Page 212 - Haematologica - Vol. 105 n. 6 - June 2020

I. Scheller et al.
activation and aggregation responses under static condi- tions in vitro.
In sharp contrast, however, Cotl1 deficiency markedly affected platelet aggregation and thrombus formation under shear flow conditions in vitro. We used a flow adhe- sion assay where the perfusion of whole anticoagulated WT blood over a collagen-coated surface leads to rapid platelet adhesion, activation and three-dimensional aggre- gate formation. While aggregate formation at low shear (150 s-1) was comparable between Cotl1-/- and WT samples, we observed significantly reduced platelet adhesion, sur- face coverage and thrombus volume in blood from Cotl1-/- animals at medium and high shear rates (1 000 s-1, *P<0.05; 3 000 s-1, **P<0.01) (Figure 2A-C and Online Supplementary Figure S5). Strikingly, thrombus volume and platelet sur- face coverage were also significantly reduced in blood from Cotl1-/- mice when the flow adhesion assay was car- ried out in the presence of coagulation (Online Supplementary Figure S6).
Platelet adhesion and aggregate formation under medi- um and high shear rates is dependent on the interaction between the mechanoreceptor GPIb and immobilized vWF.24 To investigate a possible involvement of Cotl1 in GPIb-mediated tethering/adhesion, we perfused blood from WT and Cotl1-/- animals over a vWF-coated surface at high shear (3,000 s-1). The velocity of individual rolling Cotl1-/- platelets on immobilized vWF was comparable to the WT (Figure 2E, right); however, the number of adher- ent Cotl1-/- platelets was significantly reduced (**P<0.01) (Figure 2D and E). Our results thus indicated that Cotl1 is required to ensure GPIb function during platelet adhesion and aggregate formation under conditions of high shear.
Growing experimental evidence suggests that signaling induced by the GPIb-vWF interaction involves mechan- otransduction and transmission of forces to the actin cytoskeleton.3,25,26 Therefore, to assess the impact of Cotl1 deficiency on platelet biomechanical properties more gen- erally, we subjected Cotl1-/- platelets to the recently described real-time deformability cytometry (RT-DC),27 a method for continuous mechanical characterization of cells which are deformed by shear forces in a microfluidics chamber (Figure 2F). Strikingly, we found a significantly increased deformability of Cotl1-/- platelets (*P<0.05) as compared to WT (Figure 2G and H, right). Importantly, we could exclude the possibility that the increased deforma- bility was due to an increased platelet size; by contrast, the measured size of Cotl1-/- platelets in this assay was even slightly decreased as compared to the control (Figure 2H, left). Together, these results indicate that Cotl1 sup- ports GPIb function and thus the formation of stable platelet aggregates under shear conditions, and that this function may, in part, be mediated by the modulation of platelet biomechanical properties.
Cotl1 regulates leukotriene biosynthesis in platelets
Leukotrienes are pro-inflammatory lipid mediators mainly produced by immune competent cells such as leukocytes, e.g. mast cells, eosinophils, neutrophils, monocytes and basophils, and are implicated in a variety of inflammatory responses. Interestingly, besides its func- tion as an actin-regulatory protein, Cotl1 was shown to bind and modulate the activity of the enzyme 5-lipoxyge- nase (5-LO).15,17,28 5-LO catalyzes the two initial steps of LT biosynthesis: (1) the oxygenation of AA to 5-HPETE; and (2) the subsequent dehydration into leukotriene A4 (LTA4)
which is then further converted to LTB4 (Online Supplementary Figure S7).29,30 Besides serving as substrate for LT biosynthesis, AA is converted to thromboxanes (TxA/B2), prostacyclin (PGI2), and prostaglandines (PGE/F2) by cyclo-oxygenases in platelets (Online Supplementary Figure S4).31,32
To investigate the effect of Cotl1 deficiency on LT pro- duction in platelets, we assessed the release of 5,12- diHETE and LTB4 in the supernatant of washed CRP- or thrombin-stimulated platelets. Strikingly, the secretion of both lipid mediators was significantly reduced in Cotl1-/- platelets upon CRP activation as compared to WT controls and a similar tendency was observed for thrombin-stimu- lated platelets (*P<0.05) (Figure 3A). Of note, the total AA amount was comparable to WT platelets, demonstrating that the abundance of this initial metabolite was not affected by Cotl1 deficiency (Figure 3A, left). To assess whether AA was consumed by other pathways, we ana- lyzed TxB2 levels by ELISA. Strikingly, we found signifi- cantly increased TxB2 release in thrombin-stimulated and, to a lesser extent, CRP-stimulated Cotl1-/- platelets com- pared to the control (*P<0.05), indicating that the excess of available AA in Cotl1-/- platelets was consumed by an upregulation of prostaglandin biosynthesis (Online Supplementary Figure S8).
To assess whether, indeed, Cotl1 directly influences 5- LO activity, lysates of CRP- or thrombin-stimulated platelets were probed for active 5-LO (S663 phosphoryla- tion) by western blotting (Figure 3B). Of note, basal levels of active 5-LO were comparable between Cotl1-/- and WT platelets. Strikingly, while activation induced pronounced S663 phosphorylation in WT platelets, this process was significantly reduced in Cotl1-/- platelets (thrombin: *P<0.05; CRP: **P<0.01) (Figure 3B and C). Together, these results demonstrated that Cotl1 directly influences 5-LO activity, ultimately resulting in reduced biosynthesis and, subsequently, release of LT from Cotl1-/- platelets.
Defective shear-dependent thrombus formation in Cotl1-deficient mice is rescued by exogenous addition of leukotriene B4
We next investigated whether the reduced release of lipid mediators in Cotl1-/- mice contributed to defective platelet aggregate formation of Cotl1-/- platelets under flow. We decided to focus on LTB4, one of the end products of the LT biosynthesis pathway downstream of LTA4, which was shown to stimulate neutrophil chemotaxis33 and acti- vation.34 First, we tested whether LTB4 was able to directly induce platelet activation. As LTB4 was described to induce neutrophil aggregation and degranulation at con- centrations of 0.1 μM35,36 and leukocyte aggregation, chemotaxis and chemokinesis at a subnanomolar range of 0.39 nM,35 we used concentrations of 0.025-250 nM LTB4 (Cayman Chemicals) in our assays. None of the tested LTB4 concentrations induced platelet activation under stat- ic conditions in vitro (Online Supplementary Figures S9A and S10A). Likewise, LTB4 addition did not further enhance integrin αIIbβ3 activation, degranulation or aggregation of WT or Cotl1-/- platelets (Online Supplementary Figures S9B and S10B).
Using the in vitro flow adhesion assay (Figure 2A-C), we next investigated the effect of LTB4 on platelet aggregate formation under flow. Adding concentrations of 2.5 nM or higher interfered with aggregate formation (1,700s-1) in WT blood, whereas lower concentrations had no effect
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