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)

Phospholipid-covered solid supports have been used successfully as model membranes in studies on blood coagulation and other research fields. In order to produce such membranes, simple exposure of the support to suspensions of phospholipid vesicles was recently introduced, but questions have remained about the process of vesicle adherence to the surface and the physico-chemical properties of the resulting membranes. Using a new technique, mixing of phospholipids in such membranes was demonstrated. A rotating, hydrophilic, silicon disc was exposed in a two-step procedure to vesicles prepared from mixtures of dioleoylphosphatidylserine (DOPS) and dioleoylphosphatidylcholine (DOPC). Factor Xa, factor Va and prothrombin were added and the transport-limited production rate of thrombin was measured. For low surface coverage with 40% DOPS/60% DOPC, a much higher conversion rate was found if, prior to addition of coagulation factors, excess DOPC vesicles were added to fill up vacant surface area. It is concluded that DOPS is spread over the entire surface and that confluent bilayers are formed. The presented technique may also be used to measure lateral diffusion constants.
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PMID:Production of thrombin as a probe for mixing of phospholipids in membranes on solid supports. 761 41

This study was designed to elucidate the participation of endothelin-1(ET-1) in vivo and in vitro coagulation. The microvascular hemodynamic changes in terms of intravascular thrombus formation in rat mesentery induced by the superfusion of ET-1 (0.5, 1 and 2 pmol) were visualized by an intravital microscope system assisted by television-video tape recorder system. In addition to vasoconstriction we observed the blockade of circulation by clumps resembling thrombus in a dose dependent fashion by ET-1. Thrombus formation could be attenuated by pretreatment with superfusion of 3.8% Na citrate solution but not by the prior superfusion of 1 to 3 ng of nitroglycerine. Thrombus formation was found after the administration of 10 microliters of CaCl2 (100 nM) solution in Na citrate (3.8%, 20 microliters) and ET-1 treated field. In vitro study, a dose dependent increase in TAT (thrombin-antithrombin complexes) and decrease in AT III (antithrombin III) (%) activity, the prolongation of PT (prothrombin time) and APTT (activated partial thromboplastin time) was found by administering ET-1 immediately in native (unanticoagulated) blood in silicon coated test tubes (p < 0.05; n = 6). However in citrated blood, TAT complexes, AT III (%) activity, PT and APTT were not significantly changed after administration of the same doses of ET-1 (p > 0.05; n = 6). Therefore, this study suggested that endothelin-1 caused intravascular thrombosis and enhanced intra test tube coagulation which could be attenuated by blocking ionic calcium.
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PMID:Coagulation in vivo microcirculation and in vitro caused by endothelin-1. 830 59

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

Microstructured silicon wafers were employed as miniaturized solid-phase reaction vessels as well as miniaturized micro titer plates. Employing piezoelectric drop-on-demand liquid jets, a combinatorial library of 256 Peptides was synthesized on single beads. The synthesis protocol was associated to the location in the silicon nano-well arrangement. Products were photolytically cleaved in the same well that was used for synthesis and subsequently interrogated for thrombin inhibition in a homogeneous competition assay. The assay procedure was based on drop-on-demand liquid delivery and laser induced fluorescence imaging. The novel format proved useful for the integration of both synthesis and screening into one platform, a prerequisite for an iterative, evolutionary approach towards drug discovery.
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PMID:Single bead parallel synthesis and screening. 1219 67

Antibody mediated inhibition of tissue factor (TF) function reduces thrombus size in ex vivo perfusion of human blood over a TF-free surface at venous shear rates suggesting that TF might be involved in the mechanism of deep vein thrombosis. Moreover, TF-bearing monocytes and polymorphonuclear (PMN) leukocytes were identified in human ex vivo formed thrombi and in circulating blood. To understand the role of TF in thrombus growth, we applied a rabbit venous thrombosis model in which a collagen-coated thread was installed within the jugular vein or within a silicon vein shunt. The effect of an inhibitory monoclonal antirabbit TF antibody (AP-1) or Napsagatran, a specific inhibitor of thrombin, was quantified by continuously monitoring 125I-fibrinogen incorporation into the growing thrombi. The antithrombotic effect obtained with the anti-TF antibody was comparable to the effect observed with the thrombin inhibitor napsagatran suggesting that in this animal model the thrombus propagation is highly TF dependent. Immunostaining revealed that TF was mostly associated with leukocytes within the thrombi formed in the jugular vein or in the silicon vein shunt. Ex vivo perfusion experiments over collagen-coated coverslips demonstrated the presence of TF-bearing PMN leukocytes in circulating blood. The results suggest that in rabbits venous thrombus growth is mediated by clot-bound TF and that blocking the TF activity can inhibit thrombus propagation.
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PMID:Inhibition of tissue factor limits the growth of venous thrombus in the rabbit. 1287 49

Osteogenic injectable bone substitutes may be useful for many applications. We developed a novel injectable bone substitute based on osteoblast-fibrin glue suspension and calcium phosphate bone cement (BC). Human osteoblasts were isolated from trabecular bone samples and cultured under standard conditions. Osteoblasts were suspended in fibrinogen solution (FS). BC was cured with thrombin solution. 8 x 4 mm injectable bone discs were prepared using silicon molds and a custom-made applicator device. Discs containing BC, BC/FS, or BC/FS/osteoblasts were implanted subcutaneously into athymic nude mice. After 3, 9 and 24 weeks, specimens were explanted and subjected to morphologic and biomechanical evaluation. In vitro fibrin gel-embedded osteoblasts displayed a differentiated phenotype as evidenced by alkaline phosphatase, collagen type 1 and von Kossa stains. A proportion of osteoblasts appeared morphologically intact over a 3-day in vitro period following application into the BC. BC/FS and BC/FS/osteoblast discs were sparsely infiltrated with vascularized connective tissue. There was no bone formation in implants from all groups. However, positive von Kossa staining only in BC/FS/osteoblast groups suggests engraftment of at least some of the transplanted cells. Biomechanical evaluation demonstrated initial stability of the composites. Young's modulus and maximal load did not differ significantly in the BC/FS and BC/FS/osteoblast groups. The practicability of osteoblast-containing injectable bone could be demonstrated. The dense microstructure and the suboptimal initial vascularization of the composites may explain the lack of bone formation. Modifications with regard to enhanced osteoblast survival are mandatory for a possible application as injectable osteogenic bone replacement system.
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PMID:Fibrin gel-immobilized primary osteoblasts in calcium phosphate bone cement: in vivo evaluation with regard to application as injectable biological bone substitute. 1604 62

A numerical model of thrombosis/thromboembolism (T/TE) is presented that predicts the progression of thrombus growth and thromboembolization in low-shear devices (hemodialyzers, oxygenators, etc.). Coupled convection-diffusion-reaction equations were solved to predict velocities, platelet agonist (ADP, thromboxane A2, and thrombin) concentrations, agonist-induced and shear-induced platelet activation, and platelet transport and adhesion to biomaterial surfaces and adherent platelets (hence, thrombus growth). Single-platelet and thrombus embolization were predicted from shear forces and surface adhesion strengths. Values for the platelet-biomaterial reaction constant and the platelet adhesion strength were measured in specific experiments, but all other parameter values were obtained from published sources. The model generated solutions for sequential time steps, while adjusting velocity patterns to accommodate growing surface thrombi. Heparinized human blood was perfused (0.75 ml/min) through 580 microm-ID polyethylene flow cells with flow contractions (280 microm-ID). Thrombus initiation, growth, and embolization were observed with videomicroscopy, while embolization was confirmed by light scattering, and platelet adhesion was determined by scanning electron microscopy. Numerical predictions and experimental observations were similar in indicating: 1) the same three thrombotic locations in the flow cell and the relative order of thrombus development in those locations, 2) equal thrombus growth rates on polyethylene and silicon rubber (in spite of differing overall T/TE), and 3) similar effects of flow rate (1.5 ml/min versus 0.75 ml/min) on platelet adhesion and thrombosis patterns.
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PMID:Computational model of device-induced thrombosis and thromboembolism. 1607 18

We report an interferometric method to detect chemical binding at an interface. The interference layer consists of the thin native oxide on silicon, and we utilize nearly opposite phase shifts of light at the oxide/water and oxide/silicon interfaces to achieve near-complete destructive interference. We measure selective binding of thrombin in solution to DNA aptamers covalently bound to the oxide. The technique can be used to detect and quantify surface binding of less than 1 A of material, sensitivity similar to that of surface plasmon resonance imaging or arrayed imaging reflectometry. Results are in quantitative agreement with what is predicted theoretically. The method is very convenient to implement since it utilizes unmodified silicon wafers as substrates and is extremely insensitive to both probe light bandwidth and collimation.
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PMID:Label-free sensing of binding to microarrays using Brewster angle straddle interferometry. 1784 4

Abstract Chronic subdural hematoma (CSH) is a disease frequently seen in the neurosurgical department. CSH also has a high rate of recurrence. Our hypothesis is that thrombin solution irrigation reduces recurrence in high-risk CSH patients. We define high risk as follows: use of anti-platelets, use of anticoagulants, recurrent CSH, renal failure, liver cirrhosis, and hematological disease. From January 1, 1998, to March 31, 2008, we compared a saline solution irrigation group (43 patients) and a thrombin solution (100 unit/ml) irrigation group (36 patients) prospectively and randomly. Surgical procedures were the same: one burr hole craniostomy, drainage of hematoma, irrigation of cavity, frontal insertion of silicon tube, replacement of air with solution, and removal of tube at 24 h after surgery. We define recurrence as an additional drainage operation due to neurological deficit within six months of surgery. Recurrences of CSH arose in two patients (5.5%) with thrombin irrigation and in 11 patients (25.6%) with saline irrigation (p < 0.05). Saline irrigation patients with anti-platelet medication experienced recurrence in five of 19 patients, although no thrombin-irrigated side recurred with the same drug. No complication occurred in relation to thrombin irrigation. Irrigation of CSH with thrombin solution is an effective treatment option for high-risk cases of CSH without complication.
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PMID:Irrigation with thrombin solution reduces recurrence of chronic subdural hematoma in high-risk patients: preliminary report. 1942 8

An aptamer-functionalized silicon-nanowire (Si-NW) field effect transistor (FET) biosensor was successfully fabricated, characterized and applied to real-time electrical detection of binding with the target protein for biomedical applications. Surface modifications were carried out using 3-aminopropyl diethoxysilane and succinic anhydride to introduce amine and carboxyl groups onto Si substrates. Anti-thrombin aptamers with 5'-end amine groups were chemically grafted onto the surface-modified Si substrates through amide bond formation. Atomic force microscopic (AFM) analyses confirmed the successful immobilization of anti-thrombin aptamers on Si-NWs and their binding with thrombin samples. The anti-thrombin aptamers bound to Si-NWs through the linker appeared to have a mean height of approx. 4 nm and the thrombin/aptamer complex to have a mean height of approx. 8 nm. Fluorescence micrographs visualized the FITC-labeled thrombin after binding to anti-thrombin aptamers immobilized on Si-NWs. Furthermore, the anti-thrombin Si-NW FET biosensor was successfully applied to the real-time detection of electronic signals during and after binding with a thrombin sample at a concentration of approx. 330 pmol l(-1) and the thrombin in blood samples.
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PMID:The fabrication, characterization and application of aptamer-functionalized Si-nanowire FET biosensors. 1944 97


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