Cosmc Is Essential for O-Glycosylation of Platelet Glycoproteins and Platelet Function

Abstract 383

Platelets express a large number of glycoproteins involved in adhesion and signaling events, but the specific roles of the Asn-linked (N-glycans) and Ser/Thr-linked (O-glycans) pathways are not well understood. While O-glycans are known to play important roles as selectin ligands important in leukocyte recruiting and lymphocyte homing, little is known about the functions of O-glycans in platelet biology. O-glycan biosynthesis requires sequential actions of glycosyltransferases in the Golgi apparatus. Key among these is the T-synthase, which converts the Tn antigen (GalNAcα1-Ser/Thr) to the core 1 O-glycan Galβ1-3GalNAcα1-Ser/Thr. Core 1 O-glycans are the common precursor for complex O-glycans on secreted and membrane glycoproteins on hematopoietic cells. Biosynthesis of an active T-synthase requires a unique and T-synthase-specific molecular chaperone termed Cosmc. In mice, disruption of either Cosmc or T-synthase is embryonic lethal due in part to impaired angiogenesis. In humans, somatic mutations in Cosmc on Xq24 result in Tn syndrome. Tn syndrome, which is characterized by the Tn antigen expression on blood cells of all lineages, is associated with thrombocytopenia, indicating a potential role of Cosmc and complex O-glycans in platelet biology and hemostasis.

To further explore the roles of O-glycans in hemostasis we generated mice with deletions of Cosmc in hematopoietic and endothelial cells (EHC-Cosmc^-/y) by breeding floxed (Cosmc^flox/flox) females with Tie2-Cre transgenic male mice. Any EHC-Cosmc^-/y mice are null, since the Cosmc gene is X-linked. Cre-mediated excision of the Cosmc gene was effective as demonstrated by the almost complete absence of Cosmc transcript, T-synthase activity, and the expression of Tn antigen on EHC-Cosmc^-/y platelets. A high incidence of post-natal lethality (89%) due to hemorrhage was observed in EHC-Cosmc^-/y mice along with a smaller body size, and the few survivors died within 3–5 months as a result of either pulmonary or GI hemorrhage. Tail-bleeding times were markedly prolonged in the surviving EHC-Cosmc^-/y mice in comparison to both littermate controls and heterozygous females.

Consistent with the human phenotype observed in patients with Tn syndrome, EHC Cosmc^-/y mice were markedly macrothrombocytopenic (166±57 10³/mm³) compared with their littermate controls (787±124 10³/mm³). EHC Cosmc^-/y platelets were more than twice as large as controls and appeared circular in contrast with the normal discoid shape of control platelets. The severity of the bleeding phenotype led us to more closely examine the platelet phenotype and function. In EHC Cosmc^-/y platelet extracts both GPIbα and integrinα_IIb appeared as proteolytically cleaved fragments, and platelets from EHC Cosmc^-/y mouse had low expression of GPIbα on their cell surface as measured by flow cytometry. Levels of transcripts encoding GPIbα, GPIIb, GPV, and P-selectin in platelets were normal compared to those from control mice. EHC Cosmc^-/y platelets have remarkably reduced adhesion and spreading on Von Willebrand factor coated plates, consistent with the low levels of GPIbα on the platelet surface. A similar defect in adhesion and binding was observed to a fibrinogen surface. Additionally, EHC Cosmc^-/y platelets had significantly impaired activation in response to thrombin with decreased binding of antibodies to P-selectin and the activation-specific integrin α_IIbβ₃ antibody JON/A following stimulation. Female heterozygotes (Cosmc^-/+) appeared normal on gross examination, however occasionally dysfunctional megathrombocytes were observed in peripheral blood consistent with lyonization. This study is the first to demonstrate the essential role of Cosmc in regulating correct platelet O-glycosylation, and to indicate the importance of complex O-glycans in platelet morphogenesis, biogenesis, and function.