Activation of the early components of the classical complement pathway under physiologic conditions.

Abstract

Abstract We studied the early events in activation of the classical complement (C) pathway by treating normal human serum (NHS) with different C activators and measuring the following parameters as a function of time: C1 activation, C2 consumption, C4 consumption, and C1 inactivation. C1 activation and inactivatidn were quantitated with SDS-PAGE radioassays, whereas C2 and C4 consumption were measured by hemolytic titration. In undiluted NHS at 37°C, C1 activation as well as C2 and C4 consumption were turned off within 3 min after the addition of limiting quantities of activator. The binding of C1-inactivator (C1-ln) to C1 was so rapid that no free C1 was detected at any time during the reaction. Direct binding studies with purified proteins revealed that, under physiologic conditions, 50% of the C1 bound to C1-In within 13 sec and 90% within 55 sec of activation. Thus, the activation of C1 is the rate limiting step for this 3-min reaction sequence. C1 turned over a surprisingly low number of C2 and C4 molecules in NHS. This was due to the fact that C1 was so rapidly inactivated by C1-ln. An average of only 4 C2 molecules and 35 C4 molecules Were activated per C1 molecule when soluble agglgG, tetanus-anti-tetanus precipitate, EAIgG, or EAIgM were added to NHS. The nonimmune activator LPS R595 differed from these immune activators in that it induced the activation of 5 times less C2 per C1. The Michaelis-Menten constant (0.96 × 10−6 M) and turnover number (303 C4/C1/min) were calculated for the activation of purified C4 by purified C1. These values are consistent with the observed turnover of C4 in NHS when the short half-life of C1 is taken into account. The early classical pathway was reconstituted from purified C1q, C1r, C1s, C1-ln, C2, and C4, all at physiologic concentrations. When treated with agglgG, this purified component mixture and NHS were indistinguishable with respect to consumption of C2 and C4. Thus, C1-In is the only serum factor involved in the control of C1 activities. Our results together demonstrate that the control protein C1-In is extraordinarily efficient.