Regulation of factor V by the anticoagulant protease activated protein C: Influence of the B-domain and TFPIα

Abstract

Activated protein C (APC) is an important anticoagulant protein that regulates thrombin generation through inactivation of factor V (FV) and activated factor V (FVa). The rate of APC inactivation of FV is slower compared to FVa, although proteolysis occurs at the same sites (Arg³⁰⁶, Arg⁵⁰⁶, and Arg⁶⁷⁹). The molecular basis for FV resistance to APC is unknown. Further, there is no information about how FV-short, a physiologically relevant isoform of FV with a shortened B-domain, is regulated by APC. Here, we identify the molecular determinants which differentially regulate APC recognition of FV versus FVa and uncover how FV-short can be protected from this anticoagulant pathway. Using recombinant FV derivatives and B-domain fragments, we show that the conserved basic region (BR; 963-1008) within the central portion of the B-domain plays a major role in limiting APC cleavage at Arg⁵⁰⁶. Derivatives of FV lacking the BR, including FV-short, were subject to rapid cleavage at Arg⁵⁰⁶ and were inactivated like FVa. The addition of a FV-BR fragment reversed this effect and delayed APC inactivation. We also found that anticoagulant glycoprotein TFPIα, which has a C-terminal BR homologous to FV-BR, protects FV-short from APC inactivation by delaying cleavage at Arg⁵⁰⁶. We conclude that the FV-BR plays a major role in protecting FV from APC inactivation. Using a similar mechanistic strategy, TFPIα also shields FV-short from APC. These findings clarify the resistance of FV to APC, advance our understanding of FV/FVa regulation, and establish a mechanistic framework for manipulating this reaction to alter coagulation.

Keywords: acidic and basic residues; coagulation factor; cofactor; factor V; factor Va; hemostasis; procofactor; protein complex; prothrombinase; thrombin; tissue factor pathway inhibitor.

Figure 1

Schematic representation and SDS-PAGE of purified FV species.A, schematic representation of FV shows the HC, LC, and intervening B-domain (residues 710–1545) with the conserved BR (shaded blue), AR1, and AR2 (both shaded red). FV-1033 and FV-s46 have residues 1034 to 1491 truncated and FV-s46 has BR residues 963 to 1008 replaced with nonhomologous FVIII B-domain sequences (1032–1077; shaded purple). FV-810 and FV-short are truncated at residues 811 to 1491 and 756 to 1458, respectively, and are missing the conserved BR. FV-B152 is truncated at residues 811 to 963 and 1008 to 1538 and is missing AR2 but retains the BR. FVa is truncated at 710 to 1545 and is lacking BR and AR2. B, protein purity was assessed by SDS-PAGE under reducing conditions and stained with Coomassie Blue R-250. Lanes are: 1, plasma-derived FV, PD-FV; 2, recombinant FV, rFV; 3, FV-1033; 4, FV-s46; 5, FV-810; 6, FV-810-R506Q; 7, FV-810-R306Q; 8, FV-810-QQ; 9, FV-short; and 10, rFVa. FV-B152 was not included on the gel. The apparent molecular weights of protein standards and FVa HC and LC are indicated. BR, basic region; FV, factor V; FVa, factor Va; HC, heavy chain; LC, light chain.

Figure 2

APC-catalyzed proteolysis of FV and FV species. Proteolysis of 20 nM PD-FV (A), rFV (B), FVa (C), FV-1033 (D), and FV-s46 (E) by 1.0 nM APC in reaction mixtures containing 20 μM PCPS in assay buffer was monitored over time (0–15 min, 37 °C). Samples were subjected to SDS-PAGE and cleavage products visualized by immunoblotting using an antihuman FV-HC antibody. Apparent molecular weight markers and key proteolytic fragments are indicated. In (F), the intensity of the starting material (uncleaved FV or FV species; A–E) was plotted as a function of time; PD-FV (○), rFV (Δ), FV-1033 (□), FV-s46 (■), and FVa (●). These data are representative of at least two independent experiments. FV, factor V; FVa, factor Va.

Figure 3

Proteolysis of FV-810 is impaired when bound to FV-BR. Cleavage of 20 nM FV-810 by 1.0 nM APC in the absence (A) or presence of FV-BR at a fixed concentration (250 nM; B) over time or increasing FV-BR concentration (0–5 μM; C) at a single end point (3 min) was monitored in assay buffer. Samples were resolved by SDS-PAGE and immunoblotted as in Figure 2. In panel (D), band density of starting material (A & B) was expressed as a function of time; FV-810 (□) and FV-810 + FV-BR (■). Data are representative of two or more similar experiments. BR, basic region; FV, factor V.

Figure 4

FV-BR impacts susceptibility to APC proteolysis and loss of cofactor activity. APC (1.0 nM) inactivation of 20 nM FV-810 (A) or FVa (B) was assessed in the absence (○) or presence (●) of 250 nM FV-BR in assay buffer over time. In panel (C), FV-1033 and FV-s46 inactivation by APC was followed in the absence of exogeneous FV-BR. Residual cofactor activity was assessed in a calcium-initiated TGA using FV-DP containing 2 pM TF/4 μM phospholipid and fluorogenic thrombin substrate (Z-GGR-AMC). Data are shown as percentage peak and are an average of three similar experiments (mean ± SD)%. BR, basic region; FV, factor V; TGA, thrombin generation assay.

Figure 5

TFPIα, TFPIα-BR, and FV-BR impair FV-short proteolysis by APC. Immunoblots of FV-short (20 nM) proteolysis by APC (1.0 nM) in the absence (A) or presence of 250 nM TFPIα-BR (B), FV-BR (C), or TFPIα (D) are indicated. Panel (E) shows quantitative densitometry data of panels (A–D) expressed as a percentage of starting material remaining over time for FV-short (●) with TFPIα-BR (○), TFPIα (□), or FV-BR (■). Panel (F) is data from TGA functional assay expressed as peak thrombin. Plotted data are an average of three similar experiments (mean ± SD)%. BR, basic region; FV, factor V.

Figure 6

Cleavage at Arg⁵⁰⁶is altered by BR fragments. Proteolysis of APC cleavage site mutants in FV-810 was followed in two assays in the absence (●) and presence of TFPIα-BR (○) or FV-BR (■) as described in ‘‘Experimental Procedures.’’ Data are expressed as percentages of residual cofactor activity (peak thrombin) in TGAs and starting material remaining (densitometry of immunoblots) over time for FV-810-R506Q (A & D), FV-810-R306Q (B & E), and FV-810-QQ (C & F), respectively (n = 3; mean ± SD) %. In panel (G), representative immunoblots for the data in panel (E) are shown. BR, basic region; FV, factor V; TGA, thrombin generation assay.