant properties of a modified form of protein S. 297 8

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

Protein C inhibitor (PCI), antithrombin, and heparin cofactor II are members of the serine proteinase inhibitor (serpin) superfamily that inhibit proteinases at rates which increase in the presence of the glycosaminoglycan heparin. These studies were undertaken to understand how PCI activity is modulated by various substances that are found in or interact with the vascular endothelium/basement membrane. The effects of antithrombin-heparin, thrombomodulin, vitronectin and leukocyte elastase on PCI-thrombin and PCI-activated protein C (APC) interactions were investigated. Antithrombin, which does not inhibit APC but which does bind to heparin/heparan sulphate with higher affinity than PCI, caused only a small decrease in the inhibition rate of PCI-APC in the presence of unfractionated heparin. Thrombomodulin, a chondroitin sulphate-containing proteoglycan, accelerated PCI inhibition of thrombin and APC. PCI-thrombin in the presence or absence of heparin bound plastic absorbed vitronectin, but neither PCI alone nor PCI-APC bound. Vitronectin also decreased the inhibition rate of PCI-thrombin and PCI-APC in the presence of low concentrations of heparin. Leukocyte elastase proteolytically inactivated PCI in a reaction that was accelerated by heparin. Overall, these results indicate that PCI activity is modulated by these endothelial cell/basement membrane-based substances in similar ways as other heparin-binding serpins, especially antithrombin.
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PMID:Modulation of protein C inhibitor activity. 753 47

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

Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, has been characterized as an extremely potent anticoagulant and reversible tight-binding inhibitor of human factor Xa (FXa). The ecotin gene was cloned by PCR, highly expressed in E. coli, and purified from the E. coli periplasm. The binding of ecotin to FXa was stoichiometric with an equilibrium dissociation constant Ki of 54 pM. The association rate constant was 1.35 x 10(6) M-1 s-1, and the dissociation rate constant, measured in the presence of human leukocyte elastase (HLE) to prevent reassociation of ecotin with FXa, was 6.5 x 10(-5) s-1. Ecotin prolonged clotting time ca. 10-fold at 0.3 microM and at 2 microM in activated partial thromboplastin time and prothrombin time assays, respectively. Ecotin did not effectively inhibit the human plasma proteases thrombin, tissue factor.factor VIIa, factor XIa, activated protein C, plasmin, or tissue plasminogen activator (t-PA); however, it did potently inhibit factor XIIa, plasma kallikrein, HLE, and bovine trypsin and chymotrypsin. Coincubation of ecotin and FXa at 10 microM each resulted in a (ecotin)2.(FXa)2 complex as determined by gel filtration. Dimerization of ecotin alone was measured by fluorescence titration which yielded a Kd of ca. 390 nM. FXa cleaved ecotin slowly at pH 4.0 between M84 and M85. Replacement of the P1 Met84 residue with Arg and Lys led to FXa inhibitors with Ki values of 11 and 21 pM, respectively. The P1 Arg and Lys mutants also significantly inhibited thrombin, factor XIa, activated protein C, plasmin, factor XIIa, kallikrein, and bovine trypsin and chymotrypsin but did not inhibit tissue factor.factor VIIa, t-PA, or HLE.
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PMID:Ecotin is a potent anticoagulant and reversible tight-binding inhibitor of factor Xa. 814 99

Leukocytes release lysosomal proteases and reactive oxygen species in response to various stimuli that damage the adjacent endothelial cells. We investigated the effects of granulocyte lysosomal proteases (granulocyte elastase [GE] and cathepsin G [CG] and/or hydrogen peroxide (H2O2) on thrombomodulin (TM) activity of endothelial cells. We wished to determine whether the activated leukocytes damage the nonthrombogenic systems of endothelial cells. When cultured human umbilical vein endothelial cells (HUVECs) were incubated with GE, TM activity of the cells (as judged by the protein C activation capacity) decreased to about 10% of control with the concomitant increase of immunoreactive TM concentration in the conditioned media. CG also decreased TM activity to about 20% of control with the concomitant increase in immunoreactive TM concentration in the conditioned media. The GE- or CG-induced inactivation of TM was not observed in the presence of alpha 1-proteinase inhibitor. Immunoblot analysis showed that CG cleaved purified TM to yield one major fragment with an M(r) of 43,000; TM activity of this fragment was about 10% of the control activity. When purified TM was incubated with GE, TM activity decreased to 10% of control, and no detectable band was found on immunoblotting, suggesting that CG and GE cleave TM into inactive fragments and that GE degrades the epitope structure of TM. Although H2O2 (1.0 mmol/L) enhanced chromium 51 release from prelabeled HUVECs after 30 minutes of incubation, it decreased TM activity only slightly.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Granulocyte proteases and hydrogen peroxide synergistically inactivate thrombomodulin of endothelial cells in vitro. 820 Dec 66

In a previous report, we described the molecular cloning, expression, and partial characterization of a second human tissue factor pathway inhibitor (TFPI), which we designated as TFPI-2 [Sprecher, C. A., et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3353-3357]. Recombinant TFPI-2 inhibited the amidolytic activity of trypsin as well as that of factor VIIa in complex with tissue factor. TFPI-2 recently has been shown to be identical to placental protein 5 (PP5), a glycoprotein originally isolated from placenta that exhibits serine protease inhibitory activity. In the present study, we have examined TFPI-2/PP5 for its ability to inhibit a number of serine proteases involved in blood coagulation and fibrinolysis, inasmuch as TFPI-2/PP5 prolonged the coagulation time of human plasma induced by either tissue factor or contact activation in a dose-dependent manner. In addition to its ability to inhibit the amidolytic and proteolytic activities of the factor VIIa-tissue factor complex, TFPI-2/PP5 strongly inhibited the amidolytic activities of human factor XIa, human plasma kallikrein, and human plasmin with Ki values of 15, 25, and 3 nM, respectively. TFPI-2/PP5 was also a weak inhibitor of the activation of factor X by a complex of human factor IXa and poly(lysine) with an apparent Ki of 410 nM. Heparin markedly enhanced the ability of TFPI-2/PP5 to inhibit factor VIIa-tissue factor both in the solution phase and on cell surfaces. In addition, heparin augmented the inhibition of human factor Xa amidolytic activity at relatively high levels (10-100 nM) of TFPI-2/PP5. No significant inhibition of glandular kallikrein, urinary plasminogen activator, tissue plasminogen activator, human activated protein C, human factor Xa, human thrombin, or leukocyte elastase was observed when these proteases were incubated with TFPI-2 in the absence of heparin.
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PMID:Inhibitory properties of a novel human Kunitz-type protease inhibitor homologous to tissue factor pathway inhibitor. 855 84

We investigated the effect of activated protein C (APC) on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats to investigate the possible usefulness of APC as a treatment for adult respiratory distress syndrome. Intravenously administered LPS (5 mg/kg) significantly increased pulmonary vascular permeability. APC prevented the LPS-induced increase in pulmonary vascular permeability observed at 6 hours. Heparin plus antithrombin III (ATIII) and active site-blocked factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, inhibited LPS-induced coagulopathy but did not prevent LPS-induced pulmonary vascular injury. LPS-induced pulmonary vascular injury was significantly attenuated in rats with nitrogen mustard-induced leukocytopenia and in rats treated with ONO-5046, a potent granulocyte elastase inhibitor. Administration of LPS also increased pulmonary accumulation of leukocytes, as evaluated by measurement of myeloperoxidase activity in the lungs. APC significantly reduced LPS-induced increases in pulmonary accumulation of leukocytes at 1 hour. Neither ATIII plus heparin nor DEGR-Xa inhibited leukocyte accumulation. Active site-blocked APC (DIP-APC) prevented neither the LPS-induced pulmonary accumulation of leukocytes nor the LPS-induced increase in pulmonary vascular permeability. These results suggest that the mechanism of APC inhibition of LPS-induced pulmonary vascular injury was independent of its anticoagulant activity and was related to its ability to inhibit accumulation of leukocytes. In addition, these findings suggest that the serine protease activity of APC may be essential to its inhibitory effect on LPS-induced pulmonary accumulation of leukocytes and subsequent pulmonary vascular injury.
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PMID:Activated protein C attenuates endotoxin-induced pulmonary vascular injury by inhibiting activated leukocytes in rats. 855 86

Adult respiratory distress syndrome (ARDS) is a serious complication of disseminated intravascular coagulation (DIC) or multiple organ failure. To determine whether recombinant soluble human thrombomodulin (rsTM) may be useful in treating ARDS due to sepsis, we investigated the effect of rsTM on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats. The intravenous administration of rsTM prevented the increase in pulmonary vascular permeability induced by LPS. Neither heparin plus antithrombin III (AT III) nor dansyl Glu Gly Arg chloromethyl ketone-treated factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, prevented LPS-induced vascular injury. The agents rsTM, heparin plus AT III, and DEGR-Xa all significantly inhibited the LPS-induced intravascular coagulation. Recombinant soluble TM pretreated with a monoclonal antibody (moAb) that inhibits protein C activation by rsTM did not prevent the LPS-induced vascular injury; in contrast, rsTM pretreated with a moAb that does not affect thrombin binding or protein C activation by rsTM prevented vascular injury. Administration of activated protein C (APC) also prevented vascular injury. LPS-induced pulmonary vascular injury was significantly reduced in rats with leukopenia induced by nitrogen mustard and by ONO-5046, a potent inhibitor of granulocyte elastase. Results suggest that rsTM prevents LPS-induced pulmonary vascular injury via protein C activation and that the APC-induced prevention of vascular injury is independent of its anticoagulant activity, but dependent on its ability to inhibit leukocyte activation.
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PMID:Recombinant human soluble thrombomodulin reduces endotoxin-induced pulmonary vascular injury via protein C activation in rats. 860 7


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