{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T16:58:45Z","timestamp":1790614725207,"version":"4.1.0"},"reference-count":34,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2022,4,4]],"date-time":"2022-04-04T00:00:00Z","timestamp":1649030400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec><jats:title>Background<\/jats:title><jats:p>Triple knockout (TKO) donor pigs lacking alpha-1,3-galactose (Gal),<jats:italic>N<\/jats:italic>-glycolylneuraminic acid (Neu5Gc), and Sd(a) expressions were developed to improve the clinical success of xenotransplantation. Neu5Gc, a sialic acid expressed on cell surfaces, recruits factor H to protect cells from attack by the complement system. Lack of Neu5Gc expression may cause unwanted complement activation, abrogating the potential benefit of gene-modified donor pigs. To investigate whether TKO porcine cells display increased susceptibility to complement activation in human serum, pathway-specific complement activation, apoptosis, and human platelet aggregation by porcine cells were compared between<jats:italic>alpha-1,3-galactosyltransferase<\/jats:italic>gene-knockout (<jats:italic>GT<\/jats:italic>KO) and TKO porcine cells.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Primary porcine peripheral blood mononuclear cells (pPBMCs) and endothelial cells (pECs) from<jats:italic>GT<\/jats:italic>KO and TKO pigs were used. Cells were incubated in human serum diluted in gelatin veronal buffer (GVB<jats:sup>++<\/jats:sup>) or Mg<jats:sup>++<\/jats:sup>-EGTA GVB, and C3 deposition and apoptotic changes in these cells were measured by flow cytometry. C3 deposition levels were also measured after incubating these cells in 10% human serum supplemented with human factor H. Platelet aggregation in human platelet-rich plasma containing<jats:italic>GT<\/jats:italic>KO or TKO pECs was analyzed.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>The C3 deposition level in<jats:italic>GT<\/jats:italic>KO pPBMCs or pECs in GVB<jats:sup>++<\/jats:sup>was significantly higher than that of TKO pPBMCs or pECs, respectively, but C3 deposition levels in Mg<jats:sup>++<\/jats:sup>-EGTA-GVB were comparable between them. The addition of factor H into the porcine cell suspension in 10% serum in Mg<jats:sup>++<\/jats:sup>-EGTA-GVB inhibited C3 deposition in a dose-dependent manner, and the extent of inhibition by factor H was similar between<jats:italic>GT<\/jats:italic>KO and TKO porcine cells. The percentage of late apoptotic cells in porcine cell suspension in GVB<jats:sup>++<\/jats:sup>increased with the addition of human serum, of which the net increase was significantly less in TKO pPBMCs than in<jats:italic>GT<\/jats:italic>KO pPBMCs. Finally, the lag time of platelet aggregation in recalcified human plasma was significantly prolonged in the presence of TKO pECs compared to that in the presence of<jats:italic>GT<\/jats:italic>KO pECs.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>TKO genetic modification protects porcine cells from serum-induced complement activation and apoptotic changes, and delays recalcification-induced human platelet aggregation. It does not hamper factor H recruitment on cell surfaces, allowing the suppression of alternative complement pathway activation.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fimmu.2022.859261","type":"journal-article","created":{"date-parts":[[2022,4,4]],"date-time":"2022-04-04T12:31:47Z","timestamp":1649075507000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":3,"title":["Effect of Factor H on Complement Alternative Pathway Activation in Human Serum Remains on Porcine Cells Lacking N-Glycolylneuraminic Acid"],"prefix":"10.3389","volume":"13","author":[{"given":"Haneulnari","family":"Lee","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eun 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