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:3.4.21.6 (thromboplastin)
13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new protein, salmorin, that inhibits fibrinogen clotting was purified to homogeneity from the snake venom of Agkistrodon halys brevicaudus. Salmorin was characterized to be a heterodimeric protein composed of 15 kDa and 14 kDa subunits. Purified salmorin inhibited not only thrombin-induced fibrinogen clotting but also factor Xa-induced prothrombin activation. Experimental evidence indicated that the hetero-dimeric protein does not bind to the thrombin catalytic site but binds to thrombin and prothrombin exosites. Analyses of the cDNA sequences encoding the two separate polypeptide chains revealed that the mature subunits are composed of 131 and 122 amino acids, respectively. In the isolated cDNAs, N-termini of both chains are preceded by hydrophobic signal peptides of 23 residues. The deduced amino acid sequence exhibited considerable identity with other snake venom C-type lectin-like proteins derived from various snake venoms.
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PMID:Purification and cDNA cloning of salmorin that inhibits fibrinogen clotting. 1096 89

Recombinant factor VIII SQ (r-VIII SQ), ReFacto, is a recombinant factor VIII product similar to the smallest active factor VIII protein found in plasma-derived factor VIII (p-VIII) concentrates. The protein comprises two polypeptide chains of 80 and 90 kDa and lacks the major part of the heavily glycosylated B-domain i.e. amino acids Gln744 to Ser1637. r-VIII SQ retains six potential glycosylation sites for N-linked oligosaccharides at asparagine residues 41, 239, 582, 1685, 1810 and 2118. We describe a thorough comparison of the characteristics of r-VIII SQ with those of p-VIII. The primary and secondary structures of r-VIII SQ were in good agreement with that of B-domain-deleted p-VIII (p-VIII-LMW) as shown by SDS-PAGE, Western blotting with antifactor VIII antibodies, tryptic mapping, amino acid sequence analysis and circular dichroism spectroscopy. A few divergences also existed. Thus r-VIII SQ was shown to contain a small amount of the single chain primary translation product of 170 kDa and also the product specific sequence of 14 amino acids, the SQ-link, in the C-terminal end of the 90 kDa chain. It was shown that r-VIII SQ had a high specific activity of about 14,000 IU VIII:C/mg as determined by use of a chromogenic substrate assay. The r-VIII SQ protein was comparable to p-VIII forms with a retained B-domain, in terms of potency measured by a chromogenic substrate or a two-stage clotting assay, in interactions with thrombin, and with activated protein C (APC) in combination with Protein S. The ability of r-VIII SQ to participate as a cofactor in factor Xa generation in a mixture of factors IXa and X, phospholipid and calcium was in conformity with that of p-VIII. Furthermore r-VIII SQ had a good binding capacity for phospholipid vesicles and von Willebrand factor (vWF) as shown in gel filtration studies. The same kinetics in binding to von Willebrand factor was found for r-VIII SQ and p-VIII as determined by real-time biospecific interaction analysis (BIA) with use of the BIAcore instrument. The apparent association rate constant was 4 x 10(6) M(-1)s(-1). Two dissociation rate constants were found, 1 X 10(-2)s(-1) and 4 x 10(-4)s(-1). The results extend the present knowledge that the factor VIII B-domain is dispensable for the factor VIII cofactor function in hemostasis.
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PMID:Structural and functional characteristics of the B-domain-deleted recombinant factor VIII protein, r-VIII SQ. 1120 95

In order to study arginine decarboxylase regulation, we produced an antiserum against a hybrid of a 615 amino acid residue fragment of grapevine arginine decarboxylase cDNA with maltose-binding protein. The antiserum generated recognized mainly a protein band of ca. 80 kDa in extracts from grapevine tissues. Extracts from leaves and internodes in different developmental stages showed differences in the quantity of the 80 kDa band recognized by the antiserum. However, these differences did not correspond with changes in arginine decarboxylase specific activity. Furthermore, western blot analysis of extracts from cell cultures, where enzyme-specific activity was induced or repressed, did not reveal respective changes in the quantity of the 80 kDa protein band. Digestion of the hybrid by the specific protease factor Xa resulted in a polypeptide of 90 kDa instead of the expected two polypeptides of 43 and 66 kDa. Finally, western blot analysis of shoot extract incubated with factor Xa or the hybrid protein previously digested by factor Xa revealed that factor Xa-digested hybrid protein cleaved the 80 kDa band, resulting in two bands of ca. 38 and 40 kDa, whereas factor Xa alone did not affect it. These results suggest that arginine decarboxylase protein levels and/or activity is post-translationally regulated, as has been shown for other enzymes of polyamine biosynthesis.
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PMID:Indications for post-translational regulation of Vitis vinifera L. arginine decarboxylase. 1143 Apr 29

Hirudin, the anticoagulatory polypeptide of the leech Hirudo medicinalis, strongly inhibits thrombus formation by specifically interacting with thrombin. For diagnostic purposes, hirudin should be superior to other anticlotting compounds because it only minimally alters the mineral, protein, and cellular blood constituents. To test this hypothesis, hirudinized and routinely processed venous blood from 80 healthy volunteers and patients was subjected to a variety of automated blood tests. A strong correlation was found between the results of automated complete blood counts obtained from K(2)-ethylenediaminetetraacetic acid (EDTA) anticoagulated and hirudinized blood (1000 antithrombin units [ATU] hirudin/ml). In addition, clinical chemistry and serological infection parameters (asparlat amintransferase [ASAT], lactate dehydrogenase [LDH], sodium, and so on, and antibodies against hepatitis B and C and human immunodeficiency virus [HIV]1/2, respectively) correlated well when measured in serum as compared with hirudinized plasma. Contrary to single clotting factors, global coagulation parameters (activated partial thromboplastin time [aPTT], prothrombin time [PT]) could not be measured in hirudinized blood. Recombinant hirudin neither interfered with immunophenotyping of mononuclear cells using FACScan analysis, nor did it alter the detection of Wilms' tumor gene expression by RT-PCR technology even at high doses (5000 ATU hirudin). Thus, a hirudin-containing blood sampling tube can be designed as a universal blood sampling tube (UBT) for testing the majority of diagnostic blood parameters.
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PMID:Measurement of hematological, clinical chemistry, and infection parameters from hirudinized blood collected in universal blood sampling tubes. 1154 57

Lentiviral vectors represent an attractive technology platform from which to develop a targetable injectable gene delivery system for transduction of specific cell populations in vivo, irrespective of their cell cycle status. Targeted HIV-1-based lentiviral vectors were generated by pseudotyping them with chimeric murine leukemia virus (MLV) envelope glycoproteins displaying N-terminal targeting polypeptides. Vectors displaying an EGF polypeptide were fully infectious on EGF receptor-negative cells, but were inactive on cells with abundant EGF receptors (inverse targeting). Receptor-mediated inactivation of gene transfer was overcome by competing the EGF receptors on the target cells with soluble EGF or by removing the displayed EGF domain from the surface of the vector particles by factor Xa cleavage of a specific protease substrate engineered into its tethering linker (protease targeting). Intravenous infusion of nontargeted HIV-1 vectors led to maximal luciferase activity in liver and spleen with moderate or minimal activity in heart, skeletal muscle, lung, brain, kidney, ovaries and bone marrow. In contrast, intravenous EGF-displaying vectors were expressed maximally in spleen with very low level luciferase expression detectable in liver (EGF-receptor rich). Liver transduction by the EGF-displaying vector was restored by pretreating the animals with soluble EGF suggesting that these vectors are inversely targeted to spleen.
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PMID:Organ distribution of gene expression after intravenous infusion of targeted and untargeted lentiviral vectors. 1159 58

An inhibitor of factor Xa (FXa) was isolated from the nymphs of the camel tick Hyalomma dromedarii by a combination of chromatography on DEAE-cellulose and Sephacryl S-300 columns. The isolated nymphal FXa inhibitor turned out to be a homogenous preparation of a single polypeptide chain (15 kDa) as judged by both the native and denatured SDS-PAGE. Its pI value ranged from 7.7 to 7.9. The inhibitor is a potent anticoagulant since it prolonged both the activated partial thromboplastin time (APTT) and the prothrombin time (PT) of the camel plasma in a concentration-dependent manner. Its activity was threefold lower toward thrombin than FXa, but it did not inhibit any of the proteases; trypsin, alpha-chymotrypsin, papain, pepsin and subtilisin. The inhibitor binds at two sites on FXa uncompetitively with an inhibition constant (K(i)) value of 134 nM.
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PMID:Factor Xa (FXa) inhibitor from the nymphs of the camel tick Hyalomma dromedarii. 1169 27

We report upon the purification and characterization of a novel prothrombin-activating enzyme from the body fluid (total homogenates of isolated digestive tract without eggs, spinnerets and silk glands) of the spider, Nephila clavata by a combination of acetone fractionation, ion exchange, and Soybean trypsin inhibitor-Sepharose chromatography. Analysis of the purified enzyme with SDS-PAGE and gel filtration revealed a single polypeptide chain with an apparent molecular weight of 24kDa. The proteolytic activity of the enzyme was stable up to 50 degrees C, however, it became unstable over 55 degrees C. The enzyme had an optimum pH of 8, and Ca(2+) was not required for the enzyme activity. According to inhibition profiles obtained with several serine protease inhibitors such as PMSF and benzamidine, the purified protease is a member of the serine proteases. Bz-Ile-Glu(gamma-OR)- Gly-Arg-pNA and Z-Arg-Gly-Arg-pNA which are known as substrates for factor Xa, were hydrolyzed favorably by the enzyme. And the Nephila protease could produce thrombin from prothrombin at nM range, and form the turbid ring using fibrinogen-agarose plate. The results obtained confirmed that the purified protease is a potent prothrombin-activating activity belonging to the family of serine protease.
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PMID:Purification and characterization of a prothrombin-activating protease from Nephila clavata. 1171 Nov 26

Inflammatory cytokines are suspected to play an important role in the pathophysiology of left ventricular (LV) remodeling. We investigated whether high-sensitivity C-reactive protein (CRP) (hs-CRP) is a predictor for LV remodeling in patients with acute myocardial infarction (AMI) with successful reperfusion, and also whether such a situation can be avoided by the administration of angiotensin-converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARB). The subjects were 139 patients with an initial attack of anterior myocardial infarction successfully treated by reperfusion therapy. They were randomly divided into the following two groups: an angiotensin (AG) group (91 patients treated with ACEI/ARB) and a NON-AG group (48 patients not treated with ACEI/ARB). Levels of hs-CRP, creatine kinase, human atrial natriuretic polypeptide, brain natriuretic peptide (BNP), fasting blood glucose, serum lipids, fibrinogen, fibrin degradation product, prothromloin time, and activated partial thromboplastin time were measured immediately after 1, 2, 3, and 7 days, and 1 months after the onset of AMI. ACEI or ARB administration lowered hs-CRP levels and prevented the development of LV remodeling. Peak CRP levels significantly correlated with BNP levels during the acute stage (r = +0.54, P < 0.0001), end-diastolic volume index (r = +0.78, P < 0.0001), end-systolic volume index (r = +0.36, P = 0.0405), ejection fraction (r = -0.45, P = 0.0052), left ventricular end-diastolic diameter (r = +0.61, P < 0.0001), cardiac output (r = -0.52, P = 0.0005), cardiac index (r = -0.41, P = 0.0099), and systolic pulmonary arterial pressure (r = +0.48, P = 0.0017) 1 month after the onset of AMI in the NON-AG group but not in the AG group. Logistic multivariate analysis revealed that peak CRP alone was an independent risk factor for the development of LV remodeling in the NON-AG group (odds ratio = 1.79, P = 0.002). These results suggest that hs-CRP is a useful factor for predicting LV remodeling. Furthermore, ACEI or ARB administration to AMI patients showing increased hs-CRP levels during the early stage of the disease could prevent LV remodeling.
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PMID:High-sensitivity C-reactive protein and left ventricular remodeling in patients with acute myocardial infarction. 1275 2

Caesalpinia echinata is a tree belonging to the Leguminosae family. The red color of the trunk, looking like burning wood ('brasa' in Portuguese), is the origin of the name Brazil. Seeds of leguminous plants contain high amounts of serine proteinase inhibitors that can affect different biological processes. Here we show that a protein isolated from seeds of C. echinata is able to inhibit enzymes that participate in blood coagulation and fibrinolysis. This inhibitor (CeKI) was purified to homogeneity by ion exchange and reversed-phase chromatography. SDS-PAGE indicated a single polypeptide chain with a molecular mass of 20 kDa. CeKI inhibits human plasma kallikrein ( K i =3.1 nM), plasmin ( K i =0.18 nM), factor XIIa ( K i =0.18 nM), trypsin ( K i =21.5 nM) and factor Xa ( K i =0.49 mM). CeKI inhibited kinin release from highmolecular- mass kininogen by kallikrein in vitro . The N-terminal sequence, determined by automatic Edman degradation, identified the inhibitor as a member of the Kunitz family. The secondary structure, determined by circular dichroism, is mainly a random coil followed by beta-sheet structure. The action of CeKI on enzymes of the blood-clotting intrinsic pathway was confirmed by prolongation of the activated partial thromboplastin time.
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PMID:A proteinase inhibitor from Caesalpinia echinata (pau-brasil) seeds for plasma kallikrein, plasmin and factor XIIa. 1557 29

In this study, we isolated a novel prothrombin activator from the venom of Bothrops cotiara, a Brazilian lance-headed pit viper (Cotiara, Jararaca preta, Biocotiara), which we have designated "cotiaractivase" (prefix: cotiar- from B. cotiara; suffix: -activase, from prothrombin activating activity). Cotiaractivase was purified using a phenyl-Superose hydrophobic interaction column followed by a Mono-Q anion exchange column. It is a single-chain polypeptide with a molecular weight of 22,931 Da as measured by mass spectroscopy. Cotiaractivase generated active alpha-thrombin from purified human prothrombin in a Ca2+-dependent manner as assessed by S2238 chromogenic substrate assay and SDS-PAGE. Cotiaractivase cleaved prothrombin at positions Arg271-Thr272 and Arg320-Ile321, which are also cleaved by factor Xa. However, the rate of thrombin generation by cotiaractivase was approximately 60-fold less than factor Xa alone and 17 x 10(6)-fold less than the prothrombinase complex. The enzymatic activity of cotiaractivase was inhibited by the chelating agent EDTA, whereas the serine protease inhibitor PMSF had no effect on its activity, suggesting that it is a metalloproteinase. Interestingly, S2238 inhibited cotiaractivase activity non-competitively, suggesting that this toxin contains an exosite that allows it to bind prothrombin independently of its active site. Tandem mass spectrometry and N-terminal sequencing of purified cotiaractivase identified peptides that were identical to regions of the cysteine-rich and disintegrin-like domains of known snake venom metalloproteinases. Cotiaractivase is a unique low molecular weight snake venom prothrombin activator that likely belongs to the metalloproteinase family of proteins.
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PMID:Isolation and characterization of cotiaractivase, a novel low molecular weight prothrombin activator from the venom of Bothrops cotiara. 1664 9


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