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The interaction of guinea pig or rat serum with Z at 0 degrees C leads to the formation of properdin pathway intermediate, ZPI, whose activity is assessed by its capacity to deplete the titer of cobra venom inducible lysis in diluted rat serum. The formation of ZPI does not require C1 or C2 and is not diminished by prior absorption of the serum with Z. In contrast, removal of P suppresses ZPI formation. Both Ca++ and Mg++ are essential cofactors for the formation of this intermediate whose function is abrogated in the presence of anti-C3, anti-P, but not by anti-Factor B. Since C4-deficient guinea pig serum is also effective in ZPI formation, the data suggest that ZPI is an alternative pathway intermediate containing both P and C3.

Mg++, Mn++ and Co++ activate the C system as judged by the reduction in cobra venom factor indeed hemolysis, and cleavage of properdin factor B and C3. The maximum effect requires the addition of 5 to 10 mM of these cations to dialyzed sera respond in similar fashion indicating that these divalent cations can trigger the alternative pathway without the addition other activating agents.