Interaction of plasminogen activator inhibitor type-1 (PAI-1) with vitronectin (Vn): mapping the binding sites on PAI-1 and Vn

The serpin plasminogen activator inhibitor type-1 (PAI-1), as the primary physiological inhibitor of both urokinase-type (uPA) and tissue-type (tPA) plasminogen activator, plays an important role in the regulation of the fibrinolytic system as well as in extracellular remodeling in both physiological and pathophysiological processes. In plasma as well as in the extracellular matrix PAI-1 binds to vitronectin (Vn), an interaction that affects the function of both proteins. As PAl-1/Vn interaction has a significant regulatory function in fibrinolysis, thrombolysis, and cell adhesion in cancer spread, there is a strong interest in defining the binding sites on PAI-1 and Vn as the basis of a rational design of novel drugs that may modulate PAI-1/Vn-mediated effects. In this minireview, we give an overview on the approaches to define the Vn binding site of PAI-1 and vice versa. Although in the case of PAI-1 the region around alpha-helix E and alpha-helix F of PAI-1 has been demonstrated to be important for its interaction with Vn, the precise location of the Vn-binding region has not completely been resolved. The major high-affinity PAI-1 binding region of Vn is localized within the N-terminal somatomedin B (SMB) domain of Vn. There are indications for at least one other low-affinity PAI-1 binding site in the C-terminal region of Vn, which seems to be involved in the formation of larger PAI-1/Vn complexes.     

Evidence that type 1 plasminogen activator inhibitor binds to the somatomedin B domain of vitronectin.

The interaction between type 1 plasminogen activator inhibitor (PAI-1) and fragments of vitronectin (Vn) was investigated. The PAI-1-binding domain was not destroyed when Vn was cleaved by treatment with either acid or CNBr. Acid-cleaved Vn was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by PAI-1 ligand binding. The smallest fragment (Mr 40,000) that retained PAI-1 binding function was sequenced and shown to contain the NH2 terminus of the molecule. Further cleavage of this fragment by treatment with CNBr generated a Mr 35,000 fragment (Pro52-Asp239) that did not interact with PAI-1, and a Mr 6,000 NH2-terminal fragment (Asp1-Met51) that spanned the somatomedin B domain and contained the RGD (cell binding) sequence. The purified Mr 6,000 fragment competed with immobilized Vn for PAI-1 binding, and formed complexes with activated PAI-1. These complexes could be immunoprecipitated by antibodies to PAI-1. Synthetic peptides containing the RGD sequence had no effect on the binding of this fragment to PAI-1. These results suggest that the cell-binding and PAI-1 binding sequences of Vn occupy distinct regions in the NH2-terminal somatomedin B domain of the molecule.     

The PKA phosphorylation of vitronectin: effect on conformation and function.

Vitronectin (Vn) stabilizes the inhibitory form of plasminogen activator inhibitor-1 (PAI-1), an important modulator of fibrinolysis. We have previously reported that Vn is specifically phosphorylated by PKA (at Ser378), a kinase we have shown to be released from platelets upon their physiological activation. Here we describe the molecular consequences of this phosphorylation and show (by circular dichroism, and by phosphorylation with casein kinase II) that it acts by modulating the conformation of Vn. The PKA phosphorylation of Vn is enhanced in the presence of either PAI-1, or heparin, or both. This enhanced phosphorylation occurs exclusively on Ser378 as shown with the Vn mutants Ser378Ala and Ser378Glu. The binding of PKA phosphorylated Vn to immobilized PAI-1 and to immobilized plasminogen is shown to be lower than that of Vn. The evidence compiled here suggests that this phosphorylation of Vn can modulate plasminogen activation and consequently control fibrinolysis.     

Localization of protein kinase A and vitronectin in resting platelets and their translocation onto fibrin fibers during clot formation

Physiological stimulation of platelets with thrombin brings about the release of protein kinase A (PKA) into the plasma. In human blood, this kinase singles out and phosphorylates vitronectin (Vn), a multifunctional regulatory protein, which was proposed to play an important role in the control of fibrinolysis. Here we present immuno-cytochemical evidence to show: (i) that intact platelets possess on their surface an ecto-PKA which can preferentially phosphorylate Vn; (ii) that in the resting platelet, both the catalytic and the regulatory subunits of PKA are present on the platelet surface, in the surface-connected canalicular system, and within the alpha-granules of the platelets; (iii) that the process initiated upon platelet activation, which leads to the formation of fibrin fibers and consequently forms the fibrin net, is accompanied by a translocation of PKA, of Vn, and of PAI-1 onto the fibrin fibers. We propose that the localization and the translocation of these proteins in the fibrin net, together with our finding that PKA phosphorylation of Vn reduces its grip of PAI-1, can unleash PAI-1 in its free form. The free PAI-1 can then assume its latent (non inhibitory) conformation, allow plasminogen activators to trigger the formation of active plasmin, and to initiate fibrinolysis.     

Truncated vitronectins: binding to immobilized fibrin and to fibrin clots, and their subsequent interaction with cells.

The plasminogen activator inhibitor-1 (PAI-1) is stabilized in its inhibitory conformation by binding to Vitronectin (Vn). The anchorage of PAI-1 to the fibrin fibers was recently shown to be mediated by Vn, and as such to modulate fibrinolysis. Here we report the mapping of the fibrin binding sites in Vn using truncated recombinant Vns, and show that two segments of Vn are involved: one at its carboxyl terminus (within residues 348-459) and one at its amino terminus (within residues 1-44). This mapping sets the stage for (i) the design of specific inhibitors for the Vn-fibrin interaction; (ii) for studying the role of this interaction in the anchoring of endothelial cells and platelets onto the fibrin clot; and (iii) for getting a deeper insight into the mechanism of the Vn-fibrin interaction in fibrinolysis. (c)2002 Elsevier Science.     

Interaction of plasminogen activator inhibitor type-1 (PAI-1) with vitronectin.

The serpin plasminogen activator inhibitor type 1 (PAI-1) plays an important role in physiological processes such as thrombolysis and fibrinolysis, as well as pathophysiological processes such as thrombosis, tumor invasion and metastasis. In addition to inhibiting serine proteases, mainly tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, PAI-1 interacts with different components of the extracellular matrix, i.e. fibrin, heparin (Hep) and vitronectin (Vn). PAI-1 binding to Vn facilitates migration and invasion of tumor cells. The most important determinants of the Vn-binding site of PAI-1 appear to reside between amino acids 110-147, which includes alpha helix E (hE, amino acids 109-118). Ten different PAI-1 variants (mostly harboring modifications in hE) as well as wild-type PAI-1, the previously described PAI-1 mutant Q123K, and another serpin, PAI-2, were recombinantly produced in Escherichia coli containing a His(6) tag and purified by affinity chromatography. As shown in microtiter plate-based binding assays, surface plasmon resonance and thrombin inhibition experiments, all of the newly generated mutants which retained inhibitory activity against uPA still bound to Vn. Mutant A114-118, in which all amino-acids at positions 114-118 of PAI-1 were exchanged for alanine, displayed a reduced affinity to Vn as compared to wild-type PAI-1. Mutants lacking inhibitory activity towards uPA did not bind to Vn. Q123K, which inhibits uPA but does not bind to Vn, served as a control. In contrast to other active PAI-1 mutants, the inhibitory properties of A114-118 towards thrombin as well as uPA were significantly reduced in the presence of Hep. Our results demonstrate that the wild-type sequence of the region around hE in PAI-1 is not a prerequisite for binding to Vn.     

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.     

Activation of platelet caspases by TNF and its consequences for kinetics

TNF is known to induce a thrombocytopenia, due to a reduced platelet life span. Injection of TNF (10 microg) to mice did markedly increase the number of platelet-derived microparticles in plasma, most pronounced 1h after injection. Injection of TNF induced a transient activation of platelet caspases, -1, -3, -6, -8, -9, as seen by the binding of caspases probes detected by flow cytometry, most pronounced 1h after injection. Activation of caspase-3 was also evidenced by antibodies. Injection of the caspases inhibitor ZVAD-fmk decreased TNF-induced generation of microparticles and thrombocytopenia, indicating a causal role of caspases in platelet fragmentation. Activation of platelet caspases was also evident in platelets exposed to TNF in vitro, indicating that TNF acts on platelets directly. Comparison of platelets from +/+, TNFR1 -/- and TNFR2 -/- mice showed that caspases are activated mainly by the TNFR1. These observations indicate that TNF activates platelet caspases via the TNFR1, which results in platelet fragmentation and thrombocytopenia.     

Abrogation of Plasminogen Activator Inhibitor-1-Vitronectin Interaction Ameliorates Acute Kidney Injury in Murine Endotoxemia

Sepsis-induced acute kidney injury (AKI) contributes to the high mortality and morbidity in patients. Although the pathogenesis of AKI during sepsis is poorly understood, it is well accepted that plasminogen activator inhibitor-1 (PAI-1) and vitronectin (Vn) are involved in AKI. However, the functional cooperation between PAI-1 and Vn in septic AKI has not been completely elucidated. To address this issue, mice were utilized lacking either PAI-1 (PAI-1^−/−) or expressing a PAI-1-mutant (PAI-1^R101A/Q123K) in which the interaction between PAI-1 and Vn is abrogated, while other functions of PAI-1 are retained. It was found that both PAI-1^−/− and PAI-1^R101A/Q123K mice are associated with decreased renal dysfunction, apoptosis, inflammation, and ERK activation as compared to wild-type (WT) mice after LPS challenge. Also, PAI-1^−/− mice showed attenuated fibrin deposition in the kidneys. Furthermore, a lack of PAI-1 or PAI-1-Vn interaction was found to be associated with an increase in activated Protein C (aPC) in plasma. These results demonstrate that PAI-1, through its interaction with Vn, exerts multiple deleterious mechanisms to induce AKI. Therefore, targeting of the PAI-1-Vn interaction in kidney represents an appealing therapeutic strategy for the treatment of septic AKI by not only altering the fibrinolytic capacity but also regulating PC activity.     

Binding of anticoagulant vitamin K-dependent protein S to platelet-derived microparticles.

Vitamin K-dependent protein S is an anticoagulant plasma protein serving as cofactor to activated protein C in degradation of coagulation factors Va and VIIIa on membrane surfaces. In addition, it forms a noncovalent complex with complement regulatory protein C4b-binding protein (C4BP), a reaction which inhibits its anticoagulant function. Both forms of protein S have affinity for negatively charged phospholipids, and the purpose of the present study was to elucidate whether they bind to the surface of activated platelets or to platelet-derived microparticles. Binding of protein S to human platelets stimulated with various agonists was examined with FITC-labeled monoclonal antibodies and fluorescence-gated flow cytometry. Protein S was found to bind to membrane microparticles which formed during platelet activation but not to the remnant activated platelets. Binding to microparticles was saturable and maximum binding was seen at approximately 0.4 microM protein S. It was calcium-dependent and reversed after the addition of EDTA. Inhibition experiments with monoclonal antibodies suggested the gamma-carboxyglutamic acid containing module of protein S to be involved in the binding reaction. An intact thrombin-sensitive region of protein S was not required for binding. The protein S-C4BP complex did not bind to microparticles or activated platelets even though it bound to negatively charged phospholipid vesicles. Intact protein S supported binding of both protein C and activated protein C to microparticles. Protein S-dependent binding of protein C/activated protein C was blocked by those monoclonal antibodies against protein S that inhibited its cofactor function. In conclusion, we have found that free protein S binds to platelet-derived microparticles and stimulates binding of protein C/activated protein C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum-derived vitronectin influences the pericellular distribution of type 1 plasminogen activator inhibitor

Bovine aortic endothelial cells (BAEs) were used as a model system to study the nature and origin of protein(s) in the extracellular matrix that bind to type 1 plasminogen activator inhibitor (PAI-1). Matrix samples were fractionated by SDS-PAGE and analyzed by PAI-1 ligand binding and by immunoblotting using antibodies to vitronectin (Vn). PAI-1 bound primarily to two Vn-related polypeptides of Mr 63,000 and 57,000, and both of these partially degraded polypeptides were present in the culture serum. Radiolabeling experiments failed to detect significant Vn biosynthesis by BAEs (less than 0.03% of total), or by human umbilical vein endothelial cells and HT 1080 cells. The binding of PAI-1 to Vn was relatively specific since direct binding studies failed to demonstrate significant interactions between PAI-1 and other matrix proteins (e.g., fibronectin, type IV collagen, laminin, or matrigel). Kinetic studies indicate that PAI-1 rapidly accumulates in the matrix when BAEs are plated on Vn, appearing in the conditioned medium only after a significant lag period (1-2 h). However, no PAI-1 was detected in the matrix when the cells were plated on fibronectin-coated dishes, and there was no lag period for PAI-1 accumulation in the medium. These results indicate that PAI-1 binds specifically to serum-derived Vn in the matrix, and suggest that the composition of both the matrix and serum itself may influence the pericellular distribution of this important inhibitor.     

The urokinase/PAI-2 complex: a new high affinity ligand for the endocytosis receptor low density lipoprotein receptor-related protein

The efficient inactivation of urokinase plasminogen activator (uPA) by plasminogen activator inhibitor type 2 (PAI-2) at the surface of carcinoma cells is followed by rapid endocytosis of the uPA-PAI-2 complex. We now show that one pathway of this receptor-mediated endocytosis is mediated via the low density lipoprotein receptor-related protein (LRP) in prostate cancer cells. Detailed biochemical analyses using ligand binding assays and surface plasmon resonance revealed a novel and distinct interaction mechanism between native, human LRP and uPA-PAI-2. As reported previously for PAI-1, inhibition of uPA by PAI-2 significantly increased the affinity of the complex for LRP (K(D) of 36 nm for uPA-PAI-2 versus 200 nm for uPA). This interaction was maintained in the presence of uPAR, confirming the validity of this interaction at the cell surface. However, unlike PAI-1, no interaction was observed between LRP and PAI-2 in either the stressed or the relaxed conformation. This suggests that the uPA-PAI-2-LRP interaction is mediated by site(s) within the uPA molecule alone. Thus, as inhibition of uPA by PAI-2 resulted in accelerated clearance of uPA from the cell surface possibly via its increased affinity for LRP, this represents a mechanism through which PAI-2 can clear proteolytic activity from the cell surface. Furthermore, lack of a direct interaction between PAI-2 and LRP implies that downstream signaling events initiated by PAI-1 may not be activated by PAI-2.     

Interaction of type 1 plasminogen activator inhibitor with the enzymes of the contact activation system

The interaction between type 1 plasminogen activator inhibitor (PAI-1), a serine protease inhibitor, and the three serine proteases generated during contact activation of plasma was studied using functional and immunologic approaches. Incubation of Factor XIIa, Factor XIa, and plasma kallikrein with either purified PAI-1 or platelet-derived PAI-1 resulted in the formation of sodium dodecyl sulfate-stable complexes as revealed by immunoblotting techniques. Functional assays indicated that Factor XIa and, to a lesser extent, Factor XIIa and plasma kallikrein neutralized the ability of purified PAI-1 to bind to immobilized tissue-type plasminogen activator (t-PA). Immunoblotting demonstrated that these enzymes also neutralized the ability of PAI-1 to form complexes with fluid-phase t-PA. Clot lysis assays employing purified proteins and 125I-fibrinogen were used to investigate the profibrinolytic effect of these contact activation enzymes. At enzyme concentrations that did not result in direct activation of plasminogen, only Factor XIa was capable of stimulating the lysis of clots supplemented with both t-PA and PAI-1. As a consequence of their interactions with PAI-1, the amidolytic activity of Factor XIIa, Factor XIa, and plasma kallikrein was neutralized by this inhibitor in a time-dependent and concentration-dependent manner. Minimum values estimated for the apparent second-order rate constant of inhibition were 1.6 x 10(4), 2.1 x 10(5), and 6.0 x 10(4) M-1 s-1 for Factor XIIa, Factor XIa, and plasma kallikrein, respectively. These data define new reactions between coagulation and fibrinolysis proteins and suggest that a major mechanism for stimulation of the intrinsic fibrinolytic pathway may involve neutralization of PAI-1 by Factor XIa.     

Tissue-type plasminogen activator binding to human endothelial cells. Evidence for two distinct binding sites

The endothelium may contribute to fibrinolysis through the binding of plasminogen activators or plasminogen activator inhibitors to the cell surface. Using a solid-phase radioimmunoassay, we observed that antibodies to recombinant tissue-type plasminogen activator (rt-PA) and plasminogen activator inhibitor type 1 (PAI-1) bound to the surface of cultured human umbilical vein endothelial cells (HUVEC). HUVEC also specifically bound added radiolabeled rt-PA with apparent steady-state binding being reached by 1 h at 4 degrees C. When added at low concentrations (less than 5 nM), rt-PA bound with high affinity mainly via the catalytic site, forming a sodium dodecyl sulfate-stable 105-kDa complex which dissociates from the cell surface over time and which could be immunoprecipitated by a monoclonal antibody to PAI-1. rt-PA bound to this high affinity site retained less than 5% of its expected plasminogen activator activity. At higher concentrations, binding did not require the catalytic site and was rapidly reversible. rt-PA initially bound to this site retained plasminogen activator activity. These studies suggest that tissue-type plasminogen activator and PAI-1 are expressed on the surface of cultured HUVEC. HUVEC also express unoccupied binding sites for exogenous tissue-type plasminogen activator. The balance between the expression of plasminogen activator inhibitors and these unoccupied binding sites for plasminogen activators on the endothelial surface may contribute to the regulation of fibrinolysis.     

Platelet-derived microparticles express high affinity receptors for factor VIII.

Factor VIII is a cofactor in the tenase enzyme complex which assembles on the membrane of activated platelets. A critical step in tenase assembly is membrane binding of factor VIII. Platelet membrane factor VIII-binding sites were characterized by flow cytometry using either fluorescein maleimide-labeled recombinant factor VIII or a fluorescein-labeled monoclonal antibody against factor VIII. Following activation by thrombin, most platelets bound factor VIII within 90 s. In addition, over the course of several minutes, membranous vesicles (microparticles) were shed from the platelet plasma membrane and each microparticle bound as much factor VIII as a stimulated platelet. Over 30 min, stimulated platelets (but not microparticles) lost the capacity to bind factor VIII. Factor VIII bound saturably to microparticles from platelets stimulated with thrombin, thrombin plus collagen, or the complement proteins C5b-9. The binding of factor VIII was compared to factor V, a structurally homologous coagulation cofactor. Analysis of microparticle binding kinetics yielded similar on and off rates for factor VIII and factor Va and KD values of 2-10 nM. In the presence of 20 nM factor Va, the binding of factor VIII to microparticles was increased, and there was a comparable increase in platelet tenase activity. At higher factor Va concentrations, factor VIII binding and tenase activity were inhibited. Conversely, factor VIII had a similar dose-dependent effect on factor Va binding and platelet prothrombinase activity. Synthetic phospholipid vesicles containing phosphatidylserine competed with microparticles for binding of factor VIII and factor Va. These studies indicate that activated platelets express a transient increase in high affinity receptors for factor VIII, whereas platelet-derived microparticles express a sustained increase in receptors. The binding characteristics of platelet membrane receptors for factor VIII are similar to those for factor Va.     

Platelets retain high levels of active plasminogen activator inhibitor 1

The vascular fibrinolytic system is crucial for spontaneous lysis of blood clots. Plasminogen activator inhibitor 1 (PAI-1), the principal inhibitor of the key fibrinolytic enzyme tissue-type plasminogen activator (tPA), is present in platelets at high concentrations. However, the majority of PAI-1 stored in platelets has been considered to be inactive. Our recent finding (Brogren H, et al. Blood 2004) that PAI-1 de novo synthesized in platelets remained active for over 24 h, suggested that PAI-1 stored in the α-granules might be active to a larger extent than previously reported. To re-evaluate this issue, we performed experiments where the fraction of active PAI-1 was estimated by analyzing the tPA-PAI-1 complex formation. In these experiments platelets were lysed with Triton X-100 in the presence of serial dilutions of tPA and subsequently the tPA-PAI-1 complex was evaluated by Western blot. Also, using a non-immunologic assay, tPA was labeled with (125)I, and (125)I-tPA and (125)I-tPA-PAI-1 was quantified by scintigraphy. Interestingly, both methods demonstrated that the majority (>50%) of platelet PAI-1 is active. Further analyses suggested that pre-analytical procedures used in previous studies (sonication or freezing/thawing) may have substantially reduced the activity of platelet PAI-1, which has lead to an underestimation of the proportion of active PAI-1. Our in vitro results are more compatible with the role of PAI-1 in clot stabilization as demonstrated in physiological and pathophysiological studies.     

Collagen-induced exposure of anionic phospholipid in platelets and platelet-derived microparticles.

We have shown recently that the calcium-dependent phospholipid-binding protein annexin V (placental anticoagulant protein I) can be used to study the exposure of anionic phospholipid after platelet activation. In this study we have further examined the mechanism of this process. Collagen-induced exposure of annexin V binding sites correlated directly with increased ability to support activity of the reconstituted prothrombinase complex. The potency of annexin V as an inhibitor of platelet prothrombinase was the same as its Kd for platelets. Prior incubation of platelets with 5'-p-fluorosulfonylbenzoyladenosine or p-chloromercuribenzenesulfonate had no significant effect on annexin V binding. Similarly, inhibition of platelet cyclic endoperoxide synthesis by acetylsalicylic acid or indomethacin did not inhibit annexin V binding. Staurosporine inhibited collagen-induced, but not A23187-induced, annexin V binding. Agents that increase intraplatelet cyclic nucleotides partially inhibited collagen-induced annexin V binding. Thus, collagen-induced exposure of anionic phospholipid appears to depend primarily on increases in intraplatelet free calcium and may be independent of ADP- or endoperoxide-mediated pathways. Binding sites for annexin V on microparticles derived from collagen-stimulated platelets were demonstrated by flow cytometry and gel filtration. In addition, prior incubation of platelets with 100 nM annexin V inhibited factor Va binding to both platelets and platelet-derived microparticles. These results support the concept that the procoagulant effect of platelets and platelet-derived microparticles is mediated by calcium-induced exposure of anionic phospholipids.     

Structure-function studies of the SERPIN plasminogen activator inhibitor type 1. Analysis of chimeric strained loop mutants.

Three chimeric mutants of plasminogen activator inhibitor 1 (PAI-1) have been constructed where the strained loop of wild type PAI-1 (wtPAI-1) has been replaced with a 19-amino acid region from either plasminogen activator inhibitor 2 (PAI-2), antithrombin III, or with an artificial serine protease inhibitor superfamily consensus strained loop. The inhibitors were expressed in Escherichia coli, and the purified proteins had specific activities toward urokinase-type plasminogen activator (uPA) or the single- and two-chain forms of tissue type plasminogen activator (tPA) that were similar to wtPAI-1. Experiments suggest that the strained loop of PAI-1 is not responsible for the transition between the latent and the active conformations or for binding to vitronectin. Second-order rate constants for the interactions with uPA and single- or two-chain tPA were similar to those of wtPAI-1. Values range from a low of 1.8 x 10(5) M-1 s-1 for the interaction of the PAI-2 chimera with single-chain tPA to a high value of 1.6 x 10(7) M-1 s-1 for the consensus mutant with two-chain tPA. This former value is 200 times higher than the reported rate constant for the interaction between PAI-2 and single-chain tPA, suggesting that structures outside of the strained loop are responsible for the major differences in specificity between PAI-1 and PAI-2.     

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.     

Catabolism of tissue-type plasminogen activator by the human hepatoma cell line Hep G2. Modulation by plasminogen activator inhibitor type 1

Catalytic activity of tissue-type plasminogen activator (t-PA) in plasma is regulated in part by formation of complexes with specific inhibitors as well as by hepatic clearance. Potential interaction of these two regulatory mechanisms was examined in the human hepatoma cell line Hep G2. These cells secrete plasminogen activator inhibitor type-1 (PAI-1) and initiate catabolism of exogenous t-PA by receptor-mediated endocytosis. Specific binding of 125I-t-PA to cells at 4 degrees C results in dose-dependent formation of a 95-kDa species recognized by monospecific anti-PAI-1 and anti-t-PA antibodies and stable in the presence of low (0.2%) concentrations of sodium dodecyl sulfate (SDS). Specific binding of 125I-t-PA and formation of the 95-kDa SDS-stable species are inhibited in a concentration-dependent manner following preincubation of cells with anti-PAI-1 antibodies. High and low molecular weight forms of urokinase plasminogen activator (u-PA) capable of forming specific complexes with PAI-1 complete for 125I-t-PA binding sites. However, the proenzyme form of u-PA (scu-PA), incapable of forming complexes with PAI-1, does not compete for 125I-t-PA binding sites. The role of the serine protease active site of t-PA in mediating both interaction with PAI-1 and specific binding was examined using 125I-t-PA that had been functionally inactivated with D-phenylalanyl-L-propyl-L-arginyl-chloromethyl ketone (PPACK). 125I-t-PA-PPACK, despite a 6-fold lower affinity than active 125I-t-PA, exhibited specific binding to cells without detectable formation of SDS-stable complexes with PAI-1. Both surface-bound 125I-t-PA and 125I-t-PA-PPACK are internalized and degraded by cells at 37 degrees C. 125I-t-PA is internalized as a stable complex with PAI-1, whereas 125I-t-PA-PPACK is internalized with similar kinetics but without the presence of an SDS-stable complex. Thus, PAI-1 appears capable of modulating t-PA catabolism in the human hepatocyte.