INTERACTION OF THROMBIN AND FACTOR Xa WITH BOVINE VASCULAR ENDOTHELIAL CELLS, SMOOTH MUSCLE CELLS AND RAT HEPATOMA CELLS

The inhibition of thrombin as well as of factor Xa has been thought to occur primarily in plasma through the neutralizing action of the serine protease inhibitor antithrombin III (AT-III). However, inhibition of thrombin and Xa by this mechanism may not be sufficient for effective elimination of these clotting factors in states of increased coagulation activity. The potential role of the vascular endothelium in the inhibition of clotting factor activities has therefore received attention in recent years. The aim of the present investigation was to characterize the binding and inhibition of thrombin and factor Xa to the vascular endothelial cell (EC), smooth muscle cell (SMC) and rat hepatoma cell (RHC) in vitro, as well as to evaluate the effects of plasma constituents upon the inhibition of these factors. Purified bovine thrombin and factor Xa were used. The enzymatic activities of both factors were assayed using chromogenic substrates. The cells were exposed for 5 U/ml thrombin or 0.5 U/ml factor Xa. After 10 minutes incubation, the initial thrombin activity in the solution had decreased by about 20% in case of EC and SMC and about 11% when incubated with RHC. Thrombin activity recovered from the cell surface amounted to 0.02 U/cm2. When the cells with the surface bound enzyme were incubated with defibrinogenated plasma or AT-III for 30 seconds, only about 10% and 25-40%, respectively, of initial activity could be found. In similar experiments with factor Xa, after 10 minutes incubation, the initial activity in the solution had decreased by 10%. Factor Xa activity recovered from the cell surface was 0.001 U/cm2. After 30 seconds exposure to AT-III, no cell surface related factor Xa activity was recovered, whereas 10% of the cell surface activity was recovered after incubation with defibrinogenated plasma. It is concluded that thrombin and factor Xa are taken up and inhibited by EC, SMC and RHC cell surfaces in similar ratios suggesting that cell surface-mediated inactivation of activated clotting factors is not restricted to vascular wall cells. The inactivation of factor Xa was dependent on AT-III, however, the inactivation of thrombin was further promoted by an additional unidentified plasma constituent

Interaction of Thrombin and Factor Xa with Bovine Vascular Endothelial Cells, Smooth Muscle Cells and Rat Hepatoma Cells

10.1055/s-0038-1647020 ◽

1988 ◽

Vol 60 (02) ◽

pp. 148-152 ◽

Cited By ~ 1

Author(s):

Maciej Dryjski ◽

Be-Sheng Kuo ◽

Thorir D Bjornsson

Keyword(s):

Endothelial Cells ◽

Smooth Muscle ◽

Vascular Endothelial Cells ◽

Factor Xa ◽

Vascular Endothelial ◽

Initial Activity ◽

Cell Surfaces ◽

At Iii ◽

Rat Hepatoma ◽

Rat Hepatoma Cells

SummaryThe aims of the present investigation were to characterize the binding and inhibition of thrombin and factor Xa to bovine vascular endothelial cells (EC), bovine smooth muscle cells (SMC), and rat hepatoma cells (RHC), and to evaluate the effects of plasma constituents on their inhibition. The enzymatic activities of bovine thrombin and factor Xa were assayed using chromogenic substrates. After 10 min incubation with the cells, thrombin activity in the solution had decreased by about 20% and was subsequently recovered on the cell surfaces. When the cells with the surface-bound thrombin were incubated with defibrinogenated plasma or antithrombin III (AT-III) for 30 sec only about 10% and 20-40%, respectively, of the initial activity could be recovered. In similar experiments with factor Xa, initial activity in the solution had decreased by 10% after 10 min incubation, and was subsequently recovered from the cell surfaces. After 30 sec incubation with AT-III, no cell surface-bound factor Xa activity was detected, whereas 10% of the bound factor Xa activity was recovered after incubation with defibrinogenated plasma. It is concluded that thrombin and factor Xa are taken up and inhibited by EC, SMC and RHC cell surfaces in similar ratios, suggesting that cell surface-mediated inhibition of clotting factors is not restricted to vascular wall cells. The inactivation of factor Xa was dependent on AT-III, however, the inactivation of thrombin was further promoted by an additional unidentified plasma constituent.

Reactivity of a Hereditary Abnormal Antithrombin III Fraction in the Inhibition of Thrombin and Factor Xa

10.1055/s-0038-1650091 ◽

1980 ◽

Vol 44 (02) ◽

pp. 092-095 ◽

Cited By ~ 8

Author(s):

T H Tran ◽

C Bondeli ◽

G A Marbet ◽

F Duckert

Keyword(s):

Antithrombin Iii ◽

Factor Xa ◽

Heparin Binding ◽

Thrombin Inhibition ◽

Superficial Thrombophlebitis ◽

Heparin Concentration ◽

Heparin Binding Site ◽

At Iii ◽

The Difference ◽

SummaryTwo different AT-III fractions were purified from the plasma of a patient with recurrent superficial thrombophlebitis. The abnormal AT-III fraction (A-AT) was compared to the normal AT-III fraction (N-AT) in the inhibition of thrombin and factor Xa. Without heparin, both inactivate proteases in a similar manner and at the same rate. However, at low heparin concentration the thrombin inhibition proceeds more slowly with A-AT than with N-AT. At high heparin concentration the difference between A-AT and N-AT becomes very small. The inhibition of factor Xa follows a similar pattern. It is suggested that the heparin binding site of A-AT differs from that of N-AT resulting in a decreased heparin cofactor activity.

GAGs-Potentiated Inhibition of Thrombin, Factor Xa and Plasmin in Plasma and in a Purified System Containing Antithrombin III – Correlation with Total Charge Density

10.1055/s-0038-1653468 ◽

1981 ◽

Vol 46 (04) ◽

pp. 749-751 ◽

Cited By ~ 3

Author(s):

E Cofrancesco ◽

A Vigo ◽

E M Pogliani

Keyword(s):

Charge Density ◽

Chemical Structure ◽

Antithrombin Iii ◽

Factor Xa ◽

Total Charge ◽

Pure System ◽

At Iii ◽

Total Charge Density ◽

The Difference ◽

SummaryThe ability of heparin and related glycosaminoglycans (GAGs) to accelerate the inhibition of thrombin, factor Xa and plasmin in plasma and in a purified system containing antithrombin III (At III) was studied using chromogenic peptide substrate assaysThere was a good correlation between the charge density of the mucopolysaccharides and the activities investigated. While the difference between potentiation of the antithrombin activity by GAGs in plasma and in the purified system was slight, the inhibition of factor Xa in plasma was more pronounced than in the presence of purified At III, indicating the mechanisms for GAGs-potentiated inhibition of thrombin and factor Xa are not identical.For the antiplasmin activity, there was a good correlation between the chemical structure and biological activity only in the pure system, confirming that the antithrombin-GAG complex plays a very limited role in the inactivation of plasmin in plasma.

Thrombin and Factor Xa Uptake and Inhibition by Cultured Bovine Aortic Endothelial Cells, Smooth Muscle Cells, and Rat Hepatoma Cells

Cardiovascular Disease ◽

10.1007/978-1-4684-5296-9_30 ◽

1987 ◽

pp. 255-263

Author(s):

Maciej Dryjski ◽

Thorir D. Bjornsson

Keyword(s):

Endothelial Cells ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Factor Xa ◽

Muscle Cells ◽

Hepatoma Cells ◽

Aortic Endothelial Cells ◽

Bovine Aortic Endothelial Cells ◽

Rat Hepatoma ◽

Rat Hepatoma Cells

EFFECT OF VARIOUS HEPARIN(OID)S ON HEPARIN COFACTOR II MEDIATED ANTI-THROMBIN ACTIVITY AND INHIBITION OF THROMBIN GENERATION IN VITRO

10.1055/s-0038-1644353 ◽

1987 ◽

Author(s):

T G van Dinther ◽

F Hol ◽

D G Meuleman

Keyword(s):

Molecular Weight ◽

Thrombin Generation ◽

Factor Xa ◽

Weight Fraction ◽

Blood Platelet ◽

Low Molecular Weight ◽

Heparin Cofactor Ii ◽

Thrombin Activity ◽

At Iii

The effects of various heparin(oid)s, standard heparin VII (SH), dermatan sulphate (DS), a low molecular weight fraction of heparin (UMW-H), FragminR (FRA), Org 10172 = low molecular weight heparinoid, the fraction of Org 10172 with high affinity for AT-III (HA-10172) and the low affinity fraction (LA-10172) respectively were examined on in vitro thrombin generation and inactivation.Thrombin inactivation in the presence of either heparin cofactor II (HC-II) or anti-thrombin III (AT-III) was assessed with two newly developed assays using the purified cofactors, thrombin and chromogenic substrate S2238 on microtiterplates. Thrombin generation in the presence of HC-II and AT-III was studied using purified factor Xa, prothrombin and blood platelet lysate and the residual thrombin activity was assessed amidolytically.The inhibition of the compounds on thrombin activity are summarized in the tableThe following conclusions can be drawn:- SH, LMW-H, HA-10172 and FRA potentiate the AT-III mediated inactivation of Ha more strongly than the HC-II mediated inactivation.- DS and LA-10172 show the reverse pattern of inactivation, while Org 10172 potentiates both inactivaton pathways to a similar extent.Thrombin generation in the presence of HC-II is inhibited by mw-heparin(oid)s at approx. 2-5 times lower concentrations than the HC-II mediated thrombin inactivation, while the inhibiting effect of SH in both assays is comparable.AT-III mediated thrombin generation inhibition and AT-III mediated thrombin inactivation is comparable as well for SH, LMW-H and FRA. In contrast, Org 10172 and its subfractions are approx. 10 times more potent on AT-III mediated thrombin generation inhibition than on AT-III mediated thrombin inactivation.Org 10172 shows low anti-thrombin activity and this activity is mainly mediated via FC-II.

A Comparison of Pentosan Polysulphate (SP54) and Heparin I: Mechanism of Action on Blood Coagulation

10.1055/s-0038-1657139 ◽

1982 ◽

Vol 47 (02) ◽

pp. 104-108 ◽

Cited By ~ 34

Author(s):

A-M Fischer ◽

T W Barrowcliffe ◽

D P Thomas

Keyword(s):

Factor Xa ◽

Factor X ◽

Factor Ixa ◽

Crossed Immunoelectrophoresis ◽

Low Concentrations ◽

At Iii ◽

Pentosan Polysulphate ◽

High Concentrations ◽

Marked Suppression ◽

SummaryThe effects of SP54 on inhibition of thrombin, factor Xa and factor IXa, in the presence and absence of antithrombin III (At III), have been examined and compared to those of heparin. SP54 potentiated inhibition of thrombin and Xa by purified At III, but crossed immunoelectrophoresis data indicated that these effects were mediated by binding to the enzyme, rather than to At III. Relatively high concentrations of SP54 were required for inhibition of thrombin and Xa in plasma, but at concentrations less than 2 μg/ml there was a marked suppression of the intrinsic activation of factor X. This effect was shown to be independent of At III, and to be due largely to inhibition of factor IXa. Prothrombin activation by factor Xa and phospholipid was also suppressed by SP54 in the absence of At III, and its effect on the APTT was also shown to be independent of At III. It is concluded that at relatively low concentrations the anticoagulant actions of SP54 are mainly due to these At III-independent pathways.

The molecular-weight dependence of the rate-enhancing effect of heparin on the inhibition of thrombin, factor Xa, factor IXa, factor XIa, factor XIIa and kallikrein by antithrombin

Biochemical Journal ◽

10.1042/bj1930395 ◽

1981 ◽

Vol 193 (2) ◽

pp. 395-400 ◽

Cited By ~ 180

Author(s):

E Holmer ◽

K Kurachi ◽

G Söderström

Keyword(s):

Molecular Weight ◽

Factor Xa ◽

Low Molecular Weight ◽

Clotting Factors ◽

Factor Ixa ◽

Molecular Weight Dependence ◽

Factor Xiia ◽

Factor Xia ◽

Heparin Fractions ◽

Heparin fractions of different molecular weight and with high affinity for antithrombin were studied with respect to their ability to potentiate the inhibition of activated clotting factors by antithrombin. Inhibition of thrombin, Factor IXa and Factor XIa showed similarities in the dependence on the molecular weight of heparin and was found to decrease with decreasing molecular weight. Inactivation of Factor Xa, Factor XIIa and kallikrein was, however, less dependent on the size of the polysaccharide and, to a great extent, was potentiated even by low-molecular-weight heparin fractions that had virtually no effect on the inhibition of thrombin, Factor IXa and Factor XIa.

HEPARIN COFACTOR II: A SIMPLE ASSAY METHOD

10.1055/s-0038-1644349 ◽

1987 ◽

Author(s):

H Vinazzer ◽

U Pangraz

Keyword(s):

Antithrombin Iii ◽

Factor Xa ◽

Tris Buffer ◽

Assay Method ◽

Healthy Individuals ◽

Heparin Cofactor Ii ◽

Complete Inactivation ◽

Thrombin Activity ◽

At Iii ◽

The Difference

A photometric assay method for heparin cofactor II (HC II) is described. In a first step antithrombin III (AT III) in plasma is blocked by an anti human AT III immunoglobuline from goats. After dilution of this plasma with Tris buffer pH 8.4 containing 3 IU/ml heparin and addition of thrombin the remaining thrombin activity is measured by use of the chromogenic substrate S-2238 Kabi. The following preliminary experiments were carried out: Variation of the amount of anti-AT III added to plasma resulted in complete inactivation of 1.25 units AT III by 1.0 ml of the inhibitor. Incubation of 1 ml anti AT III with 1 ml purified AT III ( 1 U/ml} or with 1 ml normal plasma completely abolished AT III activity within 60 sec. Incubation of the reaction mixture with thrombin resulted in maximum inactivation after 180 sec. This is in contrast to AT III activated by heparin which immediately inactivates thrombin. Anti-Xa activity after depletion of AT III was assayed in a similar way by addition of factor Xa to the reaction mixture and measuring the remaining Xa activity by the substrate S-2222. In these tests no anti Xa-activity was found after AT III depletion. From these experiments there was assumed that the anti thrombin activity measured under the following conditions was due to the action of HC II:Plasma ( 50 μl) was mixed with anti AT III (50 μl) and was incubated for 60 sec. Tris buffer with heparin pH 8.4 (900 μl) was added. From this mixture 200 μl was pipetted into a cuvette at 37°C followed by 200 μl thrombin ( 2 IU/ml). After an incubation time of 180 sec 200 μl S-2238 ( 2 mmol/1) was added and the difference in OD/min was determined at 405 nm. A calibration curve was made by series of dilutions of normal AT III depleted plasma from 20 healthy individuals. The following preliminary results ofrHC II activity as a percentage were obtained:

Factor IXa Inhibition Contributes to the Heparin Effect

10.1055/s-0038-1646412 ◽

1991 ◽

Vol 66 (03) ◽

pp. 306-309 ◽

Cited By ~ 10

Author(s):

Suzette Béguin ◽

Frédérique Dol ◽

H Coenraad Hemker

Keyword(s):

Antithrombin Iii ◽

Dermatan Sulfate ◽

Factor Xa ◽

Inhibitory Effect ◽

Heparin Cofactor Ii ◽

Factor Ixa ◽

Factor Viiia ◽

At Iii ◽

Inhibition Of Thrombin ◽

Thrombin Formation

SummaryWe investigated whether the inactivation of factor IXa contributes to the partial inhibition of thrombin formation that is observed at therapeutic concentrations of heparin. The action of standard unfractionated heparin (0.05 U/ml) on thrombin formation in the intrinsic system was compared to that of a mixture of dermatan sulfate (DS) and a synthetic pentasaccharide (PS). DS enhances the action of heparin cofactor II which inhibits thrombin only. PS specifically enhances the anti-factor Xa activity of antithrombin III (AT III). The concentrations of DS and PS were chosen so as to obtain equal anti-thrombin and anti-factor Xa activities as in 0.05 U/ml heparin. An extra inhibitory effect of heparin over the mixture is observed in situations where free factor IXa, not bound to factor VIIIa and phospholipid, limits the rate of thrombin formation, notably in contact activated plasma. We conclude that the inactivation of free factor IXa by heparin contributes importantly to the inhibition of thrombin formation in the intrinsic system such as e.g. measured in the activated partial thromboplastin time.

Homozygous Variant of Antithrombin III that Lacks Affinity for Heparin, AT III Kumamoto

10.1055/s-0038-1646520 ◽

1989 ◽

Vol 61 (01) ◽

pp. 020-024 ◽

Cited By ~ 28

Author(s):

Kenji Okajima ◽

Hidetsugu Ueyama ◽

Youichiro Hashimoto ◽

Yasuto Sasaki ◽

Keiko Matsumoto ◽

...

Keyword(s):

Affinity Chromatography ◽

Autosomal Dominant ◽

Antithrombin Iii ◽

Factor Xa ◽

Heparin Cofactor Ii ◽

At Iii ◽

Antifactor Xa ◽

Cofactor Activity ◽

Inhibition Of Thrombin ◽

Plasma Heparin

SummaryAbnormal antithrombin III (AT III) was found in the plasma of a 31-year-old female who suffered from recurrent thrombotic episodes. Heparin cofactor activity was 28% of normal and undetectable when measured by inhibition of thrombin and factor Xa (F. Xa), while both progressive antithrombin and antifactor Xa activities were normal. The concentration of plasma AT III antigen was 37 mg/dl. Analysis by crossed-immunoelec- trophoresis (CIE) in the presence of heparin and affinity chromatography on heparin-Sepharose revealed that the propositus’ AT III did not bind to heparin. When heparin cofactor II (HC II) was removed from propositus’ plasma, heparin cofactor activity of AT III was not detected. Thus, HC II seemed to account for the plasma heparin cofactor activity found in the presence of thrombin. The patient’s parents and three of her brothers demonstrated qualitative abnormality of AT III; heparin cofactor activity was 30-50% of normal levels in the presence of both thrombin and F. Xa. These findings indicate that the propositus’ AT III lacks affinity for heparin and the mode of its inheritance seems to be autosomal dominant and, hence, the propositus would be a homozygote. For this variant, the name of AT III Kumamoto is proposed.