Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Thrombin”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1979

Thrombin receptors of human platelets: thrombin binding and antithrombin properties of glycoprotein I.

Washed human platelets were solubilized and the proteins were separated by preparative gel electrophoresis in the presence of sodium dodecyl sulphate. The gel was cut into slices and the effect of the eluted proteins on the clotting of fibrinogen by thrombin was evaluated. The isolate from only one gel slice strongly inhibited the clotting of fibrinogen. The prolongation of the clotting time was dependent on the concentration of the protein and reached a plateau around 5 microgram. Gel electrophoresis of this isolate showed a prominent glycoprotein with an apparent Mr=150 000. Gel filtration studies with [125I]thrombin showed that the protein isolate bound a significant amount of thrombin which could be displaced with unlabelled thrombin. Another preparation from the same gel or purified gamma-globulin did not bind thrombin or prolong the clotting time of fibrinogen. Glycoprotein I was isolated from human platelets by affinity chromatography on lectin-Sepharose columns. The isolated glycoprotein prolonged the clotting of fibrinogen and bound [125I]thrombin which could be displaced by unlabelled thrombin. It is proposed that the high affinity receptor of thrombin on human platelets is glycoprotein I. In addition, the antithrombin activity of intact platelets is due to binding of thrombin to this glycoprotein.

Antithrombins↗
PubMed1979

Routes of thrombin action in the production of proteolytically modified, secondary forms of antithrombin-thrombin complex.

The reaction between thrombin and antithrombin results in the formation of an inactive, stable, equimolar complex between the two proteins. However, under most reaction conditions several secondary complex forms, which have lower apparent molecular weights in dodecyl sulfate/polyacrylamide gel electrophoresis, appear concomitantly with or immediately following the production of the primary form of the complex. Purification of nascent, intact complex and treatment of this complex form with thrombin demonstrated that these subsidiary forms of antithrombin-thrombin complex may arise by proteolysis of the nascent complex by excess thrombin. Dissociation of such proteolytically modified complex preparations by hydroxylamine, and examination of the dissociation products by dodecyl sulfate/polyacrylamide gel electrophoresis suggested that degradation occurs primarily in the thrombin part of the complex, and only after prolonged proteolysis in its antithrombin moiety also. Incubation of antithrombin with several autolytically modified thrombin preparations showed that formation of subsidiary complex forms can also occur by an alternative route, i.e. between premodified thrombin forms and the inhibitor. In contrast, complex formation between thrombin and active forms of antithrombin, which have been modified by thrombin before complex formation, is unlikely, since no such active forms of antithrombin could be demonstrated.

Animals↗
PubMed1977

Human thrombins. Production, evaluation, and properties of alpha-thrombin.

Human alpha-thrombin, the thromboplastin activation product of prothrombin with high clotting and esterase activity, was produced from Cohn Fraction III paste. The procedure started with 0.4 to 3.2 kg of frozen paste and was completed in 2 or 3 days. Some 23 g of thrombin were recorded for 65 quantitated preparations made from 11 lots of Fraction III paste. These preparations were obtained at protein concentrations of 3.9 +/- 1.3 mg/ml with a yield of 340 +/- 110 mg/kg of paste, which represented 48 +/- 14% of the clotting potential extracted as prothrombin. They had specific clotting activities of 2.8 +/- 0.4 U.S. (NIH) units/microng of protein and titrated to 88 +/- 8% active with p-nitrophenyl-p'-guanidinobenzoate (NPGB). Those (N - 29) examined by labeling with [14C]diisopropyl phosphorofluoridate (iPr2P-F) and electrophoresing in sodium dodecyl sulfate (SDS)-polyacrylamide gels were found to contain only (N = 4) or predominantly alpha-thrombin (97 +/- 3%) and corresponding amounts of ists degradation product, beta-thrombin (2.6 +/- 3.1%). No plasmin(ogen), prothrombin complex factors (II, VII, IX, IXalpha, X, Xalpha), or prothrombin fragments were detected in representative preparations. As produced in 0.75 M NaCl, pH approximately 6, thrombin was stable for approximately 1 week at 4 degrees and for greater than 1 year at less than or equal to 50 degrees; freeze-dried thrombin stored at 4 degrees for greater than 1 year displayed stable clotting activity and no vial to vial variation, permitting its use for reference purposes. Human thrombin generated by Taipan snake venom activation was compared with that produced by rapid thromboplastin activation: after treatment with [14C]iPr2P-F, greater than 95% of the label in both thrombins migrated at the same rate during electrophoresis in SDS; identical pairs of NH2-terminal residues were released in three consecutive Edman degradation cycles.

Blood Coagulation↗
PubMed1977

Binding of thrombin to functionally defective platelets: a hypothesis on the nature of the thrombin receptor.

The initial step in the interaction of thrombin with human platelets is binding of thrombin to specific structures on the platelet surface. Data are presented which show that platelets from thrombasthenic patients bind thrombin similar to controls. In contrast, platelets from a patient with Bernard-Soulier syndrome had lower thrombin binding capacity. It is suggested that glycoprotein one (GP-I) on the platelet surface might be the receptor for thrombin.

Blood Platelet Disorders↗
PubMed1979

The influence of thrombin on the platelet-aggregating activity of human factor VIII. A study with insolubilized thrombin.

A study was made of the influence of thrombin on the platelet-aggregating activity of human factor VIII with Ristocetin as cofactor. Purified factor VIII and different amounts of a thrombin-Sepharose 2B complex were mixed and incubated for various periods of time. The factor VIII-related platelet-aggregating activities of the filtrates of the incubation mixtures were determined in a test system using formalin-fixed platelets and Ristocetin as cofactor. The results so obtained indicate that thrombin inactivates the platelet-aggregating activity of factor VIII. The filtrates of the incubation mixtures were also tested by a two-stage test system for the clotting activities. Comparison of the influence of thrombin on the clotting- and the platelet-aggregating activities of factor VIII is presented and discussed.

Animals↗
PubMed1979

Thrombin-induced platelet secretion. Further evidence for a specific pathway.

We have studied the interaction between thrombin and washed, human platelets using prostacyclin, a reversible inhibitor of platelet secretion. The effect of thrombin is limited to those reactions that are not inhibited by an increased concentration of platelet cyclic adenosine 3',5'-monophosphate, because prostacyclin is a potent inducer of the latter. Prostacyclin-treated platelets were briefly (15-30 s) exposed to low concentrations of human thrombin (0.01-0.2 U/ml). After removal of the prostacyclin and thrombin, the platelets were incubated with fresh thrombin. Although they had not undergone the release reaction after the first thrombin incubation, these platelets had a diminished capacity to secrete [(3)H]serotonin when exposed to thrombin the second time. Refractoriness was concentration dependent: the higher the initial thrombin concentration, the greater the degree of inhibition of serotonin secretion on subsequent thrombin exposure. Inhibition was closely related to the ability of thrombin to induce platelet secretion and not to its esterase or fibrinogen clotting activity. Diisopropyl fluorophosphate-inactive thrombin did not induce refractoriness. Refractoriness to thrombin did not increase when the time of the initial incubation with thrombin was lengthened, nor was it reversible.INHIBITION WAS THROMBIN SPECIFIC: serotonin secretion induced by collagen, wheat germ agglutinin, and the ionophore A23187 was minimally affected. For an equivalent amount of thrombin bound, a decrease was observed in serotonin secretion by thrombin-pretreated platelets compared to control platelets. Thus, there is at least one step in the secretory pathway between thrombin binding and regulation of adenylate cyclase. This step appears to transmit the signal that leads to extrusion of intracellular granular contents.

Blood Platelets↗
PubMed1978

The action of immobilized thrombin on factor VIII, fibrinogen and a synthetic tripeptide.

Bovine thrombin was insolubilized by attachment to cyanogen bromide-activated Sepharose (Sepharose-thrombin) or to activated (Affi-Gel 10) agarose containing a 10 A long arm (Affi-Gel-thrombin). Coupling in both instances approximated 7,000 units of thrombin per ml packed gel as determined by 125I-thrombin incorporation. The thrombin beads hydrolyzed the synthetic tripeptide Bz-Phe-Val-Arg-pNA (S-2160) at different rates, with the Sepharose-thrombin more active (220 esterase units per ml) than Affi-Gel thrombin (20.4 units per ml). The Km was significantly higher for the insolubilized thrombins (2 X 10(-3) M) than uncoupled thrombin (Km = 8 X 10(-5) M). The Sepharose-thrombin activated factor VIII significantly more rapidly than Affi-Gel-thrombin. Neither matrix-bound thrombin clotted a fibrinogen solution or liberated significant amounts of fibrinopeptides over 48 hr. This data indicates that a proteolysis of factor VIII, rather than a complex with thrombin, is the method of activation of factor VIII and that factor VIII is more accessible to the action of immobilized thrombin than is fibrinogen.

Animals↗
PubMed1978

A catalytic role for heparin. Evidence for a ternary complex of heparin cofactor thrombin and heparin.

The interaction of heparin with chemically modified thrombin and heparin cofactor is studied. Amidinated heparin cofactor does not bind to heparin-agarose and the reaction rate of the amidinated inhibitor with unmodified thrombin is not affected by heparin. Likewise, thrombin modified with 1,2--cyclohexanedione does not bind to heparin agarose and the reaction rate of the modified enzyme with unmodified inhibitor is not affected by heparin. In the absence of heparin, the modified and unmodified proteins react at the same rate in all possible combinations. Affinity chromatography of diisopropylphosphoryl thrombin on heparin cofactor coupled to Sephadex G--50 is used to study the binding of heparin cofactor and thrombin to heparin. The thrombin for all experiments is tritium-labeled and then inactivated with diispropylfluorophosphate. Thrombin is not bound to heparin cofactor-Sephadex columns. However, after treatment of the columns with a heparin solution, thrombin binds tightly, and is eluted at high ionic strength. Bound thrombin can also be eluted with either excess non-radioactive thrombin or excess free heparin. Heparin-dependent binding of thrombin does not occur if the heparin cofactor-Sephadex is heat-denatured. The ability of heparin to couple solution-phase thrombin to solid-phase heparin cofactor indicates that a ternary complex is formed. Analysis of the binding of the proteins to heparin by a dye displacement method suggests that at least one site on heparin binds to thrombin but not to heparin cofactor. Further support for a catalytic role for heparin derives from the ability of catalytic concentrations of heparin to enhance the rate of hydrolysis of prothrombin by thrombin, another protein pair which bind mutually to heparin.

Alpha-Globulins↗
PubMed1979

Properdin factor D: effects on thrombin-induced platelet aggregation.

Factor D, when preincubated with platelet suspensions, at concentrations as low as 1.2 micrograms/ml, inhibited thrombin-induced platelet aggregation. No inhibition of collagen or arachidonic acid-induced platelet aggregation was found. Inhibition occurred, but to a lesser extent, when thrombin and factor D were added to platelets at the same time. No inhibition occurred when factor D was added after thrombin. Thrombin was able to overcome inhibition by factor D by increasing its concentration. Diisopropyl-phosphorofluoridate-inactivated factor D also inhibited thrombin-induced platelet aggregation so that enzymatic activity of factor D was not required for inhibition. Factor D absorbed with hirudin coupled to Sepharose 6B showed no decrease in inhibitory capacity. 125I-Factor D bound to platelets in a manner suggesting an equilibrium reaction similar to thrombin. At low factor D input, binding was linear, whereas at higher input, binding began to approach saturation. Binding of 125I-labeled thrombin to platelets was inhibited by factor D. Analysis of these data show that factor D does not alter the total number of thrombin molecules which bind to the platelet surface at saturation. However, the dissociation constant for thrombin is altered from 2.78 to 6.90 nM in the presence of factor D (20 micrograms/ml). Factor D is thus a competitive inhibitor of thrombin binding, although the affinity of factor D for the platelet thrombin receptor is much less than that of thrombin. These phenomena occur at physiologic concentrations of factor D. Therefore, factor D may function in vivo as an inhibitor of platelet aggregation.

Binding, Competitive↗
PubMed1979

Correlation of in vivo and in vitro inhibition of thrombin by plasma inhibitors.

Rabbit antithrombin III and thrombin were purified to homogeneity to determine the in vivo relationship of these proteins in an autologous system. These proteins, radiolabeled with Na[125I], were injected into rabbits to determine the circulatory half-life. The mean half-life values were 125I-antithrombin III, 54.75 +/- 3.10 hr; 125I-thrombin, 7.25 +/- 1.49 hr; 125I-thrombin-antithrombin III, 7.25 +/- 1.09 hr; 125I[thrombin-antithrombin III], 11.13 +/- 0.88 hr; and Tos-Lys-CH2Cl-125I-thrombin, 27.75 +/- 3.18 hr. All the mean half-life values were statistically different from that of thrombin alone except for the two forms of thrombin-antithrombin III complex. Following injection of the radiolabeled proteins, plasma samples were obtained and gel-filtered to analyze the molecular weight distribution of the radiolabel. An identical elution position on gel filtration of 125I-antithrombin III with native antithrombin III was observed. The 125I-thrombin distributed into two peaks of radioactivity, with a molecular weight of 100,000 (79%) and a molecular weight greater than 200,000 (21%). The 100,000 dalton peak is consistent with a thrombin--antithrombin III complex, and the greater than 200,000 dalton peak is consistent with a thrombin-alpha 2-macroglobulin complex as confirmed by in vitro immunochemical studies. Thrombin inactivated with Tos-Lys-CH2Cl also showed two peaks of radioactivity on gel filtration, one peak which was excluded from the column and the other peak with an elution volume that was consistent with the position of native thrombin.

Antithrombin III↗
PubMed1979

[Participation of the hypophyseal-adrenal cortex system in thrombin clearance during immobilization stress].

The examination carried out with thrombin marked by 131J resulted in a considerable increase of the thrombin clearance rate in healty male rats during the stress (caused by an immobilization lasting 30 minutes) and in an increase of thrombin deposits in the liver. A further increase of thrombin clearance occurred by the combination of immobilization and administration of ACTH. Contrary to ACTH the thrombin clearance is not stimulated in healthy animals by hydrocortisone. Thrombin clearance and thrombin deposits in the liver are lowered in adrenalectomized rats. In these animals the administration of ACTH does not result in an increase of thrombin clearance. The rate of thrombin clearance is normalized in adrenalectomized animals after administering hydrocortisone without as well as under conditions of stress. In adrenalectomized animals having received hydrocortisone as well as in healthy animals the administration of ACTH will results in an increase of thrombin clearance. From these experiments the conclusion can be drawn that ACTH will increase the intensity of thrombin clearance in stress and that hydrocortisone plays a transmitting part here.

Adrenal Cortex↗
PubMed1978

Control of proliferation of human vascular endothelial cells. Characterization of the response of human umbilical vein endothelial cells to fibroblast growth factor, epidermal growth factor, and thrombin.

Because the response of human endothelial cells to growth factors and conditioning agents has broad implications for our understanding of wound healing angiogenesis, and human atherogenesis, we have investigated the responses of these cells to the fibroblast (FGF) and epidermal growth factors (EGF), as well as to the protease thrombin, which has been previously shown to potentiate the growth response of other cell types of FGF and EGF. Because the vascular endothelial cells that form the inner lining of blood vessels may be expected to be exposed to high thrombin concentrations after trauma or in pathological states associated with thrombosis, they are of particular interest with respect to the physiological role of this protease in potentiating cell proliferation. Our results indicate that human vascular endothelial cells respond poorly to either FGF or thrombin alone. In contrast, when cells are maintained in the presence of thrombin, their proliferative response to FGF is greatly increased even in cultures seeded at a density as low as 3 cells/mm2. Human vascular endothelial cells also respond to EGF and thrombin, although their rate of proliferation is much slower than when maintained with FGF and thrombin. In contrast, bovine vascular endothelial cells derived from vascular territories as diverse as the bovine heart, aortic arch, and umbilical vein respond maximally to FGF alone and neither respond to nor bind EGF. Furthermore, the response of bovine vascular endothelial cells to FGF was not potentiated by thrombin, indicating that the set of factors controlling the proliferation of vascular endothelial cells could be species-dependent. The requirement of cultured human vascular endothelial cells for thrombin could explain why the human cells, in contrast to bovine endothelial cells, are so difficult to maintain in tissue culture. Our results demonstrate that by using FGF and thrombin one can develop cultures of human vascular endothelial cells capable of being passage repeatedly while maintaining a high mitotic index. The stock cultures used for these studies have been passed weekly with a split ratio of 1 to 10 and are currently in their 30th passage. These cultures are indistinguishable from earlier passages when examined for the presence of Weibel-Palade bodies or Factor VIII antigen. We conclude that the use of FGF and thrombin can prevent the precocious senescence observed in most human endothelial cells cultures previously described.

Animals↗
PubMed2026

Thrombin concentration shapes endothelial extracellular vesicle profiles with divergent inflammatory functions.

Thrombin, a central enzyme in the coagulation cascade, also regulates diverse cellular processes, including inflammation and vascular barrier function, primarily by activating protease-activated receptor 1. Previous studies demonstrated that thrombin elicits concentration-dependent, opposing effects; low concentrations confer anti-inflammatory and barrier-protective responses, whereas high concentrations promote inflammation and barrier disruption. The underlying mechanisms, however, remain incompletely understood. Here, we showed that thrombin stimulates extracellular vesicle (EV) release from endothelial cells across a broad concentration range and that EVs generated at low vs high thrombin concentrations carry distinct microRNA (miR) cargo. Low-thrombin EVs mediate cytoprotective responses via the transfer of miR-409-5p, which targets ubiquitin-specific protease 7 that promotes inflammation via the NF-kB signaling pathway in recipient cells, whereas high-thrombin EVs disrupt barrier integrity and promote inflammation through delivery of miR-155-5p, a regulator of suppressor of cytokine signaling 1 that acts as a crucial negative regulator of the cytokine signaling pathway. Functional manipulation of these EVs confirmed the causal roles. Incorporation of anti-miR-409-5p abrogated the protective effects of low-thrombin EVs, whereas anti-miR-155-5p suppressed the cytopathic effects of high-thrombin EVs. Moreover, control EVs engineered to carry a miR-409-5p mimic reproduced the anti-inflammatory and barrier-protective phenotype of low-thrombin EVs. Collectively, these findings identified EV-associated miRs as key mediators of the concentration-dependent dual actions of thrombin, which may open the therapeutic potential of EVs engineered to deliver selective miRs or anti-miRs for the treatment of inflammatory vascular diseases.

Thrombin↗