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C T Esmon

Publications and source records attributed to C T Esmon.

At least 199 records · Page 11Linked to original sources
PubMed1984

Effects of thrombomodulin and coagulation Factor Va-light chain on protein C activation in vitro.

Protein C activation by thrombin is significantly accelerated by the endothelial cell surface protein thrombomodulin, Factor Va, or its light chain. In this study we have compared the activation of protein C in the presence of either cofactor and examined the possibility that thrombomodulin and Factor Va-light chain act together to regulate protein C activation by thrombin. At all concentrations of protein C used, thrombomodulin was 20 times more efficient than Factor Va-light chain in accelerating protein C activation by thrombin. Protein C treated with chymotrypsin to remove the amino-terminal 41 amino acids that contain the gamma-carboxyglutamyl residues was activated by the thrombin-thrombomodulin complex at an identical rate to native protein C, whereas the modified protein C was activated by Factor Va-light chain and thrombin at only 5% of the rate obtained by using native protein C. Increasing concentrations of Factor Va-light chain, greater than or equal to 30 nM, inhibited thrombin-thrombomodulin catalyzed protein C activation with complete inhibition observed at 90 nM Factor Va-light chain. On the other hand, increasing thrombomodulin concentrations did not inhibit protein C activation by Factor Va-light chain and thrombin. These reactions in solution mimic, in part, those obtained on endothelial cells where protein C lacking the gamma-carboxyglutamyl domain is activated poorly and Factor Va-light chain at concentrations greater than 50 nM inhibited the activation of native protein C. The results of this study suggest that thrombomodulin and Factor Va-light chain may act in concert to regulate protein C activation by thrombin.

Animals↗
PubMed1984

Familial protein S deficiency is associated with recurrent thrombosis.

Recent studies have demonstrated that protein C deficiency is associated with recurrent familial thrombosis. In plasma, activated protein C functions as an anticoagulant. This anticoagulant response requires a vitamin K-dependent plasma protein cofactor, referred to as protein S. Since the anticoagulant activity of activated protein C is dependent on protein S, we hypothesized that patients lacking functional protein S might have associated thrombotic disease. Two related individuals with otherwise normal coagulation tests are described whose plasma is not effectively anticoagulated with activated protein C. Addition of purified human protein S to their plasma restores a normal anticoagulant response to activated protein C. We have developed a rapid one-stage clotting assay for protein S to quantitate the level of protein S in their plasma. Plasma is depleted of protein S by immunoadsorption with immobilized antiprotein S antibodies. The resultant plasma responds poorly to activated protein C, but is effectively anticoagulated in a dose-dependent fashion upon addition of purified protein S or small quantities of plasma. The affected individuals possess less than 5% protein S activity. Using Laurell rockets, protein S antigen was detected in the plasma but was at reduced levels of 13 and 18% in the two individuals. When the barium eluate of the patient plasma was chromatographed on quaternary aminoethyl Sephadex, a single peak of protein S antigen devoid of protein S anticoagulant cofactor activity was detected early in the chromatogram. In contrast, the barium eluate from normal donors separated into two peaks, one emerging early and also devoid of anticoagulant cofactor, and the second peak with anticoagulant activity emerging later. The first peak of protein S antigen, from both the normal donor and the patient, chromatographed in the region of the complement component C4-binding protein-protein S complex. These studies suggest that protein S deficiency may result in recurrent thrombotic disease.

Adolescent↗
PubMed1984

Protein C.

The protein C anticoagulant pathway provides many new insights into control mechanisms for regulating coagulation. The observation that protein C deficiency is associated with thrombotic tendencies in the heterozygote (106-109) and early, lethal thrombosis in the homozygote (110, 111) points to the importance of the system as a major regulatory pathway. The complexity of the system has only recently begun to emerge. Thrombin activation of protein C at the endothelial cell surface requires not only the synthesis of thrombomodulin but the coupling of the receptor to a protein C binding site. It is reasonable to assume that an inherited or acquired deficiency in thrombomodulin might lead to thrombotic tendencies. This aspect of the system may explain, in part, the association between vascular disease and thrombosis. Once activated, protein C has an almost total dependence on protein S to express anticoagulant activity. (98) This suggests that deficiencies of protein S may also be associated with thrombotic tendencies. Protein S offers an additional intriguing property. Protein S, a regulatory protein of the coagulation system, is found both free and associated with C4BP, a regulatory protein of the complement system. The high affinity, very stable interaction between these components (85) suggests that the interaction is likely to be involved in regulation. (89) The importance of the interaction remains to be demonstrated, but clearly this is a potential direct link between major control proteins of the coagulation and complement system. Clinical studies suggest that protein C and/or thrombomodulin might be effective therapeutically. Certainly, protein C supplementation during the onset of oral anticoagulant therapy would be expected to circumvent the transient rapid decrease in protein C levels that may influence the early effectiveness of oral anticoagulants. (119) In addition to the systems clinical importance, protein C, its activation, and its function offer a variety of intriguing biochemical problems. For instance, how does thrombomodulin alter the specificity of thrombin? What is the protein C binding site on the cell surface, and what role does Factor Va or its degradation products play in the formation and regulation of this site? How does protein S facilitate activated protein C anticoagulant activity and what roles do membrane surfaces play in this system? What role does beta-hydroxyaspartic acid play in protein C activation and function? How does activated protein C influence fibrinolytic activity? The answers to these questions will undoubtedly add to our understanding of the fundamental mechanisms involved in regulating blood coagulation.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Carboxyglutamic Acid↗
PubMed1984

Determination of functional levels of protein C, an antithrombotic protein, using thrombin-thrombomodulin complex.

Protein C is a vitamin K-dependent plasma protein. Activated protein C is a potent anticoagulant and enhances blood clot lysis. We have developed a functional assay for protein C in human plasma. The measurement of protein C is accomplished by the addition of thrombomodulin, an endothelial-cell-associated cofactor for protein C activation, and thrombin in a 1:1 molar complex. The activated protein C formed in the plasma is immunoadsorbed with goat anti-human protein C IgG-agarose. The immunoadsorbed activated protein C retains the ability to hydrolyze chromogenic substrates, and after unbound plasma proteins are removed by washing, the bound activated protein C is quantitated by incubation with the substrate H-D-phe-pip-arg-p-nitroanilide (S-2238). Normal individuals have functional protein C levels of 3.9-5.9 micrograms/ml, with a mean value of 4.8 micrograms/ml. Individuals undergoing warfarin anticoagulation and patients with advanced liver diseases have decreased levels, as do certain patients with evidence of intravascular clotting. Functional protein C levels correlate well with immunologic levels of the protein in the patient groups studied. Heparin enhances the rate of activated protein C inhibition, as monitored by recovery of activated protein C by immunoadsorption. A patient with recurrent venous thrombosis and abnormal functional protein C activity, but normal levels of antigen, has been identified.

Adult↗
PubMed1983

Thrombomodulin blocks the ability of thrombin to activate platelets.

When thrombin is complexed to the endothelial cell surface receptor thrombomodulin, it loses its procoagulant activities in that it no longer clots fibrinogen or activates factor V. Studies were initiated to determine if complex formation also blocks thrombin's other major procoagulant function, the activation of platelets. When bound to thrombomodulin, thrombin no longer induces platelets to either aggregate or release [14C] serotonin. Binding studies using 125I-labeled thrombin or diisopropyl phosphorothrombin indicate that the complex does not bind to the platelet. When thrombomodulin is added after thrombin has bound to the platelets, the thrombin rapidly redistributes onto the thrombomodulin. These data suggest that in addition to its other anticoagulant effects, thrombomodulin may also act to inhibit and/or reverse platelet activation by thrombin.

Animals↗
PubMed1982

Formation of a calcium-binding site on bovine activated factor V following recombination of the isolated subunits.

Calcium binding to the activated form of blood coagulation Factor V (Factor Va) has been studied by equilibrium dialysis. A single calcium-binding site was observed with a dissociation constant of 24 +/- 4.0 microM. Unlike Factor V, Factor Va consists of two subunits. Following separation and reconstitution of the isolated subunits, a single calcium-binding site with a similar dissociation constant (24 +/- 2.0 microM) was again observed. No calcium binding was detected to either isolated subunit of Factor Va. Recombination of the Factor Va subunits used in the above equilibrium binding studies resulted in reformation of the calcium-binding site. Gel filtration experiments indicated that occupancy of this site with Ca2+ was required to maintain high affinity association between the Factor Va subunits. These data indicate that the calcium-binding site is either formed or stabilized by subunit-subunit interaction and that occupancy of this single site is sufficient to stabilize the intersubunit interactions.

Animals↗
PubMed1982

Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation.

Protein C is activated rapidly when thrombin binds to a specific cell surface cofactor protein, thrombomodulin. Studies were initiated to determine the influence of thrombin-thrombomodulin complex formation on the substrate specificity of thrombin. When thrombin binds to thrombomodulin, the resultant complex retains less than 1% of the fibrinogen clotting activity of free thrombin. Permanent alteration of the thrombin molecule is not involved since full clotting activity is regenerated by incubation of the complex with excess diisopropyl phosphothrombin. Unlike the activation of protein C by the thrombin-thrombomodulin complex which is dependent on Ca2+, inhibition of fibrinogen clotting activity is not dependent on the presence of divalent metal ions. Formation of the thrombin-thrombomodulin complex also inhibits thrombin activation of factor V. Despite these changes in macromolecular substate specificity, no significant change in the hydrolysis of the synthetic substrates p-tosyl-L-arginine methyl ester and N alpha-benzoyl-L-arginine ethyl ester is detected upon formation of the thrombin-thrombomodulin complex. Formation of this complex results in a slight increase in the Km (from 9.0 +/- 0.4 to 10.2 +/- 0.6 microM) and Vmax (from 230 +/- 10 to 270 +/- 10 mol/s/mol of thrombin) for the specific thrombin substrate H-D-Phe-Pip-Arg-p-nitroanilide. These studies suggest that thrombomodulin has two distinct anticoagulant functions: 1) to inhibit the ability of thrombin to clot fibrinogen and to activate factor V; and 2) to accelerate the formation of the anticoagulant, activated protein C.

Animals↗
PubMed1982

Activation of protein C in vivo.

An endothelial cell-associated cofactor that greatly enhances the rate of protein C activation by thrombin has recently been described. The observation that the cofactor binds thrombin with unusually high affinity (K(d) = 0.5 nM) suggested that low level thrombin infusion into dogs might lead to the selective activation of protein C. Infusion of thrombin (1 U/min per kg body wt) into the jugular vein of dogs leads to the formation of a systemic anticoagulant activity within 5 min of starting the infusion. The plasma has a prolonged partial thromboplastin time and Factor X(a) clotting time, but there is no change in the thrombin clotting time. The systemic anticoagulant activity is identified as activated protein C for the following reasons: (a) anti-canine activated protein C IgG antibodies inhibit the anticoagulant activity; (b) the anticoagulant activity can be partially purified from the plasma of dogs infused with thrombin by barium citrate adsorption; (c) the anticoagulant has chromatographic properties on QAE Sephadex indistinguishable from those of activated protein C, and (d) the rate at which this anticoagulant is inhibited in citrated canine plasma is identical to that of canine activated protein C. The in vivo activation of protein C appears to be receptor mediated since it occurs at low thrombin concentration and since it can be progressively inhibited by simultaneous infusion of diisopropylphospho-thrombin with thrombin. The activation of protein C at low levels of thrombin is selective, since neither the platelet count nor the Factor V levels are altered. Thrombin infusion leads to an elevation in circulating plasminogen activator levels. This appears to be mediated through the activation of protein C since coinfusion of diisopropylphospho-thrombin with thrombin inhibits the increase in plasminogen activator levels. Pretreatment of dogs with dicumarol blocks both the formation of anticoagulant activity and the rise in plasminogen activator. When the dicumarol-treated dogs are supplemented with isolated protein C and thrombin is infused, the anticoagulant activity again appears and the circulating levels of plasminogen activator are again elevated. These studies illustrate that low levels of thrombin in vivo can activate protein C, which in turn can inhibit blood coagulation and initiate fibrinolysis by elevating circulating plasminogen activator levels.

Animals↗
PubMed1981

Functional properties of an endothelial cell cofactor for thrombin-catalyzed activation of protein C.

Thrombin-catalyzed activation of Protein C is accelerated by a human endothelial cell surface cofactor. The cofactor occurs also on mouse hemangioma cells (a transformed endothelial cell line), but not on cultured human smooth muscle cells or fibroblasts. The cofactor remains bound to the cell surface during Protein C activation. The cofactor is saturable with respect to both Protein C (Km = 0.72 +/- 0.07 microM) and thrombin (Km = 0.48 +/- 0.05 nM). Diisopropylphosphoryl-thrombin is a competitive inhibitor of the cofactor-dependent reaction with Ki = 0.56 +/- 0.1 nM. Prothrombin Fragment 1, the peptide derived from prothrombin that retains phospholipid binding capacity, does not inhibit activation of Protein C when present in a 7:1 molar excess over Protein C. Platelet Factor 4 (20 microgram/ml) also fails to inhibit Protein C activation. It is concluded that the endothelial cell provides a surface on which Protein C can be activated under physiological conditions.

Animals↗
PubMed1981

Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C.

Perfusion of the myocardium with protein C in the presence of thrombin (EC 3.4.21.5) elicits a potent anticoagulant activity, which is identified as activated protein C on the basis of synthetic substrate hydrolysis and anticoagulant properties. The rate of activated protein C formation during the transit through the myocardium is at least 20,000 times that of thrombin-catalyzed activation of protein C in the perfusion solution. The capacity of the heart to activate protein C is maintained for at least 1 hr when thrombin is present in the perfusate, but decays (half-life approximately 30 min) once thrombin is omitted. Addition of diisopropyl-phospho-thrombin increases this decay rate more than 10-fold. Coperfusing diisopropylphospho-thrombin with active thrombin lowers the amount of protein C activation in the myocardium. Cultured monolayers of human endothelium enhance the rate of thrombin-catalyzed protein C activation. As with myocardium, the activation rate is inhibited by including diisopropylphospho-thrombin in the medium. It is proposed that the surface of vascular endothelium provides a cofactor that enhances the rate of protein C activation by thrombin.

Blood Coagulation Factors↗
PubMed1981

Generation of fibrinolytic activity by infusion of activated protein C into dogs.

Bovine-activated protein C, administered intravenously to dogs, increases the rate of lysis of whole blood clots. Protein C, bovine prothrombin, and diisopropylfluorophosphate-inactivated protein Ca do not increase the rate of lysis. Repeated infusions of protein Ca sustain rapid blood clot lysis, but neither elevate circulating fibrin-split products nor decrease circulating plasminogen levels. The administration of protein Ca results in the elevation of the levels of lysine-adsorbable plasminogen activator activity in the plasma. When partially purified concentrates of this activator are added to normal dog blood at the levels seen following protein Ca injection, the rate of clot lysis is similar to that seen after protein Ca injection. The addition of protein Ca to citrated whole blood in vitro, with the subsequent neutralization of protein Ca with antibodies, results in increased rates of lysis when plasma made from the treated blood is reinjected into the animal. The generation of fibrinolytic activity is dependent on both cellular and plasma components of blood. A model of protein Ca fibrinolytic activity has a minimum of two components: a secondary messenger formed by protein Ca action on blood cells and plasma, and the subsequent appearance of plasminogen activator in the animal in response to that messenger.

Animals↗