![Figure 1. [D′D3]2 associates with FVIII primarily through the flexibly tethered D′. (A) The D1-D3 complementary DNA (cDNA) encodes the N-terminus (M1-P1247) of human or murine VWF and fuses C-terminal E and FLAG tags. Similar to VWF, D1-D3 undergoes posttranslational processing prior to its secretion as a dimer of the D′D3 domains. The propeptide catalyzes intermolecular disulfide bonds at the D3 C-terminus and is cleaved by furin to generate a new N-terminus at S764 of the D′ domain.3 (B) Class averages of murine [D′D3]2 reveal a bilobed central density with a flexibly tethered “handle” having dimensions in agreement with those of D′. (C) Fab fragments (black arrow) directed against the C-terminal FLAG tag decorate the body of [D′D3]2, distinguishing the central bilobed density as the D3 dimer and further confirming the “handle” density as D′. (D, left and right panels) Each molecule of human or murine [D′D3]2 may bind 1 to 2 human FVIII molecules and adopt various orientations. (D, center panel) Characteristic projection of a simulated map of FVIII crystal structure14 illustrates the FVIII domain architecture compared with the EM averages. In some class averages, the D′ handle can be seen in close, interacting proximity with one of the FVIII C domains (black arrows). Several averages of [D′D3]2 bound to a single FVIII clearly show a free D′ domain (white arrows) diametrically opposed to the FVIII-bound D′ domain (black arrows).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/126/8/10.1182_blood-2015-04-641696/4/939f1.jpeg?Expires=1723306958&Signature=R7lz9E19PDjpuOWrbmsflLfKU0ND2Kpzmb9ka48ZRJakt4Z6PKvhHr4MeVOnJ4-Q64bD5NxL8JfWLhBiMf7Z9sCAP5DfOpoI7la5ou-8FNB8N7SNx850F6sJoUqI3eRjuOkS1HA0JywPvwH7ew~jwg6EENdJf0xoBIOgVpszGcx~QPqala9jq7IAy717f~riWyVKL2CBfV66wrQq11BSnUj16zCVsRC8URXtBVBjhrdL6eXDNzd3L9YAf0-rw0OG6YVSOPFARf2k3jinmBmJo-klRK6WRpmwwfGl13x70TZoL1zFMYCJ4ZB-58VlenmXYN513ftayyKJIuErCdndBQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
[D′D3]2 associates with FVIII primarily through the flexibly tethered D′. (A) The D1-D3 complementary DNA (cDNA) encodes the N-terminus (M1-P1247) of human or murine VWF and fuses C-terminal E and FLAG tags. Similar to VWF, D1-D3 undergoes posttranslational processing prior to its secretion as a dimer of the D′D3 domains. The propeptide catalyzes intermolecular disulfide bonds at the D3 C-terminus and is cleaved by furin to generate a new N-terminus at S764 of the D′ domain.3 (B) Class averages of murine [D′D3]2 reveal a bilobed central density with a flexibly tethered “handle” having dimensions in agreement with those of D′. (C) Fab fragments (black arrow) directed against the C-terminal FLAG tag decorate the body of [D′D3]2, distinguishing the central bilobed density as the D3 dimer and further confirming the “handle” density as D′. (D, left and right panels) Each molecule of human or murine [D′D3]2 may bind 1 to 2 human FVIII molecules and adopt various orientations. (D, center panel) Characteristic projection of a simulated map of FVIII crystal structure14 illustrates the FVIII domain architecture compared with the EM averages. In some class averages, the D′ handle can be seen in close, interacting proximity with one of the FVIII C domains (black arrows). Several averages of [D′D3]2 bound to a single FVIII clearly show a free D′ domain (white arrows) diametrically opposed to the FVIII-bound D′ domain (black arrows).
