1. by Mark S. Hertzberg, Sandra L. Facey, and Philip J. Hogg
An Arg/Ser Substitution in the Second Epidermal Growth Factor–Like Module of Factor IX Introduces an O-Linked Carbohydrate and Markedly Impairs Activation by Factor XIa and Factor VIIa/Tissue Factor and Catalytic Efficiency of Factor IXa
by Mark S. Hertzberg, Sandra L. Facey, and Philip J. Hogg
Blood
Volume 94(1):
July 1, 1999
©1999 by American Society of Hematology

2. SDS-PAGE analysis of purified recombinant wild-type factor IX and factor IXR94S.
SDS-PAGE analysis of purified recombinant wild-type factor IX and factor IXR94S. Recombinant proteins (3 μg) were resolved on 12.5% SDS-PAGE under reducing conditions and stained with Coomassie Blue.
Mark S. Hertzberg et al. Blood 1999;94:
©1999 by American Society of Hematology

3. Comparison of the Gla-domains of plasma factor IX, wild-type factor IX, and factor IXR94S.
Comparison of the Gla-domains of plasma factor IX, wild-type factor IX, and factor IXR94S. (A) Increasing moles of factor IXpl (○), factor IXwt (•), or factor IXR94S (▴) were coated onto microtiter wells and then incubated with 15 μg/mL of IX/X-bp in the presence of 5 mmol/L Ca2+ in TBS. The binding of IX/X-bp to factor IX was measured by ELISA. The three factor IX proteins bound IX/X-bp equally well. (B) Binding of factor IX to 75:25 DOPC:DOPS vesicles. The three factor IX proteins bound phospholipid vesicles equally well. The bars and errors represent the mean and standard deviation (SD) of three experiments.
Mark S. Hertzberg et al. Blood 1999;94:
©1999 by American Society of Hematology

4. SDS-PAGE analysis of factor XIa activation of plasma factor IX and wild-type factor IX. Factor IXpl (A) or IXwt (B) (2.7 μmol/L) was incubated with factor XIa (5 nmol/L) in TBS containing 10 mmol/L Ca2+ at 37°C.
SDS-PAGE analysis of factor XIa activation of plasma factor IX and wild-type factor IX. Factor IXpl (A) or IXwt (B) (2.7 μmol/L) was incubated with factor XIa (5 nmol/L) in TBS containing 10 mmol/L Ca2+ at 37°C. At discrete time intervals, aliquots (3 μg) of the reactions were resolved on 12.5% SDS-PAGE under reducing conditions and stained with Coomassie Blue.
Mark S. Hertzberg et al. Blood 1999;94:
©1999 by American Society of Hematology

5. Western blot analysis of factor XIa activation of plasma factor IX and factor IXR94S using a factor IXa heavy chain antibody.
Western blot analysis of factor XIa activation of plasma factor IX and factor IXR94S using a factor IXa heavy chain antibody. Factor IXpl (A) or IXR94S (B) (2.7 μmol/L) was activated by factor XIa (18 nmol/L) in TBS containing 10 mmol/L Ca2+ at 37°C. The activation products were sampled at the indicated time intervals, resolved on reducing 12.5% SDS-PAGE, and transferred to Immobilon-P polyvinylidene fluoride (PVDF) membrane. The membrane was blotted with the C10D monoclonal antibody, which recognizes the heavy chain (HC) of factor IXa, and developed using chemiluminescence. The positions of intact factor IX, IX (HC + AP) and IXaHC are indicated. The positions of the molecular weight markers are indicated at left.
Mark S. Hertzberg et al. Blood 1999;94:
©1999 by American Society of Hematology

6. Western blot analysis of factor XIa activation of plasma factor IX and factor IXR94S using a factor IXa light chain antibody.
Western blot analysis of factor XIa activation of plasma factor IX and factor IXR94S using a factor IXa light chain antibody. Factor IXpl (A) or IXR94S (B) (2.7 μmol/L) was activated by factor XIa (18 nmol/L) in TBS containing 10 mmol/L Ca2+ at 37°C. The activation products were sampled at the indicated time intervals, resolved on reducing 12.5% SDS-PAGE, and transferred to PVDF membrane. The membrane was blotted with the JKIX monoclonal antibody, which recognizes the light chain (LC) of factor IXa, and developed using chemiluminescence. The positions of intact factor IX and IXaLC are indicated. The dotted arrow in (B) indicates the usual migration position of the factor IXa light chain. The positions of the molecular weight markers are indicated at left.
Mark S. Hertzberg et al. Blood 1999;94:
©1999 by American Society of Hematology

7. Western blot analysis of factor VIIa/tissue factor/DOPC:DOPS/Ca2+ enzyme complex activation of plasma factor IX and factor IXR94S using a factor IXa heavy chain antibody.
Western blot analysis of factor VIIa/tissue factor/DOPC:DOPS/Ca2+ enzyme complex activation of plasma factor IX and factor IXR94S using a factor IXa heavy chain antibody. Factor IXpl (A) or IXR94S (B) was activated by factor VIIa/tissue factor/DOPC:DOPS/Ca2+ enzyme complex in TBS at 37°C. The reaction component concentrations were: 0.5 μmol/L factor IXpl or IXR94S, 2 nmol/L factor VIIa, 4 nmol/L tissue factor, 1 mmol/L DOPC:DOPS, and 5 mmol/L Ca2+. The activation products were sampled at the indicated time intervals, resolved on reducing 5% to 15% SDS-PAGE, and transferred to PVDF membrane. The membrane was blotted with the C10D monoclonal antibody, which recognizes the heavy chain (HC) of factor IXa, and developed using chemiluminescence. The positions of intact factor IX and IXaHC are indicated. The positions of the molecular weight markers are indicated at left.
Mark S. Hertzberg et al. Blood 1999;94:
©1999 by American Society of Hematology

8. Comparison of the amidolytic activity of plasma factor IXa and factor IXaR94S.
Comparison of the amidolytic activity of plasma factor IXa and factor IXaR94S. Varying concentrations of plasma factor IXa (○) or factor IXaR94S (•) were incubated with 2.5 mmol/L CHG-GR-pNA in TBS containing 0.1% BSA, and the increase in absorbance at 405 nm with time was measured. The initial velocity of p-nitroaniline formation is expressed as a function of enzyme concentration. The solid lines represent the linear regression fit to the data. The observed rate of hydrolysis of CHG-GR-pNA by plasma factor IXa and factor IXaR94S was 1.51 ± 0.06 min−1 and 1.68 ± 0.02 min−1, respectively.
Mark S. Hertzberg et al. Blood 1999;94:
©1999 by American Society of Hematology

9. Comparison of the factor X–activating activity of plasma factor IXa and factor IXaR94S in the presence of polylysine.
Comparison of the factor X–activating activity of plasma factor IXa and factor IXaR94S in the presence of polylysine. Human factor X (600 nmol/L) was activated by 50 nmol/L plasma factor IXa (○) or factor IXaR94S (•) in the presence of polylysine (60 nmol/L) in 0.1 mol/L triethanolamine, 0.1 mol/L NaCl, 0.1% PEG 6000, pH 9.0 buffer at 37°C. At discrete time intervals, aliquots of the reactions were assayed for factor Xa concentration using CHG-GR-pNA. The results are expressed as concentration of factor Xa formed as a function of time. The solid lines represent the linear regression fit to the data. The initial rate of factor X activation by plasma factor IXa and factor IXaR94S was 2.54 ± 0.07 nmol/L/min−1 and 0.19 ± 0.01 nmol/L/min−1, respectively.
Mark S. Hertzberg et al. Blood 1999;94:
©1999 by American Society of Hematology

10. Comparison of the catalytic activity of plasma factor IXa and factor IXaR94S in the tenase enzyme complex.
Comparison of the catalytic activity of plasma factor IXa and factor IXaR94S in the tenase enzyme complex. Plasma factor IXa (0.17 nmol/L, ○, ___) or factor IXaR94S (1.5 nmol/L, •, —-) was incubated with factor X (0 to 100 nmol/L) in the presence of phospholipid vesicles (35 μmol/L), factor VIIIa (14 U/mL) and Ca2+ (5 mmol/L) in TBS containing 0.1% BSA at 37°C. At discrete time intervals, aliquots of the reactions were quenched with 50 mmol/L EDTA and factor Xa quantitated using CHG-GR-pNA. The data has been normalized with respect to enzyme concentration by expressing the ordinate axis as initial velocity, vi, divided by the factor IXa or factor IXaR94S concentration. The lines represent the best fit of the data to the Michaelis-Menten equation using nonlinear regression analysis. The kinetic parameters are summarized in Table 1.
Mark S. Hertzberg et al. Blood 1999;94:
©1999 by American Society of Hematology