Les transfusions plaquettaires ont permis par leur developpement de limiter les risques hemorragiques des thrombopenies severes au cours du traitement ou de l'evolution des maladies hematologiques. Cependant, l'allo-immunisation antiplaquettaire et l'apparition d'un etat refractaire aux transfusions sont des complications pouvant poser des problemes therapeutiques difficiles. Les alloantigenes plaquettaires tels les antigenesHLA peuvent etre communs avec d'autres cellules de l'organisme, ou bien etre specifiquement plaquettaires. Si l'allo-immunisation post-transfusionnelle anti-HLA est connue depuis longtemps, le role des antigenes specifiques est encore controverse en raison de la difficulte d'identification des anticorps specifiques tant que l'on ne disposait pas de tests analytiques. L'etat refractaire aux transfusions plaquettaires, defini par une duree de vie raccourcie des plaquettes transfusees, peut etre du a plusieurs causes telles la qualite du produit transfuse, des causes non immunes ou immunes. L'identification du mecanisme est importante pour adapter au mieux la therapeutique transfusionnelle chez ces patients. Afin de diminuer l'allo-immunisation, plusieurs strategies ont ete developpees. Il est necessaire d'en evaluer le rapport cout-efficacite. Le purpura post-transfusionnel est une complication rare mais severe de la therapeutique transfusionnelle. Classiquement, c'est devant la survenue d'une thrombopenie profonde une semaine apres une transfusion chez une femme âgee que le diagnostic doit etre evoque. Le mecanisme physiopathologique exact n'est pas connu. Mais il semble bien qu'un des facteurs declenchants soit une reponse anamnestique en rapport avec une immunisation prealable lors de grossesse anterieure. Le traitement le plus approprie est actuellement la perfusion d'immunoglobulines polyvalentes.
The human platelet alloantigen (HPA)-1 system, the first cause of alloimmune thrombocytopenia in Caucasians, results from leucine-to-proline substitution (alleles 1a and 1b) of residue 33 in β3 subunit of the integrin αIIbβ3. A third variant with a valine (V33) has been described. Although leucine and valine share similar physicochemical properties, sera containing alloantibodies to the HPA-1a antigen variably reacted with V33-β3, suggesting structural alterations of β3. To analyze the effect of the L33V transition, molecular dynamics simulations were performed on a 3D structural model of the V33 form of the whole β3 extracellular domain (690 residues). Dynamics of the PSI (carrying residue 33), I-EGF-1, and I-EGF-2 domains of β3 were compared to previously obtained dynamics of HPA-1a structure and HPA-1b structural model using classical and innovative developments (a structural alphabet). Clustering approach and local structure analysis showed that L33-β3 and V33-β3 mostly share common structures co-existing in different dynamic equilibria. The L33V substitution mainly displaces the equilibrium between common structures. These observations can explain the variable reactivity of anti-HPA-1a alloantibodies suggesting that molecular dynamic plays a key role in the binding of these alloantibodies. Unlike the L33P substitution, the L33V transition would not affect the structure flexibility of the β3 knee, and consequently the functions of αIIbβ3.
The use of agonist monoclonal antibodies (mAbs) to probe the signalling function of platelet membrane proteins is severely limited by the dependence of the mAb effect on Fc–FcγRII interaction. Furthermore, in addition to its anchoring role, the FcγRII receptor itself generates a stimulation signal resulting in granule secretion. Platelet stimulation by the released granule contents can then further obscure the original activation signal. Here we demonstrate that these problems are largely overcome by the use of platelets which had been degranulated with thrombin prior to stimulation with mAbs. We found that, like intact cells, degranulated platelets could also be activated and induced to aggregate by mAbs against a 67 kD membrane protein (known as PTA1) and CD9, and by crosslinked CD32 (FcγRII). However, the signal generated by crosslinked FcγRII was weak compared with that induced by the other monoclonal antibodies. Thus, by diminishing the FcγRII signal contribution, we have succeeded for the first time to clearly dissect the target antigen signal from that generated by FcγRII. In addition to differences in the degree of aggregation, analysis of the signals generated by each mAb showed differences in Ca 2+ fluxes and protein phosphorylation. Moreover, the signals generated by CD9 and PTA1 antigens differed significantly in their sensitivity to PKC inhibition or ADP‐ribosylation of the small GTP‐binding protein rhoA. Despite these differences, the signals initiated by all three antigens converged to a common signalling pathway which included activation of tyrosine kinase(s). The pattern of protein phosphorylation strongly resembled that induced by gpIIb/IIIa‐mediated platelet interaction with macromolecular ligands and by mutual cell contact. The multiple intercellular links formed by mAb would have a similar effect since the Fc‐receptor anchorage required for antigen stimulation is already known to be provided by adjacent cells. The present findings suggest that the function of both CD9 and PTA1 antigens is closely associated with gpIIb/IIIa activation.
Human platelet antigens (HPA) polymorphisms may cause HPA alloimmunization, platelet (PLT) refractoriness, fetomaternal alloimmune thrombocytopenia, and posttransfusion purpura. Characterized by significant racial admixture, the Brazilian population might benefit from the knowledge about HPA frequency to guide decision-making concerning PLT transfusion.HPA frequencies were determined in 158 DNA samples from Brazilian blood donors by microarray for HPA-1 to -9, -11, and -15. A HPA-2 discrepancy was solved by polymerase chain reaction with sequence-specific primers (PCR-SSP) and sequencing.While a alleles were predominant for HPA-1 to -9 and -11, b alleles were absent for HPA-6, -7, -8, and -11. HPA-3 and HPA-15 had a higher prevalence of ab genotypes. One case of HPA-4ab and two cases of HPA-9abw were detected, the latter not previously described in Brazilian blood donors. One sample was not interpretable for HPA-2 due to a GPIb 468 C>G mutation; this donor was characterized as HPA-2ab by PCR-SSP and sequencing.Allele frequencies were comparable to those described in other Brazilian studies. Rare HPA-9 alleles were described in Brazilians for the first time. A mutation near the HPA-2 polymorphism suggests that complementary methods might be necessary in specific cases. PLT genotyping by microarray proved to be fast, accurate, and reliable.
BACKGROUND: A new platelet antigen, Cab2 a+ , was identified in a case of severe neonatal alloimmune thrombocytopenia (<8 × 10 9 /L) in twins. STUDY DESIGN AND METHODS: Coding sequences of αIIb and β3 genes from parents were amplified and sequenced. CHO cell lines expressing wild‐type or mutated forms of the complex were established to study the role of the mutation in alloimmunization and in αIIbβ3 functions. RESULTS: The father and twins were heterozygous for a single αIIb c.1508G>A mutation leading to a Ser472Asn substitution. Immunologic assays with transfected CHO cells revealed the Asn472 form of αIIbβ3 responsible for the Cab2 a+ epitope but not an Ala472 form. Using these cells lines we demonstrated that both Ser472Asn and Ser472Ala substitutions produced limited structural alteration as revealed by the reactivity of a panel of anti‐αIIbβ3 monoclonal antibodies (MoAbs). Activated Asn472 and Ala472 forms of αIIbβ3 supported 1) binding of soluble fibrinogen and of the ligand mimetic MoAb PAC‐1, 2) ligand‐induced binding site epitopes exposure (MoAbs AP‐5 and D3GP3), and 3) cell aggregation. Adhesion onto adsorbed fibrinogen was conserved and was specifically inhibited by MoAb AP‐2 or peptide RGDS. Finally outside‐in signaling was not affected. CONCLUSION: We have characterized a new low‐frequency alloantigen (<1%) resulting from the Ser472Asn substitution in αIIb and shown this polymorphism to have a limited effect, if any, on the αIIbβ3 complex functions.
Alloimmune thrombocytopenia resulting from the destruction of platelets by an alloantibody elicited during the recipient's immune response against platelets from a genetically different individual is not a rare event. The major clinical condition is the fetal and neonatal alloimmune thrombocytopenia affecting 1:800 to 1:2000 live births in Caucasians. In case of severe thrombocytopenia, the most feared complication is intracranial hemorrhage leading to death or neurological impairment (10% and 20% of the reported cases in retrospective studies respectively). Important developments have been made in diagnosis and therapy in the recent years. However, there are still questions addressing the pathophysiology and the optimal management. Development of animal models and large collaborative studies are of importance for the better understanding of the implicated mechanisms, prevention, and specific therapies of this condition and its deleterious consequences.
