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)

In this study we sought to extend the plasma half-life while maintaining the potent antithrombin activity of hirudin. We hypothesized that gene fusion of hirudin to albumin would result in the expression of a slowly cleared hirudin molecule. A hirudin variant 3 (HV3) cDNA was obtained by gene synthesis, while a 1,996-bp full-length rabbit serum albumin (RSA) cDNA was selected from a rabbit liver cDNA library. Expression of the former in COS-1 cells conferred antithrombin activity on media conditioned by the cells, while expression of the latter resulted in the secretion of a 67-kD protein that reacted with mono-specific anti-RSA antibodies. Having shown independent expression of the two proteins, we next expressed two fusion proteins: HV3 linked via its C-terminus to albumin (HLA), and HV3 linked via its N-terminus to albumin (ALH). The former, but not the latter, inhibited both the amidolytic and fibrinogenolytic activities of thrombin. HLA also retained the dye-binding characteristics of RSA, as judged by Affi-Gel Blue chromatography. Highly similar concentrations of either commercial HV1 (40 nmol/L) or HLA (30 nmol/L) were required to halve the initial rate of thrombin reaction with chromogenic substrate S2238, suggesting the retention of high-affinity inhibition of thrombin by the fusion protein. An His-tagged form of HLA was purified by Ni2+-chelate affinity and heparin-Sepharose chromatography. The purified, radioiodinated protein was injected into rabbits, and demonstrated a catabolic half-life of 4.60 +/- 0.16 days. This represents an extension of hirudin half-life in vivo of greater than two orders of magnitude; gel analysis of HLA(H)6 recovered from rabbits showed that it circulated in intact form. Our results provide a rationale for future testing of the biological effects of HLA, and support our initial hypothesis.
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PMID:Potent antithrombin activity and delayed clearance from the circulation characterize recombinant hirudin genetically fused to albumin. 912 29

Thrombus formation and intimal hyperplasia on the surface of implantable biomaterials such as poly(ethylene terepthalate) (Dacron) vascular grafts are major concerns when utilizing these materials in the clinical setting. Thrombin, a pivotal enzyme in the blood coagulation cascade primarily responsible for thrombus formation and smooth muscle cell activation, has been the target of numerous strategies to prevent this phenomenon from occurring. The purpose of this study was to covalently immobilize the potent, specific antithrombin agent recombinant hirudin (rHir) to a modified Dacron surface and characterize the in vitro efficacy of thrombin inhibition by this novel biomaterial surface. Bovine serum albumin (BSA), which was selected as the "basecoat' protein, was reacted with various molar ratios of the cross-linker sulphosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulpho-SMCC; 1:5-1:50). These BSA-SMCC complexes were then covalently linked to sodium hydroxide-hydrolysed Dacron (HD) segments via the cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Covalent linkage of these complexes to HD (HD-BSA-SMCC) was not affected by any of the sulpho-SMCC cross-linker ratios assayed. rHir, which was initially reacted with 2-iminothiolane hydrochloride (Traut's reagent) in order to create sulphydryl groups, was then covalently bound to these HD-BSA-SMCC surfaces (HD-BSA-SMCC-S-rHir). The 1:50 (BSA: sulpho-SMCC) HD-BSA-SMCC-S-rHir segments bound 22-fold more rHir (111 ng per mg Dacron) compared to control segments and also possessed the greatest thrombin inhibition of the segments evaluated using a chromogenic substrate assay for thrombin. Further characterization of the HD-BSA-SMCC-S-rHir segments demonstrated that maximum thrombin inhibition was 20.43 NIHU, 14.6-fold greater inhibition than control segments (1.4 NIHU). Thrombin inhibition results were confirmed by 125I-thrombin binding experiments, which demonstrated that the 1:50 HD-BSA-SMCC-S-rHir segments had significantly greater specific thrombin adhesion compared to control segments. Non-specific 125I-thrombin binding to and release from the 1:50 HD-BSA-SMCC-S-rHir segments was also significantly less than the control segments. Thus, these results demonstrate that rHir can be covalently bound to a clinically utilized biomaterial (Dacron) while still maintaining its ability to bind and inhibit thrombin.
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PMID:Covalent linkage of recombinant hirudin to poly(ethylene terephthalate) (Dacron): creation of a novel antithrombin surface. 915 59

To determine whether decreases in plasma antithrombin (AT) level, as seen in non-gestational acquired AT deficiency, result from a hypercoagulable state and/or liver/kidney damage, AT activity was measured in 24 uncomplicated and 30 preeclamptic women. The fifth percentile of AT levels in the normal pregnancies was used as a cut-off value to subdivide the preeclamptic patients into two groups. Markers of activated coagulation, i.e, levels of thrombin-antithrombin complex (TAT), fibrin D-dimer, soluble fibrin, von Willebrand factor (vWF) and platelet counts, were determined. Indicators of hepatic or renal function, i.e. concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, urinary albumin (U-albumin) and serum albumin (S-albumin), were assayed. AT levels were lower in those with preeclampsia than in the normal pregnancy group (P < 0.01). In the group with AT levels less than the cut-off point, levels of fibrin D-dimer (P < 0.05), soluble fibrin (P < 0.05), vWF (P < 0.05), ALT (P < 0.05), AST (P < 0.05), creatinine (P < 0.01) and U-albumin (P < 0.01) were increased, whereas platelet counts (P < 0.05) and S-albumin (P < 0.05) were decreased. All patients with ALT levels > 0.46 mu kat/1, AST > 0.58 mu kat/1, S-albumin < 23 g/1 and/or U-albumin > 4.9 g/24 h had AT levels < or = cut off. AT levels correlated with vWF (rs = - 0.73, P < 0.01) and creatinine (Rs = -0.70, P < 0.01). It is suggested that in preeclampsia, acquired AT deficiency is secondary to a hypercoagulable state, and/or associated with impaired hepatic and/or renal function.
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PMID:Acquired deficiency of antithrombin in association with a hypercoagulable state and impaired function of liver and/or kidney in preeclampsia. 919 20

alpha-Thrombin (AT) and bradykinin (BK) are endogenous mediators that are released during an inflammatory response, and could have a synergistic effect on endothelial permeability. Human umbilical vein endothelial cells (HUVEC) were grown on Transwell membranes and then tested for alterations in permeability to fluorescein isothiocyanate-labeled human serum albumin. Addition of 1 microM AT produced a significant increase in the permeability coefficient at 30 minutes from control levels of 1.59 x 10(-6) cm/sec to 4.92 x 10(-6) cm/sec. BK (1 microM) produced a similar increase to 4.46 x 10(-6) cm/sec. For both compounds, permeability remained elevated for 90 minutes. Pre-treatment of the HUVEC with the bradykinin receptor antagonist, Na-adamantaneacetyl-bradykinin (NA-BK) (1 microM), prior to addition of AT, reduced the AT permeability coefficient to 2.69 x 10(-6) cm/sec. Addition of NA-BK (1 microM) for 5 minutes, then BK (1 microM) for 5 minutes, inhibited the effect of BK and of AT (1 microM on permeability, decreasing the permeability coefficient of the endothelial monolayer to control levels (1.62 x 10(-6) cm/sec). AT (1 microM) increased HUVEC intracellular calcium mobilization, as monitored by FURA-2, to 245 nM from control (70 nM), however, pre-treatment with either BK or the bradykinin receptor antagonist decreased the AT induced intracellular calcium mobilization compared to AT alone. Pre-treatment of the HUVEC with bradykinin (1 microM) for 2 minutes also inhibited the effects of alpha-thrombin (1 microM) on f-actin distribution examined by BODIPY-phallodin staining and increased the clotting times for an alpha-thrombin dependent fibrinogen to fibrin clotting assay. However, incubation of bradykinin (1 microM) with alpha-thrombin (1 microM) for either 10 minutes or 100 minutes produced no detectable hydrolysis products. These data strongly suggest that the inflammatory mediators alpha-thrombin and bradykinin when released together, rather than being synergistic, are antagonistic.
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PMID:Bradykinin antagonizes the effects of alpha-thrombin. 924 71

The intracellular calcium indicator dye Fura-2 has been widely used for the study of human platelet thrombin receptor-mediated calcium mobilisation in disease states. In general, authors (a) use a Fura-2/AM concentration of 2-3 mumol/l and (b) calibrate fluorescence signals on the basis of maximum and minimum ratios of fluorescence (Rmax and Rmin) and the ratio of the calcium-free and calcium-bound fluorescence at an excitation wavelength of 380 nm ("C2/B2"). In the present study, it is found (a) that a greater peak response to thrombin is seen when 1 mumol/l Fura-2/AM rather than 2 or 3 mumol/l is used; and (b) that calibration leads to a poorer test-retest reliability and in general a greater variability of the obtained calcium signal than when the simple measurement of the 340 nm/380 nm fluorescence ratio is used. It is suggested that this poor variability is due to the presence of an extracellular factor that can quench the Fura-2 signal once the platelets have been permeabilised by detergent treatment. Consistent with this, addition of bovine serum albumin to the assay medium has no significant effect on the fluorescence ratio response to thrombin, but greatly increases the observed calibrated calcium signal.
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PMID:Calibration of Fura-2 signals introduces errors into measurement of thrombin-stimulated calcium mobilisation in human platelets. 938 66

Recent work has shown that osteopontin expression is upregulated at sites of cardiovascular injury. It has been hypothesized that osteopontin provides an adhesive matrix for endothelial and smooth muscle cells during remodeling of the vascular wall following injury. Osteopontin has also been found to be synthesized by monocytes and macrophages within injury sites. Here, we present data showing that osteopontin can promote leukocyte adhesion through the alpha4beta1 integrin. In the presence of physiologic concentrations of Mg2+ and Ca2+, osteopontin purified from bovine milk promoted cell-substrate adhesion of HL-60 and Ramos cells, two model leukocyte cell lines. As with other adhesive ligands, adhesion to osteopontin required leukocyte activation. Under these conditions, no adhesion to control substrates such as bovine serum albumin was observed. Leukocyte adhesion was inhibited by anti-integrin antibodies directed at either the alpha4 or beta1 integrin subunits but not by control antibodies directed to other integrins. Further adhesion experiments revealed that leukocyte binding to osteopontin was completely inhibited by an alpha4beta1-binding peptide containing the leucine-aspartate-valine (LDV) sequence, while a control, non-binding peptide containing leucine-glutamate-valine (LEV) had minimal effects. Affinity chromatography using either surface labeled HL-60 or Ramos cell extracts revealed that the alpha4beta1 integrin specifically bound to osteopontin. Immunoprecipitation of eluted fractions from these columns positively identified the alpha4beta1 integrin. In order to localize potential alpha4beta1-binding sites within osteopontin, the protein was proteolytically cleaved with thrombin. A 30 kDa N-terminal osteopontin fragment purified using fast protein liquid chromatography promoted alpha4beta1 dependent leukocyte adhesion in a manner similar to that of the intact protein. These data collectively demonstrate that the alpha4beta1 integrin is a new adhesion receptor for osteopontin and that an alpha4beta1 binding site exists in the NH2-terminal thrombin fragment of osteopontin.
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PMID:Osteopontin is a ligand for the alpha4beta1 integrin. 954 93

In a previous report we demonstrated that the blood compatibility of poly(ether urethane) (PEU) was improved by grafting phosphorylcholine (PC) groups on the surface. The improved blood compatibility was indicated by decreased platelet adsorption/activation and reduced thrombin formation at the polymer surface in experiments in which the surfaces were contacted with platelet-rich plasma in vitro. In the present study, we investigated the effect of grafted PC groups at a PEU surface on protein and phospholipid adsorption. Adsorption of human fibrinogen (Fg), human serum albumin (Alb), human high-molecular-weight kininogen (HMWK), and dioleoyl phosphatidylcholine (DOPC) vesicles was measured by ellipsometry. For this purpose, thin PEU films were cast on silicon wafers. The polymer film was photochemically modified with a PC-containing aryl azide. The presence of PC groups on the polymer surface was demonstrated by ESCA (Electron Spectroscopy for Chemical Analysis). The hydrophilicity of the polymer surface increased by the surface modification, as indicated by a decrease of the contact angle from 59 degrees before to 43 degrees after modification. Our data show that the presence of PC groups has little effect on the adsorption of proteins to a PEU surface. The highest adsorption was observed for Fg (0.49 microgram/cm2 on PC-modified PEU and 0.50 microgram/cm2 on PEU), followed by HMWK (0.28 microgram/cm2 on both PC-modified PEU and PEU), and Alb (0.16 microgram/cm2 on PC-modified PEU and 0.18 microgram/cm2 on PEU). Protein adsorption was further studied on a "biomembrane-like" DOPC bilayer formed on hydrophilic silicon. We found no protein adsorption on this DOPC bilayer. The adsorption of small unilamellar DOPC vesicles on the polymer surfaces amounted to about 0.06 microgram/cm2 (corresponding to circa 30% of monolayer coverage) and was similar for PC-modified PEU and PEU. Despite this partial surface coverage, preadsorbed DOPC on the polymer surface diminished the subsequent adsorption of proteins considerably. These results show that the mere presence of phosphorylcholine groups on a PEU surface is insufficient to suppress protein adsorption. The highly ordered structure of natural phospholipid bilayers seems to be required to suppress protein adsorption effectively.
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PMID:Adsorption of proteins onto poly(ether urethane) with a phosphorylcholine moiety and influence of preadsorbed phospholipid. 954 14

Surface thrombus formation on implantable biomaterials such as polyurethane is a major concern when utilizing these materials in the clinical setting. Thrombin, which is responsible for thrombus formation and smooth muscle cell activation, has been the target of numerous surface modification strategies in an effort to prevent this phenomenon from occurring. The purpose of this study was to covalently immobilize the potent, specific antithrombin agent recombinant hirudin (rHir) onto a novel polyurethane polymer synthesized with carboxylic acid groups which served as protein attachment sites. The in vitro efficacy of thrombin inhibition by this novel biomaterial surface was then evaluated. Bovine serum albumin (BSA), which was selected as the basecoat protein, was reacted with sulfo-SMCC in a 1:50 molar ratio. This BSA-SMCC complex was then covalently linked to the carboxylated polyurethane (cPU) surface via the crosslinker EDU (cPU-BSA-SMCC). This cPU-BSA-SMCC surface was then reacted with Traut's-modified 125I-rHir, a procedure which created free sulfhydryl groups on rHir (cPU-BSA-SMCC-S-125I-rHir). Using these crosslinking procedures, the cPU-BSA-SMCC-S-125I-rHir segments bound 188 +/- 40 ng/cm2 (n = 60) whereas the controls with non-specifically bound 125I-rHir (Mitrathane + EDC + BSA + 125I-rHir-SH and cPU-BSA + 125I-rHir-SH) bound 13 +/- 8 ng/cm2 and 4 +/- 8 ng/cm2, respectively. Evaluation of these cPU-BSA-SMCC-S-125I-rHir segments for 131I-thrombin inhibition using a chromogenic assay for thrombin showed that a maximum of 2.64 NIHU thrombin was inhibited in contrast to the controls which inhibited bound 0.76 and 0.70 NIHU. Controls with nonspecifically bound 125I-rHir also had 0.31 and 0.76 NIHU 131I-thrombin adherent to their respective surfaces whereas the maximum 131I-thrombin binding to the cPU-BSA-SMCC-S-rHir segments was 1.51 NIHU. Exposure to 131I-thrombin did not result in any release of covalently bound 125I-rHir from the cPU-BSA-SMCC-S-125I-rHir segments. Thus, these results demonstrate that rHir can be covalently bound to this novel polyurethane surface and still maintain potent antithrombin activity.
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PMID:Covalent linkage of recombinant hirudin to a novel ionic poly(carbonate) urethane polymer with protein binding sites: determination of surface antithrombin activity. 970 17

Development of a small diameter prosthetic vascular graft with surface based antithrombin properties should aid in maintaining early graft patency in small vessel reconstruction. The purpose of this study was to bind covalently a basecoat protein (canine serum albumin [CSAJ) and a potent antithrombin agent (recombinant hirudin [rHir]) to 4 mm inner diameter poly(carbonate urea) urethane grafts with reactive carboxylic acid groups (cPU). 125I-CSA was covalently bound to 1 cm length segments of cPU grafts using the carbodimide cross-linker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). To bind 125I-rHir covalently, CSA was modified with the heterobifunctional cross-linker sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) before linkage to the cPU surface with EDC (cPU-CSA-SMCC). 125I-rHir was modified with Traut's reagent and reacted with the cPU-CSA-SMCC surface, covalently linking 125I-rHir to surface bound CSA. 125I-CSA binding to the cPU graft surface (34,235 ng/segment) was ninefold, sevenfold, and 10-fold greater than controls with nonspecifically bound 125I-CSA. Covalent linkage of 125I-rHir to the cPU-CSA-SMCC surface (9,974 ng/segment) was 172, 192, and 142-fold greater than controls with nonspecifically bound 125I-rHir. Surface antithrombin properties were characterized using a chromogenic assay to measure residual thrombin activity. Evaluation of surface antithrombin activity showed significantly greater 131I-thrombin inhibition and binding by the cPU surface with covalently bound 125I-rHir, as compared with controls. Release of 125I-rHir from the cPU surface was minimal as compared with controls. Therefore, rHir can be covalently linked to a novel small diameter polyurethane vascular graft surface while maintaining its potent antithrombin properties.
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PMID:Bioengineering of a novel small diameter polyurethane vascular graft with covalently bound recombinant hirudin. 980 16

When human platelets are stimulated with collagen or thrombin, the asymmetric distribution of membrane lipids is disrupted as phosphatidylserine and phosphatidylethanolamine translocate from the inner monolayer to the outer monolayer. Coincident with the stimulus-dependent rearrangement of membrane phospholipids is a rapid redistribution of cholesterol from the outer to the inner membrane monolayer. This redistribution of cholesterol was observed when the stimulus was collagen or ADP. The data presented here show that epinephrine stimulation does not promote cholesterol translocation but does potentiate collagen-promoted movement of cholesterol. To investigate the process of cholesterol translocation, experiments were performed to determine whether collagen stimulated reverse cholesterol movement; i.e. from the inner to the outer monolayer. For this study, the fluorescent sterol cholestatrienol (C-3) was incorporated into platelet membranes by exchange from cholesterol-containing phosphatidylcholine small unilamellar vesicles. C-3 was then removed selectively from the outer monolayer by treatment of the platelets with bovine serum albumin (BSA). During the subsequent incubation of BSA-treated platelets, C-3 moved spontaneously into the outer from the inner monolayer. This translocation had an apparent half-time of approximately 25 min and was unaltered by the presence of collagen. These results suggest that collagen treatment of platelets selectively facilitates the inward movement of the sterol. We have hypothesized that cholesterol translocation may be thermodynamically driven as a result of an unfavorable entropy, resulting in cholesterol translocation out of an environment becoming enriched in phosphatidylethanolamine. The unidirectional nature of collagen-promoted cholesterol movement from the phosphatidylethanolamine-rich outer monolayer is consistent with this interpretation.
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PMID:Collagen-stimulated unidirectional translocation of cholesterol in human platelet membranes. 991 52


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