RAT HEPAT0CYTES IN CULTURE INACTIVATE FACTOR Xa.

1987 ◽  
Author(s):  
G D Qureshi ◽  
M Sun ◽  
C Gervin ◽  
H Evans
Keyword(s):  
Serine Proteases ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Rat Hepatocytes ◽  
Factor X ◽  
Clotting Factors ◽  
Hepatocyte Cultures ◽  
The Stability ◽  
Stabilization Period

Plasma contains zymogens of clotting factors, which under various stimuli are activated to serine proteases. Whereas much knowledge has been gained about the activation of clotting factors, relatively little is known about inactivation of these proteases. Antithrombin III has been shown to inactivate some activated clotting factors in plasma. Studies in intact animals have suggested that activated clotting factors are mainly inactivated in the liver. To investigate more fully the role of liver in inactivating the activated factors, we studied the stability of activated factor X(Xa) in hepatocyte cultures. Monolayer cultures on non-proliferating rat hepatocytes were prepared according to the method of Bissell et al. The culture medium was chemically defined and was free from serum or serum products. After the 24 h stabilization period, 0.5 units/ml of 100% activated bovine factor Xa was co-cultured with hepatocytes for 8 h. Samples were collected at 0, ½, 1 2, 4 and 8 h and tested for Xa activity using chromogenic substrate S-2222. At the end of 8 h only 41.07% of the initial Xa activity remained. Xa inactivation was not affected by a commercially prepared unfractionated heparin (1 unit/ml) and estradiol at 12.5, 25, 125 nM, a potentiator and inhibitor of antithrombin III, respectively. Inactivation of Xa in hepatocyte cultures was inhibited by the addition of cycloheximide (10-4M). Our data suggests that factor Xa is inactivated in hepatocyte cultures by one or more hepatic derived factors which do not meet the functional characteristics of antithrombin III.

scholarly journals Activated clotting factors in factor IX concentrates

Blood ◽  
1979 ◽  
Vol 54 (5) ◽  
pp. 1028-1038 ◽  
Author(s):  
MB Hultin
Keyword(s):  
Antithrombin Iii ◽  
Initial Rate ◽  
Factor Xa ◽  
Factor Ix ◽  
Clinical Settings ◽  
Factor X ◽  
Clotting Factors ◽  
Factor Ixa ◽  
Human Factor Ix ◽  
Factor X Activation

Abstract The precise quantitation of activated factors in human factor IX concentrates has been accomplished with the use of recently developed, specific assays for factors IXa, Xa, and thrombin. The assay for factor IXa, which measures the initial rate of 3H-factor-X activation, was shown to be specific for factor IXa in the concentrates. Activated factor IX concentrates contained 1.0–2.3 microgram/ml of factor IXa; whereas the assays of unactivated concentrates were negative (less than 0.2 microgram/ml). The assays of factor Xa and thrombin, which measure the initial rate of p-nitroaniline release from S-2222 and S-2238, respectively, showed similar small amounts of factor Xa (4–34 ng/ml) and thrombin (12–76 ng/ml) in the activated and unactivated concentrates. The nonactivated partial thromboplastin time of the concentrates correlated significantly with the factor IXa content, but not with factor Xa or thrombin. Antithrombin III antigen in 3 of 4 concentrates was several-fold higher than antithrombin III activity, suggesting the presence of antithrombin III complexed with activated factors. These results support the hypothesis that the degree of activation of factor IX concentrates is related primarily to the concentration of factor IXa, which may be responsible for the thrombogenicity of these concentrates in some clinical settings.

The Effects Of Heparin On The Activation Of Factor X And Prothrombin In Antithrombin III-Depleted Plasma

1981 ◽  
Author(s):  
F Ofosu ◽  
A Cerskus ◽  
J Hirsh ◽  
M A Blajchman
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Clotting Factor ◽  
Factor X ◽  
Independent Action ◽  
Clotting Factors ◽  
Factor Ixa ◽  
Normal Plasma ◽  
High Concentrations ◽  
Independent Effects

The predominant mode of the anticoagulant action of heparin is considered to be the enhancement of the rate of inactivation, by antithrombin-III, of several activated clotting factors. Recent evidence, however, suggests that heparin can reversibly inhibit the activation of prothrombin and factor X even in the absence of anti- thrombin-III. This antithrombin-III-independent action of heparin has been demonstrated only in purified clotting factor systems. In order to determine the significance of the antithrombin-III-independent effects of heparin in plasma, the effects of heparin on the activation of factor X and prothrombin were studied in antithrombin-III- depleted plasma produced by affinity chromatography of normal plasma on heparin-Sepharose. Heparin partially inhibited the activation of factor X and prothrombin on the addition of either factor IXa or factor Xa in anti- thrombin-III-depleted plasma. This inhibition was demonstrable only when high concentrations (1 or 10 units/ ml) of heparin were used. In contrast, when as little as 1% antithrombin-III was added to antithrombin-III-depleted plasma containing 1.0 u of heparin per ml of plasma, no factor X or prothrombin activation could be demonstrated. Thus, it appears that in comparison with the magnitude of the antithrombin-III-dependent effect, the contribution of the antithrombin-III-independent anticoagulant effect of heparin on the activation of factor X and prothrombin in normal plasma is limited.

scholarly journals The Role of Factor IXa and Factor VIII in the Activation of Factor X.

1977 ◽  
Author(s):  
Earl W. Davie ◽  
Gordon Vehar ◽  
Kazuo Fujikawa ◽  
Richard Di Scipio
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Serine Proteases ◽  
Factor Xa ◽  
Basic Mechanism ◽  
Factor X ◽  
Amino Terminal ◽  
Factor Ixa ◽  
A Charge

Factor IXa and factor VIII participate in the middle phase of blood coagulation. These two proteins convert factor X to factor Xa in the presence of calcium ions and phospholipid. The coagulant activity of factor VIII is increased 50-100 fold by the addition of thrombin, and this activity is stabilized in the presence of CaCl2. The activated product (tentatively identified as activated factor VIII) was readily inhibited by diisopropyl phosphorofluoridate or antithrombin III, suggesting that it is a serine enzyme. The exact role of this enzyme in the conversion of factor X to factor Xa, however, is not known. When factor X (bovine or human) is converted to factor Xa, an activation peptide is cleaved from the amino-terminal end of the heavy chain. This gives rise to a new amino-terminal sequence of Ile-Val-Gly-Gly-in the heavy chain. No change occurs in the light chain during the activation reaction. These data indicate that the basic mechanism involved in the conversion of human and bovine factor X to factor Xa appears to be essentially identical and probably involves the formation of a charge relay system characteristic of the pancreatic serine proteases.

scholarly journals Activated clotting factors in factor IX concentrates

Blood ◽  
1979 ◽  
Vol 54 (5) ◽  
pp. 1028-1038 ◽  
Author(s):  
MB Hultin
Keyword(s):  
Antithrombin Iii ◽  
Initial Rate ◽  
Factor Xa ◽  
Factor Ix ◽  
Clinical Settings ◽  
Factor X ◽  
Clotting Factors ◽  
Factor Ixa ◽  
Human Factor Ix ◽  
Factor X Activation

The precise quantitation of activated factors in human factor IX concentrates has been accomplished with the use of recently developed, specific assays for factors IXa, Xa, and thrombin. The assay for factor IXa, which measures the initial rate of 3H-factor-X activation, was shown to be specific for factor IXa in the concentrates. Activated factor IX concentrates contained 1.0–2.3 microgram/ml of factor IXa; whereas the assays of unactivated concentrates were negative (less than 0.2 microgram/ml). The assays of factor Xa and thrombin, which measure the initial rate of p-nitroaniline release from S-2222 and S-2238, respectively, showed similar small amounts of factor Xa (4–34 ng/ml) and thrombin (12–76 ng/ml) in the activated and unactivated concentrates. The nonactivated partial thromboplastin time of the concentrates correlated significantly with the factor IXa content, but not with factor Xa or thrombin. Antithrombin III antigen in 3 of 4 concentrates was several-fold higher than antithrombin III activity, suggesting the presence of antithrombin III complexed with activated factors. These results support the hypothesis that the degree of activation of factor IX concentrates is related primarily to the concentration of factor IXa, which may be responsible for the thrombogenicity of these concentrates in some clinical settings.

Scanning Electron Microscopic and Electrophoretic Observations on Barium Sulphate Used to Adsorb Clotting Factors

1975 ◽  
Vol 33 (02) ◽  
pp. 256-270
Author(s):  
R. M Howell ◽  
S. L. M Deacon
Keyword(s):  
Electron Microscopy ◽  
Factor Xa ◽  
Barium Sulphate ◽  
High Density ◽  
Low Density ◽  
Factor X ◽  
Clotting Factors ◽  
Electron Microscopic ◽  
Complementary Techniques ◽  
Scanning Electron

SummaryElectron microscopy and particle electrophoresis were found to be complementary techniques with which to complete the physical data from an earlier study on barium sulphates used to adsorb clotting factors from serum. The differences revealed by scanning electron microscopy (S. E. M.) in the physical shape of low and high density grades of barium sulphate particles appear to be of greater significance than charge as expressed by electrophoretic mobility, in determining whether or not precursor or preformed factor Xa is eluted.This conclusion was based on the finding that at pH values close to 7, where the adsorption from serum occurs, all samples with the exception of natural barytes were uncharged. However as the high-density, or soil-grade, was found by S. E. M. to consist of large solid crystals it was suggested that this shape might induce activation of factor X as a result of partial denaturation and consequent unfolding of the adsorbed protein. In contrast, uptake of protein into the centre of the porous aggregates revealed by S. E. M. pictures of low-density or X-ray grade barium sulphate may afford protection against denaturation and exposure of the enzyme site.The porous nature of particles of low-density barium sulphate compared with the solid crystalline forms of other grades accounts not only for its lower bulk density but also for its greater surface/gram ratio which is reflected by an ability to adsorb more protein from serum.Neither technique produced evidence from any of the samples to indicate the presence of stabilising agents sometimes used to coat particles in barium meals.

The rise of factor X level in blood plasma of patients at severe burn injuries

2021 ◽  
Author(s):  
George P Kozynets ◽  
Volodymyr P Tsyhankov ◽  
Daria S Korolova ◽  
Olga V Gornytska ◽  
Olexiy M Savchuk ◽  
...  
Keyword(s):  
Protein C ◽  
Antithrombin Iii ◽  
Burn Injury ◽  
Activated Partial Thromboplastin Time ◽  
Burn Injuries ◽  
Factor X ◽  
Severe Burn ◽  
Clotting Factors ◽  
Thrombotic Complications ◽  
Severe Burn Injuries

Abstract This work is dedicated to the detection of imbalance between the pro- and anti-coagulant branches of hemostasis at severe burn injuries by evaluating the content or activity of individual clotting factors. To select the targets for accurate diagnostics we measured the concentrations of soluble fibrin monomeric complexes and fibrinogen, levels of total prothrombin, factor X, protein C and antithrombin III, and recorded the time of clotting in activated partial thromboplastin time and prothrombin time tests. Factor X level was increased in 26 % of patients on the first day after the burn and it rose further in 62 % patients on the 14 th day of recovery. Increasing factor X level is assumed to be a risk factor of thrombotic complications. We propose to use it as a marker of predisposition to thrombosis at severe burn injury.

scholarly journals Active site-specific immunoassays

Blood ◽  
1990 ◽  
Vol 76 (4) ◽  
pp. 755-766 ◽  
Author(s):  
KG Mann ◽  
EB Williams ◽  
S Krishnaswamy ◽  
W Church ◽  
A Giles ◽  
...  
Keyword(s):  
Active Site ◽  
Serine Proteases ◽  
Solid Phase ◽  
Activated Protein C ◽  
Factor Xa ◽  
Immune Recognition ◽  
Factor X ◽  
Zymogen Activation ◽  
Prothrombinase Complex ◽  
Active Site Histidine

Abstract This study describes a process by which serine proteases that contain an S-1 arginine subsite and active site histidine may be inactivated and subsequently quantitated using a combination of peptidyl chloromethylketone chemistry and immune recognition technology. Active site labeling and inactivation of proteases is attained by modification of the active site histidine with a peptidyl chloromethylketone. In the specific illustrations demonstrated, we used the compound biotinyl- epsilon-aminocaproyl-phenylalanylprolylarginyl chloromethylketone. This reagent reacts quantitatively and specifically with the active site histidine of a wide variety of proteases that are elaborated in the coagulation and fibrinolytic system. The inactivated enzyme(s) may be quantitated by combinations of antiprotein antibodies and avidin binding technology using the biotin moiety on the peptide inhibitor. We have demonstrated the capability of capture of inactivated enzyme products directly on to solid-phase avidin with subsequent quantitation of bound protein using specific antibodies. In the converse system we have captured specific proteases using antiprotein antibodies in the solid phase and have quantitated bound enzyme by using avidin. Subsequent detection and quantitation has been achieved using the enzymatic activity of horseradish peroxidase conjugated either to the antibody or to avidin. Both types of assays are feasible, with avidin capture being the preferred mode when enzyme is evaluated in the presence of excess zymogen, as would be common in the evaluation of most blood-clotting enzymes. Assays are illustrated for tissue plasminogen activator, plasmin, thrombin, factor Xa, and activated protein C, which can measure protease concentrations as low as 50 pmol/L. Specific applications of the assays are provided in studies of the activation of prothrombin by the prothrombinase complex and of factor X with Russell's viper venom factor X activator. These assays measure the mass of active site present in the reaction mixture and are relatively independent of subspecies of enzyme or the environment in which the activity is generated. These assay systems provide powerful tools for elucidating product-precursor relationships in multienzyme feedback reactions involving zymogen activation.

Antithrombin III and Venous Thrombosis

1979 ◽  
Author(s):  
Duncan P. Thomas
Keyword(s):  
Venous Thrombosis ◽  
Antithrombin Iii ◽  
Physiological Role ◽  
Factor Xa ◽  
Original Description ◽  
Clear Cut ◽  
Naturally Occurring ◽  
At Iii ◽  
General Studies

Increasing interest in the physiological role of inhibitors of coagulation has highlighting the role of antithrombin III (AT III) as the most important naturally occurring inhibitor of venous thrombosis. Since Egeberg’s original description in 1965, it has been recognized that inherited deficiency of AT III is associated with an increased incidence of venous thromboembolism. The role of acquired deficiency of AT III in the pathogenesis of thromboembolism remainsless clear-cut, partly due to methodological differences. While low values have been reported in groups of patients with thromboembolism, estimations of AT III in individual patients are not allways abnormal. In general, studies which have measured protein concentration rather than functional activity, or cl otting assays which measure total antithrombin activity and not specific anti-Factor Xa activity have failed to demonstrate a clear relationship between AT III and thromboembolism. However, in two groups of patients, namely women on oral contraceptives and patients undergoing total hipreplacement, an acquired deficiency of AT III, particularly when measured by anti-Xa clotting assays, correlates highly with postoperative venous thrombosis. Although venous thrombosis may develop in patients despite normal AT III values, an activity below approximately 80% in an anti-Xa clotting assay has been found to be of predictive value in patients subjected to the stress of trauma or surgery.

HEPARIN IS NOT AN EFFICIENT INHIBITOR OF THE FACTOR Xa-DEPENDENT ACTIVATION OF FACTOR V AND FACTOR VIII

1987 ◽  
Author(s):  
F A Ofosu ◽  
G J Modi ◽  
M R Buchanan ◽  
J Hirsh ◽  
M A Blajchman
Keyword(s):  
Factor Viii ◽  
Antithrombin Iii ◽  
Specific Inhibitor ◽  
Factor Xa ◽  
Factor V ◽  
Factor X ◽  
Inhibitory Effects ◽  
Efficient Inhibitor ◽  
System 1 ◽  
Prothrombin Activation

We have previously proposed that the steps in coagulation most sensitive to inhibition by heparin are the thrombin-dependent activation of factor V and factor VIII. This observation was based on the demonstration that therapeutic concentrations of heparin or 1μM of the thrombin specific inhibitor, phe-pro-arg CH2Cl (PPACK) completely inhibited the activation of prothrombin when contact-activated plasma (CAP) was recalcified for up to 1 min. Under similar conditions, heparin and PPACK only partially inhibited the activation of factor X. Moreover, the addition of thrombin (lOnM) to CAP 1 min before that of heparin or PPACK reversed their inhibitory effects. We now provide further support for our hypothesis by showing that when the activity of thrombin is suppressed by heparin or PPACK, efficient activation of radiolabelled prothrombin occurs only when the factor Xa then present activates factor V and factor VIII. We compared the effects of HEP of PPACK on the following four systems for initiating the activation of prothrombin: (1) CAP; (2) CAP + lOnM thrombin; (3) CAP + InM Xa and (4) unactivated plasma + InM Xa + InM Va + coagulant phospholipids. In each system, the enzymes were added 1 min before the heparin or PPACK. In the absence of heparin or PPACK, all four systems generated the same amount of thrombin activity in 45s. Complete inhibition of prothrombin activation by heparin and PPACK was observed only in system 1 which did not contain exogenous thrombin or factor Xa. No inhibition by heparin or PPACK was observed when thrombin or factor Xa was added to CAP in systems (2) and (3). Only partial inhibition was observed in system (4) which contained exogenous prothrombi-nase complex. Factor Xa thus provides an effective by-pass mechanism for the activation of factor VIII and factor V in plasma containing therapeutic concentrations of heparin. Our data provide further evidence that the heparin-antithrombin III system is not effective in inactivating factor Xa. These results support the hypothesis that in unactivated normal plasma, the primary anticoagulant effect of heparin is the inhibition of the thrombin-dependent activation of factor V and factor VIII.

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