Normas de origen preferenciales

It is known that atherosclerotic plaque rupture exposes high levels of type I collagen which induces localised thrombus formation via the adhesion of platelets to exposed subendothelial matrix components in vascular walls. This initiates a cascade process that leads to coagulation. To examine how CEACAM2 contributes to platelet aggregation and pathological thrombus formation, two methods for inducing arterial injury were used, for wild type and Cc2-deficient mice, and for two types of vascular beds. FeCl3 was added, to

induce injury and expose the subendothelial matrix in mesenteric arterioles and laser was utilised to induce injury in cremaster arterioles. The resulting formation of thrombi was examined over a 10 minute time period, using in vivo intravital microscopy and Z-stack images to provide a spatial context.

Unlike CEACAM1, the physiologic role of platelet CEACAM2 in thrombus formation in vivo had not been determined. These two receptors share some structural similarities, but differ in the number of extracellular Ig-Domains and in the ligand binding properties of their distal variable N-terminal Ig-Domain. The fact that CEACAM2 has different ligand properties than CEACAM1 would suggest unique biological roles as the avidity of ligand interactions (activation versus clustering) appears fundamental to drive cell signalling responses. In recent studies, CEACAM2 appears to function in a similar way as CEACAM1, at least in the context of insulin metabolism [188].A more recent study has demonstrated that a closely related Ig-ITIM superfamily member, CEACAM1 serves as a negative regulator of platelet collagen GPVI-FcR -chain mediated signalling [187]. Using intravital microscopy to FeCl3-injured mesenteric arterioles, we have shown that thrombi in

Cc2–/– mice are larger and are more stable over time than wild-type mice providing evidence that CEACAM2 negatively regulates platelet-collagen interactions and thrombus growth in

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their ligands and have the ability to negatively modulate downstream signalling events and thrombus formation.

This chapter describes real time observation via intravital microscopy to reveal how CEACAM2 influences thrombus formation in vitro and in vivo, and highlights the importance of CEACAM2 in regulating thrombus formation on immobilised collagen under arterial flow conditions produced in vitro. Under in vitro conditions of physiological arterial shear flow, absence of CEACAM2 results in increased surface coverage of platelets on immobilised type I collagen (Figure 5-1). There was no evidence of sexual dimorphism in this enhanced thrombus growth phenotype of Cc2–/– platelets. Moreover, to observe the processes under different conditions, we included vascular injury of mesenteric arterioles induced by FeCl3 and laser-induced vascular injury of cremaster arterioles. FeCl3-induced

injury exposed a number of sub-endothelial collagen types, allowing collagen-GPVI platelets to interact with the exposed type I collagen. Thus, FeCl3-induced injury led to

larger thrombi, in terms of area and volume, among mice lacking CEACAM2 than in wild- type mice (Figure 5-2). Importantly, these differences were consistent over time, and the thrombi in Cc2–/– null mice were more stable than in the wild-type control group (Figure 5- 3). The in vivo thrombus stability phenotype observed in Cc2–/– mice required platelets with GPVI receptor sites (Figure 5-6). Using the laser-induced vascular injury model, Cc2–/– cremaster muscular arterioles displayed an increase in thrombus volume and thrombi that were more stable compared to wild-type arterioles (Figure 5-4D, F). Therefore, using several models of microvascular thrombosis in vivo, we have shown that CEACAM2 deletion results in larger and more stable thrombi than wild-type mice and that this is at least partly depends on the presence of GPVI (Figure 5-6). This suggests that CEACAM2 negatively regulates thrombus growth, in vivo, in both mesenteric and cremaster muscle arterioles. Future studies could compare the relative importance of platelet-expressed

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CEACAM2 with the endothelial type in regulating the in vivo growth of thrombi. CEACAM2 is a third example of the Ig-ITIM superfamily that negatively regulates the stationary adhesive signalling of platelet-collagen interactions and thrombus growth in vivo.

Previous research into arteriole thrombus formation in PECAM-1 and CEACAM1– deficient mice, using the FeCl3 vascular injury model, revealed slight differences in the time

taken to reach the 75% vessel occlusion level [178, 187]. Overall, the mean rate of vessel occlusion in PECAM-1–/– and Cc1–/– mice was slightly shorter than for the control group. By contrast, the laser-induced injury model, which does not expose type I collagen but promotes tissue factor generated platelet/fibrin thrombi, produced a larger response [178]. This suggests that PECAM-1, CEACAM1 and CEACAM2 all contribute to negative regulation during in vivo platelet-collagen interactions and thrombus formation.

Whether CEACAM2 regulates thrombin signalling in platelets remains unknown. In response to thrombin (at 0.25-1.0 U/mL), Cc2–/– platelets had comparable amplitude and slope of thrombin-mediated aggregation responses compared to wild-type platelets (Figure 3-5). In addition, GPCR platelet aggregation mediated by ADP, thrombin and PAR-4 agonist peptide were comparable in Cc2–/– and wild-type platelets (Figure 3-3). Moreover,

Cc2–/– platelets had similar alpha and dense granules release upon thrombin and PAR-4 agonist peptide stimulation compared to wild-type platelets (Figure 3-6A and 3-7A). While these results would argue against CEACAM2 regulating thrombin signalling, more studies using thrombin at subthreshold concentrations are needed to fully address this question. Moreover, the laser injury model used in this study is mediated by thrombin:tissue factor inflammatory process and the Cc2–/– arterioles had increased thrombi that were more stable under these conditions (Figure 5-5). Thus, more studies are needed to investigate whether CEACAM2 affects thrombin signalling pathway.

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Unlike human platelets, murine platelets lack the low-affinity IgG receptor, FcgammaRIIa, but contain two ITAM-bearing receptors, GPVI/FcR gamma chain and CLEC-2. Based on our in vivo mouse thrombus models, we show that Cc2–/– arterioles have larger thrombi that are more stable in response to type I collagen exposure (i.e: FeCl3-

induced vascular injury of mesenteric arterioles; Figure 5-2) and thrombin-driven processes without type I collagen exposure (i.e: laser induced injury of cremaster muscle arterioles [224, 275]; Figure 5-5). This was also the case for Cc1–/– (in this Chapter) and PECAM-1–/– arterioles [178]. While GPVI depletion resulted in the reversal of thrombus growth, thrombus stability was less affected indicating the involvement of other potential mechanisms regulating thrombus stability (Figure 5-3E).

Like CEACAM1, CEACAM2 plays a role in limiting thrombi formed under conditions of type I collagen exposure such as in the pathological process of atherosclerotic plaque rupture. Further studies are required to decipher its role in other mechanisms in platelet function, thrombus stabilisation and wound healing.

In conclusion, the chapter has defined the importance of CEACAM2 as an inhibitory co-receptor in murine platelets in platelet-collagen interactions in vivo. CEACAM2 is a negative regulator of platelet-collagen interactions and platelet thrombus formation and growth in vivo. CEACAM2 restricts arteriolar thrombus growth and postpones the development of thrombosis linked to type I collagen. Ultimately, this could be beneficial as it could lower the rate of thrombus formation in vessels with disease or with ruptured atherosclerotic plaques that expose the type I collagen matrix.

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6 Chapter Six: In vitro studies of CEACAM2 positive