Structural implications of the substitution of Val for met at residue 239 in the alpha chain of human platelet glycoprotein IB

Abstract A new monoclonal antibody (MoAb), SZ 2, reactive with the human platelet glycoprotein Ib complex has been produced by the hybridoma technique. SZ 2 immunoprecipitated the components of the glycoprotein Ib complex, glycoprotein Ib and glycoprotein IX, from Triton-X-100- solubilized, periodate-labeled platelets. Western blot analysis indicated that the epitope for SZ 2 was on the alpha-subunit of glycoprotein Ib. Scatchard analysis of SZ 2 binding to formaldehyde- fixed, washed platelets revealed a single class of binding sites with Kd = 6.6 +/- 3.3 X 10(-10) mol/L and 15,200 +/- 4,100 binding sites per platelet (mean +/- SD, n = 10). Intact antibody and its purified (Fab')2 fragments not only inhibited the ristocetin-dependent binding of von Willebrand factor to platelets and ristocetin-induced platelet agglutination but also inhibited platelet aggregation induced by Type I collagen and platelet-activating factor (PAF). SZ 2 inhibited platelet serotonin and beta-thromboglobulin release in response to these stimuli and also platelet thromboxane A2 formation in response to ristocetin and collagen. SZ 2 was without effect on platelet aggregation or release in response to other platelet stimuli such as ADP, thrombin, or arachidonic acid. The inhibition by SZ 2 of collagen- and PAF-induced platelet aggregation is surprising in that Bernard-Soulier syndrome platelets, which lack the glycoprotein Ib complex, respond normally to both these stimuli. SZ 2 was unreactive toward Bernard-Soulier syndrome platelets, as evaluated by fluorescence-associated cell sorting, and had no effect on the collagen- and PAF-induced aggregation of Bernard- Soulier syndrome platelets. The combined results suggest that the inhibition by SZ 2 of collagen- and PAF-induced aggregation of normal platelets is steric and are consistent with the glycoprotein Ib complex and the platelet collagen and PAF receptor(s) being adjacent in the human platelet plasma membrane.

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5624817 Mutations in the gene encoding the alpha chain of platelet glycoprotein Ib

Biotechnology Advances ◽

10.1016/s0734-9750(97)87571-4 ◽

1997 ◽

Vol 15 (3-4) ◽

pp. 680-681

Keyword(s):

Platelet Glycoprotein ◽

Glycoprotein Ib ◽

Gene Encoding ◽

Alpha Chain

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MOLECULAR CLONING OF HUMAN PLATELET GLYCOPROTEIN Ib

10.1055/s-0038-1642927 ◽

1987 ◽

Author(s):

J A Lopez ◽

D W Chung ◽

K Fujikawa ◽

F S Hagen ◽

T Papavannopoulou ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Human Platelet ◽

Repetitive Sequences ◽

Platelet Glycoprotein ◽

Robert Wood Johnson Foundation ◽

Predicted Amino Acid Sequence ◽

Activation Process ◽

Glycoprotein Ib ◽

Proteolytic Fragment

Glycoprotein Ib (GPIb) mediates von Willebrand factor-dependent platelet adhesion and participates in the resulting platelet activation process. In the present investigation, the primary structure of human platelet GPIb was studied. GPIb and its proteolytic fragment glycocalicin were purified to near homogeneity from human platelets by affinity chromatography using wheat germ agglutinin and anti-GPIb monoclonal antibody (D. Nugent, University of Washington) coupled to Sepharose. GPIba chain, β chain, and glycocalicin were isolated, reduced and carboxymethylated, and then fragmented by trypsin and S. aureus V8 protease. Peptides were isolated by HPLC and subjected to amino acid sequence analysis. Approximately 200 amino acid residues were identified. Affinity purified rabbit antibodies directed against the a chain, the ft chain, and glycocalicin were prepared and shown to be monospecific by Western blot analysis. Total RNA was prepared from human erythroleukemia cells grown in the presence of phorbol acetate. Poly(A)+ RNA was selected and used to prepare a cDNA library in λgt11. The library was screened with [125]I-labeled polyclonal antibody to glycocalicin. The clone with the largest cDNA insert was sequenced and shown to code for amino acid sequences corresponding to those determined by Edman degradation of glycocalicin. The predicted amino acid sequence contains at least six tandem repeats of 24 amino acids that are highly homologous with 13 repeats present in leucine rich α2 glycoprotein of human plasma. Another region in the protein contains a second repeat rich in threonine and serine, which shows some homology to a 9 amino acid repeat in the connecting region of human factor V. This region is probably the major site of attachment of clusters of O-linked carbohydrate in GPIbα. These results indicate that human platelet glycoprotein Ibα has a multi-domain structure composed of a number of repetitive sequences. Supported in part by grants from the American Heart Association, Robert Wood Johnson Foundation, Veterans Administration, and National Institutes of Health.

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