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Thrombin As an Anticoagulant Enrico Di Cera

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Thrombin As an Anticoagulant Enrico Di Cera Thrombin as an Anticoagulant Enrico Di Cera Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri, USA I. Preamble ...................................................................................... 146 II. Thrombin Interactions ..................................................................... 147 III. Thrombin Structure ........................................................................ 148 IV. Thrombin is an Allosteric Enzyme ...................................................... 151 þ V. Structures of E*, E, and E:Na .......................................................... 153 VI. Dissociating Procoagulant and Anticoagulant Activities ............................ 158 VII. WE: A Prototypic Anticoagulant Thrombin ........................................... 163 VIII. Molecular Mechanism of Anticoagulant Activity of Thrombin Mutants ........ 165 IX. Beyond WE .................................................................................. 170 X. Conclusions................................................................................... 175 References.................................................................................... 175 Thrombosis is the most prevalent cause of fatal diseases in developed countries. An antithrombotic agent that can be administered to patients with severe acute thrombotic diseases without the risk of causing hemorrhage, as experienced with antithrombotic/thrombolytic therapy in the treatment of acute ischemic stroke or systemic anticoagulants like heparin, would likely revolutionize the treatment of cardiovascular and cerebrovascular diseases. Thrombin remains at the forefront of cardiovascular medicine and a major target of antithrombotic and anticoagulant therapies, due to its involvement in thrombotic deaths. Heparins and direct thrombin inhibitors currently used in the treatment of acute thrombotic complications, especially in the venous circulation, are plagued by complications related to dosage and bleeding. A new strategy of intervention has been proposed in recent years aiming at modulating, rather than inhibiting, thrombin function. Specifically, efforts have been directed toward finding ways of exploiting the anticoagulant function of thrombin unleashed by the activation of protein C, either using small modula- tors or protein engineering. The ability of thrombin to activate protein C coexists with its procoagulant and prothrombotic functions, mediated re- spectively by cleavage of fibrinogen and the protease-activated receptor 1 (PAR1). A strategy that inhibits thrombin at the active site abrogates the procoagulant and prothrombotic functions, but also shuts down activity toward the anticoagulant protein C. On the other hand, a strategy that selectively compromises fibrinogen and PAR1 recognition may take advantage of the anticoagulant and cytoprotective functions of activated protein C and prove Progress in Molecular Biology Copyright 2011, Elsevier Inc. and Translational Science, Vol. 99 145 All rights reserved. DOI: 10.1016/S1877-1173(11)99004-8 1877-1173/11 $35.00 146 ENRICO DI CERA of interest for in vivo applications. This chapter summarizes current protein engineering efforts to convert thrombin into a potent and safe anticoagulant for in vivo applications. I. Preamble Although substantial progress has been made in the prevention and treatment of cardiovascular disease and its major risk factors, it has been predicted that thrombotic complications will remain the leading cause of death and disability and will represent a major burden to productivity worldwide well into the year 2020.1 Indeed, thrombosis is the most prevalent cause of fatal diseases in devel- oped countries. More than 80% of stroke cases are of thrombotic origin, and stroke is among the three leading causes of mortality and severe chronic disability in the US.2 One of the causal treatment options for acute ischemic stroke is early antithrombotic/thrombolytic therapy. A critical problem with such therapy is that the currently available potent antithrombotic agents, most notably tissue-type plasminogen activator, are not specific for thrombosis. They disable the hemo- static system at their most efficacious doses and, for this reason, cannot be administered at their fully effective doses.3 Systemic anticoagulants like heparin prevent comorbidity from deep vein thrombosis and reduce the progression of acute cerebral thrombosis. However, their bleeding side effects in the acute phase outweigh the benefits.4 An antithrombotic agent that can be administered to patients with severe acute thrombotic diseases, such as heart attack and stroke, without the risk of causing hemorrhage, would be so significant as to revolutionize the treatment of cardiovascular and cerebrovascular diseases. Progress in the developments of new anticoagulants and antithrombotics, therefore, remains a top medical and social priority and has the potential to impact the lifestyle and life expectancy of millions of people worldwide. Due to its involvement in thrombotic deaths, thrombin remains at the forefront of cardiovascular medicine and a major target of antithrombotic and anticoagulant therapies.5 Because heparin and direct thrombin inhibitors are plagued with complications related to dosage and bleeding,4,5 an entirely new strategy of intervention has been proposed in recent years to take advan- tage of the so-called protein C anticoagulant pathway.6 Protein C requires thrombin for activation and activated protein C acts as a potent anticoagulant and cytoprotective agent.6,7 A thrombin mutant engineered for exclusive activ- ity toward protein C and devoid of activity toward fibrinogen and the platelet receptor PAR1 could represent an innovative and potentially powerful tool to achieve anticoagulation without disruption of the hemostatic balance. THROMBIN AS AN ANTICOAGULANT 147 Proof-of-principle that such a strategy could benefit the treatment of thrombotic diseases has come from in vivo studies in nonhuman primates.8–11 Specifically, the anticoagulant thrombin mutant, W215A/E217A (WE), has been shown to elicit an antithrombotic effect that is more efficacious than the direct adminis- tration of activated protein C and safer than the administration of low molecu- lar weight heparins.10 Substantial room for optimization remains. The procoagulant activity of the mutant can be abrogated by further protein engi- neering to eliminate any possible side effects due to fibrinogen clotting or platelet aggregation. This problem presents a challenging task for basic science with tremendous relevance to the medical community. Developments toward the goal of dissociating thrombin functions and optimizing its anticoagulant activity toward protein C are the main focus of this chapter. II. Thrombin Interactions Blood coagulation is initiated by the exposure of tissue factor that forms a complex with factor VIIa and results in the generation of small quantities of factors IXa and Xa.12,13 The small quantities of Xa generate minute concentra- tions of thrombin that result in the activation of factor XI and the cofactors VIII and V. At this point, the VIIIa–IXa complex generates sufficient quantities of Xa þ to form the prothrombinase complex, composed of factors Va, Xa, Ca2 , and phospholipids, which leads to the explosive generation of thrombin from prothrombin.14 Thrombin is a trypsin-like protease endowed with important physiological functions that are mediated and regulated by interaction with numerous macromolecular substrates, receptors, and inhibitors.15–18 Activity of the enzyme toward synthetic and physiological substrates is enhanced þ allosterically by the binding of Na to a site located > 15 A˚ away from residues þ of the catalytic triad H57, D102, and S19519,20 (Fig. 1). Na activation is present in all vitamin K-dependent clotting enzymes and many complement þ factors.21 The effect of Na is seen not only on cleavage of fibrinogen and PAR1,22–24 the primary procoagulant and prothrombotic substrates,16,25,26 but also on PAR3 and PAR415,27 and activation of factors V,28 VIII,29 and XI30 that ensure the build-up of coagulation factors responsible for the explosive gener- þ ation of thrombin from prothrombin.14,31 Importantly, Na binding has no effect on protein C activation in the absence or presence of thrombomodu- þ lin,23,24 which makes Na an exquisite procoagulant/prothrombotic cofactor of þ thrombin. Evidence that Na plays an important role in blood coagulation through its specific interaction with thrombin comes from the observation that several naturally occurring mutations, like prothrombin Frankfurt (E146A),32 Salakta (E146A),33 Greenville (R187Q),34 Scranton (K224T),35 Copenhagen þ (A190V),36 and Saint Denis (D221E),37 affect residues linked to Na binding20 148 ENRICO DI CERA Exosite II D102 H57 60 loop W215 S195 Exosite I D189 W141 Na+ site Autolysis loop þ 20 FIG. 1. The structure of thrombin in the E:Na form (PDB ID code 1SG8 ) rendered as a ribbon in spectrum color. The enzyme is composed of two polypeptide chains of 36 (A chain) and 259 (B chain) residues that are covalently linked through a disulfide bond.17,44 In this standard orientation,44 the A chain (blue) runs in the back of the molecule, opposite to the front hemisphere of the catalytic B chain. Catalytic residues (H57, D102, S195) and the S1 site (D189) are labeled, þ along with the two major fluorophores, W215 and W141. The bound Na (red ball) is 15 A˚ away from the residues of the catalytic triad and within
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