Characterization of abnormalities in MYH9-RD patient platelets. (A) Peripheral blood from a patient expressing c.4270G>A (D1424N) MYH9-RD mutation was collected in EDTA. Buffy coat was incubated with CD41-APC, anti-MIIA or anti-pS1943, and Hoechst 33342. Platelets were identified as CD41⁺ Hoechst^– (scale bar = 5 μm). (B) Peripheral MIIA is disrupted in platelets with the MYH9-RD mutation. Intensity line scans were made along perpendicular major and minor axes as shown. MIIA distribution is quantified by the ratio of average peripheral intensity to average center intensity. Total MIIA is peripheral in normal platelets and diffuse in mutant platelets, and phospho-MIIA is diffuse in either case. MYH9-RD mutation results in a decrease in total MIIA (C) but not in pS1943 MIIA expression (D). (E) Accounting for the maintenance of pS1943 density, but reduction in total MIIA, the patient platelets show an approximately twofold increase in percent of phosphorylated MIIA. (F) Patient platelets show an increase in projected area. (G) MYH9-RD mutation has a dramatic effect on observed platelet shape (Normal: n = 83; D1424: n = 78; RBC: n = 32). Normal RBCs were used as a calibration control for (E-F). RBCs, red blood cells. (H) Thrombocrit values calculated from mean platelet volume (MPV), and platelet count data show a weak dependence on MPV (linear regression: thrombocrit = −0.01 * MPV + 0.33, R² = 0.12, n = 49).³¹  The red diamond is the value for the D1424N patient. If a compensatory mechanism of increased MK number applies as described for mouse,¹⁸  the MYH9-RD patients’ thrombocrit values also fit the trend of normal thrombocrit values. (I) Proposed model of platelet generation accounting for MYH9-RD induced macrothrombocytopenia, with MK shedding of preplatelets being analogous to erythroblast enucleation, where the circulating reticulocyte becomes a discocyte through the aid of hydrodynamic shear force. MIIA evidently has no role because RBCs appear normal in MYH9-RD blood. Blood shear forces likewise assist the transition from preplatelet to proplatelet, but MIIA drives cleavage furrow formation and fission, resulting in small normal platelets. MYH9-RD mutations abrogate normal MIIA activity, preventing cleavage furrow formation and platelet fission, thus causing an increase in circulating preplatelets and the phenotypic large platelets.