Abstract
Lamprey angiotensinogen (l-ANT) is a hormone carrier in the regulation of blood pressure, but it is also a heparin-dependent thrombin inhibitor in lamprey blood coagulation system. The detailed mechanisms on how angiotensin is carried by l-ANT and how heparin binds l-ANT and mediates thrombin inhibition are unclear. Here we have solved the crystal structure of cleaved l-ANT at 2.7 Å resolution and characterized its properties in heparin binding and protease inhibition. The structure reveals that l-ANT has a conserved serpin fold with a labile N-terminal angiotensin peptide and undergoes a typical stressed-to-relaxed conformational change when the reactive center loop is cleaved. Heparin binds l-ANT tightly with a dissociation constant of ∼10 nm involving ∼8 monosaccharides and ∼6 ionic interactions. The heparin binding site is located in an extensive positively charged surface area around helix D involving residues Lys-148, Lys-151, Arg-155, and Arg-380. Although l-ANT by itself is a poor thrombin inhibitor with a second order rate constant of 500 m-1 s-1, its interaction with thrombin is accelerated 90-fold by high molecular weight heparin following a bell-shaped dose-dependent curve. Short heparin chains of 6-20 monosaccharide units are insufficient to promote thrombin inhibition. Furthermore, an l-ANT mutant with the P1 Ile mutated to Arg inhibits thrombin nearly 1500-fold faster than the wild type, which is further accelerated by high molecular weight heparin. Taken together, these results suggest that heparin binds l-ANT at a conserved heparin binding site around helix D and promotes the interaction between l-ANT and thrombin through a template mechanism conserved in vertebrates.
Highlights
Lamprey angiotensinogen (l-ANT) is a hormone carrier in the regulation of blood pressure, but it is a heparin-dependent thrombin inhibitor in lamprey blood coagulation system
The interaction between l-ANT and thrombin was enhanced by high molecular weight (HMW) heparin, with significantly more l-ANT1⁄7thrombin complexes formed (Fig. 2C, lane 4); the well characterized H5* that promotes factor Xa inhibition by antithrombin had no effect on the inhibition of thrombin by l-ANT. l-ANT did not inhibit factor Xa, but the P1R variant did form a complex with factor Xa
Both HMW heparin and H5* had little effect on the activity of P1R variant toward factor Xa. These data confirm that l-ANT is a relatively specific thrombin inhibitor, and its activity is enhanced by HMW heparin but not heparin pentasaccharide
Summary
Lamprey angiotensinogen (l-ANT) is a hormone carrier in the regulation of blood pressure, but it is a heparin-dependent thrombin inhibitor in lamprey blood coagulation system. An l-ANT mutant with the P1 Ile mutated to Arg inhibits thrombin nearly 1500-fold faster than the wild type, which is further accelerated by high molecular weight heparin. Taken together, these results suggest that heparin binds l-ANT at a conserved heparin binding site. Lamprey angiotensinogen (l-ANT) has an Ile as the P1 residue instead of a thrombin-preferred Arg. The interactions between serpins and proteases of blood coagulation are often regulated by cofactors such as heparin [23]. This study provides information on how the angiotensin system and the blood coagulation system overlapped and diverged during evolution
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