Contribution of amino acid region 334-335 from factor Va heavy chain to the catalytic efficiency of prothrombinase

Abstract

We have demonstrated that amino acids E (323), Y (324), E (330), and V (331) from the factor Va heavy chain are required for the interaction of the cofactor with factor Xa and optimum rates of prothrombin cleavage. We have also shown that amino acid region 332-336 contains residues that are important for cofactor function. Using overlapping peptides, we identified amino acids D (334) and Y (335) as contributors to cofactor activity. We constructed recombinant factor V molecules with the mutations D (334) --> K and Y (335) --> F (factor V (KF)) and D (334) --> A and Y (335) --> A (factor V (AA)). Kinetic studies showed that while factor Va (KF) and factor Va (AA) had a K D for factor Xa similar to the K D observed for wild-type factor Va (factor Va (WT)), the clotting activities of the mutant molecules were impaired and the k cat of prothrombinase assembled with factor Va (KF) and factor Va (AA) was reduced. The second-order rate constant of prothrombinase assembled with factor Va (KF) or factor Va (AA) for prothrombin activation was approximately 10-fold lower than the second-order rate constant for the same reaction catalyzed by prothrombinase assembled with factor Va (WT). We also created quadruple mutants combining mutations in the amino acid region 334-335 with mutations at the previously identified amino acids that are important for factor Xa binding (i.e., E (323)Y (324) and E (330)V (331)). Prothrombinase assembled with the quadruple mutant molecules displayed a second-order rate constant up to 400-fold lower than the values obtained with prothrombinase assembled with factor Va (WT). The data demonstrate that amino acid region 334-335 is required for the rearrangement of enzyme and substrate necessary for efficient catalysis of prothrombin by prothrombinase.

Figure 1

Proteolytic processing of human factor V. Factor V is composed of 2196 amino acids and has three A domains (A1, A2, and A3), a connecting B region, and two C domains (C1 and C2). The procofactor is activated following three cleavages by thrombin at Arg⁷⁰⁹, Arg¹⁰¹⁸, and Arg¹⁵⁴⁵. The latter cleavage is the last to occur and is a prerequisite for light chain formation and maximum cofactor activity generation. These cleavages release the active cofactor composed of heavy and light chains associated in the presence of divalent metal ions, and two activation fragments. The mutations within a 45-amino acid region of the A2 domain of the heavy chain of the molecule that is involved in cofactor activity (hatched box) are indicated together with the designation for the recombinant mutant factor V molecules created and used throughout the paper. The black box preceding cleavage at Arg⁷⁰⁹ depicts the acidic COOH-terminal region of factor Va that is essential for optimum cofactor activity.

Figure 2

Factor Va heavy chain peptides. At the top, the sequence of N42R is illustrated (part of the hatched box in Figure 1). The underlined sequence (323−335, 13 amino acids) is the regulatory amino acid sequence of the factor Va heavy chain. Overlapping peptides from the central portion of the factor Va heavy chain [AP3−AP7 (23)] are also shown. The role of the underlined amino acid region included in the box (i.e., Arg³³⁴-Tyr³³⁵) and common to IWDYA, AP5 and AP6, is under investigation here. Bold and underlined residues are the amino acid residues mutated in this study.

Figure 3

Inhibitory potential of factor Va heavy chain peptides. (A) Inhibition of prothrombinase activity. Increasing concentrations of AP5, AP6, P15H, IWDYA, and AP5^DY→KF were preincubated with factor Xa and assayed for prothrombinase activity as described in . P15H (◼) represents the control peptide containing amino acids 337−351 of the human factor Va heavy chain (see Figure 2 for details). AP5 (◆) represents amino acids 327−336 of factor Va. AP5^DY→KF (△) represents amino acid residues 327−336 with residues 334 and 335 mutated from D and Y to K and F, respectively. IWDYA (◻) represents amino acid residues 332−336 of the human factor Va heavy chain. Data for AP6 are shown with ●. The concentration of peptide given on the x axis represents its final concentration in the prothrombinase mixture. The data represent the average of the results found in three independent experiments. The apparent inhibition constant (K_i) reported in the text is the value calculated from the formula IC₅₀ = K_i(1 + S₀/K_m) (53), where K_m is the Michaelis−Menten constant of the reaction in the absence of inhibitor, S₀ is the concentration of prothrombin used, and IC₅₀ is the half-maximal inhibition of prothrombinase by a given peptide. (B) Inhibition of prothrombinase assembly. Prothrombinase was assembled with [OG₄₈₈]-EGR-hXa (10 nM), PCPS vesicles (10 µM), and increasing concentrations of human factor Va (up to 25 nM) in the presence of 2 mM Ca²⁺. Once a plateau was obtained, the preformed complexes were titrated with increasing concentrations of P15H (◼), AP3 (▲), AP4′ (▼), AP5 (◆), and AP6 (●). Δr was calculated as described in and plotted as extensively detailed previously (23). The data represent all the data points from two independent experiments performed in triplicate.

Figure 4

Kinetic analyses of prothrombin activation by prothrombinase in the presence of AP5. (A) Michaelis−Menten plots. Prothrombin generation experiments were performed in the absence (◼) and presence of increasing concentrations of AP5 [10 (▼), 15 (◆), and 20 µM (▲)], as described in using prothrombin concentrations varying from 40 nM to 1 µM. Initial rates of thrombin formation are plotted as a function of substrate concentration. (B) Analysis of the data using the Dixon plots. The data were analyzed and plotted as 1/V_o as a function of inhibitor concentration (AP5, Dixon plots). For mixed-type inhibition, a Dixon plot of 1/V_o as a function of increasing concentration of inhibitor is linear at a fixed enzyme and substrate concentration (53). The apparent inhibition constant (K_i) reported in the text is the value derived from the intercept of each of the four graphs (∼10.5 µM). The lines drawn represent the best fit through the points with an R² varying from 0.9812 (worst) to 0.9881 (best). The following concentrations of prothrombin were used in the experiments: 80 (◆), 100 (●), 150 (◻), and 200 nM (△).

Figure 5

Prothrombin titrations to determine the kinetic parameters of prothrombinase assembled with the various recombinant factor Va species. Thrombin generation experiments were carried out as described in by varying the substrate concentration. The raw data (hyperbolas) are shown in panel A: (◼ and ◻) two different preparations of wild-type factor Va, (○) factor Va^AA, (●) factor Va^FF-KF, (▽ and ▼) two different preparations of factor Va^KF, (crossed boxes) factor Va^MI-AA, (◆) factor Va^MI-KF, (◼) factor Va^FF-AA, (+) factor Va^I332A, and (×) factor Va^W333A. The K_m values for all cofactor molecules are given in Table 1, while the k_cat values are shown in panel B (min⁻¹). Panel C provides the second-order rate constants (M⁻¹ s⁻¹). The data represent the average values found in at least three different titrations for each recombinant factor Va molecule.

Scheme 1. Thermodynamic Cycle for Prothrombinase Activity following Substitutions in the Factor Va Heavy Chain

ΔΔG_int is the free energy of interaction between the side chains of D³³⁴Y³³⁵ and E³²³Y³²⁴/E³³⁰V³³¹ of the factor Va heavy chain and was calculated with eqs 3 and 4 described in . The values illustrated on the arrows are the ΔΔG^⧧ values between recombinant species as indicated. Parallel arrows show the difference in catalytic efficiency between prothrombinase assembled with different mutant factor Va molecules. Different forms of prothrombinase are compared for their ability to activate prothrombin.

Figure 6

Structural analyses of the factor V heavy chain. A portion of the prothrombin interactive site containing amino acids D⁶⁹⁵−Q^699,, is shown as magenta spheres. The α-helix contained in S⁵²⁸−F⁵³⁸ is colored red. This region contains a secondary binding site for factor Xa (24). The β-sheet contained in the R³²¹−V³³¹/K³⁶⁵−T³⁶⁹ sequence that is important for expression of factor Va cofactor activity (22,23,29) is colored yellow. The loop contained within the sequence of amino acid residues 400−420 is shown with cyan ribbons. Amino acid residues F⁵³⁸ and H³⁶² are shown as green sticks. S⁴¹² is shown as cyan sticks. N⁵³⁴ and K³⁶⁴ are shown as lime sticks. Amino acid residues Y⁵³⁰ and E³³⁰ are shown as olive sticks, and E³²³ is shown as orange sticks. The final snapshot at 2 ns of factor Va^WT is shown in panel A with amino acid residues D³³⁴ and Y³³⁵ represented as blue sticks. Panel B shows the final snapshot at 3.5 ns of factor Va^KF with the mutated amino acid residues K³³⁴ and Phe³³⁵ as blue sticks and R⁴¹³ and S⁴¹² as cyan sticks. Panel C is a snapshot at 2 ns of factor Va^AA with the mutated amino acid residues A³³⁴ and A³³⁵ colored blue.