Cross Reaction of Antibody Against Helicobacter Pylori Urease B with Platelet Glycoprotein IIIa and Its Significance in the Pathogenesis of ITP.

Abstract

The data about association of Idiopathic Thrombocytopenic Purpura (ITP) with Helicobacter pylori (H pylori) infection are always contradictory, and the factors that incite ITP remain uncertain. Several hypotheses have been suggested about the striking discrepancies between the reported results, one of which is variable effects of genetically diverse H pylori strains in remote geographic regions. The sequence of gene for H pylori urease B is relatively constant, and its protein production is believed to be a crucial factor resulting in some diseases. We therefore conducted a study to assess the role of H pylori in the pathogenesis of ITP, and to investigate its immune mechanisms. Firstly, bacteria H pylori were isolated from gastric mucosal tissue of a patient with peptic ulcer and cultured. H pylori urease B gene was amplified, then ligated into expression plasmid. The latter was transfected into E coli. The recombinant urease B was used to develop monoclonal antibody (MoAb), 1F11, against urease B of H pylori. In Western blot analysis, both 1F11 and SZ21 (MoAb against GP IIIa) bound to the band of protein 95 KDa of normal platelet lysate, but not to that of platelet lysate prepared from a patient with Glanzmann thrombobasthenia (GT), in whom platelet membrane GP IIb/IIIa were absent. Flow cytometry analysis also showed that normal platelets were reacted with both 1F11 and SZ21, whereas platelets of the GT patient showed a low reactivity with 1F11 or SZ21. In immunoradiometric assay, in which four MoAbs against different platelet membrane GPs (GP Ib, GP IIb, GP IIIa and GP IV) were each coated on microtiter plates, and platelet lysate and then 125I-labeled 1F11 were separately added, the radioactivity value (count per minute) in plates coated with SZ21 was the highest among all coated plates. All the three different experimental results showed that there exists a cross reaction between MoAb 1F11 against Helicobacter pylori urease B and platelet glycoprotein IIIa. In addition, MoAb 1F11 could inhibit ADP induced platelet aggregation in a dose-dependent manner. However, 1F11 had no effect on plasma P-selectin and TXB2 levels induced by ADP, because platelet P-selectin expression and TXB2 synthesis could occur before aggregation, the “final step” of platelet aggregation, just like what reported in Glanzmann thrombocytopenia. Finally, we performed a prospective clinical study on H pylori infection by means of immuno-radiometric assay of urease B in 164 ITP patients, 88 (54%) were found H pylori-positive. platelet counts in 15 of 38 H pylori-positive patients were significantly increased after eradication treatment, and were completely recovered in 7 patients. In conclusion, our results first indicate that antibidy against H pylori urease B could cross-react with platelet GP IIIa, and inhibit platelet aggregation. H pylori eradication may improve the platelet counts in some chronic ITP patients.