Role of Platelet Purinergic Receptors in LPS induced pulmonary inflammation

Rationale: Platelets express P2Y1, P2Y12 and P2X1 receptors, which are activated by ADP (P2Y1 & P2Y12), and ATP (P2X1) respectively (1, 2). Recent work has described a requirement for platelet-leukocyte complexes in driving leukocyte recruitment to inflammatory tissues which is mediated by P2Y1 receptors in allergic inflammation (3). Furthermore, platelets have been demonstrated to be necessary for pulmonary leukocyte recruitment after LPS challenge (4). Using the specific purinergic receptor antagonists MRS2500 and AR-C66096 (P2Y1 and P2Y12 respectively) the role of these receptors in LPS induced pulmonary inflammation were investigated.

Methods: Balb/c mice were pretreated with MRS2500 or AR-C66096 (3mg/kg i.v.) 30 minutes prior to provocation with 200µg/ml LPS i.n. Mice were culled 4 hours post LPS challenge blood collected for circulating platelet counts and bronchoalveolar lavage performed for inflammatory cell enumeration.

Results: LPS challenge induced a significant increase in both total leukocyte counts (Sham: 20.93 ± 3.99x104 cells/ml vs. LPS: 63.14 ± 8.86x104 cells/ml, P < 0.001) and PMN counts (Sham: 0.13±0.06x104 cells/ml vs. LPS: 45.49 ± 7.52x104 cells/ml, P < 0.001). This LPS induced increase was reduced in animals pre-treated with the 3mg/kg of the P2Y1 antagonist MRS2500 (Total cells: 36.75 ± 4.66; PMNs: 24.01 ± 3.60, P < 0.01) but not in animals pre-treated with 3mg/kg of the P2Y12 receptor antagonist AR-C66096 where cell numbers remained significantly elevated compared to sham levels (P < 0.001) (Total Cells: 67.14 ± 8.06x104 cells/ml; PMNs: 49.16 ± 4.49x104 cells/ml). Furthermore, circulating platelet levels were quantified post LPS challenge. Here we observed a mild systemic thrombocytopenia following LPS challenge (Sham: 1.13 ± 0.04x109 platelets/ml vs. LPS: 0.821 ± 0.04x109 platelets/ml, P < 0.001. Pretreatment with 3mg/kg MRS2500 attenuated the LPS induced decrease in circulating platelets (MRS2500: 0.99 ± 0.04x109 platelets/ml). In contrast despite pre-treatment with 3mg/kg AR-C66096, a mild systemic state of thrombocytopenia remained (AR-C66096: 0.83 ± 0.04x109 platelets/ml, P < 0.001).

Conclusions: This data reveals that P2Y1 receptor plays an important role in pulmonary leukocyte recruitment and platelet dynamics in a model of LPS induced pulmonary leukocyte recruitment, whilst the P2Y12 receptor appears to have no significant role following LPS challenge. This is in agreement with studies performed in a murine model of allergic inflammation suggesting a consistent mechanism of platelet interactions with leukocytes in inflammatory conditions (3,5). Furthermore, our data demonstrates a mild systemic thrombocytopenia following LPS challenge which is consistent with similar work performed in allergic studies. The reversal of the mild systemic thrombocytopenia following pretreatment with the P2Y1 receptor antagonist MRS2500 but not the P2Y12 receptor antagonist AR-C66096 suggests that platelet recruitment to the pulmonary architecture may also be regulated by the P2Y1 receptor in a similar manner to platelet-induced leukocyte recruitment.

References:

1. Nylander, S. et al. (2003) Thromb Res. 111, 65–73

2. Dovlatova, N. et al. (2008): Thromb Haemost. 100(2), 261-70

3. Amison, R. et al. (2015): J. Allergy Clin. Immunol. 135(2), 528-538

4. Pan, D. et al. (2015): Blood. 125(7), 1146-58

5. Pitchford, S. et al. (2008): Am. J. Hosp. Crit. Care. Med. 177(6), 604-612