Studies On The Autoimmune Disease And Anti-platelet Autoantibodies

Autoimmune disease (AID) is a group of disorders in which tissue and organ were damaged due to autoimmune reaction. The thrombocytopenia and thrombosis are common manifestation of autoimmune disease. The thrombocytopenia is not only due to autoimmune destruction of platelets by anti-platelets antoantibodies, but also due to consumption of platelets by vascular endothelial cell impairment, platelet activation and thrombus formation. Here, we intend to elucidate the role of anti-platelet membrane glycoprotein autoantibody in the pathogenesis of autoimmune disease. Meanwhile, we construct human Fab antibody phage display library in order to select completely human antibodies directed against platelet GPIIb/IIIa. I Studies on anti-platelet membrane glycoprotein autoantibodies in autoimmune disease Background /Objective The thrombocytopenia is common manifestation of autoimmune disease. Anti-platelet autoantibody plays a key role in the pathogenesis of autoimmune disease. Primary Sj?gren syndrome (pSS) is an autoimmune disease that mainly affects exocrine glands and usually presents as a persistent dryness of the mouth and eyes due to functional impairment of the salivary and lacrimal glands, it affects non-exocrine glands tissue simultaneously, the incidence rate is 0.29%-0.77% in China. Patients with pSS have a wide variety of hematologic abnormalities, including anemia, leucocytopenia and thrombocytopenia. Thrombocytopenia was found in 23% of Chinese pSS patients. This study is aiming at detecting anti-platelet membrane glycoprotein autoantibodies in pSS, and to explore the pathogenesis of thrombocytopenia in pSS. Methods By utilizing the monoclonal antibodies to human platelet membrane glycoproteins(GP), SZ-2 (anti-GPⅠb),SZ-21(anti-GP Ⅲa ), SZ-51(anti-P-selectin) and 7E3(anti-GPⅡb/Ⅲa).To detect plasma level of anti-platelet membrane GP autoantibodies by ELISA procedure in 70 patients with pSS (including 38 pSS patients with normal platelet counts and 32 pSS patients with thrombocytopenia), 32 patients with idiopathic thrombocytopenic purpura (ITP) and 35 normal controls. Results The level of anti-GPⅠb,GPⅢa ,GPⅡb/Ⅲa autoantibodies in the pSS patients with normal platelet counts[(0.695±0.134),(0.802±0.069) ,(0.782±0.104)] had no significant difference with that of normal controls [(0.663 ±0.126),(0.771 ±0.064) ,(0.781±0.100)] (P>0.05),while the level of anti-P-selectin autoantibody [(0.738±0.090)] was significantly higher than that of normal controls [(0.684±0.110)] (P<0.05),the positive rate of the anti-GPⅢa and anti-P-selectin autoantibody was 10.52%(4/38) and 7.89% (3/38);the level of anti-GPⅠb,GPⅢa ,GPⅡb/Ⅲa and P-selectin autoantibodies in the pSS patients with thrombocytopenia[(0.812 ±0.164),(0.915±0.093) ,(0.910±0.125) ,(0.854±0.142)] was significantly higher than that of normal controls(P<0.01), the positive rate of the anti-GPⅠb,GPⅢa ,GPⅡb/Ⅲa and P-selectin autoantibody were 25%(8/32),65.63%(21/32),28.13%(9/32),40.63% (13/32); the level of anti-GPⅠb,GPⅢa ,GPⅡb/Ⅲa and P-selectin autoantibodies in the ITP patients [(0.775±0.138),(0.846±0.071) ,(0.878±0.133) ,(0.796±0.097)] were significantly higher than that of normal controls(P<0.01), the positive rate of the anti-GPⅠb,GPⅢa ,GPⅡb/Ⅲa and P-selectin autoantibody were 15.63%(5/32),25% (8/32),21.88%(7/32),15.63% (5/32) ; the level of anti-GP Ⅰb,GP Ⅱb/ Ⅲa autoantibodies in the pSS patients with thrombocytopenia had no significant difference with that of ITP(P>0.05), while the level of anti-GP Ⅲa and P-selectin autoantibodies was significantly higher than that of ITP (P<0.01,P<0.05)Conclusion The level of anti-GPⅠb,GPⅢa ,GPⅡb/Ⅲa and P-selectin autoantibodies in the pSS patients with thrombocytopenia was significantly higher than that of normal controls and that of the pSS patients with normal platelet counts .This suggested platelet membrane glycoprotein autoantibodies played a role in the pathogenic mechanism of thrombocytopenia in pSS. Meanwhile, there were anti-GPⅢa and anti-P-selectin autoantibody in plasma of pSS patients who had normal platelet counts , the level of anti-GPⅢa and anti-P-selectin autoantibodies in the pSS patients with thrombocytopenia were significantly higher than that of ITP, we could also draw the conclusion that there was activation of platelet and endothelial cell simultaneously in pSS. II Study on human anti-platelet membrane glycoprotein IIb/IIIa phage display antibody. Background/Objectives With purpose of preparing Fab antibodies for the therapy of thromboembolic diseases , we construct human Fab antibody phage display library in order to select completely human antibodies against platelet GPIIb/IIIa by using the spleen of ITP patient whose anti-GPIIb/IIIa autoantibody is positive. Materials and Methods 1 Extraction of total RNA: Total RNA was extracted from the spleen of ITP patient whose anti-GPIIb/IIIa autoantibody was positive by using Gibco Trizol Reagent. To generate first-strandcDNA, total RNA was reverse transcribed by using the primers OligodT (12 ). 2 Amplifying the fragments of heavy (Fd) and light chain genes of antibodies by RT-PCR: Synthesize Fd and light chain genes by using first-strand cDNA . The amplified products were purified on a 1.5% agarose gel. DNA was extracted and purified with the QIAEXII gel extraction kit and quantitated. 3 Construction of light chain library: The light (κ,λ)chains genes were first ligated into the SacI/XbaI site of pComb3H and transformed into Escherichia coli XL-1 blue by electroporation. The transformed bacteria were amplified, then isolated the plasmid DNA , which was a light chain library. At the same time, the light chain genes'insertion was detected. 4 Construction of Fab antibody phage display library: The Fd fragment product of PCR and isolated plasmid DNA containing the light chain library were cut with XhoI/SpeI, gel-purfied. Ligated them and transformed into Escherichia coli XL-1 blue by electroporation. E.coli was amplified and superinfected with helper phage VCSM13, yielded a library of Fd+light chain(Fab) antibody. determination of transformation rate was also carried out. 5 Panning and enrichment of Fab antibody phage display library: For panning and enrichment, ELISA plate was coated with Chinese hamster ovary (CHO) cells expressing GPIIb/IIIa, CHO123 cell line. Freshly prepared phage suspension was added. E.coli XL-1 blue were then infected with the eluted phage which was absorbed specifically. By superinfected with helper phage VCSM13, flimentous phage was produced for the next round of panning. The panning, affinity absorption-elution-amplification, was repeated three times. Identified Fab antibody from phage display library at different rank, and measured its titre. 6 Production of soluble Fab fragments: For the production of soluble Fab fragments, DNA was isolated from plasmid after the third round of panning. The plasmid DNA was digested with SpeI/NheI, recovered from a 1.0% agarose gel, self-ligated, and retransformed into E.coli XL-1 blue byelectroporation. E.coli containing the correctly recombinated plasmid were then used to produce soluble Fab fragments. 7 Screening and identification of specific positive clones: Screened positive clones by cell-ELISA, rejecting the positive clones which cross-reacted with wild type CHO cells, identifying their specificity by Western-blot. 8 Sequencing specific positive clone genes: Amplified two clones which bound with CHO cells expressing GPIIb/IIIa better. Their sequences of Fd and light chain DNA fragments were assayed and their homology with human Ig were analyzed. 9. Identification of specific antibody's function: Platelet aggregation induced by ADP was measured by using Born's method, prepared platelet rich plasma(PRP),adjusted platelet concentration to 3×108 /L, added specific antibody, induced by ADP,PRP without antibody as control. Results 1.Amplification of light chain and heavy (Fd) chain genes: Total RNA was extracted from spleen of ITP patient, the RNA was pure (A260/280 =1.9～2.0). Fd and κ,λDNA fragments (with size about 700bp) of immunoglobulin chains were amplified by PCR .then mixed the PCR products of κ,λand Fd chains for constructing phage display library. 2.Construction and identification of human light chain library: The genes of light chain were cloned into the vector pComb3H, transformed into Escherichia coli XL-1 blue by electroporation. Then plated 10,1,0.1μl transformed E. coli on LB plates/Carb. The quantity of the transformants reached 8.6×106. Picked out 9 clones randomly, cut with SacI/XbaI and identified them. There were fragments (with size about 700bp) in 7 clones, the recombination rate was 78%, so the practical volume of light chain library was 6.7×106. 3 Construction and identification of human Fab antibody phage display library: The genes of Fd were cloned into the light chain library,then yielded a Fabantibody phage display library. The quantity of the transformants reached 9.4×106. Picked out 13 clones randomly, cut with SacI/SpeI and identified them.The recombination rate was 85%, so the practical volume of light chain library was 6.2×106. 4 Panning and enrichment of Fab antibody phage display library: For panning and enrichment, ELISA plate was coated with CHO cells expressing GPIIb/IIIa. After three rounds of panning, the libraries were enriched about 100-fold., 5 Production of soluble Fab fragments: The recombinated DNA was isolated from plasmid after the third round of panning. The part of gene III in recombinated plasmid DNA was cut out , self-ligated and transformed into E.coli XL-1 blue by electroporation. The soluble Fab fragments were induced by IPTG. There were soluble Fab fragments in supernatant. 6 Screening and identification of specific positive clone: Screened 60 clones by cell-ELISA, meanwhile rejected the positive clones which cross-reacted with wild type CHO cells. Gained 2 positive clones with anti-GPIIb/IIIa specificity. Western-blot showed that there was specific band in culture supernatant and frozen-thawn supernatant of positive clone. 7 Sequencing specific positive clone genes: Amplified two clones which bound with CHO cells expressing GPIIb/IIIa better. Their sequences of Fd and light chain DNA fragments were assayed and their homology with human Ig were analyzed according to Gene Bank. Results showed that sequence of light chain had the homology of 97% with κregion and Fd fragmen had the homology of 94% with human immunoglobulin. Translated the sequences of Vκand VH nucleotidyl into the sequences of amino acid, and located their complementary determining regions (CDRs). 8 Identification of specific antibody's function: The specific antibody significantly inhibited platelet aggregation induced by ADP, it showed high activity of anti-thrombosis。Conclusions 1 The human Fab antibody phage display library has been constructed successfully by using RT-PCR and phage antibody library technology. 2 The positive clones specifically binding with GPIIb/IIIa have been selected . The sequence was consistent with classical antibody variable region structure. And the soluble antibody was expressed by IPTG. The specificity was proved by Western-blot. 3 The specific antibody significantly can inhibit platelet aggregation in vitro and it will be a useful anti-thrombotic agent. 4 The constructed human Fab antibody phage display library provided a useful source for further screening of antibodies against other platelet membrane glycoproteins.