Metals affect the structure and activity of human plasminogen activator inhibitor‐1. I. Modulation of stability and protease inhibition

Abstract

Human plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor with a metastable active conformation. Under physiological conditions, half of the inhibitor transitions to a latent state within 1-2 h. The interaction between PAI-1 and the plasma protein vitronectin prolongs this active lifespan by~50%. Previously, our group demonstrated that PAI-1 binds to resins using immobilized metal affinity chromatography (Day, U.S. Pat. 7,015,021 B2, March 21, 2006). In this study, the effect of these metals on function and stability was investigated by measuring the rate of the transition from the active to latent conformation. All metals tested showed effects on stability, with the majority falling into one of two types depending on their effects. The first type of metal, which includes magnesium, calcium and manganese, invoked a slight stabilization of the active conformation of PAI-1. A second category of metals, including cobalt, nickel and copper, showed the opposite effects and a unique vitronectin-dependent modulation of PAI-1 stability. This second group of metals significantly destabilized PAI-1, although the addition of vitronectin in conjunction with these metals resulted in a marked stabilization and slower conversion to the latent conformation. In the presence of copper and vitronectin, the halflife of active PAI-1 was extended to 3 h, compared to a half-life of only~30 min with copper alone. Nickel had the largest effect, reducing the half-life to~5 min. Together, these data demonstrate a heretofore-unknown role for metals in modulating PAI-1 stability.

Metals affect the structure and activity of human plasminogen activator inhibitor‐1. II. Binding affinity and conformational changes

“…This was accomplished initially via steady‐state binding experiments using surface plasmon resonance (SPR) and then was expanded to provide insight into the binding mechanism using transient‐state kinetic measurements. Consistent with our previous metal categorization,35 the Type I and II metals promote different changes in PAI‐1 intrinsic protein fluorescence upon binding, suggesting that each group of metals binds via unique interactions that induce differing effects on PAI‐1 structure. These data also indicate that cobalt and nickel bind to PAI‐1 with a dissociation constant in the low micromolar range, whereas copper binds with ∼200‐fold stronger affinity.…”

“…The concentrations of metals used in our previous PAI‐1 stability measurements were intentionally set to high values35 to ensure that the effects were measured under saturating conditions. Thus, it is now important to evaluate binding directly to determine whether metal affinities are in a physiological range.…”

“…These two proteins are located primarily in plasma and the extracellular space where they inhibit fibrinolysis or extracellular proteolysis, control cellular adhesion and migration, and assist in the inflammatory response 24–34. In our previous report35, we have shown that calcium, magnesium, and manganese (referred to as Type I metals) have a modest stabilizing effect on the t 1/2 of PAI‐1, whereas cobalt, copper, and nickel (referred to as Type II metals) effect a vitronectin‐ dependent modulation of the t 1/2 of PAI‐1. The Type II metals induce a substantial destabilization of PAI‐1, reducing t 1/2 to a few minutes.…”

Crystal Structure of Plasminogen Activator Inhibitor-1 in an Active Conformation with Normal Thermodynamic Stability

“…It should be noted that adding submicromolar concentrations of either divalent zinc or copper ions to the crystallization drops caused immediate precipitation, supporting a specific interaction of the metals with PAI-1. This specific metal binding that leads to a conformational change may be relevant to recently published work that shows a destabilization of the active conformation of PAI-1 by copper, nickel, or cobalt and metal effects that are modulated by vitronectin binding to PAI-1 (32,33).…”

Vitronectin Inhibits Efferocytosis through Interactions with Apoptotic Cells as well as with Macrophages

“…Vitronectin lacking the SMB domain, delta SMB mutant (ΔSMB), or isolated SMB domain were expressed in Drosophila S2 cells using methods reported by Schar et al (38), and proteins purified as described by Thompson et al (39). Cyclo(Arg-Gly-Asp-D-Phe-Val) RGDfv and cyclo(Arg-Ala-Asp-D-Phe-Val) RADfv were purchased from Enzo Life Science (Plymouth Meeting, PA), whereas RGD-FITC was from AnaSpec (Fremont, CA).…”