Induction of megakaryocytes to synthesize and store a releasable pool of human factor VIII

Abstract

Summary. von Willebrand factor (VWF) is a complex plasma glycoprotein that modulates platelet adhesion at the site of a vascular injury, and it also serves as a carrier protein for factor (F)VIII. As megakaryocytes are the only hematopoietic lineage to naturally synthesize and store VWF within a-granules, this study was performed to determine if expression of a FVIII transgene in megakaryocytes could lead to traf®cking and storage of FVIII with VWF in platelet a-granules. Isolex 1 selected CD34 cells from human G-CSF mobilized peripheral blood cells (PBC) and murine bone marrow were transduced with a retrovirus encoding the B-domain deleted form of human FVIII (BDD-FVIII). Cells were then induced with cytokines to form a population of multiple lineages including megakaryocytes. Chromogenic analysis of culture supernatant from FVIIItransduced human cells demonstrated synthesis of functional FVIII. Treatment of cells with agonists of platelet activation (ADP, epinephrine, and thrombin receptor-activating peptide) resulted in the release of VWF antigen and active FVIII into the supernatant from transduced cells. Immuno¯uorescence analysis of cultured human and murine megakaryocytes revealed a punctate pattern of staining for FVIII that was consistent with staining for VWF. Electron microscopy of transduced megakaryocytes using immunogold-conjugated antibodies colocalized FVIII and VWF within the a-granules. FVIII retained its association with VWF in human platelets isolated from the peripheral blood of NOD/SCID mice at 2±6 weeks post-transplant of transduced human PBC. These results suggest feasibility for the development of a locally inducible secretory pool of FVIII in platelets of patients with hemophilia A.

Factor VIII ectopically targeted to platelets is therapeutic in hemophilia A with high-titer inhibitory antibodies

“…FVIII normally circulates in plasma and is not present in platelets or other blood cells (22,23). Our laboratory has previously demonstrated in vitro costorage of FVIII with VWF in megakaryocyte α-granules (22,23) and endothelial Weibel-Palade bodies (21) when FVIII is ectopically expressed in these cells, and VWF enhances this expression. Clinically, 25-30% of hemophilia patients develop inhibitory antibodies in response to FVIII replacement therapy (43,44), and thus gene therapy by constitutive synthesis and release of recombinant FVIII into plasma (i.e., specifying gene therapy to liver or endothelial cells) might not be therapeutic in these patients in the presence of such inhibitory antibodies.…”

“…This failure may be attributed in part to the fact that none of these approaches directed FVIII synthesis to cells that synthesize and store VWF, even though VWF is known to stabilize FVIII in plasma (40) and in the commercial production of recombinant FVIII products (41,42). FVIII normally circulates in plasma and is not present in platelets or other blood cells (22,23). Our laboratory has previously demonstrated in vitro costorage of FVIII with VWF in megakaryocyte α-granules (22,23) and endothelial Weibel-Palade bodies (21) when FVIII is ectopically expressed in these cells, and VWF enhances this expression.…”

“…We have previously demonstrated that coexpression of FVIII in a cell that stores VWF results in the costorage, and release, of FVIII (4,21). More specifically, directing FVIII expression to megakaryocytes results in storage of FVIII with VWF in the α-granules of platelets (22,23). A megakaryocytic, lineage-specific promoter would direct FVIII expression only to that blood cell lineage where VWF is normally endogenously synthesized and stored (20,24).…”

Extrahepatic sources of factor VIII potentially contribute to the coagulation cascade correcting the bleeding phenotype of mice with hemophilia A

“…25,[31][32][33] The advantages of expressing FVIII in platelets are these cells' involvement in early hemostasis and the fact that they serve as a major site for storage of FVIII. 34 In megakaryocytes and endothelial cells the presence of von Willebrand factor should be helpful for stabilizing FVIII. It is possible that FVIII in platelets may not cause the development of neutralizing antibodies.…”

Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells

“…Recent studies have suggested that it may be beneficial to express FVIII in cells that also express its natural carrier protein von Willebrand factor (VWF). [5][6][7][8][9][10][11] In the circulation, VWF protects FVIII from premature clearance and proteolytic degradation by virtue of its ability to bind to it with high affinity. [12][13][14] VWF is expressed in megakaryocytes and vascular endothelial cells.…”