Amino acid region 1000-1008 of factor V is a dynamic regulator for the emergence of procoagulant activity

Abstract

Single chain factor V (fV) circulates as an Mr 330,000 quiescent pro-cofactor. Removal of the B domain and generation of factor Va (fVa) are vital for procoagulant activity. We investigated the role of the basic amino acid region 1000-1008 within the B domain of fV by constructing a recombinant mutant fV molecule with all activation cleavage sites (Arg(709)/Arg(1018)/Arg(1545)) mutated to glutamine (fV(Q3)), a mutant fV molecule with region 1000-1008 deleted (fV(ΔB9)), and a mutant fV molecule containing the same deletion with activation cleavage sites changed to glutamine (fV(ΔB9/Q3)). The recombinant molecules along with wild type fV (fV(WT)) were transiently expressed in COS-7L cells, purified, and assessed for their ability to bind factor Xa (fXa) prior to and following incubation with thrombin. The data showed that fV(Q3) was severely impaired in its interaction with fXa before and after incubation with thrombin. In contrast, KD(app) values for fV(ΔB9) (0.9 nM), fVa(ΔB9) (0.4 nM), and fV(ΔB9/Q3) (0.7 nM) were similar to the affinity of fVa(WT) for fXa (0.3 nM). Two-stage clotting assays revealed that although fV(Q3) was deficient in its clotting activity, fV(ΔB9/Q3) had clotting activity comparable with fVa(WT). The kcat value of prothrombinase assembled with fV(ΔB9/Q3) was minimally affected, whereas the Km value of the reaction was increased 57-fold compared with the Km value obtained with prothrombinase assembled with fVa(WT). These findings strongly suggest that amino acid region 1000-1008 of fV is a regulatory sequence protecting the organisms from spontaneous binding to fXa and unnecessary prothrombinase complex formation, which in turn results in catastrophic physiological consequences.

Keywords: Coagulation Factors; Factor V; Factor Va; Factor Xa; Phospholipid Vesicle; Protein Chemistry; Prothrombin; Prothrombinase; Thrombin.

FIGURE 1.

FV structure and mutant molecules. The pro-cofactor fV is composed of three A domains (red), a connecting B domain (yellow), and two C domains (blue). fV undergoes three sequential cleavages by thrombin at Arg⁷⁰⁹, Arg¹⁰¹⁸, and Arg¹⁵⁴⁵ to generate the active cofactor fVa. The deletion within the basic homologous region of the B domain (amino acid residues 1000–1008) is coupled with mutations at each one of the thrombin activation sites (Arg → Gln). The recombinant mutant fV molecules created are indicated with specific designations used throughout this work.

FIGURE 2.

Comparison of the basic region of amino acid sequences 1000–1008 from fV B domain among multiple species. The databases GenBank^TM and NCBI Trace Archive were used to derive sequences of fV among various mammalian species to compare homology. The basic amino acid sequence of interest is shown in red. The following species are included (from top to bottom): Homo sapiens, human; Pan troglodytes, chimpanzee; Pongo abelii, Sumatran orangutan; Bos taurus, cattle; Sus scrofa, pig; Callithrix jacchus, white-tufted-ear marmoset; Rattus norvegicus, Norway rat; Mus musculus, western European house mouse.

FIGURE 3.

Electrophoretic analyses of the purified recombinant molecules. Purified recombinant fV^WT and purified recombinant fV molecules fV^ΔB9, fV^ΔB9/Q3, fV^RQQ, and fV^Q3 were incubated with thrombin as described under “Experimental Procedures” and analyzed by SDS-PAGE followed by staining with silver. Panels A–C show the molecules before (−) and after activation by thrombin (+). Panels D–F show the molecules before (0 min) and after incubation with thrombin for 10 and 20 min. The identity of each fV molecule is shown below each panel. HC, heavy chain; LC, light chain.

FIGURE 4.

Determination of the dissociation constant of recombinant fV^ΔB9/Q3 and fV^Q3 for plasma fXa. Thrombin generation experiments were carried out as described under “Experimental Procedures.” Prothrombinase assembled with varying concentrations of recombinant purified fV^Q3 is depicted by open squares, prothrombinase assembled with purified recombinant fV^ΔB9/Q3 by filled squares, and prothrombinase assembled with purified recombinant fVa^RQQ by filled triangles. Prothrombinase assembled with varying concentrations of recombinant purified fVa^WT is depicted by filled circles, and prothrombinase assembled with purified recombinant fVa^plasma is depicted by open circles. The solid lines represent a nonlinear regression fit of the data using Prism® GraphPad software and the equation describing the one binding site model. Titrations shown herein were performed with multiple preparations of recombinant proteins as detailed in Table 1. The kinetic constants derived directly from the plotted data are also reported in Table 1. The assay was performed with fV/fVa species varying from 0 to 15 nm. However, for the easy plotting of the data, only points from 0 to 10 nm are shown.

FIGURE 5.

Analyses of back activation of wild type fV and recombinant fV molecules within a prothrombinase assay. fVa^WT, fVa^ΔB9, fV^ΔB9, fVa^RQQ, and fV^ΔB9/Q3 were incubated in a prothrombin activation assay mixture before (lane 1) and 1 h after (lane 2) the addition of fXa as described under “Experimental Procedures.” The reaction was stopped as described under “Experimental Procedures.” After SDS-PAGE and transfer to a PVDF membrane, fV fragments were detected using monoclonal antibodies αHFVa_HC17 and αHFVa_LC9 recognizing the heavy chain (HC) and light chain (LC), respectively. At right, the positions of fV and the heavy and light chains of fVa are shown.

FIGURE 6.

Determination of kinetic parameters of prothrombinase assembled with various fV/Va species. Initial rates of thrombin generation were determined using the K_D(app) for fXa found in Fig. 4 to determine the concentration of fVa necessary to obtain over 98% fXa saturation as described under “Experimental Procedures” in the presence of PCPS vesicles and fXa (32, 33). Data for prothrombinase assembled with fVa^WT are shown by filled circles (20 nm, 98.6% fXa saturation). Data for prothrombinase assembled with 60 nm fV^ΔB9/Q3 are depicted by filled squares (98% fXa saturation), and data for prothrombinase assembled with 80 nm fV^ΔB9/Q3 are depicted by open inverse triangles (99% fXa saturation). Data for prothrombinase assembled with fVa^RQQ are shown by filled diamonds. The titration shown with 60 nm fV^ΔB9/Q3 is the average of results from experiments performed with five different preparations of purified recombinant protein, although the titration with 80 nm fV^ΔB9/Q3 is the average from two different preparations of recombinant protein. The kinetic constants were derived directly from the plotted data and are reported in Tables 1 and 2.

FIGURE 7.

SDS-PAGE analyses of prothrombin-activated fragments by prothrombinase assembled with various recombinant molecules. Panel A, plasma-derived prothrombin (1.4 μm) was activated by prothrombinase assembled with fVa^WT (final concentration of 20 nm, 96.6% fXa saturation); panel B, prothrombinase assembled with fV^ΔB9/Q3 (final concentration of 60 nm, 99% fXa saturation). Aliquots were withdrawn at various time intervals and treated as described (18, 40). M represents the lane with molecular weight markers (from top to bottom): 98,000, 64,000, 50,000, and 36,000, respectively. Lanes 1–19 show samples from the reaction mixture before (0 min) the addition of fXa and 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, and 240 s and 5, 6, 10, 20, 30, and 60 min, respectively, after the addition of fXa. Positions of prothrombin-derived fragments are shown to the right of panel B.

FIGURE 8.

Analysis of prothrombin consumption by prothrombinase assembled with recombinant fV/fVa molecules. The two gels shown in Fig. 7 were scanned, and prothrombin consumption was recorded as described under “Experimental Procedures.” Following scanning densitometry, the data representing prothrombin consumption as a function of time (seconds) were plotted using nonlinear regression analysis according to the equation representing a first-order exponential decay using the software Prism®. The apparent first-order rate constant, k (s⁻¹), was obtained directly from the fitted data. Prothrombinase was assembled with recombinant fVa^WT (filled circles), fV^ΔB9/Q3 (filled squares), and factor Va^RQQ (gel not shown in Fig. 7, filled triangles).

FIGURE 9.

Schematic interpretation of the data. In full-length fV, the basic amino acid region 1000–1008 from the B domain (blue) covers the fXa-binding site(s) located on the A2 and A3 of the molecule (yellow). The acidic 680–709 region is shown in purple. From the kinetic and binding data obtained, we can hypothesize that fV^ΔB9/Q3 and fVa^RQQ are able to have a productive interaction with prothrombin because both the fXa and prothrombin-bindings sites are available.