Relationship between post-translational glycosylation and anticoagulant function of secretable recombinant mutants of human thrombomodulin.
Two glycoforms of a soluble mutant of recombinant human thrombomodulin (rec.TM) were used to identify critical N- and O-linked glycans of the endothelial cell thrombin receptor. While N-linked glycans were not found to be involved in any function of rec.TM, an acidic chondroitin sulphate-like glycosaminoglycan (CSGAG) was found to be critical for all the direct anticoagulant functions of rec.TM, including inhibition of thrombin-mediated platelet aggregation. A glycoform of rec.TM lacking CSGAG had very poor anticoagulant activity. Furthermore, the glycoform of rec.TM possessing CSGAG showed strong inhibition by and had high affinity for poly-cationic basic proteins, whereas the CSGAG-deficient rec.TM did not. Monoclonal antibody binding as well as lectin mapping of rec.TM with agglutinins identified sialic acid containing O-linked glycans in both glycoforms additional to the CSGAG in high molecular weight rec.TM These findings define important molecular interactions modulating the anticoagulant function of TM, which appear to be critically regulated by CSGAG, and also showed that the overall post-translational glycosylation pattern of the two glycoforms was very similar except for the presence of CSGAG. The possibility exists that differently expressed glycoforms of TM may be crucial for the expression of endothelial cell-related anticoagulant potential in different vascular beds.
Koyama
T
T
Haemostasis Research Unit, Kerckhoff-Klinik, Max-Planck-Gesellschaft, Bad Nauheim, Germany.
Parkinson
J F
JF
Aoki
N
N
Bang
N U
NU
Müller-Berghaus
G
G
Preissner
K T
KT
D000911:Antibodies, Monoclonal / Q000378:metabolism; D001777:Blood Coagulation / Q000502:physiology*; D015153:Blotting, Western; D006031:Glycosylation; D006801:Humans; D010974:Platelet Aggregation / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D018179:Receptors, Thrombin; D011994:Recombinant Proteins / Q000235:genetics; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D011994:Recombinant Proteins