Platelet biogenesis and functions require correct protein O-glycosylation

Abstract

Platelets express a variety of membrane and secreted glycoproteins, but the importance of glycosylation to platelet functions is poorly understood. To explore the importance of O-glycosylation, we generated mice with a targeted deletion of Cosmc in murine endothelial/hematopoietic cells (EHC) (EHC Cosmc(-/y)). X-linked Cosmc encodes an essential chaperone that regulates protein O-glycosylation. This targeted mutation resulted in lethal perinatal hemorrhage in the majority of mice, and the surviving mice displayed severely prolonged tail-bleeding times and macrothrombocytopenia. EHC Cosmc(-/y) platelets exhibited a marked decrease in GPIb-IX-V function and agonist-mediated integrin αIIbβ3 activation, associated with loss of interactions with von Willebrand factor and fibrinogen, respectively. Significantly, three O-glycosylated glycoproteins, GPIbα, αIIb, and GPVI normally on platelet surfaces that play essential roles in platelet functions, were partially proteolyzed in EHC Cosmc(-/y) platelets. These results demonstrate that extended O-glycans are required for normal biogenesis of the platelets as well as the expression and functions of their essential glycoproteins, and that variations in O-glycosylation may contribute to altered hemostasis.

Fig. 1.

Cosmc disruption (EHC Cosmc^−/y) results in hemorrhage and loss of T-synthase activity. (A) E15.5 embryos (EHC Cosmc^−/y) reveal hemorrhaging, denoted by arrows. (Scale bars, 1 mm.) (B) RT-PCR analysis of Cosmc transcription level in different platelet genotypes. (C and D) T-synthase activity in platelets isolated from EHC Cosmc^+/y, EHC Cosmc^+/−, and EHC Cosmc^−/y. α-Mannosidase II activity shown as a control. Error bars indicate ±1 SEM of two independent experiments. (E) Flow cytometry analysis of EHC Cosmc^+/y and EHC Cosmc^−/y platelets, stained with anti-CD41 and anti-Tn antigen antibodies. (F) Western blot of platelet lysates from wild-type mice (Cosmc^+/y), EHC Cosmc^+/− mice, and EHC Cosmc^−/y mice, using biotin labeled anti-Tn antigen antibody. GAPDH was used as a loading control.

Fig. 2.

EHC Cosmc-deficient animals have prolonged bleeding times and macrothrombocytopenia. (A) Tail-bleeding time (s) in three different mouse genotypes (n = 6–7). Cautery of mice with continued bleeding was performed at 600 s (red arrow). (B) Peripheral platelet counts in EHC Cosmc^+/y, EHC Cosmc^+/−, and EHC Cosmc^−/y mice. *P = 0.00023, **P = 0.0000002 (equal variance). Error bars indicate ± 1 SD (n = 6). (C) Panels C-1 and C-3 (EHC Cosmc^+/y), and C-2 and C-4 (EHC Cosmc^−/y) show images of platelets (arrows) from blood smears stained by Wright-Giemsa (C-1 and C-2), or viewed by transmission electron microscopy (C-3 and C-4).

Fig. 3.

Cosmc-deficient platelets have impaired platelet GPIbα expression and function. (A) Platelet surface GPIbα expression as measured by flow cytometry (n = 4, *P = 0.0122, equal variance). (B) Western blot of GPIbα in platelet lysates in reducing and nonreducing conditions. (C) Flow cytometry of botrocetin-treated EHC Cosmc^+/y and EHC Cosmc^−/y platelets in the presence of VWF and incubated with FITC-labeled anti-VWF antibody. (D) Botrocetin-activated EHC Cosmc^+/y and EHC Cosmc^−/y platelets were allowed to adhere to BSA or VWF. Adherent platelets were stained with fluorescein-labeled phalloidin.

Fig. 4.

Altered expression and function of integrin αIIb and other platelet glycoproteins in Cosmc-deficient platelets. (A) Fluorescent microscopy: EHC Cosmc^+/y and EHC Cosmc^−/y platelet binding to fibrinogen detected by fluorescein-labeled phalloidin; EHC Cosmc^+/y platelets without (A-1) or with (A-3) thrombin stimulation. EHC Cosmc^−/y platelets without (A-2) or with (A-4) thrombin stimulation. SEM: thrombin-stimulated EHC Cosmc^+/y and EHC Cosmc^−/y platelets (A-5 and A-6). (B and C) In the absence of Cosmc, thrombin fails to stimulate activated αIIbβ3 measured by JON/A antibody (B) and P-selectin expression on the surface of platelets (C). Comparing resting to thrombin-stimulated platelets, *P = 0.0388, **P = 0.0219 (B), *P = 0.0102, **P = 0.00037 (C), NS, not significant as noted in graph (*, unequal variance; NS and **, equal variance). (D) Western blots of EHC Cosmc^+/y and EHC Cosmc^−/y platelets with anti-GPIIb and anti-GPIIIa antibodies. (E) Western blot of GPVI in platelet lysates in reducing conditions.