Abstract
Coagulation factor V exists in two pools in human blood. One is plasma-derived, originating from synthesis in the liver. The other is platelet-derived, originating from endocytosis of the plasma-derived cofactor by megakaryocytes and consisting of both unactivated and activated cofactor. Studies have demonstrated the presence of a non-dissociable, membrane-bound form of platelet-derived factor Va. When washed platelets were activated with thrombin and subjected to additional washing in the presence of EDTA to disrupt the calcium-dependent interaction between the factor Va heavy and light chains, western blotting and flow cytometric analyses revealed that ~35% of the heavy chain could not be removed from the platelet surface. Similarly, in a prothrombin time-based clotting assay, ~25% of the factor Va cofactor activity remained on the activated platelet surface after extensive washing, demonstrating that this platelet-bound cofactor pool functions in Prothrombinase. The mechanism by which this factor Va pool is bound to the membrane was investigated. Sequence analysis of factor Va has revealed a consensus sequence for glycosylphosphatidylinositol (GPI) anchor addition at Ser692 in the C-terminus of the heavy chain. Lipid raft domains in cell membranes are enriched in GPI-anchored proteins and are resistant to solubilization in Triton X-100 at 4°C but are soluble at 37°C. Compared to lysis at 4°C, ~50% more factor Va heavy chain was solubilized when platelets were lysed at 37°C. When cells are solubilized in Triton X-114, membrane-anchored and trans-membrane proteins segregate to the detergent phase, and when activated platelets were subjected to this procedure, a portion of the factor Va heavy chain segregated to the detergent phase. Flow cytometric analyses of activated platelets have demonstrated that phosphatidylinositol-specific phospholipase C (PI-PLC), which can cleave GPI-anchored proteins from cell surfaces, is able to remove ~45% of the non-dissociably bound factor Va heavy chain from the platelet surface in a dose-dependent manner. Subsequent analysis of these samples by Triton X-114 phase separation corroborated these results, indicating that ~50% of the heavy chain was removed from the detergent phase upon treatment with PI-PLC. After cleavage by PI-PLC, GPI-anchored proteins express an epitope known as the cross-reacting determinant (CRD), which encompasses the remainder of the GPI anchor on the modified protein. Western blotting analyses of platelet supernatants after PI-PLC treatment have demonstrated that the platelet-derived factor Va heavy chain contains the CRD epitope. To investigate the origin of the non-dissociably bound pool of factor Va on the platelet surface, platelets were isolated from a factor V-deficient individual with undetectable levels of the cofactor, who receives therapeutic transfusions of fresh frozen plasma. When the individual’s platelets were subjected to Triton X-114 phase separation, a population of factor Va heavy chain was detected in the detergent phase. These results are consistent with the non-dissociable portion of the platelet-derived factor Va pool being formed by post-translational modification of factor V from plasma subsequent to its endocytosis by megakaryocytes. The presence of a GPI anchor consensus sequence in the factor Va heavy chain, combined with the washing, solubilization, phase separation, PI-PLC treatment, and anti-CRD immunoblotting data strongly suggest that non-dissociably bound, platelet-derived factor Va is linked to the activated platelet membrane via a GPI anchor on its heavy chain. GPI-anchored, platelet-derived factor Va is functional in Prothrombinase, and, as it is retained on the platelet surface at the site of vascular injury, it is likely a physiologically significant source of cofactor activity.
Defining the Factor Xa-binding Site on Factor Va by Site-directed Glycosylation