Summary of results with wild-type and mutant human rFVa2 and C6PS
| Proteins* . | Kd, C6PS, μM . | Kd, FXa, nM . | Stoichiometry of binding to C6PS, no. . | Cofactor activity,‡ nM/sec . |
|---|---|---|---|---|
| rFVa2 | 20 ± 121 | 3.1 ± 0.4 | 4.1 ± 0.1 | 15 000 ± 455 (3) |
| rFVa2 (W2063,W2064)A | 37 ± 121 | 4.0 ± 0.6 | 3.1 ± 0.2 | 14 300 ± 470 (3) |
| rFV C2 | 2.8 ± 0.321 | NA | 1.1 ± 0.217 | NA |
| rFV C2 (W2063,W2064)A | >300† | NA | <0.0617 | NA |
| rFVa2 (Y1956,L1957)A | 47 ± 5 (3) | 564 ± 35 (3) | 3.04 ± 0.1 | 1.1 ± 0.4 (3) |
| rFVa2 (Y1956,L1957,W2063,W2064)A | NM | 624 ± 40 (3) | 2.1 ± 0.2 | 1.0 ± 0.2 (3) |
| Proteins* . | Kd, C6PS, μM . | Kd, FXa, nM . | Stoichiometry of binding to C6PS, no. . | Cofactor activity,‡ nM/sec . |
|---|---|---|---|---|
| rFVa2 | 20 ± 121 | 3.1 ± 0.4 | 4.1 ± 0.1 | 15 000 ± 455 (3) |
| rFVa2 (W2063,W2064)A | 37 ± 121 | 4.0 ± 0.6 | 3.1 ± 0.2 | 14 300 ± 470 (3) |
| rFV C2 | 2.8 ± 0.321 | NA | 1.1 ± 0.217 | NA |
| rFV C2 (W2063,W2064)A | >300† | NA | <0.0617 | NA |
| rFVa2 (Y1956,L1957)A | 47 ± 5 (3) | 564 ± 35 (3) | 3.04 ± 0.1 | 1.1 ± 0.4 (3) |
| rFVa2 (Y1956,L1957,W2063,W2064)A | NM | 624 ± 40 (3) | 2.1 ± 0.2 | 1.0 ± 0.2 (3) |
Two or (where indicated in parentheses) three determinations in the present study were made. The average standard error from the nonlinear regression analysis is also given (as ±).
NM indicates not measured (not performed because the two light chain sites have been eliminated and binding of C6PS to the heavy chain of bovine FVa has previously been characterized [data fit by two linked site model: Kd1 ∼2.2 and Kd2 ∼260 μM22 ]); NA, not applicable (not applicable because C2 domain has no activity and its binding to FXa is not the subject of this study).
Kd of C1 (Y1956,L1957)A and C1C2 (Y1956,L1957,W2063,W2064)A mutants are similar to the Kd of FVa:FXa in absence of C6PS.
Kd estimated from sensitivity of the stoichiometry measurement.
We note an error in our previous papers where we first defined FVa2 “cofactor activity” in the presence of C6PS.16,22 In those papers, we mistakenly used different units for the initial rates of prothrombin activation by FXa-FXa-C6PS and FXa-FVa, resulting in values 103 times too small.