1003 – MEMBRANE BUDDING AS A MECHANISM OF PLATELET BIOGENESIS

Abstract

How platelets are produced by megakaryocytes in vivo remains unclear. Megakaryocytes readily produce proplatelets (long pseudopodia processes from megakaryocytes) in vitro and analogous structures are found in vivo. Proplatelets are thought to be the precursors to circulating platelets. The current generations of in vitro platelet producing technologies are designed to recapitulate proplatelet formation. These platforms can generate at 70 – 80 platelets per megakaryocyte. However, the in vivo platelet biomass is thought to be sustained by the production of approximately 1,000 – 3,000 platelets per megakaryocyte. This indicates that substantial additional refinement of in vitro proplatelet formation is required, or that other mechanisms drive the high rate of in vivo platelet production. Using quantitative 3D imaging, 4D intra-vital imaging, and the use of a genetic model of severe thrombocytopenia, we show that within the native prenatal and adult environments, the frequency and rate of proplatelet formation is incompatible with the physiological demands of platelet production. We resolve this inconsistency by performing in-depth analysis of plasma membrane budding, a cellular process that has previously been dismissed as a source of platelet production. Our studies demonstrate that membrane budding results in the sustained release of platelets directly into the peripheral circulation during both fetal and adult life without induction of cell death or proplatelet formation. We propose that membrane budding, rather than proplatelet formation, provides a mathematically adequate explanation for how the majority of the in vivo platelet biomass in sustained. How platelets are produced by megakaryocytes in vivo remains unclear. Megakaryocytes readily produce proplatelets (long pseudopodia processes from megakaryocytes) in vitro and analogous structures are found in vivo. Proplatelets are thought to be the precursors to circulating platelets. The current generations of in vitro platelet producing technologies are designed to recapitulate proplatelet formation. These platforms can generate at 70 – 80 platelets per megakaryocyte. However, the in vivo platelet biomass is thought to be sustained by the production of approximately 1,000 – 3,000 platelets per megakaryocyte. This indicates that substantial additional refinement of in vitro proplatelet formation is required, or that other mechanisms drive the high rate of in vivo platelet production. Using quantitative 3D imaging, 4D intra-vital imaging, and the use of a genetic model of severe thrombocytopenia, we show that within the native prenatal and adult environments, the frequency and rate of proplatelet formation is incompatible with the physiological demands of platelet production. We resolve this inconsistency by performing in-depth analysis of plasma membrane budding, a cellular process that has previously been dismissed as a source of platelet production. Our studies demonstrate that membrane budding results in the sustained release of platelets directly into the peripheral circulation during both fetal and adult life without induction of cell death or proplatelet formation. We propose that membrane budding, rather than proplatelet formation, provides a mathematically adequate explanation for how the majority of the in vivo platelet biomass in sustained.