Activation of pro-urokinase and plasminogen on human sarcoma cells: a proteolytic system with surface-bound reactants

Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serum-containing medium, bound plasmin activity could be eluted from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited by an anticatalytic monoclonal antibody to u-PA, indicating that this enzyme was responsible for the activation. Preincubation of the cells with diisopropyl fluorophosphate-inhibited u-PA led to a decrease in surface-bound plasmin, indicating that a large part, if not all, of the cell surface plasminogen activation was catalyzed by surface-bound u-PA. In the absence of plasminogen, most of the cell surface u-PA was present in its single-chain proenzyme form, while addition of plasminogen led to formation of cell-bound two-chain u-PA. The latter reaction was catalyzed by cell-bound plasmin. Cell-bound u-PA was accessible to inhibition by endogenous PAI-1 and by added PAI-2, while the cell-bound plasmin was inaccessible to serum inhibitors, but accessible to added aprotinin and an anticatalytic monoclonal antibody. A model for cell surface plasminogen activation is proposed in which plasminogen binding to cells from serum medium is followed by plasminogen activation by trace amounts of bound active u-PA, to form bound plasmin, which in turn serves to produce more active u-PA from bound pro-u-PA. This exponential process is subject to regulation by endogenous PAI-1 and limited to the pericellular space.     

One-chain urokinase-type plasminogen activator from human sarcoma cells is a proenzyme with little or no intrinsic activity

We have compared the plasminogen activating capacity of one- and two-chain urokinase-type plasminogen activator (u-PA). In a 125I-plasminogen conversion assay in the presence of high amounts of a plasmin inhibitor, one-chain u-PA pretreated with diisopropyl fluorophosphate had no detectable activity, the detection limit corresponding to the activity of a 400-fold lower amount of two-chain u-PA. In coupled assays in which generated plasmin was measured with a synthetic substrate, activity was clearly observed with the one-chain preparation, but the initial rate of plasminogen activation was lower than that of a 250-fold smaller concentration of two-chain u-PA. The coupled assays for one-chain u-PA are self-activating because plasmin catalyzes conversion of one- to two-chain u-PA, and it is not possible to decide whether the low activity of one-chain u-PA observed with this type of assay is intrinsic or due to contaminations. On the basis of these findings and a discussion of previous studies, it is concluded that one-chain u-PA has a variety of properties similar to the one-chain proenzyme forms of other serine proteases and that it should, therefore, be considered as a genuine proenzyme form of u-PA.     

Plasminogen activation by t-PA on the surface of human melanoma cells in the presence of alpha 2-macroglobulin secretion.

Several human melanoma cell lines produced tissue-type plasminogen activator (t-PA), as detected by zymography and immunocapture assay of culture media and cell lysates. Urokinase (u-PA) was found at only less than or equal to 1% the level of t-PA. Acid eluates of the cell surface indicated that the melanoma cells had t-PA bound on their surface, but no u-PA, and also had a very low capacity to bind exogenous u-PA. After incubation of the melanoma cells with 10% plasminogen-depleted fetal calf serum and human plasminogen, bound plasmin activity could be eluted from the cell surface with tranexamic acid, an analogue of lysine. This indicated that plasminogen was activated on the cell surface. The cell-surface plasmin formation was inhibited by an anti-catalytic monoclonal antibody to human t-PA, and not by an anti-catalytic antibody to u-PA. The melanoma cells also synthesized and secreted alpha 2-macroglobulin (alpha 2M), as shown by alpha 2M-specific mRNA in Northern blotting and detection of alpha 2M protein in conditioned cell culture media. The media were found to inhibit u-PA but not t-PA. This inhibition was related to their alpha 2M content, and immunoabsorption of alpha 2M removed the inhibitory activity. These studies suggest that t-PA can bind to the surface of melanoma cells and generate surface-bound plasmin. Because t-PA and cell-bound plasmin are unaffected by alpha 2M, t-PA may, in the case of melanoma cells, serve an analogous function to u-PA in supporting tumor cell invasion.     

Anticoagulant low molecular weight heparin does not enhance the activation of plasminogen by tissue plasminogen activator

The activity of tissue plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) is stimulated by heparin. Heparin binds tightly to t-PA, u-PA, and plasminogen and decreases the usual stimulatory effect of fibrin on t-PA activity. In the present study we have found that low molecular weight heparin (LMW-heparin) preparations obtained by nitrous acid depolymerization or heparinase treatment of standard heparin have different properties with respect to their interaction with the fibrinolytic system. LMW-heparin prepared by either method does not stimulate plasmin formation by t-PA. However, these preparations of heparin still efficiently accelerate the inhibition of thrombin by antithrombin III. Binding data show that LMW-heparin does not bind t-PA and Glu-plasminogen and only binds very weakly to Lys-plasminogen. These results illustrate that it is possible to selectively destroy the fibrinolytic stimulating properties of heparin while leaving the classical anticoagulant characteristics intact.     

Identification and characterization of human endothelial cell membrane binding sites for tissue plasminogen activator and urokinase

Cultured human endothelial cells synthesize and secrete two types of plasminogen activator, tissue plasminogen activator (t-PA) and urokinase (u-PA). Previous work from this laboratory (Hajjar, K.A., Hamel, N. M., Harpel, P. C., and Nachman, R. L. (1987) J. Clin. Invest. 80, 1712-1719) has demonstrated dose-dependent, saturable, and high affinity binding of t-PA to two sites associated with cultural endothelial cell monolayers. We now report that an isolated plasma membrane-enriched endothelial cell fraction specifically binds 125I-t-PA at a single saturable site (Kd 9.1 nM; Bmax 3.1 pmol/mg membrane protein). Ligand blotting experiments demonstrated that both single and double-chain t-PA specifically bound to a Mr 40,000 membrane protein present in detergent extracts of isolated membranes, while high molecular weight, low molecular weight, and single-chain u-PA associated with a Mr 48,000 protein. Both binding interactions were reversible and cell-specific and were inhibitable by pretreatment of intact cells with nanomolar concentrations of trypsin. The relevant binding proteins were not found in subendothelial cell matrix, failed to react with antibodies to plasminogen activator inhibitor type 1 and interacted with their respective ligands in an active site-independent manner. The isolated t-PA binding site was resistant to reduction and preserved the capacity for plasmin generation. In contrast, the isolated u-PA binding protein was sensitive to reduction, and did not maintain the catalytic activity of the ligand on the blot. The results suggest that in addition to sharing a matrix-associated binding site (plasminogen activator inhibitor type 1), both t-PA and u-PA have unique membrane binding sites which may regulate their function. The results also provide further support for the hypothesis that plasminogen and t-PA can assemble on the endothelial cell surface in a manner which enhances cell surface generation of plasmin.     

Vascular origin determines plasminogen activator expression in human endothelial cells. Renal endothelial cells produce large amounts of single chain urokinase type plasminogen activator

Positioned at the boundary between intra- and extravascular compartments, endothelial cells may influence many processes through their production of plasminogen activators (PA). Available data have shown that tissue-type plasminogen activator (t-PA) is the major form produced by human endothelial cells. We have compared the molecular forms of PA produced by human endothelial cells from different microvascular and large vessel sources including two different sites within the circulation of the kidney. Using combined immunoactivity assays specific for u-PA and t-PA activity and antigen, we found that both human renal microvascular and renal artery endothelial cells produced high levels of u-PA antigen (60.48 ng/10(5) cells/24 h and 50.42 ng/10(5) cells/24 h, respectively) and corresponding levels of u-PA activity after activation with plasmin. Activity was not evident before plasmin activation, showing that the u-PA produced is almost exclusively as single chain form U-PA. In contrast, human omental microvascular endothelial cells and human umbilical vein endothelial cells produced exclusively t-PA (8.80 ng/10(5) cells/24 h and 2.17 ng/10(5) cells/24 h, respectively). Neither endothelial cell type from human kidney produced plasminogen activator inhibitor, as determined by reverse fibrin autography and titration assays. Agents including phorbol ester, thrombin, and dexamethasone were shown to regulate the renal endothelial cell production and mRNA expression of both u-PA and t-PA. Among the macro- and microvascular endothelial cells tested, only those from the renal circulation produced high levels of single chain form U-PA, suggesting the vascular bed of origin determines the expression of plasminogen activators.     

Thrombospondin is a slow tight-binding inhibitor of plasmin.

Thrombospondin is a multifunctional glycoprotein of platelet alpha-granules and a variety of growing cells. We demonstrate that thrombospondin is a slow tight-binding inhibitor of plasmin as determined by loss of amidolytic activity, loss of ability to cleave fibrinogen, and decreased lysis zones in fibrin plate assays. Stoichiometric titrations indicate that approximately 1 mol of plasmin interacts with 1 mol of thrombospondin, an unexpected result considering the trimeric nature of thrombospondin. Plasmin in a complex with streptokinase or bound to epsilon-aminocaproic acid is protected from inhibition by thrombospondin, thereby implicating the lysine-binding kringle domains of plasmin in the inhibition process. Thrombospondin also inhibits urokinase plasminogen activator, but more slowly than plasmin, stimulates the amidolytic activity of tissue plasminogen activator, and has no effect on the amidolytic activity of alpha-thrombin or factor Xa. These results, therefore, identify thrombospondin as a new type of serine proteinase inhibitor and potentially important regulator of fibrinolysis.     

Purification of epidermal plasminogen activator inhibitor

A plasminogen activator inhibitor was purified from human cornified cell extract by DEAE-Sepharose, Sephacryl S-200, and high-performance liquid chromatographies on hydroxyapatite HPHT and anion-exchanger Mono Q at pH 7.2 and 8.0. The purified inhibitor showed Mr 43,000 and pI 5.2 50% inhibition of fibrinolytic activity (1.5 IU) of urokinase and tissue-type plasminogen activator was attained by 0.60 ng and 11.0 ng purified inhibitor, respectively. Synthetic substrate assay demonstrated slow tight-binding inhibition to both urokinase and tissue-type plasminogen activator. The inhibitor did not inactivate plasmin, thrombin, glandular kallikrein or trypsin.     

The novel anti-tumour agent oxamflatin differentially regulates urokinase and plasminogen activator inhibitor type 2 expression and inhibits urokinase-mediated proteolytic activity

Cell surface, urokinase (u-PA)-mediated, plasminogen activation has recently been recognised as a process integral to extracellular matrix degradation. The primary inhibitor of u-PA activity in the extracellular matrix is plasminogen activator inhibitor type 2 (PAI-2), a serine protease inhibitor. The malignant metastatic phenotype is associated with excessive and uncontrolled, tumour cell-associated, u-PA-mediated, extracellular matrix degradation. Inhibition of the malignant metastatic phenotype via induction of PAI-2 expression and/or inhibition of u-PA expression may represent a novel means via which the metastatic phenotype can be arrested. Agents capable of inducing PAI-2 and/or inhibiting u-PA activity may restrict u-PA-mediated tumour cell proteolysis and facilitate in the development of therapeutic strategies to combat malignant disease. We have identified the hydroxamic acid derivative oxamflatin, previously noted to revert the malignant phenotype in K-ras-transformed NIH-3T3 cells, as capable of upregulating PAI-2 and simultaneously suppressing u-PA expression in two different cell systems. In addition, zymographic analysis indicated that oxamflatin treatment results in a significant reduction in u-PA proteolytic activity in both HT-1080 fibrosarcoma and U-937 histiocytic lymphoma cells. We postulate that oxamflatin represents a novel means by which induction of PAI-2 and concomitant inhibition of u-PA gene and protein expression can be achieved and may be of benefit in inhibiting the malignant metastatic phenotype.     

Characterization of a tissue-type plasminogen activator from porcine urine

Porcine urine, unlike human urine, does not contain detectable amounts of urokinase-type plasminogen activator (u-PA). The plasminogen activator present in porcine urine is of tissue-type (t-PA) as identified by the following criteria. (1) Porcine urine PA exhibits an Mr of 65,000 similar to the Mr of human t-PA (64-70,000) but distinct from the Mr of human u-PA (55,000). (2) Antibodies against human t-PA bind and inhibit crude and purified porcine urine PA, while human u-PA-specific antibodies do not react with porcine urine PA. (3) Plasminogen activation by porcine urine PA is markedly stimulated in the presence of fibrinogen fragments. (4) Porcine urine PA activity is not affected by concentration of amiloride substantially suppressing human u-PA activity.     

Urokinase-type plasminogen activator and its inhibitor secreted by cultured human monocyte-macrophages

Human blood monocytes in culture differentiate to macrophagelike cells within 1 week. Coinciding with this morphological transition the cells started releasing increasing amounts of the serine proteinase plasminogen activator (PA; Mr 56,000) of the urokinase (u-PA) type and the proteinase inhibitor alpha-2-macroglobulin (alpha 2M). Unlike the cell-associated PA activity, which was also readily detected in fresh monocytes, the activity secreted into the serum-free culture medium could be measured only after treatment of the samples with sodium dodecyl sulphate. Heat or acid treatment of the medium was not sufficient to reveal the PA activity, suggesting that, apart from alpha 2M, another PA-inhibiting substance was present in the culture medium. The inhibitor (Mr 65,000) was found to be synthesized by macrophages and specifically inhibited u-PA activity but not tissue-type PA (t-PA) or plasmin activity. Dexamethasone decreased the secretion of PA by differentiated macrophages without affecting the production of alpha 2M or the PA inhibitor. Dexamethasone also inhibited the morphological differentiation of the cells when added to the monocyte-phase cells.     

In vitro decreases of the fibrinolytic potential of cultured human fibrosarcoma cell line, HT1080, by Nigella sativa oil

It is generally accepted that the fibrinolytic potential of tumor cells is related to their malignant phenotype. In the present study, Nigella sativa oil (NSO) was studied to evaluate its effect on the fibrinolytic potential of the fibosarcoma cell line HT1080 to elucidate whether this oil might have an antitumor activity through its modulation of the fibrinolytic potential of such cells. NSO produced a concentration-dependent inhibition of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 (PAI-1). When subconfluent HT1080 cells were conditioned with oil, a concentration (0.0-200 microg oil/ml)-dependent decrease in t-PA, u-PA and PAI-1 antigen was observed. There was also a concentration-dependent decrease (from 0.0 to 112.5 microg oil/ml) in the confluent cultures. The results showed that blackseed oil decreases the fibrinolytic potential of the human fibrosarcoma cell line (HT1080) in vitro, implying that inhibition of local tumor invasion and metastasis may be one such mechanism.     

Thrombin increases proliferation and decreases fibrinolytic activity of kidney glomerular epithelial cells.

Human glomerular epithelial cells (GECs) in culture synthesize single-chain, urokinase-type plasminogen activator (SC-uPA), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor 1 (PAI-1) and possess specific membrane-binding sites for u-PA. Using purified 125I-alpha thrombin, we demonstrate here the presence of two populations of specific binding sites for thrombin on GECs (1.Kd = 4.3 +/- 1.0 x 10(-10) M, 5.4 +/- 1.4 x 10(4) M sites per cell, 2. Kd = 1.6 +/- 0.5 x 10(-8) M, 7.9 +/- 1.8 x 10(5) sites per cell). Purified human alpha thrombin promoted the proliferation of GECs and induced a time- and dose-dependent increase of SC-uPA, t-PA, and PAI-1 antigens released by GECs. Thrombin-mediated increase in antigen was paralleled by an increase in the levels of corresponding u-PA and PAI-1 messenger RNA. In contrast, thrombin decreased u-PA activity in conditioned medium. This discrepancy between u-PA antigen and u-PA activity was explained by a limited proteolysis of SC-uPA by thrombin, leading to a two-chain form detected by immunoblotting and that could not be activated by plasmin. Thrombin also decreased the number of u-PA binding sites on GECs (p less than 0.05) without changing receptor affinity. Hirudin inhibited the binding and the cellular effects of thrombin, whereas thrombin inactivated by diisopropylfluorophosphate had no effect, indicating that both membrane binding and catalytic activity of thrombin were required. We conclude that thrombin, through specific membrane receptors, stimulates proliferation of GECs and decreases the fibrinolytic activity of GECs both at the cell surface and in the conditioned medium. These results suggest that thrombin could be involved in the pathogenesis of extracapillary proliferation and persistency of fibrin deposits in crescentic glomerulonephritis.     

Involvement of the plasminogen activation system in cow endometritis

The objectives of this study were to investigate the: (a) presence and activity of components of the "plasminogen activators/plasmin" system in dairy cows with or without endometritis; (b) variations in enzyme activity according to the degree of endometritis; and (c) associations between these enzymes and changes in endometrial histology after intrauterine antibiotic treatment. Endometrial biopsies were collected from anestrus (no palpable ovarian structures and milk progesterone <1 ng/ml) Holstein cows, 30-40 days postpartum. On the basis of a vaginoscopic examination, rectal palpation of the cervix and uterus, and endometrial histology, there were 92 cows with endometritis and 20 cows without endometritis. After biopsy collection, each cow was given an intrauterine infusion of 1.5x10(6) IU of procaine penicillin G. In cows with endometritis, genital tract examinations and biopsies were repeated 2 weeks later. Both plasminogen activators (PAs), tissue type (t-PA) and urokinase (u-PA), were immunologically identified in all uterine biopsies. Plasminogen activator activity (PAA) increased, whereas plasminogen activator inhibition (PAI) and plasmin inhibition (PI) decreased in proportion to the degree of inflammation. Two weeks after intrauterine treatment, PAA had decreased significantly in all cows that had reduced severity of endometrial inflammation and had increased significantly in all cows with increased severity of inflammation. The change in the degree of inflammation depended upon plasminogen activator activity; cows with higher PAA were more likely to improve. In conclusion, there was evidence for a role of the plasminogen activation proteolytic system in bovine endometritis.     

The stratagem utilized by the plasminogen activator from the snake Trimeresurus stejnegeri to escape serpins

The plasminogen activator isolated from the venom of the snake Trimeresurus stejnegeri (TSV-PA) triggers plasmin production, along with tissue-type plasminogen activators (t-PA) and urokinase (u-PA). The half-life of TSV-PA in plasma is remarkable. We unveil in this paper two of the molecular mechanisms allowing TSV-PA to escape inhibition by plasma serpins. The first involves a phenylalanine at position 193 (chymotrypsinogen numbering system). Phe(193) distinguishes TSV-PA from nearly all trypsin-like proteinases, having glycine at this position. A mutant of TSV-PA (F193G), in which Phe(193) had been replaced by a glycine, was inactivated by plasminogen activator inhibitor 1 (PAI-1) and alpha(2)-antiplasmin 100-fold more rapidly than the wild-type enzyme. The second mechanism originates from the 37-loop of TSV-PA. Swapping the 37-loop of TSV-PA for either that of t-PA or that of u-PA also increased dramatically the rate of inactivation by PAI-1. Loop swapping and F193G mutations were additive, resulting in a rate of inactivation by PAI-1 that was 4 orders of magnitude higher than for the wild-type enzyme. The potential role of Phe(193) and of the 37-loop in the immunity of TSV-PA toward alpha(1)-antitrypsin and antithrombin is also discussed.     

Human endothelial cells produce a plasminogen activator inhibitor and a tissue-type plasminogen activator-inhibitor complex

Serum-free conditioned media and cell extracts from cultured human umbilical vein endothelial cells were analyzed for plasminogen activator by SDS-polyacrylamide gel electrophoresis and enzymography on fibrin-indicator gels. Active bands of free and complexed tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA) were identified by the incorporation of specific antibodies against, respectively, t-PA or u-PA in the indicator gel. The endothelial cells predominantly released a high-molecular-weight t-PA (95000–135000). This t-PA form was converted to M_r-72000 t-PA by 1.5 M NH₄OH/39 mM SDS. A component with high affinity for both t-PA and u-PA could be demonstrated in serum-free conditioned medium and endothelial cell extract. The complex between this component and M_r-72000 t-PA comigrated with high-molecular-weight t-PA. From the increase in M_r of t-PA or u-PA upon complex formation, the M_r of the endothelial cell component was estimated to be 50000–70000. The reaction between t-PA or u-PA and the plasminogen activator-binding component was blocked by 5 mM p-aminobenzamidine, while the complexes, once formed, could be cleaved by 1.5 M NH₄OH/39 mM SDS. These observations indicated that the active center of plasminogen activator was involed in the complex formation. It was further noted that serum-free conditioned medium of endothelial cell extract inhibited plasminogen activator activity when assayed by the fibrin-plate method. Evidence is provided that the plasminogen activator-binding component was different from a number of the known plasma serine proteinase inhibitors, the placenta inhibitor and the fibroblast surface protein, proteinase-nexin. We conclude that cultured endothelial cells produce a rapid inhibitor of u-PA and t-PA as well as a t-PA-inhibitor complex.     

Mutant and chimeric recombinant plasminogen activators. Production in eukaryotic cells and preliminary characterization

Mutant urokinase-type plasminogen activator (u-PA) genes and hybrid genes between tissue-type plasminogen activator (t-PA) and u-PA have been designed to direct the synthesis of new plasminogen activators and to investigate the structure-function relationship in these molecules. The following classes of constructs were made starting from cDNA encoding human t-PA or u-PA: 1) u-PA mutants in which the Arg156 and Lys158 were substituted with threonine, thus preventing cleavage by thrombin and plasmin; 2) hybrid molecules in which the NH2-terminal regions of t-PA (amino acid residues 1-67, 1-262, or 1-313) were fused with the COOH-terminal region of u-PA (amino acids 136-411, 139-411, or 195-411, respectively); and 3) a hybrid molecule in which the second kringle of t-PA (amino acids 173-262) was inserted between amino acids 130 and 139 of u-PA. In all cases but one, the recombinant proteins, produced by transfected eukaryotic cells, were efficiently secreted in the culture medium. The translation products have been tested for their ability to activate plasminogen after in situ binding to an insolubilized monoclonal antibody directed against urokinase. All recombinant enzymes were shown to be active, except those in which Lys158 of u-PA was substituted with threonine. Recombination of structural regions derived from t-PA, such as the finger, the kringle 2, or most of the A-chain sequences, with the protease part or the complete u-PA molecule did not impair the catalytic activity of the hybrid polypeptides. This observation supports the hypothesis that structural domains in t-PA and u-PA fold independently from one to another.     

Concentration of plasminogen activators and inhibitor in the human endometrium at different phases of the menstrual cycle.

The concentrations of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA) and plasminogen activator inhibitor (PAI-1) have been determined in endometrial curettings obtained from 46 subfertile women during proliferative, early or late secretory phases of the menstrual cycle. t-PA activity and antigen concentrations was significantly higher (P < 0.001) in late secretory endometrium than in proliferative or early secretory endometrium. Higher concentrations of PAI-1 antigen (P < 0.05) were also noted in late secretory phase than in proliferative and early secretory endometrium. However, u-PA concentration was not significantly different and no PAI activity could be demonstrated in the menstrual phases studied. Zymography studies confirmed the presence of both t-PA and u-PA in the endometrium. Ovarian hormonal patterns may therefore influence the activity of plasminogen activators especially of t-PA in the endometrium during various phases of the menstrual cycle.     

Plasminogen activator activity is inhibited while neuroserpin is up‐regulated in the Alzheimer disease brain

Amyloid‐beta plaques are a pathological hallmark of Alzheimer’s disease. Several proteases are known to cleave/remove amyloid‐beta, including plasmin, the product of tissue plasminogen activator cleavage of the pro‐enzyme plasminogen. Although plasmin levels are lower in Alzheimer brain, there has been little analysis of the plasminogen activator/plasmin system in the brains of Alzheimer patients. In this study, zymography, immunocapture, and ELISAs were utilized to show that tissue plasminogen activator activity in frontal cortex tissue of Alzheimer patients is dramatically reduced compared with age‐matched controls, while tissue plasminogen activator and plasminogen protein levels are unchanged; suggesting that plasminogen activator activity is inhibited in the Alzheimer brain. Analysis of endogenous plasminogen activator inhibitors shows that while plasminogen activator inhibitor‐1 and protease nexin‐1 levels are unchanged, the neuroserpin levels are significantly elevated in brains of Alzheimer patients. Furthermore, elevated amounts of tissue plasminogen activator‐neuroserpin complexes are seen in the Alzheimer brain, and immunohistochemical studies demonstrate that both tissue plasminogen activator and neuroserpin are associated with amyloid‐beta plaques in Alzheimer brain tissue. Thus, neuroserpin inhibition of tissue plasminogen activator activity leads to reduced plasmin and may be responsible for reduced clearance of amyloid‐beta in the Alzheimer disease brain. Furthermore, decreased tissue plasminogen activator activity in the Alzheimer brain may directly influence synaptic activity and impair cognitive function.