Medicine (RMH) - Theses
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ItemThe HIT epitope( 2001)Heparin-induced thrombocytopenia (HIT) is a disorder found in some patients who developed thrombocytopenia after given heparin and similar glycosaminoglycans, with an incidence of 3%. There is little experimental study on the predisposition to HIT, while the pathogenesis of HIT has been investigated more widely. The currently favoured model is based on the activation of platelets FeγRII by HIT IgG, which only occur when the HIT IgG is in complex with PF4 and glycosaminoglycan. However, no study has looked into platelets activation by the HIT antibody beyond the binding to FeγRII. As the signal transduction pathways of platelets activation become increasingly understood, the activation of platelets by the HIT antibody await further in-depth studies. The involvement of endothelial cells in HIT is also an area that demands further detailed studies. Almost nothing is known about the immunogenesis of HIT, apart from isolated animal study based on idiotype. The possibility of involvement of cellular immunity in HIT is only studied very recently. HIT may be diagnosed by a functional assay based on activation of platelets or immunoasaay based on the detection of anti-PF4-hcparin antibody. A map of the HIT epitope is essential in elucidating the various aspects of HIT. It is now known that the majority, if not all HIT antibodies bind to a complex of hPF4 and heparin. Although the role of heparin or similar glycosaminoglycan is not fully understood, it is plausible that the HIT epitope is presented entirely on the PF4 tetramer. Heparin presumably serves to modify the presentation of the HIT epitope on the PF4 tetramers. One model contends that heparin alters the conformation of each PF4 tetramer, such that a neo-epitope is exposed on binding to heparin. On the other hand, when heparin is absent, the HIT epitope is hidden within the PF4 tetramer. An alternative model is based on the alignment of many PF4 tetramers by chains of heparin polysaccharides. Knowledge of the HIT epitope is crucial in unravelling the interaction between the HIT antibody and its antigen, and the subsequent platelets activation. This study uses site directed mutagenesis to create a comprehensive set of hPF4 mutant proteins. By measuring the binding avidity of these mutant PF4s with a batch of HIT sera, the contribution of the surface residues of PF4 tetramer is estimated. Subsequently, a single HIT population epitope is mapped on hPF4. Using graphical molecular modelling, several possible ways in which the HIT antibody, PF4 and heparin may interact with each other are examined. Viewing PF4 as a six faceted cuboid, the HIT epitope is found to be presented predominantly on one pair of opposite faces. The heparin binding helical pairs are located on a different pair of opposite faces. When a curve is drawn around the PF4 tetramer along the longitudinal axes of the two pairs of α-helices, the HIT epitope bearing faces are the pair that are crossed by this curve. This finding is significant because it is consistent with a model of interaction between PF4 and heparin, in which heparin chains bind to four pairs of lysine residues on each pair of α-helices in a orthogonal orientation between the longitudinal axes of the heparin chain and the helical pair. The finding is also consistent with a modified model in which heparin binds to the pair of opposite faces whose normal axes are directed perpendicular to the longitudinal axes of the paired α-helices. It is of significance to note that the pair of faces containing the HIT epitope are the only pairs which have a paucity of positively charged amino acid residues. If the HIT epitope resides on any of the other two pairs of faces, the HIT antibody will be forced to compete with heparin for binding to regions containing positively charged residues. The localisation of the HIT epitope on the pair of faces poor in positively charged residues is also significant, as PF4 tetramers are free to be aligned into arrays via cross-linking by heparin chains. These arrays of heparin cross-linked PF4 arrays have their HIT epitope bearing facets exposed. HIT IgG are facilitated to bind onto arrays of HIT epitopes aligned in optimal positions for coordinated binding of HIT IgG. The net effect of such coordinated binding is the oligomerisation of HIT IgG. When the oligomerised HIT IgG bind to Feγ receptors, the latter were forced to undergo lateral movement in the cell membrane in order to reach the binding targets. The resultant clustering of Feγ receptors on the cell membrane is expected to serve as trigger for downstream signal transduction which eventually leads to the activation of platelets. It may be some time yet before the finding of this study can be confirmed by 3D structural mapping of the HIT epitope using X-ray crystallography or NMR study. The tetrameric nature of PF4 and the tri-molecular interaction pose enormous barrier to this approach. In the meantime, the functionally determined HIT epitope will serve as a model for further experimental investigation and understanding of HIT.