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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.

Keywords

P-selectin - P-selectin polymorphism - P-selectin glycosylation - soluble P-selectin - platelet-monocyte aggregate

 

  • References

  • 1 Stenberg PE, McEver RP, Shuman MA. et al. A platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation. J Cell Biol 1985; 101: 880-886.
    CrossrefPubMedGoogle Scholar
  • 2 McEver RP, Beckstead JH, Moore KL. et al. GMP-140, a platelet alpha-granule membrane protein, is also synthesized by vascular endothelial cells and is localized in Weibel-Palade bodies. J Clin Invest 1989; 84: 92-99.
    CrossrefPubMedGoogle Scholar
  • 3 Hattori R, Hamilton KK, Fugate RD. et al. Stimulated secretion of endothelial von Willebrand factor is accompanied by rapid redistribution to the cell surface of the intracellular granule membrane protein GMP-140. J Biol Chem 1989; 264: 7768-7771.
    PubMedGoogle Scholar
  • 4 Moore KL, Eaton SF, Lyons DE. et al. The P-selectin glycoprotein ligand from human neutrophils displays sialylated, fucosylated, O-linked poly-N-acetyllactosamine. J Biol Chem 1994; 269: 23318-23327.
    PubMedGoogle Scholar
  • 5 Moore KL, Stults NL, Diaz S. et al. Identification of a specific glycoprotein ligand for P-selectin (CD62) on myeloid cells. J Cell Biol 1992; 118: 445-456.
    CrossrefPubMedGoogle Scholar
  • 6 Celi A, Pellegrini G, Lorenzet R. et al. P-selectin induces the expression of tissue factor on monocytes. Proc Natl Acad Sci USA 1994; 91: 8767-8771.
    CrossrefPubMedGoogle Scholar
  • 7 Andre P, Hartwell D, Hrachovinova I. et al. Pro-coagulant state resulting from high levels of soluble P-selectin in blood. Proc Natl Acad Sci USA 2000; 97: 13835-13840.
    CrossrefPubMedGoogle Scholar
  • 8 Mayadas TN, Johnson RC, Rayburn H. et al. Leukocyte rolling and extravasation are severely compromised in P selectin-deficient mice. Cell 1993; 74: 541-554.
    CrossrefPubMedGoogle Scholar
  • 9 Ushiyama S, Laue TM, Moore KL. et al. Structural and functional characterization of monomeric soluble P-selectin and comparison with membrane P-selectin. J Biol Chem 1993; 268: 15229-15237.
    PubMedGoogle Scholar
  • 10 Dunlop LC, Skinner MP, Bendall LJ. et al. Characterization of GMP-140 (P-selectin) as a circulating plasma protein. J Exp Med 1992; 175: 1147-1150.
    CrossrefPubMedGoogle Scholar
  • 11 Chong BH, Murray B, Berndt MC. et al. Plasma P-selectin is increased in thrombotic consumptive platelet disorders. Blood 1994; 83: 1535-1541.
    PubMedGoogle Scholar
  • 12 Katayama M, Handa M, Araki Y. et al. Soluble P-selectin is present in normal circulation and its plasma level is elevated in patients with thrombotic thrombocytopenic purpura and haemolytic uraemic syndrome. Br J Haematol 1993; 84: 702-710.
    CrossrefPubMedGoogle Scholar
  • 13 Herrmann SM, Ricard S, Nicaud V. et al. The P-selectin gene is highly polymorphic: reduced frequency of the Pro715 allele carriers in patients with myocardial infarction. Hum Mol Genet 1998; 7: 1277-1284.
    CrossrefPubMedGoogle Scholar
  • 14 Carter AM, Anagnostopoulou K, Mansfield MW. et al. Soluble P-selectin levels, P-selectin polymorphisms and cardiovascular disease. J Thromb Haemost 2003; 1: 1718-1723.
    CrossrefPubMedGoogle Scholar
  • 15 Kee F, Morrison C, Evans AE. et al. Polymorphisms of the P-selectin gene and risk of myocardial infarction in men and women in the ECTIM extension study. Etude cas-temoin de l'infarctus myocarde. Heart 2000; 84: 548-552.
    CrossrefPubMedGoogle Scholar
  • 16 Tregouet DA, Barbaux S, Escolano S. et al. Specific haplotypes of the P-selectin gene are associated with myocardial infarction. Hum Mol Genet 2002; 11: 2015-2023.
    CrossrefPubMedGoogle Scholar
  • 17 Bugert P, Vosberg M, Entelmann M. et al. Polymorphisms in the P-selectin (CD62P) and P-selectin glycoprotein ligand-1 (PSGL-1) genes and coronary heart disease. Clin Chem Lab Med 2004; 42: 997-1004.
    PubMedGoogle Scholar
  • 18 Barbaux SC, Blankenberg S, Rupprecht HJ. et al. Association between P-selectin gene polymorphisms and soluble P-selectin levels and their relation to coronary artery disease. Arterioscler Thromb Vasc Biol 2001; 21: 1668-1673.
    CrossrefPubMedGoogle Scholar
  • 19 Ay C, Jungbauer LV, Kaider A. et al. P-selectin gene haplotypes modulate soluble P-selectin concentrations and contribute to the risk of venous thromboembolism. Thromb Haemost 2008; 99: 899-904.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 20 Reiner AP, Carlson CS, Thyagarajan B. et al. Soluble P-selectin, SELP polymorphisms, and atherosclerotic risk in European-American and African-African young adults: the Coronary Artery Risk Development in Young Adults (CARDIA) Study. Arterioscler Thromb Vasc Biol 2008; 28: 1549-1555.
    CrossrefPubMedGoogle Scholar
  • 21 Volcik KA, Catellier D, Folsom AR. et al. SELP and SELPLG genetic variation is associated with cell surface measures of SELP and SELPLG: the Atherosclerosis Risk in Communities Carotid MRI Study. Clin Chem 2009; 55: 1076-1082.
    CrossrefPubMedGoogle Scholar
  • 22 Miller MA, Kerry SM, Dong Y. et al. Association between the Thr715Pro P-selectin gene polymorphism and soluble P-selectin levels in a multiethnic population in South London. Thromb Haemost 2004; 92: 1060-1065.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 23 Volcik KA, Ballantyne CM, Coresh J. et al. P-selectin Thr715Pro polymorphism predicts P-selectin levels but not risk of incident coronary heart disease or ischemic stroke in a cohort of 14595 participants: the Atherosclerosis Risk in Communities Study. Atherosclerosis 2006; 186: 74-79.
    CrossrefPubMedGoogle Scholar
  • 24 Ghazouani L, Abboud N, Khlifa SB. et al. P-selectin gene polymorphisms and risk of coronary heart disease among Tunisians. J Thromb Thrombolysis 2009; 28: 314-319.
    CrossrefPubMedGoogle Scholar
  • 25 Uitte de Willige S, De Visser MC, Vos HL. et al. Selectin haplotypes and the risk of venous thrombosis: influence of linkage disequilibrium with the factor V Leiden mutation. J Thromb Haemost 2008; 6: 478-485.
    CrossrefPubMedGoogle Scholar
  • 26 Kuge O, Dascher C, Orci L. et al. Sar1 promotes vesicle budding from the endoplasmic reticulum but not Golgi compartments. J Cell Biol 1994; 125: 51-65.
    CrossrefPubMedGoogle Scholar
  • 27 Bhalla-Gehi R, Penuela S, Churko JM. et al. Pannexin1 and pannexin3 delivery, cell surface dynamics, and cytoskeletal interactions. J Biol Chem 2008; 285 (12) 9147-9160.
    PubMedGoogle Scholar
  • 28 Marie M, Sannerud R, Avsnes Dale H. et al. Take the 'A' train: on fast tracks to the cell surface. Cell Mol Life Sci 2008; 65: 2859-2874.
    CrossrefPubMedGoogle Scholar
  • 29 Chen WQ, Kang SU, Lubec G. Protein profiling by the combination of two independent mass spectrometry techniques. Nat Protoc 2006; 1: 1446-1452.
    CrossrefPubMedGoogle Scholar
  • 30 Suzuki K, Ota H, Sasagawa S. et al. Assay method for myeloperoxidase in human polymorphonuclear leukocytes. Anal Biochem 1983; 132: 345-352.
    CrossrefPubMedGoogle Scholar
  • 31 Rosin C, Brunner M, Lehr S. et al. The formation of platelet-leukocyte aggregates varies during the menstrual cycle. Platelets 2006; 17: 61-66.
    CrossrefPubMedGoogle Scholar
  • 32 Koedam JA, Cramer EM, Briend E. et al. P-selectin, a granule membrane protein of platelets and endothelial cells, follows the regulated secretory pathway in AtT-20 cells. J Cell Biol 1992; 116: 617-625.
    CrossrefPubMedGoogle Scholar
  • 33 Fujimoto T, Stroud E, Whatley RE. et al. P-selectin is acylated with palmitic acid and stearic acid at cysteine 766 through a thioester linkage. J Biol Chem 1993; 268: 11394-11400.
    PubMedGoogle Scholar
  • 34 Charollais J, Van Der Goot FG. Palmitoylation of membrane proteins (Review). Mol Membr Biol 2009; 26: 55-66.
    CrossrefPubMedGoogle Scholar
  • 35 Greaves J, Salaun C, Fukata Y. et al. Palmitoylation and membrane interactions of the neuroprotective chaperone cysteine-string protein. J Biol Chem 2008; 283: 25014-25026.
    CrossrefPubMedGoogle Scholar
  • 36 Sim DS, Dilks JR, Flaumenhaft R. Platelets possess and require an active protein palmitoylation pathway for agonist-mediated activation and in vivo thrombus formation. Arterioscler Thromb Vasc Biol 2007; 27: 1478-1485.
    CrossrefPubMedGoogle Scholar
  • 37 Nagy Jr. B, Csongradi E, Bhattoa HP. et al. Investigation of Thr715Pro P-selectin gene polymorphism and soluble P-selectin levels in type 2 diabetes mellitus. Thromb Haemost 2007; 98: 186-191.
    Article in Thieme ConnectPubMedGoogle Scholar