Parenteral anticoagulants: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines.

This article describes the pharmacology of approved parenteral anticoagulants. These include the indirect anticoagulants, unfractionated heparin (UFH), low-molecular-weight heparins (LMWHs), fondaparinux, and danaparoid, as well as the direct thrombin inhibitors hirudin, bivalirudin, and argatroban. UFH is a heterogeneous mixture of glycosaminoglycans that bind to antithrombin via a unique pentasaccharide sequence and catalyze the inactivation of thrombin, factor Xa, and other clotting enzymes. Heparin also binds to cells and plasma proteins other than antithrombin causing unpredictable pharmacokinetic and pharmacodynamic properties and triggering nonhemorrhagic side effects, such as heparin-induced thrombocytopenia (HIT) and osteoporosis. LMWHs have greater inhibitory activity against factor Xa than thrombin and exhibit less binding to cells and plasma proteins than heparin. Consequently, LMWH preparations have more predictable pharmacokinetic and pharmacodynamic properties, have a longer half-life than heparin, and are associated with a lower risk of nonhemorrhagic side effects. LMWHs can be administered once daily or bid by subcutaneous injection, without coagulation monitoring. Based on their greater convenience, LMWHs have replaced UFH for many clinical indications. Fondaparinux, a synthetic pentasaccharide, catalyzes the inhibition of factor Xa, but not thrombin, in an antithrombin-dependent fashion. Fondaparinux binds only to antithrombin. Therefore, fondaparinux-associated HIT or osteoporosis is unlikely to occur. Fondaparinux exhibits complete bioavailability when administered subcutaneously, has a longer half-life than LMWHs, and is given once daily by subcutaneous injection in fixed doses, without coagulation monitoring. Three additional parenteral direct thrombin inhibitors and danaparoid are approved as alternatives to heparin in patients with HIT.

Designing nonsaccharide, allosteric activators of antithrombin for accelerated inhibition of factor Xa.

Antithrombin is a key regulator of coagulation and prime target of heparins, clinically used anticoagulants. Heparins induce a two-step conformational activation of antithrombin, a process that has remained challenging to target with molecules devoid of the antithrombin-binding pentasaccharide DEFGH. Computational screening of a focused library led to the design of two tetra-sulfated N-arylacyl tetrahydroisoquinoline variants as potential nonsaccharide activators of antithrombin. A high yielding synthetic scheme based on Horner-Wadsworth-Emmons or Pictet-Spengler reactions was developed to facilitate the functionalization of the tetrahydoisoquinoline ring, which upon further amidation, deprotection, and sulfation gave the targeted nonsaccharide activators. Spectrofluorometric measurement of affinity displayed antithrombin binding affinities in the low to high micromolar range at pH 6.0, I 0.05, 25 °C. Measurement of second-order rate constants of antithrombin inhibition of factor Xa in the presence and absence of the designed activators showed antithrombin activation in the range of 8-80-fold in the pH 6.0 buffer. This work puts forward 20c, a novel tetra-sulfated N-arylacyl tetrahydroisoquinoline-based molecule, that activates AT only 3.8-fold less than that achieved with DEFGH, suggesting a strong possibility of rationally designing sulfated organic molecules as clinically relevant AT activators.

Antithrombin activation by nonsulfated, non-polysaccharide organic polymer.

Accelerated antithrombin inhibition of procoagulant enzymes has been exclusively achieved with polysulfated polysaccharides. We reasoned that antithrombin activation should be possible with nonsulfated activators based only on carboxylic acid groups. As a proof of the principle, linear poly(acrylic acid)s were found to bind to antithrombin and accelerate inhibition of factor Xa and thrombin. Our work demonstrates that molecules completely devoid of sulfate groups can activate antithrombin effectively and, more importantly, suggests that it may be possible to develop orally bioavailable, carboxylate-based antithrombin activators.

Heparin-activated antithrombin interacts with the autolysis loop of target coagulation proteases.

A unique pentasaccharide fragment of heparin can enhance the reactivity of antithrombin with coagulation proteases factors IXa and Xa by 300- to 600-fold through a conformational activation of the serpin, without having a significant effect on the reactivity of antithrombin with thrombin. In this study, it was hypothesized that differences in the structure of the autolysis loop of coagulation proteases (residues 143-154 in chymotrypsin numbering) may be responsible for their differential reactivity with the native and heparin-activated antithrombin. To test this hypothesis, the autolysis loops of both thrombin and the anticoagulant serine protease-activated protein C were replaced with the corresponding loop of factor Xa. Inhibition studies revealed that in contrast to the approximately 1.5-fold difference in the reactivity of thrombin with antithrombin in the absence and presence of pentasaccharide, the difference in reactivity was increased to approximately 37-fold for the mutant thrombin. In the case of the activated protein C mutant, similar to factor Xa, pentasaccharide accelerated the reaction 375-fold. These results suggest that structural differences in the autolysis loop of coagulation proteases play a key role in their differential reactivity with the native and heparin-activated conformations of antithrombin.

Designing small, nonsugar activators of antithrombin using hydropathic interaction analyses.

Conformational activation of antithrombin is a critical mechanism for the inhibition of factor Xa, a proteinase of the blood coagulation cascade, and is typically achieved with heparin, a polyanionic polysaccharide clinically used for anticoagulation. Although numerous efforts have been directed toward the design of better activators, a fundamental tenet of these studies has been the assumed requirement of an oligo- or a polysaccharide backbone. We demonstrate here a concept that small nonsaccharidic nonpolymeric molecules may be rationally designed to interact with and activate antithrombin for enhanced inhibition of factor Xa. The rational design strategy is based on a study of complexes of natural and mutant antithrombins with heparin-based oligosaccharides using hydropathic interaction (HINT) technique, a quantitative computerized tool for analysis of molecular interactions. A linear correlation was observed between the free energy of binding for antithrombinminus signoligosaccharide complexes and the HINT score over a wide range of approximately 13 kcal/mol, indicating strong predictive capability of the HINT technique. Using this approach, a small, nonsugar, aromatic molecule, (minus sign)-epicatechin sulfate (ECS), was designed to mimic the nonreducing end trisaccharide unit DEF of the sequence specific heparin pentasaccharide DEFGH. HINT suggested a comparable antithrombin-binding geometry and interaction profile for ECS and trisaccharide DEF. Biochemical studies indicated that ECS binds antithrombin with equilibrium dissociation constants of 10.5 and 66 microM at pH 6.0, I 0.025, and pH 7.4, I 0.035, respectively, that compare favorably with 2 and 80 microM observed for the natural activator DEF. ECS accelerates the antithrombin inhibition of factor Xa nearly 8-fold demonstrating for the first time that conformational activation of antithrombin is feasible with appropriately designed small nonsugar organic molecules. The results present unique opportunities for de novo activator design based on this first-generation lead.

Conformational changes in serpins: II. The mechanism of activation of antithrombin by heparin.

Antithrombin, uniquely among plasma serpins acting as proteinase inhibitors in the control of the blood coagulation cascade, circulates in a relatively inactive form. Its activation by heparin, and specifically by a pentasaccharide core of heparin, has been shown to involve release of the peptide loop containing the reactive centre from partial insertion in the A sheet of the molecule. Here we compare the structures of the circulating inactive form of antithrombin with the activated structure in complex with heparin pentasaccharide. We show that the rearrangement of the reactive centre loop that occurs upon activation is part of a widespread conformational change involving a realignment of the two major domains of the molecule. We also examine natural mutants that possess high affinity for heparin pentasaccharide, and relate the kinetics of their interaction with heparin pentasaccharide to the structural transitions occuring in the activation process.