Proplatelet formation deficit and megakaryocyte death contribute to thrombocytopenia in Myh9 knockout mice.

BACKGROUND: Inactivation of the mouse Myh9 gene (Myh9Δ) or its mutation in MYH9-related diseases leads to macrothrombocytopenia. Paradoxically, previous studies using in vitro differentiated megakaryocytes showed an increased capacity for proplatelet formation when myosin was absent or inhibited. METHODS: To explore the origin of the thrombocytopenia induced by myosin deficiency, we studied proplatelet formation using bone marrow explants of wild-type (WT) and Myh9Δ mouse where megakaryocytes have matured in their native environment. RESULTS AND DISCUSSION: A dramatic decrease in the number and complexity of proplatelets was observed in megakaryocytes from Myh9Δ mice, while inhibition of myosin activity by blebbistatin increased proplatelet formation from WT mature megakaryocytes. Moreover, Myh9Δ megakaryocytes had a smaller size than the WT cells. These data indicate that myosin deficiency acts negatively on proplatelet formation, probably by impairing in situ megakaryocyte maturation, while myosin activity is dispensable at the latest stage of proplatelet formation. In addition, ultrastructural examination of Myh9Δ bone marrow revealed an increased proportion of megakaryocytes exhibiting signs of non-apoptotic cell death as compared with the WT mice. CONCLUSION: These data indicate that thrombocytopenia in Myh9Δ mice results from defective development of megakaryocyte size, impaired proplatelet formation and increased cell death.

Arterial thrombosis: relevance of a model with two levels of severity assessed by histologic, ultrastructural and functional characterization.

BACKGROUND: We previously described a model of laser-induced thrombosis in mesenteric arterioles with superficial and deep levels of injury producing a transient thrombus resolving within 2 min and a larger almost occlusive thrombus, respectively. Both types of lesion were sensitive to platelet GPIIb-IIIa and P2Y(12) inhibition, whereas only deep injuries were sensitive to thrombin blockade. OBJECTIVE: The aim of the present study was to use histologic methods and electron and intravital microscopy to characterize the lesions and thrombi and to extend our knowledge of the sensitivity of this model to genetic and pharmacologic inhibition. RESULTS: A superficial injury was found to detach the endothelial cells and expose a collagen III- and IV-rich subendothelium where platelets could adhere. Tissue factor and fibrin were not detected. Deeper penetration of the external elastic lamina occurred in deep injuries, with exposure of collagen I, III and IV. Here the thrombus was composed of platelets exhibiting a decreasing gradient of degranulation from the deepest lesion area to the surface. Fibrin was found close to the most activated platelets. Consistently, glycoprotein VI (GPVI)-collagen and GPIb-von Willebrand factor (VWF) interactions were found to be critical in superficial injuries. After deep lesion, thrombus formation was modestly reduced in GPVI-immunodepleted mice and still strongly inhibited in VWF(-/-) mice. Combined hirudin infusion and GPVI depletion further inhibited thrombosis after deep injury. CONCLUSIONS: This study confirms the feasibility of inducing arterial thrombosis with distinct levels of severity and establishes the central roles of collagen and VWF in thrombus formation after superficial injury. Collagen, VWF and thrombin all appear to contribute to thrombosis after deep arterial lesion.