Pathogenetic and clinical study of a patient with thrombocytopenia due to the p.E527K gain-of-function variant of SRC

The SRC gene was the first proto-oncogene to be discovered. Its product SRC is a nonreceptor protein tyrosine kinase that is the prototype, and a ubiquitously expressed member, of the SRC family kinases. SRC has been investigated for decades in mouse and in vitro models: these studies have indicated that SRC signalling has a central role in many cellular functions and in oncogenesis. 1 In platelets, SRC mediates signal activation pathways downstream different integrins and G protein-coupled receptors.2 However, much remains to be understood about SRC functions in human megakaryocytes and platelets. Recently, the first germline mutation in SRC causing human disease was reported in two families. Here we report the investigation of a new unrelated individual carrying the p.E527K variant that provides additional information on the clinical and pathogenetic features of the disorder and the role of SRC in human megakaryocytes

Studies of the interaction of ticagrelor with the P2Y13 receptor and with P2Y13-dependent pro-platelet formation by human megakaryocytes

Ticagrelor is an antagonist of the platelet P2Y12 receptor for ADP, approved for the prevention of thromboembolic events in patients with acute coronary syndrome. Previous studies showed that ticagrelor has no significant activity versus P1 receptors for adenosine and other known P2Y receptors, with the exception of P2Y13, which was not tested. The P2Y12 antagonist cangrelor has been shown to also inhibit P2Y13 and to decrease the P2Y13-regulated capacity of megakaryocytes to produce pro-platelets. We tested whether or not ticagrelor inhibits P2Y13 signalling and function. The in vitro effects of ticagrelor, its active (TAM) and inactive (TIM) metabolites, cangrelor and the P2Y13 antagonist MRS2211 were tested in two experimental models: 1) a label-free cellular response assay in P2Y13-transfected HEK293 T-REx cells; and 2) pro-platelet formation by human megakaryocytes in culture. Ticagrelor, TAM, cangrelor and MRS2211, but not TIM, inhibited the cellular responses in P2Y13-transfected cells. In contrast, only MRS2211 and cangrelor, confirming previous results, inhibited pro-platelet formation by megakaryocytes in vitro. The platelet count of patients randomised to treatment with ticagrelor in the PLATO trial did not change during treatment and was comparable to those of patients randomised to clopidogrel. In conclusion, ticagrelor and TAM act as P2Y13 antagonists in a transfected cell system in vitro but this does not translate into any impact on pro-platelet formation in vitro or altered platelet count in patients

Constitutively released adenosine diphosphate regulates proplatelet formation by human megakaryocytes

Background The interaction of adenosine diphosphate with its P2Y(1) and P2Y(12) receptors on platelets is important for platelet function. However, nothing is known about adenosine diphosphate and its function in human megakaryocytes. DESIGN AND METHODS: We studied the role of adenosine diphosphate and P2Y receptors on proplatelet formation by human megakaryocytes in culture. RESULTS: Megakaryocytes expressed all the known eight subtypes of P2Y receptors, and constitutively released adenosine diphosphate. Proplatelet formation was inhibited by the adenosine diphosphate scavengers apyrase and CP/CPK by 60-70% and by the P2Y(12) inhibitors cangrelor and 2-MeSAMP by 50-60%, but was not inhibited by the P2Y(1) inhibitor MRS 2179. However, the active metabolites of the anti-P2Y(12) drugs, clopidogrel and prasugrel, did not inhibit proplatelet formation. Since cangrelor and 2-MeSAMP also interact with P2Y(13), we hypothesized that P2Y(13), rather than P2Y(12) is involved in adenosine diphosphate-regulated proplatelet formation. The specific P2Y(13) inhibitor MRS 2211 inhibited proplatelet formation in a concentration-dependent manner. Megakaryocytes from a patient with severe congenital P2Y(12) deficiency showed normal proplatelet formation, which was inhibited by apyrase, cangrelor or MRS 2211 by 50-60%. The platelet count of patients with congenital delta-storage pool deficiency, who lack secretable adenosine diphosphate, was significantly lower than that of patients with other platelet function disorders, confirming the important role of secretable adenosine diphosphate in platelet formation. Conclusions This is the first demonstration that adenosine diphosphate released by megakaryocytes regulates their function by interacting with P2Y(13). The clinical relevance of this not previously described physiological role of adenosine diphosphate and P2Y(13) requires further exploration