The role of SERPIN citrullination in thrombosis.

Aberrant protein citrullination is associated with many pathologies; however, the specific effects of this modification remain unknown. We have previously demonstrated that serine protease inhibitors (SERPINs) are highly citrullinated in rheumatoid arthritis (RA) patients. These citrullinated SERPINs include antithrombin, antiplasmin, and t-PAI, which regulate the coagulation and fibrinolysis cascades. Notably, citrullination eliminates their inhibitory activity. Here, we demonstrate that citrullination of antithrombin and t-PAI impairs their binding to their cognate proteases. By contrast, citrullination converts antiplasmin into a substrate. We recapitulate the effects of SERPIN citrullination using in vitro plasma clotting and fibrinolysis assays. Moreover, we show that citrullinated antithrombin and antiplasmin are increased and decreased in a deep vein thrombosis (DVT) model, accounting for how SERPIN citrullination shifts the equilibrium toward thrombus formation. These data provide a direct link between increased citrullination and the risk of thrombosis in autoimmunity and indicate that aberrant SERPIN citrullination promotes pathological thrombus formation.

Plasminogen binding inhibitors demonstrate unwanted activities on GABAA and glycine receptors in human iPSC derived neurons.

Plasminogen binding inhibitors (PBIs) reduce the risk of bleeding in hemorrhagic conditions. However, generic PBIs are also associated with an increased risk of seizures, an adverse effect linked to unwanted activities towards inhibitory neuronal receptors. Development of novel PBIs serve to remove compounds with such properties, but progress is limited by a lack of higher throughput methods with human translatability. Herein we apply human induced pluripotent stem cell (hiPSC) derived neurons in combination with dynamic mass redistribution (DMR) technology to demonstrate robust and reproducible modulation of both GABAA and glycine receptors. These cells respond to GABA (EC50 0.33 ±â€¯0.18 µM), glycine (EC50 11.0 ±â€¯3.7 µM) and additional ligands in line with previous reports from patch clamp technologies. Additionally, we identify and characterize a competitive antagonistic behavior of the prototype inhibitor and drug tranexamic acid (TXA). Finally, we demonstrate proof of concept for effective counter-screening of lead series compounds towards unwanted GABAA receptor activities. No activity was observed for a previously identified PBI candidate drug, AZD6564, whereas a discontinued analog, AZ13267257, could be characterized as a potent GABAA receptor agonist.

Urokinase-type plasminogen activator modulates mammalian circadian clock phase regulation in tissue-type plasminogen activator knockout mice.

Glutamate phase shifts the circadian clock in the mammalian suprachiasmatic nucleus (SCN) by activating NMDA receptors. Tissue-type plasminogen activator (tPA) gates phase shifts by activating plasmin to generate m(ature) BDNF, which binds TrkB receptors allowing clock phase shifts. Here, we investigate phase shifting in tPA knockout (tPA-/- ; B6.129S2-Plattm1Mlg /J) mice, and identify urokinase-type plasminogen activator (uPA) as an additional circadian clock regulator. Behavioral activity rhythms in tPA-/- mice entrain to a light-dark (LD) cycle and phase shift in response to nocturnal light pulses with no apparent loss in sensitivity. When the LD cycle is inverted, tPA-/- mice take significantly longer to entrain than C57BL/6J wild-type (WT) mice. SCN brain slices from tPA-/- mice exhibit entrained neuronal activity rhythms and phase shift in response to nocturnal glutamate with no change in dose-dependency. Pre-treating slices with the tPA/uPA inhibitor, plasminogen activator inhibitor-1 (PAI-1), inhibits glutamate-induced phase delays in tPA-/- slices. Selective inhibition of uPA with UK122 prevents glutamate-induced phase resetting in tPA-/- but not WT SCN slices. tPA expression is higher at night than the day in WT SCN, while uPA expression remains constant in WT and tPA-/- slices. Casein-plasminogen zymography reveals that neither tPA nor uPA total proteolytic activity is under circadian control in WT or tPA-/- SCN. Finally, tPA-/- SCN tissue has lower mBDNF levels than WT tissue, while UK122 does not affect mBDNF levels in either strain. Together, these results suggest that either tPA or uPA can support photic/glutamatergic phase shifts of the SCN circadian clock, possibly acting through distinct mechanisms.

Cytotoxicity of the Urokinase-Plasminogen Activator Inhibitor Carbamimidothioic Acid (4-Boronophenyl) Methyl Ester Hydrobromide (BC-11) on Triple-Negative MDA-MB231 Breast Cancer Cells.

BC-11 is an easily synthesized simple thiouronium-substituted phenylboronic acid, which has been shown to be cytotoxic on triple negative MDA-MB231 breast cancer cells by inducing a perturbation of cell cycle when administered at a concentration equal to its ED50 at 72 h (117 µM). Exposure of cells to BC-11, either pre-absorbed with a soluble preparation of the N-terminal fragment of urokinase-plasminogen activator (uPa), or in co-treatment with two different EGFR inhibitors, indicated that: (i) BC-11 acts via binding to the N-terminus of the enzyme where uPa- and EGF receptor-recognizing sites are present, thereby abrogating the growth-sustaining effect resulting from receptor binding; and (ii) the co-presence of the EGFR inhibitor PD153035 potentiates BC-11's cytotoxicity. Exposure of cells to a higher concentration of BC-11 corresponding to its ED75 at 72 h (250 µM) caused additional impairment of mitochondrial activity, the production of reactive oxygen species and promotion of apoptosis. Therefore, BC-11 treatment appears to show potential for the development of this class of compounds in the prevention and/or therapy of "aggressive" breast carcinoma.

Inhibition of plasminogen activator inhibitor-1 is a potential therapeutic strategy in ovarian cancer.

Plasminogen activator inhibitor (PAI)-1 is predictive of poor outcome in several types of cancer. The present study investigated the biological role for PAI-1 in ovarian cancer and potential of targeted pharmacotherapeutics. In patients with ovarian cancer, PAI-1 mRNA expression in tumor tissues was positively correlated with poor prognosis. To determine the role of PAI-1 in cell proliferation in ovarian cancer, the effects of PAI-1 inhibition were examined in PAI-1-expressing ovarian cancer cells. PAI-1 knockdown by small interfering RNA resulted in significant suppression of cell growth accompanied with G2/M cell cycle arrest and intrinsic apoptosis. Similarly, treatment with the small molecule PAI-1 inhibitor TM5275 effectively blocked cell proliferation of ovarian cancer cells that highly express PAI-1. Together these results suggest that PAI-1 promotes cell growth in ovarian cancer. Interestingly, expression of PAI-1 was increased in ovarian clear cell carcinoma compared with that in serous tumors. Our results suggest that PAI-1 inhibition promotes cell cycle arrest and apoptosis in ovarian cancer and that PAI-1 inhibitors potentially represent a novel class of anti-tumor agents.

L-mimosine and dimethyloxaloylglycine decrease plasminogen activation in periodontal fibroblasts.

BACKGROUND: The use of prolyl hydroxylase inhibitors such as l-mimosine (L-MIM) and dimethyloxaloylglycine (DMOG) to improve angiogenesis is a new approach for periodontal regeneration. In addition to exhibiting pro-angiogenic effects, prolyl hydroxylase inhibitors can modulate the plasminogen activator system in cells from non-oral tissues. This study assesses the effect of prolyl hydroxylase inhibitors on plasminogen activation by fibroblasts from the periodontium. METHODS: Gingival and periodontal ligament fibroblasts were incubated with L-MIM and DMOG. To investigate whether prolyl hydroxylase inhibitors modulate the net plasminogen activation, kinetic assays were performed with and without interleukin (IL)-1. Moreover, plasminogen activators and the respective inhibitors were analyzed by casein zymography, immune assays, and quantitative polymerase chain reaction. RESULTS: The kinetic assay showed that L-MIM and DMOG reduced plasminogen activation under basal and IL-1-stimulated conditions. Casein zymography revealed that the effect of L-MIM involves a decrease in urokinase-type plasminogen activator activity. In agreement with these findings, reduced levels of urokinase-type plasminogen activator and elevated levels of plasminogen activator inhibitor 1 were observed. CONCLUSION: L-MIM and DMOG can reduce plasminogen activation by fibroblasts from the gingiva and the periodontal ligament under basal conditions and in the presence of an inflammatory cytokine.

Effect of thyroxine on fibrinolytic system in rat.

Thyroid hormones have many effects on the cardiovascular system. Thyroid dysfunction accelerates atherosclerosis not only through conventional risk factors (dyslipidemia) but they also show a very close relationship with hemodynamic parameters. Thyroxine is determinant of the several components of fibrinolytic system even though the exact relationship is far from clear. Present study was carried out to determine the effect of thyroxine on fibrinolytic parameters such as plasminogen activators (PA) in rat heart, levels of PA and plasminogen activator inhibitor (PAI), glucose in plasma and serum lipid profile. Rats were injected with 50 ug eltroxine/100 gm(-1) body weight intraperitoneally for one week. Compared with controls, thyroxine treatment increased PA activity significantly in rat heart. No changes were seen in PA, PAI and glucose in plasma of two groups of rats. A significant decrease in total cholesterol and LDL-cholesterol levels was seen in serum of treated group resulting in the decrease of LDL/HDL and Total cholesterol/HDL-cholesterol ratios. These results suggest that thyroxine treatment may have considerable clinical significance. It raised PA activity in heart as well as reduced cholesterol content in blood. It is possible that thyroxine treatment may confer a beneficial effect on cardiovascular risk.

Inactivation of plasminogen activator inhibitor type 1 by activated factor XII plays a role in the enhancement of fibrinolysis by contact factors in-vitro.

AIMS: Several activated coagulation factors have been reported to enhance fibrinolysis by inactivating plasminogen activator inhibitor type 1 (PAI-1), a serine protease inhibitor. We analyzed the interaction between PAI-1 and the three serine proteases generated during contact activation of plasma, activated factor XII (FXIIa), FXIa, and kallikrein, and evaluated their effects on fibrinolysis in-vitro. MAIN METHODS: Effects of kaolin on euglobulin clot lysis time (ECLT) and behavior of PAI-1 in factor-depleted plasma were analyzed. KEY FINDINGS: The ECLT of pooled plasma obtained from normal volunteers (designated as 100%) was shortened to 62.1+/-3.1% by Ca(2+) (5 mM) and 29.9+/-3.1% by kaolin. Activated protein C reversed the ECLT shortened by Ca(2+)-supplementation (86.3+/-17.4%), but did not affect the ECLT shortened by kaolin (31.4+/-2.1%). Thus, in contrary to Ca(2+)-supplementation, kaolin appeared to shorten the ECLT by a mechanism independent of thrombin generation. In three kinds of contact factor-depleted plasma, kaolin did not shorten ECLT only in FXII-depleted plasma. PAI-1 was cleaved to its inactive form in the Ca(2+) as well as the kaolin-supplemented euglobulin fraction in normal plasma, the latter of which, however, was not observed in FXII-depleted plasma. Similarly, a high molecular weight complex between FXIIa and PAI-1, as well as a cleaved form of PAI-1, was observed in kaolin-supplemented normal plasma, but neither was found in kaolin-supplemented FXII-depleted plasma. SIGNIFICANCE: PAI-1 inactivation by FXIIa appears to be a mechanism by which contact phase coagulation factors enhance fibrinolysis independently of thrombin generation.

The urokinase receptor and its structural homologue C4.4A in human cancer: expression, prognosis and pharmacological inhibition.

The urokinase-type plasminogen activator receptor (uPAR) and its structural homologue C4.4A are multidomain members of the Ly6/uPAR/alpha-neurotoxin protein domain family. Both are glycosylphosphatidylinositol-anchored membrane glycoproteins encoded by neighbouring genes located on chromosome 19q13 in the human genome. The structural relationship between the two proteins is, however, not reflected at the functional level. Whereas uPAR has a well-established role in regulating and focalizing uPA-mediated plasminogen activation to the surface of those cells expressing the receptor, the biological function of C4.4A remains elusive. Nonetheless, both uPAR and C4.4A have been implicated in human pathologies such as wound healing and cancer. A large body of experimental evidence thus demonstrates that high levels of uPAR in resected tumour tissue as well as in plasma are associated with poor prognosis in a number of human cancers including colon adenocarcinoma and pulmonary squamous cell carcinoma. Targeting uPAR in experimental animal models has also provided promising results regarding the interference with pathogenic plasminogen activation. In the case of C4.4A, very recent data have demonstrated that high protein expression in tumour cells of non-small cell pulmonary adenocarcinomas is associated with a particularly severe disease progression. This review will evaluate structural-functional and disease-related aspects of uPAR and C4.4A with a view to possible pharmacological targeting strategies for therapy and for non-invasive bioimaging.

Inhibition of plasminogen activators attenuates the death of differentiated retinal ganglion cells and stabilizes their neurite network in vitro.

PURPOSE: Although previous studies have indicated that elevated levels of the tissue plasminogen activator (tPA) and the urokinase plasminogen activator (uPA) associate with the death of retinal ganglion cells (RGCs), it was unclear whether these proteases directly cause cell death. With the use of a transformed and undifferentiated retinal ganglion cell line, RGC-5, which does not express tPA, and by treating this cell line with staurosporine, which induces not only the differentiation of RGC-5 cells but also the expression of uPA and tPA in other neuronal cells, the authors sought to determine whether these proteases regulate the differentiation of RGC-5 cells and whether elevated levels of these proteases directly cause the death of RGC-5 cells. METHODS: Transformed RGC-5 cells were cultured in serum-free medium and were treated with 0.5 muM to 2.0 muM staurosporine to induce their differentiation. Neurite outgrowth was assessed by phase-contrast microscopy and calcein AM staining and quantified with imaging software. Proteolytic activities of tPA and uPA were determined by zymography assays. Cell viability was determined by LIVE/DEAD viability assay kit. RESULTS: Compared with untreated RGC-5 cells, cells treated with staurosporine differentiated as early as 1 to 6 hours. However, proteolytic activities of neither tPA nor uPA were observed within this time frame. Differentiated RGC-5 cells expressed detectable levels of uPA proteolytic activity starting at 24 hours and tPA proteolytic activity only at 48 hours. RGC-5 cells synthesized and secreted uPA and tPA into the conditioned medium, depending on staurosporine concentration and treatment time. At lower concentrations of staurosporine, differentiated RGC-5 cells had longer neurites and expressed lower levels of tPA and uPA. At higher concentrations of staurosporine, differentiated RGC-5 cells expressed higher levels of tPA and uPA, had smaller neurites, and most of them died. In contrast, when RGC-5 cells were treated with staurosporine along with inhibitors specific to tPA and uPA, proteolytic activities of both PAs were significantly reduced. Under these conditions, a significant number of RGC-5 cells survived, showed increased neurite outgrowth, and established their neurite network in vitro. CONCLUSIONS: Results presented in this study indicate that RGC-5 cells do not require tPA and tPA for their differentiation. In fact, differentiated RGC-5 cells synthesize elevated levels of tPA and uPA, and elevated levels of these proteases acting in an autocrine-fashion in turn lead to the death of RGC-5 cells.

Changes in coagulation and fibrinolysis of post-SARS osteonecrosis in a Chinese population.

The purpose of this study was to detect changes in coagulation and fibrinolysis of post-severe acute respiratory syndrome (SARS) Chinese patients with osteonecrosis, investigate the aetiology of post-SARS osteonecrosis (ON), and select the sensitive molecular markers for identifying the susceptible population. For this study, blood samples were collected from 88 patients with post-SARS ON and 52 healthy people. Activated partial thromboplastin time (APTT), protein C (PC), antithrombin III (AT-III), plasminogen activator inhibitor (PAI), activated protein C resistance (APC-R), plasminogen (PLG), von Willebrand's factor(vWF), D-dimer (D-D), fibrinogen (Fib), and homocysteine (HCY) were examined by enzyme-linked immunosorbent assay (ELISA). We noted that blood agents of patients with ON changed obviously. APTT, PC, AT-III, PAI, APC-R, and PLG were significantly different between the two groups. Hypercoagulation and hypofibrinolysis were found in patients with post-SARS ON. Therefore, these examinations can be used to screen a population susceptible to ON. Measurements of APTT, PC, AT-III, PAI, APC-R, and PLG are sensitive blood tests for screening purposes.

Beneficial effect of low-molecular-weight heparin against lipopolysaccharide-induced disseminated intravascular coagulation in rats is abolished by coadministration of tranexamic acid.

OBJECTIVE: We examined the role of coagulation and fibrinolysis in lipopolysaccharide (LPS) induced disseminated intravascular coagulation (DIC) in rats, studying their contribution to fibrin deposition and organ failure in rats with LPS-induced DIC by concurrent administration of low molecular weight heparin (LMWH) with or without tranexamic acid (TA). METHODS: DIC was induced in male Wistar rats by a 4-h infusion of LPS (30 mg/kg) via the tail vein (LPS group). In the LPS+LMWH group LMWH (200 u/kg) was administered to rats from 30 min before the infusion of LPS for 4.5 h. In the LPS+LMWH+TA group LMWH (200 microg/kg) and TA (50 mg/kg) were administered to rats from 30 min before the infusion of LPS for 4.5 h. RESULTS: In the LPS+LMWH group lower plasma levels of TAT, D dimer, creatinine, and alanine aminotransferase were observed, along with less glomerular fibrin deposition and improved survival over rats administered LPS alone. However, these effects of LMWH were completely eliminated and damage beyond that observed in rats administered LPS alone resulted from combined administration of TA (LPS+LMWH+TA group), except that TAT and D dimer levels remained lower than in the group administered LPS alone. CONCLUSIONS: Suppression of fibrinolysis by TA (despite coadministration of LMWH) resulted in increased organ damage in this study, suggesting that depressed fibrinolysis plays a large role in organ failure resulting from LPS-induced DIC, even though hemostatic activation is moderately suppressed by LMWH.

Characterization and comparative evaluation of a structurally unique PAI-1 inhibitor exhibiting oral in-vivo efficacy.

Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Elevated levels of PAI-1 are associated with thrombosis and vascular disease, suggesting that high plasma PAI-1 may promote a hypercoagulable state by disrupting the natural balance between fibrinolysis and coagulation. In this study, we identify WAY-140312 as a structurally novel small molecule inactivator of PAI-1, compare its inhibitory activity with other previously identified small molecule inhibitors, and investigate the mechanism of inactivation of PAI-1 in the presence of both tPA and uPA. In an immunofunctional assay, WAY-140312 inhibited PAI-1 with an estimated inhibitory concentration (IC(50)) of 11.7 micro m, which was the lowest value obtained of the four different PAI-1 inactivators tested. Surface activity profiling indicated that the critical micelle concentration for WAY-140312 was 95.8 micro m, and that each inhibitor exhibited unique physical chemical properties. Using a sensitive direct activity assay, the IC(50) for WAY-140312 was similar when either tPA or uPA was used as the target protease. Immunoblot analysis demonstrated that WAY-140312 near the IC(50) inhibited the complex formation between either tPA or uPA and PAI-1. After oral administration, WAY-140312 exhibited 29% bioavailability with a plasma half-life of approximately 1 h. In an in-vivo model of vascular injury, a 10 mg kg(-1) oral dose of WAY-140312 was associated with improvement in arterial blood flow and reduction in venous thrombosis. Thus, WAY-140312 represents a structurally novel small molecule inhibitor of PAI-1, and is the first such molecule to exhibit efficacy in animal models of vascular disease following oral administration.

Extravascular fibrin, plasminogen activator, plasminogen activator inhibitors, and airway hyperresponsiveness.

Mechanisms underlying airway hyperresponsiveness are not yet fully elucidated. One of the manifestations of airway inflammation is leakage of diverse plasma proteins into the airway lumen. They include fibrinogen and thrombin. Thrombin cleaves fibrinogen to form fibrin, a major component of thrombi. Fibrin inactivates surfactant. Surfactant on the airway surface maintains airway patency by lowering surface tension. In this study, immunohistochemically detected fibrin was seen along the luminal surface of distal airways in a patient who died of status asthmaticus and in mice with induced allergic airway inflammation. In addition, we observed altered airway fibrinolytic system protein balance consistent with promotion of fibrin deposition in mice with allergic airway inflammation. The airways of mice were exposed to aerosolized fibrinogen, thrombin, or to fibrinogen followed by thrombin. Only fibrinogen followed by thrombin resulted in airway hyperresponsiveness compared with controls. An aerosolized fibrinolytic agent, tissue-type plasminogen activator, significantly diminished airway hyperresponsiveness in mice with allergic airway inflammation. These results are consistent with the hypothesis that leakage of fibrinogen and thrombin and their accumulation on the airway surface can contribute to the pathogenesis of airway hyperresponsiveness.

3D-QSAR CoMFA/CoMSIA studies on Urokinase plasminogen activator (uPA) inhibitors: a strategic design in novel anticancer agents.

Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) was performed on a series of indole/benzoimidazole-5-carboxamidines as urokinase plasminogen activator (uPA) inhibitors. The ligand molecular superimposition on template structure was performed by atom/shape-based RMS fit, multifit, and RMSD fit methods. The removal of two outliers from the initial training set of 30 molecules improved the predictivity of the models. The statistically significant model was established from 28 molecules, which were validated by evaluation of test set of nine compounds. The atom-based RMS alignment yielded best predictive CoMFA model (r2(cv) = 0.611, r2(cnv) = 0.778, F value = 43.825, r2(bs) = 0.842, r2(pred) = 0.616 with two components) while the CoMSIA model yielded (r2(cv) = 0.499, r2(cnv) = 0.976, F value=96.36, r2(bs) = 0.993, r2(pred) = 0.694 with eight components). The contour maps obtained from 3D-QSAR studies were appraised for the activity trends of the molecules analyzed. The results indicate that the steric, electrostatic, and hydrogen bond donor/acceptor substituents play significant role in uPA activity and selectivity of these compounds. The data generated from the present study can be used as putative pharmacophore in the design of novel, potent, and selective urokinase plasminogen activator inhibitors as cancer therapeutics.

The fibrinolytic system attenuates vascular tone: effects of tissue plasminogen activator (tPA) and aminocaproic acid on renal microcirculation.

1. The renal medulla is a major source of plasminogen activators (PA), recently shown to induce vasodilation in vitro. Treatment with PA inhibitors has been associated with renal dysfunction, suggesting compromised renal microvasculature. We investigated the impact of the PA inhibitor epsilon amino-caproic acid (EACA) upon vascular tone in vitro, and studied the effect of both tPA and EACA upon intrarenal hemodynamics in vivo. 2. In vitro experiments were carried out in isolated aortic rings and with cultured vascular smooth muscle cells. Studies of renal microcirculation and morphology were conducted in anesthetized Sprague-Dawley rats. 3. In isolated aortic rings, EACA (but not the other inhibitors of the fibrinolytic system PAI-1 or alpha-2 antiplasmin) reduced the half-maximal effective concentration of phenylephrine (PE) required to induce contraction (from 32 nm in control solution to 2 and 0.1 nm at EACA concentrations of 1 and 10 microm, respectively). Using reteplase (retavase) in the same model, we also provide evidence that the vasoactivity of tPA is in part kringle-dependent. In cultured vascular smooth muscle cells, Ca(2+) internalization following PE was enhanced by EACA, and retarded by tPA. 4. In anesthetized rats, EACA (150 mg x kg(-1)) did not affect systemic blood pressure, total renal or cortical blood flow. However, the outer medullary blood flow declined 12+/-2% below the baseline (P<0.03). By contrast, tPA (2 mg x kg(-1)), transiently increased outer medullary blood flow by 8+/-5% (P<0.02). Fibrin microthrombi were not found within the renal microvasculature in EACA-treated animals. 5. In conclusion, both fibrinolytic and antifibrinolytic agents modulate medullary renal blood flow with reciprocal effects of vasodilation (PA) and vasoconstriction (EACA). In vitro studies suggest that these hemodynamic responses are related to direct modulation of the vascular tone.

Enhancement of endogenous fibrinolysis does not reduce local fibrin deposition, but modulates inflammation upon intestinal ischemia and reperfusion.

This study investigated the contribution of endogenous suppression of fibrinolysis and increased fibrin deposition to intestinal dysfunction and injury in a rat model of intestinal ischemia/reperfusion (I/R), as fibrinolytic inhibition may lead to thrombotic obstructions that compromise microcirculation and promote intestinal injury. Circulatory fibrinolysis was enhanced by intravenous administration of recombinant tissue plasminogen activator (rt-PA) or by inhibition of PAI-I by administration of MA-33H1F7. Coagulation and fibrinolysis parameters obtained from portal blood were correlated to fibrin deposition (determined by anti-rat fibrin antibody staining), intestinal function (glucose/water clearance) and intestinal injury (histological evaluation by Park/Chiu score). Enhanced circulatory fibrinolytic activity, as evidenced by increased portal plasma plasminogen activator activity, elevated fibrin degradation products and decreased levels of PAI-I, did not reduce mucosal fibrin deposition and microthrombosis in postischemic intestinal tissue. Furthermore, rt-PA or anti-PAI-I antibody administration did not attenuate I/R-induced intestinal injury or dysfunction, as demonstrated by intestinal histopathology scores of 4.8+/-0.2 and 4.7+/-0.3 (control I/R group 4.7+/-0.2) and glucose clearances of 47+/-6 and 46+/-9 micro L/min g (control I/R group 30+/-8 micro L/min. g) after 40 minutes of intestinal ischemia and 3 hours of reperfusion, respectively. However, both interventions resulted in decreased levels of interleukin-6, which may indicate fibrin-induced modulation of inflammation. Attempts to enhance the fibrinolytic activity (either by rt-PA or by anti-PAI-I administration), indicated by increased portal plasma levels of released FDP, failed to decrease mucosal fibrin deposition and to attenuate intestinal I/R injury. Based on our observations and previous reports, the contribution of suppressed endogenous fibrinolysis to microcirculatory fibrin deposition and I/R-injury may be of limited importance.

Inhibition of plasminogen activation protects against ganglion cell loss in a mouse model of retinal damage.

PURPOSE: The mechanisms that trigger ganglion cell death in ischemic retinal diseases are not clearly understood. Using a mouse optic nerve ligation model, the objective of this study was to test the hypothesis that extracellular matrix (ECM) modulating plasminogen activators (PAs) potentiate ganglion cell loss. METHODS: Optic nerve ligation was performed to initiate ganglion cell loss in the retina. Urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) activity in retinal extracts was determined by plasminogen/fibrinogen zymography. Immunostaining and western blot analysis was performed to detect uPA and tPA proteins. Plasmin activity was determined by casein gel-zymography. Plasminogen and plasmin proteins were detected and quantified by western blotting. Morphology was assessed using hematoxylin and eosin stained retinal cross sections, and programmed cell death was monitored by an apoptotic assay. Laminin degradation in retinal extracts was assessed by western blot analysis. RESULTS: Optic nerve ligation led to a transient increase in uPA and plasmin proteolytic activity in the retina. Urokinase inhibitor, amiloride, blocked uPA activity in retinal extracts. We found a correlation between the increased uPA activity, and conversion of zymogen plasminogen to active plasmin in retinal extracts with laminin degradation in the retina and apoptosis of ganglion cells. We found that by adding exogenous plasmin, in vitro, laminin present in control retinal extracts could be degraded in similar fashion. In addition, uPA or tPA failed to degrade laminin in control retinal extracts unless plasminogen was added, indicating that plasminogen activation is necessary for laminin degradation, in vitro. After intravitreal injection of plasmin inhibitor, alpha-2 antiplasmin, we found a significant protection against optic nerve ligation-induced ganglion cell loss. CONCLUSIONS: Optic nerve ligation-induced plasmin(ogen) activation that precedes ganglion cell loss suggest that specific targeting of plasmin activity may have therapeutic potential in preventing ganglion cell loss in retinal diseases.

Protease nexin 1 is a potent urinary plasminogen activator inhibitor in the presence of collagen type IV.

Protease nexin 1 (PN1) in solution forms inhibitory complexes with thrombin or urokinase, which have opposing effects on the blood coagulation cascade. An initial report provided data supporting the idea that PN1 target protease specificity is under the influence of collagen type IV (1). Although collagen type IV demonstrated no effect on the association rate between PN1 and thrombin, the study reported that the association rate between PN1 and urokinase was allosterically reduced 10-fold. This has led to the generally accepted idea that the primary role of PN1 in the brain is to act as a rapid thrombin inhibition and clearance mechanism during trauma and loss of vascular integrity. In studies to identify the structural determinants of PN1 that mediate the allosteric interaction with collagen type IV, we found that protease specificity was only affected after transient exposure of PN1 to acidic conditions that mimic the elution protocol from a monoclonal antibody column. Because PN1 used in previous studies was purified over a monoclonal antibody column, we propose that the allosteric regulation of PN1 target protease specificity by collagen type IV is a result of the purification protocol. We provide both biochemical and kinetic data to support this conclusion. This finding is significant because it implies that PN1 may play a much larger role in the modeling and remodeling of brain tissues during development and is not simply an extravasated thrombin clearance mechanism as previously suggested.