Exosites 1 and 2 are essential for protection of fibrin-bound thrombin from heparin-catalyzed inhibition by antithrombin and heparin cofactor II.
Assembly of ternary thrombin-heparin-fibrin complexes, formed when fibrin binds to exosite 1 on thrombin and fibrin-bound heparin binds to exosite 2, produces a 58- and 247-fold reduction in the heparin-catalyzed rate of thrombin inhibition by antithrombin and heparin cofactor II, respectively. The greater reduction for heparin cofactor II reflects its requirement for access to exosite 1 during the inhibitory process. Protection from inhibition by antithrombin and heparin cofactor II requires ligation of both exosites 1 and 2 because minimal protection is seen when exosite 1 variants (gamma-thrombin and thrombin Quick 1) or an exosite 2 variant (Arg93 --> Ala, Arg97 --> Ala, and Arg101 --> Ala thrombin) is substituted for thrombin. Likewise, the rate of thrombin inhibition by the heparin-independent inhibitor, alpha1-antitrypsin Met358 --> Arg, is decreased less than 2-fold in the presence of soluble fibrin and heparin. In contrast, thrombin is protected from inhibition by a covalent antithrombin-heparin complex, suggesting that access of heparin to exosite 2 of thrombin is hampered when ternary complex formation occurs. These results reveal the importance of exosites 1 and 2 of thrombin in assembly of the ternary complex and the subsequent protection of thrombin from inhibition by heparin-catalyzed inhibitors.
Department of Medicine, McMaster University and Hamilton Civic Hospitals Research Centre, Hamilton, Ontario L8V 1C3, Canada.
D L
DL
Becker
J C
JC
Fredenburgh
A R
AR
Stafford
J I
JI
Weitz
D000925:Anticoagulants / Q000378:metabolism* / Q000494:pharmacology; D000991:Antithrombins / Q000378:metabolism* / Q000494:pharmacology; D001777:Blood Coagulation; D005337:Fibrin / Q000737:chemistry / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism* / Q000494:pharmacology; D015844:Heparin Cofactor II / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D011485:Protein Binding; D011487:Protein Conformation; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism*
D000925:Anticoagulants; D000991:Antithrombins; D015842:Serine Proteinase Inhibitors; D015844:Heparin Cofactor II; D005337:Fibrin; D006493:Heparin; D013917:Thrombin