Table 2.

C1 constructs and predicted accessibility of C1 residues

No.	Construct name		Residue	Substitution	Accessible surface area values
	hC1pfVIII	HSA-C1			2R7E	3CDZ	4BDV
1	MHI-82	MHI-128	K2020	E	0.88	0.92	0.96
2			T2023		0.16	0.29	0.19
3	MHI-170	MHI-171	H2031	R	0.58	0.26	0.40
4	MHI-83	MHI-129	E2066	D	0.80	0.51	0.42
5	MHI-84	MHI-130	F2068	H	1.00	0.90	0.92
6	MHI-85	MHI-131	K2085	M	0.29	0.31	0.26
7	MHI-86	MHI-132	K2110	R	0.89	0.86	0.85
8	MHI-87	MHI-133	K2111	N	0.64	0.76	0.69
9	MHI-155	MHI-172	K2110/K2111	R/N	—	—	—
10			T2114		0.49	0.17	0.22
11	MHI-88	MHI-134	S2132	A	0.31	0.22	0.22
12			I2145		0.30	0.32	0.25
13			Q2087		0.02	0.06	0.02
14	N/A	MHI-173	R2090	A	0.62	0.69	0.76
15			I2098		0.00	0.02	0.01
16	N/A	MHI-174	S2119	A	0.59	0.45	0.40
17			N2129		0.14	0.06	0.03
18	MHI-89	MHI-135	R2150	H	0.13	0.24	0.15
19			P2153		0.11	0.16	0.10
20	N/A	MHI-138	R2159	A	0.21	0.05	0.08
21	N/A	MHI-136	Q2042	A	0.61	0.63	0.47
22	N/A	MHI-137	Y2043	A	0.30	0.39	0.46
23	N/A	MHI-139	K2065	A	0.27	0.67	0.63
24	N/A	MHI-140	P2067	A	0.47	0.61	0.64
25	N/A	MHI-141	Y2156	A	0.30	0.39	0.43

Overview of plasmid constructs encoding HSA-fVIII C1 domain and hC1pfVIII variants. The names of the plasmid constructs, the position and substitution of respective amino acid residue in the C1 domain (2020-2172), and the values for solvent-accessible surface area (ASA) are listed. ASA values were calculated for the BDD-fVIII structures 2R7E,³⁶ 3CDZ,³⁷ and 4BDV,³⁸ using the program ASAView (www.abren.net/asaview/).³⁹ Rows 1-12, nonconserved residues among human and porcine fVIII C1 domains; rows 13-20, HA missense mutations in the C1 domain associated with reduced VWF binding²⁵ and identified in the FVIII Variant Database (www.factorviii-db.org) by Chiu et al²⁴; rows 21-22, residues additionally identified by site-directed mutagenesis to be involved in VWF binding²⁶; rows 23-25, residues identified by eye screening of fVIII structures to be in close proximity to the crucial antibody binding residues E2066 and F2068. All mutant constructs supported expression and secretion of the respective protein similar to the native hC1pfVIII and HSA-hC1 constructs. N/A, not available.