Exploring potential anticoagulant drug formulations using thrombin generation test

Abstract

SummaryMany anticoagulant drugs inhibiting proteins of the coagulation cascade have been developed. The main targets of anticoagulant drugs are thrombin and factor Xa; inhibiting these factors delays thrombus growth, thus preventing thrombosis while increasing bleeding risk. A balance between thrombosis and bleeding is ensured in the ‘therapeutic window’ of the anticoagulant drug concentration range. Novel anticoagulant drugs and combinations thereof are being developed. We rank coagulation factors as potential anticoagulant drug targets in combination with thrombin inhibitors, aptamer HD1 and bivalirudin, providing a background for several promising dual target treatment strategies.The thrombin generation test was used to assess the whole coagulation cascade in normal and factor-deficient human blood plasma. Potential therapeutic windows were estimated for coagulation factors, ranking them as targets for anticoagulant drugs. Thrombin and factor Xa have been revealed as the most promising targets, which fully agrees with the current drug development strategy. Inhibitors of factors Va and VIIa are expected to have narrow therapeutic windows. Inhibitors of factors VIIIa and IXa are expected to have a moderate anticoagulant effect. Factors XI and XII are poor targets for anticoagulant drugs. Compared with plasma that is deficient in factor II, the thrombin inhibitors bivalirudin and aptamer HD1 had increased activity. Both inhibitors were tested in deficient plasma providing a model of potential drug combination. The most promising combinations were anti-thrombin with anti-V/Va and also anti-thrombin with anti-IX/IXa. Each combination had an incremental dose-effect dependence that is promising from the standpoint of the therapeutic window.

<b>Structural and physical-chemical analyses of sulfated polysaccharides from the sea lettuce <i>Ulva lactuca</i> and their effects on thrombin generation

“…Regarding U. lactuca food safety, elucidation of molecular mechanisms behind the biological effects on the coagulation in vitro (Pomin, 2012;Mourão, 2015) is still needed as a critical step to antithrombotic drug formulation (Zavyalova & Kopylov, 2016).…”

“…Its anticoagulant mechanism is proved by antithrombin (AT)-binding specific pentasaccharide sequence, which it is not found in other natural sources (Pomin, 2012;Mourão, 2015). The main targets of the algal anticoagulants are the thrombin (Rodrigues et al, 2013;Mourão, 2015), the final enzyme that converts fibrinogen into fibrin clot (Castoldi & Rosing, 2011), and the factor Xa (Athukorala et al, 2006;Glauser et al, 2009;Mourão, 2015), a coagulation protease that generates thrombin (Zavyalova & Kopylov, 2016), and/or by independent effect of serpins (Quinderé et al, 2014), preventing thrombosis without producing hemorrhage (Rodrigues et al, 2011;Quinderé et al, 2014;Mourão, 2015). However, at high dose, SPs from seaweeds may lead to thrombosis due to activation of factor XII in the intrinsic coagulation system (Rodrigues et al, 2011;Quinderé et al, 2014;Mourão, 2015).…”

“…The anticoagulant effects of the seaweeds SPs are routinely measured by two tests: 1) the activated partial thromboplastin time (APTT); and 2) the prothrombin time (PT), which distinguish the anticlotting potential on both intrinsic/extrinsic coagulation pathways, respectively (Rodrigues et al, 2009;Mourão, 2015); but, they do not indicate the normal function status and thrombin generation (TG)-based coagulation assays have been introduced to develop more accurately antithrombotic drugs (Jung et al, 2014;Zavyalova & Kopylov, 2016) and for clinical prognostics (bleeding disorders and surgical procedures), and epidemiology of thrombosis (Castoldi & Rosing, 2011;Duarte et al, 2017).…”

<b><i>In vitro</i> inactivation of thrombin generation by polysulfated fractions isolated from the tropical coenocytic green seaweed <i>Caulerpa racemosa</i> (Caulerpaceae, Bryopsidales)

“…Observation suggested that our system could also constitute as an useful tool to guide complementary analyses for antivirally active algal SPs development (Cardozo et al, 2007) in parallel to prevention of thrombosis in vitro (Nishiro et al, 1999;Mourão et al, 2001;Rodrigues et al 2016Rodrigues et al , 2017b as support to predict the risk of bleeding or coagulopathy (Castoldi & Rosing, 2011;Zavyalova & Kopylov, 2016) on the focus of patients subjected to antiviral treatment (Ghosh et al, 2004;Ghosh et al, 2009).…”

“…Biological Sciences Maringá, v. 39, n. 3, p. 283-292, July-Sept., 2017 time (PT), respectively, although emphasizing that these two in vitro tests do not reflect the hemostasis (Ferreira, Sousa, Dusse, & Carvalho, 2010). Given these limitations and the complexity of the coagulation process, thrombin generation (TG)-based assays would offer a better understanding of the in vivo event to measure TG in a blood plasma sample (Castoldi & Rosing, 2011;Xin, Chang, & Ovanesov, 2016) and analyze anticoagulants Zavyalova & Kopylov, 2016). Circulatory dysfunctions are usually associated with the sedentary lifestyle, poor eating habits and stress that potentially lead to venous thromboembolism, heart attack and pulmonary embolism as the most prevalent cardiovascular diseases worldwide, leading causing to patient death or else partial or total disability.…”