Nelly Kieffer

Identification of platelet membrane thrombospondin binding molecules using an anti-thrombospondin antibody

A rat monoclonal IgG2a antibody, 5G11, was raised against native human platelet thrombospondin (TSP). Western blot analysis revealed that 5G11 bound (i) to TSP before and after disulfide reduction, and (ii) to a 15-kDa fragment released after prolonged trypsin digestion. Crossed immunoelectrophoresis confirmed that the binding epitope was expressed in the presence of Ca2+ and after treatment of TSP with EDTA. Since 5G11 had no effect on platelet aggregation, the antibody was used to immunoprecipitate Ca2+-dependent and Ca2+-independent TSP-binding molecules on the surface of thrombin-activated surface-labeled 125I-platelets. The experimental basis was that ligand-receptor interactions are of high affinity and that anti-ligand antibodies should precipitate the ligand-receptor complex. With platelets activated in the presence of EDTA, 5G11 predominantly precipitated a 125I-labeled band of Mr 88,000, identified as glycoprotein (GP) IV. In contrast, in the presence of 2 mM Ca2+ and 1 mM Mg2+, 5G11 precipitated a complex of five radiolabeled proteins, among which GPIIb, GPIIIa and GPIV were the most prominent.

Early Immunization against Platelet Glycoprotein IIIa in a Newborn Glanzmann Type I Patient

Abstract. Alloimmunization against platelet glycoprotein lib and/or IIIa is a complication rarely observed during the evolution of type I Glanzmanns thrombasthenic patients [1,2]. The occurrence of such alloantibodies is usually due to repeated blood transfusions [2, 3] and greatly complicates the treatment of these patients since they prevent effective platelet transfusion and might, theoretically, cause posttransfusion purpura. We describe the case of a newborn thrombasthenic patient who developed an IgG platelet allo‐antibody 1 month after birth. The diagnosis of Glanzmanns thrombasthenia was complicated by the rare platelet phenotype (PLAt‐negative PLA2‐positive) of the healthy mother, which was probably heterozygous for the abnormal thrombasthenic gene. Immunofluorescence and immunoblotting techniques demonstrated that the patient antibody was principally directed against the platelet glycoprotein IIIa. Surprisingly, this patient had only received four blood transfusions (fresh frozen plasma on days 1 and 2, and standard red blood cell concentrates on days 5 and 6) before the discovery of the antibody, suggesting prior in utero sensitization. This study emphasizes the need for early diagnosis of the disease. Thrombasthenic patients should be transfused with deleukocyted platelet‐free blood products.