Essential Thrombocythemia Patients with CALR Mutations Have Increased Platelet Activation Markers Compared to JAK2V617F Mutated Patients

Essential thrombocythemia (ET) is associated with an increased risk for thrombo-hemorrhagic complications. The presence of the JAK2V617F mutation, found in approximately 50% of ET patients, has been associated with increased indices of platelet (PLT) activation suggesting its casual role in thrombus formation. Mutations in CALR were recently described in the majority of JAK2V617F negative ET patients, and are associated with a decreased rate of thrombotic events. This has led us to hypothesize that CALR mutations have a different influence on PLT activation compared to JAK2V617F.

To evaluate the PLT activation state, surface expression of two PLT activation markers - p-selectin (CD62P) and PAC1 was studied using specific antibodies. MFI was analyzed by flow cytometry at baseline, as well as following ADP addition to PLT rich plasma. Monocyte-platelet aggregates were studied in whole blood samples by gating CD45+/CD14+ cells and calculating the percentage of CD41+ cells in the monocytes population. The immature PLT fraction (IPF) was analyzed with the XE-5000 hematology analyzer (Sysmex UK Ltd., Milton Keynes, UK), and the absolute number of immature PLT (nIP) was calculated from the total PLT count.

Low risk ET patients (N-13, M/F-5/8) and healthy controls (N-10, M/F-4/6) are included in this analysis. JAK2V617F and CALR mutations were present in 8 and 5 patients, respectively; low dose aspirin (range 75-100mg) was taken by 85% of patients and 90% of controls. Median PLT count in CALR mutated, JAK2V617F mutated and healthy subjects was 913, 579 and 247 K/uL, respectively (p=0.0002), and it was higher in CALR compared to JAK2V617F positive patients (p=0.09). Both patient subgroups had a lower baseline MFI of p-selectin and PAC1 compared to healthy controls (p-selectin: 2.8, 3 and 4.5 for JAK2V617F [p=0.01], CALR [p=0.05] and controls; PAC1: 3, 3.3 and 5.2 for JAK2V617F [p=0.01], CALR [p=0.02] and controls, respectively) with no difference between CALR and JAK2V617F mutated patients. CALR compared to JAK2V617F mutated patients had higher median number of immature PLT (30 and 10.6 K/uL, p=0.04), and a higher fraction of monocyte- platelet aggregates (90 and 58%, p=0.05). nIP and monocyte- platelet aggregates were also significantly higher in CALR mutated but not in JAK2V617F mutated patients compared to healthy controls. Interestingly, there was no difference in post ADP PLT activation (post/baseline ratio) between ET patients and healthy controls. Finally, there were correlations between the PLT counts and nIP (R=0.8, p<0.0001), monocyte- platelet aggregates (R=0.5, p=0.02), baseline p-selectin MFI (R=-0.5, p=0.02) and PAC1 MFI (R=-0.5, p=0.01).

Our preliminary results suggest a correlation between PLT activation markers and the PLT numbers, which can explain why CALR mutated patients in our cohort had higher nIP and monocyte- platelet aggregates fractions. The absence of an increased ADP induced PLT activation between patients and controls in this cohort compared with previous reports could be explained by the use of aspirin in the majority of patients and the high ADP concentration used for PLT activation. These results will be further studied in a lager cohort of patients.