The use of sequence-specific antibodies to identify a secondary binding site in thrombin.
The peptide comprising residues 62-73 of the B-chain of human alpha-thrombin was synthesized and polyclonal antibodies raised against it. These antibodies were found to bind to the synthetic peptide, a CNBr fragment, and a proteolytic subfragment containing this sequence, as well as the entire thrombin molecule. The purified antibodies had no effect on the hydrolysis by thrombin of D-Phe-pipecolyl-Arg-p-nitroanilide and caused only a minimal decrease (20%) in the second-order rate constant for inactivation by antithrombin III. On the other hand, the antibodies competitively inhibited the binding of hirudin over the concentration range tested (0-43 nM), and a dissociation constant of 3.4 +/- 0.5 nM was found for the antibodies. The release of fibrinopeptide A from the A alpha-chain of fibrinogen by thrombin was competitively inhibited with an inhibition constant of 11.7 +/- 0.4 nM. The activation of protein C by thrombin in the presence of thrombomodulin was also inhibited by the antibodies, and an apparent inhibition constant of 10.7 +/- 1.5 nM was found. In contrast, the antibodies had no effect on the activation of protein C in the absence of thrombomodulin. These results are discussed in relation to data obtained recently on the interaction of well defined proteolytic derivatives of human alpha-thrombin with the ligands described above.
Noé
G
G
Friedrich Miescher Institut, Basel, Switzerland.
Hofsteenge
J
J
Rovelli
G
G
Stone
S R
SR
D000906:Antibodies / Q000502:physiology; D000990:Antithrombin III / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D003488:Cyanogen Bromide; D005340:Fibrinogen / Q000378:metabolism; D005344:Fibrinopeptide A / Q000378:metabolism; D006629:Hirudins / Q000378:metabolism; D006801:Humans; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism*; D011486:Protein C / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000494:pharmacology; D018179:Receptors, Thrombin; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D006629:Hirudins; D010446:Peptide Fragments; D011486:Protein C; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D005344:Fibrinopeptide A; D000990:Antithrombin III; D005340:Fibrinogen; D013917:Thrombin; D003488:Cyanogen Bromide