Effects of multiple myeloma therapy on haemostasis and thrombosis

Multiple myeloma (MM) is a malignancy of plasma cells characterised by lesions in multiple bones involving transformed, matured post-follicular B cells. In MM, uncontrolled clonal cell proliferation leads to accumulation of mutated plasma cells throughout bone marrow, and despite many novel therapies being reported, MM remains incurable. Current management of MM involves different combinations of treatments including proteasome inhibitors (PIs), such as bortezomib (BOR) and carfilzomib (CAR), and immunomodulatory drugs (IMiDs), such as thalidomide (Thal), lenalidomide (LEN), and pomalidomide, and anti-human CD38 (daratumumab or DARA). Patients with MM experience haemostatic imbalances and increased risk of thromboembolic events. Treatments with IMiDs, PIs, and anti-human CD38 antibody, in combination with steroids or cyclophosphamide (CYCLO), are clinically reported to further exacerbate thrombotic risk and produce adverse side effects including venous and arterial thrombosis, and thrombocytopenia. In this study the effect of single MM therapies (BOR, CAR, LEN, dexamethasone (DEX), CYCLO, and DARA as intact IgG or DARA Fab fragment) and combination treatments (BOR, CYCLO, and DEX; LEN, CYCLO, and DEX; BOR, LEN, and DEX; CAR and DARA; and DEX and intact DARA (IgG)) were investigated on healthy human platelet function and thrombus formation in vitro on type I collagen. In addition, a C57BL/6 mouse model was pretreated with PIs (BOR or CAR) or IMiDs (Thal or LEN) to explore the effect of these treatments on platelet function by observing ex vivo thrombus formation on type I collagen and by measuring platelet activation markers. The main goal of this study was to determine the haemostatic or thrombotic potential of single and combination MM therapies. Experiments involving healthy human platelets demonstrated that CAR, LEN, DEX, and CYCLO, but not BOR, increased thrombus formation in vitro on immobilised type I collagen under arterial flow in a dose-dependent manner. Findings also showed increased thrombin-mediated P-selectin expression and PAC-1 binding in response to CAR, LEN, DEX, and CYCLO, whilst BOR produced no effect on P-selectin expression or PAC-1 binding following thrombin stimulation. Furthermore, intact DARA (IgG) and DARA Fab fragment, increased thrombus formation in vitro on immobilised type I collagen under arterial flow, as well as thrombin-mediated P-selectin expression and PAC-1 binding. Likewise, all combination treatments increased thrombus formation, P-selectin expression, and PAC-1 binding. These studies also showed that neither single MM therapies, nor combination therapies, affected resting human platelet glycoprotein expression of integrin ¿IIbß3, GPVI, or GPIX. Pretreatment of the C57BL/6 mouse model with different MM therapies revealed an increase in ex vivo thrombus formation on type I collagen under arterial flow in response to CAR or LEN. These findings were also accompanied by increased thrombin-mediated P-selectin expression and JON/A binding. Conversely, no effect on P-selectin expression or JON/A binding following thrombin stimulation was observed following pretreatment with BOR or Thal. Again, no treatments impacted glycoprotein expression of integrin ¿IIbß3, GPIb¿, CD9, or GPVI in resting mouse platelets. Taken together, the in vitro and ex vivo findings from this study indicate that single MM therapies, like CAR, LEN, CYCLO, DEX, or DARA, have the potential to increase human platelet activation and thrombus formation on type I collagen under physiological conditions of arterial shear flow rates. In addition, the results from this study also suggest that combination treatments containing LEN, CYCLO, DEX, CAR, or DARA, are more likely to be prothrombotic and require concurrent prophylactic anti-platelet therapy. Furthermore, the effects of PIs and IMiDs were investigated ex vivo using mouse platelets and confirmed the potential of CAR and LEN to activate platelets and induce thrombus formation. In conclusion, this study presented a novel effect of MM therapies on platelet activation and revealed that BOR+LEN+DEX combination treatment has a lower risk in triggering platelet activation.