Fig 2: MACF1 is not important for thrombus formation, clot retraction and hemostasis. (A) Thrombus formation was assessed on collagen at a wall shear rate of 1,000 s−1. Shown are representative bright field (upper panel) and fluorescence (lower panel) pictures of platelets stained with a Dylight-488 anti-GPIX antibody. (n = 6, representative for two independent experiments) (B) Surface coverage and relative thrombus volume was quantified according to fluorescence distribution and fluorescence intensity, respectively. (C) Clot formation was observed over time and (D) residual serum volume was measured. (n = at least 3, representative for three independent experiments) (E) Determination of tail bleeding time performed on filter paper of Macf1+/+ and Macf1−/− mice.

Fig 3: Lack of MACF1 does not affect microtubule and actin arrangement. (A) Images of the platelet cytoskeleton ultrastructure on poly-L-lysine (n = 2). (B) TEM images of resting platelets (n = 4) and (C) evaluation of microtubule number. (D) Resting or thrombin-activated platelets were fixed, permeabilized, stained with phalloidin-FITC, and analyzed by flow cytometry. (E) The ratio of polymerized actin in activated vs. resting platelets was determined (n = 3, representative for two independent experiments). (F) Representative transmission electron micrographs of the cytoskeleton ultrastructure of platelets spread on fibrinogen in the presence of 0.01 U/ml thrombin (n = 2). (G) Representative images and (H) statistical analysis of the different spreading phases of fixed Macf1+/+ and Macf1−/− platelets on fibrinogen at different time points (n = 3). *P < 0.05.

Fig 4: MACF1 deficiency has no effect on platelet count and size. (A) Western blot analysis of MACF1 expression in platelets. Loading control: Filamin A. (B) Platelet count per μL and (C) platelet size given as mean forward scatter (FSC) were determined via flow cytometry. (D) Immunostaining of resting platelets on Poly-L-lysine. Green: MACF1/ACF7, cyan: α-tubulin, purple: F-actin. Scale bar: 3 µm. (E) Representative transmission electron micrographs (TEM) from Macf1+/+ and Macf1−/− platelets. (F) Determination of glycoprotein expression by flow cytometry. Diluted whole blood was incubated with FITC-labelled antibodies and measured as mean fluorescence intensity (MFI); (n = 3); **P < 0.01. (A,D,E) Representative of at least 3 versus 3 samples.