PB0739 - Interaction of Antiplatelet and Anticoagulant APAC with Complement Proteins Attenuates Complement Activation via C1q and Factor H

Background: Antiplatelet, anticoagulant APAC, conjugate of human albumin and unfractionated heparin (UFH) differs from UFH as superior antithrombotic in vascular injury models. APAC targets vascular injury sites by interacting with extracellular proteins. Unlike UFH, APAC inhibits collagen- and thrombin-induced platelet activity and is a stronger anticoagulant. Heparin and heparan sulphates interact with complement system prominently involved in injury responses, by inducing apoptosis, promoting local inflammation, and recruiting inflammatory cells to the injury site.

Aims: Our aim was to compare the effects of APAC with UFH in complement activation and its regulation in solution and on cell surfaces.

Methods: Pathway-specific functional assays were used to determine complement activities in plasma. Binding of complement regulators C1q and factor H (FH) to APAC- and UFH-coated on polystyrene surfaces was analyzed by ELISAs. Interactive partners were evaluated by serum pulldowns with biotinylated APAC, with follow-up western-blot detection of C1q and FH. APAC interaction and inhibitory capability on cell surfaces were assessed with activation assays with zymosan and cells.

Results: APAC (1-30 µg/mL) is more potent (1.3-3-fold) anticoagulant than UFH in plasma and whole blood. In comparison to UFH, APAC (100-300 µg/mL) potently inhibits classical (CP) and alternative (AP) pathways. Serum pulldown showed selective binding between APAC and CP initiator C1q and AP regulator FH. On APAC-coated surfaces, C1q and FH have detectable binding already at 0.01% serum dilution, whereas 1% serum is needed for binding to UFH-coating. APAC binds to necrotic cells, and at the level of C5b-9 activation, APAC decreases complement activation on zymosan up to 90%.

Conclusion(s): Overall, APAC may modulate fluid phase and surface-localized complement activation through combined effects of regulators C1q and FH. Compared to UFH, APAC has more potential to regulate the complement system. As a net effect on APAC targeted sites inflammatory response may be reduced and phagocytic clearance of debris promoted.