Ternary vitronectin-thrombin-antithrombin III complexes in human plasma. Detection and mode of association.
Radiolabeled antithrombin III (ATIII) was incubated at 37 degrees C with purified vitronectin (VN) or fibrinogen-deficient plasma before thrombin was added to initiate complex formation. Incorporation of radiolabeled ATIII was detected using polyacrylamide gel electrophoresis (PAGE) and autoradiography. The PAGE conditions appeared to be crucial for the detection of VN.TAT complexes. In the absence of SDS, ternary complexes formed instantaneously, whereas in the presence of SDS, only 50% of the TAT was associated with VN after a 60-min incubation. Formation of ternary complexes could be confirmed by gel filtration of the plasma to which thrombin was added. Furthermore, TAT in patient plasmas (disseminated intravascular coagulation and sepsis) was found to bind to heparin-Sepharose, indicating that this endogenously formed TAT was also associated with VN. The amino-terminal region of VN and the thrombin moiety of the TAT complex were found to be responsible for their interaction, which was stabilized by disulfide bridges. These results indicate that in normal plasma all TAT is complexed with VN. This association alters the conformational state of plasma VN, which appears to be responsible for the clearance of thrombin complexes from the circulation.
de Boer
H C
HC
Department of Haematology, University Hospital, Utrecht, The Netherlands.
de Groot
P G
PG
Bouma
B N
BN
Preissner
K T
KT
D000911:Antibodies, Monoclonal; D000990:Antithrombin III / Q000302:isolation & purification / Q000378:metabolism*; D001345:Autoradiography; D001798:Blood Proteins / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000097:blood* / Q000302:isolation & purification; D006801:Humans; D015151:Immunoblotting; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D013917:Thrombin / Q000302:isolation & purification / Q000378:metabolism*; D019096:Vitronectin
D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D006023:Glycoproteins; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D019096:Vitronectin; D000990:Antithrombin III; D013917:Thrombin