This article reports a Glanzmann thrombasthenia (GT) patient, N.M., with a point mutation in the third cysteine-rich repeat of ␤3-integrin or platelet glycoprotein (GP) IIIa, leading to the expression of a constitutively activated fibrinogen receptor. The diagnosis of GT was based on a severely reduced platelet-aggregation response to a series of agonists and approximately 20% of surface-expressed GPIIb-IIIa. The patient's GPIIb-IIIa constitutively expressed epitopes recognized by antibodies to ligandinduced binding sites (LIBS) and also spontaneously bound the fibrinogen-mimetic antibody, PAC-1. Furthermore, significant amounts of bound fibrinogen were detected on his platelets ex vivo. No signs of platelet activation were observed on sections of unstimulated platelets from N.M. by electron microscopy. Immunogold labeling highlighted the presence of surface-bound fibrinogen but revealed platelet heterogeneity with regard to the surface density. When the patient's platelets were stimulated by thrombin-receptor activating peptide, amounts of surface-expressed GPIIb-IIIa increased and the aggregation response improved, although it failed to normalize. Platelets from N.M. were able to adhere and spread on immobilized fibrinogen. Sequence analysis of genomic DNA from N.M. revealed a homozygous g1776T>C mutation in GPIIIa, leading to a Cys560Arg amino acid substitution. A stable Chinese hamster ovary (CHO) cell line was prepared expressing surface GPIIbArg560IIIa. Like platelets from the patient, GPIIb-Arg560IIIa-transfected CHO cells constitutively bound LIBS antibodies and PAC-1. They also showed an enhanced ability to adhere on surface-bound fibrinogen. Overall, these data demonstrate that a gain-of-function mutation can still be associated with a thrombasthenic phenotype even though platelets show spontaneous fibrinogen binding. IntroductionIntegrins are heterodimeric transmembrane proteins that mediate cell adhesion and cell migration. [1][2][3] They are involved in numerous fundamental biologic processes, including development, homing, inflammation, angiogenesis, and wound healing, all processes in which integrin function is subjected to a highly sophisticated regulation. [4][5][6][7][8] The platelet fibrinogen receptor, the ␣ IIb ␤ 3 integrin (glycoprotein [GP] IIb-IIIa), has constituted an ideal model for the study of an integrin's role in cell contact interactions. [9][10][11][12][13] This receptor has the capacity to shift through conformational changes from a low-affinity state unable to bind macromolecular ligands to a ligand-competent state on activated platelets. This process of affinity modulation is an essential part of integrin function. Furthermore, ligand binding to GPIIb-IIIa itself modifies the conformation of this integrin, exposing neoepitopes known as ligand-induced binding sites (LIBS) and leading to postreceptor occupancy events such as clustering of GPIIb-IIIa complexes, generation of secondary signals, and cytoskeletal rearrangement, all of which contribute to maximal platelet aggregatio...

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These data suggest that in 77% of the patients, BCM positivity was not related with anti-HLA antibodies, and, in this case, graft survival was similar to that of the -BCM controls. In a minority of patients, anti-HLA class II antibodies were responsible for the +BCM, and their presence was associated with lower early, but not long-term, graft survival. Consequently, a +BCM should not systematically contraindicate kidney transplantation.

To assess the potential contribution of the cysteine-rich domain of human platelet glycoprotein (GP) IIIa to the structure of the PlA1 epitope, we used site-directed mutagenesis to substitute alanines for cysteines at key positions potentially involved in PlA1 alloantigen formation. One of these GPIIIa isoforms in which alanine replaced Cys435 reacted normally with a series of well-characterized murine MoAbs directed against the 250 amino-terminal residues of GPIIIa, whereas binding of MoAbs specific for residues 348–692 was diminished or lost. Interestingly, of eight PlA1-specific antibodies tested, two recognized Ala435GPIIIa, two did not, and four bound to it less well than to wild-type (WT) GPIIIa. However, disruption of disulfide bonds located at or near the N-terminus of GPIIIa abolished the binding of all the anti-PlA1 alloantibodies tested. These findings provide evidence that the humoral response to the PlA1 antigen is (1) heterogenous, ie, the binding site on GPIIIa for human anti-PlA1 antibodies differs from one individual to another and (2) complex--although some anti-PlA1 alloantibodies bind to an epitope comprised solely of the amino-terminus of GPIIIa, others combine most efficiently with a more complex determinant that involves the cysteine-rich domain of GPIIIa. These findings may have implications for diagnostic and therapeutic use of synthetic or recombinant PlA1 mimetics.

Background Transfusion-related acute lung injury (TRALI) is currently one of the most common causes of transfusion-related major morbidity and death. Among the many TRALI mediators, leucocyte antibodies have been identified as important triggers of severe TRALI.