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Conflicting reports exist in the literature concerning the primary mechanism for heparin's inhibition of C1 hemolytic function. We provide evidence here that heparin's most effective inhibition is mediated through interaction with and potentiation of C1-INH. The molecular nature of the interaction between heparin and the C1-INH molecule is evidenced by an anodal shift in the electrophoretic mobility of C1-INH upon the addition of heparin.l We also demonstrate that C3 and C4 conversions in normal human serum by classical pathway activators such as heat-aggregated IgG are inhibited by addition of heparin, yet heparin does not prevent similar C3 conversion in serum depleted of C1-INH Heparin's inhibitory action is restored to C1-INH-depleted serum by the addition of purified C1-INH.

The mechanism by which heparin inhibits the alternative complement pathway (ACP) by a fluid-phase activator, CoVF, has been studied. Results presented here indicate that heparin's major (rate-limiting) effect on the fluid-phase activation of the ACP was to potentiate Factor H activity. Such an effect results in a very efficient inhibition of C3b and C3bBb function and restriction of subsequent complement activation and hemolytic activity. Evidence was obtained to indicate that soluble heparin H shifted anodally. Assuming that the rate-limiting inhibitory effect of heparin is to potentiate Factor H, then C3-converting complexes such as CoVF-Bb, which do not require C3b for activity, should not be effected by heparin. Indeed, the inhibitory effect of heparin on C3 conversion in EGTA-Mg2+ serum-CoVF mixtures was lost with a prolonged incubation time (i.e. 60-90 min at 37 C). This finding indicated that with time ACP-mediated cleavage of C3 was able to bypass the heparin-mediated inhibitory step. From these studies it is suggested that heparin restricts the C3-converting activity due to soluble C3bBb complexes but not the C3 conversion due to CoVF-Bb complexes. Heparin-mediated restriction of the ACP activation by CoVF was used to calculate the relative percentages of C3 conversion due to C3bBb or CoVF-Bb complexes. In carefully controlled experiments, heparin could not prevent the spontaneous conversion of C3 which occurs upon removing functional Factor H from the sera. Addition of isolated Factor H restored heparin's inhibitory effect on the ACP. Kinetic studies of heparin's inhibition of ACP-mediated lysis of rabbit erythrocytes indicated that heparin's inhibitor functions did not occur until after the addition of an ACP activator. Each of these findings is consistent with the postulate that the major (rate-limiting) effect of heparin on the ACP is to potentiate the function of Factor H on activated C3b.

Acidic proline-rich salivary proteins, PRPI, PRPII, PRPIII, PRPIV, upper Db and Statherin, were isolated from parotid saliva and tested for interaction with complement. It was determined that each of the isolated proline-rich proteins blocked C1 hemolytic activity in assay systems where C1 was rate-limiting. Further studies comparing the specific activity of the proline-rich proteins to unfractionated parotid saliva indicated that other salivary substances (sensitive to urea treatment of parotid saliva) were also interacting with the first complement component but in a time-dependent manner. Based on a series of experiments examining the effect of the sequence of addition of the proline-rich proteins in the complement assay systems, it is postulated that these salivary proteins are able to block the proper interaction of C1 with EAC4 cells (sheep erythrocytes coated with antibody and C4gp). The proline-rich salivary proteins had no effect on C1 once C1 was bound to the immune complexes on the EAC4 cells. The C1 macromolecular complex undergoes conformational changes upon interaction with immune complexes resulting in a more avid binding of the C1q-Clr-Cls subunits with one another. Thus it is speculated that the EAC4-bound C1 becomes resistant to disruption by the proline-rich salivary proteins. Although the urea-sensitive factors had the highest specific C1-fixing activity, the activity of the acidic proline-rich proteins on C1 is important because of their relatively high concern in salivary secretions. Since complement-containing serous exudates and transudates are present on inflamed mucosal tissues, salivary substances which interact with C1 may play a role in regulating the initiation of the classical complement pathway, particularly at those mucosal sites where there is a high ratio of salivary secretion to serous exudate.