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Ana Cvejic,
Lonneke Haer-Wigman,
Jonathan C Stephens,
Myrto Kostadima, Peter A Smethurst,
Mattia Frontini,
Emile van den Akker,
Paul Bertone,
Ewa Bielczyk-Maczyńska,
Samantha Farrow, [......],
Harm-Jan Westra,
Derek Stemple,
Lude Franke,
Nicole Soranzo,
Hendrik G Stunnenberg,
Nick Goldman,
Pim van der Harst,
C Ellen van der Schoot,
Willem H Ouwehand,
Cornelis A Albers
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ABSTRACT: The blood group Vel was discovered 60 years ago, but the underlying gene is unknown. Individuals negative for the Vel antigen are rare and are required for the safe transfusion of patients with antibodies to Vel. To identify the responsible gene, we sequenced the exomes of five individuals negative for the Vel antigen and found that four were homozygous and one was heterozygous for a low-frequency 17-nucleotide frameshift deletion in the gene encoding the 78-amino-acid transmembrane protein SMIM1. A follow-up study showing that 59 of 64 Vel-negative individuals were homozygous for the same deletion and expression of the Vel antigen on SMIM1-transfected cells confirm SMIM1 as the gene underlying the Vel blood group. An expression quantitative trait locus (eQTL), the common SNP rs1175550 contributes to variable expression of the Vel antigen (P = 0.003) and influences the mean hemoglobin concentration of red blood cells (RBCs; P = 8.6 × 10(-15)). In vivo, zebrafish with smim1 knockdown showed a mild reduction in the number of RBCs, identifying SMIM1 as a new regulator of RBC formation. Our findings are of immediate relevance, as the homozygous presence of the deletion allows the unequivocal identification of Vel-negative blood donors.
Nature Genetics 04/2013; · 35.53 Impact Factor
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Sylvia T Nürnberg,
Augusto Rendon, Peter A Smethurst,
Dirk S Paul,
Katrin Voss,
Jonathan N Thon,
Heather Lloyd-Jones,
Jennifer G Sambrook,
Marloes R Tijssen,
Joseph E Italiano,
Panos Deloukas,
Berthold Gottgens,
Nicole Soranzo,
Willem H Ouwehand
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ABSTRACT: We recently identified 68 genomic loci where common sequence variants are associated with platelet count and volume. Platelets are formed in the bone marrow by megakaryocytes, which are derived from hematopoietic stem cells by a process mainly controlled by transcription factors. The homeobox transcription factor MEIS1 is uniquely transcribed in megakaryocytes and not in the other lineage-committed blood cells. By chromatin immunoprecipitation we show that 5 of the 68 loci pinpoint a MEIS1 binding event within a group of 252 MK-overexpressed genes. In one such locus in DNM3, regulating platelet volume, the MEIS1 binding site falls within a region acting as an alternative promoter that is solely used in megakaryocytes, where allelic variation dictates different levels of a shorter transcript. The importance of dynamin activity to the latter stages of thrombopoiesis was confirmed by the observation that the inhibitor Dynasore reduced murine proplatelet formation in vitro.
Blood 09/2012; · 9.90 Impact Factor
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Cornelis A Albers,
Dirk S Paul,
Harald Schulze,
Kathleen Freson,
Jonathan C Stephens, Peter A Smethurst,
Jennifer D Jolley,
Ana Cvejic,
Myrto Kostadima,
Paul Bertone, [......],
Claudia A L Ruivenkamp,
Jennifer G Sambrook,
Kenneth Smith,
Derek L Stemple,
Gabriele Strauss,
Chantal Thys,
Chris van Geet,
Ruth Newbury-Ecob,
Willem H Ouwehand,
Cedric Ghevaert
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ABSTRACT: The exon-junction complex (EJC) performs essential RNA processing tasks. Here, we describe the first human disorder, thrombocytopenia with absent radii (TAR), caused by deficiency in one of the four EJC subunits. Compound inheritance of a rare null allele and one of two low-frequency SNPs in the regulatory regions of RBM8A, encoding the Y14 subunit of EJC, causes TAR. We found that this inheritance mechanism explained 53 of 55 cases (P < 5 × 10(-228)) of the rare congenital malformation syndrome. Of the 53 cases with this inheritance pattern, 51 carried a submicroscopic deletion of 1q21.1 that has previously been associated with TAR, and two carried a truncation or frameshift null mutation in RBM8A. We show that the two regulatory SNPs result in diminished RBM8A transcription in vitro and that Y14 expression is reduced in platelets from individuals with TAR. Our data implicate Y14 insufficiency and, presumably, an EJC defect as the cause of TAR syndrome.
Nature Genetics 02/2012; 44(4):435-9, S1-2. · 35.53 Impact Factor
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Marloes R Tijssen,
Ana Cvejic,
Anagha Joshi,
Rebecca L Hannah,
Rita Ferreira,
Ariel Forrai,
Dana C Bellissimo,
S Helen Oram, Peter A Smethurst,
Nicola K Wilson,
Xiaonan Wang,
Katrin Ottersbach,
Derek L Stemple,
Anthony R Green,
Willem H Ouwehand,
Berthold Göttgens
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ABSTRACT: Hematopoietic differentiation critically depends on combinations of transcriptional regulators controlling the development of individual lineages. Here, we report the genome-wide binding sites for the five key hematopoietic transcription factors--GATA1, GATA2, RUNX1, FLI1, and TAL1/SCL--in primary human megakaryocytes. Statistical analysis of the 17,263 regions bound by at least one factor demonstrated that simultaneous binding by all five factors was the most enriched pattern and often occurred near known hematopoietic regulators. Eight genes not previously appreciated to function in hematopoiesis that were bound by all five factors were shown to be essential for thrombocyte and/or erythroid development in zebrafish. Moreover, one of these genes encoding the PDZK1IP1 protein shared transcriptional enhancer elements with the blood stem cell regulator TAL1/SCL. Multifactor ChIP-Seq analysis in primary human cells coupled with a high-throughput in vivo perturbation screen therefore offers a powerful strategy to identify essential regulators of complex mammalian differentiation processes.
Developmental cell 05/2011; 20(5):597-609. · 13.36 Impact Factor
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Cornelis A Albers,
Ana Cvejic,
Rémi Favier,
Evelien E Bouwmans,
Marie-Christine Alessi,
Paul Bertone,
Gregory Jordan,
Ross N W Kettleborough,
Graham Kiddle,
Myrto Kostadima,
Randy J Read,
Botond Sipos,
Suthesh Sivapalaratnam, Peter A Smethurst,
Jonathan Stephens,
Katrin Voss,
Alan Nurden,
Augusto Rendon,
Paquita Nurden,
Willem H Ouwehand
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ABSTRACT: Gray platelet syndrome (GPS) is a predominantly recessive platelet disorder that is characterized by mild thrombocytopenia with large platelets and a paucity of α-granules; these abnormalities cause mostly moderate but in rare cases severe bleeding. We sequenced the exomes of four unrelated individuals and identified NBEAL2 as the causative gene; it has no previously known function but is a member of a gene family that is involved in granule development. Silencing of nbeal2 in zebrafish abrogated thrombocyte formation.
Nature Genetics 01/2011; 43(8):735-7. · 35.53 Impact Factor
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ABSTRACT: Exposed subendothelial collagen acts as a substrate for platelet adhesion and thrombus formation after vascular injury. Synthetic collagen-derived triple-helical peptides, designated collagen-related peptide (CRP), GFOGER, and VWF-III, can specifically engage the platelet collagen receptors, glycoprotein VI and integrin alpha(2)beta(1), and plasma von Willebrand factor (VWF), respectively. Hitherto, the role of these 3 collagen-binding axes has been studied indirectly. Use of these uniform peptide substrates, rather than collagen fibers, provides independent control of each axis. Here, we use confocal imaging and novel image analysis techniques to investigate the effects of receptor-ligand engagement on platelet binding and activation during thrombus formation under flow conditions. At low shear (100s(-1) and 300s(-1)), both GFOGER and CRP are required for thrombus formation. At 1000s(-1), a combination of either CRP or GFOGER with VWF-III induces comparable thrombus formation, and VWF-III increases thrombus deposition at all shear rates, being indispensable at 3000s(-1). A combination of CRP and VWF-III is sufficient to support extensive platelet deposition at 3000s(-1), with slight additional effect of GFOGER. Measurement of thrombus height after specific receptor blockade or use of altered proportions of peptides indicates a signaling rather than adhesive role for glycoprotein VI, and primarily adhesive roles for both alpha(2)beta(1) and the VWF axis.
Blood 03/2010; 115(24):5069-79. · 9.90 Impact Factor
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ABSTRACT: We have analyzed the adhesion of human and murine platelets, and of recombinant human and murine GpVI ectodomains, to synthetic triple-helical collagen-like peptides. These included 57 peptides derived from the sequence of human type III collagen and 9 peptides derived from the cyanogen bromide fragment of bovine type III collagen, alpha1(III)CB4. We have identified several peptides that interact with GpVI, in particular a peptide designated III-30 with the sequence GAOGLRGGAGPOGPEGGKGAAGPOGPO. Both human and murine platelets bound to peptide III-30 in a GpVI-dependent manner. III-30 also supported binding of recombinant GpVI ectodomains. Cross-linked III-30 induced aggregation of human and murine platelets, although with a lower potency than collagen-related peptide. Modifications of the peptide sequence indicated that the hydroxyproline residues play a significant role in supporting its GpVI reactivity. However, many peptides containing OGP/GPO motifs did not support adhesion to GpVI. These data indicate that the ability of a triple-helical peptide to bind GpVI is not solely determined by the presence or spatial arrangement of these OGP/GPO motifs within the peptides.
Blood 06/2008; 111(10):4986-96. · 9.90 Impact Factor
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[show abstract]
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ABSTRACT: Collagen-related peptide is a selective agonist for the platelet collagen receptor Glycoprotein VI. The triple helical peptide contains ten GPO triplets/strand (single letter amino acid nomenclature, where O is hydroxyproline) and so over-represents GPO compared with native collagen sequence. To investigate the ability of Glycoprotein VI to recognize GPO triplets in a setting more representative of the collagens, we synthesized a set of triple helical peptides containing fewer GPO triplets, varying their number and spacing within an inert (GPP)n backbone. The adhesion of recombinant human Glycoprotein VI ectodo-main, like that of human platelets, to these peptides increased with their GPO content, and platelet adhesion was abolished by the specific anti-Glycoprotein VI-blocking antibody, 10B12. Platelet aggregation and protein tyrosine phosphorylation were induced only by cross-linked peptides and only those that contained two or more GPO triplets. Such peptides were less potent than cross-linked collagen-related peptide. Our data suggest that both the sequences GPOGPO and GPO.........GPO represent functional Glycoprotein VI recognition motifs within collagen. Furthermore, we propose that the (GPO)4 motif can support simultaneous binding of two glycoprotein VI molecules, in either a parallel or anti-parallel stacking arrangement, which could play an important role in activation of signaling.
Journal of Biological Chemistry 02/2007; 282(2):1296-304. · 4.77 Impact Factor
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[show abstract]
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ABSTRACT: Collagen-related peptide is a selective agonist for the platelet collagen receptor Glycoprotein VI. The triple helical peptide
contains ten GPO triplets/strand (single letter amino acid nomenclature, where O is hydroxyproline) and so over-represents
GPO compared with native collagen sequence. To investigate the ability of Glycoprotein VI to recognize GPO triplets in a setting
more representative of the collagens, we synthesized a set of triple helical peptides containing fewer GPO triplets, varying
their number and spacing within an inert (GPP)n backbone. The adhesion of recombinant human Glycoprotein VI ectodo-main, like that of human platelets, to these peptides
increased with their GPO content, and platelet adhesion was abolished by the specific anti-Glycoprotein VI-blocking antibody,
10B12. Platelet aggregation and protein tyrosine phosphorylation were induced only by cross-linked peptides and only those
that contained two or more GPO triplets. Such peptides were less potent than cross-linked collagen-related peptide. Our data
suggest that both the sequences GPOGPO and GPO.........GPO represent functional Glycoprotein VI recognition motifs within
collagen. Furthermore, we propose that the (GPO)4 motif can support simultaneous binding of two glycoprotein VI molecules, in either a parallel or anti-parallel stacking arrangement,
which could play an important role in activation of signaling.
Journal of Biological Chemistry 01/2007; 282(2):1296-1304. · 4.77 Impact Factor
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ABSTRACT: Platelet activation by collagen relies on the interaction of the receptor glycoprotein VI (GPVI) with collagen helices. We have previously generated two recombinant single chain human antibodies (scFvs) to human GPVI. The first, 10B12, binds to the collagen-binding site on the apical surface between the two immunoglobulin-like domains (D1D2) of the receptor and so directly inhibits GPVI function. The second, 1C3, binds D1D2 independently of 10B12 and has been shown to have a more subtle effect on platelet responses to collagen. Here we have shown that 1C3 potentiates the effect of 10B12 on platelet aggregation induced by collagen and cross-linked collagen-related peptide (CRP-XL). We investigated this by measuring the effect of both scFvs on the binding of D1D2 to immobilized collagen and CRP. As expected, 10B12 completely inhibited binding of GPVI to each ligand in a dose-dependent manner. However, 1C3 inhibited only a proportion of GPVI binding to its ligands, implying that it interferes with another aspect of ligand recognition by GPVI. To further understand the mode of inhibition, we used a unique set of CRPs in which the content of critical glycine-proline-hydroxyproline (GPO) triplets was varied in relation to an "inert" scaffold sequence of GPP motifs. We observed that a stepwise increase in D1D2 binding with (GPO)(2) content was blocked by 1C3. Together these results indicate that 1C3 inhibits clustering of the immunoglobulin-like domains of GPVI on collagen/CRPs, a conclusion that is supported by mapping the 1C3 epitope to the region including isoleucine 148 in D2.
Journal of Biological Chemistry 12/2006; 281(44):33505-10. · 4.77 Impact Factor
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Nicola S Jennings, Peter A Smethurst,
C Graham Knight,
Marie N O'Connor,
Lotta Joutsi-Korhonen,
Prachi Stafford,
Jonathan Stephens,
Stephen F Garner,
Ian J Harmer,
Richard W Farndale,
Nicholas A Watkins,
Willem H Ouwehand
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ABSTRACT: We report the development of an expression system for the production of soluble, calmodulin (CaM)-tagged proteins in Drosophila Schneider S2 cells and the subsequent use of these proteins for the selection of phage displayed antibodies. The CaM-tag permitted the purification of recombinant protein to >90% purity in a single step at yields of >20 mg/l. Using platelet glycoprotein VI (GP6) as a model, we demonstrated that the recombinant CaM-tagged protein was post-translationally N-glycosylated and had identical ligand specificity to native protein. A novel selection strategy, exploiting the CaM tag, was then used to isolate four single chain Fv fragments (scFvs) specific for GP6 from a non-immune phage display library. In contrast to other selection methods, which can result in antibodies that do not recognise native protein, all of the scFvs we selected bound cell surface expressed GP6. In conclusion, the production of CaM-tagged proteins in Drosophila Schneider S2 cells and the selection strategy reported here offer advantages over previously published methods, including simple culture conditions, rapid protein purification, specific elution of phage antibodies and preferential selection of phage antibodies that recognise native, cell surface expressed protein.
Journal of Immunological Methods 10/2006; 316(1-2):75-83. · 2.20 Impact Factor
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Sandra Penz,
Armin J Reininger,
Richard Brandl,
Pankaj Goyal,
Tamer Rabie,
Isabell Bernlochner,
Enno Rother,
Christine Goetz,
Bernd Engelmann, Peter A Smethurst,
Willem H Ouwehand,
Richard Farndale,
Bernhard Nieswandt,
Wolfgang Siess
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ABSTRACT: Lipid-rich atherosclerotic plaques are vulnerable, and their rupture can cause the formation of a platelet- and fibrin-rich thrombus leading to myocardial infarction and ischemic stroke. Although the role of plaque-based tissue factor as stimulator of blood coagulation has been recognized, it is not known whether plaques can cause thrombus formation through direct activation of platelets. We isolated lipid-rich atheromatous plaques from 60 patients with carotid stenosis and identified morphologically diverse collagen type I- and type III-positive structures in the plaques that directly stimulated adhesion, dense granule secretion, and aggregation of platelets in buffer, plasma, and blood. This material also elicited platelet-monocyte aggregation and platelet-dependent blood coagulation. Plaques exposed to flowing blood at arterial wall shear rate induced platelets to adhere to and spread on the collagenous structures, triggering subsequent thrombus formation. Plaque-induced platelet thrombus formation was observed in fully anticoagulated blood (i.e., in the absence of tissue factor-mediated coagulation). Mice platelets lacking glycoprotein VI (GPVI) were unable to adhere to atheromatous plaque or form thrombi. Human platelet thrombus formation onto plaques in flowing blood was completely blocked by GPVI inhibition with the antibody 10B12 but not affected by integrin alpha2beta1 inhibition with 6F1 mAb. Moreover, the initial platelet response, shape change, induced by plaque was blocked by GPVI inhibition but not with alpha2beta1 antagonists (6F1 mAb or GFOGER-GPP peptide). Pretreatment of plaques with collagenase or anti-collagen type I and anti-collagen type III antibodies abolished plaque-induced platelet activation. Our results indicate that morphologically diverse collagen type I- and collagen type III-containing structures in lipid-rich atherosclerotic plaques stimulate thrombus formation by activating platelet GPVI. This platelet collagen receptor, essential for plaque-induced thrombus formation, presents a promising new anti-thrombotic target for the prevention of ischemic cardiovascular diseases.
The FASEB Journal 07/2005; 19(8):898-909. · 5.71 Impact Factor
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ABSTRACT: Only three recognition motifs, GFOGER, GLOGER, and GASGER, all present in type I collagen, have been identified to date for collagen-binding integrins, such as alpha(2)beta(1). Sequence alignment was used to investigate the occurrence of related motifs in other human fibrillar collagens, and located a conserved array of novel GER motifs within their triple helical domains. We compared the integrin binding properties of synthetic triple helical peptides containing examples of such sequences (GLSGER, GMOGER, GAOGER, and GQRGER) or the previously identified motifs. Recombinant inserted (I) domains of integrin subunits alpha(1), alpha(2) and alpha(11) all bound poorly to all motifs other than GFOGER and GLOGER. Similarly, alpha(2)beta(1) -containing resting platelets adhered well only to GFOGER and GLOGER, while ADP-activated platelets, HT1080 cells and two active alpha(2)I domain mutants (E318W, locked open) bound all motifs well, indicating that affinity modulation determines the sequence selectivity of integrins. GxO/SGER peptides inhibited platelet adhesion to collagen monomers with order of potency F >/= L >/= M > A. These results establish GFOGER as a high affinity sequence, which can interact with the alpha(2)I domain in the absence of activation and suggest that integrin reactivity of collagens may be predicted from their GER content.
Journal of Biological Chemistry 11/2004; 279(46):47763-72. · 4.77 Impact Factor
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Lotta Joutsi-Korhonen,
Sandy Preston, Peter A Smethurst,
Martin Ijsseldijk,
Elisabeth Schaffner-Reckinger,
Kathryn L Armour,
Nicholas A Watkins,
Michael R Clark,
Philip G de Groot,
Richard W Farndale,
Willem H Ouwehand,
Lorna M Williamson
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ABSTRACT: Recombinant HPA-1a antibodies with Fc, mutated to remove destructive effector functions, have been developed as a potential therapy for fetomaternal alloimmune thrombocytopenia (FMAIT), via blockade of binding of human HPA-1a polyclonal antibodies to fetal HPA-1a1b platelets. We have assessed the effect of the IgG1 HPA-1a antibody B2G1 and two mutated derivatives in various functional assays in resting and agonist-stimulated platelets of the three HPA-1 genotypes. With HPA-1a1b platelets (fetal genotype), the antibodies did not activate signalling or CD62P expression in resting platelets, did not change in vitro bleeding time (IVBT), and did not inhibit platelet adhesion to collagen in flowing blood. Adhesion of HPA-1a1b platelets to fibrinogen was reduced by 20%, and aggregation induced by ADP by 50%, but collagen-related peptide (CRP-XL)-induced aggregation was normal. With HPA-1a1a platelets, aggregation to both ADP and CRP-XL was inhibited, with total blockade of adhesion to fibrinogen and of IVBT responses. Interestingly, a monovalent antibody fragment with identical specificity had no inhibitory effect on aggregation. In static adhesion assays using human alphaIIbbeta3 or alphaVbeta3 transfectants of HPA-1a (Leu(33)) phenotype, attachment to fibrinogen of the latter but not of the former was completely blocked by the HPA-1a antibodies. These observations are best explained by antibody-mediated blockade of the RGD binding site on beta3 by a mechanism of steric hindrance. As the effect on platelet function is modest with HPA-1a1b (fetal type) platelets, the mutated HPA-1a antibodies described here could be developed further for FMAIT therapy.
Thrombosis and Haemostasis 05/2004; 91(4):743-54. · 5.04 Impact Factor
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ABSTRACT: The platelet glycoproteins (GPs) Ib, integrin alpha(2)beta(1), and GPVI are considered central to thrombus formation. Recently, their relative importance has been re-evaluated based on data from murine knockout models. To examine their relationship during human thrombus formation on collagen type I fibers at high shear (1000 s(-1)), we tested a novel antibody against GPVI, an immunoglobulin single-chain variable fragment, 10B12, together with specific antagonists for GPIb alpha (12G1 Fab(2)) and alpha(2)beta(1) (6F1 mAb or GFOGER-GPP peptide). GPVI was found to be crucial for aggregate formation, Ca(2+) signaling, and phosphatidylserine (PS) exposure, but not for primary adhesion, even with more than 97% receptor blockade. Inhibiting alpha(2)beta(1) revealed its involvement in regulating Ca(2+) signaling, PS exposure, and aggregate size. Both GPIb alpha and alpha(2)beta(1) contributed to primary adhesion, showing overlapping function. The coinhibition of receptors revealed synergism in thrombus formation: the coinhibition of adenosine diphosphate (ADP) receptors with collagen receptors further decreased adhesion and aggregation, and, crucially, the complete eradication of thrombus formation required the coinhibition of GPVI with either GPIb alpha or alpha(2)beta(1). In summary, human platelet deposition on collagen depends on the concerted interplay of several receptors: GPIb in synergy with alpha(2)beta(1) mediating primary adhesion, reinforced by activation through GPVI, which further regulates the thrombus formation.
Blood 03/2004; 103(4):1333-41. · 9.90 Impact Factor
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Peter A Smethurst,
Lotta Joutsi-Korhonen,
Marie N O'Connor,
Erica Wilson,
Nicola S Jennings,
Stephen F Garner,
Yanjun Zhang,
C Graham Knight,
Timothy R Dafforn,
Ashley Buckle,
Martin J W IJsseldijk,
Philip G De Groot,
Nicholas A Watkins,
Richard W Farndale,
Willem H Ouwehand
[show abstract]
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ABSTRACT: Glycoprotein (GP) VI is the major receptor responsible for platelet activation by collagen, but the collagen-binding surface of GPVI is unknown. To address this issue we expressed, from insect cells, the immunoglobulin (Ig)-like ectodomains (residues 1-185) of human and murine GPVI, called hD1D2 and mD1D2, respectively. Both proteins bound specifically to collagen-related peptide (CRP), a GPVI-specific ligand, but hD1D2 bound CRP more strongly than did mD1D2. Molecular modeling and sequence comparison identified key differences between hD1D2 and mD1D2. Ten mutant hD1D2s were expressed, of which 4 had human residues replaced by their murine counterpart, and 6 had replacements by alanine. CRP binding studies with these mutants demonstrated that the exchange of lysine at position 59 for the corresponding murine glutamate substantially reduced binding to CRP. The position of lysine59 on the apical surface of GPVI suggests a mode of CRP binding analogous to that used by the related killer cell Ig-like receptors to bind HLA. This surface was confirmed as critical for collagen binding by epitope mapping of an inhibitory phage antibody against GPVI. This anti-GPVI, clone 10B12, gave dose-dependent inhibition of the hD1D2-collagen interaction. Clone 10B12 inhibited activation of platelets by CRP and collagen in aggregometry and thrombus formation by the latter in whole blood perfusion. Antibody 10B12 showed significantly reduced binding to the hD1D2-E59, and, on that basis, the GPVI:10B12 interface was modeled.
Blood 03/2004; 103(3):903-11. · 9.90 Impact Factor
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Lotta Joutsi-Korhonen, Peter A Smethurst,
Angela Rankin,
Elaine Gray,
Martin IJsseldijk,
Catherine M Onley,
Nicholas A Watkins,
Lorna M Williamson,
Alison H Goodall,
Philip G de Groot,
Richard W Farndale,
Willem H Ouwehand
[show abstract]
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ABSTRACT: Interaction of platelets with collagen under conditions of blood flow is a multi-step process with tethering via glycoprotein IbIXV (GPIbIXV) over von Willebrand factor, adhesion by direct interaction with the integrin GPIaIIa, and signaling via GPVI. GPVI can be specifically agonized by cross-linked collagen-related peptide (CRP-XL), which results in a signaling cascade very similar to that evoked by native collagen. The GPVI gene has 2 common alleles that differ by 3 replacements in the glycosylated stem and 2 in the cytoplasmic domain. We used CRP-XL to elucidate the variation in responses observed in platelet function in different individuals. We observed a 3-fold difference in the response to CRP-XL in platelet aggregation when comparing platelets from 10 high-frequency allele homozygotes with 8 low-frequency ones (2-way analysis of variance [ANOVA], P <.0001). The difference in functional responses was reflected in fibrinogen binding and in downstream signaling events as measured by tyrosine phosphorylation, the expression of P-selectin, and the binding of annexin V and the generation of thrombin on the platelet surface (2-way ANOVA, P <.001). Platelets homozygous for the low-frequency allele tended to be less able to form a thrombus on a collagen surface in flowing whole blood or in the platelet function analyzer-100 (t test, P =.065 and P =.061, respectively). The functional difference was correlated to a difference in total and membrane-expressed GPVI measured by monoclonal and polyclonal antibodies. This study demonstrates for the first time that platelet function may be altered by allelic differences in GPVI.
Blood 06/2003; 101(11):4372-9. · 9.90 Impact Factor
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ABSTRACT: Calreticulin is an abundant protein in the endoplasmic reticulum of most cells. In this study, flow cytometry and immunoprecipitation from surface-biotinylated platelets each provided direct evidence that calreticulin is also expressed on the surface of human platelets. Anti-calreticulin antibodies caused platelet activation, inducing FcgammaRIIa-independent platelet aggregation. In addition, these antibodies inhibited platelet adhesion to the integrin alpha2beta1-specific ligands, GFOGER-GPP and monomeric collagen I, and to the glycoprotein VI-specific ligand, CRP. Inhibition of platelet adhesion to these ligands was independent of integrin alphaIIbbeta3. In resting platelets, calreticulin was shown to interact with integrin alpha2beta1 and glycoprotein VI. Together, these data demonstrate that surface calreticulin is associated with collagen receptors on the platelet surface, where it may play a role in the modulation of the platelet-collagen interaction.
Thrombosis and Haemostasis 11/2002; 88(4):648-54. · 5.04 Impact Factor
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ABSTRACT: The biallelic human platelet alloantigen (HPA) 1 system encodes a leucine to proline substitution at position 33 in the beta3 integrin. Homozygous individuals can be immunized by the non-self allele-encoded protein following transfusion or during pregnancy. In post-transfusion purpura (PTP), a subsequent recall alloantibody response against the non-self form of beta3 is paralleled by the destruction of autologous platelets, leading to profound thrombocytopenia. Although serological evidence suggests platelet autoantibodies are responsible, such autoantibodies are poorly defined. We used variable gene phage display to isolate alphaIIbbeta3 autoantibodies formed in the acute phase of PTP and determined the epitopes recognized. An immunoglobulin G (IgG)-encoded variable heavy-chain domain (VH) gene repertoire containing 4.7 x 10(7) single-chain Fv fragments was cloned and three alphaIIbbeta3 antibodies were isolated (clones 2F2, E3 and B12). All three used different VH genes with a low level of somatic mutation for genes derived from gamma-encoding mRNA. Two (2F2 and E3) recognized an overlapping epitope and their binding was inhibited by sera from patients with PTP; all three recognized Ca2+-dependent compound epitopes on alphaIIbbeta3. Our results support the theory that a transient loss of tolerance for alphaIIbbeta3 with autoantibody formation occurs in PTP.
British Journal of Haematology 04/2002; 116(3):677-85. · 4.94 Impact Factor
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ABSTRACT: The collagen type I-derived fragment α1(I)CB3 is known to recognize the platelet collagen receptor integrin α2β1 as effectively as the parent collagen, although it lacks platelet-aggregatory activity. We have synthesized the fragment
as seven overlapping peptides that spontaneously assemble into triple helices. On the basis of their capacity to bind purified
α2β1 and the recombinant α2A-domain, and their ability to support α2β1-mediated cell adhesion, we identified two peptides, CB3(I)-5 and -6, which contain an α2β1 recognition site. Synthesis of the peptide CB3(I)-5/6, containing the overlap sequence between peptides 5 and 6, allowed
us to locate the binding site within the 15-residue sequence, GFP*GERGVEGPP*GPA (where P* represents hydroxyproline), corresponding
to residues 502–516 of the collagen type I α1 chain. The Glu and Arg residues in the GER triplet were found to be essential for recognition since substitution of either
residue with Ala caused a loss of α2 A-domain binding. By contrast, substitution of the Glu in GVE did not reduce binding, but rather enhanced it slightly. We
were unable to detect significant recognition of α2β1 by the peptide CB3(I)-2 containing the putative α2β1 recognition sequence DGEA. Peptides CB3(I)-1 to -6, together with peptide CB3(I)-5/6, exhibited good platelet-aggregatory
activity, in some cases better than collagen. However, peptide CB3(I)-7 was inactive, suggesting the presence of an inhibitory
element that might account for the lack of aggregatory activity of the parent α1(I)CB3 fragment.
Journal of Biological Chemistry 12/1998; 273(50):33287-33294. · 4.77 Impact Factor
