Abstract

Factor V (fV) binds to phospholipid (PL) membranes via a motif localized to the C2 domain. We and others have shown that PL binding is mediated by two pairs of hydrophobic residues, each displayed at the tips of β-hairpin turns. The homologous hydrophobic residues in the C2 domain of factor VIII also contribute to PL binding. We hypothesized that the solvent-exposed hydrophobic residues of the fV C2 domain make specific contacts that influence membrane affinity and activity of fV. To test this hypothesis we have prepared fVIII/fV hybrid mutants in which amino acid(s) of the fV C2 domain were changed to the homologous residues of fVIII (Mutants #1 W/W 2063/2064 M/F, #2 L/S 2116/2117 L/L, and #3 W/W/S 2063/2064/2117 M/F/L). Mutants were expressed in COS-1 cells and purified by FPLC. The specific activity of the fV/FVIII hybrids #2 and #3 exceeded those of wild type factor V by approx. 10-fold and approx. 4-fold, respectively, in a prothrombin time assay with factor V deficient plasma. Apparent PL affinites were evaluated in a prothrombinase complex assay with limiting phospholipid. The apparent affinities are 9.8 and 21-fold higher than wild type factor V for mutants #2 and #3 which contain the S→L change in the second hydrophobic spike. An unexpected result was that mutants 1–3 supported prothrombinase activity in the absence of added phospholipid, in contrast to wild type fV. We hypothesized that this activity resulted from phospholipid that was not dissociated from fV during purification. This hypothesis was supported because activity was eliminated by incubation of the factor V mutants with phospholipase A2 or by incubation with lactadherin. The tenacity of the phospholipid binding was further investigated by washing immobilized wild type fV and fV mutants with CHAPS prior to elution from an FPLC column. fV mutants #2 and #3 retained activity after the CHAPS wash, free of added PL and activity remained inhibitable by lactadherin, further illustrating the tenacious phospholipid affinity. We utilized Mutants #1 & 2 in the absence of added PL to evaluate the mechanism through which soluble phosphatidylserine with 6-carbon acyl chains (C6PS) enhances activity of the prothrombinase complex. In the absence of phospholipid vesicles C6PS enhanced activity of Mutant 1 equivalent to wild type fV (> 20 fold). However, enhancement of mutant 2 activity, which presumably retained phospholipid via residues 2116/2117, was < 3-fold. This suggests that C6PS functions to activate wild type fV, in part, by engaging the free LS 2116/2117 hydrophobic spike. Together, these data indicate that the hydrophobic spikes of factor V influence the specific activity of factor V, that the high affinity reversible binding of fV to phospholipid membranes is readily perturbed by mutations, and that activation of the prothrombinase complex by C6PS and phospholipid membranes likely involves engagement of amino acids 2116/2117.

Author notes

Disclosure: No relevant conflicts of interest to declare.