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Logo of jcinvestThe Journal of Clinical Investigation
J Clin Invest. 1989 July; 84(1): 7–17.
PMCID: PMC303946

Isolation and characterization of a membrane protein from normal human erythrocytes that inhibits reactive lysis of the erythrocytes of paroxysmal nocturnal hemoglobinuria.


The observation that type III erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) are susceptible to hemolysis initiated by activated cobra venom factor complexes (CoFBb), whereas normal erythrocytes are resistant, implies that the PNH III cells are deficient in a membrane constituent that regulates this process. To isolate the inhibitory factor from normal erythrocytes, membrane proteins were first extracted with butanol and then subjected to sequential anion exchange, hydroxylapatite, and hydrophobic chromatography. Analysis by SDS-PAGE and silver stain of the inhibitory fractions showed a single band corresponding to a protein with an apparent Mr of 18 kD. PNH erythrocytes were incubated with incremental concentrations of the radiolabeled protein and then washed. In a dose-dependent fashion, the protein incorporated into the cell membrane and inhibited CoFBb-initiated lysis. This protein inhibitor functioned by restricting the assembly of the membrane attack complex at the level of C7 and C8 incorporation. By using a monospecific antibody to block the function of the inhibitor, it was shown that normal erythrocytes are rendered susceptible to CoFBb-initiated hemolysis. Analysis by Western blot of membrane proteins revealed that PNH III erythrocytes are deficient in the 18-kD protein. By virtue of its molecular weight and inhibitory activity, the 18-kD protein appears to be discrete from other previously described erythrocyte membrane proteins that regulate complement. These studies also indicate that the susceptibility of PNH III erythrocytes to reactive lysis is causally related to a deficiency of the 18-kD membrane inhibitor.

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Selected References

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  • Vogel CW, Smith CA, Müller-Eberhard HJ. Cobra venom factor: structural homology with the third component of human complement. J Immunol. 1984 Dec;133(6):3235–3241. [PubMed]
  • Parker CJ, Wiedmer T, Sims PJ, Rosse WF. Characterization of the complement sensitivity of paroxysmal nocturnal hemoglobinuria erythrocytes. J Clin Invest. 1985 Jun;75(6):2074–2084. [PMC free article] [PubMed]
  • Rosse WF, Parker CJ. Paroxysmal nocturnal haemoglobinuria. Clin Haematol. 1985 Feb;14(1):105–125. [PubMed]
  • Parker CJ, Stone OL, Bernshaw NJ. Characterization of the enhanced susceptibility of paroxysmal nocturnal hemoglobinuria erythrocytes to complement-mediated hemolysis initiated by cobra venom factor. J Immunol. 1989 Jan 1;142(1):208–216. [PubMed]
  • Nicholson-Weller A, March JP, Rosenfeld SI, Austen KF. Affected erythrocytes of patients with paroxysmal nocturnal hemoglobinuria are deficient in the complement regulatory protein, decay accelerating factor. Proc Natl Acad Sci U S A. 1983 Aug;80(16):5066–5070. [PubMed]
  • Pangburn MK, Schreiber RD, Müller-Eberhard HJ. Deficiency of an erythrocyte membrane protein with complement regulatory activity in paroxysmal nocturnal hemoglobinuria. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5430–5434. [PubMed]
  • Nicholson-Weller A, Burge J, Fearon DT, Weller PF, Austen KF. Isolation of a human erythrocyte membrane glycoprotein with decay-accelerating activity for C3 convertases of the complement system. J Immunol. 1982 Jul;129(1):184–189. [PubMed]
  • Medof ME, Gottlieb A, Kinoshita T, Hall S, Silber R, Nussenzweig V, Rosse WF. Relationship between decay accelerating factor deficiency, diminished acetylcholinesterase activity, and defective terminal complement pathway restriction in paroxysmal nocturnal hemoglobinuria erythrocytes. J Clin Invest. 1987 Jul;80(1):165–174. [PMC free article] [PubMed]
  • Kinoshita T, Rosenfeld SI, Nussenzweig V. A high m.w. form of decay-accelerating factor (DAF-2) exhibits size abnormalities in paroxysmal nocturnal hemoglobinuria erythrocytes. J Immunol. 1987 May 1;138(9):2994–2998. [PubMed]
  • Medof ME, Kinoshita T, Silber R, Nussenzweig V. Amelioration of lytic abnormalities of paroxysmal nocturnal hemoglobinuria with decay-accelerating factor. Proc Natl Acad Sci U S A. 1985 May;82(9):2980–2984. [PubMed]
  • Hänsch GM, Hammer CH, Vanguri P, Shin ML. Homologous species restriction in lysis of erythrocytes by terminal complement proteins. Proc Natl Acad Sci U S A. 1981 Aug;78(8):5118–5121. [PubMed]
  • Schönermark S, Rauterberg EW, Shin ML, Löke S, Roelcke D, Hänsch GM. Homologous species restriction in lysis of human erythrocytes: a membrane-derived protein with C8-binding capacity functions as an inhibitor. J Immunol. 1986 Mar 1;136(5):1772–1776. [PubMed]
  • Zalman LS, Wood LM, Frank MM, Müller-Eberhard HJ. Deficiency of the homologous restriction factor in paroxysmal nocturnal hemoglobinuria. J Exp Med. 1987 Feb 1;165(2):572–577. [PMC free article] [PubMed]
  • Hänsch GM, Schönermark S, Roelcke D. Paroxysmal nocturnal hemoglobinuria type III. Lack of an erythrocyte membrane protein restricting the lysis by C5b-9. J Clin Invest. 1987 Jul;80(1):7–12. [PMC free article] [PubMed]
  • Parker CJ, White VF, Falk RJ. Site-specific activation of the alternative pathway of complement. Synthesis of a hybrid molecule consisting of antibody and cobra venom factor. Complement. 1986;3(4):223–235. [PubMed]
  • Parker CJ, Baker PJ, Rosse WF. Increased enzymatic activity of the alternative pathway convertase when bound to the erythrocytes of paroxysmal nocturnal hemoglobinuria. J Clin Invest. 1982 Feb;69(2):337–346. [PMC free article] [PubMed]
  • Parker CJ, Soldato CM, Telen MJ. Increased efficiency of binding of nascent C3b to the erythrocytes of chronic cold agglutinin disease. J Clin Invest. 1984 Sep;74(3):1050–1062. [PMC free article] [PubMed]
  • Parker CJ, Baker PJ, Rosse WF. Comparison of binding characteristics of factors B and H to C3b on normal and paroxysmal nocturnal hemoglobinuria erythrocytes. J Immunol. 1983 Nov;131(5):2484–2489. [PubMed]
  • Parker CJ, Soldato CM, Rosse WF. Abnormality of glycophorin-alpha on paroxysmal nocturnal hemoglobinuria erythrocytes. J Clin Invest. 1984 Apr;73(4):1130–1143. [PMC free article] [PubMed]
  • Weber K, Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed]
  • Parker CJ, Frame RN, Elstad MR. Vitronectin (S protein) augments the functional activity of monocyte receptors for IgG and complement C3b. Blood. 1988 Jan;71(1):86–93. [PubMed]
  • Blake MS, Johnston KH, Russell-Jones GJ, Gotschlich EC. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem. 1984 Jan;136(1):175–179. [PubMed]
  • Hoffman EM. Inhibition of complement by a substance isolated from human erythrocytes. I. Extraction from human erythrocyte stromata. Immunochemistry. 1969 May;6(3):391–403. [PubMed]
  • Hoffmann EM. Inhibition of complement by a substance isolated from human erythrocytes. II. Studies on the site and mechanism of action. Immunochemistry. 1969 May;6(3):405–419. [PubMed]
  • Seya T, Farries T, Nickells M, Atkinson JP. Additional forms of human decay-accelerating factor (DAF). J Immunol. 1987 Aug 15;139(4):1260–1267. [PubMed]
  • Brauch H, Roelcke D, Rother U. Glycophorin A inhibits lysis by the complement attack phase. Immunobiology. 1983 Aug;165(2):115–120. [PubMed]
  • Sugita Y, Nakano Y, Tomita M. Isolation from human erythrocytes of a new membrane protein which inhibits the formation of complement transmembrane channels. J Biochem. 1988 Oct;104(4):633–637. [PubMed]
  • Stewart JL, Kolb WP, Sodetz JM. Evidence that C5b recognizes and mediates C8 incorporation into the cytolytic complex of complement. J Immunol. 1987 Sep 15;139(6):1960–1964. [PubMed]
  • Medof ME, Kinoshita T, Nussenzweig V. Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes. J Exp Med. 1984 Nov 1;160(5):1558–1578. [PMC free article] [PubMed]
  • Davitz MA, Low MG, Nussenzweig V. Release of decay-accelerating factor (DAF) from the cell membrane by phosphatidylinositol-specific phospholipase C (PIPLC). Selective modification of a complement regulatory protein. J Exp Med. 1986 May 1;163(5):1150–1161. [PMC free article] [PubMed]
  • Medof ME, Walter EI, Roberts WL, Haas R, Rosenberry TL. Decay accelerating factor of complement is anchored to cells by a C-terminal glycolipid. Biochemistry. 1986 Nov 4;25(22):6740–6747. [PubMed]
  • Hänsch GM, Weller PF, Nicholson-Weller A. Release of C8 binding protein (C8bp) from the cell membrane by phosphatidylinositol-specific phospholipase C. Blood. 1988 Sep;72(3):1089–1092. [PubMed]
  • Low MG, Saltiel AR. Structural and functional roles of glycosyl-phosphatidylinositol in membranes. Science. 1988 Jan 15;239(4837):268–275. [PubMed]
  • Selvaraj P, Dustin ML, Silber R, Low MG, Springer TA. Deficiency of lymphocyte function-associated antigen 3 (LFA-3) in paroxysmal nocturnal hemoglobinuria. Functional correlates and evidence for a phosphatidylinositol membrane anchor. J Exp Med. 1987 Oct 1;166(4):1011–1025. [PMC free article] [PubMed]
  • Burroughs SF, Devine DV, Browne G, Kaplan ME. The population of paroxysmal nocturnal hemoglobinuria neutrophils deficient in decay-accelerating factor is also deficient in alkaline phosphatase. Blood. 1988 Apr;71(4):1086–1089. [PubMed]
  • Haziot A, Chen S, Ferrero E, Low MG, Silber R, Goyert SM. The monocyte differentiation antigen, CD14, is anchored to the cell membrane by a phosphatidylinositol linkage. J Immunol. 1988 Jul 15;141(2):547–552. [PubMed]
  • Selvaraj P, Rosse WF, Silber R, Springer TA. The major Fc receptor in blood has a phosphatidylinositol anchor and is deficient in paroxysmal nocturnal haemoglobinuria. Nature. 1988 Jun 9;333(6173):565–567. [PubMed]
  • Nicholson-Weller A, March JP, Rosen CE, Spicer DB, Austen KF. Surface membrane expression by human blood leukocytes and platelets of decay-accelerating factor, a regulatory protein of the complement system. Blood. 1985 May;65(5):1237–1244. [PubMed]
  • Nicholson-Weller A, Spicer DB, Austen KF. Deficiency of the complement regulatory protein, "decay-accelerating factor," on membranes of granulocytes, monocytes, and platelets in paroxysmal nocturnal hemoglobinuria. N Engl J Med. 1985 Apr 25;312(17):1091–1097. [PubMed]
  • Kinoshita T, Medof ME, Silber R, Nussenzweig V. Distribution of decay-accelerating factor in the peripheral blood of normal individuals and patients with paroxysmal nocturnal hemoglobinuria. J Exp Med. 1985 Jul 1;162(1):75–92. [PMC free article] [PubMed]
  • Asch AS, Kinoshita T, Jaffe EA, Nussenzweig V. Decay-accelerating factor is present on cultured human umbilical vein endothelial cells. J Exp Med. 1986 Jan 1;163(1):221–226. [PMC free article] [PubMed]
  • Medof ME, Walter EI, Rutgers JL, Knowles DM, Nussenzweig V. Identification of the complement decay-accelerating factor (DAF) on epithelium and glandular cells and in body fluids. J Exp Med. 1987 Mar 1;165(3):848–864. [PMC free article] [PubMed]
  • Blaas P, Berger B, Weber S, Peter HH, Hänsch GM. Paroxysmal nocturnal hemoglobinuria. Enhanced stimulation of platelets by the terminal complement components is related to the lack of C8bp in the membrane. J Immunol. 1988 May 1;140(9):3045–3051. [PubMed]

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