A refined kinetic analysis of plasminogen activation by recombinant bovine tissue-type plasminogen activator indicates two interconvertible activator forms

Bovine tissue-type plasminogen activator (tPA) was heterologously expressed in the methylotrophic yeast Pichia pastoris and characterized structurally and kinetically. The bovine single-chain tPA-mediated activation of bovine plasminogen was studied in the presence and absence of fibrinogen fragments. We have proposed a refined new method of kinetic analysis which allows examination of both stationary and prestationary phases of this process. The investigation revealed the presence of two interconvertible forms of the recombinant bovine tPA being in equilibrium at a 1 to 50 ratio. Only the minor form was able to bind and activate plasminogen. Saturation of the whole pool of tPA required high plasminogen concentration (Km >/= 5 microM) in order to reverse the equilibrium between the two forms. Fibrinogen fragments activated the single-chain tPA due to preferential binding and stabilization of the minor "active" form of the enzyme until all the molecules of tPA were converted. The same mechanism could be applied to human tPA as well. The Km values, obtained for recombinant bovine and human tPA in the presence of fibrinogen fragments, were found to be similar (Km = 0.1 microM) while kcat of human tPA was 5-10 times higher.     

Plasmin, substilisin-like endoproteases, tissue plasminogen activator, and urokinase plasminogen activator are involved in activation of latent TGF-beta 1 in human seminal plasma

OBJECTIVES: To compare the effects of simulated and mild actual hemorrhage on parameters used traditionally to assess hemorrhaging patients: heart rate (HR), blood pressure (BP), and Shock Index (SI = HR/systolic BP), with stroke distance (SD) measured ultrasonically as an index of cardiac stroke volume. MATERIALS and METHODS: Hemorrhage was simulated in 19 healthy volunteers by the application of graded lower-body negative pressure (LBNP) (0, -20, -40, and -60 mm Hg) to pool blood in the lower body and reduce venous return. Measurements were also made before and after a standard blood donation (450 mL) in nine healthy volunteers. MEASUREMENTS and MAIN RESULTS: SD decreased significantly and progressively from the baseline level of 23.8+/-5.7 cm (mean+/-SD) at each level of LBNP: by 3.4+/-1.9, 7.4+/-2.5, and 11.8+/-3.2 cm at LBNP of -20, -40, and -60 mm Hg, respectively. Neither HR nor SI changed significantly at the lowest level of LBNP (-20 mm Hg), but they showed progressive, significant increases thereafter. Mean BP did not change significantly at any level of LBNP. Similarly, after a controlled hemorrhage of 450 mL, SD decreased significantly by 3.3+/-1.6 cm from 22.2+/-2.8 cm, whereas HR and SI remained unchanged and mean BP increased slightly. CONCLUSION: Changes in SD may provide an earlier indication of progressive blood loss than either HR or BP alone or in combination.     

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.     

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.     

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.     

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.     

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.     

Pulmonary thrombo-embolism in nephrotic syndrome treated with tissue plasminogen activator

We describe the case of a boy with steroid sensitive nephrotic syndrome and left pulmonary artery thrombo-embolism. clinical presentation initially suggested sepsis and respiratory signs were minor. Treatment with tissue plasminogen activator infused into the pulmonary artery was successful. CONCLUSION: Pulmonary thrombo-embolism should be considered in unwell children with nephrotic syndrome.     

A molecular variant of plasminogen activator inhibitor of rat decidual tissue

Artificially induced rat decidual tissue expresses plasminogen activator inhibitor (PAI). This PAI, isolated and purified employing chromatographic techniques, is a low molecular weight protein unlike the known PAIs. The final purified preparation resolves into a single band following SDS-PAGE and has an approximate molecular weight of 29 kDa. The properties studied include specificity for urokinase-type (uPA) and tissue-type (tPA) plasminogen activators, binding to conA and heparin, inhibition of thrombin, plasmin and trypsin. Decidual PAI is immunogenic in rabbit and a monospecific antiserum raised against the decidual inhibitor cross reacts with an extract of human placenta.     

An autopsy case of intracranial hemorrhage during tissue plasminogen activator infusion

A comprehensive classification system has been proposed by the AO/ASIF Foundation for the classification of long bone fractures. After an explanatory talk and with the aid of an illustrated pamphlet, 18 orthopaedic surgeons were asked to classify 10 long bone fractures according to the AO system. Three of the participating surgeons had previous experience of the classification system. After individual classification, a consensus classification was derived and the results of the individual and consensus codings were compared. Only 32 per cent of all codings agreed with the final consensus. There was no difference between the surgeons with previous experience of the system (66 per cent) and novice coders (69 per cent) in the number of inaccurate codes when compared with the consensus codes. Reasons for error in coding are discussed. It is recommended that if the AO system is used for the purposes of research and computer-based audit, a consensus of opinion is used as the basis of classification.     

Atheroembolic disease following administration of tissue plasminogen activator (TPA)

Atheroembolic disease is an uncommon condition with many interesting manifestations and has been reported following various procedures. Its occurrence following thrombolytic therapy is extremely rare, with only a few case reports in the literature. However, with a widespread application of thrombolysis in patients with acute myocardial infarction, its incidence is likely to increase and therefore this entity needs to be recognized. Early recognition of the illness may help avoid further expensive and unnecessary investigations. We report two cases of atheroembolic disease following the administration of human recombinant tissue plasminogen activator (TPA).     

Recombinant murine cell lines, transformed by various vectors based on bovine type 1 papillomavirus and expressing human tissue plasminogen activator. II. Analysis of cell lines, producing recombinant tissue plasminogen activator

We have characterized a number of recombinant cell lines established with BPV1 and Lx1 (containing duplication of LCR-E6-E7 sequence) vectors on the basis of C127 cells. It had been shown that Lx1 based vectors possess the higher number of intracellular copies than analogous vectors on the basis of wtBPV, and most part of them is integrated into the host genome. Using various concentrations of heavy metal salts we have developed the optimized procedure for induction of recombinant tPA synthesis which is controlled by the mouse MT1 promoter. A 8-fold increase of rtPA concentration was reached in the course of induction. It had been shown that native and non-glucosylated forms of recombinant and human tPA are identical in their properties.     

Inhibitory mechanism of serpins: loop insertion forces acylation of plasminogen activator by plasminogen activator inhibitor-1

Serpin inhibitors are believed to form an acyl enzyme intermediate with their target proteinases which is stabilized through insertion of the enzyme-linked part of the reactive center loop (RCL) as strand 4 in beta-sheet A of the inhibitor. To test critically the role and timing of these steps in the reaction of the plasminogen activator inhibitor PAI-1, we blocked the vacant position 4 in beta-sheet A of this serpin with an octapeptide. The peptide-blocked PAI-1 was a substrate for both tissue-type plasminogen activator (tPA) and trypsin and was hydrolyzed at the scissile bond. The reactivity of the peptide-blocked substrate PAI-1 was compared to that of the unmodified inhibitor by rapid acid quenching as well as photometric techniques. With trypsin as target, the limiting rate constants for enzyme acylation were essentially the same with inhibitor and substrate PAI-1 (21-23 s-1), as were also the associated apparent second-order rate constants (2.8-2.9 microM-1 s-1). With tPA, inhibitor and substrate PAI-1 reacted identically to form a tightly bound Michaelis complex (Kd approximately Km approximately 20 nM). The limiting rate constant for acylation of tPA, however, was 57 times faster with inhibitor PAI-1 (3.3 s-1) than with the substrate form (0.059 s-1), resulting in a 5-fold difference in the corresponding second-order rate constants (13 vs 2.5 microM-1 s-1). We attribute the ability of tPA to discriminate between the two PAI-1 forms to exosite bonds that cannot occur with trypsin. The exosite bonds retain specifically the distal part of the PAI-1 RCL in the substrate pocket, which favors a reversal of the acylation step. Acylation of tPA becomes effective only by separating the products of the acylation step. With substrate PAI-1, this depends on passive displacement of bonds, whereas with inhibitor PAI-1, separation is accomplished by loop insertion that pulls tPA from its docking site on PAI-1, resulting in faster acylation than with substrate PAI-1.     

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.     

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.