We report the first identified mutation in the gene encoding human cytochrome c (CYCS). Glycine 41, invariant throughout eukaryotes, is substituted by serine in a family with autosomal dominant thrombocytopenia caused by dysregulated platelet formation. The mutation yields a cytochrome c variant with enhanced apoptotic activity in vitro. Notably, the family has no other phenotypic indication of abnormal apoptosis, implying that cytochrome c activity is not a critical regulator of most physiological apoptosis.

Platelets originate from megakaryocytes (MKs) by cytoplasmic elongation into proplatelets. Direct platelet release is not seen in bone marrow hematopoietic islands. It was suggested that proplatelet fragmentation into platelets can occur intravascularly, yet evidence of its dependence on hydrodynamic forces is missing. Therefore, we investigated whether platelet production from MKs could be up-regulated by circulatory forces. Human mature MKs were perfused at a high shear rate on von Willebrand factor. Cells were observed in real time by videomicroscopy, and by confocal and electron microscopy after fixation. Dramatic cellular modifications followed exposure to high shear rates: 30% to 45% adherent MKs were converted into proplatelets and released platelets within 20 minutes, contrary to static conditions that required several hours, often without platelet release. Tubulin was present in elongated proplatelets and platelets, thus ruling out membrane tethers. By using inhibitors, we demonstrated the fundamental roles of microtubule assembly and MK receptor GPIb. Secretory granules were present along the proplatelet shafts and in shed platelets, as shown by P-selectin labeling. Platelets generated in vitro were functional since they responded to thrombin by P-selectin expression and cytoskeletal reorganization. In conclusion, MK exposure to high shear rates promotes platelet production via GPIb, depending on microtubule assembly and elongation. (Blood. 2009;114:1875-1883) IntroductionMegakaryocyte (MK) differentiation is a continuous process characterized by sequential steps. MK ploidy increases through endomitosis, with parallel increase in cell size. Synthesis of storage organelles is enhanced and plasma membrane invaginates, resulting in the formation of demarcation membranes. Finally, mature MKs undergo complete cytoskeleton reorganization with microtubule involvement, to induce pseudopodial elongations called proplatelets. 1,2 Platelets are released from the tips of proplatelets that contain the organelles. 3 In static conditions, platelets are formed from mature MKs cultured in the presence of thrombopoietin (TPO). 3 MK fragmentation into platelets does not occur in the extravascular, hematopoietic space in normal bone marrow. To achieve platelet formation, MKs need to migrate into the sinusoid capillaries and mature MKs are generally located in the vicinity of sinusoids, more precisely on their abluminal side. 4 MK fragmentation occurs only in the intravascular space, and mature MKs have been identified in the blood circulation where they may interact via their surface receptors with proteins expressed on endothelial cells. [4][5][6] Adhesion of MKs to endothelial cells is essential for robust platelet production in vitro, a process that is believed to mimic what occurs in vivo when MKs interact with bone marrow endothelial cells within the vascular niche. 7 Recently, the process of proplatelet formation was examined in mouse skull bone marrow in vivo: the authors observed large MK fragments resembling imma...