Thromb Haemost 2012; 108(05): 963-972
DOI: 10.1160/TH12-01-0047
Platelets and Blood Cells
Schattauer GmbH

The Thr715Pro variant impairs terminal glycosylation of P-selectin

Hariharan Subramanian
1   Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria
2   Institute of Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Wuerzburg, Germany
,
Stepan Gambaryan
2   Institute of Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Wuerzburg, Germany
,
Simon Panzer
3   Clinical Department of Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Vienna, Austria
,
Thomas Gremmel
4   Department of Internal Medicine II, Division of Angiology, Medical University of Vienna, Vienna, Austria
,
Ulrich Walter
2   Institute of Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Wuerzburg, Germany
,
Christine Mannhalter
1   Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria
› Author Affiliations

Financial support: This study is a part of the PhD program, Cell Communication in Health and Disease (CCHD) funded by Austrian Science Fund (FWF) and the Medical University of Vienna. Experimental work in Wuerzburg was supported by the Deutsche Forschungsgemeinschaft (DFG, SFB688).
Further Information

Publication History

Received: 26 January 2012

Accepted after major revision: 21 August 2012

Publication Date:
29 November 2017 (online)

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Summary

P-selectin variant 715Pro is associated with lower concentrations of plasma P-selectin and reduced risk for thrombosis. We examined the influence of 715Pro on P-selectin synthesis, post-translational processing, surface expression and function using HEK293 cells, which do not express endogenous P-selectin. Mass spectrometry revealed that HEK293 cells produced recombinant P-selectin which has a glycosylation pattern comparable to platelet P-selectin. Compared to wild-type transfectants, 715Pro transfectants have ~50% less terminally glycosylated P-selectin and accumulate more immature P-selectin in Golgi. Following Brefeldin A treatment, the majority of 715Pro P-selectin is not modified by Golgi enzymes, while wild-type P-selectin undergoes complete modification. Flow cytometry revealed that 715Pro transfectants have ~20% less P-selectin on the cell surface compared to wild-type transfectants. Secretion of P-selectin by 715Pro transfectants was about 38% lower compared to wild-type transfectants. Binding of HL-60 cells to 715Pro transfectants was ~29% lower than to wild-type transfectants. This observation was confirmed by the presence of fewer platelet-monocyte aggregates (PMA) in the blood of healthy individuals and patients with angiographically proven atherosclerosis, carrying 715Pro P-selectin compared to individuals with wild-type P-selectin. We conclude that the 715Pro variant impairs terminal glycosylation of P-selectin in Golgi, leading to reduced amounts of mature P-selectin and subsequently less surface expression and secretion of P-selectin. The reduced surface expression of 715Pro P-selectin contributes to inefficient adhesion to HL-60 cells or monocytes.