Pexelizumab fails to inhibit assembly of the terminal complement complex in patients with ST-elevation myocardial infarction undergoing primary percutaneous coronary intervention. Insight from a substudy of the Assessment of Pexelizumab in Acute Myocardial Infarction (APEX-AMI) trial.

BACKGROUND: Reasons for pexelizumab lack of benefit in ST-elevation myocardial infarction patients undergoing primary percutaneous coronary intervention remain unclear. In a substudy of the APEX-AMI trial, we explored the hypothesis that early complement activation preceding drug administration explained the failure. METHODS: A panel of terminal complement complex proteins and fragments and biomarkers of inflammation, apoptosis, and high-risk features were assessed in serum obtained before and 24 hours after administration of placebo or pexelizumab and primary percutaneous coronary intervention (n = 356) and in human umbilical vein endothelial cell cultures coincubated with serum (n = 45). RESULTS: In the placebo group, C5a and sC5b-9 levels increased by 37% (7.9-14.2 ηg/mL, P = .007) and 96% (442-845 ηg/mL, P < .0001), respectively, during the first 24 hours. Pexelizumab prevented the increase in C5a (P = .01 vs placebo), but not that of sC5b-9 (502-1,157 ηg/mL, not significant vs placebo). Levels of C-reactive protein, interleukin (IL) 6, IL-1ß, Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES) or Chemokine C-C motif ligand 5 (CCL5), and N-terminal probrain natriuretic peptide increased significantly in both groups; those of IL-10, IL-12, IL-1ra, and Interferon gamma-induced protein 10 (IP-10) or C-X-C motif chemokine 10 (CXCL10) decreased. Pexelizumab halved the increase in IL-6 (+92% vs 156%, P = .01) without effects on other markers, including C-reactive protein and N-terminal probrain natriuretic peptide. In cell culture, pexelizumab inhibited C5a, sC5b-9, and membrane-bound C5b-9 by 92%, 75%, and 78%, respectively (all P < .0001), without influencing cytokine levels and cell apoptosis. CONCLUSIONS: The blockage of both C5a and terminal complement in cell culture, but of C5a only in vivo with minimal effects on inflammation and risk biomarkers, supports the hypothesis that late administration of pexelizumab after the ischemia/reperfusion insult precluded adequate myocardial protection, resulting in a negative trial.

Requirements for membrane attack complex formation and anaphylatoxins binding to collagen-activated platelets.

BACKGROUND: The activation of complement during platelet activation is incompletely understood. OBJECTIVES: We sought to explore the formation of C5b-9 and anaphylatoxins binding to collagen-activated platelets. METHODS: C5b-9, anaphylatoxins C3a, C4a and C5a, and anaphylatoxin receptors C3aR1 and C5aR were measured by flow cytometry and/or confocal microscopy. Platelet microparticles were quantified by flow cytometry, and their C5b-9 content was determined by western blot analyses. In all experiments, sodium citrate was used for blood anticoagulation. RESULTS: C5b-9 rapidly formed on the platelet surface following activation with collagen, TRAP, ADP or A23187, but was surprisingly restricted to a subset of platelets (1 to 15%) independently of P-selectin or phosphatidylserine exposure. Following collagen activation, C5b-9-positive platelets in thrombi were found associated with collagen fibres. C5b-9 formation was obliterated by Mg(2+)-EGTA and significantly reduced by the thrombin inhibitor hirudin (-37%, p<0.05), but was unaffected by chondroitinase, compstatin, SCH79797 (PAR-1 inhibitor), or in the PRP of a MBL-deficient donor. Compstatin and Mg(2+)-EGTA, but not hirudin, SCH79797 or chondroitinase, inhibited the formation of collagen-induced microparticles (-71% and -44%, respectively, p<0.04). These microparticles contained greater amounts of C5b-9 compared with the other agonists. Platelet activation by collagen or convulxin resulted in the strong binding of anaphylatoxins and the exposure of receptors C3aR1 and C5aR (CD88) on their surface. CONCLUSIONS: C5b-9 formation on collagen-activated platelets is i) partially controlled by thrombin, ii) restricted to a subset of platelets, and iii) can occur without P-selectin expression or phosphatidylserine exposure. Activated platelets bind anaphylatoxins on their surface and express C3a and C5a receptors, which may contribute to the localization of inflammatory processes during thrombosis.

Matching the evaluation of the clinical efficacy of clopidogrel to platelet function tests relevant to the biological properties of the drug.

OBJECTIVES: This study aimed to explore platelet function tests relevant to the biological effects of clopidogrel that could help the clinical monitoring of drug efficacy. BACKGROUND: Clopidogrel selectively inhibits the P2Y12 receptor, the major role of which is stabilization of aggregation, whereas initiation of aggregation depends on activity of both P2Y1 and P2Y12 receptors. METHODS: Tests used were peak aggregation (Agg(max)) and late aggregation (Agg(6min)), and disaggregation, relating to P2Y1 and P2Y12 activity, respectively; and monoclonal antibody binding activated glycoprotein (GP) IIb/IIIa receptors (PAC-1) and P-selectin, measuring activation and secretion. A first study compared hirudin/PPACK (r-hirudin and D-phenylalanyl-prolyl-arginine chloromethyl ketone) with citrate as blood anticoagulant (16 patients), and a second control study compared the effects of clopidogrel, aspirin, or both (20 normal controls). RESULTS: Clopidogrel similarly inhibited adenosine 5'-diphosphate (ADP)-induced Agg(max) with either anticoagulant, but significantly more Agg(6min) (75% vs. 31%), P-selectin (72% vs. 53%), and PAC-1 (62% vs. 24%) in hirudin/PPACK. In the control study, it inhibited Agg(max) by 22%, and Agg(6min), P-selectin, and PAC-1, by 69%, 66%, and 55%, respectively (all p < 0.05). Disaggregation at six min reached 62% with clopidogrel, but was virtually absent with placebo and aspirin. Non-responsiveness as evaluated by inhibition of Agg(max) in citrate was diagnosed in 35% of patients; in half this rate by Agg(6min), P-selectin, and PAC-1; and in 6% to 12% with the latter tests performed in hirudin/PPACK. CONCLUSIONS: The evaluation of clopidogrel responsiveness by platelet function tests is largely influenced by the choice of blood preservative and functional tests. Measures of aggregation stabilization, and of consequent secretion and activation, identified most patients as responders, contrasting with measures of peak aggregation, by likely reflecting better the interactions clopidogrel and the P2Y12 receptor.