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Absence of collagen-induced platelet activation caused by compound heterozygous GPVI mutations

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Absence of collagen-induced platelet activation caused by compound heterozygous GPVI mutations
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  doi:10.1182/blood-2009-03-213504 Prepublished online Jun 23, 2009; Oudin, Nadine Ajzenberg, Bernard Grandchamp and Martine Jandrot-Perrus Benedicte Dumont, Dominique Lasne, Chantal Rothschild, Maxime Bouabdelli, Veronique Ollivier, Claire  heterozygous GPVI mutationsAbsence of collagen-induced platelet activation caused by compound   http://bloodjournal.hematologylibrary.org/misc/rights.dtl#repub_requests Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/misc/rights.dtl#reprints Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/subscriptions/index.dtl Information about subscriptions and ASH membership may be found online at: . Hematology; all rights reservedCopyright 2007 by The American Society of DC 20036.by the American Society of Hematology, 1900 M St, NW, Suite 200, Washington Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published semimonthly  For personal use only.at INSERM DISC on June 24, 2009. www.bloodjournal.orgFrom    1 Absence of collagen-induced platelet activation caused by compound heterozygous GPVI mutations Short title: GPVI GENETIC ABNORMALITIES Bénédicte Dumont, 1,2  Dominique Lasne, 3,4  Chantal Rothschild, 5  Maxime Bouabdelli, 1  Véronique Ollivier, 1  Claire Oudin, 6  Nadine Ajzenberg, 1,2 Bernard Grandchamp, 6  and Martine Jandrot-Perrus 1,2 1 INSERM, U698, Hôpital Bichat, Paris, France; 2 Laboratoire d’Hématologie, Hôpital Bichat, AP-HP, Paris, France;  3 Service d’Hématologie Biologique, Hôpital Necker, AP-HP, Paris, France; 4 INSERM, U765, Paris, France; 5 Centre d'Hémophilie, Hôpital Necker, AP-HP, Paris, France; and 6 Biochimie B, Hôpital Bichat, AP-HP, Paris, France Corresponding author : Martine Jandrot-Perrus, INSERM U698, CHU Xavier Bichat 46 rue Henri Huchard 75877 Paris Cedex 18, France E-mail : martine.jandrot-perrus@inserm.fr. Phone : 33 (1) 40 25 75 31; Fax : 33 (1) 40 25 86 02 Blood First Edition Paper, prepublished online June 23, 2009; DOI 10.1182/blood-2009-03-213504   Copyright © 2009 American Society of Hematology  For personal use only.at INSERM DISC on June 24, 2009. www.bloodjournal.orgFrom    2 Abstract The GPVI/FcR γ   complex is a key receptor for platelet activation by collagen. We describe for the first time two genetic abnormalities in one patient. This 10 year-old girl presented ecchymoses since infancy, a prolonged bleeding time despite a normal platelet count and no antiplatelet antibodies. Collagen-induced platelet activation was null whereas GPVI quantification by flow cytometry evidenced an incomplete deficiency. Immunoblotting showed an abnormal migration of residual GPVI, and no FcR γ   defect. GPVI DNA sequencing revealed (i) a R38C mutation in exon 3 of one allele (ii) an insertion of 5 nucleotides in exon 4 of the other allele, leading to a premature nonsense codon and absence of the corresponding mRNA. Introduction of the R38C mutation into recombinant GPVI-Fc resulted in abnormal protein migration and a loss of collagen binding. Thus, this composite genetic GPVI deficiency and dysfunction causes absence of platelet responses to collagen and a mild bleeding phenotype. For personal use only.at INSERM DISC on June 24, 2009. www.bloodjournal.orgFrom    3 Introduction Collagen is a major thrombogenic constituent of the vessel wall. Platelets principally react with collagen via  integrin α 2 β 1 and GPVI, 1  a member of the immunoglobulin receptor family. 2,3  GPVI plays a major role in platelet activation through signaling mediated by the non-covalently associated ITAM-containing FcR γ   chain. 1,4  The extracellular domain of GPVI comprises two Ig-like domains important for collagen binding and receptor dimerisation respectively. 5-8  GPVI deficiencies 9  have been described mostly in patients with immune thrombocytopenic purpura, where autoantibodies are assumed to induce GPVI shedding. 10,11  We report the first case of congenital GPVI deficiency with a mild bleeding disorder. This patient, whose platelets failed to respond to collagen, is a compound heterozygote for two GPVI mutations: one leads to an incomplete protein deficiency; the second is a loss of function Arg to Cys substitution in the first GPVI Ig-like loop. Methods See on line supplement Patient The patient is a 10 year-old female who consulted for easy bruising since infancy. Her parents were non-consanguineous and there was no family history of bleeding. She had a prolonged bleeding time (Ivy > 15 min, PFA-100 closure time on collagen/epinephrine 210 sec), a normal platelet count (280 G/L), no morphological abnormalities on blood smears and no detectable anti-platelet antibodies. Von Willebrand factor exploration was in the normal range. All studies were performed with written informed consent according to the Declaration of Helsinki and with approval of the ethical board of Necker hospital (AP-HP). For personal use only.at INSERM DISC on June 24, 2009. www.bloodjournal.orgFrom    4 Platelet function studies Platelet aggregation was measured on platelet-rich plasma (PRP) and washed platelets. 12  Dense granule secretion was quantified using an ATP determination kit. Platelet adhesion to collagen under flow conditions and thrombin generation were measured mainly as described. 13,14  GPVI expression was analyzed by flow cytometry and immunoblot using the moAb 3J24.2 or 9O12.2 and a human polyclonal antibody. Genetic studies Each of the eight exons and intron-exon junctions of GPVI were amplified by polymerase chain reaction (PCR); products were sequenced on both strands. Total mRNA was extracted; cDNA corresponding to exons 3 and 4 was amplified and PCR products sequenced. R38C mutant Arg38 was changed to Cys to produce R38C GPVI-Fc. Wild type and mutant GPVI-Fc were produced and purified. 3,15   Results and discussion The patient's platelets (PRP and washed platelets) failed to respond to collagen up to 4 µg/mL but aggregated normally in response to ADP, TRAP, arachidonic acid and ristocetin (Figures 1A, 1S). Activation induced by the GPVI-specific agonist convulxin was also impaired (Figure 1S). ATP secretion was low in response to 4 µg/mL collagen (404 vs  4727 nM for the patient and control respectively). The patient's PPP did not inhibit collagen aggregation responses of control platelets or induce spontaneous aggregation. The capacity of the patient’s platelets to form thrombi on collagen in flow conditions was strongly impaired (Figure 1B) with a 43% decreased surface coverage as compared to control. For personal use only.at INSERM DISC on June 24, 2009. www.bloodjournal.orgFrom 
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