Linking in silico and in vitro experiments to identify and evaluate a biomarker for enoxaparin activity
Abhishek Gulati (1)*, James M Faed (2), Geoffrey K Isbister (3, 4), Stephen B Duffull (1)
(1) School of Pharmacy, University of Otago, Dunedin, New Zealand, (2) Department of Pathology, School of Medicine, University of Otago, Dunedin, New Zealand, (3) Department of Clinical Toxicology and Pharmacology, Calvary Mater Newcastle, NSW, Australia, (4) School of Medicine and Public Health, University of Newcastle, NSW, Australia
Background: Enoxaparin is a low molecular weight heparin (LMWH) anticoagulant and is widely used in thromboprophylaxis for both prevention of primary thrombosis and at higher doses for treatment of patients with pulmonary embolism, deep vein thrombosis and acute coronary syndromes [1-2]. Dosing of enoxaparin, like other anticoagulants, may result in bleeding following excessive doses and clot formation if the dose is too low. For warfarin and unfractionated heparin (UFH), it is usual to measure the time for a blood sample to clot after stimulation with activating agent(s). The standardised tests, prothrombin time (PT), expressed as the international normalised ratio (INR), and the activated partial thromboplastin time (aPTT) assess multiple steps in the clotting system and have been found to provide a good prediction of the risk for bleeding, or clotting and thrombosis, when used with warfarin and UFH respectively [3-4]. Neither the PT nor aPTT produce significant dose‑response changes with enoxaparin and so do not provide meaningful evaluation of bleeding or thrombotic risks [5]. Currently there is no multi‑step clotting time test for LMWHs such as enoxaparin and assessment of dose effect is seldom performed. Rarely anti‑Xa activity is used to assess the dose for enoxaparin, particularly where renal impairment is present, but its utility to predict for clotting or bleeding remains uncertain. We consider there is a need for an alternative, simple, stable, diagnostic clotting time‑based test to monitor treatment with enoxaparin.
Aim: The overarching aim of this study was to identify and evaluate plausible activating agent(s) for a clotting time test to assess the anticoagulant effect of enoxaparin. Four specific objectives were identified: (1) in silico assessment of standard clotting time tests (aPTT and PT) when applied to enoxaparin, (2) in silico identification of new targets for activating a clotting time test, (3) in vitro assessment of Xa as a new target for activating a clotting time test and (4) in silico predictions of the kinetics of activation in the test. These specific objectives were designed to provide a proof-of-mechanism of the clotting time test where the in silico experiments provide the mechanistic framework and the in vitro experiments show a realisation of the mechanism.
Methods: (1) A previously developed mathematical model of the coagulation network [6] was used to assess current clotting time tests, PT and aPTT, when applied to enoxaparin. The influences of various initial conditions for the tests were investigated. Effect of enoxaparin was simulated at its therapeutic concentration (taken as 0.5 IU/mL of anti-Xa activity). Time courses of X and Xa in the absence as well as presence of enoxaparin (0.5 IU/mL) were also simulated using the model. (2) The mathematical model was used to identify new targets for monitoring enoxaparin therapy. To identify an activating agent for a clotting time test with enoxaparin, the in silico clotting system was activated using a range of activated clotting factors or complexes, including: IIa, Va, VIIa, TF, VII‑TF, VIIa‑TF, VIIIa, IXa, IXaVIIIa, Xa, XaVa, XIa, XIIa, XIIIa, over a range of concentrations, individually and in combination with each other. The aim of the simulations was to identify an activating agent, in the form of a clotting factor, that provides a measurable clotting time (<60 seconds) which was prolonged by at least two‑fold in the presence of a therapeutic concentration of enoxaparin (0.5 IU/mL). (3) Xa was identified in silico (from method 2) as the best option as an activating agent for a clotting time test to detect enoxaparin effect. In vitro experiments were then carried out to demonstrate proof of mechanism of the clotting time test activated by Xa. Clotting times were measured in three different sets of experiments: (i) where the concentration of Xa was varied in the absence of enoxaparin, (ii) where the concentration of Xa was varied in the presence of a therapeutic concentration of enoxaparin (0.5 IU/mL) and (iii) where the concentration of enoxaparin was varied in the presence of a specific Xa concentration. (4) In silico assessment of the new target was then used to assess whether the mathematical model supports the findings from the experimental observations on clotting in the presence of varying concentrations of Xa and enoxaparin.
Results: (1) In silico assessment of standard clotting time tests (aPTT and PT) when applied to enoxaparin: The simulations suggested that both the PT and aPTT tests used high concentrations of their respective activating agents which result in excessive Xa concentrations that overcome the anticoagulant effect of therapeutic enoxaparin concentration (0.5 IU/mL). Hence therapeutic enoxaparin was predicted to cause only a small prolongation in clotting times in currently manufactured versions of the PT and aPTT. (2) In silico identification of new targets for activating a clotting time test: Low concentrations of Xa or tissue factor were identified as plausible activating agents for a clotting time test for enoxaparin. Xa appeared more appropriate as it produced shorter clotting times. (3) In vitro assessment of Xa as a new target for activating a clotting time test: A clotting time of 15 seconds, similar to the upper end of the physiological range for the PT was obtained with a Xa concentration of 10 nM and this concentration was used to activate the clotting system to assess the effect of varying enoxaparin concentrations (0.1‑1.0 IU/mL). In the presence of 1.0 IU/mL enoxaparin the clotting time was prolonged 10‑fold to 153 seconds; in contrast, 0.1 IU/mL enoxaparin caused only a 1.7‑fold prolongation to 26 seconds, compared to the control with no enoxaparin. (4) In silico predictions of the kinetics of activation in the test: There was good agreement between the in silico and in vitro results after scaling for Xa concentration.
Conclusions: Using both simulations from the in silico model and in vitro experiments we show that a Xa clotting time test (we have called this the "XaCT Test") can potentially assess the effect of enoxaparin on the clotting system. The next stage of the development of the prototype "XaCT Test" will be a proof‑of‑concept study that would validate the novel "XaCT Test" using plasma from wide range of healthy volunteers. A successful proof‑of‑concept study would mean that this "XaCT Test" could then be evaluated in patients receiving therapeutic LMWH to assess the predictive performance of the XaCT test for reduced risk of thrombotic and bleeding events. We suggest that the XaCT test might provide a missing direct link for dose optimisation of drugs like LMWH and fondaparinux.
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