Increased platelet sensitivity to ristocetin is predicted by the binding characteristics of a GPIb/IX determinant

Abstract

Platelets from patients with platelet-type von Willebrand disease (vWD) were used as immunogens for the production of murine monoclonal antibodies (MoAbs). One such MoAb, C-34, inhibited ristocetin-induced aggregation of patient or normal platelets, but not aggregation induced by other aggregating agents. As demonstrated by crossed-immunoelectrophoresis, C-34 recognized an epitope within the GPIb/IX complex. In indirect immunofluorescence studies on fresh platelets, the ratio of any of four different anti-GPIb MoAbs to one another was near unity (0.88-1.14) both for normals and for patients. In contrast, the ratio of the binding of C-34 to such a MoAb (AP-1) was 0.31 +/- 0.02 (means +/- SE) for normal platelets and significantly increased to 0.54 +/- 0.01 for patient platelets (P less than 0.001). In NP-40 lysates of 3H-labelled platelets, saturating concentrations of C-34 produced much fainter bands than did AS-2 or other anti-GPIb MoAbs in the GPIb and GPIX regions. In contrast to the other anti-GPIb MoAbs, C-34 did not bind to the purified 125I-labelled glycocalicin fragment of GPIb, to the glycocalicin derivative identified by crossed-immunoelectrophoresis, or to the amino-terminal approximately 40 kDa portion of GPIb alpha cleaved from 3H-labelled platelets by trypsin. C-34 appears to be the first MoAb that is quantitatively informative in identifying the abnormal platelets in platelet-type vWD. The observed differences between the patient and normal platelets may reflect an abnormality in the primary structure of the GPIb/IX complex. Alternatively, patient platelets may have an abnormality of other structures near this region that impose less of a steric hindrance upon binding of antibody to the C-34 epitope.

Glycoprotein Ib Can Mediate Endothelial Cell Attachment to a von Willebrand Factor Substratum

“…Platelet cell surface sialic acid residues were labeled by periodate oxidation followed by reduction with Na3HBH4 (23,24). Platelets at a concentration of 5 X 109/ml were oxidized with 2 mM sodium metaperiodate at pH 6.5 for 10 min in the dark and washed twice with PBS-EDTA (20 mM sodium phos phate, 130 mM NaCl, 2mM EDTA), pH 6.8.…”

Mutation in the gene encoding the alpha chain of platelet glycoprotein Ib in platelet-type von Willebrand disease.

“…Competitive oligonucleotide primer assay for this mutation showed a homozygous wild-type pattern in genomic DNA from the 161 normal volunteers studied and from 6 phenotypically normal members of a PT-vWD family. All 7 affected members of this family studied were heterozygous for the mutant allele. Platelet GP Iba mRNA reverse-transcribed and studied by competitive oligonucleotide primer assay showed similar expression of the mutant and wild-tpe alleles in the affected PT-vWD patients.…”

“…The unique ability of desialylated vWF (asialo-vWF) to agglutinate patient platelets in the presence of the divalent-cation chelator EDTA has additionally been demonstrated (6). Platelets from patients with PT-vWD also show a characteristically increased binding of the monoclonal antibody C-34, which is directed against an epitope within the platelet glycoprotein (GP) lb/IX complex (7). Although this complex is known to constitute the platelet's ristocetin-dependent receptor for vWF (8), identification of a unique structural abnormality within this complex that might underlie the functional abnormalities seen in PT-vWD has not yet been achieved.…”

Mimotope/anti-mimotope probing of structural relationships in platelet glycoprotein Ib alpha.

“…By indirect immunofluorescence, the ratio of binding of C-34 compared to four other anti-GPIba mAbs was 0.31 for normal platelets, but significantly increased to 0.54 for PT-vWD platelets (12). The epitope for C-34 is present within the GPIb/IX complex (12) and recently has been localized to the extracellular portion of the GPIba chain expressed on the surface of Chinese hamster ovary cells (14). The failure of C-34 to bind to denatured GPIba in Western blots (13,15) (17), using the decapeptide library described by Christian et al (19) and kindly provided by Bruce Malcolm (Univ.…”