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. Author manuscript; available in PMC: 2020 Apr 1.
Published in final edited form as: J Thromb Haemost. 2019 Mar 19;17(4):681–694. doi: 10.1111/jth.14404

Figure 4. Internalization of FVIII by CLEC4M by a clathrin-coated pit-dependent pathway.

Figure 4.

CLEC4M-expressing cells were preincubated with endocytosis inhibitors for 1 hour as described in Methods. (A and B) Association of rFVIII (2 U/mL) or pdVWF (2 U/mL VWF, 2.4:1 VWF-FVIII complex) with CLEC4M-expressing cells (1 hour) pre-treated with inhibitors of endocytic pathways, methyl-β-cyclodextrin (MBCD), pitstop-2 (PS), dynasore hydrate (DH) as described in Methods. Images are representative from 4–5 independent experiments. Cells were imaged with immunofluorescence; CLEC4M (red), FVIII (green), DAPI (blue), colocalization (yellow). (C and D) Images from 4–5 independent experiments were quantified using ImagePro software and cell-bound VWF or FVIII was normalized to the number of CLEC4M expressing cells per field of view. ± SEM, p<0.05 = *, p<0.001 = **.