Human Platelet Membrane Glycoprotein Research Articles

Frequency of Human Platelet Antigens -1 to -5 and -15 in Turkmen Blood Donors.

Due to the presence of platelet antigen polymorphisms, human platelet membrane glycoproteins can be identified as an alloantigen or autoantigen. The aim of this study was to determine the frequencies of human platelet antigens (HPAs)-1 to-5 and-15 in Turkmen blood donors and establish a panel of accredited HPAs negative donors as well as an HPA-typed platelet donor registry. HPA-1 to-5 and-15 typing was performed by the polymerase chain reaction-sequence-specific primer techniques on 80 unrelated Turkmen donors who were referred to Aq-Qala Blood Transfusion Center in Golestan Province from September 2018 to October 2019. The frequencies of HPA phenotypes were determined as follows: HPA-1aa: 92.5%, HPA-1ab: 7.5%, HPA-2aa: 77.5%, HPA-2ab: 20.0%, HPA-2bb: 2.5%, HPA-3aa: 75.3%, HPA-3ab: 50%, HPA-3bb: 11.2%, HPA-4aa: 100%, HPA-5aa: 78.5%, HPA-5ab: 21.5%, HPA-15aa: 41.2%, HPA-15ab: 56.2% and HPA-15bb: 17.5%. Determining the genotype of HPAs that play an important role in platelet refractory can improve the management of alloimmunization due to the incompatibility of HPAs between the recipients and donors. Therefore, the registration process for national platelet donors can help patients accelerate and improve the quality of transfused platelets.

A Novel Method for Flow Cytometric Quantification of Human Platelet Membrane Glycoproteins in Citrated Whole Blood

Introduction: Diagnosis of platelet receptor defects can be confirmed by flow cytometry using a panel of monoclonal antibodies. The conventional method utilizing platelet rich plasma (PRP) has many limitations such as time-consuming preparation, requirement of a large sample volume or limitations in samples from patients with thrombocytopenia or abnormally large platelets. In order to overcome these difficulties a novel method was developed using a second antibody directed against an erythrocyte protein which allows differentiation between platelets and erythrocytes in citrated whole blood (CWB). Methods: CWB or PRP from healthy blood donors and outclinic patients without platelet disorder (n= 18) as well as from two patients with Glanzmann thrombasthenia (GT) was stained with fluorescein isothiocyanate (FITC)-conjugated antibodies against CD42b (GP Ib), CD41a (GP IIb/IIIa), or CD62. CWB was stained in addition with a phycoerythrin (PE)-conjugated antibody against CD235a, a sialoglycoprotein expressed by erythroid precursors and erythrocytes. Platelet activation was induced by addition of 2 µM phorbol 12-myristate 13-acetat (PMA). The expression of CD42b, CD41a and CD62 on CD235a-negative cells was quantified by flow cytometry with and without PMA stimulation. Results: CWB and PRP samples showed no differences of mean (± standard deviation) fluorescence intensity (MFI)(GP Ib 74.78 ± 21.39 vs. 85.67 ± 26.03, GP IIb/IIIa 250.33 ± 59.08 vs. 234.50 ± 56.96, GP IIb/IIIa with PMA 349.50 ± 76.87 vs. 328.67 ± 72.92, GP IIb 26.10 ± 6.17 vs. 29.15 ± 8.29, GP IIb with PMA 35.58 ± 8.86 vs. 37.50 ± 8.98, CD62 1.91 ± 0.49 vs. 2.49 ± 1.14, CD62 with PMA 43.88 ± 10.89 vs. 37.78 ± 7.20, respectively). The increase of the fluorescence signal with PMA vs. without PMA was similar in CWB and PRP with a percentage increase of 141.17 ± 10.97 vs. 142.74 ± 15.08 % for GP IIb/IIIa, 136.76 ± 16.65 vs. 131.83 ± 16.64 % for GP IIb and 2432.14 ± 785.49 vs. 1701.37 ± 474.72 % for CD62. Patients with GT showed MFI values for GP IIb/IIIa and GPIIb expression less than 5 % in comparison to that of a healthy control. Conclusion: Flow cytometric analysis of platelet membrane proteins utilizing CWB is comparable to the conventional PRP method. Using an antibody against CD235a allows distinct differentiation between platelets and erythrocytes in CWB. This new method requires smaller amounts of blood, spares time-consuming preparation of PRP and is feasible to identify rare inherited platelet membrane disorders. Disclosures Berens: Baxter: Honoraria; Novo Nordisk: Honoraria; CSL Behring: Honoraria; Biotest: Honoraria; Shire: Honoraria; Pfizer: Honoraria. Müller:Roche: Membership on an entity's Board of Directors or advisory committees; Siemens: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; NovoNordisk: Membership on an entity's Board of Directors or advisory committees. Rühl:Sanofi Genzyme: Honoraria; SOBI: Honoraria; Shire: Honoraria; Bayer: Honoraria; CSL Behring: Honoraria; Grifols: Honoraria. Oldenburg:Swedish Orphan Biovitrum: Consultancy, Speakers Bureau; Grifols: Consultancy, Speakers Bureau; NovoNordisk: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Speakers Bureau; Takeda (Shire): Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy, Speakers Bureau; Bayer: Consultancy, Research Funding, Speakers Bureau; Biotest: Consultancy, Research Funding, Speakers Bureau; CSL Behring: Consultancy, Research Funding, Speakers Bureau; Octapharma: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau.

Atomic-Level Dissection of the Polyclonal Immune Response to the Human Platelet Alloantigen, HPA-1a (PlA1)

Alloantibodies to platelet-specific antigens are responsible for two clinically-important bleeding disorders: Post-transfusion purpura and fetal/neonatal alloimmune thrombocytopenia (FNAIT). The HPA-1a/1b (also known as PlA1/A2) alloantigen system of human platelet membrane glycoprotein (GP)IIIa is controlled by a Leu33Pro polymorphism, and is responsible for ~80% of the cases of FNAIT. Local residues surrounding polymorphic residue 33 are suspected to have a profound effect on alloantibody binding and subsequent downstream effector events. To define the molecular requirements for HPA-1a alloantibody binding, we generated transgenic mice that expressed murine GPIIIa isoforms harboring select humanized residues within the PSI and EGF1 domain, and examined their ability to support the binding of a series of monoclonal and polyclonal HPA-1a-specific antibodies. Humanizing the PSI domain of murine GPIIIa was sufficient to recreate the HPA-1a epitope recognized by some HPA-1a-specific antibodies, however humanizing distinct amino acids within the linearly distant, but conformationally close, EGF1 domain was required to enable binding of others. Using a series of transgenic mice and recombinant constructs, we reveal previously unsuspected, complex heterogeneity of the polyclonal alloimmune response to this clinically important human platelet alloantigen system. High-resolution mapping of this alloimmune response may improve diagnosis of FNAIT by providing cells that distinguish specific alloantibody subpopulations, and should facilitate the rational design and selection of contemplated prophylactic and therapeutic anti-HPA-1a reagents. DisclosuresCurtis:Ionis Pharmaceuticals: Consultancy.

High-resolution mapping of the polyclonal immune response to the human platelet alloantigen HPA-1a (PlA1)

AbstractAntibodies to platelet-specific antigens are responsible for 2 clinically important bleeding disorders: posttransfusion purpura and fetal/neonatal alloimmune thrombocytopenia (FNAIT). The human platelet-specific alloantigen 1a/1b (HPA-1a/1b; also known as PlA1/A2) alloantigen system of human platelet membrane glycoprotein (GP) IIIa is controlled by a Leu33Pro polymorphism and is responsible for ∼80% of the cases of FNAIT. Local residues surrounding polymorphic residue 33 are suspected to have a profound effect on alloantibody binding and subsequent downstream effector events. To define the molecular requirements for HPA-1a alloantibody binding, we generated transgenic mice that expressed murine GPIIIa (muGPIIIa) isoforms harboring select humanized residues within the plexin-semaphorin-integrin (PSI) and epidermal growth factor 1 (EGF1) domains and examined their ability to support the binding of a series of monoclonal and polyclonal HPA-1a–specific antibodies. Humanizing the PSI domain of muGPIIIa was sufficient to recreate the HPA-1a epitope recognized by some HPA-1a–specific antibodies; however, humanizing distinct amino acids within the linearly distant but conformationally close EGF1 domain was required to enable binding of others. These results reveal the previously unsuspected complex heterogeneity of the polyclonal alloimmune response to this clinically important human platelet alloantigen system. High-resolution mapping of this alloimmune response may improve diagnosis of FNAIT and should facilitate the rational design and selection of contemplated prophylactic and therapeutic anti–HPA-1a reagents.

The Collagen-binding Integrin α2β1 Is a Novel Interaction Partner of the Trimeresurus flavoviridis Venom Protein Flavocetin-A

Many snake venoms are known for their antithrombotic activity. They contain components that specifically target different platelet-activating receptors such as the collagen-binding integrin α2β1 and the von Willebrand factor receptor GPIb. In a search for an α2β1 integrin-blocking component from the venom of the habu snake (Trimeresurus flavoviridis), we employed two independent purification protocols. First, we used the integrin α2A domain, a major collagen-binding domain, as bait for affinity purification of an α2β1 integrin-binding toxin from the crude venom. Second, in parallel, we used classical protein separation protocols and tested for α2β1 integrin-inhibiting capabilities by ELISA. Using both approaches, we identified flavocetin-A as an inhibitor of α2β1 integrin. Hitherto, flavocetin-A has been reported as a GPIb inhibitor. However, flavocetin-A inhibited collagen-induced platelet aggregation even after GPIb was blocked with other inhibitors. Moreover, flavocetin-A antagonized α2β1 integrin-mediated adhesion and migration of HT1080 human fibrosarcoma cells, which lack any GPIb, on collagen. Protein chemical analyses proved that flavocetin-A binds to α2β1 integrin and its α2A domain with high affinity and in a cooperative manner, which most likely is due to its quaternary structure. Kinetic measurements confirmed the formation of a strong complex between integrin and flavocetin-A, which dissociates very slowly. This study proves that flavocetin-A, which has long been known as a GPIb inhibitor, efficiently targets α2β1 integrin and thus blocks collagen-induced platelet activation. Moreover, our findings suggest that the separation of GPIb- and α2β1 integrin-blocking members within the C-type lectin-related protein family is less strict than previously assumed.

Rapid production of quasi-stable antibody-phycoerythrin conjugates for use in flow cytometry.

A simple system is described for producing quasi-stable complexes between biotinylated antibodies and fluorochrome-labeled streptavidin. Optimal complexes were produced by mixing biotinylated antibody and phycoerythrin-streptavidin together at a molar ratio of 1:1 for 2 min, followed by the addition of soluble biotin to halt oligomerization. These complexes, which are stable for at least 4 h, exhibit fluorescence with cell binding which approximates that observed with standard two-step labeling techniques. Equally important, multiple biotinylated antibodies complexed with different streptavidin-fluorochromes can be used simultaneously.

A Dinucleotide Deletion in Exon 4 of the P1A2 Allelic Form of Glycoprotein IIIa: Implications for the Correlation of Serologic Versus Genotypic Analysis of Human Platelet Alloantigens

Platelets from a patient with a suspected case of posttransfusion purpura were subjected to alloantigen phenotyping and found to express the PlA1, but not the PlA2, allelic form of human platelet membrane glycoprotein (GP) IIIa on the platelet surface. However, genotyping showed unambiguously that the patient carried the genes for both of these GPIIa alleles. Based on these results, we postulated that the PlA2 allele was silent, ie, that this patient was a carrier for Glanzmann thrombasthenia (GT). Quantitative analysis of GPIIb-IIIa surface expression showed only 20,000 GPIIb-IIIa receptors/platelet, approximately half of the value obtained with control platelets. Southern blot analysis showed no large deletions or insertions within the GPIIIa gene, and amplification of all 14 exons encoding GPIIIa resulted in the production of normal sized polymerase chain reaction (PCR) products in all cases. DNA-sequence analysis showed an AG dinucleotide deletion affecting codons 210 and 211 within exon 4 of the GPIIIa gene, leading to a change in reading frame and the creation of a stop codon 38 nucleotides down-stream. The predicted truncated protein consists of only the first 223 of the normal 762 amino acids, thus accounting for the failure to express the PlA2 allele on the platelet surface. While encountered only rarely, carriers of either GT or Bernard Soulier syndrome that are at the same time heterozygous for human platelet alloantigenic epitopes found on GPIb, GPIIb, or GPIIIa have the possibility to give discrepant results when comparing genotypic versus phenotypic analysis. In such situations, the combination of serologic and DNA-based evaluation contributes complementary and beneficial diagnostic information than either one alone are able to provide.

5436163 Polymorphism of human platelet membrane glycoprotein IIB and diagnostic applications thereof

5436163 Polymorphism of human platelet membrane glycoprotein IIB and diagnostic applications thereof

5436163 Polymorphism of human platelet membrane glycoprotein IIB and diagnostic applications thereof

5436163 Polymorphism of human platelet membrane glycoprotein IIB and diagnostic applications thereof

5436163 Polymorphism of human platelet membrane glycoprotein IIB and diagnostic applications thereof : Newman Peter J; Aster Richard H Shorewood, WI, United States assigned to The Blood Center of Southeastern Wisconsin

5436163 Polymorphism of human platelet membrane glycoprotein IIB and diagnostic applications thereof : Newman Peter J; Aster Richard H Shorewood, WI, United States assigned to The Blood Center of Southeastern Wisconsin

5436163 Polymorphism of human platelet membrane glycoprotein IIb and diagnostic applications thereof : Newman Peter J; Aster Richard H Shorewood, WI, United States assigned to The Blood Center of Southeastern Wisconsin

5436163 Polymorphism of human platelet membrane glycoprotein IIb and diagnostic applications thereof : Newman Peter J; Aster Richard H Shorewood, WI, United States assigned to The Blood Center of Southeastern Wisconsin

5436163 Polymorphism of human platelet membrane glycoprotein iib and diagnostic applications thereof : Newman Peter J; Aster Richard H Shorewood, WI, United States assigned to The Blood Center of Southeastern Wisconsin

5436163 Polymorphism of human platelet membrane glycoprotein iib and diagnostic applications thereof : Newman Peter J; Aster Richard H Shorewood, WI, United States assigned to The Blood Center of Southeastern Wisconsin

Polymorphism of human platelet membrane glycoprotein IIIA and diagnostic applications thereof

Polymorphism of human platelet membrane glycoprotein IIIA and diagnostic applications thereof

Polymorphisms of Human Platelet Membrane Glycoproteins: Structure and Clinical Significance

The haemostatic response of platelets of any one individual will be influenced by the genetic profile of the total population of receptors expressed on the platelet surface. Among the parameters to consider will be (i) the density of each receptor, (ii) the rate at which genes are transcribed and receptors produced and (iii) the presence or not of structural polymorphisms. Already, consideration of the known polymorphisms on GP IIb and GP IIIa raises most interesting questions on the structure/function relationship for this receptor. For example, there is often no obvious pattern as to which polymorphisms will influence platelet function and which will risk giving rise to a diallelic alloantigen system. Thus, mutation at Arg214 results in a loss in the ability of the complex to support platelet aggregation, whereas a mutation at Arg143 has resulted in the production of an immune response (see above). As I have pointed out earlier, examples from inherited platelet disorders show that even a single mutated allele can influence receptor function. Others have shown that polymorphisms of plasma proteins (see 52) or membrane glycoproteins such as E-selectin of endothelial cells (see 53) can give rise to risk factors for thrombosis and/or atherosclerosis. It would be interesting to know whether polymorphisms of platelet glycoprotein receptors (and those shared with other vascular cells) also represent risk factors in cardiovascular disease.

A point mutation leads to an unpaired cysteine residue and a molecular weight polymorphism of a functional platelet beta 3 integrin subunit. The Sra alloantigen system of GPIIIa.

Recently, we described a low frequency platelet alloantigen on human platelet membrane glycoprotein (GP) IIIa, termed Sra, that was involved in neonatal alloimmune thrombocytopenia. To identify the molecular nature of the Sra alloantigen, we analyzed the nucleotide sequence of polymerase chain reaction-amplified GPIIIa mRNA, and found a C2004-->T substitution in seven Sra positive individuals which results in an Arg636-->Cys polymorphism within the cysteine-rich region of GPIIIa. Analysis of allele-specific recombinant forms of GPIIIa that differed only at amino acid residue 636 showed that anti-Sra alloantibodies reacted with the Cys636, but not the Arg636, recombinant form of GPIIIa. Interestingly, under reducing conditions, the Cys636 form of GPIIIa migrated with a slightly increased apparent molecular weight compared with the Arg636 form. Following treatment with Endoglycosidase H, both allelic forms of GPIIIa exhibited the same mobility, however the Sra epitope was lost. Sra positive platelets express the same number of GPIIb-IIIa complexes on their surface as wild-type Sra negative platelets, and also aggregate normally in response to a variety of platelet agonists. Based upon these results, we conclude that 1) GPIIIa residue 636 specifically controls the formation and expression of the Sra alloantigenic determinant, and 2) an unpaired cysteine residue alters the N-linked glycosylation pattern of the extracellular domain of GPIIIa, but affects neither the degree of surface expression nor the adhesive function of the GPIIb-IIIa complex.

An additional HpaII polymorphism in exon 2 of the human platelet membrane glycoprotein IIIa gene.

A novel HpaII polymorphic site caused by a T-->G transversion at codon 40 of the GP3a locus is described. It was found together with another polymorphic HpaII site at codon 33. Both are associated with the immunologically defined HPA-1b antigen.

Demonstration of feline and canine platelet glycoproteins by immuno- and lectin histochemistry

Canine and feline platelet cytocentrifuge preparations (CCPs), cryostat and paraffin-embedded bone marrow sections were used in this study. We evaluated whether platelets, megakaryocytes and megakaryocyte precursor cells could be labelled by monoclonal antibodies (Y2/51, CLB-thromb/1, HPL1) against human platelet membrane glycoprotein GP IIIa and the GP IIb/IIIa complex or by the following 10 biotinylated lectins: concanavalin A (Con A), Lens culinaris agglutinin (LCA), Pisum sativum agglutinin (PsA), wheat germ agglutinin (WGA), peanut agglutinin (PNA), Phaseolus vulgaris lectin (PHA-L), Ricinus communis agglutinin 120 (RCA120), Ulex europaeus agglutinin-I(UEA-1), soybean agglutinin (SBA) and Dolichos biflorus agglutinin (DBA). Monoclonal antibodies Y2/51 and HPL1 cross reacted with platelets and megakaryocytic cells from both species, whereas CLB-thromb/1 was unreactive with canine preparations. Only Y2/51 labelled megakaryocytic cells in paraffin-embedded samples. LCA, PSA, WGA and PHA-L labelled feline and canine platelets and different numbers of morphologically identifiable megakaryocytes and numerous other, mostly myeloid, cells. Immunoblots of dog and cat platelet lysates using Y2/51 visualized a single protein of 95 kDa (unreduced), a mol.wt value within the range of those reported for GP IIIa. Some of the platelet (but not necessarily megakaryocyte) glycoproteins reacting with LCA, PSA and WGA could be identified in lectin blots following one- or two (nonreduced/reduced)-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Thus in dogs and cats, the immunohistochemical detection of GP IIIa (and eventually GP IIb/IIIa) rather than lectin binding patterns could be important for the diagnosis of megakaryoblastic leukaemias.

An amino acid polymorphism within the RGD binding domain of platelet membrane glycoprotein IIIa is responsible for the formation of the Pena/Penb alloantigen system.

The human Pena/Penb alloantigen system represents a naturally occurring polymorphism of human platelet membrane glycoprotein (GP) IIIa, and has previously been implicated in the onset of two important clinical syndromes, neonatal alloimmune thrombocytopenic purpura and posttransfusion purpura. To investigate the molecular basis of the polymorphism underlying the Pen alloantigen system, we used the polymerase chain reaction to amplify platelet-derived GPIIIa mRNA transcripts. DNA sequence analysis of amplified GPIIIa cDNAs from nucleotides 161 to 1341 (encompassing amino acid residues 22-414) revealed a G526<==>A526 polymorphism that segregated precisely with Pen phenotype in twelve other individuals examined. This nucleotide substitution results in an Arg (CGA) to Gln (CAA) polymorphism at amino acid 143 of GPIIIa. Interestingly, this polymorphic residue is located within the putative RGD binding site (residues 109-171) of GPIIIa. Platelet aggregation patterns of a Penb/b individual, however, were nearly normal in response to all physiological agonists tested, indicating that this polymorphism does not grossly affect integrin function. Short synthetic peptides encompassing residue 143 were unable to mimic either the Pena or Penb antigenic determinants, suggesting that the Pen epitopes are dependent upon proper folding of the polypeptide chain. Finally, we constructed allele-specific recombinant forms of GPIIIa that differed only at amino acid residues 143. Whereas anti-Pena alloantibodies were able to recognize the Arg143 recombinant form of GPIIIa, anti-Penb antibodies were not. Conversely, anti-Penb alloantibodies were reactive only with the Gln143 isoform of the GPIIIa molecule. It thus appears that amino acid 143 of GPIIIa is not only associated with Pen phenotype, but specifically controls the formation and expression of the Pen alloantigenic determinants.

5091302 Polymorphism of human platelet membrane glycoprotein iha and diagnostic and therapeutic applications thereof: Peter J Newman, Richard Aster assigned to The Blood Center of Southeastern Wisconsin Inc

5091302 Polymorphism of human platelet membrane glycoprotein iha and diagnostic and therapeutic applications thereof: Peter J Newman, Richard Aster assigned to The Blood Center of Southeastern Wisconsin Inc

Similar Effects of Toluene and Dibucaine on Human Platelet Membrane Glycoprotein Ib and Actin‐Binding Protein

Annals of the New York Academy of SciencesVolume 625, Issue 1 p. 835-836 Similar Effects of Toluene and Dibucaine on Human Platelet Membrane Glycoprotein Ib and Actin-Binding Protein ANNE SMITH-KIELLAND, Corresponding Author ANNE SMITH-KIELLAND National Institute of Forensic Toxicology P.O. Box 16 Gaustad, N-0320 Oslo 3, NorwaycAuthor to whom correspondence should be addressed.Search for more papers by this authorANNE MARGRETHE AAKHUS, ANNE MARGRETHE AAKHUS Research Institute for Internal Medicine University of Oslo Rikshospitalet, N-0027 Oslo 1, NorwaySearch for more papers by this authorÅSE RIPEL, ÅSE RIPEL National Institute of Forensic Toxicology P.O. Box 16 Gaustad, N-0320 Oslo 3, NorwaySearch for more papers by this authorNILS O. SOLUM, NILS O. SOLUM Research Institute for Internal Medicine University of Oslo Rikshospitalet, N-0027 Oslo 1, NorwaySearch for more papers by this author ANNE SMITH-KIELLAND, Corresponding Author ANNE SMITH-KIELLAND National Institute of Forensic Toxicology P.O. Box 16 Gaustad, N-0320 Oslo 3, NorwaycAuthor to whom correspondence should be addressed.Search for more papers by this authorANNE MARGRETHE AAKHUS, ANNE MARGRETHE AAKHUS Research Institute for Internal Medicine University of Oslo Rikshospitalet, N-0027 Oslo 1, NorwaySearch for more papers by this authorÅSE RIPEL, ÅSE RIPEL National Institute of Forensic Toxicology P.O. Box 16 Gaustad, N-0320 Oslo 3, NorwaySearch for more papers by this authorNILS O. SOLUM, NILS O. SOLUM Research Institute for Internal Medicine University of Oslo Rikshospitalet, N-0027 Oslo 1, NorwaySearch for more papers by this author First published: June 1991 https://doi.org/10.1111/j.1749-6632.1991.tb33930.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume625, Issue1Molecular and Cellular Mechanisms of Alcohol and AnestheticsJune 1991Pages 835-836 RelatedInformation