Thrombin generation and formation of thrombin-antithrombin III complexes in congenital antithrombin III deficiency

Two families with quantitative congenital AT III deficiency and a high incidence of thromboembolism are reported. In two unrelated patients (one from each family) thrombin generation in whole blood occured more rapidly than in the control, as demonstrated by the kinetics of prothrombin consumption, AT III disappearance and thrombin-antithrombin III complexes formation. Similar results were obtained in plasma and can be experimentally reproduced with a plasma depleted of AT III by immunoadsorption using a rabbit anti-AT III antiserum. The addition of purified AT III in vitro leads to a complete correction of the abnormalities when the level of AT III is greater than 0.8 unit/ml.     

Management of anti-thrombin III deficiency during pregnancy without administration of anti-thrombin III

We report a patient with hereditary antithrombin III deficiency who was successfully treated with heparin throughout pregnancy. Functional antithrombin III levels fell to 0.32 U/ml during heparin treatment, but it was possible to achieve a heparin effect, measured by the activated partial thromboplastin time, thrombin clotting time and heparin assay with subcutaneous heparin in doses of 30,000 U to 35,000 U/24 hours. This achieve an long term heparin effect was obtained without the need for antithrombin III infusions.     

Treatment of venous thromboembolism in patients with congenital deficiency of antithrombin III.

The treatment course of all thromboembolic events in the patients with congenital deficiency of antithrombin III (AT III) in the national Swedish register was reviewed in order to assess the appropriate medical therapy in this situation. The medical treatment of 70 events of venous thromboembolism was evaluated. There were eight cases with signs of clinical progression. The risk of therapeutic failure with heparin could be as low as 1.5% or as high as 9.2%. It would not be cost-effective to substitute with concentrates of AT III in every case with congenital deficiency thereof in connection with acute venous thromboembolism. "Heparin resistance" does not seem to be a problem in the vast majority of these patients.     

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.     

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.     

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.     

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.     

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.     

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.     

Plasma antithrombin III deficiency in ischaemic stroke in the young.

A deficiency of plasma antithrombin III has been identified as a potential risk factor for thrombosis. In a pilot study of 56 patients aged less than 40 years who presented with ischaemic stroke of unknown etiology, we detected only one case of plasma antithrombin III deficiency. Antithrombin III activity was estimated by a chromogenic assay. Hence, antithrombin III deficiency, though rare, should be considered while evaluating young patients with stroke of unknown etiology.     

Management of pregnancy in women with antithrombin III congenital defect: report of four cases

Four pregnant women with antithrombin III congenital deficiency underwent thrombosis prophylaxis including oral anticoagulants administered from the 16-18th week to the 36-37th week of pregnancy, subcutaneous heparin before the 16-18th week and after the 36-37th week, and a single infusion of AT III concentrate in the peripartum period in order to obtain a minimal level of 0.8 U/ml of AT III functional activity. The level of circulating AT III after the concentrate infusion needs to be evaluated by functional methods, because of a consistent amount in the concentrates of inactive AT III immunoreactive material. No thrombotic or haemorrhagic complication occurred after starting prophylaxis in any woman either in any newborn.     

Further studies of the turnover of dog antithrombin III. Study of 131I-labelled antithrombin protease complexes

Fresh plasma containing 131I-antithrombin III (*I-AT) was coagulated and incubated at 37 degrees C for 2 hr. A "complex peak," separated on heparin-agarose contained AT and *I-AT antigen but no heparin cofactor activity. Crossed immunoelectrophoresis showed only AT complexes. SDS PAGE showed 80% of the *I-AT in a major band (approximately 80,000 daltons), 15% in a minor band (approximately 100,000 daltons) and the rest in trace bands (approximately 60,000 and/or 115,000 daltons). Ammonia treatment of the complex peak released alpha-thrombin. After i.v. injection 80% of the complexed *I-AT, chiefly as the major band, left the plasma with t 1/2 approximately 15 min and was almost immediately catabolized to low molecular weight breakdown products. A major catabolic site was the liver. A simple kinetic model describes the findings approximately.     

Quantitative and qualitative congenital deficiency of antithrombin III: a new molecular variant called ATIII-Barcelona 2

A Spanish family with a quantitative-qualitative antithrombin III (ATIII) deficiency and thrombotic tendency is reported. The qualitative defect was suggested by the crossed immunoelectrophoresis (CIE) in the presence of heparin in plasma of all those affected. However, the crossed immunoelectrofocusing (CIEF) showed the same ATIII pattern in controls and affected members. Two populations of ATIII were detected by affinity chromatography on heparin-sepharose from affected members' plasma. The ATIII unbound to sepharose beads was devoid of heparin cofactor activity and showed a lack of anodal migration in CIE in the presence of heparin. The ATIII eluted corresponded to normal ATIII. Our data supports the view that an abnormal ATIII molecule is present in all affected family members in the heterozygous state. This is the first reported ATIII variant in which a molecular abnormality produces a lack of affinity for heparin but no changes in its isoelectric point. This familial ATIII deficiency was named ATIII- Barcelona 2.