Identification and initial evaluation of a prototype clotting time test for enoxaparin

Background: Enoxaparin is a low molecular weight heparin anticoagulant and is widely used in thromboprophylaxis for both prevention of primary thrombosis and at higher doses for treatment in patients with pulmonary embolism, deep vein thrombosis and acute coronary syndromes. Dosing of enoxaparin, like other anticoagulants, may result in bleeding following excessive doses and clot formation if the dose is too low. There is no standard measure of enoxaparin clinical effectiveness.

Aim: The aim of this study was to identify and assess plausible activating agent(s) for a clotting time test to assess the anticoagulant effect of enoxaparin.

Methods: (1) A previously developed mathematical model of the coagulation network was used to evaluatein silico why enoxaparin does not result in significant (>2 fold) prolongation of the common clotting times, the prothrombin time (PT) and the activated partial thromboplastin time (aPTT). (2) The model was then used to identify suitable targets for activating coagulation in vitro that might permit monitoring of enoxaparin therapy. (3) In vitro experiments were then carried out to demonstrate proof of mechanism of the clotting time test activated by the new target activator. (4) In silico assessment of the new target was then used to predict the clotting times for the in vitro experiments to provide a comparison with the in silico predictions.

Results: (1) In silico assessment of activation of standard clotting time tests: The simulations suggested that both the PT and aPTT used high concentrations of the respective activating agents which resulted in excess Xa concentration that largely overcame the anticoagulant effect of therapeutic enoxaparin concentrations (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: 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 the new target: Thein vitro experiments showed that the clotting time was prolonged 4.6 fold in the presence of enoxaparin (0.5 IU/mL) where 10 nM Xa was used to activate clotting. (4) In silico assessment of the new target: There was good agreement between the in silico and in vitro results after scaling for Xa concentration.

Conclusion: 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.