Bound thrombin-antithrombin

Table II. Displacement of Bound Thrombin-Antithrombin III Experimental Parameters...

These agents interact directly with thrombin and do not require antithrombin to have antithrombotic activity. They are capable of inhibiting both circulating and clot-bound thrombin, which is a potential advantage over UFH and the LMWHs. They also do not induce immune-mediated thrombocytopenia and are widely used for the treatment of HIT. 

Weitz JI, Hudoba M, Messel D, Maraganore J, Hirsh J. (1990) Clot-bound thrombin is protected from inhibition by heparin-antithrombin 111 but is susceptible to inactivation by antithrombin Ill-independent inhibitors. J Clin Invest 86 385-391. 

Heparin binds to antithrombin III and induces a conformational change that accelerates the interaction of antithrombin III with the coagulation factors. Heparin also catalyzes the inhibition of thrombin by heparin cofactor II, a circulating inhibitor. Smaller amounts of heparin are needed to prevent the formation of free thrombin than are needed to inhibit the protease activity of clot-bound thrombin. Inhibition of free thrombin is the basis of low-dose prophylactic therapy. 

Micromedex, lepirudin directly inhibits all actions of thrombin. It inhibits free and clot-bound thrombin without requiring endogenous cofactors. Lepirudin is not inhibited by platelet factor 4 and acts independently of antithrombin III and heparin cofactor II. It has no direct effect on platelet function, except inhibition of thrombin-induced platelet activation. No physiological inhibitor of lepirudin is known. 

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... 

Fondaparinux is a chemically synthesized pentasaccharide that mimics the antithrombin-binding site of heparin and LMWH. Its molecular size (1728Da) is too small to bind to thrombin molecules while it is bound to antithrombin, Therefore, it is a pure anti-Xa inhibitor. It binds very little to platelets, proteins, or endothelium and is excreted in the urine, It does not form a complex with PF4 or other positively charged molecules. It is not neutralizable by protamine sulfate, Recent clinical trials have resulted in FDA approval for prophylaxis of deep vein thrombosis in orthopedic surgery, It has been shown to be effective and safe for the treatment of pulmonary embolism (20,21) and ACS (non-ST-elevation Ml) (OASIS 5—Michelangelo Trial) (17). 

Direct inhibitors of thrombin inactivate fibrin-bound thrombin which may promote thrombus extension (as opposed to heparin which acts indirectly through antithrombin) as follows ... 

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. 

Desorption of Bound Thrombin by Antithrombin III, Heparin or Hep-AT III Complex. Thrombin was injected onto PAOM or PSSO chromatographic supports in 0.1 M NaCl solution. To minimize possible kinetic effects, the flow-rate was reduced to 0.2 ml/min. After washing with 0.1 M NaCl buffer, an excess of either AT III, Hep, or AT Ill-Hep complex was injected. The flow was then stopped for five minutes in order to obtain a better exchange of macromolecules between the mobile and the stationary phases. The objective was to determine whether AT III, Hep or AT Ill-Hep complex could act as eluents for the "biospecific desorption" of the bound enzyme at 0.1 M NaCl. 

The comparison between the chromatogram of Th-AT complex (Figure 4A) and the desorption of bound thrombin by antithrombin III from the PAOM resins (Figure 5A) indicates that the active... 

Figure 5. Desorption of bound thrombin from PAOM (A) by AT III, Hep and AT Ill-Hep and from PSSO resins by antithrombin III (B), AT Ill-Hep complex (C) and heparin (D), at 25 °C. Eluent 0.05 M phosphate buffer (pH = 7.4), NaCl 0.1 M and 2 M, flow rate 0.2 ml/min.

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. 

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 has the disadvantages of a variable anticoagulant effect, sensitivity to platelet factor 4, a relative inability to inhibit clot-bound thrombin, and the potential to cause thrombocytopenia and/or heparin-induced thrombocytopenia syndrome (28). Low molecular weight hep-arins (LMWHs) and direct thrombin inhibitors are the antithrombins... 

Because clot-bound thrombin is less effectively inhibited by UFH (attachment of fibrin to the fibrin-binding domain makes the heparinbinding domain inaccessible), it was proposed that the direct antithrombins had an advantage over UFH in STEMI patients treated with a fibrinolytic because of their greater ability to block both fluid-phase and clotbound thrombin. This thrombin hypothesis was the basis for several randomized trials. After the TIMI 9A, GUSTO Ila, and HIT 3 trials that tested hirudin as an adjunct to fibrinolytic therapy were... 

The results confirmed that sulfated N-carboxymethyl chitosan bound to antithrombin inhibits the thrombin present according to the following reactions ... 

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