Recombinant human thrombomodulin. Regulation of cofactor activity and anticoagulant function by a glycosaminoglycan side chain

Abstract

Two glycoforms of a secretable human thrombomodulin mutant [TMD1-105 and TMD1-75; Parkinson, Grinnell, Moore, Hoskins, Vlahos & Bang (1990) J. Biol. Chem. 265, 12602-12610] were expressed in human 293 cells and used to study the role of glycosylation in the functions of this endothelial-cell thrombin receptor. Carbohydrate content analysis and intrinsic labelling with [3H]glucosamine and [35S]sulphate showed that TMD1-105 contained a chondroitin sulphate whereas TMD1-75 did not. Other than chondroitin sulphate, the carbohydrate contents of the two glycoforms were identical, indicating similar glycosylation patterns at other O-linked and N-linked sites in the two glycoforms. The properties of TMD1-105 were converted into those of TMD1-75 by chondroitin ABC lyase digestion. Trypsin digestion of labelled TMD1-105 permitted isolation of two overlapping peptides that contained chondroitin sulphate, spanned the entire O-glycosylation domain and had O-glycosylation sites at Ser-492, Ser-498, Thr-500, Thr-504 and Thr-506. The chondroitin sulphate-attachment site was assigned to Ser-492 as this residue is conserved in mouse and bovine thrombomodulin and lies within a sequence Ser-Gly-Ser-492-Gly-Glu-Pro, which has strong similarity to chondroitin sulphate attachment sites in other proteoglycans. Five peptides with N-linked carbohydrate were also isolated and contained glycosylation sites in the lectin-like domain (Asn-47, Asn-115, Asn-116) and in the fourth (Asn-382) and fifth (Asn-409) epidermal growth factor domains. The role of N-linked and simple O-linked carbohydrates in the functions of human thrombomodulin remain unclear. The present studies demonstrate, however, that the presence of chondroitin sulphate in human thrombomodulin has profound effects on all of the anticoagulant properties of this important anticoagulant thrombin receptor.

Novel Proteoglycan Linkage Tetrasaccharides of Human Urinary Soluble Thrombomodulin, SO4-3GlcAβ1–3Galβ1–3(±Siaα2–6)Galβ1–4Xyl

“…They have also shown that uTM improves disseminated intravascular coagulation without excessive prolongation of the activated partial thromboplastin time (6). The protein possesses five potential N-linked glycosylation sites (7) as deduced from its amino acid sequence, whereas the detection of GalNAc suggests the presence of Olinked sugar chains (8). Although uTM does not contain a glycosaminoglycan, recombinant TM and some TMs obtained from cultured human endothelial cells are expressed in both a high molecular weight TM containing chondroitin sulfate and a low molecular weight TM lacking this modification (9,10).…”

Eosinophil cationic granule proteins impair thrombomodulin function. A potential mechanism for thromboembolism in hypereosinophilic heart disease.

“…Similarly, as shown in Fig. 5 (10)(11)(12)(14)(15)(16)(17)(18)(19)(20). This unique GAG is comprised of chondroitin sulfate-like dissaccharides, some of which are unusually hypersulfated (and hence more anionic) because they contain two rather than one sulfate per disaccharide unit ( 14).…”

“…To investigate directly the role of the TM GAG domain in MBP inhibition ofTM function, we used a pair ofrecombinant human TM mutant proteins, both containing the entire extracellular domain and differing only in the presence or absence of the chondroitin sulfate-like GAG moiety ( 19,20,22). These proteins migrate on SDS-PAGE with apparent molecular masses 105 kD (TMD-105) and 75 kD (TMD-75).…”

“…With regard to the second possibility, we have found in preliminary experiments that MBP appears to unmask a high affinity calcium-binding site on TMD-105, converting its APC-generating calcium concentration optimum to resemble that of TMD-75 (22), chondroitin ABC Iyase-cleaved TMD-105 (20), and elastase-cleaved rabbit TM (48), with maximum activity at subphysiologic (0.3 mM) calcium concentrations. Finally, given the relative potency of "platelet releasate" as an inhibitor of TM-dependent APC generation ( 18) and the pronounced similarities of platelet Factor 4 and MBP with regard to cationicity, heparin binding, and inhibition of angiogenesis (49,50), it will be of great interest to see if platelet Factor 4 or some other cationic component released by activated platelets also inhibits TM function of endothelial monolayers, a finding of obvious physiologic as well as pathologic relevance.…”

“…APC, in turn, is a powerful anticoagulant serine protease that, in conjunction with protein S, terminates the procoagulant activity of Factors Va and VIIla (10)(11)(12). Of note, the complete TM molecule is quite anionic (pI 4 [131]), in part because ofextensive posttranslational glycation of the large extracellular domain of TM with an unusual hypersulfated, chondroitin sulfate E-like moiety (14)(15)(16)(17)(18)(19)(20). This bulky polyanionic domain strongly influences all three known anticoagulant functions of TM (14)(15)(16)(17)(18)(19)(20)(21)(22)(23).…”