Molecular defects in the Bernard-Soulier syndrome: assessment of receptor genes, transcripts and proteins.

Bernard-Soulier syndrome involves a multicomponent adhesion receptor on the surface of human platelets. Patients with this disorder bleed excessively from the skin and mucous membranes; and in occasional cases, the bleeding is fatal. At a molecular level, the Bernard-Soulier defect affects the structure and/or function of a receptor that mediates platelet adhesion in the arterial circulation. This receptor, termed glycoprotein (GP) Ib-IX-V, consists of 4 distinct polypeptides (GPs: Ib alpha-143 kDa, Ib beta-22 kDa, IX-20 kDa, V-83 kDa) that share features such as physical associations and leucine-rich glycoprotein (LRG) repeats. All 4 genes and cDNAs have now been cloned and characterized, and the genes have been localized to distinct chromosomal sites. A number of Bernard-Soulier syndrome kindreds have been defined at the molecular genetic level; and in most instances, the defect proved to be a point mutation in either the GP Ib alpha or the GP IX gene. Study of the genetic defects provides insight into both the expression and the function of the receptor. Expression requires the co-ordinated synthesis of the Ib-IX polypeptides with a contribution from GPV. Function of the receptor entails the effect of shear forces generated by blood flow in the artificial circulation. The current challenge in this field is to understand the structure-function relationships within the receptor and its cognate adhesive ligand, von Willebrand factor (vWf).

Laboratory assessment of von Willebrand factor. Use of different assays can influence the diagnosis of von Willebrand's disease, dependent on differing sensitivity to sample preparation and differential recognition of high molecular weight VWF forms.

Three separate laboratory assays for von Willebrand Factor (VWF), a standard "antigen" (antisera-ELISA-based) assay (VWF:Ag), a standard ristocetin-dependent-platelet-agglutination procedure (VWF:RCof), and an ELISA-based collagen-VWF binding assay (VWF:CBA), have been evaluated for their ability to detect alterations in VWF levels following differential processing of blood for testing, and specifically in (1) serum compared to plasma and (2) filtered plasma compared to nonfiltered plasma. Although all assays tended to detect some change, sensitivity of detection varied between assays, with the VWF:CBA most consistently able to detect large decreases in VWF levels in serum and filtered plasma. The authors propose that the increased sensitivity of the VWF:CBA assay to VWF depleted in these circumstances is that this assay selectively detects higher molecular weight forms (ie, those known to be more functionally relevant), and that assay results reflect the preferential incorporation of these forms in in the platelet-fibrin-gel during the clotting process, and onto the filter matrix during filtration. To confirm this, multimer analysis was performed and showed a reduction in high molecular weight forms of VWF in these cases. Finally, direct evidence that the VWF:CBA assay preferentially detects high molecular weight forms of VWF was obtained following fractionation of normal plasma VWF (separation according to molecular weight using size exclusion matrix; confirmed by specific multimer analysis) and assessment of eluted VWF. Using a standard VWF:Ag assay, detection of eluted VWF was unrelated to molecular size. In contrast, the VWF:CBA showed selective detection, and was able to preferentially discriminate high and intermediate forms of VWF from low molecular weight forms. The findings are of particular relevance to diagnostic pathology laboratories because filtered plasma or serum can be inappropriately (and unknowingly) provided for the clinically queried diagnosis of von Willebrand's disease (VWD). As outlined in this report, these samples can yield VWF results that closely mimic those of a Type 2A or Type 2B VWD individual, and thus, VWD may be incorrectly diagnosed.