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)

Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. We isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO4/PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated fVIII to function as a cofactor in the activation of factor X by factor IXa, we found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by light-chain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function.
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PMID:Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin. 250 52

Sympathetic neurons release both urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA). A number of inhibitors of serine proteases have been tested to determine their effects on neurite outgrowth from rat sympathetic neurons. Some inhibitors increase neurite outgrowth while others have little or no effect on outgrowth. Inhibition of plasminogen activator (PA) activity but not other serine protease activity correlates with the increase in neurite outgrowth (uPA, r = 0.89; tPA, r = 0.86; plasmin, r = 0.015; thrombin, r = 0.025). Antibodies that inhibit uPA activity increase neurite outgrowth, while antibodies that bind to uPA but do not inhibit activity do not alter outgrowth. Time-lapse videomicroscopy of neurite outgrowth indicates that about 85% of the neurites increase their rate of outgrowth following exposure to inhibitors of PA. Routinely, 1-2 min after exposure of a growth cone to an inhibitor, there is an increase in lamellipodial activity at the leading edge of the growth cone and a decrease in lamellipodial activity on the sides and base of the growth cone. The increase in the rate of outgrowth combined with the decrease in lamellipodial activity on the sides of the growth cones results in neurites being very long and straight in the presence of inhibitors (persistence time P = 3.7 and 15.3 hr for controls and in the presence of inhibitors of PA, respectively). PAs released from sympathetic neurons and PC12 cells interact with 3 different binding sites on the cell surface: (1) an inhibitor of serine proteases (including uPA and tPA) is bound to the surface via a heparinase-sensitive site; (2) a uPA-selective binding site is present in patches on the bottom surface of PC12 cells; and (3) a tPA-selective binding site with high affinity (KD = 23 +/- 10 nM) and high capacity (340,000 +/- 130,000 sites/neuron) for 125I-tPA is homogeneously distributed over the entire surface. Data in the present study are consistent with PA being involved in neurite outgrowth and open the possibility of other PA-dependent functions occurring when tPA and/or uPA interacts with cell surface binding sites.
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PMID:Neuronal plasminogen activators: cell surface binding sites and involvement in neurite outgrowth. 251 75

5'-p-Fluorosulfonylbenzoyl adenosine (FSBA), a nucleotide analog of ADP, has been shown to inhibit ADP-induced shape change, aggregation and exposure of fibrinogen binding sites concomitant with covalent modification of a single surface membrane polypeptide of Mr 100,000 (aggregin). Since thrombin can aggregate platelets which have been modified by FSBA and are refractory to ADP, we tested the hypothesis that thrombin-induced platelet aggregation might involve cleavage of aggregin. At a low concentration of thrombin (0.05 U/ml), platelet aggregation, exposure of fibrinogen receptors and cleavage of aggregin in FSBA-modified platelets did not occur, indicating ADP dependence. In contrast, incubation of [3H]FSBA-labeled intact platelets with a higher concentration of thrombin (0.2 U/ml) resulted in cleavage of radiolabeled aggregin, aggregation, and exposure of fibrinogen binding sites. Under identical conditions, aggregin in membranes isolated from [3H]FSBA-labeled platelets was not cleaved by thrombin. Thrombin-induced platelet aggregation and cleavage of aggregin were concomitantly inhibited by a mixture of 2-deoxy-D-glucose, D-gluconic acid 1,5-lactone, and antimycin A. These results suggest that thrombin cleaves aggregin indirectly by activating an endogeneous protease. Thrombin is known to elevate intracellular Ca2+ concentration and thereby activates intracellular calcium dependent thiol proteases (calpains). In contrast to serine protease inhibitors, calpain inhibitors including leupeptin, antipain, and ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (chelator of Ca2+) inhibited platelet aggregation and cleavage of aggregin in [3H]FSBA-labeled platelets. Leupeptin, at a concentration of 10-20 microM, used in these experiments, did not inhibit the amidolytic activity of thrombin, thrombin-induced platelet shape change, or the rise in intracellular Ca2+. Purified platelet calpain II caused aggregation of unmodified and FSBA-modified platelets and cleaved aggregin in [3H]FSBA-labeled platelets as well as in isolated membranes. The latter is in marked contrast to the action of thrombin on [3H]FSBA-labeled membranes. Thus, thrombin-induced platelet aggregation may involve intracellular activation of calpain which proteolytically cleaves aggregin thus unmasking latent fibrinogen receptors, a necessary prerequisite for platelet aggregation.
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PMID:Thrombin-induced platelet aggregation involves an indirect proteolytic cleavage of aggregin by calpain. 254 93

Thrombin stimulates polyphosphoinositide hydrolysis in embryonic chick heart cells and in 1321N1 astrocytoma cells and increases intracellular Ca2+ in the 1321N1 cells. The serine protease trypsin mimics these actions in a dose-dependent fashion, whereas the proteolytically inactive thrombin derivatives diisopropyl fluorophosphate-thrombin (DIP-thrombin) and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-thrombin (PPACK-thrombin) are ineffective in this regard. The phosphoinositide responses to thrombin or trypsin and the muscarinic agonist carbachol are additive, but no additivity is observed between the responses to thrombin and trypsin. Unlike the response to carbachol, the phosphoinositide and Ca2+ responses to thrombin and trypsin desensitize, with no recovery of the calcium response even when Ca2+ stores are replenished. Cross-desensitization of phospholipase C activation and calcium mobilization between these proteases is also observed. In addition, PPACK-thrombin, which elicits no response itself, effectively inhibits trypsin-stimulated phosphoinositide hydrolysis. It is proposed that thrombin and trypsin act through the same receptor. Proteolysis appears to be important in the mechanism by which these agonists elicit phosphoinositide hydrolysis, calcium mobilization, and, perhaps, subsequent receptor desensitization.
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PMID:Thrombin and trypsin act at the same site to stimulate phosphoinositide hydrolysis and calcium mobilization. 254 47

The possibility that thrombin-induced platelet reactivity could occur via both a receptor-related and a proteolytic process was examined. Thrombin elicited the formation of considerably more [32P]phosphatidic acid (an index of phospholipase C catalysed phosphoinositide metabolism) than did platelet activating factor, 5-hydroxytryptamine, ADP, and the thromboxane A2 analogue EP171, when these agents were added either alone or in combination. Co-addition of thrombin and EP171 did not evoke significantly more [32P]phosphatide acid than did thrombin alone. The protease inhibitor leupeptin, decreased but did not abolish [32P]phosphatidic acid formation elicited by either thrombin alone or thrombin in combination with EP171. The serine protease, trypsin, stimulated an increase in [32P]phosphatidic acid and this effect was additive with that of EP171. This augmentation by trypsin of EP171-induced [32P]phosphatidic acid formation was inhibited by leupeptin. These results are consistent with the concept that thrombin-induced activation of phospholipase C occurs by two distinct mechanisms: one via proteolysis, which is sensitive to leupeptin, and the other via receptor activation, a process shared by EP171. The individual components of this dual mechanism can be mimicked by the co-addition of a receptor-directed agonist (EP171) and a proteolytic agent (trypsin).
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PMID:Evidence for two mechanisms of thrombin-induced platelet activation: one proteolytic, one receptor mediated. 255 47

The thromboresistant function of a surface with end-point attached heparin is based upon interaction among the immobilized heparin, antithrombin, and at least factor Xa or thrombin. Heparinized arteriovenous shunts were implanted in dogs. By compressing a segment of the shunt, high and low wall shear rate regions were obtained in each shunt. After removal, the tubings were tested for their factor Xa and thrombin inhibitory capacity. It was found that on a molar basis, the factor Xa and thrombin inhibitory capacity were similar in low wall shear rate segments. In high wall shear rate segments, the thrombin inhibitory capacity was decreased, thus indicating that the AT-mediated inhibition of the serine protease is dependent on the wall shear rate.
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PMID:Influence of high and low wall shear rates on the inhibition of factor Xa and thrombin at surfaces coated with immobilized heparin. 260 95

Biological glue is obtained by mixing different specific plasma proteins including a serine protease, thrombin. Surprisingly at present the thrombin used in such a mixture is from equine or bovine origin while all other components are from human. In this paper we described a particular efficient and specific chromatographic method for the purification of human thrombin usable as a serine protease in the preparation of biological glue. A pure and active thrombin is obtained from a plasma fraction after adsorption on benzamidine-Spherodex followed by an elution with non specific (sodium chloride gradient) or biospecific competitors (arginine methylester or benzamidine). The obtained thrombin with a yield close to 80% and a purification factor close to 160, showed good properties in the replacement of animal thrombin in the condition of biological glue.
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PMID:[Purification of human thrombin by affinity chromatography for its use in preparations for biological coagulation]. 261 44

We have previously shown that a serum protein, termed differentiation reversal factor (DRF), is responsible for neurite retraction in differentiated cultures of an adenovirus 12 (Ad12) transformed human retinoblast cell line. Data is presented here to show that DRF is identical to the serine protease prothrombin. Both proteins have been immunoprecipitated using an antibody raised against purified prothrombin and have been shown to hydrolyse a specific thrombin substrate only after activation by the snake venom ecarin. Following addition to Ad12 HER 10 cells, which had previously been differentiated by culture in the presence of 2 mM dibutyryl cAMP in serum-free medium, thrombin and prothrombin caused half-maximal retraction of neurites at concentrations of 0.5 ng/ml and 20 ng/ml respectively. Interestingly, activation of prothrombin was shown to be unnecessary for biological activity. Using the inhibitor di-isopropylfluorophosphate (DIP), we have shown that abrogation of the proteolytic activity of thrombin also results in a loss (greater than 2000 fold) of differentiation reversal activity. Thrombin and its zymogen both stimulated the mitosis of differentiated Ad12 HER 10 cells to a similar extent. In addition, differentiation reversal was highly specific since, at physiologically significant concentrations, closely related serine proteases did not cause neurite retraction. Prothrombin and thrombin also reversed morphological differentiation in the SK-N-SH neuroblastoma cell line and in heterogeneous cultures of cells from various regions in the human foetal brain.
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PMID:Modulation of morphological differentiation of human neuroepithelial cells by serine proteases: independence from blood coagulation. 279 85

We have analyzed the binding of thrombin, a serine protease with central roles in hemostasis, to the subendothelial extracellular matrix (ECM) produced by cultured endothelial cells. This substrate provides a thrombogenic surface where hemostasis is initiated. Binding was saturable and equilibrium was achieved after 3 h incubation with 125I-alpha-thrombin. Scatchard analysis of thrombin binding revealed the presence of 5.1 X 10(9) binding sites per squared millimeter ECM, with an apparent Kd of 13 nM. The catalytically blocked enzyme, diisofluorophosphate (DIP)-alpha-thrombin competed efficiently with 125I-alpha-thrombin, indicating that the binding was independent of its catalytic site. Moreover, high concentrations of the synthetic tetradecapeptide, representing residues 367-380 of thrombin B chain (the macrophage mitogenic domain of thrombin), competed with thrombin binding to ECM, indicating that the binding site may reside in the vicinity of "loop B" region. Thrombin binds to dermatan sulfate in the ECM, as demonstrated by the inhibition of 125I-alpha-thrombin binding to ECM pretreated with chondroitinase ABC, but not with heparitinase or chondroitinase AC. This stands in contrast to 125I-FGF (fibroblast growth factor) binding to ECM, which was inhibited by heparitinase but not by chondroitinase ABC, ECM-bound thrombin exhibits an exposed proteolytic site as monitored by the Chromozyme TH assay and by its ability to convert fibrinogen to a fibrin clot and to induce platelet activation as indicated by 14C-serotonin release. ECM-bound thrombin failed to form a complex with its major circulating inhibitor-antithrombin III (AT III), compared with rapid complex formation with soluble thrombin. We propose that thrombin binds to subendothelial ECM where it remains functionally active, localized, and protected from inactivation by circulating inhibitors.
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PMID:Binding of thrombin to subendothelial extracellular matrix. Protection and expression of functional properties. 279 47

The genes of seven structural mutants of antithrombin III (ATIII), presenting either defective serine protease reactivity or abnormal heparin binding, were analyzed. The polymerase chain reaction (PCR) was used to amplify the corresponding gene exon and the mutation was identified by either dot blot analysis using a battery of allele-specific oligonucleotide probes or sequencing. Variants Paris and Paris 2 were identified as Arg 47 Cys mutations, and Clichy, Clichy 2, and Franconville were found to be Pro 41 Leu mutations. All five are heparin binding-site variants. ATIII Avranches is an Arg 393 His mutation and ATIII Charleville is an Ala 384 Pro mutation. These two mutations impair the reactive site of the molecule. ATIII Charleville is a new mutation of the reactive center, as predicted by previous biochemical data. The position of this new mutation, together with the other previously described mutations of the reactive center, sheds light on the molecular function of this site in inhibiting thrombin. Finally, genomic amplification by PCR is a powerful technique for the fast identification of antithrombin III mutations and their homozygous/heterozygous status, and should be useful for predicting thrombotic risk.
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PMID:Molecular characterization of antithrombin III (ATIII) variants using polymerase chain reaction. Identification of the ATIII Charleville as an Ala 384 Pro mutation. 279 60


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