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)

To determine in vivo functional roles for thrombin's structural domains, we have compared the relative antithrombotic and antihemostatic effects of (i) catalytic-site antithrombin peptide, D-Phe-Pro-Arg; (ii) exosite antithrombin peptide, the C-terminal tyrosine-sulfated dodecapeptide of hirudin; and (iii) bifunctional antithrombin peptide, a 20-mer peptide combining catalytic-site antithrombin peptide and exosite antithrombin peptide with a polyglycyl linker. All three peptides inhibited thrombin-mediated platelet aggregation and fibrin formation in vitro. In vivo thrombus formation was measured in real time as 111In-labeled platelet deposition and 125I-labeled fibrin accumulation on thrombogenic segments incorporated into chronic exteriorized arteriovenous access shunts in baboons. Under low flow conditions, the continuous infusion of peptides reduced thrombus formation onto collagen-coated tubing by half at doses (ID50) and corresponding concentrations (IC50) of 800 nmol per kg per min and 400 nmol/ml for catalytic-site antithrombin peptide, greater than 1250 nmol per kg per min and greater than 1500 mumol/ml for exosite antithrombin peptide, and 50 nmol per kg per min and 25 nmol/ml for bifunctional antithrombin peptide. Under arterial flow conditions, systemically administered bifunctional antithrombin peptide decreased thrombus formation in a dose-dependent manner for segments of collagen-coated tubing or prosthetic vascular graft ID50 and IC50 values of 120 nmol per kg per min and 15 nmol/ml; this dose also produced intermediate inhibition of hemostatic function [bleeding time, 21 +/- 3 min vs. 4.5 +/- 0.5 min (baseline values); P less than 0.001; activated partial thromboplastin time, 285 +/- 13 sec vs. 31 +/- 3 sec (baseline), P less than 0.001]. In contrast, thrombus formation onto segments of endarterectomized aorta was potently decreased by bifunctional antithrombin peptide with an ID50 value of 2.4 nmol per kg per min and an IC50 value of 0.75 nmol/ml, a systemic dose that failed to affect hemostasis. Thus, inhibiting both thrombin's catalytic and exosite domains increases antithrombotic potency by several orders of magnitude over the inhibition of either domain alone, particularly at sites of deep arterial injury.
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PMID:Antithrombotic effects of synthetic peptides targeting various functional domains of thrombin. 138 67

Secretion from recombinant yeast was used as a potential source of large quantities of the leech protein antistasin (ATS), a potent and highly specific inhibitor of the serine protease coagulation factor Xa. Mature recombinant ATS (r-ATS) is obtained after intracellular cleavage by the yscF protease of the mating factor alpha-1 pre-proleader from the fusion protein at the Lys-Arg sequence junction. Production levels are relatively low (ca. 1 mg/liter). Purification of the secreted product from a complex growth medium involved cell removal by microfiltration and diafiltration, cation-exchange capture and concentration on S-Sepharose Fast Flow, C-4 reverse-phase high-performance liquid chromatography (RP-HPLC), and HPLC cation-exchange chromatography step, and RP-HPLC concentration and desalting. The process was scaled up from the 16- to the 250-liter level with a corresponding increase in amount of r-ATS. From the 250-liter fermentation two major forms, r-ATS-I and r-ATS-II, distributed approximately 60:40, and a minor form, r-ATS-minor (ca. 1% of the purified r-ATS), were characterized. Limited N-terminal sequence analysis by Edman degradation indicated that r-ATS-I has the predicted mature N-terminus starting with Gln, that r-ATS-II is N-terminally blocked with pyroglutamate, and that r-ATS-minor is an incompletely processed form. RP-HPLC, hydrophilic-interaction HPLC, cation-exchange HPLC analysis, and electrophoresis results are consistent with the differences observed by sequencing. Preliminary in vitro characterization by intrinsic Ki determination for factor Xa inhibition indicated that the yeast r-ATS forms are indistinguishable from each other as well as from r-ATS expressed by the insect baculovirus host-vector system. Nevertheless, r-ATS-I and r-ATS-II appear less potent than insect-derived r-ATS in the activated partial thromboplastin time clotting assay. Further characterization indicated that C-terminal cleavage at Pro-116 had occurred in r-ATS-I and r-ATS-II as well as oxidation of methionine residues to methionine sulfoxide. The possible role of the C-terminus in inhibition of the prothrombinase complex is discussed.
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PMID:Characterization of recombinant antistasin secreted by Saccharomyces cerevisiae. 139 15

Recombinant tick anticoagulant peptide (rTAP) is a highly selective inhibitor of blood coagulation factor Xa. rTAP has been characterized kinetically as a slow, tight-binding, competitive inhibitor of the enzyme. We used an approach consisting of both recombinant, site-directed mutagenesis and solid-phase chemical synthesis to generate 31 independent mutations in rTAP to identify those regions of the molecule which contribute to the specific, high-affinity binding interaction with factor Xa. Our results demonstrate that the four amino-terminal residues of rTAP constitute the primary recognition determinant necessary for the formation of the high-affinity enzyme-inhibitor complex. The Arg residue in position three is probably not interacting with the S1-specificity pocket of factor Xa in a substrate-like manner since substitution at this position with a D-Arg amino acid produced only a modest decrease in affinity (5-fold). An additional domain in the rTAP molecule located between residues 40 and 54 was identified as a probable secondary binding determinant. Interestingly, this region in rTAP shares significant amino acid sequence homology with a sequence in prothrombin immediately amino-terminal to the factor Xa cleavage site that generates meizothrombin. These observations indicate that specific segments within two different regions of the rTAP molecule contribute to the potent binding interaction between rTAP and factor Xa.
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PMID:Site-directed analysis of the functional domains in the factor Xa inhibitor tick anticoagulant peptide: identification of two distinct regions that constitute the enzyme recognition sites. 145 8

A recent report hypothesized that an Arg79-->Gln mutation in the first epidermal growth factor-like domain of human factor VII is the molecular basis for a severe (< 1%) factor VII functional deficiency. In the present study, a site-specific mutant human factor VII cDNA (Arg79-->Gln) was constructed, subcloned and expressed in baby hamster kidney cells. Mutant factor VII was purified to homogeneity and characterized with respect to gamma-carboxyglutamic acid content, ability to activate, tissue factor-dependent amidolytic activity and expression of factor VIIa proteolytic activity on tissue factor-bearing cells. Mutant factor VII was fully carboxylated and exhibited the same molecular weight and coagulant activity as plasma factor VII. Mutant factor VII was activated by factor Xa at the same rate, and to the same extent, as plasma factor VII. In the presence of tissue factor, mutant factor VII was converted to factor VIIa in an autocatalytic manner at a rate indistinguishable from that observed with plasma factor VII. In addition, the amidolytic activities of mutant factor VIIa and plasma factor VIIa towards S-2288 in the presence of relipidated tissue factor were identical. Finally, following complex formation with cell surface tissue factor, mutant factor VIIa activated factor X at essentially the same rate as plasma factor VIIa under comparable conditions. These results are not consistent with the notion that the arginine-79 residue in the first epidermal growth factor-like domain of human factor VII is essential for the expression of tissue factor-dependent factor VIIa proteolytic activity.
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PMID:Evidence that an Arg79-->Gln substitution in human factor VII is not associated with a reduction in coagulant activity. 838 4

Previous studies of human statherin showed the active region for inhibition of secondary calcium phosphate precipitation (crystal growth) to reside in the highly charged amino-terminal one-third of this molecule, and the neutral tyrosine-, glutamine- and proline-rich carboxy-terminal two-thirds of the molecule is required for maximal inhibition of primary (spontaneous) precipitation. The purpose of the present study was to define more clearly the activities of these different molecular segments of statherin with respect to the two kinds of inhibitory activities. Peptides from statherin were prepared by specific proteolysis using trypsin, endoproteinase Arg-C, and activated factor X to produce the amino-terminal hexa-, nona- and decapeptides, respectively, and carboxypeptidase-A was used to obtain a peptide extending from residue 1 to about residues 32-37. The peptides were purified by anion exchange and gel filtration chromatography, and characterized and quantified by amino-acid analysis. Serially diluted samples of statherin and derived peptides were assayed to determine the concentrations, giving a standard 50% inhibition of precipitation (C50%) in assay systems designed for this purpose using polyaspartate as a standard. Results are expressed as (C50% statherin)/(C50% peptide). For inhibition of primary precipitation, these values were peptide(1-6), 0.20; peptide(1-9), 0.15; peptide(1-31/35), 0.24. For inhibition of secondary precipitation, the values were peptide(1-6), 3.8; peptide(1-9), 2.8; peptide(1-10), 1.9; peptide(1-32/37), 1.5. These quantitative findings show that maximum inhibition of primary precipitation by statherin requires the entire molecule. Thus, removal of a relatively small segment of its carboxy-terminal region results in a substantial reduction in inhibitory activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibition of calcium phosphate precipitation by human salivary statherin: structure-activity relationships. 152 6

The design of low molecular weight thrombin inhibitors IIa-d (hirutonins) that bind concurrently with the enzyme's catalytic site and auxiliary "anion-binding exosite" for fibrinogen recognition is reported. A practical synthesis of the required homologous ketomethylene arginyl dipeptide inserts [Arg psi CO(CH2)nCO] (n = 1-4) corresponding to the P1-P1' scissile position of hirutonins is described. The substitution of the scissile amide function by a ketomethylene group is compatible with the enzyme active site and conferred complete plasma proteolytic stability. This modification also enhanced enzyme affinity up to 20-fold with hirutonin-4 (IIb, n = 4) displaying highest affinity (Ki = 140 +/- 20 pM). Hirutonins 1-4 exhibited potent inhibition of plasma prothrombin time (PT) and activated partial thromboplastin time (aPTT). The inhibition was biphasic and showed good correlation with the corresponding Ki. Hirutonin-2 inhibited thrombin-mediated platelet aggregation and exhibited a strong antithrombotic effect comparable to r-hirudin in an in vivo rat arteriovenous shunt model (ED15 = 1.20 mg/kg for hirutonin-2 and 1.14 mg/kg for r-hirudin). Lower molecular weight inhibitors were obtained by substituting the six native amino acid residues (Q-S-H-N-D-G), connecting the active site and the auxiliary exosite binding elements with a variable number of interening omega-aminopentenoyl units. In addition, the exosite component was reduced to seven amino acid residues (D-F-E-P-I-P-L). Incorporation of these modifications into the bifunctional format resulted in nanomolar thrombin inhibitory peptides (IIIa-c). The resulting inhibitors were studied by molecular modeling with alpha-thrombin, and the bimolecular interactions served to explain the retention of high enzyme affinity.
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PMID:Synthesis of a homologous series of ketomethylene arginyl pseudodipeptides and application to low molecular weight hirudin-like thrombin inhibitors. 152 82

Ketomethylene pseudopeptide analogues Aa-Pro-Arg psi (COCH2) Gly-pip, 1, where Aa are D- or L-amino acids (Dpa, beta, beta-diphenylalanine; alpha Nal, alpha-naphthylalanine; beta Nal, beta-naphthylalanine; Fgl, fluorenylglycine) with highly lipophilic side chains and psi (COCH2) is a ketomethylene pseudopeptide bond, have been synthesized through a modified Dakin-West reaction under very mild conditions with a high yield using tripeptide 4 with a labile functional group directly on the side chain. Their enzymatic assay of thrombin inhibition has been carried out. The structure-activity relationship study indicated that a lipophilic side chain on the amino acid in the P3 position is very important for binding to the apolar site of thrombin. Compound 1a with D-Dpa at the P3 position has a Ki of 0.2 microM and it doubles thrombin clotting time at only 3 times higher concentration. These values are about 7 times better than those of the corresponding D-Phe analogues. Furthermore, 1a shows poor inhibitory activity against plasmin, factor Xa, urokinase, and kallikrein. Preliminary in vivo testing (3-4-kg rabbit as the animal model) shows no observable side effect (change of blood pressure and accumulation of blood platelet in lungs) at a dose of 1 mg/kg.
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PMID:Synthesis and biological activity of ketomethylene pseudopeptide analogues as thrombin inhibitors. 152 87

Antistasin (ATS) is a 119-amino acid, leech-derived protein which exhibits selective, tight-binding inhibition of blood coagulation factor Xa. Prolonged incubation of ATS with factor Xa leads to the highly specific hydrolysis of the peptide bond between residues Arg34 and Val35, implicating this peptide bond as the putative reactive site. We report here the preparation of pure, cleaved (modified) recombinant ATS (rATS) and utilize this material to provide additional proof that the cleaved peptide bond is in fact the reactive site. Modified rATS retains strong inhibitory potency against factor Xa as evidenced by a dissociation constant of 166.3 +/- 9.6 pM; four-fold greater than that of native inhibitor, 43.4 +/- 1.4 pM. Incubation of pure, modified rATS with catalytic amounts of factor Xa results in resynthesis of the hydrolyzed peptide bond, achieving an equilibrium near unity between native and modified inhibitors. Specific removal of the newly formed carboxy-terminal Arg residue from modified rATS by carboxypeptidase B treatment obviates its conversion to native inhibitor coincident with the complete loss of inhibitory activity. These results establish that rATS inhibits factor Xa according to a standard mechanism of serine protease inhibitors and support the contention that the Arg34-Val35 peptide bond constitutes the reactive site.
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PMID:The hydrolysis and resynthesis of a single reactive site peptide bond in recombinant antistasin by coagulation factor Xa. 156 19

A series of new compounds, 6-amino-1-naphthalenesulfonamides (ANSN), were used as fluorescent detecting groups for substrates of amidases. These compounds have a high quantum fluorescent yield, and the sulfonyl moiety permits a large range of chemical modification. Fifteen ANSN substrates with the structure (N alpha-Z)Arg-ANSNR1R2 were synthesized and evaluated for their reactivity with 8 proteases involved in blood coagulation and fibrinolysis. Thrombin, activated protein C, and urokinase rapidly hydrolyzed substrates with monosubstituted sulfonamide moieties (R1 = H). The maximum rate of substrate homologue). The hydrolysis rates for substrates with branched substituents were slower than their linear analogues. Monosubstituted (N alpha-Z)Arg-ANSNR1R2 possessing cyclohexyl or benzyl groups in the sulfonamide moiety were hydrolyzed by these three enzymes at rates similar to that of the n-butyl homologue (except the cyclohexyl compound for u-PA). Factor Xa rapidly hydrolyzed substrates with short alkyl chains, especially when R1 = R2 = CH3 or C2H5. Lys-plasmin and rt-PA demonstrated low activity with these compounds, and the best results were accomplished for monosubstituted compounds when R2 = benzyl (for both enzymes). Factor VIIa and factor IXa beta exhibited no activity with these substrates. A series of 14 peptidyl ANSN substrates were synthesized, and their reactivity for the same 8 enzymes was evaluated. Thrombin, factor Xa, APC, and Lys-plasmin hydrolyzed all of the substrates investigated. Urokinase, rt-PA, and factor IXa beta exhibited reactivity with a more limited group of substrates, and factor VIIa hydrolyzed only one compound (MesD-LGR-ANSN(C2H5)2). The substrate ZGGRR-ANSNH (cyclo-C6H11) showed considerable specificity for APC in comparison with other enzymes (kcat/KM = 19,300 M-1 s-1 for APC, 1560 for factor IIa, and 180 for factor Xa). This kinetic advantage in substrate hydrolysis was utilized to evaluate the activation of protein C by thrombin in a continuous assay format. Substrate (D-LPR-ANSNHC3H7) was used to evaluate factor IX activation by the factor VIIa/tissue factor enzymatic complex in a discontinuous assay. A comparison between the commercially available substrate chromozyme TH (p-nitroanilide) and the ANSN substrate with the same peptide sequence (TosGPR) demonstrated that aminonaphthalenesulfonamide increased the specificity (kcat/KM) of substrate hydrolysis by thrombin more than 30 times, with respect to factor Xa substrate hydrolysis.
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PMID:Aminonaphthalenesulfonamides, a new class of modifiable fluorescent detecting groups and their use in substrates for serine protease enzymes. 160 66

Recombinant hirudin, a potent and specific thrombin inhibitor, is considered for anticoagulant therapy. Therefore we developed a fast and sensitive chromogenic assay for its determination in plasma. Samples can be assayed directly if aprotinin, Polybrene and urea are added to the reagent mixture. The influence of progressive inhibitors is excluded by a short incubation time. The samples (20 microliters) are diluted with 1 ml reagent mixture (0.2 M Tris, 0.025 M NaCl, pH 8.1, containing 0.833 M urea, 0.7 trypsin inhibitory units/ml aprotinin, 100 ng/ml Polybrene and 0.31 NIH units/ml bovine thrombin). After an incubation time of 1 min, 100 microliters Chromoyzm TH (Tos-Gly-Pro-Arg-pNA, 1.9 mM) is added. delta absorbance/min is linear at least up to 3 min. The calibration curve is linear up to at least 800 ng hirudin/ml plasma. The inter- and intraassay coefficient of variation in hirudin spiked normal plasma is below 5%. The detection limit is at 25 ng hirudin/ml. In plasma samples obtained from healthy subjects under hirudin therapy, a good correlation was found to the activated partial thromboplastin time (r = 0.89). In conclusion, we describe a fast and simple chromogenic substrate test to assay hirudin in plasma. Under assay conditions, the influence of endogenous thrombin inhibitors can be neglected.
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PMID:A fast photometric assay for the determination of hirudin. 171 4


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