Sulfated, low-molecular-weight lignins are potent inhibitorsof plasmin, in addition to thrombin and factor Xa: Novel opportunity for controlling complex pathologies

Abstract

Recently we prepared sulfated, low-molecular-weight lignins (LMWLs) to mimic the biological activities of heparin and heparan sulfate. Chemo-enzymatically prepared sulfated LMWLs represent a library of diverse non-sugar, aromatic molecules with structures radically different from the heparins, and have been found to potently inhibit thrombin and factor Xa. To assess their effect on the fibrinolytic system, we studied the interaction of LMWLs with human plasmin. Enzyme inhibition studies indicate that the three sulfated LMWLs studied inhibit plasmin with IC50 values in the range of 0.24 and 1.3 mM, which are marginally affected in the presence of antithrombin. Similarly, plasmin degradation of polymeric fibrin is also inhibited by sulfated LMWLs. Michaelis-Menten kinetic studies indicate that maximal velocity of hydrolysis of chromogenic substrates decreases nearly 70% in the presence of LMWLs, while the effect on Michaelis constant is dependent on the nature of the substrate. Competitive binding studies indicate that the sulfated LMWLs compete with full-length heparin. Comparison with thrombin-heparin crystal structure identifies an anionic region on plasmin as a plausible sulfated LMWL binding site. Overall, the chemo-enzymatic origin coupled with coagulation and fibrinolysis inhibition properties of sulfated LMWLs present novel opportunities for designing new pharmaceutical agents that regulate complex pathologies in which both systems are known to play important roles such as disseminated intravascular coagulation.

Figure 1. Comparison of structures of heparins (A) and sulfated LMWLs (B)

A) UFH or LMW heparin is a polysaccharide composed of alternating glucosamine and uronic acid (either iduronic or glucuronic) residues that are linked to each other in 1→ 4 manner. The saccharides residues are variably sulfated resulting in phenomenal structural heterogeneity. The average molecular weight of UFH is ~15,000, while it is 3,000–5,000 for LMW heparins. Heparins contain an average of ~2.6 sulfates per disaccharide unit. B) CDSO3, FDSO3 and SDSO3 are chemo-enzymatically prepared oligomers, which can also be thought of as a complex polymer composed of dihydrobenzofuran and phenoxy propanoic acid units connected through β-O-4 or β-5 linkages (shown as shaded ovals). The average molecular weight of sulfated LMWLs is in the range of 3,000–4,000. On average these molecules contain one sulfate group every 2 – 3 monomer units.

Figure 2. Direct inhibition of plasmin by sulfated LMWLs

The inhibition of human plasmin by CDSO3 (squares), FDSO3 (triangles) and SDSO3 (circles) was determined spectrophotometrically through a chromogenic substrate hydrolysis assay at pH 7.4 and 25 °C. Solid lines represent sigmoidal fits to the data to obtain IC₅₀, HS, Y_M, and Y_O values (equation I), as described in the Experimental Procedures.

Figure 3. Inhibition of fibrinolysis by a sulfated LMWL

Human plasmin hydrolysis of fibrin was followed in a spectrophotometric assay in the presence of varying concentrations of FDSO3 (●=1.3 μM, □=3.9 μM, ▲=4.5 μM, ○=6.4 μM) at pH 7.4 and 37 °C. The increase in transmittance at 600 nm due to plasmin cleavage of fibrin was monitored as a function of time and fitted by an exponential equation to derive the observed rate constant of inhibition (k_OBS) and maximal change in transmittance (A_max). Solid lines represent the exponential fits to the data.

Figure 4. Michaelis-Menten kinetics of Spectrozyme PL (A) and Spectrozyme (TH) hydrolysis by human plasmin in the presence of CDSO3

The initial rate of hydrolysis at various substrate concentrations was measured spectrophotometrically in pH 7.4 buffer as described in ‘Experimental Procedures’. The concentrations of CDSO3 chosen for study include 0 (○), 30 (△), 60 (□), and 90 nM (◊). Solid lines represent non-linear regressional fits to the data by the Michaelis-Menten equation III.

Figure 5. Interaction of FDSO3 with human plasmin in pH 7.4 buffer at 25 °C in the presence and absence of heparin

The decrease in intrinsic fluorescence of plasmin (λ_EX = 280 nm, λ_EM = 340 nm) that accompanies binding of FDSO3 was used to determine the K_D of FDSO3–plasmin complex. UFH was present in the titrations at 0 (□), 29 (○) and 296 nM (△) concentrations. Solid lines represent non-linear fits to the data using quadratic equation II. See Experimental Procedures for details.

Figure 6. Comparison of crystal structures of human plasmin and thrombin

Shown is a comparison of plasmin and thrombin structures from ‘1BML’ (plasmin – streptokinase complex) and 1XMN (thrombin – heparin octasaccharide complex) files. The structures were aligned and the orientation of individual residues in the two structures compared to assess similarities and differences. Several basic (shown in blue van der Waals surfaces) and hydrophobic (shown in orange van der Waals surfaces) residues in exosite II of human thrombin were found to correlate well with comparable residues present in human plasmin. Heparin oligosaccharide present in the 1XMN structure is shown in the stick representation. See text for details.