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• W2023269495 abstract "C-reactive protein (CRP) is a member of the pentraxin protein family, which have a native pentameric ring structure made up of five non-covalently associated monomers. It is an acute-phase protein produced predominantly in the liver, with plasma levels increasing up to 10 000-fold in response to infection. CRP is widely used as a marker of cardiovascular disease [1] and has been implicated in its pathogenesis [2], although its exact role is unclear [3]. Although circulating CRP exists in the native pentameric form (pCRP), it can also exist in a modified monomeric form (mCRP). These two distinct forms of CRP have been demonstrated to have opposing biological actions. CRP has been shown to modulate the interaction between platelets and neutrophils [4, 5]. mCRP was demonstrated to enhance the interaction under shear via an interaction with P-selectin and CD18, whereas pCRP inhibited the interaction via an interaction with FcγRIIa [5]. Heuertz et al. demonstrated that mCRP interacted with CD16 (FcγRIIIb) on neutrophils, but not with CD32 (FcγRIIa) [6]. CRP has been associated with procoagulant [7] and antifibrinolytic activity [8] and has also been shown to have diverse effects on platelets. Whereas heat-agglutinated CRP [9] and mCRP [10] have been shown to activate platelets, pCRP has been shown to inhibit platelet aggregation by a range of agonists [11-13]. The mechanism by which pCRP inhibits platelet aggregation is unknown, and this study was designed to elucidate the mechanism of platelet inhibition using platelet adhesion and enzyme-linked immunosorbent assay (ELISA) using purified protein. Platelet adhesion and aggregation studies were performed as previously described [14]. For this study, we used healthy volunteers who had not taken any non-steroidal anti-inflammatories for a fortnight, and we did not discriminate on the basis of age or sex. Ethical approval was obtained from the Royal College of Surgeons in Ireland ethics committee. As with previous studies, we found that recombinant pCRP (Calbiochem, Darmstadt, Germany; 10 μg mL−1) inhibited platelet aggregation induced by low-dose thrombin receptor-activating peptide (2–4 μm), collagen (38 μg mL−1) and ADP (1–5 μm) by 72 ± 10%, 95 ± 2% and 91 ± 4% respectively (P < 0.001 for all), indicating an agonist-independent effect of pCRP, similar to that seen with αIIbβ3 inhibitors. To determine whether there was a direct interaction between CRP and platelets, we investigated the ability of washed platelets to bind to immobilized CRP. Resting platelets adhered strongly to fibrinogen and to a lesser extent to CRP (30 ± 7% of the fibrinogen control; Fig. 1A). Platelets and purified αIIbβ3 adhere to immobilized C-reactive protein (CRP). (A) Platelet adhesion to CRP is potentiated by Mn2+ activation of platelets and inhibited by αIIbβ3 antagonists. (B) Purified αIIbβ3 adhered to immobilized CRP in an enzyme-linked immunosorbent assay (ELISA). Plates were coated with fibrinogen or pentameric CRP (pCRP) (50 μg mL−1) by incubating protein solutions in the wells for 2 h at 37 °C, and further blocked with 1% bovine serum albumin. Resting (washed) platelets (A) or purified αIIbβ3 (B) were treated with 1 mm MnCl2 for 10 min prior to adhesion. Where indicated, platelets or purified protein were incubated with tirofiban (1 μm) for 10 min prior to adhesion to immobilized CRP or fibrinogen. For the ELISA (B), 50 μg mL−1αIIbβ3 (Calbiochem, Darmstadt, Germany) was incubated with the plates for 1 h. Anti-CD41 clone SZ22 (2 μg mL−1) was incubated for 90 min with horseradish peroxidase-conjugated goat anti-mouse (Pierce, Rockford, IL, USA) (1 : 15 000) for 45 min. Values were normalized to the fibrinogen control (resting). Data are represented as the mean ± SEM. Statistical analysis was carried out prior to normalization using repeated-measures anova with Bonferroni’s correction for multiple comparisons (*P < 0.001, n = 3). Absolute values for the platelet adhesion assay (absorbance 405 nm) and the glycoprotein IIb/IIIa ELISA assay (absorbance 450 nm) are tabulated below (C). As αIIbβ3 is a key receptor in platelet aggregation and adhesion, we investigated its ability to bind CRP. Mn2+ alters the conformation of αIIbβ3, increasing its affinity for RGD-containing peptide ligands [15]. This is not as strong as the effect of other agonists and does not lead to signaling, which would lead to granule release and platelet shape change. All other agonists signal into the platelet (outside-in signaling), and lead not only to granule release, but also to an increase in P-selectin expression. Thus, given reports that CRP interacts with P-selectin [4, 5], we might see increased platelet adhesion or at least αIIbβ3-independent adhesion if platelets activated with soluble agonists were used. The use of MnCl2 allowed us to look at the αIIbβ3 interaction without the complications of the P-selectin interaction. Direct activation of αIIbβ3 using MnCl2 (1 mm) had little effect on platelet adhesion to fibrinogen but increased adhesion to CRP approximately 4-fold (128 ± 6% of fibrinogen control, P < 0.001). Tirofiban, a specific αIIbβ3 antagonist [16] (1 μm), completely inhibited both resting and activated platelet adhesion to fibrinogen [from 100% to 3 ± 6% (resting) and from 99 ± 3% to 3 ± 9% (MnCl2 activated), P < 0.001 for both]. Tirofiban inhibited resting platelet adhesion to CRP by 43% (from 30 ± 7% to 17 ± 2%), although this inhibition was not statistically significant, due to the variation observed in this assay at low levels of adhesion. Tirofiban completely inhibited the adhesion of MnCl2-activated platelets to CRP (from 128 ± 6% to 0 ± 12%, P < 0.001). To confirm a direct interaction with αIIbβ3, we utilized an ELISA to measure binding of purified αIIbβ3 to CRP (Fig. 1B). These data directly mirrored the results from the platelet adhesion study, with adhesion of resting αIIbβ3 to CRP at 36 ± 6% of the adhesion to fibrinogen. This was also increased 4-fold to 133 ± 25% with MnCl2 activation of αIIbβ3 (P < 0.001). Tirofiban strongly inhibited purified αIIbβ3 adhesion to CRP (resting, from 36 ± 6% to 2 ± 3%, P not significant; MnCl2-activated αIIbβ3, from 133 ± 25% to 14 ± 6%, P < 0.001). We also investigated a role for FcγRIIa, due to its role in immune-mediated platelet activation [14, 17] and previous reports of an interaction between FcγRIIa and pCRP. Adhesion to neither fibrinogen nor CRP was inhibited by the monoclonal antibody to FcγRIIa (IV.3) (data not shown), suggesting that this receptor does not play a role in platelet adhesion to CRP. We have demonstrated that immobilized CRP can support platelet adhesion and that it has a higher affinity for Mn2+-treated platelets, suggesting a greater affinity for the activated form of αIIbβ3. In support of this, we have demonstrated that immobilized CRP interacts directly with purified αIIbβ3, and also shows increased binding to Mn2+-activated αIIbβ3 in this system. This interaction was inhibited by tirofiban, suggesting that CRP interacts with αIIbβ3 through the RGD-binding site. Analysis of the surface-exposed residues of CRP identified the RGD-like motif 58RQD60. Thus, it is possible that this sequence could be responsible for the interaction with αIIbβ3 that we have demonstrated. These data may explain previous reports on the platelet inhibitory activity of pCRP. Recently, pCRP was shown to decrease fibrinogen binding to platelets preactivated with ADP [18], which our data would suggest is due to direct binding of pCRP to αIIbβ3. We suggest that soluble pCRP can interact with activated αIIbβ3, blocking fibrinogen binding, and thus acting as an αIIbβ3 antagonist in a soluble setting. This is likely to be relevant in conditions associated with infection [17] or inflammation, where the high levels of pCRP may act to suppress platelet aggregation. Atherosclerosis is probably influenced more by mCRP interactions with the vessel wall. We cannot exclude the possibility of an interaction between mCRP and αIIbβ3. Previous reports have suggested that pCRP changes conformation upon binding to cationic surfaces [19], and pCRP bound to membranes has been shown to undergo a transition to mCRPmodified (pentameric form displaying neoepitopes) and then to mCRPsoluble (soluble monomeric form) [20]. Taking into consideration the ability of soluble pCRP to inhibit aggregation induced by a wide range of platelet agonists and its ability to support platelet adhesion when immobilized, it is possible that both pCRP and mCRP can directly interact with αIIbβ3. In conclusion, we have shown that CRP at levels found during episodes of inflammation directly binds to the activated form of αIIbβ3 and inhibits platelet aggregation. This work was funded by the Health Research Board Ireland, and Science Foundation Ireland. The authors state that they have no conflict of interest." @default.
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• W2023269495 title "C‐reactive protein binds to αIIbβ3" @default.
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