Abstract 1218

Platelets are essential for hemostasis, and thrombocytopenia (platelet counts < 150×109/L) is a major clinical problem encountered across a number of conditions. Megakaryocytes (MKs) generate platelets by extending long, branching processes, designated proplatelets, into sinusoidal blood vessels. While the mechanism of proplatelet production has been studied extensively, very little is known about what initiates and regulates proplatelet formation. We used the IncuCyte system to monitor proplatelet formation in cultured megakaryocytes differentiated from murine hematopoietic stem cells. Kinetic analysis of proplatelet formation over 24 hours revealed maximum proplatelet formation after 8 hours. Inhibition of protein synthesis with puromycin and cycloheximide significantly decreased proplatelet formation from 54% ± 4.0 to 17% ± 3.8, and significantly reduced the amount of proplatelets and platelets released. Conversely, inhibition of protein synthesis with chloramphenicol had no effect on proplatelet formation or release. These data suggest eukaryotic (but not mitochondrial) proteins synthesized immediately preceding proplatelet formation are necessary for the transition from round to proplatelet-producing megakaryocytes. To identify what proteins are translated during proplatelet formation, we compared the proteome of round (puromycin and cycloheximide treated) vs. proplatelet-producing megakaryocytes by two-dimensional differential interference gel electrophoresis (2D DIGE). Interestingly, only a small number of proteomic differences were seen between the round vs. proplatelet-producing megakaryocytes, suggesting translation of a subset of proteins is sufficient to drive proplatelet production.

We focused on two proteins identified in the 2D DIGE that were up-regulated in proplatelet-producing MKs, myristoylated, alanine-rich, C kinase substrate (MARCKS) and ubiquilin. Expression of these proteins in MKs was confirmed by western blot and immunofluorescence. MARCKS is a protein kinase C (PKC) substrate that crosslinks F-actin at the plasma membrane. To examine the role of the PKC pathway in proplatelet formation, we treated MKs with the PKC inhibitor and activator Ro 32–0432 and PMA, respectively. Ro 32–0432 treatment dose-dependently augmented proplatelet production, while treatment of MKs with the PKC activator PMA dose-dependently inhibited proplatelet formation, suggesting a role for this pathway in proplatelet formation. Ubiquilin facilitates protein degradation by linking poly-ubiquitinated proteins to the 26S proteasome. We found that treatment of MKs with Bortezomib, an inhibitor of the 26S proteasome, significantly decreased proplatelet formation by 76% after 24 hours, suggesting the proteasome is necessary for proplatelet initiation.

Therefore, our preliminary data suggest that the dynamic cytoskeletal process of proplatelet formation requires both protein synthesis and degradation. In the future, targeting MARCKS and ubiquilin directly may be a viable therapeutic option to drive proplatelet production and initiate “autotransfusion” of platelets from existing bone marrow megakaryocytes.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.