Presence Of Plasminogen Activator Inhibitor-1 Research Articles

In vitro characterization of sonothrombolysis and echocontrast agents to treat ischemic stroke

The development of adjuvant techniques to improve thrombolytic efficacy is important for advancing ischemic stroke therapy. We characterized octafluoropropane and recombinant tissue plasminogen activator (rt-PA)-loaded echogenic liposomes (OFP t-ELIP) using differential interference and fluorescence microscopy, attenuation spectroscopy, and electrozone sensing. The loading of rt-PA in OFP t-ELIP was assessed using spectrophotometry. Further, it was tested whether the agent shields rt-PA against degradation by plasminogen activator inhibitor-1 (PAI-1). An in vitro system was used to assess whether ultrasound (US) combined with either Definity or OFP t-ELIP enhances rt-PA thrombolysis. Human whole blood clots were mounted in a flow system and visualized using an inverted microscope. The perfusate consisted of either (1) plasma alone, (2) rt-PA, (3) OFP t-ELIP, (4) rt-PA and US, (5) OFP t-ELIP and US, (6) Definity and US, or (7) rt-PA, Definity, and US (n = 16 clots per group). An intermittent US insonation scheme was employed (220 kHz frequency, and 0.44 MPa peak-to-peak pressures) for 30 min. Microscopic imaging revealed that OFP t-ELIP included a variety of structures such as liposomes (with and without gas) and lipid-shelled microbubbles. OFP t-ELIP preserved up to 76% of rt-PA activity in the presence of PAI-1, whereas only 24% activity was preserved for unencapsulated rt-PA. The use of US with rt-PA and Definity enhanced lytic efficacy (p < 0.05) relative to rt-PA alone. US combined with OFP t-ELIP enhanced lysis over OFP t-ELIP alone (p < 0.01). These results demonstrate that ultrasound combined with Definity or OFP t-ELIP can enhance the lytic activity relative to rt-PA or OFP t-ELIP alone, respectively.

THE PREVALENCE OF PAI-1 4G/5G POLYMORPHISM IN WOMEN WITH FETAL LOSS – FIRST DATA FOR A SERBIAN POPULATION / UČESTALOST POLIMORFIZMA PAI-1 4G/5G KOD ŽENA SA SPONTANIM POBAČAJEM - PRVI PODACI ZA SRPSKU POPULACIJU

Summary Background: Plasminogen activator inhibitor 1 (PAI-1) is an inhibitor of fibrinolysis. The PAI-1 4G/5G polymorphism is associated with elevated plasma levels of PAI-1. Over- expression of PAI-1 and impaired fibrinolysis in homozygous carriers of the 4G/4G PAI polymorphism may lead to abnor- mal placental formation and increased risk of fetal loss (FL). The aim of our study was to determine the frequency of this polymorphism in patients with FL in a Serbian population. Methods: The study was carried out in a group of 203 women (91 controls and 112 women with FL). The presence of PAI-1 4G/5G polymorphism was detected by PCR-RFLP analysis. Results: Slightly increased frequency of the PAI-1 4G/4G genotype was observed in the study group compared to the controls (32.1% vs. 30.8%). The frequency of PAI-1 was highest in women experiencing FL in the second trimester of pregnancy (50%), but this difference was not statistically sig- nificant. Conclusions: Our findings suggest that PAI-1 4G/4G might be a risk factor for FL occurring in the second trimester of pregnancy. Further studies are required in order to determine the role of PAI-1 4G/5G polymorphism in the etiology of FL.

The prevalence of PAI-1 4G/5G gene variant in Serbian population

Introduction: Plasminogen activator inhibitor 1 (PAI-1) has a major role in inhibition of firinolysis and normal haemostasis. The presence of the PAI-1 4G/4G genotype leads to increased expression of PAI-1. High blood level of PAI-1 is associated with many diseases such as thrombosis, cerebral insult, myocardial infarction, pregnancy loss, preeclampsia, insulin resistance, type 2 diabetes, breast cancer and asthma. In this study, the prevalence of PAI-1 4G/5G gene variant was determined in healthy subjects from Serbian population. Methods: The study was carried out in a group of 210 healthy subjects (105 women and 105 men). The presence of PAI-1 4G/5G gene variant was detected by PCR-RFLP analysis. Results: The prevalence of PAI-1 4G/4G genotype was 34.76% and it was increased compared to PAI-1 5G/5G genotype (19.05%). The most frequent was PAI-1 4G/5G genotype (46.19%). Allelic frequency for 4G allele was higher (0.58) compared to 5G allele (0.42). Conclusions: The prevalence of PAI-1 4G/5G gene variant in Serbian population is similar to the neighboring populations. Results of this study represent the first data for Serbian population. This study could be useful for further research where the role of PAI-1 4G/5G gene variant will be assessed in the pathogenesis of many diseases.

Inactivation of Plasminogen Activator Inhibitor-1 by RNA Aptamer Molecules

Abstract 1107 Introduction:The serine protease inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1), binds and inhibits the following plasminogen activators: tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA). This decreases plasmin production and triggers the dissolution of fibrin clots. Elevated levels of PAI-1 have been correlated with an increased risk for cardiovascular disease, as well as obesity and metabolic syndrome. Consequently, pharmacologically suppressing PAI-1 might prevent, or successfully treat vascular disease. Several PAI-1 small molecule inhibitors have recently been studied (PAI-039 is the best characterized). Since PAI-1 is a multifunctional protein, completely inhibiting PAI-1 may hinder its other functions. Therefore, it is important to independently develop inhibitors to the various regions of PAI-1. This can be accomplished by using small RNA molecules (aptamers) that bind with high affinity and specificity to individual protein domains. We recently published a paper showing how PAI-1 specific RNA aptamers bind to the heparin/vitronectin binding site of PAI-1 (Blake et al., 2009). We demonstrated that PAI-1 specific aptamers prevent cancer cells from detaching from vitronectin (in the presence of PAI-1), resulting in increased cell adhesion. These aptamers had no effect on PAI-1's other functions, particularly its antiproteolytic activity. Objective:This study's goal was to develop RNA aptamers to the active site of PAI-1; thereby, preventing the ability of PAI-1 to interact with plasminogen activators (tPA and uPA). Methods:The aptamers were generated by the systematic evolution of ligands by exponential enrichment (SELEX). Adopting the SELEX in vitro selection technique ensures the creation of nuclease-resistant RNA molecules that will bind to target proteins. We used in vitroassays to determine the effect of the aptamers on the interaction of PAI-1 with both tPA and uPA. Results:We isolated a family of aptamers that bind to wild-type PAI-1 with affinities in the nanomolar range. From this family, two aptamer clones (10–2 and 10–4) exhibited reduced binding to elastase cleaved PAI-1 and the PAI-1/tPA complex. This suggests that they bind to, or in the vicinity of, the active site. Using a chromogenic assay, we showed that the aptamer clone 10–4, and (to a lesser extent) the aptamer clone 10–2, inhibited PAI-1's antiproteolytic activity against tPA, further suggesting that these clones bind to PAI-1 within its active site region. Interestingly, neither clone was able to prevent PAI-1 from inhibiting uPA activity. Both aptamer clones disrupted PAI-1's ability to form a stable covalent complex with tPA. Increasing aptamer concentrations positively correlated with an increase in cleaved PAI-1, suggesting that these aptamer clones convert PAI-1 from an inhibitor to a substrate. Furthermore, we showed that both aptamer clones are able to inhibit PAI-1's activity in the presence of vitronectin. Conclusions:We have shown that we are able to inhibit one of PAI-1's functions without hindering its other functions. To our knowledge, this is the first report of an RNA molecule that is able to inhibit the antiproteolytic activity of PAI-1. We have generated two specific RNA aptamer molecules that hinder the ability of PAI-1 to interact with tPA, which has the potential to be used as an antithrombotic agent. Disclosures:No relevant conflicts of interest to declare.

Effects of plasminogen activator inhibitor-1-specific RNA aptamers on cell adhesion, motility, and tube formation.

The serine protease inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) is associated with the pathophysiology of several diseases, including cancer and cardiovascular disease. The extracellular matrix protein vitronectin increases at sites of vessel injury and is also present in fibrin clots. Integrins present on the cell surface bind to vitronectin and anchor the cell to the extracellular matrix. However, the binding of PAI-1 to vitronectin prevents this interaction, thereby decreasing both cell adhesion and migration. We previously developed PAI-1-specific RNA aptamers that bind to (or in the vicinity of) the vitronectin binding site of PAI-1. These aptamers prevented cancer cells from detaching from vitronectin in the presence of PAI-1, resulting in an increase in cell adhesion. In the current study, we used in vitro assays to investigate the effects that these aptamers have on human aortic smooth muscle cell (HASMC) and human umbilical vein endothelial cell (HUVEC) migration, adhesion, and proliferation. The PAI-1-specific aptamers (SM20 and WT15) increased attachment of HASMCs and HUVECs to vitronectin in the presence of PAI-1 in a dose-dependent manner. Whereas PAI-1 significantly inhibited cell migration through its interaction with vitronectin, both SM20 and WT15 restored cell migration. The PAI-1 vitronectin binding mutant (PAI-1AK) did not facilitate cell detachment or have an effect on cell migration. The effect on cell proliferation was minimal. Additionally, both SM20 and WT15 promoted tube formation on matrigel that was supplemented with vitronectin, thereby reversing the PAI-1's inhibition of tube formation. Collectively, results from this study show that SM20 and WT15 bind to the PAI-1's vitronectin binding site and interfere with its effect on cell migration, adhesion, and tube formation. By promoting smooth muscle and endothelial cell migration, these aptamers can potentially eliminate the adverse effects of elevated PAI-1 levels in the pathogenesis of vascular disease.

The Presence of PAI-1 4G/5G and ACE DD Genotypes Increases the Risk of Early-Stage AVF Thrombosis in Hemodialysis Patients

Background: In this study, we investigated the relationship between early arteriovenous fistula (AVF) thrombosis with angiotensin-converting enzyme (ACE) gene and thrombophilic factor gene polymorphisms. Methods: Thirty-five patients who suffered from three or more fistula thrombosis episodes in the early period after AVF operation and 33 control patients with no history of thrombosis for at least 3 years were enrolled in this study. Results: Factor V G1691A Leiden, factor V H1299R (R2), prothrombin G20210A, factor XIIIV34L, β-fibrinogen-455 G-A, glycoprotein IIIa L33P human platelet antigens (HPA-1), methylenetetrahydrofolate reductase C677T, and methylenetetrahydrofolate reductase A1298C gene polymorphisms were similar in both groups (p > 0.05). Plasminogen activator inhibitor 1 (PAI-1) 4G/5G genotype in the study group and 4G/4G genotype in the control group were significantly higher (p = 0.014). No significant difference was detected in terms of the 5G/5G genotype. With regard to the ACE gene polymorphism, the control group showed more ID genotype (19/33, 57.6%), whereas the study group showed more DD genotype (17/35, 48.6%). II genotype was similar in both groups (x2 = 7.40, p = 0.025). The rate of ACE inhibitor-angiotensin II receptor blockers use was 5/35 in the study group (14.3%) and 5/33 in the control group (15.2%). Individuals with PAI-1 4G/5G genotype showed 5.03 times more risk of thrombosis when compared with 4G/4G and 5G/5G genotypes [p = 0.008, OR = 5.03, 95% confidence interval (1.44:17.64)]. Individuals with ACE DD genotype showed 4.25 times more risk of thrombosis when compared with II and ID [p = 0.008, OR = 4.25, 95% confidence interval (1.404:12.83)]. Conclusion: PAI-1 4G/5G and ACE DD genotypes are associated with increased risk for early AVF thrombosis.

Effect of PAI-1 Specific RNA Aptamers On Cell Adhesion and Motility.

Abstract 2135Poster Board II-112 Introduction:The serine protease inhibitor (serpin), plasminogen activator inhibitor-1 (PAI-1) binds to and inhibits the plasminogen activators tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). This results in both a decrease in plasmin production, as well as a decrease in the dissolution of fibrin clots. PAI-1 is also associated with the pathophysiology of several diseases, including cancer and cardiovascular disease. Both experimental and clinical studies have shown that increasing the plasma and vessel wall PAI-1 levels positively correlates with an increased risk of cardiovascular-related events. Consequently, the pharmacological suppression of PAI-1 might prevent or treat vascular disease. Unfortunately, since PAI-1 is a multifunctional protein, complete inhibition of PAI-1 might hinder its ability to regulate fibrinolysis, which can provoke bleeding. However, eliminating the pathological functions of PAI-1 without hindering its physiological functions might be beneficial in treating a variety of diseases. Extracellular matrix vitronectin (VN) increases at sites of vessel injury and is also present in fibrin clots. In response to injury, vitronectin facilitates cell adhesion, thereby increasing vascular cell migration by binding to integrins and to surface-bound uPA. PAI-1 competes with integrins and the urokinase-type plasminogen activator receptor (uPAR) for VN binding, resulting in the detachment of cells from the extracellular matrix. The binding of PAI-1 to VN prevents integrins from binding to VN, and inhibits cell adhesion and migration. Objective:The goal of this study was to develop RNA aptamers to interfere with a single PAI-1 function, without obstructing its other functions. The present study concentrated on developing PAI-1 aptamers to the vitronectin binding site of PAI-1. Aptamers are single-stranded nucleic acids, either DNA or RNA, that bind to their target protein with high affinity and specificity. Methods:Our aptamers were generated by the systematic evolution of ligands by exponential enrichment (SELEX). Adopting the SELEX in vitro selection technique ensures creation of nuclease-resistant RNA molecules that will bind to target proteins. We used in vitro assays to determine the effect of the aptamers on the adhesion and migration of smooth muscle (SM) and human umbilical vein endothelial cells (EC). Results:Recently, we published a paper that showed the generation of PAI-1 specific RNA aptamers that bind to the heparin/vitronectin binding site of PAI-1 (Blake et al., 2009). We showed that PAI-1 specific aptamers prevented the detachment of cancer cells from vitronectin in the presence of PAI-1, resulting in an increase in cell adhesion. We have expanded these studies to include smooth muscle (SMC) and human umbilical vein endothelial cells (EC). We demonstrated that the PAI-1 specific aptamers (SM-20 and WT-15) dose dependently increase SMC and EC attachment in the presence of vitronectin (compared to the control aptamer). Interestingly, SM-20 (the aptamer to stable PAI-1) was more effective than WT-15 (aptamer to wild-type PAI-1). Whereas PAI-1 significantly inhibited cell migration (in the presence of vitronectin), the PAI-1 specific aptamers were able to restore migration of both SMC and EC cells. Additionally, the PAI-1 aptamers were unable to bind to the PAI-1 vitronectin binding mutant, further suggesting that these aptamers bind to the PAI-1's vitronectin binding site. Importantly, these aptamers did not affect the antiprotease activity of PAI-1. Conclusions:We have shown that we are able to inhibit one of PAI-1's functions without hindering its other functions. By promoting smooth muscle and endothelial cell migration, these aptamers can potentially eliminate the adverse effects of elevated PAI-1 levels in the pathogenesis of vascular disease. Disclosures:Sullenger:Regado Biosciences Inc.: Equity Ownership, Scientific Founder.

Antimetastatic Potential of PAI-1–Specific RNA Aptamers

The serine protease inhibitor plasminogen activator inhibitor-1 (PAI-1) is increased in several cancers, including breast, where it is associated with a poor outcome. Metastatic breast cancer has a dismal prognosis, as evidenced by treatment goals that are no longer curative but are largely palliative in nature. PAI-1 competes with integrins and the urokinase plasminogen activator receptor on the surface of breast cancer cells for binding to vitronectin. This results in the detachment of tumor cells from the extracellular matrix, which is critical to the metastatic process. For this reason, we sought to isolate RNA aptamers that disrupt the interaction between PAI-1 and vitronectin. Through utilization of combinatorial chemistry techniques, aptamers have been selected that bind to PAI-1 with high affinity and specificity. We identified two aptamers, WT-15 and SM-20, that disrupt the interactions between PAI-1 and heparin, as well as PAI-1 and vitronectin, without affecting the antiprotease activity of PAI-1. Furthermore, SM-20 prevented the detachment of breast cancer cells (MDA-MB-231) from vitronectin in the presence of PAI-1, resulting in an increase in cellular adhesion. Therefore, the PAI-1 aptamer SM-20 demonstrates therapeutic potential as an antimetastatic agent and could possibly be used as an adjuvant to traditional chemotherapy for breast cancer.

Neutralization of Plasminogen Activator Inhibitor I (PAI-1) by the Synthetic Antagonist PAI-749 via a Dual Mechanism of Action

PAI-749 is a potent and selective synthetic antagonist of plasminogen activator inhibitor 1 (PAI-1) that preserved tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) activities in the presence of PAI-1 (IC(50) values, 157 and 87 nM, respectively). The fluorescence (Fl) of fluorophore-tagged PAI-1 (PAI-NBD119) was quenched by PAI-749; the apparent K(d) (254 nM) was similar to the IC(50) (140 nM) for PAI-NBD119 inactivation. PAI-749 analogs displayed the same potency rank order for neutralizing PAI-1 activity and perturbing PAI-NBD119 Fl; hence, binding of PAI-749 to PAI-1 and inactivation of PAI-1 activity are tightly linked. Exposure of PAI-1 to PAI-749 for 5 min (sufficient for full inactivation) followed by PAI-749 sequestration with Tween 80 micelles yielded active PAI-1; thus, PAI-749 did not irreversibly inactivate PAI-1, a known metastable protein. Treatment of PAI-1 with a PAI-749 homolog (producing less assay interference) blocked the ability of PAI-1 to displace p-aminobenzamidine from the uPA active site. Consistent with this observation, PAI-749 abolished formation of the SDS-stable tPA/PAI-1 complex. PAI-749-mediated neutralization of PAI-1 was associated with induction of PAI-1 polymerization as assessed by native gel electrophoresis. PAI-749 did not turn PAI-1 into a substrate for tPA; however, PAI-749 promoted plasmin-mediated degradation of PAI-1. In conclusion, PAI-1 inactivation by PAI-749 using purified components can result from a dual mechanism of action. First, PAI-749 binds directly to PAI-1, blocks PAI-1 from accessing the active site of tPA, and abrogates formation of the SDS-stable tPA/PAI-1 complex. Second, binding of PAI-749 to PAI-1 renders PAI-1 vulnerable to plasmin-mediated proteolytic degradation.

Clinical and biochemical predictors affect the choice and the short-term outcomes of different thrombolytic agents in acute myocardial infarction

The presence of plasminogen activator inhibitor-1, angiotensin-converting enzyme and others may play a role in unsuccessful recanalization after thrombolytic therapy. To find out the clinical and biochemical predictors that may affect the choice and short-term outcomes following different thrombolytic agents in acute myocardial infarction. Angiotensin-converting enzyme and plasminogen activator inhibitor-1 plasma levels of 184 patients with acute myocardial infarction, treated with streptokinase, metalyze or reteplase, were determined. Failure of thrombolysis was assessed by noninvasive reperfusion criteria. Prolonged hospitalization, impaired left ventricular ejection fraction and reinfarction were considered as short-term outcomes. Patients who received streptokinase developed higher incidence of >50% resolution of ST-segment elevation (82.5 vs. 64.7%, P-value<0.05, in comparison with metalyze and 82.5 vs. 55.7%, P-value 0.001, in comparison with reteplase) than those who received other thrombolytic agents. High plasma angiotensin-converting enzyme was associated with prolonged hospitalization (55, 63 and 94%, P<0.02) following streptokinase, metalyze and reteplase, respectively. High plasma plasminogen activator inhibitor-1 is associated with impaired left ventricular ejection fraction (55.3, 76.7 and 68.5%, P<0.09), ST resolution<50% (13.2, 36.7 and 37.5%, P=0.03), ST resolution>50% (86.8, 63.3 and 62.5%, P=0.03) following streptokinase, metalyze and reteplase, respectively. Rapid determination of pretreatment angiotensin-converting enzyme and plasminogen activator inhibitor-1 plasma levels in patients with acute myocardial infarction may influence the choice and outcomes of the thrombolytic agents. The presence of a high plasma level of either angiotensin-converting enzyme or plasminogen activator inhibitor-1 is significantly associated with adverse short-term outcomes after treatment with reteplase or metalyze.

Vascular endothelial growth factor receptor-2-induced initial endothelial cell migration depends on the presence of the urokinase receptor.

The angiogenic response of endothelial cells initiated by different growth factors is accompanied by assembly of cell surface-bound proteolytic machinery as a prerequisite for focal invasion. We have shown previously how the vascular endothelial growth factor (VEGF) initiates proteolysis by activation of pro-urokinase (pro-PA) via the VEGF receptor-2 (VEGFR-2). We now show that the cell surface receptor of the uPA-system, the urokinase receptor (uPAR), is redistributed to focal adhesions at the leading edge of endothelial cells in response to VEGF. VEGF165 and VEGF-E, both interacting with VEGFR-2, but not PlGF exclusively stimulating VEGFR-1, induce within minutes internalization of uPAR via an LDL receptor-like molecule, dependent on generation of active uPA and the presence of plasminogen activator inhibitor-1 (PAI-1). uPAR seems to play a pivotal role in VEGFR-2-induced endothelial cell migration because cleavage of surface uPAR impaired the migratory response of endothelial cells toward VEGF-E, but not toward PlGF.

Plasminogen activator inhibitor-1 is a significant determinant of renal injury in experimental crescentic glomerulonephritis.

Crescentic glomerulonephritis is characterized by glomerular fibrin deposition, and experimental crescentic glomerulonephritis has been shown to be fibrin-dependent. Net fibrin deposition is a balance between activation of the coagulation system causing glomerular fibrin deposition and fibrin removal by the plasminogen-plasmin (fibrinolytic) system. Plasminogen activator inhibitor-1 (PAI-1) inhibits fibrinolysis by inhibiting plasminogen activators and has effects on leukocyte recruitment and matrix deposition. To test the hypothesis that the presence of PAI-1 and its levels were a determinant of injury in crescentic glomerulonephritis, accelerated anti-glomerular basement membrane glomerulonephritis was induced in mice genetically deficient in PAI-1 (PAI-1 -/-), PAI-1 heterozygotes (PAI-1 +/-), and mice engineered to overexpress PAI-1 (PAI-1 tg). Compared with strain-matched genetically normal animals, PAI-1 -/- mice with glomerulonephritis developed fewer glomerular crescents, less glomerular fibrin deposition, fewer infiltrating leukocytes, and less renal collagen accumulation at day 14 of disease. The reduction in disease persisted at day 28, when injury had become more established. In contrast, mice overexpressing the PAI-1 gene (PAI-1 tg), that have basal plasma and renal PAI-1 levels several times, normal developed increased glomerular crescent formation, more glomerular fibrin deposition, increased numbers of infiltrating leukocytes, and more renal collagen at both time points. These studies demonstrate that PAI-1 is a determinant of glomerular fibrin deposition and renal injury in crescentic glomerulonephritis.

Endothelial cells inhibit flow-induced smooth muscle cell migration: role of plasminogen activator inhibitor-1.

The endothelium may play a pivotal role in hemodynamic force-induced vascular remodeling. We investigated the role of endothelial cell (EC) plasminogen activator inhibitor-1 (PAI-1) in modulating flow-induced smooth muscle cell (SMC) migration. Human SMCs cocultured with or without human ECs were exposed to static (0 mL/min) or flow (26 mL/min; shear stress 23 dyne/cm(2)) conditions for 24 hours in a perfused capillary culture system. SMC migration was then assessed with a Transwell migration assay. In the absence but not in the presence of ECs, pulsatile flow significantly increased the migration of SMCs (264+/-26%) compared with SMCs under static conditions, concomitant with a 3- and 4-fold increase in PAI-1 mRNA and protein, respectively, in cocultured ECs. In the presence of PAI-1-/- ECs, flow increased wild-type SMC migration (226+/-25%), an effect that was reversed by exogenous PAI-1. To determine whether the antimigratory activity of PAI-1 was dependent primarily on inhibition of PAs or its association with vitronectin, experiments were conducted with PAI-1R (a mutant PAI-1 that binds to vitronectin but does not inhibit PA) and PAI-1K (a mutant that inhibits PA but has reduced affinity for vitronectin). PAI-1R inhibited both basal and flow-induced migration, whereas PAI-1K inhibited flow-induced migration in the absence of any effect on baseline migration. Flow-induced EC PAI-1 inhibits flow-induced SMC migration in vitro. EC PAI-1 expression may be one of the predominant mechanisms responsible for controlling the process of vascular remodeling.

Coordinate induction of collagenase-1, stromelysin-1 and urokinase plasminogen activator (uPA) by the 120-kDa cell-binding fibronectin fragment in fibrocartilaginous cells: uPA contributes to activation of procollagenase-1.

Specific fibronectin (Fn) fragments found in synovial fluid of arthritic joints potentially contribute to the loss of cartilage proteoglycans by inducing matrix metalloproteinase (MMP) expression. However, whether or not the Fn fragment-modulated changes in expression of MMPs result in a net increase in matrix-degradative activity through alterations in the balance between MMP activation and inhibition has not been established. To understand the mechanisms by which proteolytic Fn fragments may contribute to joint degeneration, conditioned medium from fibrocartilaginous cells exposed to Fn, its 30-kDa fragment containing the collagen/gelatin-binding domain, its 120-kDa fragment containing the central cell-binding domain, and the RGD peptide were assayed for MMPs, and MMP activators and inhibitors. We found that the 120-kDa fragment of Fn (but not intact Fn), the 30-kDa fragment, and the RGD peptide, dose-dependently induced procollagenase-1 and prostromelysin-1 and decreased levels of the tissue inhibitor of metalloproteinases (TIMPs) -1 and -2. The alpha5beta1 integrin was implicated in the induction of collagenase by the 120-kDa Fn fragment, since collagenase induction was abrogated in the presence of blocking antibody to this integrin. Conditioned medium from cells exposed to the 120-kDa Fn fragment also demonstrated increased levels of the activated collagenase-1, which resulted in significantly elevated collagen degradative activity. That the urokinase plasminogen activator (uPA) was involved in the activation of procollagenase-1 was suggested by findings that the 120-kDa Fn fragment induced uPA coordinately with procollagenase-1, and the activation of procollagenase-1 was dose-dependently inhibited in the presence of plasminogen activator inhibitor-1. These data demonstrate that the 120-kDa cell-binding fragment of Fn induces a net increase in matrix-degradative activity in fibrocartilaginous cells by concomitantly inducing MMPs and their activator, uPA, while decreasing TIMPs.

Characterization of receptors interacting specifically with the B-chain of tissue plasminogen activator on endothelial cells

Summary We have previously shown that the catalytic domain of tissue plasminogen activator (t-PA), the B-chain, binds specifically to hydrophobic components on the endothelial cell (EC) surface. The binding is mediated by amino acids within the sequence, AKHRRSPGER (B 20–29 ). The complex imparts a strong fibrinolytic potential to the EC surface, which is not inhibited by plasminogen activator inhibitor 1 (PAI-1). Here we characterize membrane proteins from EC that interact with the binding peptide and have hydrophobic properties. Specific labelling of cell surface components with affinity for the t-PA B-chain was performed using B-chain derivatized with the heterobifunctional and cleavable cross-linked SASD labelled with 125 I. Analysis of the cell extract by SDS/PAGE before and after reduction demonstrated the presence of a complex between the B-chain and a membrane protein of about 56 kDa. Radiolabelled proteins from the surface of EC were affinity purified on B 19–30 -Sepharose. The adsorbed proteins were eluted with lysine and the peptide B 20–29 . The hydrophobic fraction from the peptide eluate contained one main component with a molecular mass 56 kDa, which was absent in the hydrophilic fraction. In addition, four minor components were observed. Only traces of radiolabelled proteins were found in the hydrophobic fraction of the lysine eluate. Complexes between 125 I-t-PA and hydrophobic proteins from EC obtained by affinity chromatography on B 19–30 -Sepharose were crosslinked with disuccinylsuberate (DSS). Two radioactive complexes were identified after electroblotting to a PVDF membrane, one major component with a molecular mass of about 120 kDa and one minor 200 kDa complex. The 120 kDa compound had a significant activator activity before and after treatment with an excess of PAI-1. This is in contrast to free t-PA, which completely loses its activity in the presence of PAI-1. This indicates that t-PA (about 65 kDa) can form complexes with membrane components from EC with an approximate molecular mass of 55 kDa.