A study of the anticoagulant mechanisms of three biological compounds in vitro and in vivo

Abstract

Arterial (e.g. atrial fibrillation and acute coronary syndrome) and venous thromboembolic diseases (e.g. deep vein thrombosis and pulmonary embolism) are highly prevalent in modern society and impose a huge clinical and economic burden worldwide. According to recent U.S. data, the prevalence of atrial fibrillation (AF), the most common significant cardiac arrhythmia will increase significantly in the next 50 years with the aging of the population (Turpie 2008). To prevent stroke anticoagulation, applications of anticoagulant thromboprophylaxis agents such as vitamin K antagonists (e.g. warfarin) and heparin have been recommended as preventative treatments. However, these are not optimal due to their known side-effects and inconvenient modes of administration (Turpie 2008, Cuker, Fareed et al. 2011). More recently, newer orally available drugs, such as the direct thrombin inhibitor dabigatran and the factor Xa (FXa) inhibitors: apixaban, rivaroxaban and edoxaban have been shown in large multi-centre clinical trails to be effective anticoagulant drugs with fewer side-effect (Turpie 2012, Spyropoulos and Turpie 2013). The overall aim of this thesis is to investigate the properties of three natural anticoagulant products: a phospholipase A2 and native and lower molecular weight dermatan sulphate (DS and LMW-DS) as potential anticoagulant agents to treat venous thromboembolism. The first part of the project was to study the in vitro and in vivo anticoagulant properties and mechanism of action of the anticoagulant phospholipase A2 (PA11) from the Australian Mulga snake (Pseudechis australis) in order to determine the viability of such a product as a human therapeutic. The results show that PA11 is a very efficient inhibitor of thrombin generation (Ki = 1.58 p 0.22 nM) by binding to the prothrombinase complex reconstituted on activated platelet surface. A mathematical model of thrombin generation from this prothrombinase complex showed that PA11 binds to factor Xa (FXa) within the complex giving a Ki of 3.44 p 0.89 nM. PA11 is also an inhibitor of platelet aggregations which are introduced by thrombin, adenosine diphosphate (ADP) and calcium ionophore A23187 (calimycin) (Masci 2000). On the other hand, PA11 does not exhibit inhibitory effect on FXa in a chromogenic assay. Thus, PA11 could be a new type of FXa inhibitor with potential as a new anticoagulant agent. Studies involve the modification of the catalytic histidine and trypsin digestion show that the enzymatic core structure of PA11 is responsible for its anticoagulant activities. Furthermore, orally-delivered PA11 by gavage in a rabbit stasis thrombosis model showed antithrombotic and anticoagulant activities. Thus, digestion by rabbit stomach enzymes and acid does not affect the biological activity of this venom component. The aim of the second part of this project was to investigate the two natural products dermatan sulphate (DS) and lower molecular weight dermatan sulphate (LMW-DS), developed by the Commonwealth Scientific and Industry Research Organization (CSIRO) from the waste of bovine hide processing. DS, a sulphated polysaccharide belonging to the same family of glycosaminoglycan (GAG) as heparin, is suggested as a new anticoagulant candidate and has attracted significant attention from the pharmaceutical industry (Osborne, Daniel et al. 2008). In this study the antithrombotic and anticoagulant effect of bovine DS was compared to Clexaner (low molecular weight heparin) in vitro and in vivo in a rabbit model of stasis thrombosis (DVT). The in vivo result showed that clot formation was significantly reduced following intravenous and oral administration of both bovine DS and Clexaner. However, thromboelastography revealed that only bovine DS is capable of preventing DVT whilst still retaining clot strength following treatment. It is hypothesised that bovine DS affects platelet-fibrin interactions less than heparin, subsequently facilitating formation of a strong clot when needed and potentially minimising the bleeding risk associated with heparin treatment. In this study, we have explored the anticoagulation mechanism of three natural occurring compounds. I have shown that, in vitro, PA11 exhibits anticoagulant activity by binding to FXa in the present of phospholipids. PA11 also has anticoagulant activity, in vivo, in rabbit stasis thrombosis model after the administration by intravenous injection and gavage. Both native and lower molecular weight dermatan sufphate showed anticoagulant activity in the same rabbit model. Importantly, both dermatan sulphate and Clexaner showed anticoagulant activity by administration through gavage.e