Platelets and Heparin Induced Thrombocytopenia Antibodies Do Not Influence the Inhibitory Activity of Argatroban on Thrombin Generation.

Argatroban is a synthetic, reversible, direct thrombin inhibitor (DTI) used in patients with heparin induced thrombocytopenia (HIT). Argatroban as other DTIs prolongs PT, aPTT and ecarin clotting time. Argatroban added in vitro in normal platelet poor plasma (PPP) inhibits tissue factor (TF) triggered thrombin generation (TG) in a concentration dependent manner. We studied the influence of platelets, HIT antibodies and residual heparin, on the inhibitory effect of argatroban on TG. Argatroban (0 to 2 μM) was added in normal platelet rich plasma (PRP) and PPP, in pool PPP from three consecutive HIT patients (HIT-PPP) and in HIT-PRP prepared by mixing normal PRP with HIT-PPP (v/v). HIT-PRP and HIT-PPP were containing residual heparin (0,035 and 0,07 anti-Xa IU/ml respectively). All experiments were repeated 3 times. TG was triggered in the presence of TF (Dade-Behring Innovin; 1/1000 final dilution in plasma) and assessed with Calibrated Automated Thrombinoscope®. TG in PPP was triggered by adding PPP reagent (Thrombogram-Thrombinoscope^®). Lag time (LT), time to peak (ttP), peak (P), endogenous thrombin potential (ETP) and mean velocity index (MVI) of thrombin generation were measured. The concentrations of argatroban which prolonged 2-fold the LT and the ttP and which inhibited 50% (IC50) the P, ETP and MVI were calculated. In the presence of low argatroban concentrations (0,1 an 0,2 μM) an artifactual increase of TG was observed. This is probably due to the interference of alpha2macroglobulin-bound thrombin with the fluorogenic substrate (as shown by Hemker’s group for other reversible DTIs). Argatroban at 1 μM inhibited TG by 50% in both normal PRP and PPP. Argatroban at 1 μM induced a 2-fold prolongation of aPTT (Table 1). HIT antibodies did not modify the inhibitory activity of argatroban on TG in HIT-PRP and HIT-PPP. The presence of traces of heparin in plasma from HIT patients had a synergistic effect with argatroban on the inhibition of TG (Table 1). Our in vitro study shows that argatroban at concentration achieved in clinical practice (about 1 μM) induces 50% inhibition of TG. The inhibitory activity of argatroban on TG is not modified by the presence of platelets or HIT antibodies. In contrast, traces of residual heparin in plasma from HIT patients amplify the inhibition of thrombin generation induced by argatroban. This finding has to be confirmed in a prospective clinical study since it implicates an increased vigilance during the switch from heparin to argatroban in acute HIT patients.