Patients with ET and PV are characterized by an increased rate of thrombotic complications and by several abnormalities of platelets, more pronounced in JAK2V617F mutation carriers. Platelet function inhibitors are widely utilized for thromboprophylaxis in ET and PV patients. An increased reactivity to ADP of platelets from these patients has previously been shown by light transmission platelet aggregometry studies, which might explain the failure of aspirin to fully protect from thrombosis these patients, mainly those at high risk. While ADP activation pathway is critical for platelet aggregation, less is known about its role to the initiation of blood coagulation by platelets. In this study we wanted to evaluate in a group of patients with ET and PV, the platelet reactivity to ADP in terms of both proaggregating and procoagulant responses.
ADP-induced platelet aggregation and platelet procoagulant potential (i.e. thrombin generation capacity) were measured by the whole blood impedance aggregometry (Multiplate analyser) and by the Calibrated Automated Thrombogram (CAT) assay, respectively. Whole blood platelet aggregation was induced by 6.5 μM ADP. Whereas thrombin generation (TG) was measured in platelet rich plasma (PRP: 150,000 platelets/μl) both in resting conditions and after stimulation by 1.6 μM and 8.3 μM ADP. Fifty patients with ET (58% V617F mutation carriers) and 40 patients with PV (95% V617F mutation carriers) were enrolled into the study.
The results show that ET and PV patients presented with significantly higher ADP-induced whole blood platelet aggregation compared to control subjects (786±70 AU*min, and 777±54 AU*min, respectively, versus 465±70 AU*min, both p<0.05), the highest values being observed in ET and PV patients carriers of the JAK2V617F mutation (806±75 AU*min; p<0.001 vs controls). In resting conditions, TG in PRP from ET (133±38 nM) and PV (144±54 nM) patients was significantly greater compared to control PRP (103±21 nM; p<0.05). Similarly, in ADP-stimulated conditions, platelet TG was significantly higher (p<0.05) in ET and PV patients at both 1.6 μM ADP (140±32 nM and 154±59 nM; respectively) and 8.3 μM ADP (161±38 nM and 181±65 nM; respectively) compared to controls (1.6 μM ADP: 112±19 nM; 8.3 μM ADP: 132±26 nM). The analysis according to the JAK2 mutational status showed that platelets from JAK2V617F carriers induced significantly higher TG when stimulated by 8.3 μM ADP (185±47nM) compared to 1.6 μM ADP (146±45nM; p<0.001). Differently, this difference was not observed in JAK2 wild type patients. While in resting conditions patients on hydroxyurea (HU) generated less thrombin compared to non-HU treated patients, no effects of patients’ therapy on TG (i.e. aspirin, HU, aspirin+HU) was observed in the ADP-stimulated conditions.
In conclusion, for the first time we demonstrated that platelets from ET and PV patients are more reactive to ADP, not only in terms of increased platelet aggregation in a whole blood system, but also as an enhanced thrombin generation, particularly in those carriers of JAK2V617F mutation. These data support the hypothesis that the use of ADP receptor-inhibitors, in addition to aspirin, might be of help in the prevention of thrombosis in these conditions, by allowing a more complete inhibition of platelet functions.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.