Full Length ArticleA dichotomy in platelet activation: Evidence of different functional platelet responses to inflammatory versus haemostatic stimuli
Introduction
The central role of platelets in haemostasis is widely established and takes place through a series of specialized functions. Under flow conditions at sites of vascular damage platelets become exposed to subendothelial-bound von-Willebrand factor (VWF) and collagen. This induces platelet adhesion and activation which triggers further platelet recruitment and platelet-platelet interactions through the secretion of platelet agonists, including ADP, and the synthesis and release of thromboxane A2 (TxA2) [1]. However, platelet activation also occurs in a range of inflammatory and auto-immune disorders, including rheumatoid arthritis [2], osteoarthritis [3], and airway diseases such as asthma [4,5], acute lung injury (ALI), acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary disease (COPD) [6], although not necessarily involving classical platelet aggregation or secretion. In animal models evidence has been provided for a critical role of platelets in the recruitment of leukocytes to inflamed tissues in response to allergic and non-allergic inflammatory insults [[7], [8], [9], [10]]. Additional evidence of platelet activation in inflammatory disorders is supported by the detection of circulating platelet-leukocyte complexes in patients with allergic asthma during allergen-triggered airways obstruction, coupled with increased blood levels of the platelet-derived pro-inflammatory mediators β-thromboglobulin (β-TG), platelet activating factor (PAF), platelet factor 4 (PF-4) and RANTES [11,12]. Platelets have also been shown to migrate towards inflammatory stimuli, both in vitro and in vivo [[13], [14], [15], [16]]. However, platelets that had migrated extravascularly into tissues did not show the ultrastructural features of activation and degranulation, suggesting that classical platelet activation is not required for platelet migration [17]. Moreover, platelet migration in response to allergen in sensitized mice and humans is mediated by the binding of allergen-bound IgE to contiguous FcεRI receptors inducing their cross-linking, a phenomenon previously shown to trigger other inflammatory platelet responses, such as cytotoxicity due to the release of oxygen free radicals [18] and chemokine release [19].
Whilst the mechanisms of platelet activation in response to inflammatory stimuli remain poorly defined, recent research has highlighted the role of the small Rho GTPase RhoA and Rac-GEFs (P-Rex and Vav) in platelet dependent leukocyte recruitment, pathways only minimally involved in haemostasis [9,20]. Furthermore, distinct non-canonical (RhoA, Rac-1) signalling events downstream of platelet P2Y1 receptor activation by ADP have been shown to control platelet motility, and interactions with neutrophils, in contrast to ADP-induced aggregation and the canonical P2Y1 receptor PLC signalling cascade [21]. This functional selectivity in signalling reveals the importance of different molecular signalling pathways of platelet activation during functional activities pertinent to inflammation compared to haemostasis [21]. Stimulation of platelets with either inflammatory or haemostatic stimuli also appears to induce different patterns of phosphorylation of intracellular signalling kinases, including Akt, ERK1, ERK2 and p38 [22]. Moreover, platelets have been shown to be critical in the progression of inflammation in experimental models of arthritis by mechanisms independent from thromboxane and ADP receptors, and from aggregation, suggesting that the pathways involved may be distinct [2].
Platelets contain a range of biologically-active molecules in their α-granules and recent evidence suggests that different subsets of α-granules, differentially released depending on the stimulus, are present in platelets [23]. In vivo studies have also supported the hypothesis that the pro-inflammatory role of platelets is distinct from the role of platelets in haemostasis, as platelet-dependent inflammatory cell recruitment may be inhibited by drugs not influencing platelet aggregation [9,10].
These observations, describing a divergence in platelet functions, support the hypothesis of the existence of a 'dichotomy in platelet activation' originally proposed by one of us nearly two decades ago [24], with the triggering of different platelet functions depending on the inflammatory versus haemostatic nature of the stimulus.
The aim of the present study was therefore to systematically evaluate in vitro the effect of a panel of either agonists classically associated with haemostatic processes (ADP, collagen [Coll], convulxin [CVX], epinephrine [Epi], TRAP-6 and the TxA2 analogue U46619) or inflammatory stimuli (fMLP, histamine [Hist], interleukin-1β [IL-1β], lipopolysaccharide [LPS], macrophage-derived chemokine-[MDC/CCL22], stromal cell-derived factor-1α-[SDF-1α/CXCL12] and serotonin [5-HT]) on a series of primarily haemostatic or inflammatory functions of platelets.
Section snippets
Materials and methods
Detailed material and methods pertaining to the measurement of platelet-granulocyte, and platelet-monocyte complex formation, and platelet ultrapurification are described in the online supplementary methods.
Haemostatic, but not inflammatory, stimuli induce platelet aggregation
The aim of this study was to compare platelet responses to haemostatic versus inflammatory agonists, to examine if a different platelet response exists depending on the kind of stimulation. To test the functional effects of haemostatic versus inflammatory stimulation, we performed aggregation studies with washed platelets. After 10 min, all haemostatic stimuli tested (ADP, Coll, CVX, Epi, TRAP-6 and U46619) elicited concentration-dependent, and significant, platelet aggregation, with a variable
Discussion
This study was conducted to further investigate whether platelets have a different functional response depending on the nature of the activating stimuli, either haemostatic or inflammatory.
Our results support the existance of a 'dichotomy in platelet function', confirming a hypothesis previously formulated [24]. Using three different in vitro platelet function assays exploring the haemostatic and inflammatory activities of platelets we observed clear differences in the triggered platelet
Conclusions
The current results provide evidence for a different functional response of platelets towards haemostatic versus inflammatory stimuli supporting the existence of a dichotomy in platelet function between haemostasis and inflammation, summarized in Fig. 5. A deep understanding of the biochemical pathways behind these different functional responses may lead to the identification of novel therapeutic targets for inflammatory disorders not compromising the haemostatic functions of platelets.
The
Authorship
EP, RA, CP, PG, SM and SP were involved with the conception, hypotheses delineation, and design of the study. EP, RA, SS, SM and EP were involved with acquisition of the data, analysis and interpretation of the data. All authors contributed to the writing of the article. There are no conflicts of interest to declare.
Declaration of interest
The authors have no conflicts of interest to report.
Acknowledgements
This study was funded by the Sackler Foundation, The MIUR (protocol #2012773NE3) and the Fondazione Cassa di Risparmio Perugia (protocol #2014.0083.021).
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These authors contributed equally to this study.