ENDOTHELIAL CELLS PRODUCE PLATELET ACTIVATING FACTOR WHICH PRIMES NEUTROPHILS TO RELEASE OXIDANT PRODUCTS

Thrombin (THR) is generated during ARDS, sepsis and DIC. We wondered whether it might augment PMN/endothelial cell (EC) interaction and hence amplify EC damage by inducing platelet activating factor (PAF) (JCI 76:2235-2246;1985). To examine further this premise and the mechanisms involved, we measured intracel1ular calcium (Ca¹) in human EC (grown on glass cover slips), in a scanning spectrof1uorometer at 37°C after loading with FURA 2 (4μM). Resting Ca¹ was 148±22 nM (Mean±SEM) which increased following THR 0.5u/ml to 458±160 nM at 30s, peaking after 1 min at 559±176 nM, and returning to 273±42 nM by 5 min. Phosphatidyl inositol (PI) turnover was assessed in ³H-myoinositol-loaded EC using water-soluble extracts separated by Dowex anion exchange chromatography. Within 30s of THR (lu/ml) stimulation, PI turnover markedly increased, with production of inositol bi- and tri-phosphates showing a >5 fold rise. Associated with these perturbations, THR-treated EC monolayers enhanced O^2- generation by FMLP(10^™7)-stimulated PMNs (basal levels of 5.73±0.68 O^2-/15 min/10⁶ PMN rising to 8.01±0.85 nM (p<0.05)); moreover this enhancement could be completely inhibited with a newly described PAF antagonist BN 52021. PAF production is dependent upon phosphorylation of an acetyl transferase, and Ca¹ flux and PI hydrolysis are events known to be associated with protein kinase activation. THR-EC stimulation would seem, therefore, to initiate a sequence of events involving PMN/EC 'cross-talk' leading to contact activation of marginated PMNs by EC-derived PAF. This is an example of a novel paracrine response, and is consistent with our data showing the potent priming effect of PAF on PMN oxidant responses (Blood 68:88A; 1986) and provides evidence for a previously unsuspected pathway that promotes PMN oxidant-mediated EC injury during sepsis or other THR-generating disorders.