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:3.4.21.6 (thromboplastin)
13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this paper we describe the effects of the activation peptides prothrombin fragment 1 and fragment 1.2 on factor Xa-catalyzed prothrombin activation. Prothrombin activation in free solution by either factor Xa or factor Xa together with factor Va is unaffected by the activation fragments. When negatively charged phospholipids are present we observed considerable inhibition of prothrombin activation by both fragment 1 and fragment 1.2. For the activation of 0.25 microM prothrombin by factor Xa in the presence of 50 microM phospholipid (phosphatidylserine/phosphatidylcholine, 25/75; mol/mol) and 5 mM CaCl2 50% inhibition was obtained at 0.28 microM fragment 1 or fragment 1.2. Much higher fragment concentrations were required for 50% inhibition of a prothrombinase complex consisting of factor Xa, factor Va, Ca2+ and phospholipid. This shows that factor Va protects prothrombin activation against inhibition by its own activation peptides. Less inhibition by activation fragments was also observed at higher phospholipid and prothrombin concentrations or when the mole fraction phosphatidylserine in the phospholipid vesicles was decreased. The effects of fragment 1 and fragment 1.2 on prothrombin activation were identical throughout all experiments, indicating that the inhibition is due to the gamma-carboxyglutamic acid containing region of the activation peptides. Our observations suggest that the activation fragments inhibit prothrombin activation by competing with prothrombin and factor Xa for binding sites at the phospholipid surface. In such a model factor Va will protect against the inhibition since it is known to promote the assembly of the prothrombinase complex through interactions with factor Xa and prothrombin that are independent of the gla-residues. The kinetic properties of fragment inhibition also suggest that in vivo prothrombin activation will not be affected by the generation of activation peptides.
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PMID:The effects of bovine prothrombin fragment 1 and fragment 1.2 on prothrombin activation. 392 88

To study the dissociation of the two moieties of the factor-VIII complex during clotting, plasma, concentrate and serum were chromatographed on 4% agarose. In plasma and concentrate factor-VIII coagulant activity (VIII C), factor-VIII coagulant antigen (VIII C:Ag), and factor-VIII-related antigen (VIII R:Ag) eluted together in the void volume, but some VIII C:Ag eluted after the void volume. The amount of VIII C:Ag eluting after the void volume was made smaller by adding proteinase inhibitors. In serum nearly all VIII C:Ag eluted after the void volume. From factor-VIII complex immunoadsorbed by means of an antibody against VIII R:Ag no VIII C:Ag was dissociated by thrombin or by thrombin and physiologic CaCl2 concentrations. Radiolabelled human thrombin did not bind to the VIII C:Ag of immunoadsorbed factor-VIII complex. VIII C:Ag displaying VIII C was dissociated from immunoadsorbed factor-VIII complex by human brain thromboplastin or by phosphatidyl-serine. Our results suggest that VIII C:Ag and VIII R:Ag dissociate during clotting. This dissociation seems not to be mediated by thrombin, but may be mediated by phospholipids.
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PMID:Dissociation of the Factor-VIII complex during clotting: role of thrombin and phospholipids. 644 24

Thrombin for topical hemostasis can be prepared from bovine or human blood plasma. The prothrombin is isolated by means of adsorption on DEAE-Sephadex A-50 and consecutively activated by CaCl2 and thromboplastin. Thrombin is precipitated and purified by acetone. The specific activity of the thrombin preparation is 122 + 23 IU/mg protein while the yield is 36,360 +/- 6623 IU/liter plasma.
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PMID:A method for preparation of dry thrombin for topical application. 651 3

Factor V appears to be a procofactor with, at best, 1/400 the activity of fully activated Factor V (Factor Va). The proteolytic conversion of Factor V to Factor Va is catalyzed by thrombin. However, since Factor Va activity is required for thrombin generation, the initial participation of Factor V in the expression of prothrombinase activity is not well understood. In the present study, the activation of Factor V by Factor Xa has been investigated. Cofactor activation was assessed by monitoring the conversion of prethrombin-1 to thrombin in the presence of 5-dimethylamino-naphthalene-1-sulfonylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA). The DAPA not only provided a fluorescent signal for the formation of thrombin, but also attenuated the feedback activation of Factor V by thrombin. Trace quantities of Factor Va were removed from the Factor V preparations by immunoadsorption with immobilized murine monoclonal antibodies selective for Factor Va. The incubation of Factor V with Factor Xa in the presence of phosphatidylcholine/phosphatidylserine vesicles, CaCl2, and DAPA resulted in a time-dependent increase in cofactor activity. Phosphatidylcholine/phosphatidylserine vesicles were not absolutely required, but the rate of Factor V activation was significantly enhanced by inclusion of the vesicles. The activation was absolutely dependent upon Factor Xa and was eliminated by immunoadsorption of the Factor Xa preparation with a murine anti-Factor X (Xa) monoclonal antibody coupled to agarose. The activation was not affected by immunoadsorption of the Factor Xa and Factor V preparations with burro polyclonal anti-prothrombin IgG. Most of the products of the Factor Xa activation of Factor V differ from the products derived by the thrombin-catalyzed activation of the procofactor. The results demonstrate that Factor Xa catalyzes the activation of Factor V. Furthermore, these studies suggest that the Factor Xa activation of Factor V may be responsible for the advent of early prothrombinase activity.
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PMID:The factor Xa-catalyzed activation of factor V. 664 60

The kinetic parameters of bovine factor X activation by bovine factor IXa have been determined in the absence and presence of Ca2+, thrombin-activated bovine factor VIII (VIIIa), and phospholipid (dioleoylphosphatidylcholine/dioleoylphosphatidylserine, 75/25; mol/mol). Factor IXa in the absence of Ca2+, factor VIIIa, and phospholipid is able to catalyze factor X activation. The Km for factor X is 299 microM which is well above its concentration in bovine plasma, about 0.2 microM. The Vmax of factor Xa formation is 0.0022 mol of Xa . min-1 . mol of IXa-1 under these conditions. Addition of Ca2+ has little effect on the kinetic constants of factor X activation by factor IXa. In the presence of 10 mM CaCl2 the Km for factor X is 181 microM, and the Vmax is 0.0105 mol of Xa . min-1 . mol of IXa-1. The presence of 10 microM phospholipid dramatically decreases the Km for factor X to 0.058 microM, and the Vmax becomes 0.0025 mol of Xa . min-1 . mol of IXa-1. The Vmax of factor Xa formation slightly increases when more phospholipid is present in our experiments, and there is a considerable increase of the Km for factor X at higher phospholipid concentrations. Therefore, the Km measured in the presence of phospholipid has to be regarded as an apparent Km. The possible explanations for this phenomenon are discussed. For the complete factor X-activating complex (i.e. factor IXa, factor VIIIa, Ca2+, and 10 microM phospholipid) the Km for factor X is 0.0063 microM, and the Vmax is raised 200,000-fold to 500 mol of Xa . min-1 . mol of IXa-1. In order to exert its stimulating effect on factor X activation factor VIII has to be activated with thrombin. Our results show that factor IXa is an enzyme which can activate factor X at a very low rate. The stimulating effect of phospholipid in factor X activation is mainly due to an effect on the Km for factor X, bringing it within the range of the plasma concentration. The stimulatory effect of factor VIIIa is explained by its 200,000-fold increase of the Vmax of factor Xa formation.
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PMID:The role of phospholipid and factor VIIIa in the activation of bovine factor X. 678 1

Optimal reaction conditions for the activated partial thromboplastin time (APTT), 1-stage prothrombin time (PT), and Russell's viper venom time were studied for pooled plasmas of horses, cattle, sheep, goats, swine, dogs, cats, and persons. Changes in CaCl2 and NaCl concentrations had significant effects on the APTT and PT reactions. The APTT was more sensitive than the PT to changes in CaCl2 concentration. The CaCl2 concentration recommended by the manufacturer for the APTT was suboptimal for some of plasmas of domesticated animals in ths study. Infusorial earth (Celite, activator) concentration provided by the manufacturer was optimal for plasmas of all species, with exception of horses and swine. Optimal incubation times for the APTT for the equine, bovine, ovine, and caprine plasmas were shorter than the 5 minutes recommended for human plasma. Optimal incubations for porcine and canine plasmas were longer than 5 minutes. The APTT and PT of all plasmas were stable for up to 3 hours' storage at 4 C in all species' plasmas tested with exception of the porcine plasma.
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PMID:Coagulation studies f plasmas from healthy domesticated animals and persons. 734 May 87

A new chromogenic method has been developed and rigorously standardized for the estimation of factor VII in defibrinated diluted plasma. This method employs a mixture of CaCl2-rabbit brain thromboplastin as activator, diluted factor VII deficient plasma as source of factor X and the chromogenic substrate S2222 for the measurement of factor Xa. The chromogenic method was insensitive to cold- and kaolin-induced activation of factor VII, this in contrast to the one-stage clotting assay. Results obtained with the chromogenic method revealed good correlation with the clotting method in 33 normal subjects, in 42 patients on oral anticoagulant therapy and in five patients with severe congenital factor VII deficiency. A good correlation was also obtained with 'Thrombotest'. Comparative estimation of factor VII and of factor VII cross-reacting material in supernatants of BaCl2 adsorbed plasma of coumarin treated patients revealed that the chromogenic method does not measure decarboxy factor VII. Detailed investigations revealed a half life for decarboxy factor VII of 2.1 +/- 0.6 h.
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PMID:Evaluation of a new chromogenic assay for factor VII and its application in patients on oral anticoagulant treatment. 743 29

Activated platelets and platelet-derived microvesicles demonstrate procoagulant properties. It is known that following stimulation, negatively charged phospholipids and factor Va become located on their surfaces. The aim of this study was to see whether activated platelets and platelet-derived microvesicles also expressed some factor Xa activity on their surfaces in a system where factor Xa did not come from external sources. In order to study this question, flow cytometry, as well as the use of a chromogenic substrate to factor Xa and a clotting assay in a factor X depleted plasma, were applied. A prothrombinase assay was also applied using prothrombin, CaCl2 and a chromogenic substrate to thrombin. The platelets were gel-filtered or washed, suspended in Tris-buffered saline, and activated by calcium ionophore A23187 or the thrombin receptor agonist peptide SFLLRN. Microvesicles and activated platelets were separated by centrifugation. Flow cytometry using a monoclonal antibody against factor Xa demonstrated the presence of factor Xa on the surface of the activated platelets. In addition, platelet-derived microvesicles and activated platelets demonstrated factor Xa activity on their surfaces detected directly by splitting of the chromogenic substrate to factor Xa, or by the prothrombinase assay. The thrombin generation in the last assay could be inhibited by a selective factor Xa inhibitor (recombinant tick anticoagulant peptide (rTAP)), soybean trypsin inhibitor, and antithrombin III plus LMW-heparin, all inhibiting at the factor Xa level, as well as by leupeptin which also inhibited the thrombin-chromogenic substrate interaction as such.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Platelet-derived microvesicles and activated platelets express factor Xa activity. 754 77

The initiation and propagation of in vivo coagulation are thought to be catalyzed by factor VIIa-tissue factor (an activator of factor X and factor IX) and factor IXa-factor VIIIa (an activator of factor IX), respectively. The enzymatic activity of factor VIIa-tissue factor is abrogated by tissue factor pathway inhibitor (TFPI), which anchors a quaternary complex consisting of equimolar TFPI, factor Xa, factor VIIa, and tissue factor in which both factor Xa and factor VIIa are inactive. This study compared the anticoagulant effectiveness of TFPI (which also inactivates prothrombinase-bound factor Xa), hirudin (which inactivates thrombin), and heparin (which catalyzes the inactivation of factor Xa and thrombin by antithrombin III). Factor X and prothrombin activation were initiated by adding 5 pM r-tissue factor in a suspension of coagulant phospholipids and CaCl2 to defibrinated plasma. Compared on the basis of their ability to delay the initiation of and inhibit factor X and prothrombin activation, the anticoagulant effectiveness of 0.5 nM TFPI was greater than those of 10 nM hirudin and approximately 100 nM (0.1 unit/mL) heparin. However, a 100-fold molar excess of TFPI over tissue factor could not abrogate factor X and prothrombin activation in plasma. These results suggest that propagation of tissue-factor dependent coagulation is catalyzed by factor IXa-factor VIIIa, which unlike factor VIIa-tissue factor, is not inactivated by TFPI.
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PMID:Anticoagulant actions of tissue factor pathway inhibitor on tissue-factor-dependent plasma coagulation. 766 Jan 46

Tissue factor pathway inhibitor (TFPI) inhibits the tissue factor--factor VIIa complex and factor Xa with its first and second Kunitz domains (K1 and K2), respectively. The inhibitory activity is enhanced by heparin, and the C-terminal basic part has been shown to be a heparin-binding site (HBS-1). To characterize and localize a second heparin-binding site (HBS-2), we studied the effect of heparin on the inhibitory activity of two forms of recombinant human TFPI, the full-length TFPI (rTFPI), and TFPI lacking the C-terminal basic part (rTFPI-C), by assaying the inhibition of human factor Xa. rTFPI-C inhibited factor Xa with an initial Ki of 6.79 nM in the absence of Ca2+ and 22.3 nM in the presence of 5 mM CaCl2. Heparin decreased the initial Ki to 1.79 nM in the absence of Ca2+ and 2.68 nM in the presence of 5 mM CaCl2, indicating the presence of HBS-2 in rTFPI-C. The dissociation constant for the binding of HBS-2 with heparin was determined to be 830 nM using fluorescein-labeled heparin and rTFPI-C. Heparin enhanced the inhibitory activity of a fragment consisting of the K2 and K3 domains, but it did not stimulate the inhibitory activity of the K2 domain. A synthetic peptide mimicking from Gly212 to Phe243 in the K3 domain reduced the effect of heparin on the inhibition by rTFPI-C and rTFPI. These results defined the location of HBS-2 in the basic region of the K3 domain between Gly212 and Phe243.
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PMID:Effect of heparin on the inhibition of factor Xa by tissue factor pathway inhibitor: a segment, Gly212-Phe243, of the third Kunitz domain is a heparin-binding site. 772 33


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