Thrombin-antithrombin complex inactivation

Converse to UFH, LMWH action is primarily directed against factor Xa, because most of the chains are not sufficiently long to form the ternary complex necessary for the inactivation of thrombin (<50% of the chains contain at least the 18 saccharide units needed for the formation of the ternary heparin-thrombin-antithrombin complex) (Fig. IC). Depending on the LMWH, the antithrombin-anti-Xa activity ratio varies from 1.9 (tinzaparin) to 3.8 (enoxaparin) (2). 

H.K. Lau and R.D< Rosenberg, The inactivation and characterization of a specific antibody population directed against the thrombin-antithrombin complex, J. Biol. Chem. 255 5885 (1980). 

In addition, UFH also binds simultaneously to fibrin and thrombin. The heparin-thrombin-fibrin complex lessens the ability of the heparin-antithrombin complex to inhibit thrombin and increases the affinity of thrombin for its fibrin substrate. This results in a protection of fibrin-bound thrombin from inactivation by the heparin-antithrombin complex... 

Heparin acts indirectly at multiple points within the coagulation cascade, Its major anticoagulant effect is via interaction with its requisite co-factor, antithrombin III (AT). The heparin-AT complex inactivates factors IXa, Xa, and XIla, and binds thrombin at its active site to prevent the conversion of fibrinogen to fibrin (3). Heparin also prevents fibrin stabilization through the inhibition of fibrin stabilization factor. Heparin has no fibrinolytic activity and therefore is ineffective as a thrombolytic (4,5). 

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

Factors Ila and Xa are the most sensitive to inhibition by the UFH-antithrombin complex. In order to inactivate thrombin, the heparin molecule must form a ternary complex bridging between antithrombin and thrombin (see Fig. 19-5). Only molecules thatcontain more than 18 saccharides are able to bind to both antithrombin and thrombin simultaneously. Smaller heparin molecules cannot facilitate... 

Displacement by plasma of radiolabeled thrombin and radio-labeled thrombin-antithrombin III inactive complex from a heparinized surface was measured and found to be significant for example, removing 63% of the thrombin and 90% of the complex that could not be removed by phosphate-buffered saline alone. Heparin-poly(vinyl alcohol) (PVA) gel beads with a very low heparin release rate, prepared by acetal coupling of the heparin to the PVA, adsorbed thrombin and potentiated the inactivation of thrombin by antithrombin 111, as measured by both thrombin time and chromogenic substrate assays. 

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. 

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. 

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). 

Analysis of the radioactivity displaced by arvinized plasma indicated the presence of thrombin-anti thrombin III complex (- 70%) and what was presumed to be thrombin-a-2-macroglobulin complex (" 30%). The bound thrombin is thought to react first with antithrombin III to produce a bound inactivated thrombin-anti thrombin III complex, which is dislodged from heparin by a yet unknown plasma component(s), decomplexed by an unknown mechanism to react with a-2-macroglobulin. This mechanism is illustrated in Figure 1. After displacement, the increase in I-antithrombin III which had lost its affinity for heparin-Sepharose was attributed to the production of a post complex antithrombin III on decomplexation of the inactive complex. This modified antithrombin III has been described by Lam et al. ( ), Fish et al. (25) and Marciniak (26). Neither free I-thrombin nor I-antithrombin III were detected in the displaced eluent. 

UFH binds to exosite 2, located on antithrombin, forming a ternary complex. This ternary complex is necessary for the inhibition of thrombin by antithrombin (Fig. I A, left). Conversely to thrombin inhibition, inactivation of factor Xa does not require the formation of the ternary complex. UFH inhibits thrombin and factor Xa in the same proportion (the ratio of anti-Xa/lla activity equals I) (Fig, I A, right). The interaction of the heparins (UFH and LMWH) with antithrombin is mediated by a unique pentasaccharide sequence, which is present in approximately one-third of the UFH chains (2). 

CHAPTER 36, FIGURE 16 Proteinase inactivation by SERPINS. Proteinase inactivation occurs by reaction between proteinase and inhibitor, e.g., antithrombin. The proteinase is a stoichiometric reactant in this instance but is not a catalyst. This reaction results in the formation of a covalent bond between the reactive site residue of the inhibitor (Arg in antithrombin) and the active site residue (Ser in the proteinase). This complex formation prevents the proteinase from hydrolyzing any other peptide bond. Proteinases, thrombin, factor Xa, factor IXa, and, less effectively, factor Vila and factor XIa are all inactivated by the plasma protein inhibitor antithrombin (previously designated antithrombin III). The product AT is the cleaved form of antithrombin. It is formed in both the absence and presence of heparin, but more so in the presence. The proteinase is indicated is indicated by green, the inhibitor by red, and the inactivated proteinase by gray. Stripes on the inhibitor represent the helices (Figure 36-7). 

Specific inhibitors of clotting factors are also critical in the termination of clotting. For instance, tissue factor pathway inhibitor (TFPI) inhibits the complex ori F-VII —Xg. Separate domains in 1 FPl inhibit VII3 and X,j. Another key inhibitor is antithrombin Ilf a plasma protein that inactivates thrombin by forming an irreversible complex with it. Antithrombin III resembles... 

Small amounts of thrombin are formed all the time but are normally inactivated by formation of a complex with antithrombin III in the presence of heparin. 

Careful examination of the possible fates of the bound heparin and the bound inactive complex suggests hypothetical mechanisms which, if effective, could limit the utility of heparinized materials (Table I). For example, either the surface may become saturated with a heparin complex that can no longer accelerate the inactivation of thrombin, or consumption of antithrombin, prothrombin, or other clotting factors may result, leaving the blood systematically hypocoagulable. 

Mechanism and effects Regular heparin binds to and activates endogenous antithrombin 111 (ATlIl). The heparin-ATIll complex combines with and inactivates thrombin (activated factor 11) and several other factors, especially factor X. In the presence of heparin, antithrombin III inhibits the coagulation factors approximately 1000-fold faster than in its absence. Low doses of heparin also coat the endothelial walls of vessels and reduce the activation of clotting elements by these cells. Because it acts on preformed blood components, heparin is also active in vitro—almost instantaneously. The action of heparin is monitored with the activated partial thromboplastin time laboratory test (aPTT or PTT). 

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