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)

Selective, sensitive assays for the quantitation of serine proteases involved in coagulation and fibrinolysis have been developed employing fluorogenic substrates containing a 6-amino-1-naphthalenesulfonamide leaving group (PNS-substrates). Over one hundred substrates were evaluated for hydrolysis by the serine proteases of blood coagulation and fibrinolysis, and substrate structure-efficiency correlations were examined. PNS-substrates which contain Lys in the P1 position are specific for Lys-plasmin and are either not hydrolyzed or hydrolyzed at a relatively low rate by factor Xa, thrombin, or urokinase-type plasminogen activator (uPA). These substrates allow quantitation of Lys-plasmin at concentrations as low as 1 pM. Eighteen of over 90 substrates tested for factor XIa are hydrolyzed by this enzyme at a relatively high rate reaching a k(cat), value of 170 s(-1) and allowing quantitation of factor XIa at 10 fM. Eighteen of almost 90 PNS-substrates tested display high specificity for thrombin, some exceeding that for factor Xa by >10,000-fold and >100-fold for activated protein C (APC). Seven of these substrates have a k(cat) over 100 s(-1) and three of them have a K(M) below 1 microM. They allow the quantitation of thrombin at concentrations as low as 20 fM. For APC, uPA and the factor VIIa/tissue factor complex, quantitation is feasible at 1 pM concentration. For factor Xa and factor VIIa the limits are 0.4 pM and 40 pM respectively. The PNS-substrates presented in this study may be employed for the development of direct and sensitive serine protease assays.
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PMID:Ultrasensitive fluorogenic substrates for serine proteases. 936 84

Using enzymatic microassays, the potency of a series of new boroarginine tripeptides was determined versus thrombin and a panel of serine-proteases implicated in the coagulation and fibrinolysis pathways. The inhibition of the serine-protease complement factor I was also studied. Factor I regulates the alternate pathway of the complement and its inhibition appears to be responsible for the toxic effects of the orally available thrombin inhibitor Ac-D-Phe-Pro-boroArg (DuP-714). The structure of the new boronic acid derivatives tested was modified from that of DuP-714 by replacing the proline in the P2 position by N-cycloalkyl-glycine residues of increasing size (S18989: cyclopropyl; S18563: cyclobutyl; S18326: cyclopentyl; S18229: cyclohexyl). All compounds were found to be slow-tight binding inhibitors of thrombin versus purified human fibrinogen. Replacement of proline by N-cycloalkyl-glycines did not decrease the anti-thrombin potency of the substances up to the cyclopentyl size and this result was confirmed by classical coagulation assays with human plasma in vitro. In contrast, the inhibitory activities of the four new boronic acids were found to be lower than those of DuP-714 versus plasmin, urokinase (u-PA), plasmatic kallikrein, activated protein C (aPC) and complement factor I. The cyclopentyl derivative S18326 is a slightly more active inhibitor of thrombin than DuP-714 (initial IC50 values 3.99 +/- 0.18 nM versus 4.73 +/- 0.27 nM, respectively). Moreover S18326 was identified as the most selective compound of the series with relative potencies being 2 to 29 fold higher than that of DuP-714 versus the panel of serine-proteases tested; the rank order of potency versus the other serine-proteases for S18326 was t-PA>kallikrein>aPC>factor I>plasmin>fXa>u-PA. These results indicate that the size of the thrombin hydrophobic pocket S2 is sufficient to accept larger residues than proline in the P2 position of Ac-D-Phe-X-boroArg derivatives while this is not the case for other important serine-proteases of the fibrinolysis, coagulation and complement pathways. The N-cyclopentyl glycine containing derivative S 18326, which is the most potent and the most selective anti-thrombin compound of the series, currently undergoes major preclinical testing.
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PMID:Selection of S18326 as a new potent and selective boronic acid direct thrombin inhibitor. 936 88

Thrombomodulin (TM) expressed on endothelial cells binds thrombin and initiates anticoagulant pathways. Soluble functional proteolytic fragments of TM are also present in circulating plasma. Recently, it was reported that TM accelerated thrombin-dependent plasma procarboxypeptidase B (pro-pCPB) activation in a purified system and suggested that TM may inhibit fibrinolysis in crude plasma. The aim of present study was to evaluate any functional role of soluble TM fragments in plasma or purified TM added into plasma to the regulation of coagulation and fibrinolysis. Addition of rabbit TM (1-200 ng/ml) to plasma resulted in a concentration-dependent prolongation of urokinase (UK)- or tissue plasminogen activator (t-PA)-induced clot lysis time. The concentration of TM required for the inhibition of fibrinolysis was lower than that required for the inhibition of coagulation. Addition of anti-rabbit TM IgG or anti-human TM IgG into plasma reduced UK- or t-PA-induced clot lysis time without affecting clotting times, indicating that exogenous TM or soluble TM fragments in normal human plasma participated in regulation of fibrinolysis. Moreover, the TM-dependent inhibition of fibrinolysis was observed only in the presence of thrombin and blocked by addition of carboxypeptidase B inhibitors, but not mediated by protein C activation or direct inhibition of UK, t-PA or plasmin. Analysis of various substrates and inhibitors indicated that TM accelerated thrombin-dependent pro-pCPB activation in plasma. The present results indicate that TM, including soluble TM fragments in plasma, inhibit fibrinolysis via activation of pro-pCPB in plasma.
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PMID:Thrombomodulin in human plasma contributes to inhibit fibrinolysis through acceleration of thrombin-dependent activation of plasma procarboxypeptidase B. 949 93

A 71-year-old male, diagnosed as lung cancer, underwent unilateral pulmonary occulusion test. Through the guidewire, 7.5 Fr thermodilution catheter with occlusion balloon was introduced to the left pulmonary artery from the right internal jugular vein. Heparinized physiological saline solution was injected into the distal site of the occulusion. The occulusion time was 15 minutes. Pulmonary artery pressure and wedge pressure were within normal range. Soon after the examination, the pulmonary arteriogram (PAG) showed the defect of the branch to the lingular segment and the lower lobe. We made a diagnosis of pulmonary thrombosis. Three days after the administration of urokinase and heparin, both pulmonary perfusion scintigram and PAG exhibited the reperfusion to these areas. After the thrombolytic therapy was accomplished, antithrombin III and protein C in the serum showed within normal range. It was possible that the damage on the intima due to the thermodilution catheter or the guidewire and the following blood congestion by the pulmonary artery occulusion caused the thrombosis.
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PMID:[A case of the pulmonary thrombosis caused by unilateral pulmonary artery occulusion test]. 965 34

The trisaccharide allyl glycoside 36 and related disaccharide part structures have been prepared using the 2-trichloroacetamido-2-deoxy-alpha-D-galactopyranosyl trichloroacetimidate derivative 9 as glycosyl donor under promotion with TMSOTf or Sn(OTf)2, respectively, to produce the beta-(1-->4) linkage to suitably protected glucosamine derivatives in fair yields. Fucosylation was effected employing the ethyl 1-thio glycosyl donor 20 in the presence of IDCP. Deprotection of the intermediates afforded the disaccharide allyl glycosides beta-D-GalpNAc-(1-->4)- beta-D-GlcpNAc 13, beta-D-GalpNClAc-(1-->4)-beta-D-GlcpNAc 14, alpha-L-Fucp-(1-->3)-beta-D-GlcpNAc 24, alpha-L-Fucp-(1-->4)-beta-D- GlcpNAc 31 and the branched trisaccharide allyl glycoside beta-D-GalpNAc-(1-->4)[alpha-L-Fucp-(1-->3)]-beta-D-GlcpNAc 36. The trisaccharide which corresponds to a structural motif occurring in N-glycoprotein glycans from human urokinase, human recombinant protein C, phospholipase A2 as well as O-glycans, was converted into a neoglycoprotein following introduction of a cysteamine-derived spacer group and subsequent activation with thiophosgene.
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PMID:Synthesis of a neoglycoprotein containing the Lewis X analogous trisaccharide beta-D-GalpNAc-(1-->4)[alpha-L-Fucp-(1-->3)]-beta-D-GlcpNAc. 971 24

Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) into a virtually inactive two-chain form (tcu-PA/T), a process which may contribute to the maintenance of a fresh blood clot. We have examined the inactivation of scu-PA by thrombin in a plasma milieu to get more insight in the physiological relevance of this phenomenon. Citrated pooled normal plasma was treated with thrombin in the absence and presence of thrombomodulin. After an incubation period of 30 min the concentrations of scu-PA and tcu-PA/T were measured using specific bioimmunoassays. The inactivation of scu-PA in citrated plasma was found to be stimulated fourfold by thrombomodulin. Kinetic experiments showed that the inactivation of scu-PA by thrombin in the absence and presence of thrombomodulin occurred rapidly and declined within 1 min as a result of rapid inhibition by antithrombin III (ATIII) and other possible inhibitors. Calcium had no direct effect on the inactivation of scu-PA by exogenously added thrombin in the absence and presence of thrombomodulin. However, recalcification of plasma induced significant inactivation of scu-PA in plasma as a result of endogenous thrombin generation through the contact activation system. This calcium-induced inactivation of scu-PA was completely abolished in the presence of thrombomodulin, most likely as a result of activation of protein C by the complex formed between thrombomodulin and endogenously generated thrombin. Thrombomodulin thus appeared to play a dual role both by stimulating the inactivation of scu-PA by thrombin, and by inhibiting calcium-induced inactivation of scu-PA in plasma. In the plasma from a patient heterozygous for protein C deficiency, thrombomodulin could not prevent calcium-induced generation of tcu-PA/T, whereas the stimulating effect of thrombomodulin predominated instead. This result implied that disturbance of the protein C pathway may lead to the inactivation of substantial amounts of scu-PA in plasma under (patho)physiological circumstances and may provide an additional explanation for the association found between thromboembolism and deficiencies in the protein C pathway. This study shows that the amount of scu-PA that is inactivated in plasma depends mainly on the generation of thrombin and on thrombomodulin. We conclude that the inhibition of scu-PA-induced fibrinolysis appears to be regulated by activation of the coagulation system, providing a link between coagulation and fibrinolysis.
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PMID:The inactivation of single-chain urokinase-type plasminogen activator by thrombin in a plasma milieu: effect of thrombomodulin. 971 90

The human ovarian cancer cell line OV-MZ-19, established from a patient with cystadenocarcinoma of the ovary, expressing thrombomodulin (TM), a cell surface receptor for the serine protease thrombin, interacts with monoclonal and polyclonal antibodies having different specificity for TM. These antibodies detect TM antigen by means of flow cytofluorometry, laser scanning microscopy, immunocytochemistry, and ELISA. Therefore a highly sensitive ELISA for TM antigen was established using two different monoclonal antibodies to quantify TM in tissue extracts and biological fluids, e.g. peritoneal malignant ascites. Primary malignant ovarian tumors and metastases of the omentum and intestine contain TM antigen as determined by ELISA but in significantly lower concentrations than benign ovarian tumors (p=0.0056). In contrast, malignant ascitic fluid of patients with advanced ovarian cancer (FIGO IIIc) contain significantly elevated concentrations of soluble TM than benign peritoneal exudates (p=0.0003). Immunoaffinity purified ascites-derived TM efficiently activates protein C. Protein C activation of ascites-derived TM as well as TM expressed by the tumor cells is inhibited by the monoclonal antibodies. TM abrogates the procoagulant activity of thrombin, reduces pericellular thrombin via internalization, accelerates the thrombin-mediated inactivation of pro-uPA, and the EGF domains of TM exhibit mitogenic activity towards fibroblasts and tumor cells. Both, thrombin and pro-uPA play important roles in tumor invasion and metastasis. Therefore, downregulation and/or release of TM into ascitic fluid may play an important role in the malignant behavior of tumor cells.
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PMID:Thrombomodulin, a receptor for the serine protease thrombin, is decreased in primary tumors and metastases but increased in ascitic fluids of patients with advanced ovarian cancer FIGO IIIc. 973 90

Endothelial cells form a multifunctional cell lining that covers all of the inner surface of blood vessels and regulates several important physiological and pathological reactions. These include inflammation/immune reaction, blood vessel tonus, hemostasis/thrombosis, angiogenesis and so on. Thus, abnormalities of endothelial function may play crucial roles in the development of angitis syndrome, thrombosis/embolism, bleeding disseminated intravascular coagulation (DIC), and neovascularization in some pathological states including tumor growth and diabetic retinopathy. Research on endothelial cells now forms a new frontier termed 'Endotheliology'. Recent advances of the functional and structural aspects of endothelial cells are reviewed here mainly from the viewpoint of endothelial regulation of coagulation and the fibrinolytic system. First we show that the natural endothelial membrane protein thrombomodulin is localized not only on apical endothelial surface but also in caveolae. Since it has been reported that such factors involved in coagulation/fibrinolysis as tissue factor, tissue factor pathway inhibitor (TFPI), thrombin receptor and urokinase receptor are also localized in the caveolae, this membrane structure may act as a special component to regulate coagulation/fibrinolysis on the endothelial membrane surface. Next we demonstrate the signaling pathway of the thrombin receptor. Thrombin cleaves the N-terminus of the receptor as a substrate, exposing a new N-terminus. This newly exposed N-terminus acts as a ligand and activates platelets, endothelial cells and vascular smooth-muscle cells. We have identified that the signal from the thrombin receptor activates NF-kappaB through the activation of protein C kinase, tyrosine kinase and MAP kinase, and results in proliferation of the cells. We have also shown that the receptor is over-expressed on platelets from diabetes patients.
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PMID:Biology of endothelium. 981 71

Prof. Tage Astrup first elaborated the notion that blood fluidity involved a balance between the tendency of blood to clot and for such clots to lyse. It would seem that, at that time, this haemostatic balance involved the notion that forming fibrin orchestrated its own destruction by stimulating fibrinolytic activity. In this review, we have clarified the detail of this balance and developed the thesis that Astrup's far-sighted balance notions involve a variety of control mechanisms. These involve the notion that thrombin, being at first sight a procoagulant, can also, in conjunction with thrombomodulin, act as a stimulus of anticoagulant activity by the generation of activated protein C. The thrombin-activatable fibrinolytic inhibitor (TAFI) is also involved in this balance since the generation of thrombin provokes the neutralisation of fibrinolysis by the TAFI pathway. The kallikrein/factor XII/urokinase pathway is discussed indicating yet another aspect of balance between the generation of coagulation and fibrinolysis. The overall theme of this review, apart from an insight into various aspects of the haemostatic balance, is that blood has a strong tendency to clot when tissue is damaged, and the intact vasculature requires major anticoagulant systems to prevent clots adhering to and stabilising in the vasculature.
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PMID:The haemostatic balance -- Astrup revisited. 1049 35

Serine proteases of the chymotrypsin fold are of great interest because they provide detailed understanding of their enzymatic properties and their proposed role in a number of physiological and pathological processes. We have been developing the macromolecular inhibitor ecotin to be a "fold-specific" inhibitor that is selective for members of the chymotrypsin-fold class of proteases. Inhibition of protease activity through the use of wild-type and engineered ecotins results in inhibition of rat prostate differentiation and retardation of the growth of human PC-3 prostatic cancer tumors. In an effort to identify the proteases that may be involved in these processes, reverse transcription-PCR with PC-3 poly(A)+ mRNA was performed by using degenerate oligonucleotide primers. These primers were designed by using conserved protein sequences unique to chymotrypsin-fold serine proteases. Five proteases were identified: urokinase-type plasminogen activator, factor XII, protein C, trypsinogen IV, and a protease that we refer to as membrane-type serine protease 1 (MT-SP1). The cloning and characterization of the MT-SP1 cDNA shows that it encodes a mosaic protein that contains a transmembrane signal anchor, two CUB domains, four LDLR repeats, and a serine protease domain. Northern blotting shows broad expression of MT-SP1 in a variety of epithelial tissues with high levels of expression in the human gastrointestinal tract and the prostate. A His-tagged fusion of the MT-SP1 protease domain was expressed in Escherichia coli, purified, and autoactivated. Ecotin and variant ecotins are subnanomolar inhibitors of the MT-SP1 activated protease domain, suggesting a possible role for MT-SP1 in prostate differentiation and the growth of prostatic carcinomas.
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PMID:Reverse biochemistry: use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue. 1050 Jan 22


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