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:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The RGD-containing peptides isolated from the venoms of the Viperidae constitute a new class of small cysteine-rich peptides of variable amino acid composition and biological activity (Huang, T.-F., et al. (1987) J. Biol. Chem. 262, 16157-16163; Gan, Z.R., et al. (1988) J. Biol. Chem 263, 19827-19832; Huang, T.-F., et al. (1989) Biochemistry 28, 661-668), which it is proposed by Gould et al. (unpublished data) that we call 'disintegrins'. These peptides bind to the glycoprotein IIb-IIIa receptor on the platelet surface and inhibit aggregation induced by ADP, thrombin, platelet-activating factor and collagen. These peptides are also potent inhibitors of cell adhesion to fibrinogen (Knudsen, K.M., et al. (1988) Exp. Cell Res. 179, 42-49). We report the isolation of two further RGD-peptides from the venoms of Trimeserusus elegans and Trimeserusus albolabris, purified to homogeneity with high yield by a novel, rapid reverse-phase HPLC method. The primary structures of these two peptides were determined to be single polypeptide chains of 73 amino acids. Albolabrin differed from trigramin by eight residues whilst elegantin differed by 22 residues. The molecular mass of albolabrin calculated on the basis of amino acid sequence was 7574 Da and the pI similarly calculated was 4.27. The molecular mass of elegantin was calculated to be 7806 Da and the theoretical pI to be 4.69. RGD is maintained in the same position (51-53 AA) and all 12 cysteines are identical. Our data suggest that the presence of RGD, the conserved secondary and tertiary structure, are essential for the expression of biological activity by these peptides. Both peptides inhibited ADP-induced platelet aggregation. Extended homologies around the RGDS sequences in human von Willebrand Factor and bovine fibrinogen were found with both peptides.
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PMID:Elegantin and albolabrin purified peptides from viper venoms: homologies with the RGDS domain of fibrinogen and von Willebrand factor. 219 22

The free thiols of platelet thrombospondin (TSP) were derivatized with labeled N-ethylmaleimide (NEM) or iodoacetamide (IAM). When Ca2+ was chelated with EDTA, 2.9 mol of NEM or 2.6 mol of IAM reacted/mol of native TSP. No additional thiols were found after denaturation with urea. Since TSP has three apparently identical polypeptide chains, this suggests one free thiol/polypeptide chain. Ca2+ protected all of the thiols from reaction with IAM. In Ca2+ about half the thiols reacted normally with NEM and the others were unreactive, indicating that the thiols of TSP are not identical. The number of reactive thiols as a function of [Ca2+] revealed a sigmoidal curve with a transition midpoint of 207 microM. The ability of analogs of NEM to compete for derivatization of the thiols with labeled NEM was greater with larger, more hydrophobic agents. Gel electrophoretic separation of labeled TSP that had been partially digested with thrombin and trypsin indicated that some of the label was in the C-terminal tryptic fragment but that most was in the adjacent trypsin-sensitive region. After cyanogen bromide cleavage of the labeled and reduced protein, four labeled fractions were obtained from a gel filtration column. With subsequent combinations of tryptic digestion and reversed-phase high performance liquid chromatography, labeled peptides were purified from these four fractions, and the amino acid sequences were determined. Twelve labeled cysteines were identified, each with a specific radioactivity less than that of the thiol labeling reagent, indicating that only a fraction of that cysteine in a population of TSP molecules was a free thiol at the time of derivatization. While 2 labeled cysteines are in the non-repeating C-terminal portion of the molecule, the other 10 labeled cysteines are in the adjacent trypsin-sensitive type 3 repeats proposed (Lawler, J., and Hynes, R. O. (1986) J. Cell. Biol. 103, 1635-1648) as the calcium-binding region of the molecule. The disulfide bonds most sensitive to reduction by dithioerythritol were also stabilized by Ca2+, implying location in the Ca2(+)-sensitive part of the molecule. It is proposed that one equivalent of free thiol/polypeptide chain is distributed among 12 different cysteine residues through an intramolecular thioldisulfide isomerization.
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PMID:Free thiols of platelet thrombospondin. Evidence for disulfide isomerization. 221 66

The ability of different cell types to cooperate in the metabolism of reactive intermediates of arachidonic acid such as leukotriene A4 (LTA4) is currently receiving considerable attention. Of critical importance is the demonstration that transfer of LTA4 could occur under conditions when relatively low amounts of LTA4 are produced such as would be expected for in vitro receptor-mediated stimulation. Stimulation of human neutrophils with a combination of chemotactic factor (formyl-methionyl-leucyl-phenylalanine, FMLP) and phagocytosable particles (opsonized zymosan) resulted in little production of LTC4 alone, but measurable quantities appeared when platelets were coincubated. When these agonists were added to platelets alone in the absence of neutrophils, no LTC4 was produced. In the presence of stimulated neutrophils, the final synthesis of LTC4 was shown to occur within the platelets (from neutrophil-derived LTA4) by experiments using platelets that had been prelabeled with 35S-cysteine to label intracellular platelet glutathione. Other 35S-labeled sulfidopeptide leukotriene metabolites were also produced in this coincubation of neutrophils and platelets. The observed synergy between FMLP and opsonized zymosan in the production of LTC4 when neutrophils and platelets were coincubated may involve priming the neutrophil for LTA4 production. Activation of platelets or endothelial cells with thrombin did not alter the capacity of either cell to convert exogenously added LTA4 into LTC4. This would support the suggestion that even when platelets are activated they retain their capacity to metabolize LTA4 into LTC4. Finally, previous exposure of the platelets to LTA4 did not affect subsequent metabolism of arachidonic acid by the cyclooxygenase pathway to thromboxane A2 (TXA2) except at very high concentration of LTA4. These results suggest that cell-cell interactions may be critical determinants of the profile of eicosanoids produced in physiologic and pathophysiologic circumstances. In particular, we believe that both endothelial cells and platelets can, together with neutrophils, contribute relatively large amounts of sulfidopeptide leukotrienes to inflammatory and thrombotic events. These cell-cell interactions are aspirin-insensitive; therefore, aspirin-treated platelets are capable of synthesizing the vasoconstrictor LTC4 from neutrophil LTA4 at a time when they can no longer produce thromboxane.
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PMID:Transcellular biosynthesis of sulfidopeptide leukotrienes during receptor-mediated stimulation of human neutrophil/platelet mixtures. 222 31

Yeast KEX2 protease was examined as a potential model for a human proprotein convertase and, in all respects, mimicked the predicted properties of a proalbumin convertase. The enzyme rapidly cleaved the propeptide Arg-Gly-Val-Phe-Arg-Arg from the NH2-terminal end of proalbumin but, unlike trypsin, failed to cleave physiologically unprocessed human proalbumin variants. There was little or no cleavage of proalbumin Lille (Arg-2----His) or Christchurch (Arg-1----Gln), and there was negligible cleavage of proalbumin Blenheim (Asp1----Val), despite the fact that it retains the dibasic processing signal. Proalbumin Kaikoura (Arg-2----Cys), which appears to be partially processed in vivo, was cleaved at about half the rate of normal proalbumin despite the absence of a diarginyl sequence. Restoration of a dibasic site through aminoethylation of the new cysteine increased the rate of cleavage to near that of normal proalbumin. The KEX2-catalyzed cleavage of normal proalbumin was found to be independent of pH between pH 6.0 and 8.0. Antitrypsin Pittsburgh (Met358----Arg), a predicted specific inhibitor of in vivo proalbumin cleavage, inhibited KEX2 in a reversible manner. A molar excess of thrombin over antitrypsin Pittsburgh relieved the inhibition of KEX2, suggesting that a covalent complex is not formed between KEX2 and the inhibitor.
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PMID:Specificity of yeast KEX2 protease for variant human proalbumins is identical to the in vivo specificity of the hepatic proalbumin convertase. 225 10

Human rheumatoid synovial cells in culture secrete at least three related metalloproteinases that digest extracellular matrix macromolecules. One of them, termed matrix metalloproteinase 2 (MMP-2), has been purified as an inactive zymogen (proMMP-2). The final product is homogeneous on SDS/PAGE with Mr = 72,000 under reducing conditions. The NH2-terminal sequence of proMMP-2 is Ala-Pro-Ser-Pro-Ile-Ile-Lys-Phe-Pro-Gly-Asp-Val-Ala-Pro-Lys-Thr, which is identical to that of the so-called '72-kDa type IV collagenase/gelatinase'. The zymogen can be rapidly activated by 4-aminophenylmercuric acetate to an active form of MMP-2 with Mr = 67,000, and the new NH2-terminal generated is Tyr-Asn-Phe-Phe-Pro-Arg-Lys-Pro-Lys-Trp-Asp-Lys-Asn-Gln-Ile. However, following 4-aminophenylmercuric acetate activation, MMP-2 is gradually inactivated by autolysis. Nine endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, neutrophil elastase, cathepsin G, matrix metalloproteinase 3, and thermolysin) were tested for their abilities to activate proMMP-2, but none had this ability. This contrasts with the proteolytic activation of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). The optimal activity of MMP-2 against azocoll is around pH 8.5, but about 50% of activity is retained at pH 6.5. Enzymic activity is inhibited by EDTA, 1,10-phenanthroline or tissue inhibitor of metalloproteinases, but not by inhibitors of serine, cysteine or aspartic proteinases. MMP-2 digests gelatin, fibronectin, laminin, and collagen type V, and to a lesser extent type IV collagen, cartilage proteoglycan and elastin. Comparative studies on digestion of collagen types IV and V by MMP-2 and MMP-3 (stromelysin) indicate that MMP-3 degrades type IV collagen more readily than MMP-2, while MMP-2 digests type V collagen effectively. Biosynthetic studies of MMPs using cultured human rheumatoid synovial fibroblasts indicated that the production of both proMMP-1 and proMMP-3 is negligible but it is greatly enhanced by the treatment with rabbit-macrophage-conditioned medium, whereas the synthesis of proMMP-2 is constitutively expressed by these cells and is not significantly affected by the treatment. This suggests that the physiological and/or pathological role of MMP-2 and its site of action may be different from those of MMP-1 and MMP-3.
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PMID:Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts. Purification and activation of the precursor and enzymic properties. 226 96

Previous studies indicate that exposure of fibrinogen receptors associated with glycoprotein IIb/IIIa complex contributes to platelet loss during cardiopulmonary bypass. Recently, we isolated a number of RGD (Arg-Gly-Asp)-containing, low molecular weight, cysteine-rich peptides from viper venoms. These peptides, which we propose to call "disintegrins," block platelet-fibrinogen interaction and platelet aggregation. We compared the effect of RGDS (Arg-Gly-Asp-Ser) and four disintegrins (echistatin, flavoridin, albolabrin, and bitistatin) on platelet behavior in a membrane oxygenator. During simulated extracorporeal circulation for 2 hours, platelet count decreased to about 30% of initial values. Addition of echistatin (60-200 nM), albolabrin (60-200 nM), bitistatin (60 nM), and flavoridin (45 nM) significantly inhibited platelet loss in the circuit. RGDS (33 microM) did not show any significant inhibitory effect. ADP-induced platelet aggregation was inhibited in samples of platelet-rich plasma taken from the circuits containing disintegrins. However, echistatin appeared to be a more potent inhibitor of platelet aggregation, whereas albolabrin and flavoridin interfered more selectively with platelet loss from the circuit. Echistatin prevented the accumulation of glycoprotein IIIa on the surface of the circuit. Echistatin (60-200 nM), flavoridin (45 nM), bitistatin (60 nM), and albolabrin (200 nM) significantly inhibited the loss of beta-thromboglobulin from platelets into circulating plasma. Electron microscopy studies demonstrated shape change but not degranulation in platelets circulating in the presence of 200 nM echistatin. On the other hand, this peptide (up to 1,000 nM) did not prevent loss of alpha granules and beta-thromboglobulin from thrombin-stimulated platelets, although it prevented their aggregation. In conclusion, disintegrins protect platelets in the circuit by preventing their adhesion to surfaces and, therefore, preventing fragmentation of adhered platelets under the shear stress of flowing blood. This study indicates that disintegrins may be potential candidates for platelet protection during cardiopulmonary bypass.
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PMID:Inhibition of platelet adhesion to surfaces of extracorporeal circuits by disintegrins. RGD-containing peptides from viper venoms. 236 14

High molecular weight kininogen (HMWK) functions as a cofactor for activation of plasma serine zymogens and as an inhibitor of tissue cysteine proteases. Cell surfaces to which HMWK binds may provide sites for regulation of these systems. Localization of these HMWK-dependent processes at sites of vascular injury may depend on its binding to specific receptors on endothelial cells. In culture, passaged human umbilical vein endothelial cells (HUVEC) bind anti-HMWK antibody to the cell surface and contain 171 +/- 75 ng of HMWK/10(8) cells. [35S]Methionine-labeled HUVEC in culture synthesize a 120-kDa protein immunoisolated using an anti-kininogen antibody, and a 3500-nucleotide message for human HMWK was detected by Northern blot in RNA extracted from HUVEC. HUVEC also express unoccupied binding sites for HMWK on their surface. 125I-HMWK specifically binds to HUVEC in a reaction requiring Zn2+. 125I-HMWK binding to HUVEC is saturable at 4 degrees C but not at 23 degrees C. 125I-HMWK binds to HUVEC with equal affinity as unlabeled HMWK. Kallikrein, factor XII, fibrinogen, fibronectin, and thrombin do not inhibit 125I-HMWK binding to HUVEC. 125I-HMWK-HUVEC binding remains fully reversible at 60 min following the addition of a 50-fold molar excess HMWK. HUVEC express 9.3 +/- 2.0 X 10(5) (mean +/- S.E.) HMWK binding sites/cell (Kd = 52 +/- 13 nM). Both added and cell-bound 125I-HMWK migrate at 120 kDa on sodium dodecyl sulfate gel electrophoresis, suggesting that the protein remains uncleaved upon binding to the HUVEC surface. These studies indicate that HUVEC synthesize HMWK and the HUVEC surface has a site for its expression. By synthesizing and localizing HMWK to the cell surface, endothelial cells may contribute to the activation of plasma's contact serine zymogens and regulation of tissue cysteine proteases.
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PMID:The expression of high molecular weight kininogen on human umbilical vein endothelial cells. 246 Apr 46

We have analyzed the factor VIII (FVIII) protein and the nucleotide sequence around two thrombin cleavage sites, at arginine 372 in the FVIII heavy chain and arginine 1689 in the FVIII light chain in a naturally occurring dysfunctional FVIII variant, FVIII Okayama. The patient was a 42-year-old hemophiliac with a FVIII coagulant activity of 0.03 U/mL and a FVIII antigen level of 0.8 U/mL. The patient's FVIII was not thrombin activatable to levels seen in normal plasma. Immunoblotting of partially purified FVIII Okayama and normal FVIII showed that thrombin cleavage of the 92 kilodalton (Kd) heavy chain was impaired in the mutant protein. The patient's genomic DNA was amplified using the polymerase chain reaction with two sets of synthetic oligonucleotide primers spanning amino acid residues 319 to 400 and 1630 to 1720. Sequence analysis of the amplified DNA fragments revealed a cytosine to thymine transition, converting an arginine to a cysteine codon at residue 372. No abnormality was found in the FVIII light chain region analyzed. The patient's hemophilic brother and carrier mother revealed the same mutation. We conclude that the pathogenesis of hemophilia A in this patient is probably due to an arginine to cysteine substitution at a thrombin cleavage site in the FVIII heavy chain.
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PMID:An arginine to cysteine amino acid substitution at a critical thrombin cleavage site in a dysfunctional factor VIII molecule. 250 48

Applaggin (Agkistrodon piscivorus piscivorus platelet aggregation inhibitor) is a potent inhibitor of platelet activation. The protein is isolated from the venom of the North American water moccasin snake in three steps, including gel filtration, cation exchange, and reverse-phase HPLC procedures. The purified protein migrates as a 17,700-Da polypeptide by SDS/PAGE under nonreducing conditions and as a 9800-Da peptide in the presence of thiol. The behavior of applaggin on SDS/PAGE would indicate that the protein is a disulfide-linked dimer. Applaggin has been completely sequenced by Edman degradation and consists of 71 amino acids. The sequence is rich in cysteine and contains Arg-Gly-Asp at residues 50-52. Applaggin blocks platelet aggregation induced by ADP, collagen, thrombin, or arachidonic acid with IC50 values ranging from 12 to 128 nM (0.2-2.3 micrograms/ml) depending on the agonist and its concentration. This inhibition is found to correlate with inhibition of thromboxane A2 generation and of dense granule release of serotonin. Inhibition by applaggin of serotonin release induced by ADP, gamma-thrombin, and collagen was monitored in plasma under stirred conditions with [3H]serotonin-loaded platelets, and IC50 values for inhibition are found to range from less than 10 to 145 nM. At saturating concentrations, 125I-labeled applaggin (125I-applaggin) binds to 28,500 sites per unstimulated, washed platelet with a Kd of 1.22 x 10(-7) M. Binding of 125I-applaggin to platelets is inhibited by the synthetic undecapeptide Arg8-Gly-Asp-Val at 200 microM.
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PMID:Agkistrodon piscivorus piscivorus platelet aggregation inhibitor: a potent inhibitor of platelet activation. 251 Jan 58

The proteolytic digestion of GPIIIa on intact platelets by chymotrypsin, thrombin, plasmin, trypsin, and staphylococcal V8 protease was monitored in immunoblot studies employing three different antibodies to GPIIIa, one of which was made against a 13-residue synthetic peptide containing the amino terminus of GPIIIa. Chymotrypsin, plasmin, and trypsin gave similar patterns, from which it could be inferred that the major products after extensive digestion were two-chain molecules composed of amino-terminal fragments of Mr approximately 17,000-18,000 disulfide bonded to carboxyl-terminal remnants of Mr approximately 58,000-70,000. These patterns suggest that GPIIIa contains a large disulfide-bonded loop of at least 325 amino acids that is susceptible to proteolytic cleavage, and that the 4 cysteine residues between residues 177 and 273 bond with each other. Such a structure can also account for the presence of the PIA1 epitope, which has recently been localized to a polymorphism at position 33 on these late digestion products. Thrombin did not proteolyze GPIIIa even at 2.5 units/ml. Still to be resolved is whether the minor immunoreactive GPIIIa bands found on normal platelets are related to in vivo or in vitro proteolysis and whether GPIIIa proteolysis plays a role in chymotrypsin-induced exposure of the GPIIb/IIIa receptor.
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PMID:Evidence that platelet glycoprotein IIIa has a large disulfide-bonded loop that is susceptible to proteolytic cleavage. 252 61


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