Relationship between anticoagulant activities and polyanionic properties of rabbit thrombomodulin.
Rabbit thrombomodulin displays three distinct blood anticoagulant activities: it promotes the activation of protein C by thrombin (protein C activation cofactor activity); it promotes the inactivation of thrombin by thrombin (direct anticoagulant activity). The effects on these activities of mouse anti-thrombomodulin monoclonal antibodies and of the heparin-neutralizing proteins, platelet factor 4, histidine-rich glycoprotein, and S-protein, were investigated. One of the antibodies, which did not influence the functional properties of thrombomodulin, was used as an immunoaffinity ligand for purification of the protein. Two other antibodies, which were found to abrogate the protein C activation cofactor activity of the purified thrombomodulin, also abolished the antithrombin-dependent and the direct anticoagulant activities. The heparin-neutralizing proteins all inhibited the two latter activities, albeit to a varying extent, but did not appreciably affect the activation of protein C. These results are interpreted in relation to our previous finding that rabbit thrombomodulin contains an acidic domain, tentatively identified as a sulfated glycosaminoglycan (Bourin, M.-C., Boffa, M.-C., Björk, I., and Lindahl, U. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5924-5928). It is proposed that the acidic domain interacts with thrombin at the protein C activation site and that this interaction is a prerequisite to the expression of direct as well as antithrombin-dependent anticoagulant activity. The interaction is not essential to, but compatible with, the activation of protein C. Experiments involving treatment of thrombomodulin with various glycanases or with nitrous acid, followed by measurement of anticoagulant activities, indicated that the acidic domain is constituted by a sulfated galactosaminoglycan and not by a heparin-related polysaccharide as previously suggested.
Bourin
M C
MC
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala.
Ohlin
A K
AK
Lane
D A
DA
Stenflo
J
J
Lindahl
U
U
D000818:Animals; D000911:Antibodies, Monoclonal; D000991:Antithrombins / Q000378:metabolism; D001777:Blood Coagulation / Q000187:drug effects*; D006494:Heparin Antagonists / Q000494:pharmacology; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D011486:Protein C / Q000378:metabolism; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000494:pharmacology*; D018179:Receptors, Thrombin
D000911:Antibodies, Monoclonal; D000991:Antithrombins; D006494:Heparin Antagonists; D046911:Macromolecular Substances; D011486:Protein C; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin