Mechanosensors and redox metabolism trigger platelet production – TRIMEP

Platelets are blood cells allowing to prevent or stop hemorrhage. Transfusion of platelets from blood donors is a life-saving treatment for patients with very severe bleeding or patients with low platelet counts due to defect of platelet production from the bone marrow failure upon solid cancers or leukemia and the necessary chemotherapy.
PlatOD (Platelets-On-Demand) SME, a spin-off of INSERM, is developing ex vivo platelets as an innovative alternative to blood donor platelets to overcome supply limitations and increasing needs of the aging population. Its manufacturing process mimics the physiological production of platelet in the blood flow, creating a platelet-generating organ. In the last 4 years, an ANR-funded research project (PLASMIS) coordinated by PlatOD has successfully developed an efficient microfluidic platform consisting of a microfluidic biochip allowing platelet release in 2 hours from a large quantity of cultured cells [megakaryocytes (MK)]. However, even if this represents a progress compared to other bioreactors made by competitors, the production yield (5 to 40 collected platelets/MK) is still lower than the physiological expectation (400-500 platelets/MK).
In order to increase the platelet production yield, it became obvious that bridging this gap, which is necessary in a therapeutic perspective, requires to address new mechanistic issues of platelet production in the flow. We will decipher the molecular mechanisms of VWF-GPIb shear driven reactions by 1) identifying the minimal VWF domains that are necessary to induce both MK anchoring and shear-dependent MK membrane deformation; 2) delineating the hydrodynamic forces that promote MK capture, elongation and fragmentation in microfluidic bioreactors leading to platelet formation, 3) developing tools (time-lapse image acquisitions, protocols) to measure fluorescence at single cell level, of MK under dynamic flow, 4) analyzing in situ dynamic changes of MK cytoskeleton reorganization along MK elongation/fragmentation.
The second aspect to be studied is the effect of oxidative stress. We have recently found that an imbalance of reactive oxygen species (ROS) production could regulate platelet formation. We will thus also 5) identify the intracellular origins of ROS in mature MK, 6) address the impact of discrete modulation of MK ROS on platelet production and functions; 7) determine the link between ROS and MK cytoskeleton reorganization required for platelet release thanks to a mouse model of VWD type 2B.
PlatOD microfluidic platform will integrate all relevant findings resulting from separate analysis in static or flow conditions. We will search for a synergy between these pathways, by selecting the most prominent effects observed at single cell levels or with small amounts of cells to translate them into the protocols set-up for large platelet production in PlatOD existing biochips, expecting to reach a 10-fold increased production of functional ex vivo platelets as assessed in vitro and in vivo following infusion in immunodeficient mice.
The scientific tasks will be accomplished by a multidisciplinary consortium representing 5 of the 8 partners previously associated in PLASMIS. In this biological and biomedical research project with a high innovation potential, it is expected to generate highly competitive basic science knowledge in biology and physics and to directly transfer this knowledge in order to solve technological hurdles and overcome yield limitations. Back and forth exchanges between teams working at the molecular level and at large scale will accelerate research by selecting and confirming the relevant pathways in the company perspective of upscaling a manufacturing process.