Search PubMed⌕ Search

Biomedical subjects

G Tans

Publications and source records attributed to G Tans.

82 records · Page 5Linked to original sources
PubMed1983

Initiation of contact activation by sulfatides.

Low amount of sulfatides initiated intrinsic coagulation and the appearance of kallikrein activity in normal human plasma. The initiation of procoagulant and kallikrein amidolytic activity was dependent on the presence of Factorr XII, high molecular weight kininogen and prekallikrein. Because the activated partial thromboplastin clotting times in prekallikrein deficient plasma approach normal values upon prolonged incubation with kaolin, this phenomenon was studied and found to be even more pronounced in the presence of sulfatides. Shortening of the clotting time was essentially completed in 5 min in the presence of sulfatides whereas a pre-incubation of 15 to 20 min was required in the presence of kaolin. The limited proteolysis of 125I-Factor XII in plasma during incubation was more rapid and more extensive in the presence of sulfatides than in the presence of kaolin. Factor XII cleavage in prekallikrein deficient plasma was completed in less than 5 min in the presence of sulfatides and in less than 15 min in the presence of kaolin. Thus, the appearance of Factor XII-dependent coagulant activity correlates with the limited proteolysis of Facto XII when normal or prekallikrein deficient plasma is activated by sulfatides or by kaolin. These observations are consistent with the hypothesis that Factor XII is cleaved in plasma to generate maximal Factor XIIa activity.

Animals↗
PubMed1982

Isolation and functional properties of the heavy and light chains of human plasma kallikrein.

Human plasma kallikrein was prepared by proteolytic activation of prekallikrein with beta-Factor XIIa (Mr = 28,000). Two forms of kallikrein were generated that were each composed of two disulfide-linked polypeptide chains: a heavy chain of apparent Mr = 43,000 and a light chain of apparent Mr = either 36,000 or 33,000. Following reduction and alkylation, the heavy and light chains of kallikrein were isolated by affinity chromatography using insolubilized high molecular weight kininogen. The alkylated light chain of kallikrein did not bind to high molecular weight kininogen-Sepharose while the heavy chain did bind with high affinity and was subsequently eluted. The light chain retained the specific amidolytic activity of native kallikrein. The Km and kcat values for the hydrolysis of H-D-Pro-Phe-Arg-p-nitroanilide by kallikrein or its light chain were identical. Activation of Factor XII in solution was equally well catalyzed by kallikrein and its light chain. However, in kaolin-dependent coagulation, kallikrein was 180 times more effective than the light chain in correcting the clotting defect of prekallikrein-deficient plasma. Furthermore, the light chain was 3.5 times less potent than kallikrein in cleaving high molecular weight kininogen in solution. These observations indicate that the light chain region contains the enzymatic active site and adequately accounts for the enzymatic properties of kallikrein in solution on the protein substrate, Factor XIII, and on oligopeptide substrates. However, the heavy chain region of kallikrein is required for binding to high molecular weight kininogen, for surface-dependent activation of coagulation, and for optimal cleavage of high molecular weight kininogen.

Humans↗
PubMed1982

Activation of factor IX by factor XIa--a spectrophotometric assay for factor IX in human plasma.

The activation of Factor IX by partially purified Factor XIa was followed by active site titration, gelelectrophoresis and by a spectrophotometric assay. The assay is based on the finding that the rate of Factor X activation in the presence of phospholipid and Ca2+ is linear in time and proportional to the amount of Factor IXa present and can be determined with the chromogenic substrate S2222. Conditions were found that allowed complete activation of Factor IX in human plasma by Factor XIa. The amount of Factor IXa present in the plasma sample can be determined with the spectrophotometric assay and is proportional with the amount of plasma present. In plasma from patients receiving vitamin-K antagonists reduced Factor IX activity is found with the spectrophotometric assay and the new assay method may be useful in monitoring oral anticoagulant therapy.

Animals↗
PubMed1982

Properties of sulfatides in factor-XII-dependent contact activation.

Incubation of normal human plasma with low amounts of sulfatides resulted in the initiation of intrinsic coagulation and the appearance of kallikrein activity. The optimal initiation of procoagulant and kallikrein amidolytic activity was dependent on the presence of factor XII, high molecular weight kininogen, and prekallikrein. Since the activated partial thromboplastin clotting times in prekallikrein-deficient plasma approach normal values upon prolonged incubation with kaolin, this phenomenon of autocorrection was studied and found to be even more pronounced in the presence of sulfatides. Autocorrection was essentially completed in 5 min in the presence of sulfatides, whereas a preincubation of 15-20 min was required in the presence of kaolin. The limited proteolysis of 125I-factor XII in plasma during incubation with activating material or during clotting was determined. Cleavage of factor XII was more rapid and more extensive in the presence of sulfatides than in the presence of kaolin. In prekallikrein-deficient plasma, factor XII cleavage was completed within 5 min in the presence of sulfatides and within 15 min in the presence of kaolin. Thus, the appearance of factor-XII-dependent coagulant activity correlates with the limited proteolysis of factor XII when normal or prekallikrein-deficient plasma is activated by sulfatides or kaolin.

Blood Coagulation↗
PubMed1981

The role of phospholipid and factor VIIIa in the activation of bovine factor X.

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.

Animals↗
PubMed1980

The role of phospholipids and factor Va in the prothrombinase complex.

The kinetic parameters of the conversion of bovine prothrombin into thrombin by activated bovine blood clotting factor X (Xa) have been determined in the absence and presence of Ca2+, activated bovine factor V (Va) and phospholipid (dioleoylphosphatidylcholine/dioleoylphosphatidylserine, 1:1; mol/mol). In the absence of accessory components, the Km for prothrombin is 131 microM, which is well above its concentration in bovine plasma of about 1.5 microM. The Vmax of thrombin formation is 0.61 mol min-1 mol of Xa-1 under these conditions. In the presence of 7.5 microM phospholipid, the Km drops to 0.058 microM and the Vmax slightly increases to 2.25 mol min-1 mol of Xa. For the complete prothrombinase complex (Xa, Va, Ca2+, and 7.5 microM phospholipid), a Km for prothrombin of 0.21 microM and a Vmax of 1919 mol min-1 mol of Xa-1 is found. The Vmax of thrombin formation slightly increases when more phospholipid is present in our experiments and there is a considerable increase of the Km for prothrombin at higher phospholipid concentrations. Preliminary calculations show that the prothrombin density at the phospholipid surface at the Km is independent of the phospholipid concentration. This indicates that the Km measured in the presence of phospholipid has to be regarded as an apparent Km and the local prothrombin concentration determines the kinetics of activation. Prothrombin activation by prothrombinase complexes of different compositions was followed by gel electrophoresis in the presence of sodium dodecyl sulfate. Both in the absence and presence of phospholipid but without factor Va, prethrombin 2 is the main product formed during the initial stages of steady state prothrombin activation. In the presence of factor Va, thrombin is the main end product and minute amounts of prethrombin 2 are formed. This shift in the reaction pathway of prothrombin activation caused by factor Va will contribute to the observed increase of the Vmax measured in the presence of factor Va.

Animals↗
PubMedDate not supplied

Snake venom activators of factor X: an overview.

Activators of blood coagulation factor X have been described in the venom of many snake species belonging to the genus Viperidae and Crotalidae as well as from a few Elapid species. Based on the structural and functional properties of purified activating principles, factor X activators are either metalloproteases or serine proteases. The best known activator is RVV-X from Russell's viper (Daboia russelli), a metalloprotease consisting of a heavy chain containing the catalytic domain and two light chains which share homology with C-type lectins and which are thought to exert a regulatory function in the Ca(2+)-dependent activation of factor X. This activator is also one of the best examples of the use of exogenous activators in coagulation research and in addition it is used in many diagnostic research kits. In this paper, an overview is given of the structural and functional properties of snake venom factor X activators thus far described in the literature.

Animals↗
PubMedDate not supplied

Factor V activation and inactivation by venom proteases.

Blood coagulation factor V is a single-chain glycoprotein with M(r) = 330,000 which plays an important role in the procoagulant and anticoagulant pathways. Thrombin activates factor V into factor Va, a two-chain molecule which is composed of a heavy (M(r) = 105,000) and a light chain (M(r) = 71,000/74,000). Factor Va accelerates factor Xa-catalysed prothrombin activation more than 1,000-fold and under physiological conditions the cofactor activity of factor Va in prothrombin activation is down-regulated by activated protein C. Factor V can also be activated by a wide variety of snake venoms (e.g. from Vipera species, Naja naja oxiana, Bothrops atrox) and by proteases present in the bristles of a South American caterpillar (Lonomia achelous). Some venoms, notably of Vipera lebetina turanica and Lonomia achelous, contain proteases that are able to inactivate factor V or factor Va. Venom factor V activators are excellent tools in studying the structure-function relationship of factor V(a) and they are also used in diagnostic tests for quantification of plasma factor V levels and for the screening of defects in the protein C pathway. In this review, the structural and functional properties of animal venom factor V activators and inactivators is described.

Animals↗