StyI polymorphism at nucleotide 1610 in the human platelet glycoprotein Ib alpha gene

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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Pseudo-von Willebrand disease: a mutation in the platelet glycoprotein Ib alpha gene associated with a hyperactive surface receptor

Blood ◽

10.1182/blood.v81.7.1787.1787 ◽

1993 ◽

Vol 81 (7) ◽

pp. 1787-1791 ◽

Cited By ~ 81

Author(s):

SD Russell ◽

GJ Roth

Keyword(s):

Von Willebrand Factor ◽

Von Willebrand Disease ◽

Single Amino Acid ◽

Platelet Glycoprotein ◽

Glycoprotein Ib ◽

Platelet Surface ◽

Normal Individuals ◽

Alpha Gene ◽

Von Willebrand ◽

Willebrand Factor

Abstract Pseudo (platelet-type)-von Willebrand disease is an autosomal dominant bleeding disorder caused by the hyperfunction of a receptor on the platelet surface. The abnormal receptor, glycoprotein Ib, displays increased affinity for its ligand, von Willebrand factor. Four members (normal mother/affected father/two affected daughters) of a family with pseudo-von Willebrand disease were studied to determine the molecular genetic basis for their congenital platelet defect. Segments of the platelet glycoprotein Ib alpha gene were amplified by means of the polymerase chain reaction, cloned, and sequenced. A point mutation (A to G, codon 239) was found in segments from the affected individuals but not from the normal. The mutation results in a single amino acid substitution (valine-mutant for methionine-normal) at residue 239 within the Ib alpha binding site for von Willebrand factor. Both the mutant and the normal sequence were found in affected individuals, suggesting a heterozygous state. Amplified DNA from family members and from 58 normal individuals was analyzed by allele-specific oligonucleotide hybridization. Only the normal sequence was found in the mother and the normal individuals, whereas both the normal and the mutant alleles were found in the affected family members. The described mutation is associated with the pseudo-von Willebrand disease phenotype seen in this kindred. The resultant single amino acid substitution in glycoprotein Ib alpha relates to increased receptor function and to excessive binding of von Willebrand factor to the platelet surface.

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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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Structural characterization and chromosomal location of the gene encoding human platelet glycoprotein Ib beta

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)32456-0 ◽

1994 ◽

Vol 269 (26) ◽

pp. 17424-17427 ◽

Cited By ~ 14

Author(s):

M. Yagi ◽

S. Edelhoff ◽

C.M. Disteche ◽

G.J. Roth

Keyword(s):

Structural Characterization ◽

Chromosomal Location ◽

Human Platelet ◽

Platelet Glycoprotein ◽

Glycoprotein Ib ◽

Gene Encoding

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A murine antiglycoprotein Ib complex monoclonal antibody, SZ 2, inhibits platelet aggregation induced by both ristocetin and collagen

Blood ◽

10.1182/blood.v69.2.570.bloodjournal692570 ◽

1987 ◽

Vol 69 (2) ◽

pp. 570-577 ◽

Cited By ~ 1

Author(s):

CG Ruan ◽

XP Du ◽

XD Xi ◽

PA Castaldi ◽

MC Berndt

Keyword(s):

Monoclonal Antibody ◽

Platelet Aggregation ◽

Binding Sites ◽

Human Platelet ◽

Platelet Glycoprotein ◽

Scatchard Analysis ◽

Type I ◽

Glycoprotein Ib ◽

Induced Aggregation ◽

Bernard Soulier Syndrome

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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