Modulation of Serpin Reaction through Stabilization of Transient Intermediate by Ligands Bound to α-Helix F

Abstract

Mechanism-based inhibition of proteinases by serpins involves enzyme acylation and fast insertion of the reactive center loop (RCL) into the central ␤-sheet of the serpin, resulting in mechanical inactivation of the proteinase. We examined the effects of ligands specific to ␣-helix F (␣HF) of plasminogen activator inhibitor-1 (PAI-1) on the stoichiometry of inhibition (SI) and limiting rate constant (k lim ) of RCL insertion for reactions with ␤-trypsin, tissue-type plasminogen activator (tPA), and urokinase. The somatomedin B domain of vitronectin (SMBD) did not affect SI for any proteinase or k lim for tPA but decreased the k lim for ␤-trypsin. In contrast to SMBD, monoclonal antibodies MA-55F4C12 and MA-33H1F7, the epitopes of which are located at the opposite side of ␣HF, decreased k lim and increased SI for every enzyme. These effects were enhanced in the presence of SMBD. RCL insertion for ␤-trypsin and tPA is limited by different subsequent steps of PAI-1 mechanism as follows: enzyme acylation and formation of a loop-displaced acyl complex (LDA), respectively. Stabilization of LDA through the disruption of the exosite interactions between PAI-1 and tPA induced an increase in the k lim but did not affect the SI. Thus it is unlikely that LDA contributes significantly to the outcome of the serpin reaction. These results demonstrate that the rate of RCL insertion is not necessarily correlated with SI and indicate that an intermediate, different from LDA, which forms during the late steps of PAI-1 mechanism, and could be stabilized by ligands specific to ␣HF, controls bifurcation between the inhibitory and the substrate pathways.Serpin (1) plasminogen activator inhibitor-1 (PAI-1), 2 the major endogenous inhibitor of tissue-(tPA) and urokinase-type (uPA) plasminogen activators (2-5) is a well characterized thrombotic risk factor. High levels of PAI-1 inhibit the fibrinolytic system by preventing the formation of plasmin from plasminogen and promote thrombosis (6, 7). An elevated concentration of PAI-1 correlates with an increased rate of ischemic arterial disease (8) as follows: atherosclerosis, angina pectoris (9 -14), and myocardial infarction (15)(16)(17)(18)(19)(20)(21)(22), as well as with deep vein thrombosis and disseminated intravascular coagulation (23-25). Because complete inactivation of PAI-1 is not lifethreatening (26 -28), the development of new approaches to the therapeutic neutralization of PAI-1 is a subject of many studies (for review see Refs. 29,30).Like other serpins, PAI-1 (Scheme 1, I) (31) inactivates a target proteinase (E) through a unique conformational mechanism (Fig. 1). In this mechanism, the formation of the acylenzyme intermediate (EϳIЈ) triggers the spontaneous insertion of a reactive center loop (RCL) as a strand 4 into the central ␤-sheet A, the 70 Å translocation of the acyl-enzyme to the opposite pole of the PAI-1 molecule, and enzyme inactivation because of the mechanical distortion of its catalytic site ( Fig. 1) (32-34). Despite the fact that the structures of both the ...

Targeting of Plasminogen Activator Inhibitor 1 Improves Fibrinolytic Therapy for Tetracycline-Induced Pleural Injury in Rabbits

“…However, it does not predict the exact time of fibrinolysis needed for successful IPFT, as well as whether or not this time correlates with the fibrinolytic activity at 0-80 minutes ( Figures 6A, 6C, and E7B) or with "fibrinolytic potential" (baseline fibrinolytic activity after activation of endogenous plasminogen; Figures 6B and E6A; 0 h uPA). Although the mechanism of intrapleural tPA processing could deviate from that of scuPA (18), the similarity in the effects of MA-33H1F7 and MA-8H9D4 on the "suicide" inhibition of tPA and uPA by PAI-1 (20)(21)(22) support testing a similar approach to enhance tPA-based IPFT. The availability of a number of anti-PAI-1 mAbs, antigen-binding fragments, and single-chain variable fragments, with stoichiometries of inhibition ranging from 1.5-2.0 to greater than 100 allows interventional flexibility, which could expedite future clinical trials.…”

“…MA-33H1F7 and MA-8H9D4 additively redirect more than 95% of the reaction of human PAI-1 with uPA or tPA to the substrate branch the PAI-1 mechanism (20). Moreover, vitronectin, which binds PAI-1 with nanomolar affinity, potentiates PAI-1 neutralization by mAbs (21)(22)(23). In this study, MA-33H1F7 and MA-8H9D4 were selected after in vitro and ex vivo testing against rabbit PAI-1, and used as Protection of urokinase (uPA) (E) from inactivation by endogenous plasminogen activator inhibitor (PAI)-1 (I) by monoclonal antibody (mAb)-mediated redirection of the mechanism from the inhibitory (k i ) to the substrate (k s ) branch.…”

“…Although a number of low-molecular weight inhibitors of PAI-1 are available, monoclonal antibodies (mAbs) remain the most effective and specific for PAI-1 neutralization (19). Among several mechanisms of PAI-1 modulation by mAbs, redirection of the PAI-1 reaction from the inhibitory to the substrate branch ( Figure 1) is one of the best studied and most effective (20)(21)(22)(23). MA-33H1F7 and MA-8H9D4 additively redirect more than 95% of the reaction of human PAI-1 with uPA or tPA to the substrate branch the PAI-1 mechanism (20).…”

Intrapleural Adenoviral Delivery of Human Plasminogen Activator Inhibitor–1 Exacerbates Tetracycline-Induced Pleural Injury in Rabbits

“…The second-order rate constants for the inhibition of PAs with human and rabbit PAI-1 were determined using stopped flow fluorimetry. SDS-PAGE was used to compare the stoichiometry of inhibition (39) for the interactions between PAs and PAI-1 from both species, as previously described (23,40). The rates of mechanism-based inhibition of both uPA ( Figure E2) and tPA (data not shown) by PAI-1 were diffusion-limited (.…”

“…The concentration of PAI-1 is markedly increased in pleural loculation (20)(21)(22). Active PAI-1 spontaneously transitions to its inactive latent form (23), which is predominant under physiological conditions. Although high concentrations of PAI-1 antigen are likewise strongly correlated with the severity of human pleural injury (18,20,21,24), the proportion of PAI-1 that remains active in pleural injury remains unclear, and its contribution to the severity of pleural injury has not, to our knowledge, been previously studied.…”