Page 214 - Haematologica - Vol. 105 n. 6 - June 2020

I. Scheller et al.
(Online Supplementary Figure S11A and B). Strikingly, pre- incubation with 0.25 nM LTB4 could fully restore aggre- gate formation of Cotl1-/- platelets to WT levels (Figure 4A and B), indicating that the defect observed in untreated Cotl1-/- samples was caused by impaired platelet-derived LTB4 production. To investigate whether LTB4 has a more general function in this context, we analyzed two addi- tional knockout mouse lines with a described defect in the flow adhesion assay: Grb2fl/fl;Pf4Cre (Grb2-/-) mice have normal platelet counts but the platelets display a selective GPVI/ITAM activation defect.37 RhoAfl/fl;Pf4Cre (RhoA-/-) mice are thrombocytopenic and deficient platelets display
A
impaired G protein coupled receptor (GPCR) signaling.38 Notably, the addition of LTB4 to anticoagulated blood from Grb2-/- mice resulted in moderately increased platelet surface coverage (P=0.06) as compared to untreated sam- ples, but could not restore aggregate formation to WT lev- els (Online Supplementary Figure S12). Furthermore, LTB4 addition had no effect on aggregate formation in platelet- count adjusted blood of RhoA-/- mice (Online Supplementary Figure S13). Together, these findings demonstrated that addition of LTB4 cannot compensate for defective platelet aggregate formation under flow in the presence of promi- nent platelet activation/secretion defects. At the same
B
C
Figure 3. Cotl1 is a regulator of leukotriene biosynthesis in platelets. (A) For lipid mediator analysis platelets were either left untreated or stimulated with CRP [5 μg mL-1] or thrombin [0.01 U mL-1] for 5 minutes (min). Subsequently, samples were spun down, pellet and supernatant were separately shock-frozen in liquid nitrogen. Lipid abundance was assessed using liquid crystal mass spectrometry. Values are mean±standard deviation (SD) (n=15). (B and C) Platelets were either left untreat- ed or stimulated with CRP [5 μg mL-1] or thrombin [0.01 U mL-1] for 5 min, lysed, and processed for immunoblotting. Total 5-LO, phospho-5-LO (S663) and GAPDH (B) were probed with the respective antibodies and analyzed by densitometry (C). Values are mean±SD (n=4).
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