Abstract
Abstract 3059
Poster Board II-1035
Type 3 von Willebrand disease (VWD) is characterized by the virtual absence of von Willebrand factor (VWF) in plasma. Affected individuals are usually either homozygous or compound heterozygous for mutations that have been identified throughout the 52 exon VWF gene (listed in the ISTH SSC VWF database, http://www.sheffield.ac.uk/vwf/index.html). Most are null mutations associated with deletions, insertions, frameshifts, splicing defects and premature stop codons, however, a number of missense mutations have also been identified. VWF is normally stored in the Weibel-Palade bodies of vascular endothelial cells and within platelet alpha granules. Thus, the absence of plasma VWF in type 3 VWD patients carrying non-null mutations could be explained by one or more of: 1) premature mRNA degradation; 2) improper folding or assembly of VWF within cells resulting in proteolytic degradation; 3) rapid clearance of defective VWF molecules from plasma; 4) inability of synthesized VWF to be properly stored and/or released.
We set out to detect type 3 VWD patients with possible VWF plasma clearance, storage or release defects by measuring the relative amounts of VWF in platelets and plasma, reasoning that such defects may be associated with the accumulation of VWF in platelets. Blood was prospectively collected from 21 previously diagnosed cases. Laboratory evaluation confirmed type 3 VWD, if the VWF:Ag and VWF:RCo levels were '5 IU per dL or if the VWF:Ag or VWF:RCo levels were 6-10 IU per dL accompanied by FVIII levels of '10 IU per dL. From this cohort, 16 unrelated type 3 VWD index cases were identified. Equivalent amounts of plasma and platelet lysates were analyzed for VWF by immunoblotting. As expected, platelet and plasma VWF were observed to be very low or absent in most cases. Interestingly, 5 patients were found to have relatively more (∼10 fold) platelet than plasma VWF using semiquantitative immunoblotting and equivalent sample loading. Type 3 VWD index cases together with available type 1 VWD siblings were subjected to VWF gene sequencing analyses. VWF exons 1-52 as well as ∼1500 bp of the promoter and intron/exon boundaries were sequenced. Strategies to identify partial gene deletions are ongoing. VWF gene mutations were identified in 12/16 patients. Surprisingly, in 4 out of the 5 patients with discrepant VWF content in plasma and platelets, no mutation in the VWF gene was identified by sequencing, suggesting either that an in-frame partial deletion or intronic VWF mutation may be present or that other genes such as those regulating the assembly, storage or release of VWF could be affected. A novel homozygous frameshift mutation at position c.8418_8419 resulting from a TCCC insertion was identified in one case. The presence of platelet VWF in this patient suggests that despite this frameshift mutation, VWF is expressed and packaged into platelets. Two siblings with the same homozygous mutation had an identical pattern (no plasma VWF but platelet VWF present) whereas two heterozygous siblings had 10-30% plasma VWF:Ag with normal amounts of platelet VWF. Studies are underway to determine whether increased clearance or defects in storage and/or release from endothelial cells or platelets are the cause of absent plasma VWF. Our studies also suggest that type 3 VWD can be subcategorized into subtype 0 (no plasma no platelet VWF) versus subtype P (no plasma but platelet VWF present) by measuring plasma and platelet VWF. We hypothesize that patients with type 3 VWD with platelet-harbouring VWF may have milder clinical manifestations compared to the subtype 0, however further studies are needed to test this proposal.
Disclosures
No relevant conflicts of interest to declare.
Re-establishment of VWF-dependent Weibel-Palade bodies in VWD endothelial cells