Analysis of blood smears. Blood smears prepared at days 3 and 7 after injection. Representative examples for wt-mVWF (single platelets with no aggregates), mVWF/R1306Q (small aggregates and reduced platelet count), and mVWF/R1316M (large aggregates and reduced platelet count) are provided (A-C, respectively). Bars indicate 10 μm. Microscopy was performed using an Axioimager-A1 microscope (Carl Zeiss) with a Plan-Apochromat 100×/1.4 oil-immersion objective (Carl Zeiss). Images were acquired with an Axiocam HRc camera (Carl Zeiss) and the Axiovision 4.5 software (Carl Zeiss). (D-E) Quantitative analysis of platelet aggregates. Per mouse, 50 microscopic fields were visually inspected for the presence of small aggregates (< 10 platelets/aggregate) or large aggregates (10-40 platelets/aggregate); n = 5, and data represent mean ± SE. (F) Ratio of the number of enlarged (> 2.5 μm) over normal (< 2.5 μm) sized platelets, which was calculated by measuring the size of each individual platelet in 20 microscopic fields from blood at day 14; n = 4 for each variant. (G) Platelet volumes at day 14 were analyzed in an automated cell counter. Data are presented in a box-and-whiskers plot (F,G). *P < .05; **P < .001; all compared with wt-mVWF.