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Comparison between recombinant and platelet-derived glycoprotein (GP)Ib for human platelet antigen (HPA)-2 antibody detection. (A) An inert negative control serum and five different sera containing anti-HPA-2b tested against the Thr-145 and Met-145 forms of recombinant GPIb by enzyme-linked immunoabsorbent assay. (B) The same sera tested against platelet-derived GPIb by monoclonal antibody-specific immobilization of platelet antigens.
Source publication
Glycoprotein (GP) Ibalpha is the functionally dominant subunit of the platelet GPIb-IX-V receptor complex, with the von Willebrand factor (vWF) binding site residing on the amino-terminus. A threonine for methionine-145 replacement of GPIbalpha is associated with the human platelet antigen (HPA)-2 system. To study the structural and functional cons...
Citations
... 193 In vitro experimentation under static conditions could not demonstrate differences in VWF binding properties between the Met145 and Thr145 GPIba variant. 195 Yet, a higher binding of recombinant Thr145 GPIba to immobilized VWF was found in our group when using lower concentrations of ristocetin. 196 Moreover, we could demonstrate that platelets expressing solely Thr145 GPIba indeed bound VWF with higher affinity than their Met145 counterparts. ...
Inherited platelet disorders constitute a large group of diseases involving a wide range of genetic defects that can lead to bleeding symptoms of varying severity. They are associated with defects in surface membrane glycoproteins resulting in e.g. Bernard Soulier Syndrome and Glanzmann Thrombasthenia causing defects in platelet adhesion and aggregation, respectively, as well as in receptors for agonists (a.o. P2Y(12), TXA(2)) disrupting platelet signalling. Defects affecting platelet granules can be characterised by abnormalities of alpha-granules as in the Gray platelet syndrome or dense granules as in Hermansky-Pudlak and Chediak-Higashi syndromes, the latter two also altering other cytoplasmic organelles such as melanosomes and therefore not restricted to platelets. Finally, defects in proteins essential to signalling pathways (a.o. in Wiskott-Aldrich syndrome) or in platelet-derived procoagulant activity (Scott and Stormorken syndromes) also impair platelet function. For most of the above disorders mouse knockout models have been generated, that allowed to confirm the genotype-phenotype relationship and to further unravel the molecular causes of the disease and the mechanisms underlying primary haemostasis. More recently, interest has been growing in the effects of the more common polymorphisms that are found in the platelet glycoproteins as possible risk factors for thrombotic disorders. The new era of platelet genomics and proteomics will increase our knowledge on platelet disorders that will improve their diagnosis, but also will provide basis for new antithrombotic therapies.
... Ideally, HPA antibody detection would be based on recombinant antigens, to improve assay availability and standardization, eliminate complications caused by the coexistence of HLA antibodies and simplify the study of sera containing multiple HPA antibody specificities or antibodies against the rare HPA antigens. Previous studies highlighted the potential of this approach by demonstrating the detection of HPA-1 antibodies using Escherichia coli expressed mini-b3 (residues 1-66) and HPA-2 antibodies using a truncated version (residues 1-289) of insect cell expressed GPIba [33,34]. However, we and others have shown that the previously proposed mini-b3 recombinant protein for HPA-1 antibody is unsuitable because residues from domains distant of the PSI domain are critical to the integrity of the HPA-1 epitope [35,36]. ...
Antibodies against human platelet antigens (HPA) are clinically important in fetal-maternal alloimmune thrombocytopenia, refractoriness to platelet transfusions and post-transfusion purpura. Of the 16 HPAs, nine are located on the beta3 subunit of the alphaIIb beta3 integrin. Antibody detection is generally based on platelet-derived alphaIIb beta3 from HPA-genotyped donors. Recombinant allelic beta3 peptides, expressed at high levels would improve consistency in antibody detection, but the expression of soluble and monomeric integrins expressing complex dependent epitopes has previously proved challenging.
We aimed to generate three recombinant beta3 peptides for the detection of antibodies against HPA-4, HPA-8bw and five of the six remaining low frequency beta3 alloantigens.
The removal of the specificity-determining loop from the betaA domain and fusion of truncated beta3 to calmodulin was exploited to obtain expression of monomeric protein. Using site-directed mutagenesis, the mutations for HPA-4b and HPA-8bw were introduced in the ITGB3*001 haplotype. A third peptide for the detection of antibodies against HPA coded by non-synonymous single nucleotide polymorphisms of low frequency was generated by the introduction of five mutations forming the basis of HPA-6bw, -7bw, -10bw, -11bw, and -16bw antigens.
Reactivity of the three peptides with beta3-specific murine monoclonal antibodies and human HPA-1a phage antibodies confirmed the structural integrity of the recombinant fragments, and reactivity with a unique panel of polyclonal anti-HPA sera confirmed expression of the relevant HPA epitopes.
These data demonstrate that beta3 integrin domain-deletion fragments are suitable molecular targets for HPA antibody detection.
... The detection of HPA-1 antibodies is still based on assays requiring HPA-genotyped platelets [7]. Ideally, recombinant peptides should be developed to that end, and an earlier study from our laboratory demonstrated the feasibility of this for the detection of HPA-2 antibodies [8]. Similar studies for b 3 integrin alloantibodies have been hampered by a lack of both structural information and knowledge of the precise molecular nature of the HPA-1 epitopes. ...
... The sequences of all eight gene constructs in the plasmid pMT/Bip/CaM were confirmed by sequencing. Recombinant CaM-tagged proteins were expressed in Drosophila SchneiderÕs S2 cells and purified from culture supernatant on a W7 resin affinity column via the CaM tag, as described previously [8,14]. Recombinant proteins were analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis gel analysis, and protein concentration was measured using a bicinchonic acid assay kit (Perbio, Chester, UK). ...
The single-nucleotide polymorphism (SNP) rs5918 in the ITGB3 gene defines the human platelet antigen-1 (HPA-1) system encoding a Leu (HPA-1a) or Pro (HPA-1b) at position 33. HPA-1 antibodies are clinically the most relevant in the Caucasoid population, but detection currently requires alpha(IIb)beta3 integrin from the platelets of HPA-genotyped donors.
We set out to define the beta3 integrin domains required for HPA-1a antibody binding and produce recombinant soluble beta3 peptides for HPA-1 antibody detection.
We designed two sets (1a and 1b) of four soluble beta3 domain-deletion peptides (deltaSDL, deltabetaA, PSIHybrid, PSI), informed by crystallography studies and computer modeling. The footprints of three human HPA-1a-specific phage antibodies were defined by analyzing binding patterns to the beta3 peptides and canine platelets, and models of antibody-antigen interfaces were derived. Specificity and sensitivity for HPA-1a detection were assessed using sera from 140 cases of fetomaternal alloimmune thrombocytopenia (FMAIT).
Fusion of recombinant proteins to calmodulin resulted in high-level expression in Drosophila S2 cells of all eight beta3 peptides. Testing of FMAIT samples indicated that deltabetaA-Leu33 is the superior peptide for HPA-1a antibody detection, with 96% sensitivity and 95% specificity. The existence of type I and II categories of HPA-1a antibodies was confirmed by the study of HPA-1a phage antibody footprints and the reactivity pattern of clinical samples with the four beta3-Leu33 peptides, but there was no correlation between antibody category and clinical severity of FMAIT.
Soluble recombinant beta3 peptides can be used for detection of clinical HPA-1a antibodies.
... The detection of HPA-1 antibodies is still based on assays requiring HPA-genotyped platelets [7]. Ideally, recombinant peptides should be developed to that end, and an earlier study from our laboratory demonstrated the feasibility of this for the detection of HPA-2 antibodies [8]. Similar studies for b 3 integrin alloantibodies have been hampered by a lack of both structural information and knowledge of the precise molecular nature of the HPA-1 epitopes. ...
... The sequences of all eight gene constructs in the plasmid pMT/Bip/CaM were confirmed by sequencing. Recombinant CaM-tagged proteins were expressed in Drosophila SchneiderÕs S2 cells and purified from culture supernatant on a W7 resin affinity column via the CaM tag, as described previously [8,14]. Recombinant proteins were analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis gel analysis, and protein concentration was measured using a bicinchonic acid assay kit (Perbio, Chester, UK). ...
See also Stafford P, Garner SF, Huiskes E, Kaplan C, Kekomaki R, Santoso S, Tsuno NH, Watkins NA, Ouwehand WH. Three novel β3 domain-deletion peptides for the sensitive and specific detection of HPA-4 and six low frequency β3-HPA antibodies. This issue, pp 376–83. Summary. Background: The single-nucleotide polymorphism (SNP) rs5918 in the ITGB3 gene defines the human platelet antigen-1 (HPA-1) system encoding a Leu (HPA-1a) or Pro (HPA-1b) at position 33. HPA-1 antibodies are clinically the most relevant in the Caucasoid population, but detection currently requires αIIbβ3 integrin from the platelets of HPA-genotyped donors.Objectives: We set out to define the β3 integrin domains required for HPA-1a antibody binding and produce recombinant soluble β3 peptides for HPA-1 antibody detection.Methods: We designed two sets (1a and 1b) of four soluble β3 domain-deletion peptides (ΔSDL, ΔβA, PSIHybrid, PSI), informed by crystallography studies and computer modeling. The footprints of three human HPA-1a-specific phage antibodies were defined by analyzing binding patterns to the β3 peptides and canine platelets, and models of antibody–antigen interfaces were derived. Specificity and sensitivity for HPA-1a detection were assessed using sera from 140 cases of fetomaternal alloimmune thrombocytopenia (FMAIT). Results: Fusion of recombinant proteins to calmodulin resulted in high-level expression in Drosophila S2 cells of all eight β3 peptides. Testing of FMAIT samples indicated that ΔβA-Leu33 is the superior peptide for HPA-1a antibody detection, with 96% sensitivity and 95% specificity. The existence of type I and II categories of HPA-1a antibodies was confirmed by the study of HPA-1a phage antibody footprints and the reactivity pattern of clinical samples with the four β3-Leu33 peptides, but there was no correlation between antibody category and clinical severity of FMAIT. Conclusions: Soluble recombinant β3 peptides can be used for detection of clinical HPA-1a antibodies.
... The development of soluble recombinant HPA antigens for the detection of HPA antibodies has proven more complex than initially hoped. Early studies from our laboratory showed the feasibility of high level soluble expression of calmodulin-tagged extracellular fragments of GPIb α allowing the detection of HPA-2 antibodies [30]. The expression of soluble fragments of the two integrins α IIb β 3 and α 2 β 1 and the C3C5-like protein CD109 [8] has proven a harder nut to crack. ...
... These findings are not consistent with our results although the experimental conditions of the present study differed from those of previous studies: use of ristocetin or botrocetin or use of an assay system. Other studies have also used various methods with inconsistent results (Mazzucato et al, 1996; Li et al, 2000; Jilma-Stohlawetz et al, 2003). These reports suggest that the functional analyses of GPIba polymorphisms seem to be easily affected by several factors in relation to platelet activation or experimental conditions. ...
... In experimental studies of these polymorphisms, These findings are not consistent with our results although the experimental conditions of the present study differed from those of previous studies: use of ristocetin or botrocetin or use of an assay system. Other studies have also used various methods with inconsistent results (Mazzucato et al, 1996; Li et al, 2000; Jilma-Stohlawetz et al, 2003). These reports suggest that the functional analyses of GPIba polymorphisms seem to be easily affected by several factors in relation to platelet activation or experimental conditions. ...
Interaction of platelet glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) is essential for thrombus formation, particularly under high shear conditions. Previous case-control studies indicated that two GPIb alpha polymorphisms, (145)Thr/Met and/or variable number (1-4) tandem repeats of 13 amino-acid sequences, are associated with arterial thrombosis. The (145)Met-allele and the 3R- or 4R-allele is associated with increased risk. However, there is little clear experimental data to support this association. To elucidate the functional effects of these polymorphisms, we prepared recombinant GPIb alpha fragments and tested them in vitro. The dissociation constants of ristocetin-induced (125)I-labelled VWF binding to two forms of soluble recombinant GPIb alpha [(1)His-(302)Ala, either (145)Thr (145T) or (145)Met (145M)] were not different. Four types of Chinese hamster ovary cells expressing full-length GPIb alpha beta/IX, 145T with one repeat (T1R), 145M with one repeat (M1R), 145T with four repeats (T4R), and 145M with four repeats (M4R), were prepared, and cell interactions with immobilized-VWF were examined under various shear conditions. The cell rolling velocity of M4R under a shear condition of 114/s was significantly slower than that of T1R. Intermediate values were obtained with M1R and T4R. The results suggest that M4R interacts more strongly with VWF under flow conditions.
... Whereas the influence of VNTR on platelet reactivity in the presence of collagen under high shear stress has been postulated [48] , no such positive evidence was presented regarding the impact of the Thr/Met 145 dimorphism on the binding of vWF to GPIb. [89, 95]. However, an effect of the latter on GPIb-vWF interaction has not been examined under conditions of shear forces that regulate GPIb a function in vivo. ...
Blood platelets are not only the primary defence mechanism involved in physiological hemostasis, but also their disorders constitute a crucial risk factor in arterial thrombosis. As arterial thrombi are composed of predominantly platelets formed under conditions of elevated shear stress at sites of atherosclerotic vascular injury and disturbed blood flow, the prevention of arterial thrombosis has been for years the main target for antiplatelet therapy. Individual differences in the rate of platelet activation and reactivity markedly influence normal hemostasis and the pathological outcome of thrombosis. Such an individual variability is largely determined by environmental and genetic factors. These are known to either hamper platelets' response to agonists, and thereby mimic the pharmacological modulation of platelet function or mask therapy effect and sensitize platelets. Some clinical studies have indicated that platelet glycoprotein polymorphisms are genetic factors contributing to arterial thrombosis. In spite of some discrepancies between different studies, there is substantial evidence that the integrin beta3 P1(A2) allele, the variants GPIbalpha Met145 and GPIbalpha (-5C) haplotype or the integrin alpha2 haplotype 1 (807T) each contribute to the risk for and morbidity of thrombotic disease. In this article, we reviewed a role of the aforementioned polymorphisms in modulating platelet function and platelet response to inhibitors. The paper focuses on the association between Pl(A1/A2) polymorphism and sensitivity (or resistance) to aspirin and the inhibitory efficacy of GPIIb-IIIa antagonists. Additionally, a potential role of 807C/T polymorphism (GPIa), polymorphisms of GPIb and platelet purinoreceptor P2Y12 in affecting platelet sensitivity to blocking agents is discussed.
... The HPA-2 and VNTR polymorphisms are in strong linkage disequilibrium with each other. The HPA-2 Thrl45Met dimorphism results in a protein conformational change in a region adjacent to the vWF binding region of glycoprotein Iba, but an effect on in vitro platelet function or von Willbebrand factor binding has not been demonstrated (128,129). The VNTR polymorphism involves the macroglycopeptide region of glycoprotein Iba, 588 with the addition of each repeat resulting in an increased distan^· between the ligand binding region and the platelet surface. ...
The pathogenesis of arterial thrombotic disease involves multiple genetic and environmental factors related to atherosclerosis and thrombosis. Acute thrombosis at the site of a ruptured, lipid-rich atherosclerotic plaque is the usual precipitating event in the transition from stable or subclinical atherosclerotic disease to acute myocardial infarction (MI), stroke, or peripheral arterial occlusion (1). Pathologic studies of coronary arteries in acute MI suggest that the acute thrombosis likely involves activation of both platelets and the coagulation system.
... The amino-terminal region of GPIb␣ was expressed from baculovirus-infected insect cells (40) and purified as described previously (41). Briefly, the cDNA coding for the signal peptide and amino-terminal domain of GPIb␣ (residues Ϫ16 to 289) followed by the calmodulin (CaM) gene was inserted into the baculovirus expression vector pAcSG2 (Pharmingen, San Diego, CA). ...
The glycoprotein (GP) Ib-IX complex is a platelet surface receptor that binds thrombin as one of its ligands, although the biological significance of thrombin interaction remains unclear. In this study we have used several approaches to investigate the GPIb alpha-thrombin interaction in more detail and to study its effect on the thrombin-induced elaboration of fibrin. We found that both glycocalicin and the amino-terminal fragment of GPIb alpha reduced the release of fibrinopeptide A from fibrinogen by about 50% by a noncompetitive allosteric mechanism. Similarly, GPIb alpha caused in thrombin an allosteric reduction in the rate of turnover of the small peptide substrate d-Phe-Pro-Arg-pNA. The K(d) for the glycocalicin-thrombin interaction was 1 microm at physiological ionic strength but was highly salt-dependent, decreasing to 0.19 microm at 100 mm NaCl (Gamma(salt) = -4.2). The salt dependence was characteristic of other thrombin ligands that bind to exosite II of this enzyme, and we confirmed this as the GPIb alpha-binding site on thrombin by using thrombin mutants and by competition binding studies. R68E or R70E mutations in exosite I of thrombin had little effect on its interaction with GPIb alpha. Both the allosteric inhibition of fibrinogen turnover caused by GPIb alpha binding to these mutants, and the K(d) values for their interactions with GPIb alpha were similar to those of wild-type thrombin. In contrast, R89E and K248E mutations in exosite II of thrombin markedly increased the K(d) values for the interactions of these thrombin mutants with GPIb alpha by 10- and 25-fold, respectively. Finally, we demonstrated that low molecular weight heparin (which binds to thrombin exosite II) but not hirugen (residues 54-65 of hirudin, which binds to exosite I of thrombin) inhibited thrombin binding to GPIb alpha. These data demonstrate that GPIb alpha binds to thrombin exosite II and in so doing causes a conformational change in the active site of thrombin by an allosteric mechanism that alters the accessibility of both its natural substrate, fibrinogen, and the small peptidyl substrate d-Phe-Pro-Arg-pNA.
