by Andrew J. Gale, Mary J. Heeb, and John H. Griffin The autolysis loop of activated protein C interacts with factor Va and differentiates between the Arg506 and Arg306 cleavage sites by Andrew J. Gale, Mary J. Heeb, and John H. Griffin Blood Volume 96(2):585-593 July 15, 2000 ©2000 by American Society of Hematology

Anticoagulant activity of APC single-point mutants Anticoagulant activity of APC single-point mutants.The anticoagulant activity of the autolysis loop single mutants was tested in an APTT assay as described in “Materials and methods” over a range of APC concentration from 1.6 to 6.3 nmol/L. Anticoagulant activity of APC single-point mutants.The anticoagulant activity of the autolysis loop single mutants was tested in an APTT assay as described in “Materials and methods” over a range of APC concentration from 1.6 to 6.3 nmol/L. (A) APC: wild-type (+), 306A (▵), 307A (○), 308A (◊), 309A (□). (B) APC: wild-type (+), 311A (▵), 312A (○), 314A (◊). All curves are averaged from 4 to 8 individual experiments. Andrew J. Gale et al. Blood 2000;96:585-593 ©2000 by American Society of Hematology

Inactivation of Factor Va by APC single-point mutants Inactivation of Factor Va by APC single-point mutants.FVa was incubated at a concentration of 1 nmol/L with 50 pmol/L wild-type or mutant APC and inactivation was followed over time as described in “Materials and methods.” (A) APC: wild-type (+), 306A (▵), ... Inactivation of Factor Va by APC single-point mutants.FVa was incubated at a concentration of 1 nmol/L with 50 pmol/L wild-type or mutant APC and inactivation was followed over time as described in “Materials and methods.” (A) APC: wild-type (+), 306A (▵), 307A (○), 308A (◊), 309A (□). (B) APC: wild-type (+), 311A (▵), 312A (○), 314A (◊). Data points shown are averages of between 3 to 6 experiments for each APC. Standard deviations for all the data points shown averaged ± 3.4%. Error bars are shown for wild-type APC in panel B for illustrative purposes. Other error bars are left out because many of the curves are so close to each other. Curves were fit to the equation in “Materials and methods” according to Nicolaes and colleagues.l11 Coefficient of determination (r2) values were 0.995 or greater for all the curves except for that of 314A-APC (0.977). Andrew J. Gale et al. Blood 2000;96:585-593 ©2000 by American Society of Hematology

Anticoagulant activity of APC single-point mutants relative to wild-type APC in normal human plasma versus homozygous Q506-FV plasma.(A) Normal human plasma. Anticoagulant activity of APC single-point mutants relative to wild-type APC in normal human plasma versus homozygous Q506-FV plasma.(A) Normal human plasma. (B) Q506-FV plasma. APTT assays were done using plasma mixtures containing 10% normal plasma or 10% Q506-FV plasma and 90% FV-deficient plasma. The percentage of wild-type activity for each APC mutant was calculated as follows. The wild-type APC dose-response data were fit to a line and then that equation was used to calculate a relative amount of wild-type APC for the respective clotting time values for each mutant. For each experiment, values from 2 to 3 concentration points for each mutant were averaged together to get a percent of wild-type APC activity for that experiment. The results of 2 to 3 experiments were averaged together and the SDs are shown as error bars. Andrew J. Gale et al. Blood 2000;96:585-593 ©2000 by American Society of Hematology

Anticoagulant activity of mutants 306/312AA-APC and 306-314AAAA-APC versus wild-type APC.(A) Normal human plasma: wild-type APC (+), 306A/312AA-APC (○), 306A-314AAAA-APC (▵). Anticoagulant activity of mutants 306/312AA-APC and 306-314AAAA-APC versus wild-type APC.(A) Normal human plasma: wild-type APC (+), 306A/312AA-APC (○), 306A-314AAAA-APC (▵). (B) Q506-FV human plasma: wild-type APC (+), 306A/312AA-APC (○), 306A-314AAAA-APC (▵). APTT assays were performed using plasma mixtures containing 10% normal human plasma or 10% Q506-FV human plasma as described above. Data shown are the average of 4 experiments for each line and SDs are shown as error bars. Andrew J. Gale et al. Blood 2000;96:585-593 ©2000 by American Society of Hematology

Western blot of FVa inactivation by wild-type APC and 306-314AAAA-APC Western blot of FVa inactivation by wild-type APC and 306-314AAAA-APC.(A) Wild-type APC, 200 pmol/L. Western blot of FVa inactivation by wild-type APC and 306-314AAAA-APC.(A) Wild-type APC, 200 pmol/L. (B) 306-314AAAA-APC, 2 nmol/L. FVa at a concentration of 20 nmol/L was incubated in 50 mmol/L HEPES, pH 7.4, 100 mmol/L NaCl, 5 mmol/L CaCl2, 0.1 mmol/L MnCl2, 0.1% BSA with 25 μmol/L phospholipid vesicles. APC was added and aliquots were removed at 1 through 40 minutes. The Western blot was developed as described in “Materials and methods.” Molecular weight standards are indicated on the left in kilodalton units. On the right fragments of the FVa heavy chain are labeled. HC = intact FVa heavy chain. Andrew J. Gale et al. Blood 2000;96:585-593 ©2000 by American Society of Hematology