The von Willebrand factor (VWF) exon 4–5 deletion mutation has been described in up to 33% of type 3 von Willebrand disease (VWD) patients. The deletion of exons 4 and 5 abolish the first N-linked glycosylation site, which would most likely disrupt trafficking of the protein to the proper chaperones. Since VWF is synthesized in endothelial cells and megakaryocytes, these cells are the most representative of the pathophysiology of VWF mutations. The objective of the current study was to investigate the molecular basis of the exon 4–5 deletion mutation using two comparative cellular systems: blood outgrowth endothelial cells (BOEC) and megakaryocytes (MK). A family in the Canadian type 3 VWD population was identified as having the exon 4–5 deletion mutation. The index case (IC), T001, has type 3 VWD (VWF:Ag=0.01 IU/ml, VWF:RCo=0.02 IU/ml, FVIII:C=0.02 IU/ml, bleeding score=23) and is homozygous for the deletion and the mother, T156, has type 1 VWD (VWF:Ag=0.14 IU/ml, VWF:RCo= 0.13 IU/ml, FVIII:C=0.45 IU/ml, bleeding score=12) and is heterozygous. Peripheral blood samples were obtained from both patients in order to isolate and culture BOEC. Endothelial cell phenotype was confirmed by flow cytometry. VWF expression in patient BOEC showed a decrease in secretion by 87% in T156 and by 98% in T001 compared to control BOEC, similar decreases to those seen in previously published in vitro studies of this mutation. Confocal immunofluorescence (IF) microscopy showed both qualitative and quantitative defects in Weibel-Palade body (WPB) formation in T156 and a complete absence of WPBs in T001, with only diffuse staining of VWF present. Decreased stimulated release of VWF using PMA from T156's BOEC (26% increase) compared to control BOEC (40% increase) was observed. In T156, this degree of release is similar to the response clinically upon DDAVP administration (1 hour post VWF:Ag=0.30 IU/ml, FVIII:C=1.0 IU/ml; 2 hours post VWF:Ag =0.16 IU/ml, FVIII:C=0.76 IU/ml). Patient MKs were also isolated from peripheral blood samples and cultured by stimulating immunomagnetic isolated CD34+ cells. In T156 the VWF IF staining pattern was both diffuse and punctate and there was some co-localization with P-selectin. Despite the complete absence of VWF in platelets from T001 per immunoblot, VWF was observed in the MKs using IF microscopy. The observations that the patient's VWF IF staining pattern was diffuse rather than punctate combined with absent colocalization with the alpha granule membrane protein P-selectin within MKs, suggest that MK-synthesized VWF is not packaged into alpha granules. In conclusion we have further evaluated the molecular pathogenesis of a common and recurrent VWD mutation using two comparative cellular approaches, which demonstrates the importance of such cell types in further understanding the pathophysiology of VWD.
Walker: Baxter Corporation: Research Funding. James:CSL-Behring, Baxter, Bayer: Honoraria, Research Funding.