The Pan Histone Deacetylase Inhibitor Panobinostat and the Iso-Selective Inhibitor Romidepsin Reduce Glycoprotein VI Expression and Inhibit Platelet Activation in Response to Collagen Related Peptide

Abstract 1154

Histone deacetylase inhibitors (HDACi) are anti-cancer drugs able to induce chromatin remodelling, alter gene expression and affect function of non-histone proteins. We recently reported that the pan-HDACi panobinostat and the iso-selective inhibitor romidepsin induce thrombocytopenia by reducing megakaryocyte proplatelet number, without effects on platelet half life (Bishton et al Blood 2011). The effect of HDACis on platelet function remains unknown, and we postulated possible interference with the expression or function of key platelet activating proteins.

Platelet Glycoprotein (GP) VI is a member of the immunoglobulin superfamily, expressed exclusively on the surface of platelets and megakaryocytes, complexed with FcR g-chain dimers, predominantly responsible for adhesion of platelets to collagen. Following interaction with sub-endothelial collagen, the Src family kinases Fyn and Lyn mediate the recruitment and autophosphorylation of Syk kinase and thereby downstream signalling and platelet activation.

Following the treatment of C57BL/6 mice with 10mg/kg panobinostat or 1mg/kg romidepsin intraperitoneally (IP) daily for three days, we isolated washed murine platelets for function testing. Following stimulation with thrombin, a dose-dependant increase was seen in platelet surface expression of CD62P (P-selectin), and also the conformationally active form of the integrin αIIbbIIIa, with no difference seen between groups. When collagen related peptide (CRP) was used as a platelet agonist, and activation assessed by p-selectin and activated αIIbbIIIa expression, platelets from cohorts of mice treated with panobinostat or romidepsin failed to increase the expression of either molecule in response to CRP, compared to vehicle treated mice. Co-treatment of mice with the murine thrombopoietin mimetic, AMP-4, or the proteasome inhibitor bortezomib did not alter effects of the HDACi. Ex vivo addition of panobinostat or romidepsin to naïve platelets did not however affect platelet activation, suggesting megakaryocytes rather than platelets to be the target cell responsible for these effects.

Flow cytometric analysis of the expression of GPVI on platelets showed a consistent and statistically significant decrease in the median fluorescent intensity (MFI) of staining seen in both HDACi treated groups. No equivalent changes in the surface expression of the other collagen receptor integrin α2b1 were seen. Western blotting of murine platelets confirmed this reduction in GPVI and a ∼17kDa fragment was also seen with HDACi treated platelets, suggesting GPVI degradation. Following stimulation with CRP, Western blotting of platelets with a phospho-syk antibody showed a reduction in phospho-syk levels in platelets from mice treated with HDACi, consistent with decreased downstream signalling from the GPVI receptor. Western blotting of murine megakaryocytes differentiated from murine fetal liver cells by TPO, also demonstrated a reduction in GPVI expression following HDACi exposure, again suggesting an intrinsic megakaryocyte process to be responsible. qRT-PCR on HDACi treated megakaryocytes demonstrated a mild increase in GPVI mRNA levels post romidepsin, but no changes post panobinostat compared to vehicle treated cells, confirming transcriptional repression not to be responsible for these changes.

We show that HDACi cause a reduction in surface expression of GPVI expression by inducing its degradation and thus inhibiting murine platelet responses to CRP. There was no evidence of an effect on gene transcription. Our work suggests a potential beneficial anti-thrombotic effect of HDACi, mediated by reduction in both platelet number and function. These findings support the need to investigate the role of HDACi and their effect on GPVI in myeloproliferative neoplasms particularly with respect to their impact on thrombotic complications.