Conservative mutations in the C2 domains of factor VIII and factor V alter phospholipid binding and cofactor activity

Abstract

Factor VIII and factor V share structural homology and bind to phospholipid membranes via tandem, lectin-like C domains. Their respective C2 domains bind via 2 pairs of hydrophobic amino acids and an amphipathic cluster. In contrast, the factor V-like, homologous subunit (Pt-FV) of a prothrombin activator from Pseudonaja textilis venom is reported to function without membrane binding. We hypothesized that the distinct membrane-interactive amino acids of these proteins contribute to the differing membrane-dependent properties. We prepared mutants in which the C2 domain hydrophobic amino acid pairs were changed to the homologous residues of the other protein and a factor V mutant with 5 amino acids changed to those from Pt-FV (FV MTTS/Y ). Factor VIII mutants were active on additional membrane sites and had altered apparent affinities for factor X. Some factor V mutants, including FV MTTS/Y , had increased membrane interaction and apparent membrane-independent activity that was the result of phospholipid retained during purification. Phospholipid-free FV MTTS/Y showed increased activity, particularly a 10-fold increase in activity on membranes lacking phosphatidylserine. The reduced phosphatidylserine requirement correlated to increased activity on resting and stimulated platelets. We hypothesize that altered membrane binding contributes to toxicity of Pt-FV. (Blood. 2012;120(9):1923-1932) IntroductionFactor VIII (FVIII) is an essential cofactor for the intrinsic branch of the coagulation cascade. The clinical importance of FVIII is illustrated by hemophilia A, a disease in which deficient or defective FVIII leads to a severe clinical bleeding phenotype. FVIII has sequence homology with FV, an essential cofactor for the common final pathway of the coagulation cascade. Complete deficiency of FV is incompatible with life, 1 although a partial deficiency causes a bleeding disorder termed "parahemophilia."Both FVIII and FV bind to membranes containing phosphatidylserine (PS) and serve, respectively, as cofactors for FXase and prothrombinase enzyme complexes. [2][3][4][5] Excessive activity of the FXase 6,7 and prothrombinase complexes 8 is linked to an elevated risk of thrombosis. The importance of binding to PS-containing membranes is illustrated by nearly complete loss of activity when this interaction is blocked by an antibody 9 or when membrane binding sites are blocked by a competing protein. 10 Similarly, activity is diminished by point mutations that diminish the interaction with PS-containing membranes. 11,12 There are no reported disorders linked to increased activity related to enhanced membrane binding. The functional consequences of altered membrane binding are the foci of this investigation.To that end, we also investigated a form of FV that has less need for phospholipid membranes. The venom of the eastern brown snake (Pseudonaja textilis) contains a potent prothrombin activating protein complex (pseutarin C) that is homologous to FXa bound to FVa in the prothrombinase complex. 13 Recent results have sho...

Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising “Hot-Spot” Lysine Residues

“…In recent years, the importance of the C domains in the life cycle of FVIII has been appreciated because they are involved in lipid binding, von Willebrand factor (VWF) interaction, and cellular uptake (47)(48)(49)(50)(51)(52). The C2 domain is loosely attached to the C1 and A1 domains, and it is likely that the domain has a high degree of rotational freedom.…”

Structure of the factor VIII C2 domain in a ternary complex with 2 inhibitor antibodies reveals classical and nonclassical epitopes

“…16,17 The binding of PS and VWF to C2 is mutually exclusive, [18][19][20] and numerous studies suggest this interaction involves 3 solvent-exposed hydrophobic loops protruding from the C2 domain b-sandwich core, including residues Met2199-Phe2200, Val2223, and Leu2251-Leu2252. [21][22][23][24][25] Several positively charged, basic residues encircle the base of these b-hairpin loops, and they may contribute to the interaction with the negatively charged PS headgroup. 22 In addition to interactions with phospholipid (PL) and VWF, there are previous reports that suggest the C2 domain contributes to the binding of thrombin and factor Xa to full-length fVIII, potentially acting as a docking module that may stabilize and promote the proteolytic activity of factor Xa/thrombin cleavages elsewhere in fVIII during activation.…”

“…[21][22][23][24][25] Several positively charged, basic residues encircle the base of these b-hairpin loops, and they may contribute to the interaction with the negatively charged PS headgroup. 22 In addition to interactions with phospholipid (PL) and VWF, there are previous reports that suggest the C2 domain contributes to the binding of thrombin and factor Xa to full-length fVIII, potentially acting as a docking module that may stabilize and promote the proteolytic activity of factor Xa/thrombin cleavages elsewhere in fVIII during activation. [26][27][28] Recent studies also suggest that the C1 domain contributes to binding both VWF and PS, 29,30 and deletion studies of the C2 domain indicate that it is not essential for PL membrane binding, factor IXa binding, or clotting activity.…”

Factors VIII and V swap fatty feet

“…1 The 3-dimensional structure of these domains exposes a few characteristic loops, referred to as "fatty feet," which mediate the interaction with lipid membranes. These are similar, but not identical in FVIII and FV.…”

“…This is what Gilbert and colleagues report in their present study. 1 Their approach is a very elegant one. They focus on the C-terminal part of FVIII and FV, the C2 domain, which comprises a major lipid-binding part of these proteins.…”