Abstract
Type IIA von Willebrand disease (vWD) results from abnormalities in von Willebrand factor (vWF) characterized by absence of plasma high molecular weight (HMW) vWF multimers. In this report, 5 distinct point mutations were identified in 6 Type IIA vWD families. A total of 7 mutations, all clustered within a 124-amino acid segment of the vWF A2 domain, now account for 9 of a panel of 11 Type IIA families. In COS-7 cells, 3 single amino acid substitutions, Val844----Asp, Ser743----Leu, and Gly742----Arg, impaired the transport of vWF multimers between the endoplasmic reticulum and the Golgi complex, with more profound effects on the secretion of HMW multimers than lower molecular weight forms. In contrast, 2 substitutions, Arg834----Trp and Gly742----Glu, resulted in secretion of HMW multimers similar to wild-type vWF. The vWF structure observed within patient platelets correlated closely with the synthesis pattern seen for the corresponding mutants in COS-7 cells. These findings demonstrate that structural alterations within the A2 domain of vWF can produce the characteristic phenotype of Type IIA vWD via two distinct molecular mechanisms.
Highlights
Type IIA von Willebranddiseaseresults from abnormalities in von Willebrand factor characterized by absence of plasma high molecular weight (HMW) vWF multimers
The vWF structure observed within patient platelets correlated closely with the synthesis pattern seen for the corresponding mutants in COS-7 cells
These findings demonstrate that structural alterations within the A2 domain of vWF can produce the characteristic phenotype of Type IIAvWD via two distinct molecular mechanisms
Summary
PCR and DNA Sequence Analysis-DNA PCR was performed as medium minus methionine and cysteine with 0.5%bovine serum previously described, using allele-specific oligonucleotide primers to albumin. Including the three previously reported mutations [13, 14], we have identified 7 distinct mutations accounting for 9 of a total of 11 unrelated Type IIA vWD families studied Point Mutations Have Heterogeneous Effects on Secretion of uWF-In order to examine the biosynthesis and secretion of vWF containing the Type IIA mutations, COS cells were transfected with a vWF expression plasmid (pMT2vWF)containing each of five mutations indicated by boxes in Fig. 1.A &galactosidase expression vector (pJ3P-Gal) was co-transfected with each construct to provide an internalcontrol for transfection efficiency and immunoprecipitation. Quantitative analysis of cell media and lysates by ELISA showed that the amount of vWF present in the cell medium was similar or mildly decreased compared to wild-type for R834W and G742E, but was greatly reduced for themutants V844D, S743L, and G742R (Fig. 2).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
