Conservative mutations in the C2 domains of factor VIII and factor V alter phospholipid binding and cofactor activity by Gary E. Gilbert, Valerie A. Novakovic, Randal J. Kaufman, Hongzhi Miao, and Steven W. Pipe Blood Volume 120(9):1923-1932 August 30, 2012 ©2012 by American Society of Hematology

Crystal structures of the FVIII C2 domain (PDB entry 1D7P) and FV C2 domain (PDB entry 1CZT). Crystal structures of the FVIII C2 domain (PDB entry 1D7P) and FV C2 domain (PDB entry 1CZT). The membrane-interactive hydrophobic amino acids for spikes 1 and 3 of FVIII (left) and FV (right) are shown with side chains in dark gray, whereas the rest of the structures show the backbone only in light gray. Residue numbers relate to the full-length proteins. Gary E. Gilbert et al. Blood 2012;120:1923-1932 ©2012 by American Society of Hematology

Relative activities, membrane interaction, and affinities for FVIII mutants. Relative activities, membrane interaction, and affinities for FVIII mutants. (A) Membrane interaction for FVIIIWT (■) or FVIIILS (▾) was evaluated by mixing FIXa, FX, and various concentrations of sonicated vesicles. The quantity of FXa formed was measured with chromogenic substrate S-2765. The membrane reactivity of FVIIILS was less than FVIIIWT. The vesicles had a composition of PS:PE:PC 4:20:76; concentration of FVIII or mutants was 0.2nM; FIXa, 4nM; and FX, 130nM. Results represent the mean ± SEM for at least 3 experiments, each performed in duplicate and fitted to a single-site binding model (smooth curves). (B) Apparent KM for the FVIIIa-FIXa complex was determined as in panel A, except that the phospholipid concentration was constant (100μM) and the FX concentration varied. (A-B) Fitted results were normalized to the Bmax value for each curve to optimize comparison of the relative affinities. (C) The specific activities, apparent phospholipid interactivity, and apparent FX affinities for each mutant are displayed with reference to control experiments performed with wild-type FVIII (as in panels A and B for FVIIILS). The displayed values represent the mean ± SEM. Each fit was performed on a minimum of 3 separate experiments, each performed in duplicate. (D) Apparent VWF affinity was determined by incubating FVIIIWT and mutants with various concentrations of VWF for 60 minutes before addition to a microtiter well coated with a monoclonal antibody that competes for the VWF-binding epitope. Bound FVIII was detected with a second antibody. The fraction of VWF-bound FVIII was calculated as the difference between the maximum signal in the absence of VWF and the signal at each concentration of VWF. Smooth curves represent best-fit curves corresponding to KD values of 0.7 ± 0.1nM, 2.2 ± 0.7nM, and 4.8 ± 0.8nM for FVIIIWT (■), FVIIIWW (▴), and FVIIILS (▾), respectively. Displayed results are the mean ± SEM of at least 2 experiments, each performed in duplicate. Gary E. Gilbert et al. Blood 2012;120:1923-1932 ©2012 by American Society of Hematology

Relative activities, membrane interaction, and affinities for FV mutants. Relative activities, membrane interaction, and affinities for FV mutants. (A) Membrane reactivity for FVWT (■) or FVLL (▾) was determined by mixing with FXa, prothrombin, and various concentrations of sonicated vesicles. The quantity of thrombin formed was measured with chromogenic substrate S-2238. The apparent affinity of FVLL was higher than FVWT. The vesicles had a composition of PS:PE:PC 4:20:76; concentrations of FV and mutants were 0.2nM; FXa, 2.5pM; and prothrombin, 2μM. Displayed represent the mean ± SEM, fitted to a single-site binding model (smooth curves). (B) Apparent FXa affinity was determined as in panel A, except that the phospholipid concentration was held constant (50μM) and the FXa concentration was varied. (C) Apparent KM for the FVa-FXa complex was determined as in panel A, except that the phospholipid concentration was constant and the prothrombin concentration varied. (A-C) Fitted results were normalized to the Bmax value for each curve to optimize comparison of the relative affinities. (D) The specific activities, phospholipid vesicle reactivity, apparent FXa affinities, and apparent prothrombin affinities (KM) for each mutant are displayed with reference to control experiments performed with wild-type FV (as in panels A-C for FVLL). The displayed values represent the mean ± SEM. Each fit was performed on a minimum of 3 separate experiments, each performed in duplicate. Gary E. Gilbert et al. Blood 2012;120:1923-1932 ©2012 by American Society of Hematology

Apparent membrane-independent activity of FV mutants resulting from copurification of phospholipid. Apparent membrane-independent activity of FV mutants resulting from copurification of phospholipid. (A) The potential presence of phospholipid was evaluated by testing the capacity of the factor FVWT and mutants to support the FXase complex. FV preparations were mixed with FVIIIa, FIXa, and FX in the absence of phospholipid vesicles. FV mutants supported FXase activity, whereas FVWT supported little or no activity. Data represent the mean ± SEM for 2 experiments for FVLL and FVWT and one experiment for the other mutants each performed in duplicate. (B) Lactadherin inhibition of FVMF-supported FXase activity was evaluated using increasing concentrations of lactadherin. Activity was inhibited with half-maximal inhibition at approximately 4nM. Data represent the mean ± SEM for a single experiment performed in duplicate. (C) Prothrombinase activity supported by CHAPS-washed FVWT and FV mutants in the absence of phospholipid vesicles was evaluated in the presence of various concentrations of lactadherin. Lactadherin inhibited activity supported by FVMF (▵) with half-maximal inhibition at approximately 16nM lactadherin. Half-maximal inhibition of activity supported by FVLL (▿) occurred at approximately 1nM, whereas half-maximal inhibition of FVMF/LL (♢) occurred at an intermediate concentration. Data represent the mean ± SEM for 2 experiments. (D) Inhibition of prothrombinase activity by phospholipase A2 was evaluated for FVLL (▿) in the absence of phospholipid vesicles and for FVWT (□) in the presence of phospholipid vesicles (inset). Phospholipase A2 inhibited prothrombinase activity under both conditions. Data represent the mean ± SEM for 3 experiments performed in duplicate. Gary E. Gilbert et al. Blood 2012;120:1923-1932 ©2012 by American Society of Hematology

P textilis–motivated FV mutant. P textilis–motivated FV mutant. (Left) Sequence alignment of the C2 domains from human FVIII (H-FVIII-C2), human FV (H-FV-C2), bovine FV (B-FV-C2), and the FV-homologous subunit of pseutarin C from P textilis venom (Pt-FV-C2). Black bars represent the 4 lipid binding spikes with the hydrophobic residues marked with triangles. Amino acids that were mutated to make FVMTTS/Y are starred. (Right) Locations of the mutated amino acids on the C2 domain of human FV are shown with original side chains in dark gray with the rest of the backbone in light gray. Residue numbers relate to the full-length human FV. Numbering references follow HGVS standard, using Met of respective propeptides as 1. Gary E. Gilbert et al. Blood 2012;120:1923-1932 ©2012 by American Society of Hematology

Membrane-independent activity, apparent prothrombin and FXa affinities, and phospholipid binding affinity of FVMTTS/Y. Membrane-independent activity, apparent prothrombin and FXa affinities, and phospholipid binding affinity of FVMTTS/Y. (A) Various concentrations of FVWT or FVMTTS/Y with or without a 1% CHAPS wash during purification were mixed with FXa (0.4nM) and prothrombin (1μM) in the absence of phospholipid vesicles. Washing with 1% CHAPS during purification abolished all apparent lipid-independent activity. (B) Apparent KM for the prothrombinase complex with mutant or WT CHAPS-washed FV was determined with saturating concentrations of vesicles of composition 4:20:76 (PS:PE:PC). FVMTTS/Y showed the same apparent affinity for prothrombin as FVWT. Values represent mean ± SEM for at least 2 experiments, each performed in duplicate. (C) Direct affinity for phospholipid vesicles was evaluated for FVWT or FVMTTS/Y by immobilizing FV to mAb CBC-MOR101 covalently linked to Superose beads. Beads were incubated overnight with either FVWT or FVMTTS/Y and then mixed with various concentrations of fluorescein-labeled vesicles of composition 4:5:20:71 (PS:PE-CF:PE:PC). After 30 minutes, the quantity of vesicles bound to FVWT or FVMTTS/Y was measured by flow cytometry. The vesicle dissociation constants were 4.8 ± 1.1μM for FVMTTS/Y and 1.7 ± 0.4μM for FVWT. Data represent the mean ± SEM for at least 4 experiments. Gary E. Gilbert et al. Blood 2012;120:1923-1932 ©2012 by American Society of Hematology

Decreased PS dependence of FVMTTS/Y activity on vesicles and platelets. Decreased PS dependence of FVMTTS/Y activity on vesicles and platelets. (A) Activity on vesicles with high PS content was determined with 5pM CHAPS-washed FVWT or FVMTTS/Y and various concentrations of vesicle of composition 15:20:65 (PS:PE:PC). (B) Activity on vesicles with limiting PS was evaluated with vesicles of composition 2:20:78 (PS:PE:PC) using 10pM FV. (C) Activity on vesicles lacking PS was evaluated on vesicles of composition 20:80 (PE:PC) using 50pM FV. FVMTTS/Y showed increasingly higher relative prothrombinase activity with lower vesicle PS content. Concentrations of FXa and prothrombin were 1nM and 1μM, respectively. Data represent the mean ± SEM for 3 experiments, each performed in duplicate. (D) Prothrombinase activity of FVMTTS/Y on unstimulated platelets or platelets incubated for 10 minutes with 10μM thrombin receptor agonist protein was measured in the presence of FXa and prothrombin. Platelets were suspended at 4 × 107/mL, and concentrations of FV and mutant were 20pM; FXa, 25pM; and prothrombin 1μM. Data represent the mean ± SD for 2 experiments, each performed in duplicate. Probabilities were calculated using the Student t test. Gary E. Gilbert et al. Blood 2012;120:1923-1932 ©2012 by American Society of Hematology