Platelets are released by megakaryocytes (MKs) via cytoplasmic extensions called proplatelets, which require profound changes in the microtubule and actin organization. Here, we provide evidence that the Rho/ROCK pathway, a well-known regulator of actin cytoskeleton, acts as a negative regulator of proplatelet formation (PPF). Rho is expressed at a high level during the entire MK differentiation including human CD34 ؉ cells. Thrombopoietin stimulates its activity but at a higher extent in immature than in mature MKs. Overexpression of a dominantnegative or a spontaneously active RhoA leads to an increase or a decrease in PPF indicating that Rho activation inhibits PPF. This inhibitory effect is mediated through the main Rho effector, Rho kinase (ROCK), the inhibition of which also increases PPF. Furthermore, inhibition of Rho or ROCK in MKs leads to a decrease in myosin light chain 2 (MLC2) phosphorylation, which is required for myosin contractility. Interestingly, inhibition of the MLC kinase also decreases MLC2 phosphorylation while increasing PPF. Taken together, our results suggest that MLC2 phosphorylation is regulated by both ROCK and MLC kinase and plays an important role in platelet biogenesis by controlling PPF and fragmentation. IntroductionMegakaryocytes (MKs) are the highly specialized precursor cells that lead to platelet production. MK differentiation is a continuous process characterized by sequential steps. 1 First, MKs increase their ploidy via endomitosis and begin to increase their size. 2 Then, the synthesis of storage organelles is enhanced, as well as the synthesis of plasma membrane to form the demarcation membranes. This cytoplasmic maturation is associated with a marked increase in the MK size. Finally, mature MKs release platelets probably through cytoplasmic fragmentation at the tips of long and thin extensions called proplatelets (PPTs) that contain all the platelet organelles. 3,4 The mechanisms controlling proplatelet formation (PPF) are still incompletely understood. However, PPF is associated with remarkable morphologic changes that require a profound reorganization of the cytoskeleton. 5 Increasing evidence indicates that PPTs arise from the unfolding of demarcation membranes. The microtubule cytoskeleton provides the sliding power to unfold demarcation membranes and thus to induce the pseudopodial elongations corresponding to PPTs. 6 In addition, microtubules permit the organelle transport in the PPTs and maintain the platelet discoid shape. [7][8][9][10] Although not studied in detail, the actin cytoskeleton may also participate in PPF because cytoplasmic polymerized actin is associated with demarcation membranes and actin is highly aggregated in cultured MKs when PPF occurs. 11 In addition, a crucial role of the actin cytoskeleton has been reported in platelet functions since it regulates platelet shape in unstimulated and activated platelets. 12 Evidence suggests that actin cytoskeleton may play important roles during PPT formation at 2 different stages: (1) at early stages,...

Plasmodium sp. are obligate intracellular parasites that derive most of their nutrients from their host meaning the metabolic circuitry of both are intricately linked. We employed untargeted, global mass spectrometry to identify metabolites present in the culture supernatants of P . falciparum- infected red blood cells synchronized at ring, trophozoite and schizont developmental stages. This revealed a temporal regulation in release of a distinct set of metabolites compared with supernatants of non-infected red blood cells. Of the distinct metabolites we identified pipecolic acid to be abundantly present in parasite lysate, infected red blood cells and infected culture supernatant. Further, we performed targeted metabolomics to quantify pipecolic acid concentrations in both the supernatants of red blood cells infected with P . falciparum , as well as in the plasma and infected RBCs of P . berghei -infected mice. Measurable and significant hyperpipecolatemia suggest that pipecolic acid has the potential to be a diagnostic marker for malaria.