Short fungal fractions of -1,3 glucans affect platelet activation Hélène Vancraeyneste, 1,2,3,4 Rogatien Charlet, 1,2,3,4 Yann Guerardel, 5,6 Laura Choteau, 1,2,3,4 Anne Bauters, 7 Meryem Tardivel, 8 Nadine François, 9 Laurent Dubuquoy, 1,2,3,4 Dmitry Soloviev, 10 Daniel Poulain, 1,2,3,4,9 Boualem Sendid, 1,2,3,4,9 and X Samir Jawhara 1,2,3,4 1 Univ Lille Nord de France, Lille, France; 2 UDSL, Lille, France; 3 INSERM U995, Lille, France; 4 CHRU Lille, Lille, France; 5 Université de Lille 1, Unité de Glycobiologie Structurale et Fonctionnelle, UGSF, Villeneuve d’Ascq, France; 6 CNRS, UMR 8576, Villeneuve d’Ascq, France; 7 Laboratoire d’Hémostase, Pôle de Pathologie Génétique, Lille, France; 8 Plateforme d’Interaction Moléculaire, IMPRT-IFR114, Faculté de Médecine de Lille, Lille, France; 9 Service de Parasitologie Mycologie, Pole de Biologie Pathologie Génétique, Lille, France; and 10 Department of Molecular Cardiology, Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Cleveland Clinic, Cleveland, Ohio Submitted 30 November 2015; accepted in final form 12 May 2016 Vancraeyneste H, Charlet R, Guerardel Y, Choteau L, Bauters A, Tardivel M, François N, Dubuquoy L, Soloviev D, Poulain D, Sendid B, Jawhara S. Short fungal fractions of -1,3 glucans affect platelet activation. Am J Physiol Heart Circ Physiol 311: H725–H734, 2016. First published July 15, 2016; doi:10.1152/ajpheart.00907.2015.—Platelets are capable of binding, aggregating, and internalizing microorganisms, which enhances the elimination of pathogens from the blood. The yeast Candida albicans is a pathobiont causing life-threatening invasive infections. Its cell wall contains -1,3 glucans that are known to trigger a wide range of host cell activities and to circulate during infection. We studied the effect of -1,3 glucan fractions (BGFs) consisting of diglucosides (Glc2), tetraglucosides (Glc4), and pentaglucosides (Glc5) on human platelets, their mechanisms of action, and their possible impact on host defenses. The effect of BGFs on the coagulation process was determined by measuring thrombin generation. Platelets pretreated with BGFs were analyzed in terms of activation, receptor expression, aggregation, and adhesion to neutrophils and to C. albicans. The results show that BGFs affected the endogenous thrombin potential in a concentration-dependent manner. For platelet activation, BGFs at a low concentration (2 mol/l) reduced ATP release and prevented the phosphorylation of protein kinase C. BGFs diminished the expression of P-selectin and the activation of IIb  3 . BGFs decreased platelet aggregation and the interaction between thrombin-stimulated platelets and neutrophils, fibrinogen, and C. albicans. GLc5 decreased ATP release and TGF-1 production in response to TLR4 upregulation in thrombin-stimulated platelets, but TLR4 blockage abolished the effect of BGFs on platelets. This study provides evidence that fungal pentaglucosides modulate platelet activity mediated via TLR4 stimu- lation and reduce platelet-neutrophil interaction. platelets; coagulation; -glucans; Candida albicans; aggregation; toll- like receptors NEW & NOTEWORTHY Our study shows that the soluble short fractions of -1,3 glucans act as a shield for Candida albicans. These fractions reduce platelet-neutrophil interactions, and platelet activation through TLR4-mediated TGF-1 production and ATP release, and blocking this receptor by an anti-TLR4 antibody abolished the effect of the pentaglucosides on platelets. PLATELETS play a crucial role in hemostasis, thrombosis, and pathogen clearance (28, 38). Many pathogenic fungi can inter- act with platelets in circulating blood (33). This interaction between the fungus and the host occurs at the level of the cell wall, which consists mainly of polysaccharides associated with proteins and lipids (17). Its innermost layers are formed from a dense network of polysaccharides consisting of chitin and -1,3 glucans (17). The human pathogenic fungus Candida albicans is the predominant cause of invasive forms of candi- diasis (36). C. albicans infections continue to be a serious clinical problem with respect to increased morbidity and mor- tality (36). The cell wall components of C. albicans can interfere with platelets in circulating blood and contribute to the adherence of C. albicans to fibrin-platelet matrices (29, 33). Platelets also interact with the yeast form and hyphae of C. albicans (50). In a mouse model of invasive candidiasis, platelets adhered to C. albicans, lost their discoid shape, and generated pseudopodia against yeast cells (37). -1,3 Glucans are able to interact directly with leukocytes and platelets (39, 40). It has been demonstrated that -glucan from live yeast binds to dectin-1 associated with galectin-3 or TLR2 to modulate signaling pathways and increase the proin- flammatory cytokine response (12, 14, 16). -1,3 Glucans prevent an increase in proinflammatory cytokines in the circu- lation while stimulating IL-20 synthesis (26). The immunolog- ical activity of -1,3 glucans depends on their molecular structure, including polymer length and degree of branching, their solubility, and their influence on the activation or inhibi- tion of leukocyte receptors. Soluble -1,3 glucans could block receptors such as dectin-1 and CD11b/CD18 and prevent multivalent binding necessary for strong triggering of leuko- cyte inflammatory responses (16, 21). Clinically, -1,3 glucans derived from the fungal cell wall are released in the circulation during infection, and their detection enables the early diagnosis of an invasive fungal infection. However, the role of -1,3 glucans in the modulation of platelet activities and platelet- neutrophil interactions is unknown. The aims of this study were to assess the effect of -glucan fractions (BGFs) derived from C. albicans on platelet activity. MATERIALS AND METHODS Extraction and fractionation of -glucans. C. albicans wild-type strain SC5314 was used throughout the study (15). The fractionation and digestion procedure for extraction of BGFs from yeast cells of C. albicans has been described previously (20). Each BGF was diluted in Hanks’ balanced salt solution (HBSS). HBSS alone was used as the control. BGFs were subjected to thin-layer chromatography on silica gels. For mass spectrometry, a mixture of matrix (1 l containing 10 Address for reprint requests and other correspondence: S. Jawhara, HDR, INSERM U995/2, Université Lille Nord de France, 1 place Verdun, 59000 Lille, France (e-mail: samir.jawhara-3@univ-lille2.fr). Am J Physiol Heart Circ Physiol 311: H725–H734, 2016. First published July 15, 2016; doi:10.1152/ajpheart.00907.2015. 0363-6135/16 Copyright © 2016 the American Physiological Society http://www.ajpheart.org H725 Downloaded from journals.physiology.org/journal/ajpheart (054.162.069.248) on July 12, 2020.