Page 217 - Haematologica - Vol. 105 n. 6 - June 2020

Cotl1 regulates shear-dependent thrombus formation
the generation of intermediate LTA4 and subsequently the production of the different LT types (Online Supplementary Figure S7).35 Besides the cysteinyl (cysLT) LT (LTC4 , LTC4, LTE4), these also include LTB4, which stimulates neu- trophil chemotaxis,33 enhances neutrophil-endothelial interactions,41 and stimulates neutrophil activation, lead- ing to degranulation and the release of mediators, enzymes, and superoxides.34 LTB4 can also act on other cell types, e.g. by increasing interleukin (IL)-6 production by human monocytes.42 Platelet-derived LT were shown to contribute to inflammatory responses, e.g. during acute inflammation via activation of leukocytes,18,43 but only a few very early in vitro studies indicated an impact of LT directly on platelet aggregation.18,43
A recent comprehensive analysis of the platelet lipidome by Peng et al. revealed that the AA/5-LO/LT pathway is significantly induced by platelet activation.44 Therefore, to directly assess whether lack of Cotl1 down- regulates 5-LO activity and hence LT biosynthesis, we characterized platelet lipid mediator levels using mass spectrometry.44 Our data confirm previous findings from other cell types that Cotl1 positively regulates 5-LO,17 as lack of Cotl1 induced a shift from LT to prostaglandin biosynthesis downstream from AA, leading to reduced levels of LTA4 and LTB4, but higher levels of TxB2 in the knock-out platelets. Interestingly, CRP (but not thrombin) was able to induce significant LTB4 release in WT platelets. This is in line with findings by Peng et al. who observed that CRP, but not thrombin alone, was able to induce sig- nificant changes in the platelet lipidome.44 Thus, our detailed study further shows that the AA/LTB4 pathway is induced by GPVI/ITAM rather than GPCR signaling in platelets.
Strikingly, our results indicate that exogenous addition of LTB4 could fully rescue the defective aggregate forma- tion of Cotl1-deficient platelets on collagen under flow in vitro (Figure 4). While this finding indicates that the exoge- nous addition of LT can compensate for the GPIb function defect in Cotl1-deficient platelets, we cannot exclude that
GPIb signaling itself is involved in LT biosynthesis. Notably, exogenous addition of LTB4 did not restore aggregate formation of RhoA- or Grb2-deficient platelets, which per se display significant activation/ secretion defects. These results show that LTB4 secretion is required to fine-tune platelet function under flow rather than being a strong positive regulator of thrombus formation. However, LTB4 addition could moderately increase aggre- gate formation of Grb2-deficient platelets, which display defective GPVI/ITAM signaling. This further suggests that LTB4 is particularly relevant for platelet aggregate forma- tion induced through the GPIb/GPVI/ITAM axis. It will be important to dissect the detailed signaling mechanisms leading to LT generation, as well as the precise role of LT and their signaling pathways for platelet thrombus forma- tion in vivo in future studies.
Taken together, our study reveals that Cotl1 modulates biomechanical properties of platelets and acts as a signal- ing integrator in thrombotic processes. Given that both GPIb and LT represent potential therapeutic targets for a number of thrombo-inflammatory and autoimmune dis- eases, our findings may contribute to a better understand- ing of the molecular pathways orchestrating these processes.
Acknowledgments
We thank Stefanie Hartmann for excellent technical assis- tance and the microscopy platform of the Bioimaging Center (Rudolf Virchow Centre) for providing technical infrastructure and support. This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) (NI 556/11-2 to BN and project number 374031971 – TRR 240). MB is supported by an Emmy Noether grant of the DFG (BE5084/3-1). OO gratefully acknowledges support from the German Ministry of Education and Research (ZIK grant to OO under grant agreement 03Z22CN11). RA received further sup- port from the Federal Ministry of Education and Research (BMBF) in the German Network Bioinformatics Infrastructure (de.NBI) initiative (grants n. 031L0108A and n. 031 A 534B).
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