The role of SerpinB2 in immunity.

SerpinB2 or plasminogen activator inhibitor type 2 (PAI-2) is widely described as an inhibitor of extracellular urokinase plasminogen activator. However, the evidence that this represents the physiological role of SerpinB2 is not overly compelling. SerpinB2 is substantially up-regulated under multiple inflammatory conditions, and dysregulated expression and polymorphisms are associated with several human inflammatory diseases. A bewildering array of diverse functions and activities have been attributed to SerpinB2, but despite ≈900 publications in the field, no coherent view of what SerpinB2 does in vivo has emerged. Although SerpinB2 is abundantly expressed by activated macrophages and a range of other haematopoietic and non-haematopoietic cells, SerpinB2(-/-) mice have surprisingly few phenotypes. However, SerpinB2(-/-) mice were recently shown to generate increased Th1 responses, suggesting that at least one function of SerpinB2 is sculpting of the adaptive immune response.

Mycobacterium tuberculosis blocks crosslinking of annexin-1 and apoptotic envelope formation on infected macrophages to maintain virulence.

Macrophages infected with attenuated Mycobacterium tuberculosis strain H37Ra become apoptotic, which limits bacterial replication and facilitates antigen presentation. Here we demonstrate that cells infected with H37Ra became apoptotic after the formation of an apoptotic envelope on their surface was complete. This process required exposure of phosphatidylserine on the cell surface, followed by deposition of the phospholipid-binding protein annexin-1 and then transglutaminase-mediated crosslinking of annexin-1 through its amino-terminal domain. In macrophages infected with the virulent strain H37Rv, in contrast, the amino-terminal domain of annexin-1 was removed by proteolysis, thus preventing completion of the apoptotic envelope, which resulted in macrophage death by necrosis. Virulent M. tuberculosis therefore avoids the host defense system by blocking formation of the apoptotic envelope, which leads to macrophage necrosis and dissemination of infection in the lung.

Signaling pathways and genes that inhibit pathogen-induced macrophage apoptosis--CREB and NF-kappaB as key regulators.

Certain microbes evade host innate immunity by killing activated macrophages with the help of virulence factors that target prosurvival pathways. For instance, infection of macrophages with the TLR4-activating bacterium Bacillus anthracis triggers an apoptotic response due to inhibition of p38 MAP kinase activation by the bacterial-produced lethal toxin. Other pathogens induce macrophage apoptosis by preventing activation of NF-kappaB, which depends on IkappaB kinase beta (IKKbeta). To better understand how p38 and NF-kappaB maintain macrophage survival, we searched for target genes whose products prevent TLR4-induced apoptosis and a p38-dependent transcription factor required for their induction. Here we describe key roles for transcription factor CREB, a target for p38 signaling, and the plasminogen activator 2 (PAI-2) gene, a target for CREB, in maintenance of macrophage survival.

Inhibition of retinoblastoma protein degradation by interaction with the serpin plasminogen activator inhibitor 2 via a novel consensus motif.

Plasminogen activator inhibitor-2 (PAI-2) is well documented as an inhibitor of the extracellular serine proteinase urokinase-type plasminogen activator (uPA) and is expressed in activated monocytes and macrophages, differentiating keratinocytes, and many tumors. Here we show that PAI-2 has a novel intracellular function as a retinoblastoma protein (Rb)-binding protein. PAI-2 colocalized with Rb in the nucleus and inhibited the turnover of Rb, which led to increases in Rb protein levels and Rb-mediated activities. Although PAI-2 contains an LXCXE motif, Rb binding was primarily mediated by the C-D interhelical region of PAI-2, which was found to bind to the C pocket of Rb. The C-D interhelical region of PAI-2 contained a novel Rb-binding motif, termed the PENF homology motif, which is shared by many cellular and viral Rb-binding proteins. PAI-2 expression also protected Rb from the accelerated degradation mediated by human papillomavirus (HPV) E7, leading to recovery of Rb and inhibition of E6/E7 mRNA expression. Protection of Rb by PAI-2 begins to explain many of the diverse, uPA-independent phenotypes conferred by PAI-2 expression. These results indicate that PAI-2 may enhance Rb's tumor suppressor activity and suggest a potential therapeutic role for PAI-2 against HPV-transformed lesions.

The localization of the relaxed form of plasminogen activator inhibitor type 2 in human gingival tissues.

The plasminogen activating system is important in extracellular proteolysis. Plasmin degrades tissues and activates proteases. Plasminogen activators (tissue type; t-PA and urokinase type; u-PA) and plasminogen activator inhibitors (PAI-1, PAI-2) are found in high concentrations in gingival crevicular fluid (GCF). Previous findings indicate the significance of PAI-2 in gingival inflammation. When PAI-2 inhibits a plasminogen activator its conformation relaxes and neoepitopes can be detected with a monoclonal antibody (#2H5). Our aim was to study if and where in the gingival region PAI-2 has acted as an inhibitor. Methodological studies were performed on GCF with western blotting. Frozen sections of human gingiva were studied immunohistochemically. The methodological studies showed that our antibody #2H5 selectively detects relaxed low molecular weight non-glycosylated PAI-2. Total PAI-2 and relaxed PAI-2 were found in all gingival epithelia with a honeycomb-like staining. Relaxed PAI-2 showed the most pronounced staining in the cell layers near the surface of the epithelium and no staining in the suprabasal layers, while total PAI-2 was found throughout the epithelium, often more pronounced suprabasally. The results showed that PAI-2 indeed has acted as an inhibitor of a protease in gingival tissues, primarily in the epithelia. The results also suggest primarily an intracellular localization and thus the interaction of PAI-2 with a protein other than t-PA.

Plasminogen activator inhibitor 2 and urokinase-type plasminogen activator in plasma and leucocytes in patients with severe sepsis.

Proteins influencing plasminogen activation to plasmin, namely plasminogen activators tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) and their principal inhibitors, plasminogen activator inhibitor 1 (PAI-1) and PAI-2, were measured in the plasma, the polymorph and mononuclear cell fractions taken from patients with major sepsis who were entering a general intensive care unit. The purpose of this study was to elucidate the factors favouring the persistence of fibrin in the microvasculature and thus contributing to multiple organ failure. Levels of u-PA antigen in plasma rose in sepsis and u-PA activity, not detectable in normal plasma, appeared. Levels of u-PA antigen in the cell fractions fell concomitantly. t-PA antigen in plasma and in the mononuclear cell fraction rose in sepsis, but t-PA activity was not detectable. Plasma PAI-1 antigen levels were strikingly raised in sepsis, presumably accounting for the complete neutralization of t-PA activity. PAI-2 antigen, not normally detected in plasma, appeared in the plasma of some patients, whereas it disappeared from the cellular fractions. Appearance of PAI-2 in plasma was associated with non-survival of the patient. The observations indicate that all the agents involved in plasminogen activation are released into the plasma in major sepsis. The levels of PAI-1 reached were quantitatively sufficient to suppress all activity of the released t-PA, but the inhibitors did not prevent expression of u-PA activity in the circulation. Circulating active u-PA and PAI-2 in the plasma of patients with severe sepsis may represent material originating from leucocytes. Leucocyte release of these agents within fibrin deposits may influence the persistence of fibrin and thus the development of multiple organ failure.

Clinical significance of urokinase-type plasminogen activator activity in hepatocellular carcinoma.

BACKGROUND AND METHODS: The plasminogen activation system plays a crucial role in the process of cancer invasion and metastasis. To evaluate the most effective factor in the invasion, metastasis and prognosis of hepatocellular carcinoma (HCC), we examined urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor (PAI)-1, PAI-2 and uPA activity by enzyme-linked immunosorbent assays (ELISA) in HCC tissues obtained from 46 patients. The results were compared with the patients' clinicopathological features and prognoses. RESULTS: Of the clinicopathological features, only histological portal involvement or intrahepatic metastasis, or both (INV), was significantly correlated to the disease-free survival rates (DFS; P < 0.05). The levels of uPA, PAI-1 and PAI-2 antigens were significantly associated with INV and histological grade. The DFS was not different, however, between cases with uPA, PAI-1 and PAI-2 values above and below the median. The high levels of uPA activity were closely related to INV (P < 0.001), and the activity gradually raised histological grades (P < 0.0001). The DFS was significantly different between patients with uPA activity below and above the median (0.70 ng/mL; P = 0.0092); it was also significantly different between such patients without INV (P < 0.05). CONCLUSIONS: Urokinase-type plasminogen activator activity may be the most sensitive factor affecting HCC invasion in the plasminogen activation system and a strong predictor for the recurrence of HCC. We suggest that cases with uPA activity of more than 0.70 ng/mL should be carefully followed up for possible HCC recurrence.

Topological localization of plasminogen activator inhibitor type 2.

BACKGROUND: Plasminogen activator inhibitor type 2 (PAI-2) is a member of the serine protease inhibitor (SERPIN) superfamily and forms stable complexes with urokinase type plasminogen activator (uPA). uPA can be found on the cell surface attached to its specific receptor (uPAR), allowing for controlled degradation of the extracellular matrix by the activation of plasminogen into plasmin. The aim of this study was to evaluate if PAI-2 could also be detected on the cell surface, providing a means of regulating the activity of cell surface uPA. METHODS: Intact or permeabilized cell lines or human peripheral blood leukocytes were assayed by flow cytometry for cell surface uPA or PAI-2. Plasma membrane-enriched preparations prepared from Jurkat, HaCaT, THP-1, U937, or MM6 cells were assayed by enzyme-linked immunosorbent assay (ELISA) or Western blotting for PAI-2 antigen. RESULTS: By flow cytometry, cell surface PAI-2 was not detected on monocytes from human peripheral blood, MM6, or HaCaT cells. Cell surface PAI-2 was only detected very weakly on the surface of U937 cells. In contrast, PAI-2 could be detected in all of these cells when fixed and permeabilized. By ELISA, PAI-2 was very abundant in the cytosol-enriched preparations of U937, MM6, and HaCaT cells, but was present in lower amounts in the plasma membrane-enriched preparations. By Western blotting, monomeric nonglycosylated PAI-2, but not uPA/PAI-2 complexes, could be detected in the cytosol and plasma membrane-enriched preparations. CONCLUSIONS: These results indicate that PAI-2 cannot be detected on the surface of PAI-2-expressing cells, and confirm that PAI-2 is predominantly a cytosolic protein.

Epitope mapping of monoclonal antibodies directed to PAI-1 using PAI-1/PAI-2 chimera and PAI-1-derived synthetic peptides.

Plasminogen activator inhibitor type-1 is a key regulatory protein of the fibrinolytic system that is involved in a variety of physiological and pathophysiological processes. A panel of 14 monoclonal antibodies directed against plasminogen activator inhibitor type-1 was analyzed regarding epitope specificity on plasminogen activator inhibitor type-1. For this purpose, chimera consisting of plasminogen activator inhibitor type-1 and another plasminogen activator inhibitor, plasminogen activator inhibitor type-2, with different portions of the respective wild-type proteins, were generated and plasminogen activator inhibitor type-1-derived 20-mer and 10-mer linear peptides were synthesized. Nine of the 14 monoclonal antibodies recognized an epitope located in the region between amino acid 76-188 of plasminogen activator inhibitor type-1, which encompasses the binding sites for vitronectin, heparin, and part of the fibrin binding region. Of these nine monoclonal antibodies, six reacted with a quadruple plasminogen activator inhibitor type-1 mutant (N152H, K156T, Q321L, M356I), and seven detected a plasminogen activator inhibitor type-1 deletion mutant (DeltaF111-H114). Two of the remaining five monoclonal antibodies recognized epitopes located between amino acid 209-227 and amino acid 352-371, respectively, while the other three antibodies reacted with wild-type plasminogen activator inhibitor type-1, only. Additional experiments revealed that two of the 14 mAbs neutralized and one monoclonal antibodies increased plasminogen activator inhibitor type-1 activity toward urokinase-type plasminogen activator, one of its target proteases.

Cellular distribution and clinical value of urokinase-type plasminogen activator, its receptor, and plasminogen activator inhibitor-2 in esophageal squamous cell carcinoma.

To assess the participation of the plasminogen activation system in the invasiveness of esophageal squamous cell carcinoma, we performed immunohistochemistry and in situ hybridization to study the distribution of a urokinase-type plasminogen activator (u-PA), u-PA receptor (u-PAR), and plasminogen activator inhibitor-2 (PAI-2). u-PA and PAI-2 were expressed heterogeneously in cancer cells, and restricted expression was found in stromal cells, especially fibroblasts, that were located in the immediate proximity of the cancerous cells. u-PAR was found only in cancer cells located at the periphery of tumors. Compared with patients with u-PA-negative cancer cells, patients with u-PA-positive cancer cells more frequently showed a neoplastic invasion beyond the muscularis propria and lymph node metastases. They also showed a significantly shorter 5-year overall survival. Patients with PAI-2-positive fibroblasts showed significantly lower levels of local invasiveness, represented by a neoplastic invasion beyond the muscularis propria, than those who were PAI-2 negative. Our results suggest that the expression of u-PA in esophageal squamous cell carcinoma is predictive of poor survival, whereas the expression of PAI-2 in the fibroblasts surrounding them is protective. An analysis of u-PA and PAI-2 expression in cancer cells and their surrounding fibroblasts may be useful for predicting the prognosis of patients with esophageal squamous cell carcinoma.

Evidence for intracellular cleavage of plasminogen activator inhibitor type 2 (PAI-2) in normal epidermal keratinocytes.

Plasminogen activator inhibitor type 2 (PAI-2) is a serine proteinase inhibitor (serpin), present in high quantities in stratified squamous epithelia. Detergent extracts of human epidermis or cultured keratinocytes contain primarily active, nonglycosylated PAI-2. In keratinocytes, the vast majority of PAI-2 is retained within the cell, supporting the hypothesis that PAI-2 may serve specific intracellular function(s) through interaction with an unknown cytoplasmic proteinase. During interaction with the target proteinase, cleavage of PAI-2 within its reactive site loop leads to the formation of a more stable, "relaxed" conformation (PAI-2r). Using a monoclonal antibody specific for PAI-2r, we demonstrate here that PAI-2r is present in keratinocytes of the granular and basal layers of normal human epidermis. In addition, PAI-2r is detectable in cultured human epidermal keratinocytes, where it is concentrated in a detergent-insoluble fraction within differentiating cells. These data provide evidence for the presence of an endogenous, keratinocyte-derived proteinase that constitutively cleaves intracellular PAI-2 in normal human epidermal keratinocytes. Cleavage of PAI-2 by this proteinase may reflect specific intracellular action of PAI-2 in normal cells. Finally, we demonstrate that a commercially available anti-PAI-2 monoclonal antibody (#3750, American Diagnostica, Greenwich, CT), under native experimental conditions, preferentially recognizes the uncleaved, active form of PAI-2 and does not efficiently detect PAI-2r.

Secretion of plasminogen activator inhibitor 2 by human peripheral blood monocytes occurs via an endoplasmic reticulum-golgi-independent pathway.

Plasminogen activator inhibitor 2 (PAI-2) is a serine protease inhibitor (serpin) that is secreted and accumulated intracellularly by monocytes. We investigated PAI-2 synthesis by isolated human peripheral blood monocytes and found that a 47-kDa nonglycosylated form of PAI-2 was abundant in conditioned medium from monocytes. Secretion of PAI-2 by monocytes was not inhibited by agents that inhibit either ER-Golgi pathway-dependent secretion, brefeldin A, or N-linked glycosylation, tunicamycin. IL-1beta served as a control for a protein that is secreted by an ER-Golgi-independent pathway, and secretion of IL-1beta was not inhibited by brefeldin A. This was in contrast to secretion of TNFalpha, which was dependent on the ER-Golgi pathway. None of the treatments was cytotoxic toward monocytes, as measured by release of the intracellular enzyme lactate dehydrogenase (LDH) into the conditioned medium. Subcellular fractionation revealed that PAI-2 and IL-1beta were colocalized. The mechanism for secretion of PAI-2 was not dependent on calcium or intracellular trafficking via the classical vesicular mechanism(s), distinguishing it from IL-1beta secretion. These studies show that PAI-2 is secreted by primary human monocytes via an ER-Golgi-independent pathway.

Immunological detection of conformational neoepitopes associated with the serpin activity of plasminogen activator inhibitor type-2.

The physiological roles of plasminogen activator inhibitor-2 (PAI-2) are not yet well understood. Kinetic studies suggest a role in the regulation of plasminogen activator-driven proteolysis in many cell types. This study describes a monoclonal antibody (2H5), which uniquely recognizes neoepitope determinants on PAI-2 appearing after thermodynamic relaxation of the molecule. Enzyme-linked immunosorbent assays and native polyacrylamide gel electrophoresis immunoblotting confirmed the specificity of 2H5 for urokinase type plasminogen activator.PAI-2 complexes. Examination of the affinity of 2H5 for complexes formed between PAI-2 and a synthetic 14-mer reactive site loop peptide, PAI-2 treated with tissue plasminogen activator, or thrombin suggests that the 2H5 epitope is determined exclusively by sequences found only on PAI-2 following proteolytic cleavage of the Arg380-Thr381 bond and insertion of the reactive site loop into beta-sheet A. Peptides lacking both the P13 (Glu368) and P14 (Thr367) residues did not induce a conformational change or affect the inhibitory activity of PAI-2, indicating that one or both of these residues are critical for PAI-2 function. To our knowledge, this is the first description of a monoclonal antibody that can distinguish conformational changes in PAI-2 related specifically to its potential biological function(s).

Fibrinolysis during normal pregnancy and severe preeclampsia relationships between plasma levels of plasminogen activators and inhibitors.

The antigen levels of plasminogen activator inhibitor type 1 (PAI-1), plasminogen activator inhibitor type 2 (PAI-2), tissue-type plasminogen activator (t-PA), and urokinase-type plasminogen activator (u-PA) were measured during pregnancy and severe preeclampsia. The PAI-1 and PAI-2 antigen levels increased linearly during pregnancy and promptly returned to the level of non-pregnant women after delivery. The t-PA and u-PA antigen levels increased slowly and linearly during pregnancy. In severe preeclampsia, the PAI-2 antigen level was lower than that observed during the 3rd trimester of normal pregnancy, and the t-PA antigen level was higher than that during the 3rd trimester of normal pregnancy. Immunohistochemical examination of placental tissue showed that the PAI-2 and u-PA antigens were localized in syncytiotrophoblasts. In severe preeclampsia, the PAI-2 and u-PA antigens in placental tissue did not stain as clearly as during the 3rd trimester of normal pregnancy. The PAI-2 and u-PA antigen levels showed a positive correlation with birth weight. Therefore, PAI-2 may be an important marker of placental function and abnormality in pregnancies.

Plasminogen activation in bullous pemphigoid immunohistology reveals urokinase type plasminogen activator, its receptor and plasminogen activator inhibitor type-2 in lesional epidermis.

Keratinocytes synthesize urokinase-type plasminogen activator (uPA) and a specific cell surface receptor for uPA (uPA-R, CD 87). Plasminogen is present in plasma and interstitial fluids from where it is bound to cell surfaces via plasmin(ogen) binding sites. uPA binds to the uPA-R in an autocrine manner and activates cell-bound plasminogen: a mechanism, which provides plasmin for pericellular proteolysis. Cell-bound uPA is regulated by plasminogen activator inhibitor type-1 (PAI-1) or type-2 (PAI-2). Bullous pemphigoid is an autoimmune inflammatory skin disease characterized by subepidermal blisters. Although circumstantial evidence suggested plasminogen activation in lesional epidermis of bullous pemphigoid, immunohistological data on the type of plasminogen activators, on the uPA-receptor or the type of plasminogen activator inhibitors in the lesions of bullous pemphigoid are lacking so far. To obtain this information we have performed the present immunohistological study. The presence of uPA and its receptor as well as PAI-2 was disclosed in epidermal keratinocytes in the roof of the subepidermal blisters. Moreover, keratinocytes at the bottom of the blister, which most likely represent keratinocytes during reepithelialization were stained. Co-localization was found for uPA and its receptor, uPA and plasmin(ogen) as well as for uPA and PAI-2. In non-lesional epidermis of bullous pemphigoid only PAI-2 was found. We propose that the expression of uPA and uPA-R, as well as the upregulation of PAI-2 in keratinocytes of lesional epidermis is part of the repair and reepithelialization process following lesion formation, i.e. epidermo-dermal dyshesion, in bullous pemphigoid.

The receptor for urokinase-type plasminogen activator of a human keratinocyte line (HaCaT).

It is assumed that plasmin participates in pericellular proteolysis in the epidermis. Plasmin is generated by keratinocyte-associated plasminogen activators from the proenzyme plasminogen; plasminogen activation can proceed at the keratinocyte surface. The resultant plasmin interferes with cell to matrix adhesion and does possibly contribute to keratinocyte migration during reepithelialization. Here we describe the receptor for urokinase-type plasminogen activator (uPA-R) in the human keratinocyte cell line HaCaT, which serves to direct plasminogen activation to the cell surface; we relate the receptor to the uPA-R previously described in human myelo-/monocytes. Binding of uPA to the receptor accelerated plasminogen activation by a factor of approximately 10, compared to uPA in solution. Receptor-bound uPA was susceptible to inhibition by the plasminogen activator inhibitors 1 and 2. uPA and uPA-R antigen, as well as uPA activity, were localized to the leading front of expanding sheets of HaCaT cells. Exposure of HaCaT cells to plasminogen was followed by detachment of the cells. Detachment was prevented by an anticatalytic anti-uPA antibody, by the plasmin-specific inhibitor aprotinin, and by the lysine analogue tranexamic acid, the latter of which prevents plasmin(ogen) binding to the cell surface. Our findings support the hypothesis that uPA-mediated plasminogen activation is characteristic of mobile rather than sessile keratinocytes. Moreover, the uPA-R seems to focalize plasminogen activation to the surface of cells at the site of keratinocyte migration.

Expression of plasminogen activator inhibitor type 2 in normal and psoriatic epidermis.

The plasminogen activator (PA) proteolytic cascade has been implicated in the regulation of cell activities, including proliferation and differentiation, both of which occur continuously in normal human epidermis and are aberrant in psoriatic epidermis. To elucidate further the mechanisms by which PA is regulated in epidermis, we evaluated the levels of PA inhibitors type 1 (PAI-1) and type 2 (PAI-2) in normal and psoriatic epidermis. PAI-2, but not PAI-1, was detectable by mRNA, antigen, and activity assays, indicating that PAI-2 is the predominant epidermal PA inhibitor. In situ hybridization revealed that PAI-2 mRNA occurred throughout normal epidermis, although the signal was most intense in the granular layers. Similarly, PAI-2 antigen was most prominent in the granular layers; its distribution in these differential layers was along the cell periphery. Diffuse, fainter staining for PAI-2 was also detected in the basal cells and in some spinous layers of normal epidermis. Extracts of normal epidermis contained PA inhibitory activity identified as PAI-2 by immunoprecipitation with specific antibody. In psoriatic epidermis, PAI-2 mRNA and antigen were most prominent in the more superficial layers beneath the cornified cells. As with normal epidermis, PAI-2 assumed a pericellular distribution in the psoriatic cells. These data demonstrate that PAI-2 is constitutively expressed in vivo by keratinocytes in human epidermis and indicate that this protein is the predominant inhibitor of PA activity in normal and psoriatic human epidermis.

Microglia express the type 2 plasminogen activator inhibitor in the brain of control subjects and patients with Alzheimer's disease.

Localization of type 2 plasminogen activator inhibitor (PAI-2) was investigated immunohistochemically in postmortem brain tissue of control subjects and patients with Alzheimer's disease (AD). Both the mouse monoclonal and the goat polyclonal antibodies to PAI-2 stained microglia. Reactive microglia were stained more intensely than resting microglia. PAI-2 may regulate the plasminogen activators and plasmin system in lesions of AD. It is suggested that PAI-2 plays a role for cell migration and matrix breakdown as well as for aberrant neurite outgrowth in senile plaques where persistent microglial activation is observed.

Thrombin stimulates expression of tissue-type plasminogen activator and plasminogen activator inhibitor type 1 in cultured human vascular smooth muscle cells.

The effect of thrombin on the fibrinolytic potential of human vascular smooth muscle cells (SMC) in culture was studied. SMC of different origin responded to thrombin treatment with a dose and time dependent increase in tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1) levels in both cell lysates and conditioned media with maximum effects achieved at 10-20 IU/ml thrombin. PAI-1 antigen levels also increased in the extracellular matrix of thrombin treated SMC. PAI-2 levels in cell lysates of such SMC were not affected by thrombin. The effect was restricted to active thrombin, since DFP-thrombin and thrombin treated with hirudin showed no increasing effect on t-PA and PAI-1 levels in SMC. Enzymatically active thrombin also caused a four-fold increase in specific PAI-1 mRNA and a three-fold increase in t-PA mRNA. Furthermore we demonstrated the presence of high and low affinity binding sites for thrombin on the surface of SMC with a KD = 4.3 x 10(-10)M and 9.0 x 10(4) sites per cell and a KD = 0.6 x 10(-8) M and 5.8 x 10(5) sites per cell respectively. Thrombin could come in contact with SMC in case of vascular injury or following gap formation between endothelial cells. Our data support the idea that besides its known proliferative effect for SMC, thrombin could also modulate their fibrinolytic system.