Effects of Thalidomide on Platelets: Possible Causes of Hypercoagulability and Thrombocytopenia.

Thalidomide(Thal) is often used in hematological disorders and can predispose to thrombosis and rarely cause thrombocytopenia. The mechanisms of these effects are not well known. In order to evaluate these mechanisms, we studied a control subject and patients on thal. including 1 with thal-induced thrombocytopenia(TIT). We evaluated platelet counts, mean platelet volume, and bone marrow megakaryocyte reserve at sequential points, and also performed in vitro studies using platelet rich plasma (PRP). Platelets were studied before and after incubation with varying concentrations of thal from 10 to 20 μg/ml. Spontaneous platelet aggregation was evaluated and remaining PRP was used for studying the expression of genes for adhesion molecules, using cDNA Q-Series Adhesion molecules and ECM array from SuperArray Bioscience Corporation (Frederick, MD). RNA was extracted from untreated and in vitro thal-treated platelets from subjects in the study using mini RNAeasy kit (Qiagen, Valencia, CA) and then, reverse transcription, probe synthesis, hybridization, washes, data acquisition and analyses were performed as suggested in the supplier’s protocol. We compared expression of various genes normalized to the intensity of the ß-actin gene. The patient with TIT was a 59-year-old white male who was on thal maintenance after chemotherapy and tandem autologous stem cell transplants. Platelet count dropped from 148,000/μL to 19,000/μL within 7 days of increasing the dose from 100 to 200 mg daily. Mean platelet volume (MPV) increased from 6.4 to 9.5fL. His Bone marrow showed increased megakaryocytes without myeloma cells. There were no other causes for his thrombocytopenia, suggesting thal-induced platelet destruction, perhaps immune-mediated. In order to evaluate molecular mechanisms of thal-induced thrombocytopenia and other effects of thal on platelet function which may cause thalidomide-induced hypercoagulabilty, we studied genes controlling adhesion molecules in this patient (A) at various times during his recovery from TIT, in another thal-treated myeloma patient without thrombocytopenia (B), and in a normal control (C). Platelets of patient A showed markedly enhanced spontaneous aggregation after incubation with even low concentrations of thal. Repeat testing after cessation of thal therapy no longer demonstrated this phenomenon. No spontaneous platelet aggregation occurred in patients without TIT (B), or the control subject (C). In the control subject, there was no demonstrable expression of any adhesion molecule genes; in contrast, genes for integrin alpha10 (IGTA10), VCAM and MMP15 were expressed in platelets of both patients receiving thal (A, B), but decreased in patient A after recovery from TIT. Incubation with thal increased the gene for IGTA10 by up to 12- fold in platelets of patients on thal (A, B). Expression appeared to be dose dependent (3–4 fold with 10 μg/ml, and 12 fold with 20 μg/ml of thal). Patient B had high baseline expression of the gene for IGTA10, which did not increase on exposure to thal. VCAM and MMP 15 were also expressed on platelets of patients on thal (A, B), and further increased with exposure to in vitro thal. These results suggest that TIT is probably mediated by peripheral destruction, perhaps on immune basis, and is transient. Moreover, myeloma patients on thal may have increased expression of genes regulating some aspects of cell adhesion which may contribute to TIT or to hypercoagulability. Further studies are ongoing to verify these findings in more patients, and to delineate the mechanisms.