Disease relevance of GP9

• Patients with Bernard-Soulier syndrome demonstrate that mutations in either GPIb beta or GPIX result in an absence of platelet GPIb alpha [1].
• Using antibody screening, we cloned a cDNA encoding GPIX from a human erythroleukemia cell cDNA library constructed in phage lambda gt11 [2].
• Drug-induced thrombocytopenia: localization of the binding site of GPIX-specific quinine-dependent antibodies [3].
• In addition, Fli-1 binding of the GPIX Ets site was identified in antibody supershift experiments in nuclear extracts derived from hematopoietic human erythroleukemia cells [4].
• Flow cytometric studies, using a panel of monoclonal antibodies (MoAbs), showed that Serratia marcescens protease treatment removed greater than 97% of the glycocalicin portion of GPIb but did not affect the changes in the expression of GPIX or GMP-140 that were induced by high concentrations of alpha-thrombin (10 nmol/L) [5].

High impact information on GP9

• The predicted amino acid sequence of mature GPIX includes an NH2-terminal extracytoplasmic domain of 134 residues, a transmembrane domain of 20 residues, 6 intracytoplasmic residues, and 1 N-linked glycosylation site [2].
• To identify the structural determinants of GPIX recognized by quinine-dependent antibodies, 4 chimeric mouse/human GPIX constructs and stable Chinese hamster ovary (CHO) cell lines that expressed the chimeras in association with GPIbalpha and GPIbbeta were produced [3].
• The analysis of 6 patient sera with the chimeric cell lines provided evidence for localization of the anti-GPIX quinine-dependent antibody binding site to the C-ext region (amino acid [aa] 64-135) of human GPIX [3].
• In contrast, we demonstrate that GATA-1 and Fli-1 synergistically activate the megakaryocyte-specific promoters GPIX and GPIbalpha in transient transfections [6].
• The cysteine knot of platelet glycoprotein Ib beta (GPIb beta) is critical for the interaction of GPIb beta with GPIX [1].

Chemical compound and disease context of GP9

• We describe a new mutation in glycoprotein IX (GPIX) in a patient with Bernard-Soulier syndrome (BSS) [7].
• In the present report, we demonstrate that botrocetin does not induce agglutination of formalin-fixed platelets from a patient with Bernard-Soulier syndrome congenitally lacking GPIb and GPIX as well as GPV, a finding similar to that shown with ristocetin [8].

Biological context of GP9

• DNA-sequence analysis revealed normal sequences for GPIb alpha and GPIX [9].
• Analysis of the genes for two other components of the platelet GPIb:IX complex, namely GPIb beta and GPIX, showed two different missense mutations in the coding region of the GPIX gene: an A-->G transition in codon 21 results in conversion of an aspartic acid to glycine and an A-->G change in codon 45 converts an asparagine residue to serine [10].
• Several point mutations and deletions affecting the GPIb alpha gene have been identified as the cause of BSS, whilst in four BSS families a GPIX gene defect has been reported [11].
• Transient transfection studies revealed that the mutant GPIbbeta polypeptide was not detected in the transfected 293T cells, suggesting that null expression of the mutant GPIbbeta impairs expression of the GPIbalpha and GPIX subunits and results in a BSS phenotype in the patient [12].
• Transient transfection studies confirmed that mutant GPIX was not expressed on the transfected cells, showing that the mutation was responsible for the BSS phenotype observed in the patient [13].

Anatomical context of GP9

• Similar results were obtained with Chinese hamster ovary alpha IX cells, a stable cell line expressing GPIbalpha that retains the capacity to re-express GPIX [9].
• In this investigation, we studied the subcellular localization of GPIb, GPIX, and GPV in the resting platelets of normal subjects, patients with Bernard-Soulier syndrome, patients with Gray platelet syndrome, and human cultured megakaryocytes [14].
• The adhesion was specifically inhibited by an anti-GPIbalpha blocking antibody (AK2) and was not observed with CHO cells expressing GPIbbeta and GPIX alone [15].
• These genetic changes alter the transmembrane domain of GPIX or GPIb alpha and, therefore, would prevent proper insertion of the proteins in the plasma membrane [16].
• Intracellular GPIbalpha and GPIbbeta were detected and largely confined to the endoplasmic reticulum, and little GPIX was seen [17].

Associations of GP9 with chemical compounds

• Three affected individuals are doubly heterozygous for these mutations, which alter conserved residues in or flanking the GPIX leucine-rich glycoprotein motif [10].
• A monoclonal antibody against glycoprotein Ib, FMC 25, was negative by immunofluorescence against Bernard-Soulier platelets and immuneprecipitated both GP Ib and GPIX from Triton X100 solubilized, labeled platelets [18].
• Infusion of 1-desamino-8-D-arginine vasopressin normalized bleeding time in the two severely affected patients, although it did not modify ristocetin-induced platelet agglutination or membrane expression of GPIbalpha, GPIX, GPIIb-IIIa and GMP-140 [19].
• Both TRAP and ADP/epinephrine redistributed platelet surface GPIb, GPIX, and GPV to the SCCS [20].
• The quinidine-induced sera strongly immunoprecipitated purified GPIb-IX complex, weakly immunoprecipitated purified GPIX and the recombined complex, but did not immunoprecipitate purified GPIb [21].

Physical interactions of GP9

• This study also provides two additional lines of support for the recent report that GPV is noncovalently complexed with GPIb and GPIX in the platelet surface membrane [20].
• Inhibition studies indicated that DDAbs recognized epitopes residing on the GPIIb/IIIa complex and on the GPIX subunit, respectively [22].

Regulatory relationships of GP9

• However, Serratia treatment almost completely abolished the increase in platelet surface GMP-140 induced by low concentrations of alpha-thrombin (0.5 nmol/L) and diminished the downregulation of platelet surface GPIX by 60.9% +/- 5.6% (mean +/- SEM, n = 3) [5].

Other interactions of GP9

• This occurs through the interaction of GPIX with GPIb beta [1].
• The complex is composed of four membrane-spanning glycoproteins (GP): GPIbalpha, GPIbbeta, GPIX, and GPV [23].
• Platelet GPIb, a disulfide-linked alpha beta heterodimer (Mr 160,000) that forms a noncovalent complex with GPIX (Mr 22,000), functions as the platelet adhesion receptor for surface-bound von Willebrand factor [24].
• Sequence analysis was performed after polymerase chain reaction amplification of the coding region of the GPIX and GPI b alpha genes from the patient [25].
• Levels of some megakaryocytic messages (GPIIIa, GPIX) showed a marked rise by 12 days of incubation in the inducer combination [26].

Analytical, diagnostic and therapeutic context of GP9

• Although low levels of GPIb were demonstrable by both flow cytometry and immunoblot analysis in an affected individual's platelets, there was no evidence of GPIX immunoreactivity [10].
• We investigated the molecular basis of another case of BSS with a deficient expression of GPIX, as detected by immunofluorescence studies [11].
• By Northern blot analysis, the GPIX cDNA hybridizes with a single 1.0-kilobase species of platelet poly(A)+ RNA [2].
• Surface-labeling of the platelets and two-dimensional gel electrophoresis showed reduced but detectable amounts of glycoprotein (GP) Ib-IX-V present;however, there was markedly less GPIX (2% +/- 1% of normal) than GPIb alpha, Ib beta, or V (7% +/- 2% of normal) [25].
• Analysis of the patient's platelets by flow cytometry and Western blotting failed to detect surface expression of any of the four subunits of the glycoprotein (GP)Ib-V-IX complex and revealed small amounts of intracellular GPIbalpha, GPIbbeta and GPV but no GPIX [27].

References