Mutation of the H-helix in antithrombin decreases heparin stimulation of protease inhibition.
Blood clotting proceeds through the sequential proteolytic activation of a series of serine proteases, culminating in thrombin cleaving fibrinogen into fibrin. The serine protease inhibitors (serpins) antithrombin (AT) and protein C inhibitor (PCI) both inhibit thrombin in a heparin-accelerated reaction. Heparin binds to the positively charged D-helix of AT and H-helix of PCI. The H-helix of AT is negatively charged, and it was mutated to contain neutral or positively charged residues to see if they contributed to heparin stimulation or protease specificity in AT. To assess the impact of the H-helix mutations on heparin stimulation in the absence of the known heparin-binding site, negative charges were also introduced in the D-helix of AT. AT with both positively charged H- and D-helices showed decreases in heparin stimulation of thrombin and factor Xa inhibition by 10- and 5-fold respectively, a decrease in affinity for heparin sepharose, and a shift in the heparin template curve. In the absence of a positively charged D-helix, changing the H-helix from neutral to positively charged increased heparin stimulation of thrombin inhibition 21-fold, increased heparin affinity and restored a normal maximal heparin concentration for inhibition.
Gonzales
Patrick R
PR
Biology Department, University of Wisconsin-La Crosse, La Crosse, WI 54601, USA.
Walston
Timothy D
TD
Camacho
Laureano O
LO
Kielar
Dana M
DM
Church
Frank C
FC
Rezaie
Alireza R
AR
Cooper
Scott T
ST
D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000378:metabolism*; D000991:Antithrombins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D018411:Spodoptera
D000925:Anticoagulants; D000991:Antithrombins; D006493:Heparin