No abstract

Despite much progress in recent years, the precise signalling events triggered by the vascular-endothelial-growth-factor (VEGF) receptors, fms-like tyrosine kinase (Flt1) and kinase insert domain-containing receptor (KDR), are incompletely defined. Results obtained when Flt1 and KDR are individually expressed in fibroblasts or porcine aortic endothelial cells have not been entirely consistent with those observed in other endothelial cells expressing both receptors endogenously. It has also been difficult to demonstrate VEGF-induced phosphorylation of Flt1, which has led to speculation that KDR may be the more important receptor for the mitogenic action of VEGF on endothelial cells. In an attempt to identify physiologically important effectors which bind to KDR, we have screened a yeast two-hybrid mouse embryo library with the cytoplasmic domain of KDR. Here we describe the identification of the adaptor protein, Shc-like protein (Sck), as a binding

Summary. In chronic immune thrombocytopenic purpura (ITP), autoantibodies bind to platelet surface proteins, particularly a IIb , resulting in platelet destruction by the reticulo-endothelial system. In order to better localize the autoepitopes on a IIb , we studied the binding of antibodies to Chinese hamster ovary (CHO) cells expressing either a IIb b 3 or a IIb -a v b 3 chimaeras in which a segment of a IIb (either amino acids L1-Q459, L1-F223 or F223-Q459) was substituted for that portion of a v . We evaluated plateletassociated autoantibodies from 14 ITP patients with a IIb -dependent antibodies. Ten of 14 bound to a IIb (L1-Q459) -a v b 3 , showing that autoepitopes were often localized to this region of a IIb . In addition, each of the autoantibodies binding to a IIb (L1-Q459) -a v b 3 , also bound to CHO cells expressing either a IIb(L1-F223) -a v b 3 or a IIb(F223-Q459) -a v b 3 ). In two of the three eluates tested, > 95% of the autoantibody binding to a IIb could be adsorbed using CHO cells expressing any of the three chimaeras, showing that the epitope(s) have contact points on either side of amino acid F223; in the third eluate, only a portion ( 40%) could be adsorbed by the chimaeric cell lines showing that, in this patient, an additional antibody was also present, directed to a site distal to amino acid Q459. The remaining four eluates bound to CHO cells expressing a IIb b 3 but to none of the chimaeras, suggesting that these epitopes are also distal to amino acid Q459. We conclude that the binding of many anti-a IIb b 3 autoantibodies is dependent on the presence of a IIb amino acids L1-Q459.

Chronic immune thrombocytopenic purpura is due to platelet destruction induced by autoantibodies against platelet surface antigens. Prior studies show that some serum autoantibodies are light-chain restricted, suggesting a clonal origin. Since plasma and plateletassociated antibody from the same patient may bind to different epitopes, it is important to evaluate the clonality of platelet-associated antibody. Platelet-associated autoantibodies from 28 ITP patients were studied. Of 23 platelet-associated antibodies tested directly, 16 showed significant light chain restriction (7 complete and 9 partial) when compared to plasma IgG light chain distribution. Similarly, 9 of 12 platelet-associated antibody eluates were light chain restricted, 5 complete and 4 partial. In all cases where platelet-associated antibody and antibody eluate from the same patient were studied, the results were concordant. We conclude that a significant proportion of plateletassociated antibodies from ITP patients show apparent clonality, as evaluated by light chain restriction. These results are consistent with other studies in ITP suggesting a limited antigenic repertoire.