Activated protein C takes a bite out of factor Va Free

In this issue of Blood, Mohammed et al¹ determine the structure of coagulation factor Va (FVa) bound to activated protein C (APC). This study delineates the first structural insights into how APC engages FVa to mediate its proteolytic inactivation.

Proteolytic activation and inactivation are essential for regulating hemostasis, with FV serving as a prototypical cofactor that undergoes both processes. FV is a multidomain protein composed of A1-A2-B-A3-C1-C2 domains and circulates in plasma as an inactive procofactor. Specific cleavages by thrombin remove the large intervening B domain, which separates the heavy chain (A1-A2) from the light chain (A3-C1-C2) of FV, while preserving their tight noncovalent association.² The resulting activated form, FVa, functions as a cofactor in thrombin generation by forming a complex with its serine protease partner, FXa, on a procoagulant membrane surface. This assembly triggers an exponential amplification of prothrombin activation, increasing the reaction rate by more than 5 orders of magnitude.³ Consequently, inactivation of FVa by APC can significantly impact thrombin generation, thereby modulating the procoagulant cascade and acting as a key regulatory mechanism in the control of hemostasis.

Decades of research have elucidated the mechanism of FVa inactivation by APC, wherein APC catalyzes 2 specific proteolytic cleavages at residues R306 and R506 of FVa.⁴ These cleavages result in the spontaneous dissociation of the A2 domain, leading to the irreversible loss of cofactor activity. The peptide bond at R506 in FVa represents a primary site for proteolytic cleavage by APC. The importance of this is evident in FV Leiden, a variant of FV characterized by an R506Q mutation that impairs APC-mediated cleavage. This results in a thrombophilic disorder with an increased risk of venous thromboembolism, including deep vein thrombosis and pulmonary embolism.⁵ Biochemical studies have underscored the pivotal role of APC-mediated proteolytic cleavage and subsequent displacement of the FVa A2 domain as a key event in functional inactivation of FVa; however, structural corroboration of this mechanism has remained elusive.

In this study, the authors employed a catalytically inert variant of APC to assemble a stable FVa-APC complex in the absence of membrane, which was subsequently used for cryo-electron microscopy (cryo-EM)-based structure determination. The resulting structure illustrates how the protease domain of APC engages R506 in the A2 domain of FVa, supporting the idea that the primary interaction site of FVa with APC is localized near R506 (see figure). Notably, the contact between FVa and APC is exclusively mediated by the protease domain of APC, and the epidermal growth factor (EGF) and N-terminal γ-carboxyglutamic acid (Gla) domains show a high degree of flexibility and point away from FVa. This finding reinforces the concept that vitamin K–dependent coagulation proteases predominantly rely on their catalytic domains for mediating protein-protein interactions, whereas their ancillary domains (EGF and Gla) serve primarily structural or scaffolding functions.

The structure of FVa in the FVa-APC complex remains largely similar to that of free FVa,⁶ with a few notable changes likely induced by APC engagement. Among these alterations, the most prominent is the region spanning R306, the secondary APC cleavage site in FVa, which becomes more solvent exposed due to an upward structural shift upon APC binding. Additionally, the negatively charged patch (⁶⁵⁴VKCIPDDDEDSYEIFEP⁶⁷⁰) in the A2 domain exhibits significant deviations compared with FVa in the prothrombinase complex.⁷ This patch adopts a latchlike conformation over the protease domain of APC, functioning as an exosite ligand that precisely orients the P1 residue, R506, within the S1 pocket to facilitate efficient cleavage. This mode of interaction mirrors the exosite-dependent mechanisms that govern substrate recognition and activation in other critical coagulation reactions.⁸ 

The structure of the protease domain and EGF2 of APC in FVa-APC complex closely resembles a previously reported crystal structure of APC bound to the inhibitor (PPACK) at the active site but lacking the Gla domain.⁹ However, the 2 structures differ markedly in the orientation of the EGF1 domain, which adopts a diametrically opposite position in the complex. Additionally, the newly resolved Gla domain is misaligned relative to the principal axis of the protein, inducing a curved overall conformation of APC.

The FVa-APC complex interface is stabilized by a network of electrostatic interactions and hydrophobic contacts, which collectively facilitate efficient protease-substrate recognition. Conformational rearrangements induced by the latch region in FVa allosterically position the scissile bond at R506 into the catalytic cleft of APC. This alignment is further supported by complementary structural reorganization within the autolysis loop of APC, culminating in the formation of a catalytically competent FVa-APC complex. Beyond the autolysis loop, the additional contacts are formed by the 60-loop, short 170-helix, and the 70-loop of APC.

The structure of FVa-APC reported by Mohammed et al represents a significant advancement in our understanding of a key regulatory step in hemostasis. The structural details align well with the existing biochemical data and provide new insights into the molecular interface between FVa and APC. The detailed interaction between FVa and APC described in the article not only reinforces the existing models of APC function but also opens new avenues for targeting its anticoagulant and cytoprotective functions through structure-guided approaches.

These findings also lay the groundwork for addressing several outstanding mechanistic questions regarding APC-mediated proteolytic inactivation of FVa. Notably, how does the active site of APC access the distal cleavage site at R306, which is spatially separated from the primary cleavage site at R506? Additionally, how does association with a procoagulant membrane surface modulate the conformation and spatial organization of the FVa-APC complex. Another important consideration is whether the cleavage process is processive and, if so, whether the presence of protein S (PS), a proposed cofactor¹⁰ in APC-mediated FVa inactivation, induces conformational changes within the FVa-APC complex or modifies the interaction landscape to facilitate sequential cleavage. Addressing these questions will require structural characterization of a ternary FVa-APC-PS complex, potentially in a membrane-associated context, to further elucidate the mechanism of APC-mediated inactivation of FVa in the presence of PS.

Conflict-of-interest disclosure: S.K. declares no competing financial interests.