Identification of proteins adsorbed to hemodialyser membranes from heparinized plasma

During hemodialysis, cardiopulmonary decompensation may appear in uremic patients, possibly caused by plugging of pulmonary vessels by leukocytes. In 34 patients we noted leukopenia (20% of initial levels) during hemodialysis that in 15 was associated with impaired pulmonary function. When we infused autologous plasma, incubated with dialyzer cellophane, into rabbits and sheep, sudden leukopenia and hypoxia occurred, with doubling of pulmonary-artery pressures and quintupling of pulmonary-lymph effluent. Histologic examination showed severe pulmonary-vessel-leukostasis and interstitial edema. The syndrome was prevented by preinactivation of complement but was reproduced by infusions of plasma in which complement was activated by zymosan. Thus, acute pulmonary dysfunction from complement-mediated leukostasis may play a major part in the acute cardiopulmonary complications of cellophane-membrane hemodialysis.

The complement protein C3, when activated by limited proteolysis, forms a short-lived reactive intermediate fragment, 'nascent' C3b, which is known to bind covalently to certain surfaces. The characteristics of the covalent binding reaction have been studied by using Sepharose-trypsin as a combined proteolytic activator and binding surface for C3. Binding of C3 to Sepharose-trypsin is saturable, with a maximum of 25-26 molecules of C3b bound per molecule of trypsin. A minimum life-time of about 60 microseconds for the reactive intermediate has been calculated from binding of C3 at saturation. Initial binding efficiencies of over 30% can be obtained at physiological pH and ionic strength. The efficiency of C3 binding to Sepharose-trypsin decreases as pH increases and also shows a slight decline at high ionic strength. The covalent binding of C3 to Sepharose-trypsin can be inhibited by a range of oxygen and nitrogen nucleophiles. Activation of C3 in the presence of radioactive forms of four such nucleophiles, phenylhydrazine, methylamine, glycerol and glucosamine results in apparent covalent incorporation of the nucleophile into the C3d fragment of C3. The quantity of radioactive nucleophile bound can be predicted from the observed potency of the nucleophile as an inhibitor of the binding of C3 to Sepharose-trypsin. The radioactive nucleophiles may be considered as 'active-site' labels for C3.