Defining the Balance between the Expression of Procoagulant, Anticoagulant and Angiogenic Molecules in Myeloma Plasma Cells

Introduction: Multiple myeloma is a clonal B-cell malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow and other tissues. This disease significantly increases the risk of venous thromboembolic disorders. The interplay between Myeloma Plasma Cells (MPCs) with coagulation and angiogenesis is currently under investigation. It has been shown that MPCs promotes angiogenesis through VEGF expression. Whether MPCs express procoagulant and/or anticoagulant proteins remains to be shown.

Aim: To determine the expression of tissue factor (TF), tissue factor pathway inhibitor 1 (TFPI 1), tissue factor pathway inhibitor 2 (TFPI 2), vascular endothelial growth factor (VEGF-A) and thrombomodulin (TM) by MPCs.

Methods: Human MPCs (RPMI 8226) and human umbilical vein endothelial cells (HUVECs) that were used as control were cultured according to standard procedures. Total RNA was extracted and reversely transcribed as previously described. Briefly, 1 μg cDNA was PCR amplified using the Go TAq® qPCRMaster mix (Promega) and the Mx3000P qPCR system according to the manufacturer's protocol. Quantitative RT-PCR was used to measure the relative mRNA levels using TaqMan assays for VEGF-A, TFPI 1, TFPI 2, TF or TM (Sigma Aldrich). Ct values were normalized against the endogenous control B-Actine (from Sigma Aldrich).Relative mRNA expression was calculated using the comparative threshold method (1/PUISSANCE (2 (Ct (gene)-Ct (endogenous control))).

Results: MPCs exhibited 4-fold higher TF expression compared to HUVECs but significantly lower expression of TFPI1 and TFPI2. For both MPCs and HUVECs, the expression of TFPI 1 was more pronounced compared to the expression of TF gene. Interestingly, MPCs showed lower expression of TFPI 2 compared to TF. Therefore, the TF/TFPI 1 ratio was higher in MPCs, reflecting a potentially increased TF activity compared to HUVECs. MPCs and HUVECs expressed comparable levels of TM. Finally, VEGF expression was significantly higher in the MPCs as compared to HUVECs. Results are summarized in Table 1.

Conclusions. Plasmocytes from human multiple myeloma cells are characterized by a marked proangiogenic potential driven by high gene expression of VEGF which is accompanied by increased expression of TF. However, TF expression seems to be at least partially compensated by increased expression of TFPIs leading to the assumption that the TF-dependent procoagulant properties of plasmocytes are restricted by a balanced increase in TFPI. These data indicate that TF expression by myeloma cells may be associated with cell proliferation rather than a procoagulant

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