Inhibition of dysthrombins Quick I and II by heparin cofactor II and antithrombin.
Heparin cofactor II and antithrombin are plasma serine proteinase inhibitors whose ability to inhibit alpha-thrombin is accelerated by glycosaminoglycans. Dysfunctional thrombin mutants Quick I (Arg67-->Cys) and Quick II (Gly226-->Val) were used to further compare heparin cofactor II and antithrombin interactions. Quick I, Quick II, and alpha-thrombin were eluted at the same salt concentration from heparin-Sepharose suggesting that the putative heparin-binding site (also termed anion binding exosite-II) is functional. Antithrombin yielded similar inhibition rates for Quick I and alpha-thrombin in the absence or presence of various amounts of heparin. Also, Quick I was inhibited similarly to alpha-thrombin by heparin cofactor II in the absence of glycosaminoglycan. In contrast, glycosaminoglycan-accelerated Quick I inhibition by heparin cofactor II was greatly reduced indicating that anion binding exosite-I (where the mutation occurs in Quick I) is critical for increased inhibition by heparin cofactor II. We also found that heparin cofactor II formed a SDS-resistant bimolecular complex with Quick II and alpha-thrombin at similar rates and the rate of complex formation was accelerated in the presence of glycosaminoglycans. A three-dimensional molecular model of the Quick II active site compared to alpha-thrombin suggested that the heparin cofactor II Leu-Ser-reactive site sequence (P1-P1') is a compatible "pseudosubstrate" in contrast to the Arg-Ser sequence found in antithrombin. The importance of heparin cofactor II as a thrombin regulator will depend upon its ability to interact with glycosaminoglycans and the functional availability of thrombin exosites.
Phillips
J E
JE
Center for Thrombosis and Hemostasis, University of North Carolina School of Medicine, Chapel Hill 27599-7035.
Shirk
R A
RA
Whinna
H C
HC
Henriksen
R A
RA
Church
F C
FC
D000595:Amino Acid Sequence; D000991:Antithrombins / Q000494:pharmacology*; D002846:Chromatography, Affinity; D006493:Heparin / Q000378:metabolism; D015844:Heparin Cofactor II / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D000991:Antithrombins; D015844:Heparin Cofactor II; D006493:Heparin; D013917:Thrombin; C027648:thrombin Quick