Phospholipid binding improves plasma survival of factor VIII

Abstract

Hemophilia A is a bleeding disorder caused by the deficiency of Factor VIII (FVIII)(1). In circulation, FVIII binds to its carrier protein vWF with high affinity (Kd < 0.5nM) and this strong non-covalent interaction is critical for the normal half life and circulating plasma concentration of FVIII (2 -3). The complex protects FVIII from proteolytic degradation by proteases including activated protein C and prevents cellular uptake by LRP (2). The noncovalent interaction of vWF with FVIII involves amino acids 1672-1689 within the A3 domain and 2303-2332 of the C2 domain of . LRP binds to FVIII via A2, A3 and C2 domains (5 -6). The monoclonal antibody ESH4, directed against residues 2303-2332 of the C2 domain of FVIII, inhibits binding to both vWF and LRP (5). This implies that vWF and LRP binding sites are in close proximity and/or overlap. This same region (2303-2332) also mediates the interaction of FVIII with phosphatidylserine (PS) on platelet membranes (7). Recent evidences also suggest the participation of C1 domain of FVIII in lipid binding (8 -9). FVIII also binds to other anionic lipids including phosphatidylinositol (PI), but the specificity and affinity is highest for PS compared to other anionic lipids (10). Based on these studies it is interesting to note that within C2 domain the binding site for three macromolecular interactions of vWF, LRP and phospholipids either overlap or in close proximity ( Figure 1a). We have demonstrated previously that FVIII-PS and FVIII-PI complex reduced inhibitor development (11 -12), suggesting therapeutic potential of FVIII-lipid complex. However, the catabolism of FVIIIlipid complex is not completely understood and could be complicated due to overlapping or proximal binding sites and comparable binding affinities in nM range for both phospholipid and vWF. Further phospholipid binding can sterically block macromolecular interaction involving C1 and C2 domain of FVIII. In order to delineate the role of vWF on catabolism of FVIII-lipid complex, pharmacokinetic studies of FVIII in the presence and in the absence of PS and PI, in Hemophilia A (HA) and vWF −/− mice were carried out. Our data suggests that FVIII-lipid complex showed an improved pharmacokinetic profile in both HA mice as well as in vWF −/− mice. All animal handling was performed in accordance with the guidelines of institutional animal care and use committees (IACUC) at the University at Buffalo.FVIII-PI and FVIII-PS complexes were prepared as described previously (11). Pharmacokinetic studies and analysis were carried out as described previously (13 -14). Statistical Analysis was conducted using Minitab 15 (Minitab Inc, State College, PA). Statistical differences (p<0.05) were tested using the Student independent t-test and one-way ANOVA followed by Dunnet's post-hoc multiple comparison test. Systemic exposures were compared using Bailer-Satterwaite method(15). In order to establish the role of vWF in FVIII catabolism and to obtain relevant pharmacokinetic parameters, pharmacokinetic studies of FVIII were ...

Procoagulant activity induced by vascular injury determines contribution of elevated factor VIII to thrombosis and thrombus stability in mice

“…22 17 has comparable cofactor activity as murine FVIII, 17 and promotes coagulation after tail clipping and vessel injury in hemophilic mice. 17,[24][25][26][27] In addition, previous studies 28,29 and our findings (FVIII activity assay, see "FVIII activity assay") show FVIII has sufficient half-life in murine circulation for these experiments. The endogenous FVIII concentration in mice (1 U/mL, 100%) 30 was raised by infusing human FVIII to 285% (total murine plus human FVIII) of normal, consistent with levels associated with thrombosis in humans.…”

“…First, we used human FVIII to increase circulating levels in the mouse. However, our data and published studies demonstrate human FVIII binds murine VWF, 17 has comparable cofactor activity as murine FVIII, 17 is stable in murine circulation, 28,29 is incorporated into murine clots, 17 is inactivated by murine-activated protein C (Steve Pipe, University of Michigan, personal communication, June 21, 2011), and supports clot formation in mice. 17,[24][25][26][27] Second, the FeCl 3 /saphenous vein injury model is not commonly used as a model of venous thrombosis; however, our findings are consistent with published data from other venous models and effectively demonstrate the pathologic activity of elevated human FVIII in thrombi formed in murine veins.…”

PEGylation of a Factor VIII–Phosphatidylinositol Complex: Pharmacokinetics and Immunogenicity in Hemophilia A Mice

“…This not only masks the immunogenic epitopes on FVIII but it could also reduce FVIII exposure to plasma components, protecting FVIII from proteolysis and binding of opsonizing proteins. Administration of FVIII-PI resulted in reduced immunogenicity and prolonged half-life of FVIII in a (45). We investigated whether attachment of PEG to the complex would yield further in vivo pharmacokinetic and immunogenic improvements.…”

Alloantibodies to therapeutic factor VIII in hemophilia A: the role of von Willebrand factor in regulating factor VIII immunogenicity

“…54,[65][66][67][68] The exposed VWF-binding site on FVIII, incidentally, may be occupied by anionic phospholipids which were suggested to have additional implications for the immune response. 69,70 VWF inhibits uptake of FVIII in antigen presenting cells Qadura and colleagues were not able to confirm a protective effect of VWF in hemophilic mice, but found variant patterns of inflammatory mediators and of immune gene expression profiles in activated (CD11c + ) splenic dendritic cells (DCs) between pdFVIII-and rFVIII-treated hemophilic mice (type 1 vs. type 2 inflammatory response). 44 As the pdFVIII-treated mice in these experiments also developed a strong immune response to human VWF, these findings may have little relevance for the situation in humans.…”