Dysfunction of the PI3 kinase/Rap1/integrin αIIbβ3 pathway underlies ex vivo platelet hypoactivity in essential thrombocythemia

Patients with myeloproliferative disorders (MPDs), such as essential thrombocythemia (ET) have increased risk of thrombosis and bleeding, which are major sources of morbidity and mortality. Most MPD patients have a gain of function mutation in Janus kinase 2 (JAK2V617F), but little is known how JAK2V617F affects platelet function. Here, we demonstrate that platelets from ET patients have impaired SFLLRN-mediated fibrinogen binding and have lost the potentiating effect of thrombopoietin (which couples to JAK2) on this pathway. In contrast, SFLLRN-mediated P-selectin expression, ATP secretion, phosphorylation of the PKC substrate pleckstrin, and Ca2+ mobilization were unaffected in JAK2V617F positive platelets. In addition, thrombopoietin-mediated JAK2 phosphorylation was unchanged, suggesting that signaling pathways activated downstream of JAK2 are impaired. Indeed, we found that platelets from JAK2V617F-positive ET patients have significantly reduced phosphorylation of the PI3 kinase substrate Akt, and have reduced activation of Rap1 in response to thrombopoietin, IGF-1, ADP, SFLLRN, and thrombin. This effect was independent of Gi alpha P2Y12 purinergic receptor function as ADP-mediated inhibition of VASP phosphorylation was unchanged. These results demonstrate that the PI3 kinase/Rap1 pathway is intrinsically impaired in platelets from JAK2V617F-positive ET patients, resulting in diminished thrombin and thrombopoietin-mediated integrin alpha(IIb)beta 3 activation. (Blood. 2013; 121(7): 1209-1219)