Neutralization of thrombin by antithrombin III in the presence of cultured human fibroblasts

Recent evidence suggests that heparan sulfate on endothelial cell surfaces acts as a catalyst for the neutralization of thrombin by antithrombin III (AT III). Fibroblasts also produce heparan sulfate which is present on the cell surface and secreted into the extracellular matrix. We evaluated the ability of cultured human fibroblasts to catalyze the interaction between thrombin and AT III and found that heparan sulfate produced by post-confluent fibroblasts was anticoagulantly active. Furthermore, after initial binding of thrombin to cells, thrombin-heparan sulfate appeared in the fluid phase above the cells; this thrombin could be rapidly neutralized by AT III independent of the further presence of cells. These results indicate that fibroblasts do produce an anticoagulantly active species of heparan sulfate and that the normal interaction between AT III and thrombin may be driven by initial release of heparan sulfate from the cell surface by thrombin followed by AT III interaction with the soluble thrombin-heparan sulfate complex.     

Effect of glycosaminoglycans and antithrombin III on uptake and inhibition of thrombin by the vascular wall

Thrombin binds to and is inactivated by the endothelium. The inactivation is potentiated by plasma. The present investigation was designed to clarify the role of vessel wall glycosaminoglycans (GAG) and plasma antithrombin III (AT III) in the inactivation and binding of thrombin by endothelium. Thrombin was shown to bind to vascular endothelium and artificial surfaces containing GAG:s. The binding could be inhibited on both types of surfaces by pretreating them with protamine. Thrombin bound to endothelium was rapidly inactivated in the presence of plasma but only slowly if the plasma was replaced by AT III, AT III-depleted plasma or a balanced salt solution. It is concluded that thrombin binds to vessel wall GAG:s and is inactivated by the endothelium. Potentiation of the inhibition of the endothelially bound thrombin by plasma is dependent upon presence of AT III but an additional plasma factor is also required.     

The inactivation of thrombin and plasmin by antithrombin III in the presence of sepharose-heparin.

The inhibition of thrombin and plasmin by antithrombin III was studied in the presence of heparin conjugated to Sepharose. When either enzyme was adsorbed to the heparin conjugate in quantities insufficient to occupy all the affinity sites, subsequent passage of antithrombin III through the column invariably produced complete inhibition as measured by the response to the chromogenic substrate S2160. However, when the loading sequence was reversed (i.e. adsorbing non-saturating quantities of antithrombin III before the enzyme), antithrombin III only insignificantly inhibited either enzyme over a 20-hour period. A second load of antithrombin III following the thrombin load resulted in complete inactivation. When thrombin was rendered incapable of binding to heparin by cyclohexanedione treatment and reacted with Sepharose-heparin to which antithrombin III was adsorbed, the protease inhibitor exhibited ‘progressive’ antithrombin activity. These studies may indicate that thrombin and plasmin possess higher affinities for heparin than for the antithrombin III-heparin complex. Furthermore antithrombin III reacts more readily with the enzyme-heparin complex than with heparin. If the relative affinities of heparin, thrombin and antithrombin III in plasma are similar to those observed under these affinity chromatographic conditions, then the present data are consistent with the view that heparin promotes the inactivation of thrombin and plasmin by augmenting their reaction with antithrombin III. An allosteric effect of heparin on thrombin is apparent from the 50% increase in esterase activity of thrombin on α-N-benzoyl-L-arginine ethyl ester which is observed in the presence of optimal concentrations of heparin.     

Prevention of thrombin/antithrombin III reaction in the presence of protamine or polybrene

The rate of the thrombin/antithrombin III (AT III) reaction was decreased in the presence of free polybrene or protamine. The reaction rate was also decreased in protamine-coated tubes and tubes on which polybrene was absorbed nonspecifically. The reaction was also prevented when either thrombin or AT III was transfered into noncoated tubes after instantaneous contact with protamine-coated tubes or tubes with polybrene. These facts suggest that the thrombin/AT III reaction rate is determined with concentrations of reactive protein species.     

Inhibition of antithrombin III by lipid peroxides

A study was made of the inhibition of antithrombin III (At III) activity by lipid peroxides prepared from autoxidation of unsaturated fatty acids. Lipid peroxides markedly reduced the thrombin neutralising activity of plasma and purified At III with or without albumin carrier. Heparin Sepharose chromatography and heparin cofactor assays suggested that the primary target of lipid peroxides on the At III molecule may be the heparin binding site. Results from electrophoretic studies suggested that interaction between lipid peroxides and At III increased the negative charge of the At III molecule; however, no aggregation of the At III molecule was observed. Lipid peroxidation is being increasingly recognised as a factor in the pathogenesis of several disease states, and it is possible that local inhibition of At III by lipid peroxides could contribute towards the development of a thrombotic event.     

Inactivation of thrombin by antithrombin III on a heparinized biomaterial

Heparin covalently bonded to polyvinyl alcohol (PVA) is potentially useful as a nonthrombogenic coating in the preparation of small diameter vascular prostheses and blood sampling catheters. PVA-heparin is highly stable: the elution rate of ³⁵S-heparin from the polymer was determined to be negligible (approx. 2 × 10⁻¹¹ g/cm² min) when washed with either buffered saline (pH 7.4) or citrated human plasma. The inactivation of thrombin by antithrombin III was studied on PVA-heparin. Using small columns of PVA-heparin beads eluted by 0.14M NaCl buffered at pH 7.4 both thrombin and antithrombin III bound to the immobilized heparin. If thrombin was loaded before an excess of antithrombin III, significant inactivation of thrombin was observed; however, loading antithrombin III before thrombin did not measurably inactivate thrombin. The results suggest that the covalently-bound heparin effectively participates in the inactivation of thrombin through the formation of surface-bound heparin-thrombin, which then reacts with antithrombin III to yield a surface-bound thrombin-antithrombin III complex. The fate of this surface-bound complex has yet to be clarified.     

Prophylactic antithrombin III administration during pregnancy immediately reduces the thrombin hyperactivity of congenital antithrombin III deficiency by forming thrombin-antithrombin III complexes.

We examined the changes of haemostatic molecular markers after antithrombin III (AT III) administration in a 22-year-old woman with congenital AT III deficiency in the third trimester of pregnancy who did not have thrombosis. Various markers including fibrinopeptide A (FPA), thrombin-antithrombin III complex (TAT), prothrombin fragment F1 + 2 (F1 + 2), plasmin-alpha 2antiplasmin, D-dimer, beta-thromboglobulin, and platelet factor 4 were measured before and just after 3,000 U of AT III concentrate, which was given three times per week from the 34 week of pregnancy until delivery. Just after AT III administration, F1 + 2 and FPA levels decreased on most occasions, while TAT sometimes increased. Plasma FPA levels were markedly decreased on all 8 occasions when the plasma FPA levels was above 2.0 ng/ml before AT III administration. Plasma FPA levels were always greater than or equal to 6.4 ng/ml before AT III administration on the 4 occasions when TAT increased to above 115%. The changes of plasma F1 + 2 levels were significantly correlated with the AT III level. These results suggest that prophylactic AT III administration in the third trimester immediately inactivates intravascular thrombin to form TAT and reduce the plasma FPA level. Thus, the transient TAT elevation following AT III administration may not only be due to extraction of thrombin from the fibrin clots of thrombi but also to intravascular thrombin which is not attached to thrombi. FPA is the best molecular marker for thrombin hyperactivity and it should be monitored in AT III-deficient pregnant women in the third trimester.     

Abolition by dextran sulfate of the heparin-accelerated antithrombin III/thrombin reaction

Dextran sulfate did not inhibit the amidolytic activity of thrombin on Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide, but abolished inhibition of the enzyme with antithrombin III (AT III) in the presence of heparin.

Dextran sulfate did not bind to AT III and had less affinity than immobilized heparin for the protein. Dextran sulfate bound strongly to thrombin and had higher affinity than immobilized heparin for the enzyme.

These findings indicate that binding of dextran sulfate to a site other than the active site of thrombin to prevent the approach of AT III in the presence of heparin.     

Effect of rabbit thrombomodulin on thrombin inhibition by antithrombin in the presence of heparin

Thrombomodulin acts as a cofactor for protein C activation by thrombin (PC activation cofactor activity) and inhibits thrombin-induced fibrinogen clotting (direct anticoagulant activity). In addition, rabbit thrombomodulin has been shown to promote thrombin inactivation by antithrombin (AT-dependent anticoagulant activity). However, a non-acidic form (i.e. non-retarded on ion-exchange chromatography) of thrombomodulin generated by limited proteolysis retained only the PC activation cofactor activity. The acidic form (retarded on ion-exchange chromatography) of thrombomodulin is now shown to prevent the rapid inactivation of thrombin by antithrombin in the presence of heparin, presumably by preventing the formation of the ternary thrombin-AT-heparin complex. This effect was not observed with non-acidic thrombomodulin. When submitted to chondroitinase digestion, thrombomodulin was converted into an essentially non-acidic form that lacked both the AT-dependent and the direct anticoagulant activities but showed a PC activation cofactor function indistinguishable from that of native thrombomodulin. This chondroitinase-digested form did not prevent the catalytic effect of heparin on the inhibition of thrombin by AT. It is concluded that the acidic domain of rabbit thrombomodulin, a chondroitin (dermatan) sulfate glycosaminoglycan, interacts with a site of the thrombin molecule that is not involved in the protein C activation cofactor function, but is essential to the cleavage of fibrinogen or binding of heparin.     

Effect of NaC1 on inactivation of bovine thrombin by antithrombin III in the presence of low affinity-heparin or dextran sulfate

Heparin with low affinity (LA-heparin) to antithrombin III (AT III) enhanced the rate of inactivation of thrombin by AT III. The enhancement of the rate was saturable with AT III and was proportional to the LA-heparin concentration. Although the rate-enhancement in the presence of LA-heparin decreased with increase in NaC1 concentration, it was comparable with that in the presence of high affinity-heparin (HA-heparin) in the absence of NaC1. Inactivation of thrombin by AT III in the presence of dextran sulfate (DS) was also sensitive to NaC1 concentration. These findings indicate that free AT III is favorable for binding to the complexes of thrombin and highly sulfated polysaccharides having low affinities to AT III in the absence of NaC1.     

Inhibition of the activated Cls subunit of the first component of complement by antithrombin III in the presence of heparin

Antithrombin III, in the presence of heparin, inhibited the esterase activity of Cls. The rate of inhibition was dependent on the heparin concentration. The esterase activity of Cls was unaffected by α₁-antitrypsin.     

The effect of polycations on the inhibitory action of antithrombin III on thrombin in the absence and presence of polyanions

Polycations of biological and synthetic origin inhibit the action of AT III on thrombin activity. The effect is more pronounced with increasing molecular weight of branched polycations. Quantitatively protamine causes the same effect as quaternized polycations on the basis of charge equivalence. The accelerating effect of heparin or potassium polyvinylsulfate for the inhibitory action of AT III is abolished by charge equivalent amounts of polycation. The observations indicate a dual action of polycations in the heparin/AT III/thrombin interaction.     

The effect of human thrombomodulin on the inactivation of thrombin by human antithrombin III

The effect of human thrombomodulin (TM) on the inactivation of thrombin by human antithrombin III (ATIII) was evaluated in comparison with that produced from rabbits. Human TM did not accelerate the thrombin inhibition by ATIII but rabbit TM enhanced the activity of ATIII. Also inclusion of human TM at increasing concentration suppressed the thrombin inhibitory activity of ATIII. The intensity of ATIII activity in the presence of heparin (0.01U/ml) was also diminished by the human TM. However, this ATIII- heparin cofactor activity recovered with the addition of a 10-fold amount of heparin (0.1U/ml). In SDS-polyacrylamide gel electrophoresis and immunoblotting analysis, we found a complex formation of ATIII with both human and rabbit TM (and further confirmed their presence with isoelectrofocusing electrophoresis- data not shown). These results indicate that human TM is substantially different from rabbit TM. Our results suggest that human TM show the crucial role on protein C activation system via thrombin.     

Production of thrombin and antithrombin III by brain and astroglial cell cultures.

There is increasing evidence that some proteases and protease inhibitors are produced within the central nervous system. It has been proposed that the balance between these two classes of proteins may be an important modulator of brain cell growth and differentiation. Here we report that antithrombin III (ATIII) is produced in brain and primary astroglial cultures. In addition, we show that human astroglial cultures contain prothrombin mRNA, and secrete a thrombin-like protein that makes complexes with antithrombin III.     

Role of heparin in the inactivation of thrombin, factor Xa, and plasmin by antithrombin III

To characterize the mode of action of heparin, the kinetic behaviour of the inhibition of thrombin, factor Xa, and plasmin by antithrombin III was studied in the presence and absence of heparin. Following the concentration dependence of inactivation, in both cases a linear dependence of the apparent first-order inactivation rate constant on the antithrombin III concentration was found. These results are explained by enzyme-heparin interactions. Thus, heparin is believed to act as an activator of the enzymes that makes them more susceptible to antithrombin III.

Values of kinetic constants for the inactivation reaction of the several enzymes were determined. From these values it is concluded that heparin influences primarily the thrombin-antithrombin III interaction.     

Antithrombin III microheterogeneity in antithrombin III deficiency and in the antithrombin III abnormality, "antithrombin III Toyama"

Antithrombin III (AT III) microheterogeneity was investigated in 12 cases of congenital AT III deficiency and 2 cases of congenital AT III abnormality by isoelectric focusing (IEF) and immunofixation. In congenital AT III deficiency, IEF and immunofixation revealed AT III as 8 bands which was indistinguishable from normal control in terms of the number of bands and the isoelectric point (pI) of each band. In the proband of the congenital AT III abnormality, however, IEF and immunofixation showed AT III as 8 bands which shifted slightly but definitely to the acidic side compared to those of normal subjects. This change in pI of the abnormal AT III was considered to reflect the amino acid replacement in the polypeptide chain of the abnormal AT III molecule.     

Effect of thrombin and endotoxin on the in vivo metabolism of antithrombin III (AT III) in dogs

Effect of thrombin and endotoxin on the metabolism of I-125-labelled canine AT III was studied in mongrel dogs. Under control condition, mean total amount of intravascular AT III with standard deviation was 23.4 +/- 2.4 mg/kg, plasma half life of i.v. injected I-125-AT III was 1.7 +/- 0.2 days, and the fractional catabolic flux (j3x) was 16.3 +/- 1.6 mg/kg/day. The total amount of intra- and extra-vascular AT III was 36.0 +/- 0.34 mg/kg. Neither a 3 hour infusion of a small dose (30 units/kg/hr) of thrombin nor i.v. injection of a large amount of thrombin (5,000-15,000 units/day) with heparin significantly affected AT III metabolism except for a transient decrease in AT III concentration in the latter case, although decrease in plasma fibrinogen concentration and platelet count was observed in both cases. Two injections with 200 micrograms/kg of endotoxin resulted in an evident acceleration of AT III metabolism with significant decrease in the plasma AT III, fibrinogen concentrations and platelet count. More marked changes in AT III metabolism were induced by a single infusion with 1 mg/kg of endotoxin. Changes in hemostatic system coincided with those observed in DIC.