Inhibition of In Vitro Thrombin Generation: Another Parameter Reinforcing the LMWH Heterogeneity.

Abstract

Introduction: Low molecular weight heparins (LMWH) are derived from unfractioned heparin (UFH) by depolymerization. Thus, they present biochemical and pharmacological differences and the ratio of anti-Xa/anti-IIa activities varies from one product to another. In this study, we compared in vitro the Thrombin Generation (TG) inhibition potency of various LMWHs and UFH using the Thrombogram-Thrombinoscope® assay.Materials and Methods: TG was assessed after Tissue Factor (TF) pathway activation in Platelet Rich Plasma (PRP) (1.5x105 platelets/μl) using diluted thromboplastin (Dade Innovin®, 1:1000 final dilution). We studied five different LMWHs (Enoxaparin, Dalteparin, Nadroparin, Tinzaparin and Bemiparin), as well as UFH at five different prophylactic and therapeutic anti-Xa final concentrations. These agents were added to control plasma from 14 healthy volunteers with equivalent anti-Xa concentrations. TG was initiated by adding the triggering solution containing CaCl2 and the fluorogenic substrate. The analyzed TG parameters are the lag time, the maximal concentration of thrombin (Cmax), the time to reach Cmax (Tmax), the TG velocity and the endogenous thrombin potential (ETP).Results: Bemiparin had almost no effect on TG, with concentrations below 0.60 IUanti-Xa/ml. Dalteparin, Nadroparin and Enoxaparin showed a similar potency in inhibiting TG at equal anti-Xa concentrations. Tinzaparin proved to be the most active LMWH in inhibiting TG and had a similar potency to UFH. Tinzaparin and UFH, with the lowest anti-Xa/anti-IIa ratio, exerted their inhibitory effect mostly by prolonging lag time and Tmax and by reducing TG velocity, especially at concentration below 0.40 IU anti-Xa/ml. Besides, UFH totally inhibited TG, as expressed by ETP, at a concentration over 0.40 IU anti-Xa/ml. For a given anti-Xa/anti-IIa ratio characterizing each LMWH the IC50 for each parameter was different. The IC50 for the reduction of TG velocity was lower in comparison to the IC50 for the other parameters (Table 1).Conclusion: Our study reinforces the concept of LMWH heterogeneity and the important effect exerted by the additional anti-IIa activity combined with anti-Xa activity. Thus, their characterization can be made through their ability to inhibit TG and not only their anti-Xa/anti-IIa ratio. Nevertheless, in vitro study ignores pharmacokinetic characteristics which are important in clinical practice. Thus, Enoxaparin, at validated prophylactic concentrations (0.20–0.40 IU anti-Xa/ml) had a weak effect on TG parameters, whereas, at peak therapeutic concentrations, it showed an important inhibitory activity similar to Tinzaparin. (Table 2).The use of TG test for the biological monitoring of LMWH requires further evaluation.Table 1The IC50 for each parameter of TG (anti-Xa IU/ml)Lag timeTmaxETPCmaxVelocityBemiparin>1>10.980.850.68Enoxaparin0.620.580.550.450.38Nadroparin0.800.750.550.450.38Dalteparin0.650.650.500.420.38Tinzaparin0.350.280.350.250.18UFH0.050.100.300.250.18Table 2Effect of Enoxaparin and Tinzaparin on TG at therapeutic concentration peak in vitroTherapeutic Concentration Peak (anti-Xa)Lag TimeTmaxETPCmaxVelocityEnoxaparin1IU/ml91%94%72%82%91%Tinzaparin0.85 IU/ml>100%>100%82%90%97%