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C1 inhibitor (C1-INH) acts to inhibit active enzymes of both the classical complement and Hageman factor-dependent pathways. Previously reported C1-INH purification procedures were multistep and most have been associated with significant loss in specific functional activity. We have developed a simple chromatographic procedure which yields a pure C1-INH protein from normal human plasma with a specific activity equal to or greater than the starting sample. Briefly, protease inhibitor-treated, pooled human citrated plasma was fractionated with polyethylene glycol (PEG 4000); the supernatant fraction that remained soluble at 16% was obtained. The inhibitor was precipitated with 45% PEG. The resulting precipitate was solubilized and chromatographed on DEAE Sephacel using a linear salt gradient. The eluted fractions containing the C1-INH and other contaminants were pooled and dialyzed against the starting buffer of the next chromatographic step. A unique separation procedure using zinc ion chelate-coupled agarose was employed as the second chromatographic step. The eluted C1-INH, after zinc ion chromatography, displayed a significant enhancement in purity and maintained a specific functional activity twice that of plasma. The final procedure utilized immunoadsorption chromatography using an anti-contaminant column. Under reducing conditions on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the purified C1-INH migrated as a single band with an apparent molecular weight of 90,000-105,000, but under non-reducing conditions, a doublet with apparent molecular weights of 94,000-100,000 and 85,000-93,000 was seen. C1-INH antigenic concentrations were measured and shown to be correlated in serum, citrate plasma, and EDTA plasma from 16 normal subjects.

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An immunodiffusion assay for detecting C1 inhibitor function in human serum was described recently by Ziccardi and Cooper. In our present study, the applicability of this assay for C1 inhibitor deficiency or C1 inhibitor dysfunction was evaluated. Of the 39 patients evaluated, all eight patients with the common (C1 inhibitor deficiency) form of hereditary angioedema and all three patients with the variant (dysfunctional C1 inhibitor) form of hereditary angioedema were identified correctly. Treatment of patients with hereditary angioedema with stanozolol or danocrine increased their serum C1 inhibitor concentrations and normalized the immunodiffusion assay for C1 inhibitor function. In addition, the assay allowed the correct identification of three patients with the acquired form of C1 inhibitor deficiency, because the sera of these patients exhibited a distinctive pattern. The 25 samples from patients (chronic angioedema, chronic urticaria, or hypocomplementemic vasculitis) without C1 inhibitor deficiency had normal assays.

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Patients with hereditary angioedema lack C-1 inhibitor, a plasma alpha 2-glycoprotein that inhibits both the proteolytic action of C1, the activated first component of the complement system, and the activity of components of the contact phase of coagulation: kallikrein, factor XIa, and factor XIIa. Such patients have been shown to have low levels of C4 and C2, the natural substrates for C-1, but the levels were not correlated with the presence of symptoms. We studied three patients with angioedema for evidence of activation of the contact system and found that during a symptomatic period they had decreased levels of prekallikrein, a substrate for the activated forms of factor XII, and reductions in high-molecular-weight kininogen, a substrate for plasma kallikrein. These observations suggest that zymogens of the contact system are activated during attacks of hereditary angioedema and that some of the clinical manifestations may be mediated through products of this pathway, such as kinins.

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Six suction-induced blister fluids obtained from five patients with hereditary angioedema (HAE) contained active kallikrein, whereas only two blister fluids obtained from eight normal volunteers contained small amounts of this activity. Kallikrein was present in large amounts of HAE blister fluids as assessed by its ability to liberate smooth-muscle-contracting activity from purified high molecular weight kininogen. It was inhibited by purified antibodies specific for plasma prekallikrein and also by purified C1 inhibitor, but not by antibodies specific for C1s. These observations suggest that activation of the Hageman-factor-dependent pathways occurs in the tissues of HAE patients, and once generated, active kallikrein persists in these tissues.

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