RhoA Is Essential for Maintaining Normal Megakaryocyte Ploidy and Platelet Generation

Abstract

RhoA plays a multifaceted role in platelet biology. During platelet development, RhoA has been proposed to regulate endomitosis, proplatelet formation, and platelet release, in addition to having a role in platelet activation. These processes were previously studied using pharmacological inhibitors in vitro, which have potential drawbacks, such as non-specific inhibition or incomplete disruption of the intended target proteins. Therefore, we developed a conditional knockout mouse model utilizing the CRE-LOX strategy to ablate RhoA, specifically in megakaryocytes and in platelets to determine its role in platelet development. We demonstrated that deleting RhoA in megakaryocytes in vivo resulted in significant macrothrombocytopenia. RhoA-null megakaryocytes were larger, had higher mean ploidy, and exhibited stiff membranes with micropipette aspiration. However, in contrast to the results observed in experiments relying upon pharmacologic inhibitors, we did not observe any defects in proplatelet formation in megakaryocytes lacking RhoA. Infused RhoA-null megakaryocytes rapidly released platelets, but platelet levels rapidly plummeted within several hours. Our evidence supports the hypothesis that changes in membrane rheology caused infused RhoA-null megakaryocytes to prematurely release aberrant platelets that were unstable. These platelets were cleared quickly from circulation, which led to the macrothrombocytopenia. These observations demonstrate that RhoA is critical for maintaining normal megakaryocyte development and the production of normal platelets.

The Hippo-p53 pathway in megakaryopoiesis

“…[7][8][9][10] Moreover, megakaryocyte-specific deletion of RhoA in mice resulted in macrothrombocytopenia due to premature release of platelets. 11 Interestingly, the megakaryocytes in the animals were larger and more highly polyploidy, consistent with the inhibitor data.…”

“…Following the publication of excellent efficacy data in patients with 17p deletion (del(17p)) or TP53 mutations, 9,10 high expectations were generated in the belief that this drug was able to produce durable responses in the majority of patients, irrespective of the presence of unfavorable prognostic factors. 11 However, the median age of the patients in the trials was 64 years 8 and only 32% of them had a Cumulative Illness Rating Scale (CIRS) score of >6. 12 This reflects the inclusion criteria in the clinical trials, which required that the patients had an Eastern Cooperative Oncology Group (ECOG) performance status of less than 2, with adequate liver and kidney function, no significant neutropenia or thrombocytopenia and who did not require warfarin or strong CYP3A4/5 inhibitors.…”

Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34 + progenitors

“…Key regulators of the actin cytoskeleton CDC42 and RHOA have already been shown to be associated with thrombocytopenia, due to defects in cytoskeleton organization. [37][38][39][40][41] In affected individuals, we found abnormal tubulin organization in proplatelet-forming megakaryocytes and altered actin polymerization in platelets. Rescue experiments with CDC42 lentiviral particles were not able to fully reverse the phenotype, although the cells produced thinner extensions and swellings, which were barely observed in control cells.…”

Proteasome function is required for platelet production

“…This suggests that, similar to megakaryocyte proplatelet formation, the proteasome may control neuronal outgrowth by degrading RhoA. Moreover, RhoA signaling has been shown to maintain normal megakaryocyte development, which is critical for platelet production (18). Malfunction of the proteasome in human diseases may lead to aberrant platelet production or abnormal platelet generation.…”

“…RhoA-dependent signaling has been linked to the production of proplatelets (17,18). Therefore, we treated human megakaryocytes with Y27632, a selective inhibitor of the Rho-associated protein kinase p160ROCK, or with C3 transferase, a direct RhoA inhibitor.…”