ALBERT

Description: Introduction: Orgaran® (Org 10172) is a low molecular weight heparinoid which consists of natural sulphated glycosaminoglycans (heparan, dermatan, chondroitin sulphate). It has a mean molecular weight of approximately 6 kDa (4–10 kDa), an excellent bioavailability following subcutaneous administration and an anti-Xa/anti-IIa activity ratio superior to 22. It is the anticoagulant of choice in patients developping Heparin-Induced Thrombocytopenia (HIT), whereas its’ use is also proposed for surgical thromboprophylaxis. Orgaran® has no effect on routine coagulation tests (aPTT, PT, TT). Thrombin generation test(TG) is a global clotting assay proven to be sensitive to the anticoagulant effect of LMWHs and specific FXa inhibitors (i.e. fondaparinux and BAY-597939). In this in vitro study, we determined the tissue factor (TF)-induced TG inhibition potency of Orgaran® using the Thrombogram-Thrombinoscope® assay. Materials and Methods: TG was assessed after TF pathway activation in Platelet Rich Plasma (PRP) (1.5x105 platelets/μl) using diluted thromboplastin (Dade Innovin®, 1:1000 final dilution). The clotting process is provoked by a physiologically relevant TF concentration. Orgaran® was added to control plasma from 8 healthy volunteers at five different final concentrations (0.2, 0.4, 0.6, 0.8 and 1IU anti-Xa/ml). 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: Orgaran® prolonged significantly the lag time and the Tmax at a concentration over 0.40 IU anti-Xa/ml (p

Description: Introduction: Evolution of diabetes is associated with multiple disorders including metabolic, cellular and blood disturbances leading to various vascular complications (micro-and macro-angiopathies). Increased circulating levels of platelet-leukocyte aggregates (PLA) have been described in several thrombotic diseases. Material and Methods: In this study, we have evaluated the circulating PLA in diabetic patients and we have investigated whether they may be linked to the vascular complications currently occurring during diabetes evolution. Using flow cytometry assay, we have quantified PLA percentages in 65 diabetics (37 males/28 females, 57 ± 11 years old) including 20 patients with type I and 45 with type II diabetes, and 25 healthy subjects (15 males/10 females, 44 ± 9 years old). Labelling approach using specific monoclonal antibodies permitted us to identify platelet-polymorphonuclear aggregates (PPA) and platelet-monocyte aggregates (PMA). Results: We have observed a significant increase of PPA and PMA levels in diabetics (22 ± 12% and 45 ± 18%, respectively) compared to control subjects (7 ± 4% and 19 ± 10%, respectively). However, both PPA and PMA values were similar in the two types of diabetes and they were not correlated to the disease duration. Circulating PPA and PMA percentages were significantly enhanced in diabetics with vascular lesions (n=37; PPA: 24 ± 13%; PMA: 50 ± 18%) than in diabetics without vascular lesions (n=28; PPA: 18 ± 8%; PMA: 38 ± 15%). Patients with PPA 〉 18% and /or PMA 〉 38% had a more important vascular thrombotic damage (OR: 6; 95%; IC: 1.6; 23). The increased PMA circulating percentage seems to be more specific for micro-retinopathy occurrence (OR: 19, 95% IC: 2.3; 154). The rare patients with elevated PMA percentage and without vascular lesions have a shorter duration of diabetes (7 ± 5 years) than patients presenting retinopathy lesions (16 ± 11 years). Conclusion: Together our findings established a relationship between increased circulating PLA rate, particularly that of PMA, and the incidence of microvascular complications in diabetic patients. In addition, they reinforce the concept that pro-inflammatory cells are involved in diabetic retinopathy pathogenesis.

Description: 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 1. The IC50 for each parameter of TG (anti-Xa IU/ml) Lag time Tmax ETP Cmax Velocity Bemiparin 〉1 〉1 0.98 0.85 0.68 Enoxaparin 0.62 0.58 0.55 0.45 0.38 Nadroparin 0.80 0.75 0.55 0.45 0.38 Dalteparin 0.65 0.65 0.50 0.42 0.38 Tinzaparin 0.35 0.28 0.35 0.25 0.18 UFH 0.05 0.10 0.30 0.25 0.18 Table 2. Effect of Enoxaparin and Tinzaparin on TG at therapeutic concentration peak in vitro Therapeutic Concentration Peak (anti-Xa) Lag Time Tmax ETP Cmax Velocity Enoxaparin 1IU/ml 91% 94% 72% 82% 91% Tinzaparin 0.85 IU/ml 〉100% 〉100% 82% 90% 97%

Description: 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. Table 1. Inhibitory activity of argatroban on thrombin generation. normal PRP normal PPP HIT-PRP (0,03 aXa/ml) HIT-PPP (0,07 aXa/ml) Lag-time x2 0,7 μM 0,7 μM 0,9 μM 0,1 μM ttPeak x2 1 μM 1 μM 1 μM 0,2 μM Peak IC50 1 μM 1 μM 0,7 μM 0,3 μM ETP IC50 1 μM 1 μM 0,7 μM 0,3 μM MRI IC50 1 μM 1 μM 0,5 μM 0,3 μM PTx2 - 1 μM - - aPTT x2 - 1 μM - -

Description: Background Recently, direct thrombin inhibitors such as Hirudin, Bivalirudin, Melagatran and Argatroban have been introduced in the armamentarium of an antithrombotic drugs. Danaparoid, an anti-Xa - anti IIa agent is also used especially in Heparin induced thrombocytopenia. Objective Thrombin generation test (TGT) has been recently automated and could be useful for the evaluation of the inhibitory activity of these drugs and the measurement of their anticoagulant effects at established therapeutic concentrations. Moreover, TGT can elucidate some information about their mechanism of action. The aim was to compare Hirudin, Argatroban, Melagatran and Danaparoid while Bivalirudin was not available. Methods and results Pools of normal human plasma were spiked with increasing concentrations of the four studied drugs and TGT was determined by studying the lag time (LT) and time to peak TTP as well as the peak (P), the endogenous thrombin potential (ETP) and the mean rate of thrombin generation (velocity TG) and its inhibition once a maximal concentration has been reached (velocity TI). The mechanism of action on TGT was heterogeneous. At therapeutic concentrations, Hirudin delays dramatically thrombin generation without affecting significantly its velocity and the amount of thrombin formed. Argatroban and Melagatran induce a far less marked prolongation of LT and TTP than hirudin but they both reduce the velocity of the reaction and the amount of thrombin generated in this in vitro study. Melagatran was found more active than Argatroban but the patterns of the thrombograms were similar. Danaparoid exerts a minimal effect on LT and TTP which is associated with a very significant influence on the parameters related to the reaction of thrombin formation (velocity index, peak and ETP). In total ETP generally considered as the most informative parameter of the thrombogram was not found as relevant as predicted. Schematically, 3 different patterns for the thrombogram have been observed indicating different mechanisms of anticoagulation, all of them, clearly associated with an antithrombotic activity in vivo. Conclusion The mechanism of action of hirudin on thrombin generation is clearly different from that of Argatroban, Melagatran and Danaparoid. This work demonstrates that antithrombotic activity is associated with different alterations of TGT. The results could help to determine the blood concentrations required for an effective anticoagulation or reciprocally the alterations of the thrombogram which are associated with therapeutic efficacy. Moreover they could be useful when laboratory monitoring of the treatment with these different drugs is considered.