title: Multifunctional specificity of the protein C/activated protein C Gla domain.
abstract: Activated protein C (APC) has potent anticoagulant and anti-inflammatory properties that are mediated in part by its interactions with its cofactor protein S and the endothelial cell protein C receptor (EPCR). The protein C/APC Gla domain is implicated in both interactions. We sought to identify how the protein C Gla domain enables specific protein-protein interactions in addition to its conserved role in phospholipid binding. The human prothrombin Gla domain, which cannot bind EPCR or support protein S cofactor activity, has 22/45 residues that are not shared with the human protein C Gla domain. We hypothesized that the unique protein C/APC Gla domain residues were responsible for mediating the specific interactions. To assess this, we generated 13 recombinant protein C/APC variants incorporating the prothrombin residue substitutions. Despite anticoagulant activity similar to wild-type APC in the absence of protein S, APC variants APC(PT33-39) (N33S/V34S/D35T/D36A/L38D/A39V) and APC(PT36/38/39) (D36A/L38D/A39V) were not stimulated by protein S, whereas APC(PT35/36) (D35T/D36A) exhibited reduced protein S sensitivity. Moreover, PC(PT8/10) (L8V/H10K) displayed negligible EPCR affinity, despite normal binding to anionic phospholipid vesicles and factor Va proteolysis in the presence and absence of protein S. A single residue variant, PC(PT8), also failed to bind EPCR. Factor VIIa, which also possesses Leu-8, bound soluble EPCR with similar affinity to wild-type protein C, collectively confirming Leu-8 as the critical residue for EPCR recognition. These results reveal the specific Gla domain residues responsible for mediating protein C/APC molecular recognition with both its cofactor and receptor and further illustrate the multifunctional potential of Gla domains.
authors.affiliation: Department of Haematology, Division of Investigative Science, Hammersmith Hospital Campus, Imperial College London, London W12 0NN, United Kingdom. roger.preston@imperial.ac.uk
authors.forename: Roger J S
authors.initials: RJ
authors.lastname: Preston
authors.forename: Eva
authors.initials: E
authors.lastname: Ajzner
authors.forename: Cristina
authors.initials: C
authors.lastname: Razzari
authors.forename: Stalo
authors.initials: S
authors.lastname: Karageorgi
authors.forename: Sonia
authors.initials: S
authors.lastname: Dua
authors.forename: Björn
authors.initials: B
authors.lastname: Dahlbäck
authors.forename: David A
authors.initials: DA
authors.lastname: Lane
mesh_terms: D000595:Amino Acid Sequence; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology; D005167:Factor VII / Q000378:metabolism; D015943:Factor Va / Q000737:chemistry; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000737:chemistry; D011486:Protein C / Q000737:chemistry*; D017293:Protein S / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011516:Prothrombin / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
chemical_list: D000893:Anti-Inflammatory Agents; D010743:Phospholipids; D011486:Protein C; D017293:Protein S; D015943:Factor Va; D005167:Factor VII; D011516:Prothrombin