Regulation of platelet and neutrophil interaction in the formation of DNA extracellular traps

CARESTIA A; RIVADENEYRA L; D'ATRI LP; NEGROTTO S; SCHATTNER M

Congreso; 60th Annual Meeting of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis; 2014

Neutrophils secrete DNA extracellular traps (NETs) that kill bacteria and also exert proinflammatory and prothrombotic activities. Through the interaction with neutrophils, platelets appear to be key elements in NET formation. Considering that hyperthermia and acidosis are characteristic features of inflammation, NET generation in the presence of platelets was evaluated under these conditions. We also analyzed the effect of endothelial-derived platelet inhibitors prostacyclin (PGI2) and nitric oxide (NO), as well as the anti-inflammatory acetyl salicylic acid (ASA). Washed platelets (WP) and polymorphonuclear leukocytes (PMN) were isolated from peripheral blood of normal donors. WP were stimulated with either Gram negative (LPS 2ug/ml) or Gram positive (Pam(3)CSK(4) (Pam 1ng/ml)) bacteria wall components and then cultured with PMN at 37, 40 and 42ºC and at pH 7.4, 7 or 6.5. Confocal microscopy studies and quantification of free DNA showed that platelets potentiated NET formation triggered by LPS or Pam (PMN:0.2±0.02; PMN+WP:0.2±0.02; PMN+LPS:0.4±0.05; PMN+WP+LPS:0.8±0.1*; PMN+Pam:0.3±0.05; PMN+WP+Pam:0.6±0.1*; ug/ml of DNA X±SEM, n=3. NET generation was augmented by hyperthermia and decreased by acidosis (Figure), and under both conditions, the effects were compensated, reaching basal values. NETs were inhibited when platelets were preincubated with PGI2 (3 nM), SNP (0.1 mM) or ASA (1mM), (PGI2:76 and 65%, SNP: 95 and 82% and ASA:87 and 35%of inhibition for LPS and Pam, respectively). In conclusion our data show that hyperthermia and acidosis exert an opposite regulation on NET generation, although the effect is counterbalanced under both conditions. The inhibition of platelets could be a possible target for avoiding the exacerbated NET formation.