Neither Factor VIII Nor the Putative F8B Protein Is Detectable in Human Peripheral Blood Mononuclear Cells

Factor VIII (FVIII) is encoded by 26 exons comprising the F8 gene. The F8 mRNA transcript encodes the full-length FVIII protein, which consists of 2332 amino acids. A CpG island within intron 22 of F8 includes an initiation site for a second, shorter transcript termed F8B. F8B contains a short exon spliced in-frame to F8 exons 23-26, and this mRNA transcript is expressed in multiple human tissues. The putative protein encoded by this gene corresponds to 8 amino acids encoded by the exon within F8 intron 22, followed by the FVIII C2 domain sequence. Earlier studies have unambiguously demonstrated FVIII expression in human endothelial cells, including liver and lung. Animal model studies have indicated FVIII is expressed, possibly exclusively, in endothelial cells. Other recent reports have presented evidence for FVIII and F8B protein expression in human peripheral blood mononuclear cells (PBMCs). In this study, we utilized several methods to test the hypothesis that FVIII and/or the putative F8B protein is expressed in human PBMCs.

Frozen PBMCs from healthy human subjects were thawed and then washed 5-10X to ensure removal of plasma-derived FVIII. Samples containing 50 million PBMCs were lysed in buffer containing protease inhibitors, centrifuged, and the supernatants analyzed by immunoprecipitation (IP) followed by SDS-PAGE and Western blots. The IP experiments utilized both polyclonal anti-FVIII antibodies and cocktails of monoclonal antibodies (mAbs) specific for different FVIII domains, and Western blots used several combinations of primary and secondary antibodies. Negative controls consisted of BHK cell lysates, and positive controls were purified FVIII and lysates of BHK cells transduced with a plasmid encoding FVIII. Western blots carried out with sensitive chemiluminescence detection identified the expected bands in the positive control lanes. Bands at expected positions for FVIII or F8B proteins (~240kDa, 75 kDa, 25-30 kDa) visualized by Coomassie-blue staining of lysates analyzed by SDS-PAGE were cut from the gels, subjected to trypsin digestion and then analyzed by mass spectrometry (LC-MS). FVIII-derived peptides were detected only in the positive control samples.

Intracellular staining (ICS) of permeabilized PBMCs was carried out using mAbs specific for the FVIII A1, A2, C1, and C2 domains and a mAb specific for the FVIII light chain. The mAbs were covalently labeled with Alexa-Fluor 488 to avoid possible nonspecific binding of labeled secondary antibodies. PBMCs from (1) normal control subjects; (2) a severe hemophilia A subject with an intron-22 inversion mutation and (3) a severe hemophilia A subject with a deletion of F8 exons 3-6 were analyzed. All 5 anti-FVIII mAbs detected FVIII protein in FVIII-expressing BHK cells, while none detected FVIII in BHK cells or in any of the human PBMC samples. Our results are consistent with several recent studies reporting proteomics analysis of human PBMCs, which have identified no FVIII peptides or proteins. We conclude that if FVIII or F8B proteins are expressed in human PBMCs, their concentration is below the detection limits of these assays.