Antithrombin III complex

Schreuder, H.A., et al. The intact and cleaved human antithrombin III complex as a model for serpin-proteinase interactions. Nature (Struct. Biol.)... 

With respect to both the coagulation and fibrinolytic cascade systems, in 28 patients who developed septic shock a relation was found between lowered plasma levels of F-XII and antithrombin III and elevated levels of PAI-1 and thrombin-antithrombin III complexes at the diagnosis of sepsis and the severity of disease, expressed according to the APACHE II scoring system (L7). Nevertheless, administration of inhibitors of coagulation or enhancement of fibrinolysis did not improve the outcome in patients with sepsis (B35). 

Ikada and coworkers also studied the blood compatibility and protein denaturation properties of heparin covalently and ionically bound onto polymer surfaces [513], Both types of bound heparin gave deactivation of the coagulation process. Clotting deactivation was attributed to a heparin/ antithrombin III complex by covalently bound heparin which gave adsorbed protein denaturation and platelet deformation as compared with lack of these features with ionically bound heparin. 

Sakamoto S, Hirase T Suzuki S, et al. Inhibitory effect of argatroban on thrombin-antithrombin III complex after percutaneous transluminal coronary angioplasty. Thromb Haemost 1995 74 801-802. 

Inactive factors [Note While the heparin-antithrombin III complex readily inactivates thrombin, the complex of low molecular weight heparin with... 

A study has been carried out on the interactions of blood with plasticised poly(vinyl chloride) biomaterials in a tubular form. The influence of different factors such as the biomaterial, antithrombotic agent, blood condition and the nature of the application is represented when considering the blood response in the clinical utilisation of the plasticised PVC. The PVC was plasticised with di-(2-ethylhexyl)phthalate (DEHP) and tri-(2-ethylhexyl)trimellitate (TEHTM)and in-vitro and ex-vivo procedures used to study the biomaterial with respect to the selection of the plasticiser. The blood response was measured in terms of the measurement of fibrinogen adsorption capacity, thrombin-antithrombin III complex and the complement component C3a. X-ray photoelectron spectroscopy was used for surface characterisation of the polymers and the data obtained indicated that in comparison with DEHP-PVC, there is a higher reactivity... 

Heparin/antithrombin III complexes react also with various other factors of the coagulation/fibrinolysis cascades... 

These results indicate that surface bound heparin retains its biological activity after immobilization and does not become saturated with inactive thrombin-antithrombin III complex. 

Concern over the fate of the bound complex appears unnecessary, since radiolabeled thrombin or thrombin-antithrombin III complexes were readily displaced from the surfaces by defibrinated plasma containing crude antithrombin III. Therefore, the bound heparin apparently does not become saturated with inactive complex, enabling the bound heparin, if it remains active, to act catalytically to potentiate the inactivation of thrombin as it is generated. Whether the consumption rate of antithrombin III or prothrombin, on the other hand, can be controlled, or whether it would result in a systemic blood defect, remains to be examined. 

The results reported here, in conjunction with earlier results, indicate that immobilized heparin need not necessarily be lost from a surface in order to impart thromboresistance to that surface. For heparin-PVA, and perhaps for other covalent reactions that do not inactivate the heparin, the irreversibly bound heparin can accelerate the formation of a surface-bound inactive thrombin-antithrombin III complex. Furthermore, our results suggest that the inactive complex is not itself permanently bound to the surface, but rather can be displaced by a component or components in plasma. 

Stepwise Elution of Th-AT III Complex. It has been widely reported that thrombin inhibition by antithrombin III requires a number of critical amino acid residues in each of the two proteins (11,20). These binding sites are essentially an active-center serine of the enzyme and an arginyl residue of the inhibitor. In addition, the inherently slow formation of thrombin-antithrombin III complex is accelerated by heparin (J ). Although the mechanism of this catalysis is still under investigation (J l,, ), it has been shown that the binding sites involved in the thrombin-antithrombin III interaction differ from those of heparin for the two proteins (, , 25). 

Characterization of Displaced Protein. Two approaches were used simultaneously to characterize the I-thrombin displaced from heparin-PVA columns filtration on Sephadex G-200 and heparin-Sepharose affinity chromatography using the elution conditions described by Collen et al. (18) for the separation of antithrombin III from enzyme-antithrombin III complex. Radiolabelled antithrombin III displaced from the heparin-PVA column was characterized in a similar way by affinity chromatography on heparin-Sephrose. Detailed methods are presented elsewhere ( ... 

Radiolabelled purified bovine thrombin, adsorbed to heparin-PVA, was exposed to a variety of eluents. Compared with PBS, all eluents were effective in displacing some of the bound thrombin (or thrombin-antithrombin III complex) although arvinized plasma was the most successful in this respect. 

Characterization of Displaced Protein. With labelled antithrombin III, chromatography of the displaced radioactivity on heparin-Sepharose revealed that the bulk of the displaced radioactive material did not bind to heparin-Sepharose (Table II). With arvinized plasma as the displacing eluent, 65% of the antithrombin III eluted in the void volume, compared with 49% of the control I-antithrombin III (diluted in citrated plasma) that had not previously been used to inactivate thrombin the latter unbound fraction was likely labelled impurities or inhibitor modified by radiolabelling to lose its heparin affinity. With 5% (w/v) albumin used as a displacing eluent, 78% of the I-antithrombin III came out in the void volume. This increase in material that did not bind to heparin after displacement from heparin-PVA was attributed to post-complex antithrombin III, a modification of the original inhibitor resulting from the inactivation of thrombin. Neither thrombin-antithrombin III complex nor free antithrombin III were detected in the 5% (w/v) albumin displaced fractions while there was a barely detectable amount of complex (6%) and free antithrombin III (4%) in the material displaced by arvinized plasma. With the control I-antithrombin III, 25% of the radioactivity was determined to be free antithrombin III and 2% as complex. The remainder (22-27%) was not recovered from the column. 

By using the reverse loading sequence thrombin could interact with heparin/antithrombin III complex or with heparin alone. The colour yield obtained was similar in this sequence to that obtained by only thrombin loading, indicating that none of the thrombin on heparin-PVA gel had been neutralized by the heparin/antithrombin III complex. 

However, it is clear that thrombin adsorbed by PVA-heparin is biologically active and is inactivated by antithrombin III presumably through the formation of a surface-bound heparin-thrombin-antithrombin III complex. Furthermore, the biological activity of the heparinized gel and the mechanism of thrombin inactivation by antithrombin III on heparin-PVA have been verified using clotting assays (3). 

Ill CR, Ruoslahti E. Association of thrombin-antithrombin III complex with vitronectin in serum. J Biol Chem 1985 260 15610-15615. 

Figure 13.10. Thrombin-antithrombin III complex, TAT, concentration versus time of contact with PVC containing two plasticizers (di-(2-ethylhexyl) phtha-late, DOP, and tri-(2-ethylhexyl) trimellitate, TOM). [Adapted, by permission, from Yin FI Q Zhao X B Courtney J M Blass C R West R FI Lowe G D O, J. Mater. Sci. Mater Medicine, 10, No.9, Sept. 1999, p.527-31.]...

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