Acute phase protein alpha 1-acid glycoprotein interacts with plasminogen activator inhibitor type 1 and stabilizes its inhibitory activity.

alpha(1)-Acid glycoprotein, one of the major acute phase proteins, was found to interact with plasminogen activator inhibitor type 1 (PAI-1) and to stabilize its inhibitory activity toward plasminogen activators. This conclusion is based on the following observations: (a) alpha(1)-acid glycoprotein was identified to bind PAI-1 by a yeast two-hybrid system. Three of 10 positive clones identified by this method to interact with PAI-1 contained almost the entire sequence of alpha(1)-acid glycoprotein; (b) this protein formed complexes with PAI-1 that could be immunoprecipitated from both the incubation mixtures and blood plasma by specific antibodies to either PAI-1 or alpha(1)-acid glycoprotein. Such complexes could be also detected by a solid phase binding assay; and (c) the real-time bimolecular interactions monitored by surface plasmon resonance indicated that the complex of alpha(1)-acid glycoprotein with PAI-1 is less stable than that formed by vitronectin with PAI-1, but in both cases, the apparent K(D) values were in the range of strong interactions (4.51 + 1.33 and 0.58 + 0.07 nm, respectively). The on rate for binding of PAI-1 to alpha(1)-glycoprotein or vitronectin differed by 2-fold, indicating much faster complex formation by vitronectin than by alpha(1)-acid glycoprotein. On the other hand, dissociation of PAI-1 bound to vitronectin was much slower than that from the alpha(1)-acid glycoprotein, as indicated by 4-fold lower k(off) values. Furthermore, the PAI-1 activity toward urokinase-type plasminogen activator and tissue-type plasminogen activator was significantly prolonged in the presence of alpha(1)-acid glycoprotein. These observations suggest that the complex of PAI-1 with alpha(1)-acid glycoprotein can play a role as an alternative reservoir of the physiologically active form of the inhibitor, particularly during inflammation or other acute phase reactions.     

Immunological identity of heparin-dependent plasma and urinary protein C inhibitor and plasminogen activator inhibitor-3

Purified plasma and urinary protein C inhibitors (PCI) formed heparin-dependent complexes with activated protein C (APC) which were detected by immunoblotting after nondenaturing gel electrophoresis. Bands representing APC.PCI complexes were also seen on immunoblots after incubation of plasma with APC and heparin. The same immunoblot pattern of complexes was detected by three different methods: method A, monoclonal antibody to plasminogen activator inhibitor-3 (PAI-3, urinary urokinase inhibitor) + 125I-labeled anti-mouse IgG; method B, polyclonal antibodies to PCI + 125I-labeled purified plasma PCI; and method C, monoclonal antibody to protein C + 125I-protein C. Plasma depleted of PAI-3 by immunoadsorption with insolubilized monoclonal antibody to PAI-3 showed no detectable antigen or complexes with APC as visualized by methods A or B. This PAI-3-depleted plasma had less than 10% of the heparin-dependent inhibitory activity of normal plasma toward APC. Purified plasma PCI was fully reactive in an enzyme-linked immunoabsorbent assay for PAI-3, and plasma and urinary PCI inhibited urokinase activity in a heparin-dependent manner. These data indicate that heparin-dependent plasma and urinary PCI and PAI-3 are immunologically and functionally very similar if not identical. This observation identifies a new interrelation between the protein C anticoagulant and the fibrinolytic systems. In addition, plasma contains a heparin-independent inhibitor of APC which is not immunologically related to plasma PCI or to PAI-3.     

Purification of a protein from bovine plasma that binds to type 1 plasminogen activator inhibitor and prevents its interaction with extracellular matrix. Evidence that the protein is vitronectin

Type 1 plasminogen activator inhibitor (PAI-1) binds to the extracellular matrix of cultured bovine aortic endothelial cells. Bovine plasma and bovine lung extract contain protein(s) that bind to PAI-1 and prevent this interaction. One of these proteins was purified approximately 425-fold from ammonium sulfate-fractionated plasma using standard chromatographic procedures together with affinity chromatography on PAI-1-Sepharose. The final product consisted of a major polypeptide of Mr 65,000 and two minor polypeptides of Mr 80,000 and 57,000. NH2-terminal amino acid sequence analysis of the Mr 65,000 polypeptide revealed that it was homologous with vitronectin, and antiserum against this purified binding protein recognized vitronectin and vice versa. Immunological analysis using these antisera demonstrated that the three peptides were immunologically related, and that vitronectin was present in the extracellular matrix of bovine endothelial cells and also in bovine lung.     

Vitronectin governs the interaction between plasminogen activator inhibitor 1 and tissue-type plasminogen activator.

The "serpin" plasminogen activator inhibitor 1 (PAI-1) is the fast acting inhibitor of plasminogen activators (tissue-type (t-PA) and urokinase type-PA) and is an essential regulatory protein of the fibrinolytic system. Its P1-P1' reactive center (R346 M347) acts as a "bait" for tight binding to t-PA/urokinase-type PA. In vivo, PAI-1 is encountered in complex with vitronectin, an interaction known to stabilize its activity but not to affect the second-order association rate constant (k1) between PAI-1 and t-PA. Nevertheless, by using PAI-1 reactive site variants (R346M, M347S, and R346M M347S), we show that the binding of vitronectin to the PAI-1 mutant proteins improves plasminogen activator inhibition. In the absence of vitronectin the PAI-1 R346M mutants are virtually inactive toward t-PA (k1 less than 1 x 10(3) M-1 s-1). In contrast, in the presence of vitronectin the rate of association increases about 1,000-fold (k1 of 6-8 x 10(5) M-1 s-1). This inhibition coincides with the formation of serpin-typical, sodium dodecyl sulfide-stable t-PA.PAI-1 R346M (R346M M347S) complexes. As evidenced by amino acid sequence analysis, the newly created M346-M/S347 peptide bond is susceptible to attack by t-PA, similar to the wild-type R346-M347 peptide bond, indicating that in the presence of vitronectin M346 functions as an efficient P1 residue. In addition, we show that the inhibition of t-PA and urokinase-type PA by PAI-1 mutant proteins is accelerated by the presence of the nonprotease A chains of the plasminogen activators.     

Activated protein C mutant with minimal anticoagulant activity, normal cytoprotective activity, and preservation of thrombin activable fibrinolysis inhibitor-dependent cytoprotective functions

Activated protein C (APC) reduces mortality in severe sepsis patients and exhibits beneficial effects in multiple animal injury models. APC anticoagulant activity involves inactivation of factors Va and VIIIa, whereas APC cytoprotective activities involve the endothelial protein C receptor and protease-activated receptor-1 (PAR-1). The relative importance of the anticoagulant activity of APC versus the direct cytoprotective effects of APC on cells for the in vivo benefits is unclear. To distinguish cytoprotective from the anticoagulant activities of APC, a protease domain mutant, 5A-APC (RR229/230AA and KKK191-193AAA), was made and compared with recombinant wild-type (rwt)-APC. This mutant had minimal anticoagulant activity but normal cytoprotective activities that were dependent on endothelial protein C receptor and protease-activated receptor-1. Whereas anticoagulantly active rwt-APC inhibited secondary-extended thrombin generation and concomitant thrombin-dependent activation of thrombin activable fibrinolysis inhibitor (TAFI) in plasma, secondary-extended thrombin generation and the activation of TAFI were essentially unopposed by 5A-APC due to its low anticoagulant activity. Compared with rwt-APC, 5A-APC had minimal profibrinolytic activity and preserved TAFI-mediated anti-inflammatory carboxypeptidase activities toward bradykinin and presumably toward the anaphlatoxins, C3a and C5a, which are well known pathological mediators in sepsis. Thus, genetic engineering can selectively alter the multiple activities of APC and provide APC mutants that retain the beneficial cytoprotective effects of APC while diminishing bleeding risk due to reduction in APC's anticoagulant and APC-dependent profibrinolytic activities.     

A novel antithrombotic role for high molecular weight kininogen as inhibitor of plasminogen activator inhibitor-1 function

The adhesive glycoprotein vitronectin (VN) forms a function-stabilizing complex with plasminogen activator inhibitor-1 (PAI-1), the major fibrinolysis inhibitor in both plasma and vessel wall connective tissue. VN also interacts with two-chain high molecular weight kininogen (HKa), particularly its His-Gly-Lys-rich domain 5, and both HKa and PAI-1 are antiadhesive factors that have been shown to compete for binding to VN. In this study the influence of HKa and domain 5 on the antifibrinolytic function of PAI-1 was investigated. In a purified system, HKa and particularly domain 5 inhibited the binding of PAI-1 to VN and promoted PAI-1 displacement from both isolated VN as well as subendothelial extracellular matrix-associated VN. The sequence Gly(486)-Lys(502) of HKa domain 5 was identified as responsible for this inhibition. Although having no direct effect on PAI-1 activity itself, HKa domain 5 or the peptide Gly(486)-Lys(502) markedly destabilized the VN.PAI-1 complex interaction, resulting in a significant reduction of PAI-1 inhibitory function on plasminogen activators, resembling the effect of VN antibodies that prevent stabilization of PAI-1. Furthermore, high affinity fibrin binding of PAI-1 in the presence of VN as well as the VN-dependent fibrin clot stabilization by the inhibitor were abrogated in the presence of the kininogen forms mentioned. Taken together, our data indicate that the peptide Gly(486)-Lys(502) derived from domain 5 of HKa serves to interfere with PAI-1 function. Based on these observations potential low molecular weight PAI-1 inhibitors could be designed for the use in therapeutic interventions against thromboembolic complications.     

Type 1 plasminogen activator inhibitor binds to fibrin via vitronectin

Type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA), circulates as a complex with the abundant plasma glycoprotein, vitronectin. This interaction stabilizes the inhibitor in its active conformation In this report, the effects of vitronectin on the interactions of PAI-1 with fibrin clots were studied. Confocal microscopic imaging of platelet-poor plasma clots reveals that essentially all fibrin-associated PAI-1 colocalizes with fibrin-bound vitronectin. Moreover, formation of platelet-poor plasma clots in the presence of polyclonal antibodies specific for vitronectin attenuated the inhibitory effects of PAI-1 on t-PA-mediated fibrinolysis. Addition of vitronectin during clot formation markedly potentiates PAI-1-mediated inhibition of lysis of (125)I-labeled fibrin clots by t-PA. This effect is dependent on direct binding interactions of vitronectin with fibrin. There is no significant effect of fibrin-associated vitronectin on fibrinolysis in the absence of PAI-1. The binding of PAI-1 to fibrin clots formed in the absence of vitronectin was characterized by a low affinity (K(d) approximately 3.5 micrometer) and rapid loss of PAI-1 inhibitory activity over time. In contrast, a high affinity and stabilization of PAI-1 activity characterized the cooperative binding of PAI-1 to fibrin formed in the presence of vitronectin. These findings indicate that plasma PAI-1.vitronectin complexes can be localized to the surface of fibrin clots; by this localization, they may modulate fibrinolysis and clot reorganization.     

Additivity in effects of vitronectin and monoclonal antibodies against alpha-helix F of plasminogen activator inhibitor-1 on its reactions with target proteinases

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential therapeutic target in cardiovascular and cancerous diseases. PAI-1 circulates in blood as a complex with vitronectin. A PAI-1 variant (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-3-diazole (NBD) P9 PAI-1) with a fluorescent tag at the reactive center loop (RCL) was used to study the effects of vitronectin and monoclonal antibodies (mAbs) directed against alpha-helix F (Mab-2 and MA-55F4C12) on the reactions of PAI-1 with tissue-type and urokinase-type plasminogen activators. Both mAbs delay the RCL insertion and induce an increase in the stoichiometry of inhibition (SI) to 1.4-9.5. Binding of vitronectin to NBD P9 PAI-1 does not affect SI but results in a 2.0-6.5-fold decrease in the limiting rate constant (klim) of RCL insertion for urokinase-type plasminogen activator at pH 6.2-8.0 and for tissue-type plasminogen activator at pH 6.2. Binding of vitronectin to the complexes of NBD P9 PAI-1 with mAbs results in a decrease in klim and in a 1.5-22-fold increase in SI. Thus, vitronectin and mAbs demonstrated additivity in the effects on the reaction with target proteinases. The same step in the reaction mechanism remains limiting for the rate of RCL insertion in the absence and presence of Vn and mAbs. We hypothesize that vitronectin, bound to alpha-helix F on the side opposite to the epitopes of the mAbs, potentiates the mAb-induced delay in RCL insertion and the associated substrate behavior by selectively decreasing the rate constant for the inhibitory branch of PAI-1 reaction (ki). These results demonstrate that mAbs represent a valid approach for inactivation of vitronectin-bound PAI-1 in vivo.     

Basic residues in the 37-loop of activated protein C modulate inhibition by protein C inhibitor but not by alpha(1)-antitrypsin

The role of lysines 37-39 (chymotrypsin numbering) in the 37-loop of the serine protease activated protein C (APC) was studied by expressing acidic and neutral recombinant APC (rAPC) mutants. Activity of the APC mutants was assessed using human plasma and plasma-purified and recombinant derivatives of protein C inhibitor (PCI; also known as plasminogen activator inhibitor-3) and alpha(1)-antitrypsin, with and without heparin. The catalytic properties of the mutants to small peptidyl substrates were essentially the same as wild-type rAPC (wt-rAPC), yet their plasma anticoagulant activities were diminished. Analysis of the rAPC-protease inhibitor complexes formed after addition of wt-rAPC and mutants to plasma revealed no change in the inhibition pattern by alpha(1)-antitrypsin but a reduction in mutant complex formation by PCI in the presence of heparin. Using purified serpins, we found that inhibition rates of the mutants were the same as wt-rAPC with alpha(1)-antitrypsin; however, PCI (plasma-derived and recombinant forms) inhibition rates of the acidic mutants were slightly faster than that of wt-rAPC without heparin. By contrast, PCI-heparin inhibition rates of the mutants were not substantially accelerated compared to wt-rAPC. The mutants had reduced heparin-binding properties compared to wt-rAPC. Molecular modeling of the PCI-APC complex with heparin suggests that heparin may function not only to bridge PCI to APC, but also to alleviate putative non-optimal intermolecular interactions. Our results suggest that the basic residues of the 37-loop of APC are involved in macromolecular substrate interactions and in heparin binding, and they influence inhibition by PCI (with or without heparin) but not by alpha(1)-antitrypsin, two important blood plasma serpins.     

Purification and characterization of a plasminogen activator inhibitor 1 binding protein from human plasma. Identification as a multimeric form of S protein (vitronectin)

A binding protein for plasminogen activator inhibitor 1 (PAI-1-BP) was isolated from human plasma by a four-step procedure. 1) The 7 S globulin fraction of plasma was isolated by gel filtration on Sephacryl S-300. 2) Human endothelial cell-type plasminogen activator inhibitor (PAI-1), pretreated with 12 M urea, was added to this fraction (22 micrograms of PAI-1/ml of plasma), and a PAI-1 antigen peak with apparent mass 450 kDa (representing 65% of PAI-1 antigen and 85% of PAI activity) was isolated by gel filtration of this mixture. 3) The PAI-1.PAI-1-BP complex was further purified by immunoadsorption on an immobilized murine monoclonal antibody directed against PAI-1 (MA-7D4) and by elution with 4 M KSCN. 4) The complex was then dissociated by addition of excess human tissue-type plasminogen activator (t-PA), and t-PA and PAI-1 antigen (t-PA.PAI-1 complexes and free t-PA and PAI-1) were removed by immunoadsorption on monoclonal antibodies directed against t-PA (MA-62E8) and against PAI-1 (MA-7D4 and MA-12A4). Sodium dodecyl sulfate-gel electrophoresis of the purified material under nonreducing conditions revealed two bands with apparent mass approximately equal to 150 kDa and two bands with mass 74 and 68 kDa. Reduced sodium dodecyl sulfate-gel electrophoresis displayed two main bands with apparent masses of 73 and 64 kDa. The PAI-1-BP reacts with urea-treated, but not with inactive PAI-1. t-PA dissociates the complex between PAI-1 and PAI-1-BP. PAI-1 in complex with PAI-1-BP is 2-3-fold more stable at 37 degrees C than purified PAI-1, suggesting that PAI-1-BP may stabilize PAI-1 in blood. The concentration of PAI-1-BP in plasma determined by titration with PAI-1 is approximately 130 mg/liter. The isolated PAI-1-BP was shown to be identical to S protein (vitronectin) both by cross-reactivity with monospecific rabbit antisera and by NH2-terminal amino acid sequence analysis. The gel filtration behavior, mobility on sodium dodecyl sulfate-gel electrophoresis, and concentration in plasma suggest that PAI-1-BP is a multimer (presumably a dimer) of S protein accounting for approximately 35% of the S protein in plasma.     

The inhibition of tissue type plasminogen activator by plasminogen activator inhibitor-1. The effects of fibrinogen, heparin, vitronectin, and lipoprotein(a).

Plasminogen activator inhibitor-1 (PAI-1) regulates fibrinolysis by inhibiting tissue type plasminogen activator (t-PA). Fibrinogen, heparin, and vitronectin enhance the rate of inhibition of t-PA by PAI-1. Kinetic studies indicate that both fibrinogen and heparin increase the second-order inhibition constant by a maximum of approximately 4-fold, whereas vitronectin increases the rate constant by a maximum of approximately 6-fold. The dissociation constants of fibrinogen, heparin, and vitronectin for the inhibition reaction were 200 nM, 20 nM, and 600 pM, respectively. In addition, PAI-1 inhibition of t-PA may be regulated by the presence of lipoprotein(a) (Lp(a)). Previous studies demonstrated that Lp(a) competes with plasminogen for the active site of fibrinogen- and heparin-bound t-PA. Kinetic studies described here demonstrate that Lp(a) prevents the inhibition of t-PA by PAI-1 in the presence of fibrinogen and heparin, but has no effect on the reaction in the presence of vitronectin or in the absence of either fibrinogen or heparin. The data suggest that fibrinogen and heparin may enhance the rate of inhibition through an interaction with t-PA, and that vitronectin may enhance the inhibition through an interaction with PAI-1. In addition, these experiments indicate that Lp(a) may regulate fibrinolysis by competing with PAI-1 and plasminogen for fibrinogen- and heparin-bound t-PA. These data suggest that PAI-1 inhibition of t-PA in vivo is primarily mediated via interaction with fibrinogen, heparin, vitronectin, and Lp(a), and therefore, the functional levels of PAI-1 activity in the vasculature may be regulated by the presence of these components.     

Vitronectin regulates the synthesis and localization of urokinase-type plasminogen activator in HT-1080 cells.

The effect of extracellular matrix composition on the location, amount, and activity of cell-associated urokinase-type plasminogen activator was tested using HT-1080 cells adherent to either fibronectin or vitronectin. Specific immunoprecipitation of newly synthesized urokinase indicated that cells adherent to fibronectin synthesized 2-3-fold more urokinase than cells adherent to vitronectin. Complexes of urokinase and plasminogen activator inhibitor type 1 (PAI-1) were detected in cell layers of vitronectin-adherent but not fibronectin-adherent cells. Inhibition of PAI-1 using a neutralizing monoclonal antibody resulted in a 3-fold increase in urokinase enzymatic activity on vitronectin adherent cells. Urokinase activity on fibronectin adherent cells was only slightly increased following PAI-1 neutralization. Examination of both HT-1080 and normal human fibroblast cells by immunofluorescent microscopy localized urokinase-type plasminogen activator to discrete, focal areas underneath cells adherent to vitronectin. Urokinase was not detectable by immunofluorescence on cells adherent to fibronectin. The addition of exogenous prourokinase to locate urokinase receptors on adherent HT-1080 cells indicated that the focal localization of cell-surface urokinase resulted from the clustering of urokinase receptors following adhesion to vitronectin but not fibronectin-coated substrates. These results suggest that vitronectin can contribute to the control of cell-surface plasmin activity by regulating the synthesis of urokinase and directing the localization of urokinase receptors.     

Antithrombotic and profibrinolytic activities of eckol and dieckol

In order to develop new anticoagulant agents, two single compounds (eckol and dieckol) were isolated from Eisenia bicyclis and examined their anticoagulant activities by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT) as well as cell-based thrombin and activated factor X (FXa) generation activities. And the effects of eckol and dieckol on the expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were tested in tumor necrosis factor-α (TNF-α) activated human umbilical vein endothelial cells (HUVECs). Data showed that eckol and dieckol prolonged aPTT and PT significantly and inhibited thrombin and FXa activities. They also inhibited the generation of thrombin or FXa in HUVECs. In accordance with these anticoagulant activities, eckol or dieckol showed anticoagulant effect in vivo. Furthermore, eckol and dieckol inhibited TNF-α induced PAI-1 production and the ratio between PAI-1 and t-PA was found to be significantly decreased by eckol and dieckol. Surprisingly, these anticoagulant and profibrinolytic effects of dieckol were better than those of eckol indicating that hydroxyl group in eckol positively regulated anticoagulant function of eckol. Therefore, these results suggest that eckol or dieckol possesses antithrombotic activities and provides a possibility to develop as an agent for the anticoagulation.     

Dissociation of activated protein C functions by elimination of protein S cofactor enhancement

Activated protein C (APC) plays a critical anticoagulant role in vivo by inactivating procoagulant factor Va and factor VIIIa and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor can initiate protease-activated receptor-1 (PAR-1)-mediated cytoprotective signaling. Protein S constitutes a critical cofactor for the anticoagulant function of APC but is not known to be involved in regulating APC-mediated protective PAR-1 signaling. In this study we utilized a site-directed mutagenesis strategy to characterize a putative protein S binding region within the APC Gla domain. Three single amino acid substitutions within the APC Gla domain (D35T, D36A, and A39V) were found to mildly impair protein S-dependent anticoagulant activity (<2-fold) but retained entirely normal cytoprotective activity. However, a single amino acid substitution (L38D) ablated the ability of protein S to function as a cofactor for this APC variant. Consequently, in assays of protein S-dependent factor Va proteolysis using purified proteins or in the plasma milieu, APC-L38D variant exhibited minimal residual anticoagulant activity compared with wild type APC. Despite the location of Leu-38 in the Gla domain, APC-L38D interacted normally with endothelial cell protein C receptor and retained its ability to trigger PAR-1 mediated cytoprotective signaling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to separate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.     

Identification of a target site in plasminogen activator inhibitor-1 that allows neutralization of its inhibitor properties concomitant with an allosteric up-regulation of its antiadhesive properties

The serpin plasminogen activator inhibitor-1 (PAI-1) has a dual function: 1) it plays an important role as a direct inhibitor of the plasminogen activation system, and 2) its interaction with the adhesive glycoprotein vitronectin suggests a role in tissue remodeling and metastasis, independent from its proteinase inhibitory properties. Unique to this serpin is the close association between its conformational and functional properties. Indeed, PAI-1 can occur in an active and a latent conformation, but both functions are exclusively present in the active conformation. We report here the epitope localization and functional effects of a monoclonal antibody (MA-124K1) that inhibits rat PAI-1 activity and simultaneously increases the binding of inactive PAI-1 to vitronectin (the affinity constant of PAI-1 for vitronectin is 2 x 10(7) m(-1) in the absence of MA-124K1 and 160 x 10(7) m(-1) in the presence of MA-124K1). To the best of our knowledge, this is the first monoclonal antibody dissociating the proteinase inhibitory properties from the vitronectin binding properties in PAI-1. Mutation of Glu(212) and/or Glu(220) in rat PAI-1 to Ala results in a strongly reduced affinity or absence of binding to MA-124K1. The three-dimensional structure of PAI-1 reveals that these residues constitute a conformational epitope close to the reactive-site loop and compatible with the effect of MA-124K1 on the inhibitory properties of PAI-1. However, the vitronectin binding site is localized at the opposite site of the molecule, indicating that the effect of MA-124K1 involves an allosteric modulation of the vitronectin binding site. Cell culture experiments revealed a significant reduction of cell attachment and migration in the presence of MA-124K1, providing evidence for the functional relevance of this antibody-mediated up-regulation of the vitronectin binding properties of PAI-1. In conclusion, a novel mechanism for interference with PAI-1 functions has been identified and is of importance in the modulation of cell migration and related events (e.g. tumor metastasis).     

Molecular characterization of an extended binding site for coagulation factor Va in the positive exosite of activated protein C

The anticoagulant human plasma serine protease, activated protein C (APC), inhibits blood coagulation by specific inactivation of the coagulation cofactors factor Va (FVa) and factor VIIIa. Site-directed mutagenesis of residues in three surface loops of a positive exosite located on APC was used to identify residues that play a significant role in binding to FVa. Eighteen different residues were mutated to alanine singly, in pairs, or in triple mutation combinations. Mutant APC proteins were purified and characterized for their inactivation of FVa. Three APC residues were identified that provide major contributions to FVa interactions: Lys(193), Arg(229), and Arg(230). In addition, four residues made significant minor contributions to FVa interactions: Lys(191), Lys(192), Asp(214), and Glu(215). All of these residues primarily contribute to APC cleavage at Arg(506) in FVa and play a small role in the interaction of APC with the Arg(306) cleavage site. In conjunction with previously published work, these results define an extensive FVa binding site in the positive exosite of APC that is primarily involved in binding and cleaving at Arg(506) on FVa.     

Vimentin exposed on activated platelets and platelet microparticles localizes vitronectin and plasminogen activator inhibitor complexes on their surface.

Type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA), is found in plasma and platelets. PAI-1 circulates in complex with vitronectin (Vn), an interaction that stabilizes PAI-1 in its active conform. In this study, we examined the binding of platelet-derived Vn and PAI-1 to the surface of isolated platelets. Flow cytometry indicate that, like P-selectin, PAI-1, and Vn are found on the surface of thrombin- or calcium ionophore-activated platelets and platelet microparticles. The binding of PAI-1 to the activated platelet surface is Vn-dependent. Vn mediates the binding of PAI-1 to platelet surfaces through a high affinity (K(d) of 80 nm) binding interaction with the NH(2) terminus of vimentin, and this Vn-binding domain is expressed on the surface of activated platelets and platelet microparticles. Immunological and functional assays indicate that only -5% of the total PAI-1 in platelet releasates is functionally active, and it co-precipitates with Vn, and the vimentin-enriched cytoskeleton fraction of activated platelet debris. The remaining platelet PAI-1 is inactive, and does not associate with the cytoskeletal debris of activated platelets. Confocal microscopic analysis of platelet-rich plasma clots confirm the co-localization of PAI-1 with Vn and vimentin on the surface of activated platelets, and platelet microparticles. These findings suggest that platelet vimentin may regulate fibrinolysis in plasma and thrombi by binding platelet-derived Vn.PAI-1 complexes.     

Restoration of serine protease-inhibitor interaction by protein engineering

Tissue-type plasminogen activator (t-PA) catalyzes the rate-limiting step in the fibrinolytic cascade: conversion of plasminogen to plasmin. Plasma contains several inhibitors of t-PA that limit its activity and prevent systemic activation of plasminogen. The most important of these is endothelial cell plasminogen activator inhibitor (PAI-1), a member of the serine protease inhibitor (serpin) gene family. We have previously demonstrated that mutation of arginine 304 of t-PA to a glutamic acid residue drastically reduces the rate of interaction between the enzyme and its suicide substrate, PAI-1, without affecting the reactivity of the enzyme toward its normal substrate, plasminogen (Madison, E. L., Goldsmith, E. J., Gerard, R.D., Gething, M.J., and Sambrook, J.F. (1989) Nature 339, 721-724). We report here the use of protein modeling to design a compensatory mutation in PAI-1 (glutamic acid 350 to arginine) and create a molecule that rapidly inhibits this "serpin-resistant" variant of t-PA.     

Plasminogen activator inhibitor-1 and -3 increase cell adhesion and motility of MDA-MB-435 breast cancer cells

Plasminogen activator inhibitor-1 (PAI-1), an inhibitor of urokinase plasminogen activator, is paradoxically associated with a poor prognosis in breast cancer. PAI-1 is linked to several processes in the metastatic cascade. However, the role of PAI-1 in metastatic processes, which may be independent of protease inhibitory activity, is not fully understood. We report herein that PAI-1, when added exogenously to or stably transfected in human MDA-MB-435 breast carcinoma cells, had disparate effects on adhesion to extracellular matrix proteins and motility in vitro. Specifically, exogenously added PAI-1 inhibited cell adhesion to vitronectin but not fibronectin, in agreement with the literature. By contrast, stably transfected PAI-1 stimulated adhesion to both proteins. Wild-type PAI-1 was required for this stimulation, because expression of a non-protease inhibitory P14 (T333R) PAI-1 mutant failed to enhance adhesion. Compared with non-inhibitory PAI-1, wild-type PAI-1 also increased cell motility in chemotaxic assays. Furthermore, stable transfection of a related serine protease inhibitor, plasminogen activator inhibitor-3 (PAI-3, or protein C inhibitor) gave results similar to wild-type PAI-1. The stimulatory activity of PAI-3 was not seen with a non-protease inhibitory P14 PAI-3 mutant (T341R). We show that a downstream effect of endogenous wild-type PAI-1 and PAI-3 overexpression, but not their non-inhibitory counterparts, was the altered expression of alpha(2), alpha(3), alpha(4), alpha(5), and beta(1) integrin subunits. Additionally, blocking antibodies to beta(1) integrin inhibited PAI-1-induced adhesion. Our data provide experimental support for the stimulatory and inhibitory effects of PAI-1 in metastasis and introduce PAI-3 as another serpin potentially important in malignant disease.     

Thrombin neutralizes plasminogen activator inhibitor 1 (PAI-1) that is complexed with vitronectin in the endothelial cell matrix

Vitronectin endows plasminogen activator inhibitor 1 (PAI-1), the fast-acting inhibitor of both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), with additional thrombin inhibitory properties. In view of the apparent association between PAI-1 and vitronectin in the endothelial cell matrix (ECM), we analyzed the interaction between PAI-1 and thrombin in this environment. Upon incubating 125I-labeled alpha-thrombin with endothelial cell matrix (ECM), the protease formed SDS-stable complexes exclusively with PAI-1, with subsequent release of these complexes into the supernatant. Vitronectin was required as a cofactor for the association between PAI-1 and thrombin in ECM. Metabolic labeling of endothelial cell proteins, followed by incubation of ECM with t-PA, u-PA, or thrombin, indicated that all three proteases depleted PAI-1 from ECM by complex formation and proteolytic cleavage. Proteolytically inactive thrombin as well as anticoagulant thrombin, i.e., thrombin in complex with its endothelial cell surface receptor thrombomodulin, did not neutralize PAI-1, emphasizing that the procoagulant moiety of thrombin is required for a functional interaction with PAI-1. A physiological implication of our findings may be related to the mutual neutralization of both PAI-1 and thrombin, providing a new link between plasminogen activation and the coagulation system. Evidence is provided that in ECM, procoagulant thrombin may promote plasminogen activator activity by inactivating PAI-1.