Novel markers for constitutive secretion used to show that tissue plasminogen activator is sorted to the regulated pathway in transfected PC12 cells

Once hyperacute rejection has been prevented, the pig-to-human xenograft might be exposed to vascular cell-mediated rejection directed against vascular structures. In order to evaluate the relative importance of direct and antibody-dependent anti-endothelial cell-mediated cytotoxicity in different individuals, freshly isolated human blood leukocytes were incubated with confluent porcine aortic endothelial cells (PAEC) in a 4 h Cr-release cytotoxicity assay. Peripheral blood mononuclear cells (PBMC) and lymphocytes (PBL) of all subjects tested (but not monocytes or neutrophils) directly killed PAEC, with wide interindividual variations (from 2.8% to 32%). The addition of heat-inactivated autologous serum to PBMC and PBL (but not to myeloid cells) always enhanced cytotoxicity. This antibody-dependent cell-mediated cytotoxicity (ADCC) was also observed in the presence of adult pooled serum and cord blood pooled serum and was eliminated after adsorption of adult pooled serum to insoluble protein A, demonstrating that IgG is the only class of immunoglobulin involved in this phenomenon. Moreover, blocking Fc gamma RIII with an anti-CD16 mAb eliminated ADCC without affecting direct cytotoxicity. When the ADCC exerted by the PBL of all subjects was assessed with the same preparation of purified IgG, wide interindividual variations were again observed. Surprisingly, there was no correlation between direct cytotoxicity and ADCC although, as depletion experiments demonstrated, both were due to CD16+ natural killer (NK) cells. These results argue that CD16+ NK cells could play an important role in early vascular rejection of porcine discordant xenografts, by both a direct and an IgG xenoreactive natural antibody-dependent cell-mediated cytotoxicity.     

Plasminogen activator activity during decidualization of human endometrial stromal cells is regulated by plasminogen activator inhibitor 1

Progesterone acts on the estradiol (E2)-conditioned human endometrium to induce decidualization of stromal cells. Consistent with these differential hormone actions in vivo, progestins regulate several end points of decidualization in human endometrial stromal cell monolayers, and E2 augments the effects of progestin. This study shows that in vitro decidualization of the stromal cells is accompanied by diminished plasminogen activator (PA) expression. Polyacrylamide gel electrophoretic separation after immunoprecipitation of biosynthetically labeled PAs revealed that medroxyprogesterone acetate (MPA) lowered levels of secreted tissue type PA (tPA) at 67 kilodaltons and urokinase type PA (uPA) at 55 kilodaltons. These levels were reduced further by E2 plus MPA despite a lack of response to E2 alone. Although tPA activity was readily measured by a chromogenic assay, detection of uPA activity required prior activation, indicating that uPA is released as the pro-uPA zymogen. Comparisons of levels of immunogenic PAs, as measured by specific enzyme-linked immunosorbent assays, with the corresponding catalytic activities revealed selective progestational inhibition of PA activity vs. antigen after 3 days of experimental incubation. Thus, 10(-7) mol/L MPA produced about a 2-fold greater reduction of levels of PA activity than that of its corresponding antigen. More strikingly, 10(-8) mol/L E2 plus 10(-7) mol/L MPA virtually eliminated both tPA activity (99% inhibition; P < 0.005) and uPA activity (93% inhibition; P < 0.005); the reductions in levels of the corresponding antigens were only about 50% of the control levels and did not attain statistical significance. Only after 3-6 days of incubation with E2 plus MPA was statistically significant inhibition achieved for immunogenic levels of both tPA (P < 0.05) and uPA (P < 0.005). Preferential inhibition of levels of PA activities compared with those of the corresponding PA antigens reflects the action of the potent PA inhibitor PAI-1. Thus, the concentration of PAI-1 in the stromal cell-conditioned medium at the end of 0-3 days exceeded those of tPA and uPA, respectively, by 28- and 12-fold in response to MPA and by 52- and 25-fold in response to E2 plus MPA. Extrapolation of these in vitro results to the events of the luteal phase, whose steroidal milieu is mimicked by E2 plus MPA, indicates that decidual cell-derived PAI-1 is a key regulator of proteolytic degradation of extracellular matrix and fibrinolysis during implantation and menstruation.     

Regulation of tissue plasminogen activator production in cultured human fetal mesangial cells

This study was designed to determine the persistence of culturable bacteria versus DNA in the presence of a middle ear effusion in a chinchilla model of otitis media. Cohorts of animals were either infected with an ampicillin-resistant Haemophilus influenzae strain or injected with a tripartite inoculum consisting of freeze-thawed Streptococcus pneumoniae; pasteurized Moraxella catarrhalis; and DNA from H influenzae. The H influenzae-infected animals displayed culture positivity and polymerase chain reaction positivity through day 35. In the chinchillas infected with the low-copy number inocula of S pneumoniae, DNA was not detectable after day 1 from the co-inoculated pasteurized M catarrhalis bacteria or the purified H influenzae DNA; however, amplifiable DNA from the live low-copy number bacteria persisted through day 21 even though they were not culture-positive past day 3. These results demonstrate that DNA, and DNA from intact but nonviable bacteria, does not persist in an amplifiable form for more than a day in the presence of an effusion; however, live bacteria, while not culturable, persist in a viable state for weeks.     

Neurotrophins induce release of neurotrophins by the regulated secretory pathway

Recent studies have established that neurotrophin synthesis and secretion are regulated by activity and that these factors are involved in activity-dependent processes in the nervous system. Neurotrophins also are known to induce increases in intracellular calcium, a trigger for regulated secretion. This finding raises the possibility that neurotrophins themselves may stimulate regulated secretion of neurotrophins. To address this question, we studied the release of neurotrophins from transfected PC12 cells, a widely used model for neuronal secretion and neurotrophin signal transduction. We found that neurotrophins induced the regulated secretion of brain-derived neurotrophic factor, neurotrophin-3 (NT-3), and neurotrophin-4/5. The effect of brain-derived neurotrophic factor on release of NT-3 could be abolished by REX, a p75 blocking antibody, but not by K252a, an inhibitor of neurotrophin tyrosine kinase receptor (Trk) signaling. The nerve growth factor effect on release of NT-3 could be blocked only by simultaneous application of REX and K252a, suggesting that they are mediated by TrkA as well as p75. Our data show that neurotrophins are able to induce the regulated secretion of neurotrophins and suggest a signal-transducing role for both TrkA and p75 in this process. The neurotrophin-induced release of neurotrophins may be relevant for activity-dependent processes such as synaptic plasticity and memory formation.     

Choroidal hemorrhage associated with systemic tissue plasminogen activator

PURPOSE: To determine the cause of spontaneous choroidal hemorrhage in a 67-year-old man after a myocardial infarction and administration of tissue plasminogen activator. METHODS: The patient underwent ocular examination. RESULTS: The patient retained excellent visual acuity and the choroidal hemorrhage resolved completely within two months. CONCLUSION: The administration of tissue plasminogen activator was responsible for the large extent of hemorrhage and should be considered in the differential diagnosis of hemorrhagic choroidal detachment.     

Acceleration of plasminogen activation by tissue plasminogen activator on surface-bound histidine-proline-rich glycoprotein

Histidine-proline-rich glycoprotein (HPRG), also known as histidine-rich glycoprotein, is a major plasminogen-binding protein. In this work we characterized extensively the circumstances under which HPRG accelerates plasminogen activation and the specificity of this effect. Soluble HPRG did not significantly influence plasminogen activation. In contrast, native HPRG bound to hydrazide or nickel chelate surfaces strongly stimulated the activation of plasminogen by tissue plasminogen activator, but not by urokinase or streptokinase. The efficiency of activation on surface-bound HPRG was increased for Glu-plasminogen (41-fold), Lys-plasminogen (17-fold), and cross-linked Glu-plasminogen (11-fold) but not for mini-plasminogen, and was mainly due to a decrease in the apparent Km. A reduced susceptibility to inhibition by chloride ions contributed to the higher activation rate of Glu-plasminogen on an HPRG surface. The immobilized N- and C-terminal domains, but not the histidine-proline-rich domain of HPRG, also bound plasminogen and stimulated its activation. HPRG-enhanced plasminogen activation was proportional to the quantity of HPRG immobilized and was abolished by anti-HPRG antiserum, by low concentrations of epsilon-aminocaproic acid, by methylation of lysine residues in HPRG, and by treatment of HPRG with carboxypeptidase B. Soluble HPRG and a plasminogen fragment, kringle 1-2-3, acted as competitive inhibitors by binding to plasminogen and immobilized HPRG, respectively. The interaction of the conserved C-terminal lysine of HPRG with the high affinity lysine binding site of plasminogen is necessary and sufficient to accelerate plasminogen activation. Unlike other stimulators of plasminogen activation, the effect of HPRG on fibrinolysis is modulated by factors that influence the equilibrium between solution and surface-bound HPRG.     

Pneumatic displacement of subretinal hemorrhage without tissue plasminogen activator

PURPOSE: To analyze the results of excimer laser phototherapeutic keratectomy (PTK) combined with simple excision in recurrent pterygium to minimize the recurrence rate and obtain a smooth corneal surface. SETTING: Veni Vidi Eye Health Centre, Istanbul, Turkey. METHODS: Combined pterygium excision and excimer laser PTK was performed in 22 eyes with recurrent pterygium (22 patients). Both spot and scan modes of the Meditec MEL 60 excimer laser were used to produce a wide ablation layer (depth 40 to 80 microns). RESULTS: During the mean follow-up of 16.5 months (range 6 to 27 months), visual acuity, refraction, slitlamp, and corneal topography examinations were recorded. Pterygium recurred in only 1 eye (4.5%). Postoperative visual acuity improved in 15 eyes (68.2%). Keratometric readings were not accurately measured preoperatively because of corneal surface irregularities but could be easily taken after the surgery. Corneal astigmatism ranged from 0 to 2.00 diopters (D) (mean 1.23 D). Three months after surgery, no haze persisted in any eye. No significant intraoperative or postoperative complication was detected. CONCLUSIONS: Excimer laser PTK appears to simplify pterygium surgery because a superficial keratectomy is sufficient to remove pterygium. The excimer laser can be used to ablate the visible residual tissues and smooth the corneal surface, resulting in good postoperative refraction and visual acuity. Consequently, this procedure seems to be effective and safe.     

Gene polymorphisms for plasminogen activator inhibitor-1/tissue plasminogen activator and development of allograft coronary artery disease

BACKGROUND: Impaired fibrinolytic activity has been linked to the presence and severity of allograft vasculopathy (Tx CAD). This impairment may be associated with the presence of certain fibrinolytic protein gene polymorphisms. METHODS AND RESULTS: To investigate the relation between donor-specific fibrinolytic protein genotypes and Tx CAD, we identified donor plasminogen activator inhibitor-1 (PAI-1) HindIII and tissue plasminogen activator (TPA) EcoRI restriction fragment length polymorphisms-based genotypes by Southern blot analysis in 48 recipients of cardiac allografts and correlated these genotypes with the development of CAD. No association was found between donor TPA genotypes and the presence of Tx CAD. Among the 48 patients, 17% were homozygous for the 1/1 PAI-1 genotype, 51% for the 2/2 PAI-1 genotype, and 32% for the 1/2 PAI-1 genotype. The actuarial freedom from any CAD for the recipients with each respective donor PAI-1 genotype at 12 and 24 months was 100% and 100% for the 1/1 PAI-1 genotype, 92% and 92% for the 1/2 PAI-1 genotype, and 75% and 45% for the 2/2 PAI-1 genotype (P=0.03). Recipients with a diseased 2/2 PAI-1 genotyped allograft had longer ischemic times (P=0.02) than those recipients with a Tx CAD-free allograft. CONCLUSIONS: These data suggest that recipients with a 2/2 PAI-1 genotype are at a significant risk of developing Tx CAD. This genotype may serve as a useful screening tool for predicting the future development of Tx CAD.     

The L-arginine/nitric oxide pathway contributes to the acute release of tissue plasminogen activator in vivo in man

OBJECTIVE: To investigate focal cortical abnormalities of gamma-aminobutyric acid type A-central benzodiazepine receptors (GABA(A)-cBZRs) in patients with extratemporal partial seizures with acquired lesions and in patients with normal high-resolution MRI. METHODS: Six patients with acquired lesions and 18 patients with normal high-resolution MRI and extratemporal partial seizures, as well as 24 normal controls, were studied with 11C-flumazenil (FMZ) PET to produce voxel-by-voxel images of FMZ volume of distribution (FMZVD), which reflects density of GABA(A)-cBZRs. These images were analyzed using Statistical Parametric Mapping (SPM). Each patient was compared with the control group to reveal regions with abnormal FMZVD at p < 0.001 uncorrected, corrected to p < 0.05 for the whole brain volume. Each normal control was compared with the remaining controls in the same manner. RESULTS: All six patients with acquired lesions had a single region of reduced FMZVD. Thirteen of 18 patients with normal MRI had regions of abnormal cortical FMZVD: 10 had regions of increased FMZVD, 6 had regions of decreased FMZVD, and 3 had both regions of increased and decreased FMZVD. Seven patients had an abnormality in the lobe and 12 in the hemisphere of presumed seizure origin. CONCLUSIONS: FMZ PET analyzed with SPM is an automated, objective, sensitive, and specific means for detecting regional cortical abnormalities of GABA(A)-cBZRs in patients with partial seizures. This technique may be useful in the evaluation of patients with refractory partial seizures for surgical treatment, particularly in those patients with normal MRI.     

Use of tissue plasminogen activator factor for acute ischemic stroke

Recently, a new isoform of the type II transforming growth factor beta receptor (TGF-beta RII) was identified. This isoform (TGF-beta RII2) contains an insertion of 25 amino acids in the extracellular domain of the receptor. Using RT-PCR the authors demonstrated that both TGF-beta RII1 and TGF-beta RII2 are expressed by chondrocytes in murine and human articular cartilage. Bovine articular chondrocytes expressed TGF-beta RII1 mRNA but did not express detectable levels of TGF-beta RII2 mRNA, suggesting that the new isoform does not play an important role in normal bovine cartilage physiology. Because TGF-beta responses seem to be age related and differential TGF-beta responses have been described between normal cartilage and cartilage undergoing repair the authors studied if the relative mRNA expression between these isoforms is altered during cartilage repair and aging. No differences in the relative mRNA expression of the two isoforms of the type II TGF-beta receptor could be demonstrated in murine cartilage during aging or during the repair phase after mild PG depletion indicating that it is unlikely that age-related TGF-beta responses and differential TGF-beta responses between normal cartilage and cartilage undergoing repair are the result of differences in the relative expression of the two TGF-beta RII isoforms.     

Expression of the receptor for urokinase-type plasminogen activator in normal and neoplastic blood cells and hematopoietic tissue

Expression of the receptor for the urokinase type plasminogen activator (uPAR) has been studied by flow cytometry and immunohistology in normal blood and bone marrow cells, in vitro activated lymphoid cells, and tissue samples from reactive lymph nodes (n = 6), thymus (n = 2) and malignant lymphomas (n = 82), or leukemias (n = 32). HL-60 myeloid precursor cells and CD34-positive normal stem cells also were analyzed. In the normal cells, staining was confined to monocytes, macrophages, neutrophils, and myeloid precursors. No labelling was seen of normal or activated lymphoid cells. Purified CD34-positive hematopoietic progenitors were uPAR negative, but expressed uPAR during differentiation in short-term liquid culture stimulated in vitro by recombinant interleukin (IL)-1, IL-3, IL-6, granulocyte-macrophage colony stimulating factor (CSF), granulocyte-CSF, and stem cell factor. Enhanced uPAR expression was also seen in HL-60 cells after induction of differentiation with dimethyl sulfoxide or 1 alpha,25-dihydroxyvitamin D3. In lymphomas and leukemias, the staining pattern was similar to that seen in the normal cells with labelling of monocytic and myeloid that seen in the normal cells with labelling of monocytic and myeloid malignancies, but not of the neoplastic cells in B-cell or T-cell lymphomas or Hodgkin's disease. In conclusion, uPAR is a differentiation marker for myeloid and monocytic cells, and may act to facilitate migration of these cells in normal and pathologic conditions by cell-associated plasminogen activation. Whether expression of uPAR in myeloid and monocytic malignancies relates to their growth and behavior will be an important topic for investigations in the future.     

Analysis of tissue plasminogen activator specificity using peptidyl fluorogenic substrates

A series of 54 fluorogenic substrates have been synthesized and evaluated for tissue-type plasminogen activator (tPA) hydrolysis in an attempt to create efficient sensitive substrates for tPA and to investigate substrate structure-efficiency correlations. All substrates contain the 6-amino-1-naphthalenesulfonamide (ANSN) leaving group, Arg in the P1 position, various amino acids in the P2 and P3 positions, and various substituents in the sulfonamide moiety of the leaving group (P' position). The majority of substrates have relatively low K(M) values (< 100 microM), reaching as low as 2.6 microM, and reasonably high k(cat) values (up to 3.6 s(-1)). These substrates have higher affinity, higher hydrolysis rates, and higher efficiency for two-chain tPA than for the single-chain form of this enzyme. Analysis of the P3 structure influence on substrate efficiency demonstrates that compounds which contain D-isomers of N-blocked bulky amino acids, such as Phe, Leu, and Val, in this position are more efficient for tPA than substrates with N-unblocked small amino acids (Ser or Pro) in the P3 position. The second-order rate constants and k(cat) values for substrate hydrolysis increase with decreases in the P2 amino acid hydrophobicity in the following manner: Leu < Val and Gly < Ser < Pro. Substrates which contain an ANSN leaving group had a higher affinity for tPA than substrates with p-nitroaniline or 7-amino-4-methylcoumarin leaving groups. Analyses of substrate hydrolysis dependence on the substrate P' structure show that the k(cat) and the second-order rate constants increased with an increase in the size of monoalkyl substituent in the sulfonamide moiety, whereas substrates which contain either glycine methyl ester or a dialkyl group displayed the lowest efficiency for tPA. The substrate Boc-(p-F)Phe-Pro-Arg-ANSNHC2H5 allowed quantitation of tPA at a concentration as low as 1 pM, a concentration significantly lower than the plasma concentration of this protein. Evaluation of the activation of single-chain tPA by factor Xa demonstrates that prothrombinase is approximately 3-fold more efficient in activating sc-tPA than factor Xa alone, increasing the initial rate of activation from 0.0055 nM/s per 1 nM of factor Xa to 0.017 nM/s per 1 nM.     

Assignment of the binding site for tissue plasminogen activator on human fibronectin

The tissue-type plasminogen activator (t-PA) has been found to bind reversibly to human fibronectin (Fn). To locate the binding site on Fn for t-PA, the Fn was degraded with N-tosyl-L-phenylalanyl chloromethyl ketone-treated trypsin, and the resulting fragments were monitored by the enzyme-linked immunosorbent assay method for t-PA binding activities. A 20-kDa fragment with t-PA binding activity was identified, separated, and purified. It was subjected to further degradation with Staphylococcus aureus proteinase V8. An active 10-kDa fragment was finally purified by reverse-phase high pressure liquid chromatography on a C3 column. The dissociation constants of the binding of Fn and the 10-kDa fragment to t-PA were estimated by Scatchard plot to be 1.13 x 10(-8) and 2.08 x 10(-8) M, respectively. The 10-kDa fragment was sequenced and proved to be located at the 8-9th domains of type I homology of Fn. Based on the structural analysis of the 8-9th domains, a heptadecapeptide corresponding to the sequence Thr535-Glyl551 of Fn, which resided at the large disulfide loop of domain (I-9), was designed and synthesized. Both the 10-kDa fragment and the synthetic peptide could competitively inhibit the binding of Fn to t-PA. The synthetic peptide showed about one-tenth of the binding activity of Fn to t-PA with a dissociation constant of 1.35 x 10(-7) M and was proved to be the binding region of Fn for t-PA. In addition, like the intact Fn, both the 10-kDa fragment and the synthetic peptide could remarkably enhance the amidolytic activity of t-PA in a dose-dependent manner, as shown by using S-2288 as a chromogenic substrate.     

Recombinant tissue plasminogen activator for the treatment of spontaneous adult intraventricular hemorrhage

BACKGROUND: Intraventricular hemorrhage (IVH) has a poor prognosis with mortality rates of between 80 and 100% when all four ventricles are involved. Fibrinolytic therapy has been reported to improve overall outcome. METHODS: Patients with severe primary IVH were treated by direct intraventricular injection of recombinant tissue plasminogen activator (rt-PA) into the lateral ventricles, followed by cerebrospinal fluid (CSF) drainage if the intracranial pressure rose above 20 mm Hg. RESULTS: Over a 15-month period from 1995 through 1996, 10 patients were treated, (4 male and 6 female, mean age 35 years; range, 21-55 years). The mean Glasgow Coma Scale score on admission was 6 (range, 4-8) and the mean Graeb score for severity of IVH on the first CT scan was 10 (range, 8-12). Angiography was negative in five cases but identified arteriovenous malformations in three, a post-traumatic pseudoaneurysm in one, and Moya-moya disease in one. The mean total dose requirement of rt-PA was 8.25 mg (range, 6-12 mg) with a significant reduction in the mean Graeb score after 7 days to 3.9 (range, 2-7, p<0.0001). Outcome at 3 months was death in one case (mortality 10%), severe disability in two (20%), moderate disability in three (30%), and good result in four (40%). Four patients (40%) required subsequent CSF shunting. No complications of rehemorrhage, infection, or catheter obstruction were encountered. CONCLUSION: Intraventricular fibrinolysis with rt-PA seems to be safe and effective for the treatment of severe IVH.     

Modulation by endothelin-1 of tissue plasminogen activator and plasminogen activator inhibitor-1 release from cultured human vascular endothelial cells: interaction of endothelin-1 with cytokines

The interaction of endothelin-1 (ET-1) with either interleukin-1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF alpha) on the release of tissue plasminogen activator antigen (t-PA:Ag) and plasminogen activator inhibitor-1 antigen (PAI-1:Ag) was investigated in a culture system of vascular endothelial cells derived from human umbilical vein. The t-PA:Ag release was significantly decreased by either IL-1 beta or TNF alpha; ET-1 intensified the suppressive effect of the cytokines. In contrast, PAI-1:Ag release was significantly increased by either IL-1 beta or TNF alpha; ET-1 significantly reduced the stimulatory effect of the cytokines. The data suggest that endothelial cell-mediated fibrinolysis may be modulated by ET-1.