Heparin Binds Lamprey Angiotensinogen and Promotes Thrombin Inhibition through a Template Mechanism

Abstract

Edited by Gerald HartLamprey 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 ϳ10 nM involving ϳ8 monosaccharides and ϳ6 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.Angiotensinogen genes exist throughout all vertebrates and have evolved over 500 million years from early cyclostome (lamprey) to human (1-3). Angiotensinogen functions as the carrier of active angiotensin hormones of the renin-angiotensin system that regulates body fluid homeostasis, blood pressure, and blood vessel formation (4, 5).Early studies have shown that angiotensinogen with the angiotensin peptide located at its N terminus belongs to the serine protease inhibitor (serpin) superfamily (6, 7). All serpins are known to share similar tertiary structures with three ␤-sheets, 8 -9 ␣-helices, and an exposed reactive center loop (RCL) 4 that serves as a bait for proteolytic attack (8, 9). Once the target protease recognizes and cleaves RCL, the serpin undergoes a typical serpin stressed-to-relaxed (S-to-R) transition with the insertion of the cleaved RCL into the central ␤-sheet as a middle strand, which leads to translocation and inactivation of the covalently linked protease (10, 11).Classically, angiotensinogen is regarded as a passive hormone carrier and a non-inhibitory serpin that lacks serpin S-to-R conformational rearrangements upon RCL cleavage (12, 13); however, recent studies have found that angiotensinogen derived from lamprey, a representative of an early lineage of vertebrate, to be an inhi...

Heparin Blocks the Inhibition of Tissue Kallikrein 1 by Kallistatin through Electrostatic Repulsion

“…Stoichiometries of inhibition (SI) were measured using similar procedures as previously described [ 32 ] by incubating KLK1, KLK1-70Lm, and KLK1-90Lm (1 μM) with increasing concentrations of kallistatin variants in phosphate buffered saline (PBS) with 0.1% PEG 8000 for 2 h at 37 °C. Residual protease activity was determined by diluting the reaction mixture into 0.2 mM H-D-Val-Leu-Arg-AMC for KLK1.…”

“…The rates of inhibition by recombinant kallistatin variants were determined at room temperature (22 ± 1 °C) by a discontinuous assay procedure as previously described [ 32 ]. Briefly, under pseudo–first-order conditions, 10 μL of 10 nM proteases was mixed with 10 μL of 100–500 nM kallistatin variants in PBS with 0.1% PEG 8000.…”

“…Kallistatin belongs to a subclass of serpins known as the heparin binding serpins. In most cases, heparin binds to residues of the serpin helix D as with antithrombin [ 19 , 20 , 21 , 22 , 23 ], heparin cofactor II (HCII) [ 24 , 25 , 26 ], protease nexin 1 (PN1) [ 27 , 28 ], plasminogen activator inhibitor type 1 (PAI-1) [ 29 ], protein Z-dependent protease inhibitor (ZPI) [ 30 , 31 ], and lamprey angiotensinogen [ 32 ]. The binding of heparin promotes the interaction between serpin and protease either by an allosteric mechanism where heparin induces conformational changes in the serpin ( Figure S1B ), or by a bridging mechanism where heparin links serpin and protease together forming a transient ternary complex [ 9 , 33 , 34 ] ( Figure S1C ).…”

How serpins transport hormones and regulate their release

“…No evidence has emerged yet for any influence of the archetypal serpin conformational changes on the role of angiotensinogen in humans or other mammals. However, it is intriguing to note that, in the lamprey, angiotensinogen is an active serpin, acting as an efficient heparin-activated inhibitor of thrombin [62,63].…”

Colorimetric sensing strategy for heparin assay based on PDDA-induced aggregation of gold nanoparticles

“…2 Finally, blood clotting is prevented. 3,4 Therefore, heparin has been developed as a type of clinical anticoagulant drug, which has been widely used in medical therapies for decades. 5 For instance, in hemodialysis and cardiopulmonary bypass, clots may be created with the contact between blood and articial materials, thus, anticoagulant drugs should be injected to prevent possible clotting.…”