Philip A. Kroner

Intracellular trafficking of factor VIII to von Willebrand factor storage granules.

In plasma, von Willebrand factor (vWf) associates with Factor VIII (FVIII); however, the site at which these proteins first interact has not been defined. Administration of 1-desamino-8-D-arginine vasopressin (DDAVP) causes a rapid, concomitant elevation in plasma levels of both vWf and FVIII, suggesting the existence of a DDAVP-releasable storage pool for both proteins. To determine whether vWf and FVIII can associate intracellularly and colocalize to storage vesicles, we transfected AtT-20 cells with vWf and FVIII expression plasmids. FVIII alone was not detectable within storage granules; however, transfection of vWf cDNA into the same cell caused FVIII to alter its intracellular trafficking and to undergo granular storage, colocalizing to the vWf-containing granules. In contrast, colocalization of FVIII was not observed when these cells were transfected with plasmids encoding defective FVIII-binding vWf mutants. Transfection of bovine endothelial cells with FVIII further demonstrated vesicular storage of FVIII with vWf in Weibel-Palade bodies. Since gene therapy of hemophilia A may ultimately target endothelium or hematopoietic stem cells, the interaction between vWf and FVIII within a secretory cell is important. Thus, vWf can alter the intracellular trafficking of FVIII from a constitutive to a regulated secretory pathway, thereby producing an intracellular storage pool of both proteins.

Expression of human factor VIII under control of the platelet-specific αIIb promoter in megakaryocytic cell line as well as storage together with VWF

Hemophilia A, which results in defective or deficient factor VIII (FVIII) protein, is one of the genetic diseases that has been addressed through gene therapy trials. FVIII synthesis does not occur in normal megakaryocytes. In hemophilia patients who have inhibitors to FVIII activity, megakaryocytes could be a protected site of FVIII synthesis and subsequent release. Since von Willebrand factor (VWF) is a carrier protein for FVIII, we hypothesize that by directing FVIII synthesis to megakaryocytes, it would traffick together with VWF to storage in megakaryocyte alpha-granules and the platelets derived from these cells. Such synthesis would establish a protected, releasable alpha-granule pool of FVIII together with VWF. When platelets are activated in a region of local vascular damage, FVIII and VWF could potentially be released together to provide improved local hemostatic effectiveness. To direct FVIII expression to the megakaryocyte lineage, we designed a FVIII expression cassette where the human B-domain deleted FVIII cDNA was placed under the control of the megakaryocytic/platelet-specific glycoprotein IIb (alphaIIb) promoter. We demonstrated by means of a functional FVIII activity assay that the biosynthesis of FVIII occurred normally in Dami cells transfected with FVIII. FVIII production was higher when driven by the alphaIIb promoter compared to the CMV promoter, and was increased about 8-fold following PMA treatment of the transfected Dami cells. Immunofluorescence staining of the transfected cells showed that FVIII stored together with VWF in the granules. The data indicate that the megakaryocytic compartment of hematopoietic cells may represent a potential target of gene therapy for hemophilia A-especially in those patients who have developed inhibitors to plasma FVIII.

The human multimerin gene MMRN maps to chromosome 4q22

Multimerin is a protein present in platelets and endothelial cells and is comprised of variably sized disulfide-linked multimers (Hayward et al., 1991). In platelets multimerin is stored in ·-granules (Hayward et al., 1993). In endothelial cells multimerin is present in granules that are mostly distinct, but that sometimes co-localize with von Willebrand factor in WeibelPalade bodies (Hayward et al., 1998). During platelet activation multimerin is released with the other ·-granule proteins, although the largest multimers remain bound to the platelet surface (Hayward and Kelton, 1995). Similarly, after stimulation of endothelial cells with secretagogues, multimerin appears to redistribute from storage granules to the external cellular membrane (Hayward et al., 1998). Recently, human endothelial cell multimerin cDNA was cloned and sequenced (Hayward et al., 1995). The multimerin cDNA encodes a protein of 1,228 amino acids with a calculated molecular mass of 138 kDa. Recent studies suggest that multimerin might play an important role in the biosynthesis and storage of coagulation factor V in platelets (Hayward et al., 1995). The human factor V gene is located on chromosome 1q23, but the location of the multimerin gene has not yet been described. Utilizing two separate methods, we assign the human multimerin gene to chromosome 4q22.