Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tissue factor occurs in a dormant state on the surface of cultured normal human fibroblasts and WISH 1 amnion cells. The activity of undisturbed monolayers or cells lifted with brief trypsin treatment (0.125 per cent trypsin for 1 min) increases up to 60-fold upon prolonged digestion with dilute trypsin (0.0025 per cent trypsin for 30 min); activity appears subsequent to cell detachment. Up to 70 per cent of the total cellular tissue factor becomes active under these conditions and is released from the cells. The ruthenium red staining coat of the cells is lost during detachment, but cell viability (more than 90 per cent exclude trypan blue) and cell morphology do not change during the subsequent development of tissue factor activity. Furthermore, less than 10 percent of four intracellular enzymes and less than 20 per cent of two plasma membrane enzymes are released during this period of time. We therefore conclude that cells in culture do have tissue factor activity, that it exists in a latent form, and that total cell disruption is not necessary for this activity to initiate blood coagulation.
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PMID:Association of tissue factor activity with the surface of cultured cells. 4 34

Leukocytes can generate procoagulant (tissue factor) activity when incubated with endotoxin. These studies were undertaken to determine whether platelets could influence the procoagulant activity generated by leukocytes. Intact or disrupted platelets (rabbit or human) enhanced the clot-promoting properties of rabbit leukocytes. The enhancing effect of human platelets on human leukocytes required the presence of human serum (devoid of factor VII and X activities). When platelets were incubated with endotoxin in the absence of leukocytes, no increase in their clot-promoting properties was discernible. However, a mixture of platelets, leukocytes, and endotoxin generated procoagulant activity which appeared rapidly and was fivefold greater than that produced by leukocytes incubated with endotoxin alone. The enhancement produced by platelets was even more pronounced if homogenates were used. The platelet effect was examined in more detail by the substitution of membranes, granules, and the "soluble" fraction for whole platelets in the test system. The stimulating activity was localized to the particulate fractions, i.e., membranes and granules. Prior treatment of platelet membranes with phospholipase C or gangliosides or by extraction of lipid resulted in loss of enhancing activity, whereas no inhibition was observed after exposure to neuraminidase or trypsin. It is proposed that platelets contribute a membrane lipoprotein surface which enhances the procoagulant activity generated by leukocytes in the presence of endotoxin. This mechanism may be involved in some of the clinical and pathologic manifestations of gram-negative sepsis with disseminated intravascular coagulation.
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PMID:The stimulatory effect of platelets and platelet membranes on the procoagulant activity of leukocytes. 461 59

Endothelial cells from human umbilical veins produce a procoagulant identified as thromboplastin (tissue factor, factor III) when stimulated with the phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA), phytohaemagglutinin (PHA) or endotoxin, Inducible thromboplastin synthesis (i.e. synthesis of the protein component of thromboplastin, apoprotein III) was totally inhibited by cycloheximide and actinomycin D, indicating that de novo protein and RNA syntheses are necessary. Serum enhanced the induced apoprotein synthesis. Of the total thromboplastin activity in homogenates of stimulated endothelial cells, about 50--70% was available on the cell surface for interaction with other coagulation factors, inactivation by trypsin and neutralization with antiserum against apoprotein III. Induced synthesis of thromboplastin in endothelial cells was 2--7-fold enhanced by the presence of several other cell types in optimal ratio 4--10 cells per endothelial cell. Some of these cell types were themselves thromboplstin producers (U-937, U-937-4), some were not inducible (lymphocytes, granulocytes and the lymphoblast lines Daudi and Molt 4). This enhancing effect was also seen with cell-free culture supernatants, but these were generally somewhat less effective than the intact cells. Supernatants derived from cells cultured in the presence of TPA, PHA or endotoxin were in most cases more effective than supernatants from unstimulated cells.
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PMID:Cellular cooperation in endothelial cell thromboplastin synthesis. 684 29

Platelets are able to stimulate an increase in two distinct activities, tissue factor (thromboplastin) and fibrinolytic inhibition, in human fibroblasts in vitro. A procedure has been developed which allows the purification of a platelet macromolecule which is able to stimulate both of these changes. Washed human platelets were homogenized, sonicated, and then centrifuged at 90,000 x g for 2 h. The resulting pellet was solubilized in 0.05 M sodium carbonate, pH 10.5, and chromatographed on Sephadex G-200, then on hydroxylapatite, resulting in a 135-fold purification and a 20% yield. When the purified material was further fractionated on sodium dodecyl sulfate-polyacrylamide gels, stimulatory activity for both tissue factor and fibrinolytic inhibition was found only in the 75,000-dalton region. The active material could be inactivated by mercaptoethanol with no change in its apparent molecular weight. It was readily inactivated by trypsin with the concomitant loss of the 75,000-dalton Coomassie-staining band. Assay of the purified material for carbohydrate was negative. After isoelectric focusing, the purified material had a major band at pH 5.8 which stimulated both tissue factor and fibrinolytic inhibition. Subcellular fractionation of platelet homogenates by sucrose density gradient centrifugation resulted in a 2-fold increase in stimulatory material in the granule/mitochondrial fraction. This platelet-derived protein may represent a physiologically important regulator for both cellular procoagulant and the net fibrinolytic activity of systemic cells.
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PMID:Purification of a platelet protein which stimulates fibrinolytic inhibition and tissue factor in human fibroblasts. 711 21

Antithrombotic potency of SDZ 217-766, a potent inhibitor of thrombin and other trypsin-like serine proteases, was studied in comparison with heparin in rat models of thrombin induced lung platelet accumulation, of thrombosis in arterio-venous shunt, and of venous thrombosis induced by tissue factor. Thrombin-induced platelet accumulation in the lung was inhibited dose-dependently by SDZ 217-766 following intravenous (i.v.) administration of 0.03 mg/kg to 0.3 mg/kg as well as by intraduodenal (i.d.) administration of 3 mg/kg and 10 mg/kg. Comparable inhibitory effects were observed with heparin at 30 IU/kg and 100 IU/kg. In the rat arterio-venous shunt, following i.v. administration of SDZ 217-766, thrombus formation was inhibited by 40% at 0.1 mg/kg, by 60% at 0.3 mg/kg and was abolished at 1.0 mg/kg whilst APTT was prolonged 1.1 fold over the control value at 0.1 mg/kg and 2.7 fold at 1.0 mg/kg. Similar inhibitory effects were observed following i.d. administration of 10 and 30 mg/kg with only marginal (1.2 to 1.8 fold) APTT elevation. In the same model, heparin administered either i.v., 30-300 IU/kg, or subcutaneously, 100 and 300 IU/kg, inhibited thrombus formation dose dependently but in contrast to SDZ 217-766, the inhibitory effect was paralleled by 5-to > 10 fold APTT elevation over baseline. In the venous thrombosis model, SDZ 217-766 infused at 10 micrograms/kg/min and 20 micrograms/kg/min, reduced thrombus formation by 35% and 70%, respectively. In comparison, thrombus formation was decreased by 22% when heparin was infused at 1 IU/kg/min, and abolished at 3 IU/kg/min.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Antithrombotic activity in vivo of SDZ 217-766, a low-molecular weight thrombin inhibitor in comparison to heparin. 749 72

We have examined the effects of seven proteases on human placental tissue factor in Triton X-100, focusing on extracellular and cytoplasmic domains recognized by monoclonal antibodies HTF1, C28 1.1, and C28 2.1. Plasmin produced peptides recognized on Western blots by C281.1 but not HTF1. None of the other proteases destroyed the extracellular epitope without also removing the cytoplasmic epitope, and both trypsin and chymotrypsin removed the cytoplasmic epitope with little effect on the extracellular domain. Proteinase K destroyed both epitopes, as did neutrophil elastase when used at a relatively high concentration. When digests were sampled over time and reconstituted with lipids for determination of tissue factor activity, only proteinase K consistently produced a loss in tissue factor activity at four hours. After 24 hr, other enzymes also decreased the recovered activity, with the order of effectiveness elastase > trypsin > chymotrypsin.
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PMID:Human placental tissue factor: protease susceptibility of extracellular and cytoplasmic domains. 750 71

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

Serine proteinase inhibitors play a major role in the turnover of connective tissues. In this study, we isolated and determined partial amino-terminal amino acid sequence of trypsin/elastase/plasmin inhibitors (M(r) 33,000 and 31,000) from the extracellular matrix of SV40-transformed human skin fibroblasts. The antitrypsin activity of the inhibitors was monitored by substrate reverse zymography. Polyclonal antisera to alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 2-antiplasmin, inter-alpha-trypsin inhibitor, plasminogen activator inhibitors-1 and -2, and a monoclonal antibody to protease nexin-1 did not label the 33-, 31-, and 27-kDa inhibitors. A computer search for amino acid sequence homology indicated that the 31-kDa inhibitor is novel. In contrast, the sequence of the 33-kDa inhibitor shared 70 to 90% homology with the amino-terminal sequence of a recently characterized 32-kDa trypsin/tissue factor inhibitor called tissue factor pathway inhibitor-2. The 33- and 31-kDa inhibitors bind to heparin-Sepharose and were recovered from the affinity beads as well as from the t12 FB extracellular matrix with 1 M NaCl. Based on these results, we propose that the extracellular matrix of human mesenchymal cells sequester a family of novel serine proteinase inhibitors.
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PMID:Novel extracellular matrix-associated serine proteinase inhibitors from human skin fibroblasts. 787 99

To study the role of a putative high affinity Ca2+ binding site in the protease domain of factor X, we prepared a deletion mutant (E2FX) lacking the Gla and first epidermal growth factor-like domains. E2FX possesses a single high affinity Ca2+ binding site (Kd = 154 microM). Asp-70 or Glu-80 (chymotrypsin numbering system) was then converted to Lys. These mutants, E2FXD70K and E2FXE80K, allow examination of the Ca(2+)-dependent assembly of activation complexes involving substrate and enzyme forms of factor X that do not bind Ca2+. E2FXD70K and E2FXE80K share similar properties. They retain functional activity, but the D70K mutant no longer binds Ca2+, and neither mutant exhibits a Ca(2+)-dependent increase in peptide chromogenic substrate activity. Activation of E2FXD70K by the soluble tissue factor-factor VIIa complex was still Ca(2+)-dependent (Kd(app) = 133 +/- 38 microM), but the Kd(app) for Ca2+ was decreased relative to E2FX (Kd(app) = 205 +/- 53 microM). Prothrombin or prethrombin 1 activation by the E2FXaD70K in the presence of factor Va was also Ca(2+)-dependent (Kd(app) = 124 +/- 47 microM and 102 +/- 38 microM, respectively), and the Kd(app) was again lower than that of E2FXa (272 +/- 79 microM and 179 +/- 54 microM, respectively). In the absence of factor Va, activation of prethrombin 1 by E2FXaD70K was not influenced by Ca2+, but activation by E2FXa was enhanced (Kd(app) = 161 +/- 32 microM). The results suggest the presence of functionally important Ca2+ binding sites in components of the factor X and prothrombin activation complexes that do not involve factor X. Furthermore, the Ca2+ binding sites in factor X and trypsin appear to be structurally similar. The Asp or Glu-->Lys mutations within this site may create an internal salt bridge that conserves factor X (Xa) function even in the absence of Ca2+.
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PMID:Asp-70-->Lys mutant of factor X lacks high affinity Ca2+ binding site yet retains function. 806 84

Potent active-site inhibitors of human tissue factor-Factor VIIa (TF.FVIIa) have been selected from Alzheimer's amyloid beta-protein precursor inhibitor (APPI) Kunitz domain libraries displayed on phage. Eight randomized positions on the extended primary binding loop (P5 through P4') and positions 34 and 39 were examined in three separate libraries. Libraries contained from 3.2 x 10(5) to 3.2 x 10(6) potential variants resulting from replacing up to 5 positions with all 20 amino acids. Following 4 rounds of selection against FVIIa associated with immobilized tissue factor (TF), 12 clones from each library were sequenced. Variants were purified by trypsin affinity chromatography and reverse-phase high performance liquid chromatography, and characterized for their ability to inhibit TF.FVIIa chromogenic activity. Measured apparent equilibrium dissociation constants (Ki*) ranged from about 10 to 500 nM. From sequence and activity data, an overall consensus sequence, TF7I-C, was constructed by site-directed mutagenesis. TF7I-C differed from APPI at 4 key residues, T11P, M17L, S19L, and G39Y, and inhibited TF.FVIIa with a Ki* = 1.9 +/- 0.4 nM, which represented an increase in binding affinity of more than 150-fold compared to APPI. At 40 microM, TF7I-C prolonged the clotting times 3.5-fold in a prothrombin time assay and > 10-fold at 7 microM in an activated partial thromboplastin time assay. Prolongation of the activated partial thromboplastin time correlates with potent inhibition of FXIa (Ki* = 0.8 nM) and plasma kallikrein (Ki* = 1.2 nM). TF7I-C also inhibited plasmin (Ki* = 40 nM) and FXa (Ki* = 55 nM), but not activated protein C, thrombin, or FXIIa (Ki* > 10 microM each).
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PMID:Kunitz domain inhibitors of tissue factor-factor VIIa. I. Potent inhibitors selected from libraries by phage display. 807 37


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