Alternate Complement Pathway Induction of Aggregation and Release of 5-Hydroxytryptamine and Adenosine Diphosphate by Rabbit Platelets

Abstract

The present studies investigated patterns of rabbit platelet aggregation and release of 5-hydroxytryptamine (5HT) utilizing nine variables: three different types of challenge, soluble antigen and antibody (AG-AB), zymosan (Z), an agent known to activate the alternate complement pathway (ACP), and Z preincubated in lightly heparinized plasma so as to become coated with complement (ZC); three different types of platelet-rich plasma (PRP), lightly heparinized PRP in which both complement pathways are active, ethylene glycol tetraacetic acid-PRP (EGTA-PRP) in which only the ACP is active, and ethylene diamine tetraacetic acid PRP (EDTA-PRP), which inhibits both complement pathways; three different types of inhibitors, cobra venom factor (CoF), which causes activation of C3 proactivator (C3PA) to C3 activator (C3A) and fluid phase decomplementation of C3 and C5 through C9, adenosine monophosphate (AMP), a specific antagonist of ADP, and tosyl arginine methyl ester (TAME), an inhibitor thought to act not only on the first component of complement, but also on a platelet membrane site mediating complement-induced platelet injury as well as on C3PAse. In heparinized PRP, both AG-AB and Z produced biphasic aggregation and prompt and extensive 5HT release. A brief lag period noted with both AG-AB and Z challenge was not observed with ZC challenge, indicating that this lag period represented time required for generation of the necessary complement-dependent membrane-injuring activity. Prior decomplementation by CoF entirely prevented both aggregation and release by either AG-AB or Z but not by ZC, indicating first that fluid-phase ACP activation did not produce platelet injury, and second that ZC had on its surface an activity capable of producing immediate biphasic aggregation and prompt 5HT release without the further participation of later acting complement components. Both AMP and TAME eliminated the second phase of aggregation and diminished or eliminated 5HT release with all three challenges, suggesting that both inhibitors might be operative on similar or identical platelet membrane receptors mediating complement-dependent platelet injury. In EGTA-PRP, AG-AB and Z produced delayed monophasic aggregation and delayed and diminished 5HT release, whereas ZC produced immediate although monophasic aggregation but delayed and diminished 5HT release. This suggested that all three challenges were capable of producing ACP-mediated platelet injury. The effect of EGTA was attributed to two features of this chelating agent: a) provision of a concentration of ionized magnesium sufficient but suboptimal for ACP activation resulting in a delayed reaction: and b) provision of virtually no ionized calcium in which circumstance ADP-related second-phase aggregation and the concomitant more extensive 5HT release was inhibited. In EDTA-PRP, only ZC produced aggregation, which was immediate in onset but monophasic and was not associated with 5HT release. The data are interpreted as suggesting two operationally distinct ACP-dependent platelet 5HT release mechanisms. One is brisk, dependent on both ionized calcium and ionized magnesium, and partially inhibited by both AMP and TAME; the second is operative only under the artifical situation of chelation with EGTA, slower, independent of ionized calcium, and unaltered by AMP or TAME.