Interactions of dual antiplatelet and anticoagulant with C1q and FH regulate complement activation by injured cells.

The dual antiplatelet and anticoagulant (APAC), a heparin proteoglycan mimetic, targets vascular injury sites and alleviates thromboinflammation in models of atherosclerosis and acute ischemic reperfusion injury. Here, we establish that APAC also modulates the complement system activation. Complement activity was assessed by pathway-specific enzyme-linked immunosorbent assays. APAC-anticoagulated blood was assessed by intrinsic coagulation pathway (intrinsic pathway-activated rotational thromboelastometry [InTEM]), and APAC-plasma by prothrombin fragments 1 and 2. Interactions of APAC-biotin with serum complement components were detected by pulldown and liquid chromatography-mass spectrometry analysis. The surface effect of APAC was studied with zymosan and on THP-1 cell line-derived apoptotic and necrotic cells. Comparisons were made to unfractionated heparin (UFH). Both serum spike-in and donor APAC-plasma studies showed that APAC reduces systemic complement activity similarly to UFH, except for the classical pathway, which APAC inhibited more prominently. The effective concentration range of APAC was 30 to 100 μg/mL. In APAC-plasma, InTEM was inhibited and thrombin generation was delayed at >30 μg/mL of APAC. In APAC pulldown and in vitro complement activation tests, APAC specifically interacted with necrotic cells and recruited C1q and factor H (FH) onto the cell surfaces. Importantly, APAC promoted early complement activation at the C3 level on cell surfaces, without proportional terminal pathway (C5b-9) activation. APAC interacts with several complement components without adversely affecting systemic complement activity within the expected therapeutic range. APAC may regulate complement activation on cell surfaces even at subinhibitory concentrations, promoting early complement activation via C1q but limiting it to the level of C3 by recruiting FH.