- Top of page
- Materials and methods
ABO (H) blood group antigens are covalently linked to the oligosaccharide side-chains of von Willebrand factor (VWF). In this study, we investigated the role of the A and B antigens in the expression of VWF adhesive activity. VWF of type A, B or O was purified from fresh frozen plasma. Presence of A or B antigen on the VWF was confirmed by enzyme-linked immunosorbent assay (ELISA) and by immunoblotting with monoclonal anti-A or anti-B. The A or B antigen was also detected in the 48/52-kDa fragment of the respective VWF after trypsin digestion. Removal of A antigen with α-N-acetylgalactosaminidase or B antigen with α-galactosidase did not affect its multimer size or antigenic level, but decreased the ristocetin cofactor (RCoF) activity of the respective VWF by 33–39% (P < 0·01–0·002). Removal of A or B antigen from VWF did not affect the binding of the VWF to immobilized type III collagen. A and B antigens were not detected in platelet VWF. These results indicate that AB structures play a role in platelet aggregating activity of VWF.
von Willebrand factor (VWF), a multimeric glycoprotein (GP) synthesized in endothelial cells and megakaryocytes, is critical for normal haemostasis as it promotes platelet adhesion and aggregation at sites of vessel injury. A deficiency in VWF leads to a bleeding diathesis, which can be fatal in severe cases. Studies in animal models of von Willebrand disease have suggested that VWF is also involved in the development of arterial thrombosis and atherosclerosis (Blann & McCollum, 1994). Some studies have shown that a high plasma level of VWF in patients with coronary artery disease is an independent risk factor for myocardial infarction and death (Jansson et al, 1991; Thompson et al, 1995). VWF may also promote cancer metastasis (Nierodzik et al, 1995) and vaso-occlusion in sickle cell disease (Kaul et al, 1993).
VWF, secreted from endothelial cells as a disulphide-linked polymer of a 2050-residue polypeptide, is cleaved in the circulation by a plasma metalloproteinase to become the series of multimers found in normal plasma (Tsai, 1996). The loop structure formed by the disulphide bond between Cys509 and Cys695 in the A1 domain is critical in modulating VWF binding to platelet GP Ib/IX (Meyer & Girma, 1993). The A3 domain, which contains a disulphide-linked loop between Cys923 and Cys1109 as well as the A1 domain, is involved in VWF binding to collagen (Meyer & Girma, 1993).
Plasma VWF contains 12 N-linked and 10 O-linked oligosaccharide chains, which account for approximately 15% of the total weight (Titani et al, 1986). Recombinant VWF deficient in the O-glycans binds normally to collagen but less effectively to platelets in the presence of ristocetin (Carew et al, 1992). Although removal of sialic acid does not affect the ristocetin-induced binding of VWF to platelets, it increases the susceptibility of VWF to N-terminal proteolytic cleavage (Berkowitz & Federici, 1988). Asialo-VWF is also rapidly cleared by the liver (Sodetz et al, 1977). Platelet VWF contains 50% less carbohydrates and has a lower ristocetin cofactor activity than plasma VWF (Williams et al, 1994).
In humans and anthropoid apes, A and B antigens are found on the red cells as well as on epithelial and endothelial cells, whereas in lower mammals they are expressed only in the epithelial cells of the gastrointestinal (GI) tract (Kominato et al, 1992; Yamamoto et al, 1992). ABO (H) antigens are also present in the N-linked oligosaccharide chains of human plasma VWF and α2-macroglobulin (Matsui et al, 1993). Although the ABO (H) gene is highly conserved in the genomic DNA of various mammals (Kominato et al, 1992), the ABO (H) structures have not been associated with any biological functions.
Recombinant green coffee bean α-galactosidase and α-N-acetylgalactosaminidase purified from chicken livers have been used to produce type O cells from type B and type A cells by removing A and B antigens from the respective red blood cells (Zhu & Goldstein, 1995; Zhu et al, 1996). In this study, we investigated the role of AB antigens in the expression of VWF adhesive activity by enzymatically removing A or B antigen from blood type specific VWF.
- Top of page
- Materials and methods
A, B and O (H) blood group structures have been shown to be covalently linked to N-glycans of VWF (Matsui et al, 1992). This study confirms the presence of covalently bound AB antigens on VWF in a blood group-specific manner. The study further demonstrates that AB structures are present on the 48/52-kDa fragments of trypsin-treated VWF, which contains the A1 domain critical in regulating the binding of VWF to platelet glycoprotein Ib/IX (Meyer & Girma, 1993).
Removal of A or B antigen from type A or B VWF with Azyme or Bzyme decreased the RCoF of the VWF. The reduction of VWF activity resulted specifically from the removal of the A or B antigen because cross-treatment of type A VWF with Bzyme, type B VWF with Azyme or type O VWF with Azyme or Bzyme did not affect the RCoF activity of the VWF. Blocking of A or B antigen with polyclonal anti-A or anti-B also decreased its RCoF (data not shown).
Both RCoF- and collagen-binding activities of VWF are affected by the size of the VWF multimers. The selective decrease in RCoF but not in collagen binding suggests that the observed decrease in RCoF in association with AB antigen removal is not caused by a loss of the large multimers. SDS agarose gel electrophoresis confirms that treatment with the enzymes does not alter the multimeric structure of VWF.
How does removal of AB antigen from VWF cause a decrease in its RCoF activity? Platelet aggregation is determined not only by the binding of VWF molecules to platelets but also by their capacity to bind multiple platelets. A decrease in the VWF RCoF activity might be caused by a decrease in VWF–platelet binding. However, our data demonstrated that the same VWF or VWF at concentrations similar to those used in ristocetin cofactor assays did not exhibit a difference in platelet binding compared with control VWF. This suggests that the decrease of VWF RCoF after AB antigen removal cannot be attributed to deceased VWF–platelet binding. However, these data do not preclude the possibility that AB antigens affect VWF–platelet binding under different conditions.
Unfolding of VWF by shear stress has been shown to enhance the capacity of VWF to support ristocetin-induced platelet aggregation (Tsai & Lian, 1998), without increasing its binding to platelets (unpublished observations). Thus, we hypothesize that removal of AB antigens may cause the opposite changes in VWF; it renders the VWF more compact and less capable of binding multiple platelets. More studies are needed to determine the validity of this hypothesis.
Sialic acid or galactose moieties have been suspected to play a role in the adhesive activity of VWF (Sodetz et al, 1977; Gralnick, 1978; Kao et al, 1980). However, the observed decrease may have been due to proteolysis caused by contaminating proteases in the enzyme preparations because other studies failed to demonstrate a change in VWF activity by either neuraminidase or β-galactosidase when the experiments were conducted in the presence of protease inhibitors (Federici et al, 1984; Goudemand et al, 1985). On the other hand, recombinant VWF specifically lacking O-linked carbohydrates exhibits less binding to platelets and a diminished capacity to promote ristocetin-dependent platelet agglutination (Carew et al, 1992). Platelet VWF has been found to be less effective than plasma VWF in supporting platelet agglutination, despite its particularly large multimeric composition (Williams et al, 1994). Sweeney & Hoernig (1992) reported that platelet VWF does not contain AB antigens. Our studies confirm the absence of AB antigens on platelet VWF. Based on the findings in this study, we speculate that the lack of AB structures may contribute to the lower RCoF activity of platelet VWF than that of plasma VWF.
A difference in VWF activity may have important clinical implications. ABO blood type has been found to be an important determinant of various bleeding and thrombotic disorders. For example, high A/O and B/O relative incidences are found in patients with thrombotic strokes, whereas the opposite is found in patients with haemorrhagic strokes (Ionescu et al, 1976). Ischaemic heart disease also occurs more frequently in non-O blood group people (Medalie et al, 1971; Erikssen et al, 1980; Meade et al, 1994). On the other hand, type O is overrepresented by patients of type I von Willebrand disease (Gill et al, 1987) and by patients of Hermansky–Pudlak syndrome with bleeding manifestations (Witkop et al, 1993). Our findings provide a plausible explanation of why type O blood group, compared with non-O blood group, is associated with a lower plasma VWF RCoF activity, a higher risk of bleeding and a lower risk of thrombosis.