Plasmin Treatment Research Articles

Plasmin-Induced Lens Epithelial Cells Detachment for the Reduction of Posterior Capsular Opacification.

We sought to evaluate the efficacy and safety of plasmin injection in the capsular bag during the cataract operation for the prevention of posterior capsule opacification. Thirty-seven anterior capsular flaps taken from phacoemulsification surgery were immersed in either 1 µg/mL plasmin (plasmin group, n = 27) or phosphate-buffered saline (control group, n = 10) for 2 minutes and photographed after fixation and nuclear staining to compare the numbers of residual lens epithelial cells. In the animal experiments, the plasmin solution was injected into the capsular bag and remained for 5 minutes during hydrodissection or after lens extraction. The degree of posterior capsular opacity of the rabbits at 2 months were photographed by slit lamp biomicroscopy. In HLE-B3 cell culture, the cell detachment rate, proliferation, and apoptosis after the plasmin digestion were analyzed. The residual lens epithelial cell numbers on the capsule after plasmin treatment were 168 ± 190.7/mm2 in the 1 µg/mL plasmin group, which was significantly lower than that of the control (1012 ± 798.8/mm2; P < 0.0001). In a rabbit model, the treatment of plasmin resulted in a significantly clearer posterior capsule compared with that of the control group at 2 months postoperatively. This study suggested that plasmin injection can induce effective lens epithelial cell detachment, which could be a promising adjunctive treatment to further improve the success rate in posterior capsule opacification prevention. Plasmin injection for lens epithelial cell detachment could significantly decrease the number of residual lens epithelial cells. This approach could be a promising treatment incorporating the current treatment approach to further improve the success rate in posterior capsule opacification prevention.

Fabrication of perfusable microvessel networks by mimicking in vivo vasculogenesis using a novel scaffold-wrapping method

Due to the heterogeneous nature of microvascular structure between organs, it is important to engineer tailored capillary networks that match their physical properties for biochemical/mechanical studies. To mimic in vivo vasculogenesis, a “scaffold-wrapping” approach has been developed to create a perfusable capillary network with a tailored structure. A cylindrical fibrin scaffold with the desired pattern and dimensions was fabricated by photolithography, thermal reflow, polydimethylsiloxane (PDMS) casting, and fibrin injection. Vasculogenesis resembling “scaffold-wrapping” was achieved by culturing human umbilical vein endothelial cells (HUVECs) on a cylindrical fibrin scaffold until all branches of the scaffold were completely wrapped by HUVECs. The scaffold was encapsulated in the collagen gel followed by plasmin treatment to form a perfusable vascular lumen. The vascular endothelial markers CD31 and VE-cadherin still expressed in the HUVECs after degradation of the fibrin scaffold. The perfusability of the vascular network was also demonstrated by injecting and imaging red fluorescent beads into the networks. This customized capillary network allowed us to control its dimensions down to the micrometer level to mimic tissue-specific microvascular properties.

Heparin and Arginine Based Plasmin Nanoformulation for Ischemic Stroke Therapy.

Ischemic stroke is the most common type of stroke and thrombolytic therapy is the only approved treatment. However, current thrombolytic therapy with tissue plasminogen activator (tPA) is often hampered by the increased risk of hemorrhage. Plasmin, a direct fibrinolytic, has a significantly superior hemostatic safety profile; however, if injected intravenously it becomes rapidly inactivated by anti-plasmin. Nanoformulations have been shown to increase drug stability and half-life and hence could be applied to increase the plasmin therapeutic efficacy. Here in this paper, we report a novel heparin and arginine-based plasmin nanoformulation that exhibits increased plasmin stability and efficacy. In vitro studies revealed significant plasmin stability in the presence of anti-plasmin and efficient fibrinolytic activity. In addition, these particles showed no significant toxicity or oxidative stress effects in human brain microvascular endothelial cells, and no significant blood brain barrier permeability. Further, in a mouse photothrombotic stroke model, plasmin nanoparticles exhibited significant efficacy in reducing stroke volume without overt intracerebral hemorrhage (ICH) compared to free plasmin treatment. The study shows the potential of a plasmin nanoformulation in ischemic stroke therapy.

Zinc(II) Chelate Binds to Fibrinogen and Its αC Region

Fibrinogen is the immediate precursor of fibrin blood clots. When fibrinogen is cleaved by thrombin, it assembles into fibrin polymers through intermolecular interactions where calcium and zinc ions may both be involved. Fibrinogen’s αC domains play a major role in fibrin polymer assembly, and they contain a Zn²⁺ binding sites based on peptide studies. However, Zn²⁺ binding to natural αC and fibrin (ogen) remains to be demonstrated. The objective of this study was to directly demonstrate Zn²⁺ interactions with purified fibrinogen and its αC region. Both human and bovine αC regions were investigated due to the significant differences in their protein sequences. To detect Zn²⁺ binding, chelated Zn²⁺ in the form of Zn²⁺nitrilotriacetic acid linked to alkaline phosphatase tracer (ZnTAP) was prepared and used in ELISA and Western blot style methods. Gel electrophoresis was used to monitor changes to fibrinogen after reaction with plasmin. Human and bovine αC regions were obtained by limited plasmin digests of fibrinogen and purified by Con-A and Zn-immobilized metal ion chromatography. The results showed ZnTAP binding to fibrinogen in solution and inhibition of binding to fibrinogen coated microplates by ELISA. Inhibition concentrations (IC50) of 0.58 μM for human fibrinogen and 0.26 μM for bovine fibrinogen were determined. Blot probes revealed ZnTAP binding to fibrinogen with disulfide bonds intact and to the Aα and Bβ chains with disulfide bonds reduced. Reduced Aα chain reaction was less than that of intact fibrinogen and the reduced Bβ chain was only weakly reactive. With plasmin treatment, ZnTAP binding decreased by ELISA in parallel with the degradation of αC region by gel electrophoresis analysis. Purified αC bound ZnTAP in ELISA and on Western blots. In this study we demonstrated that Zn²⁺ binds to fibrinogen and its Aα and Bβ chains, and to the αC region of the Aα chain. The results support a role for protein conformation in Zn²⁺ binding and demonstrate the utility of the ZnTAP complex with tracer for fibrinogen binding interactions.

Neuroserpin regulates human T cell-T cell interactions and proliferation through inhibition of tissue plasminogen activator.

T cells play a key role in mounting an adaptive immune response. T cells are activated upon recognition of cognate Ag presented by an APC. Subsequently, T cells adhere to other activated T cells to form activation clusters, which lead to directed secretion of cytokines between communicating cells. T cell activation clusters have been implicated in regulating activation, proliferation, and memory formation in T cells. We previously reported the expression of the protease inhibitor neuroserpin by human T cells and showed that expression and intracellular localization is regulated following T cell activation. To gain a better understanding of neuroserpin in the proteolytic environment postactivation we assessed its role in human T cell clustering and proliferation. Neuroserpin knockdown increased T cell proliferation and cluster formation following T cell activation. This increased cluster formation was dependent on the proteases tissue plasminogen activator (tPA) and plasmin. Furthermore, neuroserpin knockdown or plasmin treatment of T cells increased the cleavage of annexin A2, a known plasmin target that regulates the actin cytoskeleton. Live cell imaging of activated T cells further indicated a role of the actin cytoskeleton in T cell clustering. The inhibition of actin regulators myosin ATPase and Rho-associated protein kinase signaling completely reversed the neuroserpin knockdown-induced effects. The results presented in this study reveal a novel role for neuroserpin and the proteolytic environment in the regulation of T cell activation biology.

Plasmin Enzyme-Assisted Vitrectomy in Pediatric Patients with Vitreoretinal Diseases

Purpose: To evaluate the efficacy and safety of using plasmin-assisted vitrectomy in pediatric patients with vitreoretinal diseases. Methods: We prospectively recruited children aged 16 years or younger who presented with vitreoretinopathies and underwent plasmin-assisted vitrectomy between 2012 and 2013. The main outcome measure was the induction of posterior vitreous detachment (PVD) using a suction power of 200 mm Hg or less during surgery. Results: Eleven eyes of 11 patients (mean age: 3.7 years; average follow-up duration: 14.1 months) were included. Of these 11 patients, there were 3 (27%) cases of stage 5 retinopathy of prematurity, 2 (18%) cases of persistent fetal vasculature, 2 (18%) cases of rhegmatogenous retinal detachment, 2 (18%) cases of idiopathic epiretinal membrane, 1 (9%) case of traumatic macular pucker, and 1 (9%) case of traumatic vitreous hemorrhage (9%). PVD was achieved in all cases (100%) during surgery using low suction after plasmin treatment (mean: 150 ± 39 mm Hg; range: 100-200). Overall, anatomical success was achieved in 8 eyes (73%). Visual acuity improved in all 5 (100%) patients for whom vision could be measured at 6 months after the operation. Cataracts were found in 4 eyes (36%), and a rise in transient intraocular pressure was observed in 1 eye (9%). Conclusions: Plasmin-assisted vitrectomy offers an effective and less traumatic intervention for a variety of pediatric vitreoretinal diseases.

Lens subluxation after plasmin and SF6 injections in rabbit eyes.

PurposeTo investigate the rate of lens subluxation following plasmin and/or SF6 injections in eyes, and whether a subsequent elevated level of vascular endothelial growth factor (VEGF) and vitreous tap would aggravate subluxation.MethodsFour groups of rabbits were used. Group 1 received an intravitreal injection (IVI) of plasmin and SF6 in the right eye; group 2 received an IVI of plasmin in the right eye; group 3 received an IVI of SF6 in the right eye; and group 4 received an IVI of balanced salt solution in the right eye. After treatment, IVIs of VEGF were given and vitreous tap was performed three times, followed by clinical observation of lens subluxation and scanning electronic microscope evaluation of the zonular fibers.ResultsAfter IVIs of plasmin and SF6, and VEGF and vitreous tap had been performed one to three times, lens subluxation was noted in 0%, 43%, 71%, 71%, and 86% of the eyes in group 1. After IVIs of plasmin, VEGF, and vitreous tap had been performed one to three times, lens subluxation was noted in 11%, 22%, 44%, 44%, and 67% of the eyes in group 2. The eyes in group 3 and 4 did not show signs of lens subluxation after VEGF IVIs and vitreous tap. Histology confirmed zonular fiber damage in the eyes treated with plasmin.ConclusionsThe incidence of lens subluxation increased following plasmin injections in the eyes, and this was aggravated by the subsequent high VEGF level in the eyes and vitreous tapping. Zonular fibers were disrupted following plasmin treatment. These effects should be kept in mind when using plasmin enzymes in patients with vitreoretinal abnormalities.

Plasmin Activation of Glial Cells through Protease-Activated Receptor 1

The objective of this study was to determine whether plasmin could induce morphological changes in human glial cells via PAR1. Human glioblastoma A172 cells were cultured in the presence of plasmin or the PAR1 specific activating hexapeptide, SFLLRN. Cells were monitored by flow cytometry to detect proteolytic activation of PAR1 receptor. Morphological changes were recorded by photomicroscopy and apoptosis was measured by annexinV staining. Plasmin cleaved the PAR1 receptor on glial cells at 5 minutes (P = 0.02). After 30 minutes, cellular processes had begun to retract from the basal substratum and by 4 hours glial cells had become detached. Similar results were obtained by generating plasmin de novo from plasminogen. Morphological transformation was blocked by plasmin inhibitors aprotinin or epsilon-aminocaproic acid (P = 0.03). Cell viability was unimpaired during early morphological changes, but by 24 hours following plasmin treatment 22% of glial cells were apoptotic. PAR1 activating peptide SFLLRN (but not inactive isomer FSLLRN) promoted analogous glial cell detachment (P = 0.03), proving the role for PAR1 in this process. This study has identified a plasmin/PAR1 axis of glial cell activation, linked to changes in glial cell morophology. This adds to our understanding of pathophysiological disease mechanisms of plasmin and the plasminogen system in neuroinjury.

Safety evaluation of a recombinant plasmin derivative lacking kringles 2-5 and rt-PA in a rat model of transient ischemic stroke

BackgroundTissue type plasminogen activator is the only approved thrombolytic agent for the treatment of ischemic stroke. However, it carries the disadvantage of a 10-fold increase in symptomatic and asymptomatic intracranial hemorrhage. A safer thrombolytic agent may improve patient prognosis and increase patient participation in thrombolytic treatment. A novel direct-acting thrombolytic agent, Δ(K2-K5) plasmin, promising an improved safety profile was examined for safety in the snare ligature model of stroke in the rat.MethodsMale spontaneously hypertensive rats were subjected to 6 hours middle cerebral artery occlusion followed by 18 hours reflow. Beginning 1 minute before reflow, they were dosed with saline, vehicle, Δ(K2-K5) plasmin (0.15, 0.5, 1.5, and 5 mg/kg) or recombinant tissue-type plasminogen activator (10 and 30 mg/kg) by local intra-arterial infusion lasting 10 to 60 minutes. The rats were assessed for bleeding score, infarct volume, modified Bederson score and general behavioral score. In a parallel study, temporal progression of infarct volume was determined. In an in vitro study, whole blood clots from humans, canines and rats were exposed to Δ(K2-K5). Clot lysis was monitored by absorbance at 280 nm.ResultsThe main focus of this study was intracranial hemorrhage safety. Δ(K2-K5) plasmin treatment at the highest dose caused no more intracranial hemorrhage than the lowest dose of recombinant tissue type plasminogen activator, but showed at least a 5-fold superior safety margin. Secondary results include: temporal infarct volume progression shows that the greatest expansion of infarct volume occurs within 2–3 hours of middle cerebral artery occlusion in the spontaneously hypertensive rat. A spike in infarct volume was observed at 6 hours ischemia with reflow. Δ(K2-K5) plasmin tended to reduce infarct volume and improve behavior compared to controls. In vitro data suggests that Δ(K2-K5) plasmin is equally effective at lysing clots from humans, canines and rats.ConclusionsThe superior intracranial hemorrhage safety profile of the direct-acting thrombolytic Δ(K2-K5) plasmin compared with recombinant tissue type plasminogen activator makes this agent a good candidate for clinical evaluation in the treatment of acute ischemic stroke.

Engineering fibrinogen-binding VSV-G envelope for spatially- and cell-controlled lentivirus delivery through fibrin hydrogels

We recently demonstrated that fibrin hydrogels can be used as vehicles for efficient lentivirus gene delivery. Gene transfer in fibrin gels was strongly dependent on matrix degradation by target cells but a fraction of lentiviral particles diffused out of the gels over time compromising spatial control of gene transfer. To overcome this challenge, we engineered lentiviral particles that bind covalently to fibrin during polymerization. To this end, we fused into the viral envelope glycoprotein (VSV-G) peptide domains that are recognized by factor XIII and protease cleavage sites that are recognized by plasmin. Lentivirus pseudotyped with the modified envelopes bound to fibrinogen in a factor XIII dose dependent manner and was released upon plasmin treatment. The peptide/VSV-G fusion envelope variants did not compromise the transduction efficiency of the resulting virus except when lacking any flexible linkers separating the peptide from the VSV-G envelope. Diffusion of virus from the gels decreased dramatically, especially at high concentrations of FXIII, even for fibrin gels with low fibrinogen concentration that were loaded with high titer virus. Lentivirus arrays prepared with fibrin-conjugated lentivirus yielded highly efficient gene transfer that was confined to virus-containing fibrin spots. As a result, signal/noise ratio increased and cross-contamination between neighboring sites was minimal. Finally, in addition to lentivirus microarrays this strategy may be used to achieve spatially-controlled gene transfer for therapeutic applications.

Effect of Plasmin Treatment on the Fibrin Gel Formation

Effect of Plasmin Treatment on the Fibrin Gel Formation

Betaglycan has Two Independent Domains Required for High Affinity TGF-β Binding: Proteolytic Cleavage Separates the Domains and Inactivates the Neutralizing Activity of the Soluble Receptor

Betaglycan is a coreceptor for members of the transforming growth factor beta (TGF-beta) superfamily. Mutagenesis has identified two ligand binding regions, one at the membrane-distal and the other at the membrane-proximal half of the betaglycan ectodomain. Here we show that partial plasmin digestion of soluble betaglycan produces two proteolysis-resistant fragments of 45 and 55 kDa, consistent with the predicted secondary structure, which indicates an intervening nonstructured linker region separating the highly structured N- and C-terminal domains. Amino terminal sequencing indicates that the 45 and 55 kDa fragments correspond, respectively, to the membrane-distal and -proximal regions. Plasmin treatment of membrane betaglycan results in the production of equivalent proteolysis-resistant fragments. The 45 and 55 kDa fragments, as well as their recombinant soluble counterparts, Sol Delta10 and Sol Delta11, bind TGF-beta, but nonetheless, compared to intact soluble betaglycan, have a severely diminished ability to block TGF-beta activity. Surface plasmon resonance (SPR) analysis indicates that soluble betaglycan has K(d)'s in the low nanomolar range for the three TGF-beta isoforms, while those for Sol Delta10 and Sol Delta11 are 1-2 orders of magnitude higher. SPR analysis further shows that the K(d)'s of Sol Delta11 are not changed in the presence of Sol Delta10, indicating that the high affinity of soluble betaglycan is a consequence of tethering the domains together. Overall, these results suggest that betaglycan ectodomain exhibits a bilobular structure in which each lobule folds independently and binds TGF-beta through distinct nonoverlapping interfaces and that linker modification may be an approach to improve soluble betaglycan's TGF-beta neutralizing activity.

Locally Applied Recombinant Plasmin Results in Effective Thrombolysis in a Porcine Model of Arteriovenous Graft Thrombosis

Arteriovenous (AV) graft thrombosis is a frequent complication in patients undergoing hemodialysis. Thrombolytic therapy with tissue plasminogen activator (TPA) is hampered by the risk of bleeding complications. Locally delivered plasmin may have similar thrombolytic efficacy with a superior safety profile, so herein it was compared with TPA in a porcine model of AV graft thrombosis. AV grafts were created bilaterally between the carotid artery and jugular vein. Graft thrombosis was induced by clamping the grafts for 1 hour. On day 3, one graft was treated with a 2-mL local recombinant plasmin injection (10 mg; n = 10) with the contralateral graft being infused with TPA (10 mg, n = 8) or saline solution (n = 2). Thrombolytic efficacy was assessed by weighing the residual clot and released clot fragments. After saline solution injection, the mean residual clot weight in the graft was 618 mg. Local administration of TPA showed the least residual clot in the graft (69 mg +/- 26), with large clot particles reaching the venous outflow (241 mg +/- 23). Plasmin treatment significantly reduced the released clot mass versus TPA (52 mg +/- 23; P < .05), whereas residual clot weight was greater compared with TPA treatment (140 mg +/- 22; P < .05). Overall thrombolytic activity of plasmin (ie, residual clot plus released clot) was significantly better than that of TPA (193 mg +/- 25 vs 310 mg +/- 35; P < .05). The thrombolytic efficacy of recombinant plasmin was shown to be superior to that of TPA in this study.

The Serine Protease Plasmin Cleaves the Amino-terminal Domain of the NR2A Subunit to Relieve Zinc Inhibition of the N-Methyl-d-aspartate Receptors

Zinc is hypothesized to be co-released with glutamate at synapses of the central nervous system. Zinc binds to NR1/NR2A N-methyl-d-aspartate (NMDA) receptors with high affinity and inhibits NMDAR function in a voltage-independent manner. The serine protease plasmin can cleave a number of substrates, including protease-activated receptors, and may play an important role in several disorders of the central nervous system, including ischemia and spinal cord injury. Here, we demonstrate that plasmin can cleave the native NR2A amino-terminal domain (NR2A(ATD)), removing the functional high affinity Zn(2+) binding site. Plasmin also cleaves recombinant NR2A(ATD) at lysine 317 (Lys(317)), thereby producing a approximately 40-kDa fragment, consistent with plasmin-induced NR2A cleavage fragments observed in rat brain membrane preparations. A homology model of the NR2A(ATD) predicts that Lys(317) is near the surface of the protein and is accessible to plasmin. Recombinant expression of NR2A with an amino-terminal deletion at Lys(317) is functional and Zn(2+) insensitive. Whole cell voltage-clamp recordings show that Zn(2+) inhibition of agonist-evoked NMDA receptor currents of NR1/NR2A-transfected HEK 293 cells and cultured cortical neurons is significantly reduced by plasmin treatment. Mutating the plasmin cleavage site Lys(317) on NR2A to alanine blocks the effect of plasmin on Zn(2+) inhibition. The relief of Zn(2+) inhibition by plasmin occurs in PAR1(-/-) cortical neurons and thus is independent of interaction with protease-activated receptors. These results suggest that plasmin can directly interact with NMDA receptors, and plasmin may increase NMDA receptor responses through disruption or removal of the amino-terminal domain and relief of Zn(2+) inhibition.

Proteolytic Release of Latent Transforming Growth Factor-.BETA. (TGF-.BETA.) Binding Protein-1 (LTBP-1) Fragment in Wound Healing

Transforming growth factor-β (TGF-β )i s ap otent growth factor that contributes to wound healing. TGF-β is usually secreted in a latent form complexed with its propeptide, latency-associated peptide (LAP), and LAP covalently binds to a molecule of latent TGF-β binding protein (LTBP). Fibrillin-1 sequesters TGF-β within connective tissue microfibrils through interaction with LTBP-1. However, it is not clear whether TGF-β bound to LTBP-1 is available during wound healing. Therefore, we further characterized LTBP-1, the extracellular regulator of TGF-β in wound healing. LTBP-1 fragments were released from skin by plasmin treatment. The LTBP-1 fragment that is similar to plasmin treatment was also detected in a wound surface. The enzymatic activity of plasmin was also detected in wound surfaces. Immunoblotting analyses showed that the LTBP-1 fragment was preferentially detected in a wound surface with proliferating granulation tissues. These results suggest that proteolytic release of LTBP-1 from a wound surface is physiological and important in regulating wound healing.

Plasmin enhances cell surface tissue factor activity in mesothelial and endothelial cells.

Mesothelial cells that line the thoracic cavity play an important role in maintaining the local balance between procoagulant and fibrinolytic activity, a role akin to the endothelial cells in blood vessels. The mechanism(s) responsible for increased tissue factor (TF) expression in mesothelial cells in response to injury are at present unclear. To investigate whether plasmin or thrombin, two major proteases that may be generated on the pleural surface upon injury, induce TF expression in human pleural mesothelial cells (HMC) and elucidate the underlying mechanism(s). Confluent monolayers of HMC and human umbilical vein endothelial cells (HUVEC) were exposed to plasmin or thrombin for varying time periods and TF expression was analyzed by measuring its activity in a factor Xa generation assay, TF antigen levels by immunoblot analysis and TF mRNA by Northern blot analysis. Both plasmin and thrombin treatments increased cell surface TF activity in HMC by 3- to 4-fold. In contrast to thrombin, plasmin-induced TF activity is not dependent on the de novo synthesis of TF. In HUVEC, plasmin had a minimal effect on unperturbed HUVEC whereas it markedly increased TF activity of activated HUVEC. Plasmin treatment neither affected anionic phospholipid levels at the cell surface nor released protein disulfide isomerase, an oxidoreductase protein that was newly described to play a role in TF activation. Plasmin cleaved cell-associated TFPI. Thrombin up-regulates TF activity in HMC through the transcriptional activation of TF whereas plasmin increases TF activity by inactivating the cell-associated TFPI by a limited proteolysis.

The p75 receptor is associated with inflammatory thermal hypersensitivity

Inflammatory pain, characterized by a decrease in the nociceptive threshold, arises through the actions of inflammatory mediators, and one of the key molecules is nerve growth factor (NGF). Here we report that the administration of neutralizing antibody to the neurotrophin receptor p75 (p75(NTR)) blocks hyperalgesia, which develops with complete Freund's adjuvant (CFA)-induced inflammation or with an intraplantar injection of NGF. Although CFA injection results in the up-regulation of calcitonin gene-related peptide (CGRP) levels in the primary sensory neurons, blocking p75(NTR) abolishes this effect. We further demonstrate that pro-NGF is the predominant ligand of p75(NTR) in vivo. Plasmin treatment, which is intended to decompose pro-NGF, ameliorates CFA-induced hyperalgesia. In addition, an intraplantar injection of pro-NGF induces hyperalgesia. These data together suggest that pro-NGF, as well as mature NGF, binding to p75(NTR) plays an important role in inflammation-induced hyperalgesia. Interference in the binding may provide a therapeutic approach for the treatment of inflammatory pain.

Anti-M9 antibodies in sera from patients with primary biliary cirrhosis recognize an epitope of glycogen phosphorylase

Anti-M9 antibodies in sera from patients with primary biliary cirrhosis (PBC) were previously found to recognize two antigenic determinants at 98 and 59 kD, using a purified antigen fraction derived from rat liver mitochondria in the Western blot. Here we show that these antibodies are directed against an epitope of the enzyme glycogen phosphorylase. By Western blotting, a determinant at 98 kD was obtained testing anti-M9 positive sera against phosphorylase from skeletal muscle, and after plasmin treatment a degradation product appeared at 59 kD. Both determinants were identical to the M9-specific determinants 98 and 59 kD as shown by absorption studies. When these antibodies were eluted from the 98 and 59 kD determinants of the M9 antigen after immunoblotting, they again recognized the same epitopes on plasmin-treated phosphorylase. Furthermore, phosphorylase enzyme activity could be also demonstrated in the purified M9 fraction, and anti-M9-positive/anti-M2-negative but not anti-M9-negative/anti-M2-positive sera could be shown to stimulate phosphorylase activity. Testing sera from 1189 patients with different hepatic and non-hepatic disorders against M9 and phosphorylase from skeletal muscle by ELISA, 20% were positive with phosphorylase and only 2% with the M9 fraction. These data indicate that the commercially available phosphorylase from skeletal muscle cannot be recommended as M9 source. It may still contain non-PBC-specific epitopes which are probably recognized by naturally occurring antibodies directed against this highly conserved protein.

Plasmin Enhances Cell Surface Tissue Factor Activity without Increasing Tissue Factor Antigen Levels at the Cell Surfaces of Mesothelial and Endothelial Cells.

Mesothelial cells that line the thoracic and peritoneal cavities play an important role in protecting the heart, lungs and internal organs and keeping the surfaces free of friction and non-adhesive. Although human mesothelial cells in culture express low levels of tissue factor (TF), TF expression was not detected in vivo in the mesothelial lining of normal pleura but was detected in the mesothelium overlying injured or inflamed lung tissue. Inflammation in the lung parenchyma predisposes to the development of inflammatory exudative pleural effusions and pleural loculation, but the mechanism(s) responsible for TF expression in mesothelial cells under such conditions remains unclear. In the present study, we investigated whether plasmin and thrombin, two major proteases present in exudative pleural effusions, induce TF expression in cultured primary human pleural mesothelial cells (HMC). Confluent monolayers of HMC were treated with plasmin (50 nM) and thrombin (5 nM) for varying time periods. TF expression was analyzed by measurement of cell surface TF activity in FX activation assay, TF antigen by Western blotting and TF mRNA by Northern blot analysis. Both plasmin and thrombin increased cell surface TF activity by 3–4 fold. Thrombin markedly increased TF antigen levels. Surprisingly, only a minimal increase in TF antigen levels was seen in the plasmin-treated cells and no increase in TF mRNA was observed in these cells. Interestingly, 75% of the increase in TF activity was observed within 30 min of plasmin treatment. In thrombin-stimulated cells, TF activity increased slowly, reaching maximum at 4–6 h. Treatment of HMC with actinomycin-D or cycloheximide prior to the addition of the protease failed to inhibit the plasmin-induced TF activity whereas they completely attenuated the thrombin-induced TF activity. These data suggest that plasmin and thrombin enhance TF activity in HMC by different mechanisms, and that plasmin-induced TF activity is independent of de novo synthesis of TF. We also examined the effect of plasmin on TF activity in human umbilical vein endothelial cells (HUVEC). Although plasmin had a minimal effect on naive HUVEC, 30 min plasmin treatment increased cell surface TF activity of thrombin-stimulated endothelial cells by more than two-fold. In our earlier studies, we showed that proteases could mobilize intracellular TF to the cell surface of fibroblasts and thus enhance TF activity independent of de novo synthesis of TF. However, FACS analysis and 125I-FVIIa binding to cell surface TF revealed no significant differences in TF antigen levels at the surfaces of control and plasmin-stimulated HMC. Since exposure of anionic phospholipids at the outer cell surface membrane can increase cell surface TF activity, we next sought to determine if plasmin increases anionic phospholipids on the outer bilayer of the HMC cell membrane. Prothrombin activation assays showed no increase in anionic phospholipids at the surface in plasmin-treated cells. Although recent studies suggest that disulfide bond formation of cysteines in TF enhances TF procoagulant activity, our recent studies in other cell model systems suggest that this mechanism is unlikely to account for plasmin-mediated induction of cell surface TF activity in HMC. While further studies are required to exclude this possibility, our present data indicate that plasmin enhances cell surface TF activity of HMC and HUVEC by a novel mechanism that is yet to be elucidated.

Plasminogen Activation–Induced Pericellular Fibronectin Proteolysis Promotes Fibroblast Apoptosis

Apoptosis of fibroblasts/myofibroblasts is a critical event in the resolution of tissue repair responses; however, mechanisms for the regulation of (myo)fibroblast apoptosis/survival remain unclear. In this study, we demonstrate counter-regulatory interactions between the plasminogen activation system and transforming growth factor-beta1 (TGF-beta1) in the control of fibroblast apoptosis. Plasmin treatment induced fibroblast apoptosis in a time- and dose-dependent manner in association with proteolytic degradation of extracellular matrix proteins, as detected by the release of soluble fibronectin peptides. Plasminogen, which was activated to plasmin by fibroblasts, also induced fibronectin proteolysis and fibroblast apoptosis, both of which were blocked by alpha2-antiplasmin but not by inhibition of matrix metalloproteinase activity. TGF-beta1 protected fibroblasts from apoptosis induced by plasminogen but not from apoptosis induced by exogenous plasmin. The protection from plasminogen-induced apoptosis conferred by TGF-beta1 is associated with the up-regulation of plasminogen activator-1 (PAI-1) expression and inhibition of plasminogen activation. Moreover, lung fibroblasts from mice genetically deficient in PAI-1 lose the protective effect of TGF-beta1 against plasminogen-induced apoptosis. These findings support a novel role for the plasminogen activation system in the regulation of fibroblast apoptosis and a potential role of TGF-beta1/PAI-1 in promoting (myo)fibroblast survival in chronic fibrotic disorders.