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Anti-HLA antibodies with complementary and synergistic interaction
SN607D8/SN230G6 induced enhanced C5b-9 on the platelet surface, which was blocked with anti-C1q (Figure 5A-B and Online Supplementary Figure S2).
Formation of a MAC is associated with cellular lysis through its end product which is pore formation.27 To
measure the permeability of platelet membranes, a live/dead marker (binding free amines, fluorescent signal increases in case of damaged membranes due to accessi- bility of intracellular amines) was added to platelets after incubation with SN607D8/SN230G6. Incubation with
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Figure 7. HLA antibody containing sera can induce complement activation on platelets. Twelve heat inactivated sera containing HLA antibodies were used to test complement activation and Syk-dependent activation on platelets from 3 different donors. (A-C) Complement activation (in presence of serum) was induced in pres- ence and absence of 25 mg/ml anti-C1q. (D-F) Syk-dependent activation (as measured by CD62P exposure) was induced in presence and absence of 5 mM Syk inhibitor IV in the absence of a complement source. HLA typing of donor 1: A2 A3 B7, donor 2: A1 A2 B15:01 B35, donor 3: A11 A24 B35 B38. (G) IgG binding meas- ured in absence of complement source employing an anti-human IgG antibody. (H) In green the antibodies present in sera matching the HLA typing of the platelet donors, and in red the expressed HLA-antigens for which no matching antibodies are present are indicated. Dotted lines in panel E-F correspond to background val- ues of heat inactivated serum controls (no anti-platelet antibodies present). MFI: mean fluorescence intensity.
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