Heparan sulfates are critical regulators of the inhibitory 1 megakaryocyte-platelet receptor G 6 b-B 2 3

46 The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical 47 for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, 48 myelofibrosis and aberrant platelet function in mice and humans. Using a combination of 49 immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular 50 matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in 51 vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is 52 specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin 53 forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from 54 humans and genetically-modified mice, we demonstrate that binding of G6b-B to HS and 55 multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream 56 signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into 57 how G6b-B is regulated and contribute to our understanding of the interaction of 58 megakaryocytes and platelets with glycans. 59 60 61 VÖGTLE et al G6b-B binds perlecan 3 INTRODUCTION 62 Platelets are highly reactive anucleated cell fragments, produced by megakaryocytes 63 (MKs) in the bone marrow, spleen and lungs. In an intact vasculature, platelets circulate in the 64 blood stream for 7-10 days and are finally cleared by the reticulo-endothelial system in the 65 spleen and liver. Upon vascular injury, however, platelets adhere to the exposed vascular 66 extracellular matrix (ECM), become activated and form a hemostatic plug that seals the wound. 67 Platelet activation must be tightly regulated to avoid hyperactivity and indiscriminate vessel 68 occlusion (Bye, Unsworth, & Gibbins, 2016; Jackson, 2011). The mechanisms that inhibit platelet 69 activation include extrinsic factors, such as endothelial-derived nitric oxide and prostacyclin, and 70 intrinsic factors, such as immunoreceptor tyrosine-based inhibition motif (ITIM)-containing 71 receptors (Coxon, Geer, & Senis, 2017; Nagy & Smolenski, 2018). 72 G6b-B is a unique platelet ITIM-containing receptor that is highly expressed in mature 73 MKs and platelets (Coxon et al., 2017; Senis et al., 2007). It is a type I transmembrane protein 74 that consists of a single N-glycosylated immunoglobulin-variable (IgV)-like domain in its 75 extracellular region, a single transmembrane domain and a cytoplasmic tail containing an ITIM 76 and an immunoreceptor tyrosine-based switch motif (ITSM). The central tyrosine residue 77 embedded in the consensus sequence of the ITIM ([I/V/L]xYxx[V/L]) and ITSM ([T]xYxx[V/I]) 78 become phosphorylated by Src family kinases (SFKs) and subsequently act as docking sites for 79 the Src homology 2 (SH2) domain-containing protein-tyrosine phosphatases (Shp)1 and 2 80 (Mazharian et al., 2012; Senis et al., 2007). The canonical mode of action of ITIM-containing 81 receptors is to position these phosphatases, as well as the SH2 domain-containing inositol 82 polyphosphate 5-phosphatase 1 (SHIP1) in close proximity to ITAM-containing receptors, 83 allowing them to dephosphorylate key components of the ITAM signaling pathway and attenuate 84 activation signals. The inhibitory function of G6b-B has been demonstrated in a heterologous 85 cell system, by antibody-mediated crosslinking of the receptor in platelets and G6b-B knockout 86 (KO) mouse models (Mazharian et al., 2012; Mori et al., 2008; Newland et al., 2007). Findings 87 VÖGTLE et al G6b-B binds perlecan 4 from these mice demonstrated that the function of G6b-B goes beyond inhibiting signaling from 88 ITAM-containing receptors (Mazharian et al., 2013; Mazharian et al., 2012). These mice 89 develop a severe macrothrombocytopenia, myelofibrosis, aberrant megakaryocyte and platelet 90 function, establishing G6b-B as a critical regulator of platelet activation and production. This 91 phenotype was also observed in a G6b-B loss-of-function mouse model (Mpig6b) in which 92 the tyrosine residues within the ITIM and ITSM were mutated to phenylalanine residues, 93 abrogating binding of Shp1 and Shp2 to G6b-B and downstream signaling (Geer et al., 2018). 94 Moreover, expression of human G6b-B in mouse platelets rescued the phenotype of G6b-B95 deficient mice, demonstrating that human and mouse G6b-B exert the same physiological 96 functions (Hofmann et al., 2018). Importantly, null and loss-of-function mutations in human 97 G6b-B have been reported to recapitulate key features of the Mpig6b KO and loss-of-function 98 mouse phenotypes, including a severe macrothrombocytopenia, MK clusters in the bone 99 marrow and myelofibrosis (Hofmann et al., 2018; Melhem et al., 2016). Despite the vital role of 100 G6b-B in regulating platelet production and function, its physiological ligand was not known. 101 Although a previous study demonstrated that G6b-B binds to the glycosaminoglycan (GAG) 102 heparin, the functional significance of this interaction was not known (de Vet, Newland, Lyons, 103 Aguado, & Campbell, 2005). 104 Proteoglycans comprise a heterogeneous family of macromolecules, consisting of a core 105 protein and associated unbranched glycosaminoglycan (GAG) side-chains. Heparan sulfates 106 (HS) are a specific subgroup of GAGs, defined by their basic disaccharide unit. They are 107 structurally-related to heparin, which is produced as a macromolecular proteoglycan by tissue108 resident mast cells (Lassila, Lindstedt, & Kovanen, 1997) and, following chemical or enzymatic 109 processing, serves as an anti-coagulant (Chandarajoti, Liu, & Pawlinski, 2016; Meneghetti et al., 11