Structure of Coagulation Factor II: Molecular Mechanism of Thrombin Generation and Development of Next-Generation Anticoagulants

Abstract

Coagulation factor II, or prothrombin, is a multi-domain glycoprotein that is essential for life and a key target of anticoagulant therapy. In plasma, prothrombin circulates in two forms at equilibrium, "closed" (~80%) and "open" (~20%), brokered by the flexibility of the linker regions. Its structure remained elusive until recently when our laboratory solved the first X-ray crystal structure of the zymogen locked in the predominant closed form. Because of this technical breakthrough, fascinating aspects of the biology of prothrombin have started to become apparent, and with this, novel and important questions arise. Here, we examine the significance of the "closed"/"open" equilibrium in the context of the mechanism of thrombin generation. Further, we discuss the potential translational opportunities for the development of next-generation anticoagulants that arise from this discovery. By providing a structural overview of each alternative conformation, this minireview also offers a relevant example of modern structural biology and establishes a practical workflow to elucidate the structural features of analogous clotting and complement factors.

Keywords: X-ray crystallography; anticoagulants; coagulation cascade; drug discovery; prothrombin; single-molecule FRET; thrombin.

Figure 1

(A) Color-coded domain architecture of prothrombin displaying the location of proteolytic sites R271 and R320 that are cleaved by the prothrombinase complex. (B) Scheme of a confocal microscope equipped for sm-PIE-FRET experiments. Briefly, the donor and acceptor dyes are excited with a ps pulsed diode laser at 532 and 633 nm, respectively. To achieve PIE, the 532 nm laser is electronically delayed (25–50 ns) relative to the 633 nm laser. Signals from single molecules are observed as bursts of fluorescence and detected with two SPAD detectors. Data are stored in the Time-tagged Time-resolved Mode (15, 16). (C) smFRET histogram for the prothrombin mutant S120C/S478C (proT120/478) in which fluorescent dyes were attached at position 120 in kringle 1 and 478 in the B-chain (16). The bottom section of the graph depicts the stoichiometry, S, vs. FRET efficiency for each diffusing molecule. The upper section shows the one-dimensional efficiency histogram of the molecules in the bottom section. ProT120/478 shows two distributions of molecules with distinct FRET efficiencies (low and high), supportive of the existence of closed and open conformations in solution. (D) Schematic models of closed and open conformations built from smFRET measurements.

Figure 2

X-ray crystal structures of closed and open conformations of prothrombin. (A) Structure of the prothrombin mutant S101C/A470C (proTCC) solved at 4.0Å resolution (PDB ID: 6C2W) (15). (B) Structure of the prothrombin mutant Δ154–167 (proTΔ154–167) solved at 2.2Å resolution (PDB ID: 5EDM) (33). Key residues Trp547 in the protease domain and Tyr93 in the kringle-1 are shown in magenta.