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Biomedical subjects

C T Esmon

Publications and source records attributed to C T Esmon.

At least 217 records · Page 12Linked to original sources
PubMed1979

The effect of prothrombin fragment 2 on the inhibition of thrombin by antithrombin III.

The effect of prothrombin fragment 2 on the inhibition of thrombin by antithrombin III has been studied. Fragment 2 was found to slow the rate of inhibition of thrombin by antithrombin III about 3-fold. The effect of prothrombin fragment 2 on antithrombin III inhibition was examined by comparing its action in the presence of either thrombin or meizothrombin (des fragment 1). The second order rate constants for antithrombin III inhibition of thrombin with saturating fragment 2 and antithrombin III inhibition of meizothrombin (des fragment 1) were the same. Prothrombin fragment 2 had no effect on either antithrombin III inhibition of meizothrombin (des fragment 1) or Factor Xa. The effect of the fragment on the reaction mechanism of thrombin inhibition was evaluated to see if the fragment altered binding of antithrombin III to thrombin or inhibited the formation of the covalent complex. The fragment was found to have no inhibitory effect on the rate of covalent complex formation, indicating that the protective effect of the fragment is by inhibiting binding of antithrombin III to thrombin. These data suggest that prothrombin fragment 2 may be an important factor in controlling the localization of clot formation by regulating the interaction between thrombin and antithrombin III.

Animals↗
PubMed1979

Interactions between heparin and factor Xa. Inhibition of prothrombin activation.

The effects of heparin on prothrombin activation have been examined. Heparin was found to inhibit the rate of prothrombin activation by Factor Xa, calcium and phospholipid. In the absence of phospholipid, heparin had no effect on the rate of prothrombin activation. In contrast, heparin was found to increase the rate of activation of prethrombin-1 and prethrombin-2. Initial velocity studies indicated that heparin blocks lipid stimulation of prothrombin activation. In accord with this, binding studies demonstrated that heparin could displace Factor Xa, and in separate experiments, prothrombin, from phospholipid vesicles.

Animals↗
PubMed1979

The subunit structure of thrombin-activated factor V. Isolation of activated factor V, separation of subunits, and reconstitution of biological activity.

Activated Factor V (Va) was prepared by treating a high molecular weight form of Factor V with thrombin. The activated Factor V was isolated by ion exchange chromatography and was composed of two polypeptide chains (Mr = 115,000 and 73,000). These chains were separated by ion exchange chromatography in the presence of EDTA. Biologically active Factor Va was restored from the inactive chains by incubation of the two chains in buffers containing MnCl2. Restoration of biological activity was correlated with formation of a complex between the chains as monitored by either disc gel electrophoresis or gel filtration chromatography. The apparent molecular weight of the activated Factor V was 290,000. Factor V was not dissociated in EDTA. However, this protein was split by thrombin to yield an activation intermediate composed of two chains (Mr = 210,000 and 115,000). Like activated Factor V, the two chains of the intermediate can be dissociated in EDTA and separated by gel filtration chromatography. The Factor V activity was restored by incubation of the two inactive chains in buffers containing MnCl2. Like Factor Va, restoration of the biological activity corresponds to formation of a complex between the chains with a higher molcular weight than either of the isolated chains. Incubation of the activation intermediate with thrombin increased the specific activity 3- to 4-fold. This increase in specific activity resulted from cleavage of the heavy chain of the Factor V intermediate by thrombin.

Animals↗
PubMed1979

The effect of aggregation and release on platelet prothrombin-converting activity.

Platelets provide a procoagulant activity for the conversion of prothrombin to thrombin during normal hemostatis. This activity designated as platelet prothrombin-converting activity (PPCA) was monitored as rate of thrombin production in a two-stage assay using gel-filtered bovine platelets, factor Xa, and prothrombin. Expression of PPCA was not associated with ADP-induced release or platelet shape change but was associated with aggregation. Release of the contents of dense bodies, measured by release of 14C-5-hydroxytryptamine, was not required for expression of PPCA during platelet aggregation. During the PPCA assay, 5-hydroxytrypamine was released, but only after onset of thrombin production. Furthermore, the release of 5-hydroxytryptamine was retarded during the assay by the addition of 2 mM theophylline and 100 nM prostaglandin E1 without a comparable reduction in PPCA. In addition, 125I-factor-Xa was bound in greater amounts to platelets (aspirin-treated) after ADP-induced aggregation (without detectable release) than to unactivated control platelets. Finally, the PPCA of the ADP-activated platelets was saturated with respect to factors Xa and Va at less than 1 nM concentrations, indicating that the aggregation induced by ADP leads to the exposure of specific procoagulant sites by some process other than dense body secretion.

Animals↗
PubMed1979

Activated protein C inhibits platelet prothrombin-converting activity.

Bovine platelets that have been activated by thrombin facilitate the conversion of prothrombin to thrombin in the presence of calcium ions and factor Xa. Activated protein C, a vitamin-K-dependent plasma protein, inhibits this platelet prothrombin-converting activity. The inhibition is time dependent and is not reversed by increasing concentrations of factor Xa. However, factor Xa is able to protect the platelet prothrombin-converting activity from inactivation by activated protein C. The activated protein C causes a parallel loss of factor Xa receptor sites and platelet prothrombin-converting activity. Activated protein C may contribute to the regulation of clotting through inactivation of the platelet prothrombin-converting activity.

Animals↗
PubMed1976

Vitamin K-dependent carboxylase. Solubilization and properties.

Vitamin K is required for an enzymatic carboxylation of glutamyl residues in a microsomal protein precursor of plasma prothrombin to form gamma-carboxyglutamic acid. The enzyme system (carboxylase) which catalyzes this reaction has now been solubilized by extraction of the microsomes with Triton X-100 and has been shown to fix H14CO3- as gamma-carboxyglutamic acid residues in biologically active prothrombin. Enzyme activity requires O2 and vitamin K hydroquinone or vitamin K + NADH. Unlike the microsomal-bound carboxylase, soluble carboxylase activity is independent of either ATP or Mg2+ addition and is unaffected by either the ATP analog, adenyl-5'-yl imidodiphosphate (AMP-P(NH)P, or EDTA. These observations suggest that the energy required to drive the carboxylation reaction is derived from the oxidation of the reduced form of vitamin K. Although the membrane-bound carboxylase is inhibited by Warfarin, this anticoagulant is ineffective as an inhibitor of the soluble enzyme. A second anticoagulant, 2-chloro-3-phytyl-1,4-natpthoquinone (chloro-K), differs from Warfarin in that it effectively inhibits both the membrane-bound and soluble carboxylases.

Adenosine Triphosphate↗
PubMed1976

A new vitamin K-dependent protein. A phospholipid-binding zymogen of a serine esterase.

Conclusive evidence is presented that a recently purified (Stenflo, J. (1976) J. Biol. Chem. 251, 355-363) vitamin K-dependent protein (arbitrarily referred to as Protein C) which is not related to prothrombin, Factors IX or X is also unrelated to Factor VII. It therefore appears to be a new, previously unrecognized vitamin K-dependent protein. In contrast to prothrombin, which binds to negatively charged phospholipid only in the presence of Ca2+ ions, Protein C, like the other vitamin K-dependent proteins, is a precursor of a serine esterase, presumably a protease, but it does not seem to be necessary for blood coagulation. Although the lipid-binding properties of Protein C may suggest that it is associated with membrane structures, its biological function remains unknown.

Blood Proteins↗
PubMed1976

Vitamin K-dependent carboxylase. Requirements of the rat liver microsomal enzyme system.

Vitamin K is required in an enzymatic reaction which carboxylates glutamyl residues in a microsomal protein precursor of plasma prothrombin to form gamma-carboxyglutamic acid residues. The partial requirements of this microsomal, vitamin K-dependent carboxylase system have been determined. A requirement of the system for cytosolic factors appears to be due primarily to the presence of reduced pyridine nucleotides or a reduced pyridine nucleotide-generating system in the cytosol. The hydroquinone of vitamin K has been demonstrated to be the enzymatically active form of the vitamin. When vitamin K1 hydroquinone is added to the carboxylase system, no NAD(P)H is needed for maximum activity. The carboxylase activity is half-maximally stimulated by 0.25 mug of vitamin K1/ml in the presence of cytosolic components but requires at least 10 times as much vitamin when microsomes are incubated in a cytosol-free buffer. Menadione is inactive as a vitamin source in this system, and the carboxylase activity is inhibited by the 2-chloro analog of vitamin K1 and by Warfarin. The ATP analog, AMP-P(NH)P, inhibited the carboxylase activity, but a dependence on exogenous ATP or an ATP-generating system could not be demonstrated. Carboxylase activity was found to be dependent on an O2-containing gas phase, and upon the HCO3- concentration.

Animals↗
PubMed1975

A new carboxylation reaction. The vitamin K-dependent incorporation of H-14-CO3- into prothrombin.

The bovine plasma zymogen prothrombin contains a number of gamma-carboxyglutamic acid residues which are not found in an abnormal prothrombin produced when cattle are given the vitamin K antagonist dicoumarol. These modified glutamic acid residues appear to be formed post-translationally by a reaction which requires vitamin K. It has been shown that postmitochondrial supernates from vitamin K-deficient rats incorporate added H-14-CO3- minus into microsomal proteins upon the addition of vitamin K. This incorporation is dependent upon the presence of the prothrombin precursor in the microsomal preparations, and upon factors which are present in the postmicrosomal supernatant. Most of the radioactive protein which can be obtained from the microsomal pellet by extraction with 0.25% Triton X-100 has been identified as prothrombin and it can be shown that all of the radioactivity is in the amino-terminal activation fragment of prothrombin. This portion of the protein has previously been shown to contain the gamma-carboxyglutamic acid residues. Hydrolysis of the purified radioactive prothrombin resulted in a loss of 50% of the radioactivity and subsequent chromatography of the amino acid hydrolyzate demonstrated that the remaining radioactivity was entirely in glutamic acid. These results are consistent with the hypothesis that all of the H-14-CO3- minus was incorporated into the carboxyl groups of gamma-carboxyglutamic acid residues.

Animals↗
PubMed1975

The functional significance of vitamin K action. Difference in phospholipid binding between normal and abnormal prothrombin.

In comparison to normal prothrombin, the abnormal prothrombin produced in response to vitamin K antagonists has been found to bind much less tightly, if at all, to phospholipid surfaces. As a consequence, the activation of abnormal prothrombin by Factor Xa and Ca2+ is not accelerated by the addition of phospholipid to the mixture while the activation of normal prothrombin under these conditions is greatly accelerated by phospholipid addition. In the absence of phospholipid, however, the rate of activation of abnormal prothrombin by Factor Xa and Ca2+ in both the presence and absence of Factor Va is indistinguishable from that of normal prothrombin. The distribution of the partial proteolysis products during activation by Factors Xa, Va, and Ca2+ also appears to be the same for both prothrombins. These observations provide an explanation for the function in prothrombin activation of the gamma-carboxyglutamate residues formed in the vitamin K-dependent carboxylation of prothrombin.

Animals↗
PubMed1975

Purification of an apparent rat liver prothrombin precursor: characterization and comparison to normal rat prothrombin.

Current evidence would suggest that prothrombin is synthesized from a liver precursor molecule in a vitamin K dependent step which involves the attachment of calcium binding groups to the precursor. A protein has now been isolated from the liver of warfarin-treated rats which has the properties predicted for this precursor. The purified precursor is a glycoprotein with a molecular weight indistinguishable from rat prothrombin. Both electrophoretic and isofocusing analyses indicate that the precursor is less negatively charged than prothrombin. Specific proteolysis of the precursor by thrombin, taipan snake venom, or clotting factor Xa yielded fragments indistinguishable from those formed by similar proteolysis of prothrombin. The rate of activation of the precursor to thrombin by factor Xa and Ca-2+ was not stimulated by the addition of phospholipid, while prothrombin activation is greatly stimulated under these conditions. All of the data obtained are consistent with the hypothesis that the protein isolated is a precursor to prothrombin, and that under the influence of vitamin K, this precursor is converted to prothrombin by the addition of a number of acidic calcium binding groups.

Animals↗