Pharmacological Study of Antimyeloma Agents on Hemostasis
Authors: Mullier François , Robert Severine , Devel Philippe , Alpan Lutfiye , Lufin Nicolas , Chatelain Bernard , Dogné Jean-Michel 
Published: October 2019

Abstract
Abstract 3175

Introduction:
Patients with multiple myeloma (MM) have an increasing risk of developing venous thromboembolism (VTE). The use of thalidomide or lenalidomide, two antimyeloma agents has been associated with an increased risk of VTE especially when associated with dexamethasone or melphalan-prednisone. This increased risk in less pronounced with the proteasome inhibitor bortezomib. The pharmacological effects of thalidomide, lenalidomide and bortezomib have never been studied on platelet aggregation or coagulation. These preliminary data are necessary to understand the differential effects of these drugs on thrombotic events.

Aims:
The aim of our study is to investigate the pharmacological effects of these drugs on primary hemostasis by light transmission agregometry (LTA) and on secondary hemostasis using thrombin generation test (TGT).

Methods:
LTA was performed with a Chrono-Log aggregometer (Kordia). Platelet-rich plasma (PRP) of healthy blood donors (n=3 to 10) was adjusted to 300,000 platelets/μl. Platelet-poor plasma (PPP) and PRP were used respectively to adjust the photometric measurement to the minimum and maximum optical density. The platelet reactivity of the healthy donors was previously checked by LTA. Thalidomide (racemic, enantiomer (+) and enantiomer (-)), lenalidomide and bortezomib were spiked at final concentration of 200 μM in PRP except for studying the influence of thalidomide on ADP-induced platelet aggregation where the maximal concentration usable was 100 μM. LTA was performed without (spontaneous aggregation) and with addition of 5μM ADP, 190μg/ml collagen, 600μM arachidonic acid (AA) and 10 μM PAR1-AP (final concentrations). Negative controls (physiological saline and DMSO at concentrations used to dissolve the drugs) were also included. TGT was performed both on PPP (from normal pool plasma (NPP)) and on PRP. We first validated the method on NPP and PRP with two anticoagulants: a thrombin direct inhibitor (i.e. argatroban) and a FXa direct inhibitor (i.e. ZK-807834). Thalidomide ((+/−), (+) and (-)), lenalidomide and bortezomid were tested at the final concentrations of 5, 10, 50, 100 and 200 μM. For the experiments on NPP, PPP reagent (5 pM TF + 4 μM PL in the final assays), PPP reagent low (1 pM TF + 4 μM PL in the final assays) and MP reagent (4 μM PL in the final assays) were used whereas for the experiments on PRP, PRP reagent (1 pM TF in the final assays) was used.

Results:
LTA Both three drugs did not induce spontaneous aggregation until 200 μM. Lenalidomide and bortezomib showed no effect on induced platelet aggregation until 200 μM, whatever the agonist used. On the contrary, for racemic thalidomide, platelet aggregation was reduced at 50 and 100 μM with 5 μM ADP and at 150 and 200 μM with 600 μM AA. These effects were more pronounced with thalidomide (+) than with thalidomide (-). So, the half maximal inhibitory concentrations (IC50) for platelet aggregation induced by 600 μM AA were 127, 143 and 221 μM for racemic, enantiomer (+) and enantiomer (-), respectively. When 190μg/ml collagen or 10 μM PAR1-AP were used as agonists, no effect was observed on platelet aggregation until 200 μM thalidomide. TGT. With argatroban and ZK-807834, we observed a concentration-dependent decrease and delay of the thrombin activity profiles in PRP and NPP, whatever the inducer used. No significant effect on thrombin activity in NPP or PRP has been observed for antimyeloma drugs at all concentrations tested, whatever the inducer used.

Conclusions:
Lenalidomide, thalidomide and bortezomid do not induce spontaneous platelet aggregation and do not affect platelet aggregation induced by collagen or PAR1-AP. Conversely to lenalidomide and bortezomib, thalidomide, and especially its enantiomer (+), moderately inhibits platelet aggregation induced by ADP and AA. Moreover, these antimyeloma agents have no effect on thrombin generation. The increased risk of VTE by thalidomide or lenalidomide in patients with multiple myeloma is thus not mediated by a direct impact on primary or secondary hemostasis at therapeutic concentrations.

Disclosures:
No relevant conflicts of interest to declare.