Proplatelet Formation Is Regulated by the Rho/Rock Pathway.

Abstract

Megakaryopoiesis is a highly specialized cellular process which sustains platelet production. At the end of megakaryopoiesis, megakaryocyte (MKs) fragments into platelets via long and thin cytoplasmic extensions called proplatelets. Proplatelet formation (PPF) is associated essentially with cytoskeleton changes, including actin dynamics. The Rho/Rock pathway is a well characterized regulator of the actin reorganization. In the present study, we have tried to understand the precise role of the Rho/Rock pathway in PPF from human CD34+ derived MKs. Our results show that Rho is expressed in MKs and that its expression and activity remain stable during megakaryopoiesis. Overexpression of a RhoA dominant negatif (RhoA N19) in MKs leads to an increase in PPF. Conversely overexpression of a RhoA spontaneous active (RhoA V14) in MKs leads to a decrease in PPF. These results indicate that Rho activation could inhibit PPF in vitro. It is known that Rho/ROCK promotes actin cytoskeleton dynamics by regulating myosin light chain 2 (MLC2) phosphorylation. To demonstrate that Rho/Rock inhibits PPF through MLC2 phosphorylation, we added MLC kinase inhibitor (P18), Rho inhibitor (TatC3) and ROCK inhibitor (Y27362) in MKs culture just before PPF. Western blot analysis shows that MLC2 phosphorylation was inhibited by these 3 compounds, in contrast, PPF was significantly increased. Moreover, the platelet produced have an identical size and ultrastructure as control platelets and could be normally activated. These results suggest that Rho/ROCK could inhibit PPF through MLC phosphorylation during megakaryopoiesis.

A novel heterozygous ITGB3 p.T720del inducing spontaneous activation of integrin αIIbβ3 in autosomal dominant macrothrombocytopenia with aggregation dysfunction

“…Megakaryocytes extend long cytoplasmic projections called proplatelets, and the tips of the proplatelets are released as platelets [32]. The RhoA-ROCK-myosin-IIA pathway suppressed proplatelet formation in premature megakaryocytes [33,34]. Partially activated αIIbβ3 decreased RhoA activity and induced proplatelet-like protrusion in CHO cells [35].…”

Anagrelide Modulates Proplatelet Formation Resulting in Decreased Number and Increased Size of Platelets

“…30 The FAK-RhoA-ROCK-MLC2-myosin IIA pathway plays an important role controlling actin dynamics in the process of proplatelet formation. 22,23,25 An association between decreased platelet count and increased platelet size was reported in congenital macrothrombocytopenia, in which the FAK-RhoA-ROCK-MLC2-myosin IIA pathway was disregulated. [23][24][25][26] Heterozygous mutation at the membrane proximal region of ITGA2B or ITGB3 causes constitutional partial activation of the integrin aIIbb3 complex and premature release of platelets, resulting in production of giant platelets.…”

Megakaryocyte Cytoskeletal Proteins in Platelet Biogenesis and Diseases

“…140 Surprisingly, myosin deficiency or inhibition of its activity has been reported to increase the number of cells extending proplatelets in culture. 61 141 142 Activation of NMII-A activity through the Rho/ROCK/MLC-2 pathway is supposed to inhibit proplatelets in vitro. 142 In living mice, NMII-A has been shown to regulate the protrusive and retraction forces during proplatelet extension.…”

“…61 141 142 Activation of NMII-A activity through the Rho/ROCK/MLC-2 pathway is supposed to inhibit proplatelets in vitro. 142 In living mice, NMII-A has been shown to regulate the protrusive and retraction forces during proplatelet extension. 130 Several mechanisms, including increased cell death, defective proplatelet formation, and premature platelet release in the bone marrow compartment, are supposed to contribute to macrothrombocytopenia in MYH9-RD patients.…”