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Background: Tissue‐type plasminogen activator (t‐PA) is approved for treatment of ischemic stroke patients, but it may increase the risk of intracranial bleeding (ICB). Matrix metalloproteinases (MMPs), which can be activated through the plasminogen/plasmin system, may contribute to ICB after ischemic stroke. Objectives: To explore the contribution of plasminogen, MMP‐3 and MMP‐9 to ICB associated with t‐PA treatment after ischemic stroke. Methods: Using a thrombotic middle cerebral artery occlusion (MCA‐O) model, ICB was studied in mice with genetic deficiencies of plasminogen (Plg−/−), stromelysin‐1 (MMP‐3−/−), or gelatinase B (MMP‐9−/−) and their corresponding wild‐type (WT) littermates. The induction of MMP‐3 and MMP‐9 was also studied in C57BL/6 WT mice. Results: ICB induced by t‐PA (10 mg kg−1) was significantly less than WT in Plg−/−(P< 0.05) and MMP‐3−/−(P < 0.05) but not in MMP‐9−/−mice. Furthermore, administration of the broad‐spectrum MMP inhibitor GM6001 after t‐PA treatment reduced ICB significantly (P< 0.05) in MMP‐3+/+mice, but had no effect on MMP‐3−/−mice. MMP‐3 expression was significantly enhanced at the ischemic hemisphere; with placebo treatment, it was expressed only in neurons, whereas it was up‐regulated in endothelial cells with t‐PA treatment. Although MMP‐9 expression was also significantly enhanced at the ischemic brain, the amount and the distribution were comparable in mice with and without t‐PA treatment. Conclusions: Our data with gene‐deficient mice thus suggest that plasminogen and MMP‐3 are relatively more important than MMP‐9 for the increased ICB induced by t‐PA treatment of ischemic stroke.

Background:Adeno‐associated viral (AAV) and lentiviral vectors are promising vectors for gene therapy for hemophilia because they are devoid of viral genes and have the potential for long‐term gene expression. Objectives:To compare the performance of different AAV serotypes (AAV8 and AAV9) vs. lentiviral vectors expressing factor (F) IX. Methods and results:AAV‐based and lentiviral vectors were generated that express FIX from the same hepatocyte‐specific expression cassette. AAV9 transduced the liver as efficiently as AAV8 and resulted in supra‐physiological FIX levels (3000–6000% of normal) stably correcting the bleeding diathesis. Surprisingly, AAV9 resulted in unprecedented and widespread cardiac gene transfer, which was more efficient than with AAV8. AAV8 and AAV9 were not associated with any proinflammatory cytokine induction, in accordance with their minimal interactions with innate immune effectors. In contrast, lentiviral transduction resulted in modest and stable FIX levels near the therapeutic threshold (1%) and triggered a rapid self‐limiting proinflammatory response (interleukin‐6), which probably reflected their ability to efficiently interact with the innate immune system. Conclusions:AAV8 and 9 result in significantly higher FIX expression levels and have a reduced proinflammatory risk in comparison with lentiviral vectors. The unexpected cardiotropic properties of AAV9 have implications for gene therapy for heart disease.

Background:Thrombomodulin (TM) is predominantly a vascular endothelial cell plasma membrane glycoprotein that, via distinct structural domains, interacts with multiple ligands, thereby modulating coagulation, fibrinolysis, complement activation, inflammation and cell proliferation. We previously reported that by mediating signals that interfere with mitogen-activated protein kinase and nuclear factor κB pathways, the amino-terminal C-type lectin-like domain of TM has direct anti-inflammatory properties. Methods:In the current study, we use murine models of acute inflammatory arthritis and biochemical approaches to assess the mechanism by which the lectin-like domain of TM modifies disease progression. Results:Mice lacking the lectin-like domain of TM (TMLeD/LeDmice) develop inflammatory arthritis that is more rapid in onset and more severe than that developed in their wildtype counterparts. In two models of arthritis, treatment of mice with recombinant soluble lectin-like domain of TM significantly suppresses clinical evidence of disease and diminishes monocyte/macrophage infiltration into the synovium, with weaker expression of the pro-inflammatory high mobility group box chromosomal protein 1. While thrombin-TM mediated activation of thrombin activatable fibrinolysis inhibitor inactivates complement factors C3a and C5a, we show that TM has a second independent mechanism to regulate complement: the lectin-like domain of TM directly interferes with complement activation via the classical and lectin pathways. Conclusions:These data extend previous insights into the mechanisms by which TM modulates innate immunity, and highlight its potential as a therapeutic target for inflammatory diseases.

Background: N‐glycosylation occurs in the variable region of about 10% of antibodies but the role of carbohydrate at this location is still poorly understood. Objectives: We investigated the function of N‐glycosylation in the variable region of the heavy chain of a human monoclonal antibody, mAb‐LE2E9, that partially inhibits factor VIII (FVIII) activity during coagulation. Methods and results: Enzymatic deglycosylation indicated that the oligosaccharides do not determine the affinity of the antibody but enhance its FVIII neutralizing activity. A mutant antibody lacking the N‐glycosylation site in the variable region of the heavy chain inhibited FVIII activity by up to 40%, while inhibition by the native antibody was 80%. To evaluate the physiological effect of such a FVIII inhibition, we investigated the ability of the mutant antibody devoid of N‐glycosylation in the variable region to prevent thrombosis in mice with a strong prothombotic phenotype resulting from a type II deficiency mutation in the heparin binding site of antithrombin. Despite its moderate inhibition of FVIII activity, the mutant antibody significantly prevented thrombosis in treated animals. We also carried out glycan analysis of native and mutant antibodies. Conclusions: Modification of glycosylation in the variable region of antibodies contributes to the diversity of FVIII type II inhibition possibly by steric hindrance of the active site of FVIII by glycans, and may provide a novel strategy to modulate the functional activity of therapeutic antibodies.

Background: Apoptosis of vascular cells is considered to be a major determinant of atherosclerotic plaque vulnerability and potential rupture. Plasmin can be generated in atherosclerotic plaques and recent in vitrodata suggest that plasminogen activation may trigger vascular smooth muscle cell (VSMC) apoptosis. Aim: To determine whether plasminogen activation may induce aortic VSMC apoptosis ex vivoand in vivo. Methods and results: Mice with single or combined deficiencies of apolipoprotein E (ApoE) and plasminogen activator inhibitor‐1 (PAI‐1) were used. Ex vivoincubation with plasminogen of isolated aortic tunica media from PAI‐1‐deficient mice induced plasminogen activation and VSMC apoptosis, which was inhibited by α2‐antiplasmin. In vivo, levels of plasmin, active caspase 3 and VSMC apoptotic index were significantly higher in atherosclerotic aortas from mice with combined ApoE and PAI‐1 deficiencies than in those from littermates with single ApoE deficiency. A parallel decrease in VSMC density was observed. Conclusions: These data strongly suggest that plasminogen activation may contribute to VSMC apoptosis in atherosclerotic plaques.

Background and Objectives: Tissue plasminogen activator (t‐PA) improves the outcome of ischemic stroke by recanalization of occluded vessels, but has neurotoxic side effects in experimental stroke models. Here, the effect of plasminogen activator inhibitor‐1 (PAI‐1), an endogenous inhibitor of t‐PA, on ischemic infarct volume was studied. Methods: After either permanent ligation or thrombotic occlusion of the middle cerebral artery (MCA), infarct volume, spontaneous reperfusion of thrombosed MCA, t‐PA/PAI‐1 complex level, and blood–brain barrier (BBB) permeability in the ischemic region was studied in transgenic mice with overexpression of PAI‐1 and wild‐type littermate controls and in mice with intracerebroventricular injection of human PAI‐1. Results: Infarct volume was smaller in PAI‐1 transgenic mice (2.9 ± 3.7 mm3, mean ± SD) than in controls (8.9 ± 5.0 mm3, P < 0.05) after permanent MCA ligation (plasma PAI‐1 level 39 ± 23 ng mL−1in transgenic mice vs. 1.5 ± 0.6 ng mL−1in controls), whereas after MCA thrombosis it was larger in transgenics (13.1 ± 3.1 mm3) than in controls (8.0 ± 3.2 mm3, P < 0.05). Spontaneous reperfusion of the thrombosed MCA was significantly delayed in transgenic vs. control mice. In the ligation model, t‐PA/PAI‐1 complex levels were higher and BBB disruption was more pronounced in the ischemic region. Human PAI‐1 injection reduced infarct volume by about 50% in wild‐type mice but not in t‐PAgene deficient mice. Conclusions: High PAI‐1 levels reduced infarct volume in the permanent MCA ligation model, but enhanced it in the MCA thrombosis model.

Objectives: To investigate the role of plasminogen activator inhibitor‐1 (PAI‐1) in adipose tissue development and insulin metabolism. Methods: Aged male wild‐type (WT) or transgenic mice with adipose tissue overexpression of PAI‐1 (45–55 weeks) in 50% C57Bl/6: 50% Friend Virus B‐strain (FVB) genetic background, kept on normal chow, were used without or with administration of a synthetic low molecular weight PAI‐1 inhibitor (PAI‐039) to the food (1 mg g−1) for 4 weeks. Results: The PAI‐1 transgenic mice showed somewhat lower body weight and adipose tissue mass than WT mice, whereas fasting insulin levels were higher. Glucose and insulin tolerance tests did not reveal significant differences between both genotypes. Addition of PAI‐039 to the food did not significantly affect total body fat, weight of the isolated s.c. and gonadal fat territories or their adipocyte size and blood vessel composition in either genotype. Fasting glucose levels and glucose tolerance tests were, for both genotypes, comparable with those without inhibitor treatment. Insulin levels and insulin tolerance tests in WT, but not in PAI‐1 transgenic mice, suggested a higher insulin sensitivity after inhibitor treatment (insulin level 30 min after glucose injection of 2.0 ± 0.17 ng mL−1vs. 3.2 ± 0.48 ng mL−1without inhibitor treatment; P = 0.028). Conclusions: In this model, overexpression of PAI‐1 moderately impaired adipose tissue formation without affecting glucose or insulin tolerance. Administration of a synthetic PAI‐1 inhibitor for 4 weeks did not affect adipose tissue development in WT or PAI‐1 transgenic mice, but induced a higher insulin sensitivity in WT mice.

Background: Thrombolytic therapy of ischemic stroke with tissue‐type plasminogen activator (tPA) improves clinical outcome which may, however, be partially offset by significant intracerebral bleeding (ICB). Objects: The comparative effects of microplasmin (µPli) and tPA on ICB were evaluated in a thrombotic middle cerebral artery (MCA) occlusion model in mice. Methods: A dose of µPli (5 mg kg−1) or tPA (4 mg kg−1) which are comparably effective for reduction of brain damage, or a double dose (10 or 8 mg kg−1, respectively) or the µPli excipient as a control were intravenously administered as a bolus at 30 min or 4 h after MCA occlusion. ICB was measured at 24 h by hemoglobin assay of exsanguinated brain extracts. Bleeding time was measured by tail cutting. Results: In controls given solvent at 4 h, ICB was on average 8.8 µL, which was significantly increased with 10 mg kg−1µPli and with 4 and 8 mg kg−1tPA to 12–13 µL (P < 0.05 each vs. controls, n = 7–9), whereas 5 mg kg−1µPli did not affect bleeding (8.5 µL P = NS vs. controls, n = 7). When given at 30 min, neither µPli nor tPA altered ICB (6.3–6.8 µL, mean; n = 7–9). tPA but not µPli increased bleeding time; from 2.4 min in controls to 5.9 min (median, P < 0.05 vs. controls) and 8.7 min (P < 0.01 vs. controls) with 4 and 8 mg kg−1, respectively, and to 2.3 and 2.9 min with 5 and 10 mg kg−1µPli, respectively (n = 10). Conclusions: µPli at a dose comparably effective as tPA for brain damage reduction induced significantly less ICB, and less bleeding time prolongation in mice with thrombotic MCA occlusion.

Over the past two decades tissue-type plasminogen activator (t-PA), the main physiological plasminogen activator, has been developed as a fibrin-specific thrombolytic agent for the treatment of various thromboembolic diseases. Milestones in this development include: first purification of human t-PA from uterine tissue, elucidation of the interactions regulating physiological fibrinolysis, thus providing a molecular basis for the concept of fibrin-specific plasminogen activation, first animal models of thrombosis and pilot studies in patients supporting the therapeutic potential of t-PA, cloning and expression of recombinant t-PA providing sufficient amounts for large scale clinical use, and demonstration of its therapeutic benefit in large multicenter clinical trials, mainly in patients with acute myocardial infarction (AMI), but also in patients with massive pulmonary embolism, ischemic stroke, deep vein thrombosis and peripheral arterial occlusion. Genetically modified variants of t-PA have been developed for bolus administration in patients with AMI.

Objective: To investigate the influence of transmembrane tumor necrosis factor (TNF)‐α on adipose tissue development and insulin‐mediated glucose metabolism. Methods and results: TNF‐α and lymphotoxin‐α‐deficient mice expressing non‐cleavable transmembrane TNF‐α (Tg‐tmTNF‐α) and TNF‐α/lymphotoxin‐α double knockout (control) mice were kept on high‐fat diet for 15 weeks. The food intake and feeding efficiency of Tg‐tmTNF‐α mice were significantly higher compared with control mice. At the end of the study, Tg‐tmTNF‐α mice had a significantly higher total body weight, as well as subcutaneous and gonadal adipose tissue mass. Histological analysis revealed that the expression of Tg‐tmTNF‐α resulted in a significantly increased adipocyte area and blood vessel density. Plasma leptin levels correlated positively with adipose tissue mass. The plasma levels of total cholesterol and HDL‐cholesterol were significantly increased and LDL‐cholesterol levels significantly decreased in Tg‐tmTNF‐α mice. Fasting blood glucose and plasma insulin levels were not different between the two genotypes and intraperitoneal glucose and insulin tolerance tests did not show significant differences. Conclusions: Transmembrane TNF‐α enhances adipose tissue formation without altering insulin‐mediated glucose metabolism in mice with nutritionally induced obesity.

Venous thromboembolic disease is a major cause of morbidity and mortality, necessitating antithrombotic therapy. A human monoclonal anti‐factor (F)VIII antibody, LCL‐mAb‐LE2E9, produced by a lymphoblastoid cell line derived from a hemophilia A patient with inhibitor to wild‐type but not mutant self FVIII, was previously reported to achieve efficient inhibition of thrombosis in an experimental vena cava thrombosis model in mice. Here, the antithrombotic efficacy of a recombinant DNA‐derived version of this anti‐FVIII antibody (rec‐mAb‐LE2E9) was tested in mice which carry a type II heparin binding site antithrombin deficiency mutation and display spontaneous chronic thrombosis in several sites including the penile vein of sexually active males. The recombinant anti‐FVIII antibody (100 µg, repeated after 3 days) prevented thrombotic priapism in all treated males, whereas all control animals treated with saline (group of four animals) developed priapism within 6 days after mating (P < 0.05 for treated vs. saline). The rec‐mAb‐LE2E9 and the original LCL‐mAb‐LE2E9 were equally effective (five and seven males/group, respectively). These results confirm that FVIII inhibition represents a potent antithrombotic strategy, and show that both LCL‐mAb‐LE2E9 and rec‐mAb‐LE2E9 efficiently prevent thrombosis in a physiological model representative of thrombosis in patients with a severe prothrombotic risk.

The controversial role of plasminogen activator inhibitor‐1 (PAI‐1) in neointima formation and restenosis was studied with the use of a vascular injury model in transgenic mice overexpressing murine PAI‐1 (PAI‐1 Tg) and in wild‐type (WT) controls. Despite the high circulating PAI‐1 levels in the PAI‐1 Tg mice (52 ± 9.8 ng mL−1vs. 0.76 ± 0.17 ng mL−1in WT mice), no significant fibrin deposition was observed in non‐injured femoral arteries of 8‐ to 12‐week‐old mice. Two weeks after severe electric injury, extensive and comparable fibrin deposition was observed in both genotypes, despite a significantly reduced in situfibrinolytic activity in arterial sections of the PAI‐1 Tg mice. The neointimal and medial areas were similar in WT and PAI‐1 Tg mice, resulting in comparable intima/media ratios (e.g. 0.94 ± 0.25 and 1.04 ± 0.17 at the center of the injury). Nuclear cell counts in cross‐sectional areas of the neointima of the injured region were also comparable in arteries from WT and PAI‐1 Tg mice (224 ± 63, 233 ± 20), and the distribution pattern of α‐actin‐positive smooth muscle cells was similar. These findings indicate that in a vascular injury model that induces extensive and persistent fibrin deposition in femoral arteries of mice, overexpression of PAI‐1 does not affect neointima formation.

Significant progress has recently been made in the development of gene therapy for the treatment of hemophilia A and B. These advances parallel the development of improved gene delivery systems. Long-term therapeutic levels of factor (F) VIII and FIX can be achieved in adult FVIII- and FIX-deficient mice and in adult hemophiliac dogs using adeno-associated viral (AAV) vectors, high-capacity adenoviral vectors (HC-Ad) and lentiviral vectors. In mouse models, some of the highest FVIII or FIX expression levels were achieved using HC-Ad vectors with no or only limited adverse effects. Encouraging preclinical data have been obtained using AAV vectors, yielding long-term FIX levels above 10% in primates and in hemophilia B dogs, which prevented spontaneous bleeding. Non-viral ex vivogene therapy approaches have also led to long-term therapeutic levels of coagulation factors in animal models. Nevertheless, the induction of neutralizing antibodies (inhibitors) to FVIII or FIX sometimes precludes stable phenotypic correction following gene therapy. The risk of inhibitor formation varies depending on the type of vector, vector serotype, vector dose, expression levels and promoter used, route of administration, transduced cell type and the underlying mutation in the hemophilia model. Some studies suggest that continuous expression of clotting factors may induce immune tolerance, particularly when expressed by the liver. Several gene therapy phase I clinical trials have been initiated in patients suffering from severe hemophilia A or B. Some subjects report fewer bleeding episodes and occasionally have low levels of clotting factor activity detected. Further improvement of the various gene delivery systems is warranted to bring a permanent cure for hemophilia one step closer to reality.

The effect of recombinant human microplasmin was studied in ischemic stroke models in mice and in an extracorporeal loop thrombosis model in rabbits. Human microplasminogen (µPlg), which lacks the five ‘kringle’ domains of plasminogen was expressed with high yield in Pichia pastoris. It was purified, converted to microplasmin (µPli) and equilibrated with 5 mmol L−1citrate, pH 3.1, yielding a stable preparation. In mice with middle cerebral artery (MCA) ligation, an intravenous (i.v.) bolus of 5.0 mg kg−1µPli reduced infarct size at 24 h from 27 (26–30) to 25 (21–28) mm3(median and range, n= 16 each, P= 0.0001), whereas 4.0 mg kg−1rt-PA and 40 mg kg−1µPlg had no effect. Infarct reduction was observed with administration at 4 h after occlusion. In mice with MCA, infarct size at 24 h was reduced from 20 (14–30) to 9.1 (3.1–25) mm3with 5.0 mg kg−1µPli (n = 15 each, P< 0.002) and to 11 (5.2–27) mm3with 4.0 mg kg−1rt-PA (n = 6; P= 0.02). Infarct reduction was still observed at 10 h after occlusion with µPli but not with t-PA. In rabbits with radiolabeled clots in an extracorporeal arteriovenous loop, local infusion of 2.5 mg kg−1µPli over 2 h, induced 51 ± 15% lysis (mean ± SD, n= 11) vs. a control value of 23 ± 5.5%. µPli did not prolong template bleeding times, whereas equipotent doses of rt-PA were associated with extensive rebleeding. The potency of µPli in both models was similar to that of intact plasmin. These findings indicate that recombinant µPli may be useful for treatment of ischemic stroke and arterial thrombosis.

α2-antiplasmin (α2-AP) is the main physiologic plasmin inhibitor in mammalian plasma. Inactivation of the murine α2-APgene was achieved by replacing, through homologous recombination in embryonic stem cells, a 7-kb genomic sequence encoding the entire murine protein (exon 2 through part of exon 10, including the stop codon) with theneomycin resistanceexpression cassette. Germline transmission of the mutated allele was confirmed by Southern blot analysis. Mendelian inheritance of the inactivated α2-APallele was observed, and homozygous deficient (α2-AP−/−) mice displayed normal fertility, viability, and development. Reverse transcription-polymerase chain reaction confirmed the absence of α2-APmRNA in kidney and liver from α2-AP−/−mice, in contrast to wild-type (α2-AP+/+) mice. Immunologic and functional α2-AP levels were undetectable in plasma of α2-AP−/−mice, and were about half of wild-type in heterozygous littermates (α2-AP+/−). Other hemostasis parameters, including plasminogen activator inhibitor-1, plasminogen, fibrinogen, hemoglobin, hematocrit, and blood cell counts were comparable for α2-AP+/+, α2-AP+/−, and α2-AP−/−mice. After amputation of tail or toe tips, bleeding stopped spontaneously in α2-AP+/+, as well as in α2-AP+/−and α2-AP−/−mice. Spontaneous lysis after 4 hours of intravenously injected 125I-fibrin–labeled plasma clots was significantly higher in α2-AP−/−than in α2-AP+/+mice when injecting clots prepared from α2-AP+/+plasma (78% ± 5% v46% ± 9%; mean ± SEM, n = 6 to 7; P= .02) or from α2-AP−/−plasma (81% ± 5% v46% ± 5%; mean ± SEM, n = 5; P= .008). Four to 8 hours after endotoxin injection, fibrin deposition in the kidneys was significantly reduced in α2-AP−/−mice, as compared with α2-AP+/+mice (P≤ .005). Thus, α2-AP−/−mice develop and reproduce normally; they have an enhanced endogenous fibrinolytic capacity without overt bleeding.

?2-antiplasmin (?2-AP) is the main physiologic plasmin inhibitor in mammalian plasma. Inactivation of the murine ?2-AP gene was achieved by replacing, through homologous recombination in embryonic stem cells, a 7-kb genomic sequence encoding the entire murine protein (exon 2 through part of exon 10, including the stop codon) with theneomycin resistance expression cassette. Germline transmission of the mutated allele was confirmed by Southern blot analysis. Mendelian inheritance of the inactivated ?2-AP allele was observed, and homozygous deficient (?2-AP-/-) mice displayed normal fertility, viability, and development. Reverse transcription-polymerase chain reaction confirmed the absence of ?2-AP mRNA in kidney and liver from ?2-AP-/- mice, in contrast to wild-type (?2-AP+/+) mice. Immunologic and functional ?2-AP levels were undetectable in plasma of ?2-AP-/- mice, and were about half of wild-type in heterozygous littermates (?2-AP+/-). Other hemostasis parameters, including plasminogen activator inhibitor-1, plasminogen, fibrinogen, hemoglobin, hematocrit, and blood cell counts were comparable for ?2-AP+/+, ?2-AP+/-, and ?2-AP-/- mice. After amputation of tail or toe tips, bleeding stopped spontaneously in ?2-AP+/+, as well as in ?2-AP+/- and ?2-AP-/- mice. Spontaneous lysis after 4 hours of intravenously injected 125I-fibrin–labeled plasma clots was significantly higher in ?2-AP-/- than in ?2-AP+/+ mice when injecting clots prepared from ?2-AP+/+ plasma (78%?±?5% v 46%?±?9%; mean?±?SEM, n?=?6 to 7; P?=?.02) or from ?2-AP-/-plasma (81%?±?5% v 46%?±?5%; mean?±?SEM, n?=?5; P?=?.008). Four to 8 hours after endotoxin injection, fibrin deposition in the kidneys was significantly reduced in ?2-AP-/- mice, as compared with ?2-AP+/+ mice (P?=?.005). Thus, ?2-AP-/- mice develop and reproduce normally; they have an enhanced endogenous fibrinolytic capacity without overt bleeding.

Basal expression of the human plasminogen activator inhibitor-1 (PAI-1) is mediated by a promoter element named B box that binds the helicase-like transcription factor (HLTF), homologous to SNF/SWI proteins. Electrophoretic mobility shift assays performed on a set of B box point mutants demonstrated two HLTF sites flanking and partially overlapping with a GT box binding Sp1 and Sp3. Mutations affecting either the Sp1/Sp3 or the two HLTF sites inhibited by 6- and 2.5-fold, respectively, transient expression in HeLa cells of a reporter gene fused to the PAI-1 promoter. In Sp1/Sp3-devoid insect cells, co-expression of PAI-1-lacZ with Sp1 or Sp3 led to a 14-26-fold induction while HLTF had no effect. Simultaneous presence of Sp1 or Sp3 and the short HLTF form (initiating at Met-123) provided an additional 2-3-fold synergistic activation suppressed by mutations that prevented HLTF binding. Moreover, a DNA-independent interaction between HLTFMet123 and Sp1/Sp3 was demonstrated by co-immunoprecipitation from HeLa cell extracts and glutathione S-transferase pull-down experiments. The interaction domains were mapped to the carboxyl-terminal region of each protein; deletion of the last 85 amino acids of HLTFMet123 abolished the synergy with Sp1. This is the first demonstration of a functional interaction between proteins of the Sp1 and SNF/SWI families.

A murine monoclonal antibody (MA-12E6A8) was raised against human urokinase-type plasminogen activator (u-PA), which, in an enzyme-linked immunosorbent assay (ELISA), reacted 15,000-fold better with recombinant two-chain u-PA (rtcu-PA) than with recombinant single-chain u-PA (rscu-PA). The antibody had no effect on the activity of rtcu-PA or on its inhibition by a chloromethylketone, but reduced the inhibition of rtcu-PA by recombinant plasminogen activator inhibitor-1 (rPAI-1) at least 10-fold. The dissociation constant of the rtcu-PA/MA-12E6A8 complex was 7 nmol/L. An ELISA was developed using MA-12E6A8 as capture antibody and a horseradish peroxidase conjugated u-PA specific antibody for tagging. It recognized free and active site blocked rtcu-PA but not rtcu-PA in complex with rPAI-1 or with α2-antiplasmin. This ELISA was used to monitor the generation of rtcu-PA during fibrin clot lysis with rscu-PA in human plasma. Addition of 5 μg/mL rscu-PA to 3 mL plasma containing a 0.2 mL 125l-fibrin labeled plasma clot caused 50% clot lysis in 62 ± 13 minutes (mean ± SD, n = 6), at which time 99 ± 28 μg/mL rtcu-PA was detected but no fibrinogen breakdown had occurred. Fifty percent fibrinogen breakdown did occur only when rtcu-PA had reached a level of 1,000 ± 270 μg/mL (at 150 ± 21 minutes). rscu-PA, 2 μg/mL, induced 50% clot lysis in 160 ± 41 minutes (n = 6); no fibrinogen degradation occurred within 4 hours and rtcu-PA levels did not exceed 80 μg/mL. In the absence of a fibrin clot, 5 μg/mL rscu-PA added to human plasma did not result in significant generation of rtcu-PA (<50 μg/mL after 4 hours) and no fibrinogen degradation was observed. These results indicate that clot lysis with rscu-PA in a plasma milieu does not require extensive systemic conversion of rscu-PA to rtcu-PA, and that fibrinogen degradation occurs secondarily to systemic conversion of rscu-PA to rtcu-PA.

The recombinant chimeric plasminogen activator, rt-PA-AFE/ scu-PA-e, consisting of amino acids 1 to 3 and 87 to 274 of tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of single-chain urokinase-type plasminogen activator (scu-PA), has a markedly increased thrombolytic potency following its continuous intravenous infusion in animal models of venous thrombosis (Collen et al. Circulation,in press). In the present study, the thrombolytic potencies of intravenous bolus injections of rt-PA-AFE/scu-PA-e, of recombinant t-PA (rt-PA), and of recombinant scu-PA (rscu-PA), given alone or in combination, were compared with those of intravenous infusions in a hamster pulmonary embolism model. Dose-dependent clot lysis was obtained in the absence of systemic activation of the fibrinolytic system and fibrinogen breakdown. In bolus injection experiments, the maximal rate of clot lysis, expressed in percent clot lysis per milligrams per kilogram compound administered, was 120 ± 10 for rt-PA, 54 ± 8 for rscu-PA, and 2,100 ± 500 for rt-PA-AFE/scu-PA-e {P≪ .01 vrt-PA or rscu-PA). Comparative results with continuous infusion over 1 hour were 270 ± 64, 99 ± 18, and 1,500 ± 250 [P≪ .01 vrt-PA or rscu-PA) percent lysis per mg/kg compound infused for rt-PA, rscu-PA, and rt-PA-AFE/scu-PA-e, respectively. Thus, rt-PA and rscu-PA are more potent when administered as an infusion than as a bolus, whereas rt-PA-AFE/scu-PA-e is at least as potent when administered as a bolus. Combined bolus injections of rt-PA and rscu-PA had a 2.2-fold synergistic effect on clot lysis, but no synergism was observed with combined bolus injections or with combined infusions of rt-PA and rt-PA-AFE/ scu-PA-e, or of rscu-PA and rt-PA-AFE/scu-PA-e. The present study thus shows that rt-PA-AFE/scu-PA-e is much more potent for clot lysis than rt-PA or rscu-PA when administered as a bolus injection, but no synergistic interaction is observed between the chimera and either rt-PA or rscu-PA.

A murine monoclonal antibody (MA-12E6A8) was raised against human urokinase-type plasminogen activator (u-PA), which, in an enzyme-linked immunosorbent assay (ELISA), reacted 15,000-fold better with recombinant two-chain u-PA (rtcu-PA) than with recombinant single-chain u-PA (rscu-PA). The antibody had no effect on the activity of rtcu-PA or on its inhibition by a chloromethylketone, but reduced the inhibition of rtcu-PA by recombinant plasminogen activator inhibitor-1 (rPAI-1) at least 10-fold. The dissociation constant of the rtcu-PA/MA- 12E6A8 complex was 7 nmol/L. An ELISA was developed using MA-12E6A8 as capture antibody and a horseradish peroxidase conjugated u-PA specific antibody for tagging. It recognized free and active site blocked rtcu- PA but not rtcu-PA in complex with rPAI-1 or with alpha 2-antiplasmin. This ELISA was used to monitor the generation of rtcu-PA during fibrin clot lysis with rscu-PA in human plasma. Addition of 5 micrograms/mL rscu-PA to 3 mL plasma containing a 0.2 mL 125I-fibrin labeled plasma clot caused 50% clot lysis in 62 +/- 13 minutes (mean +/- SD, n = 6), at which time 99 +/- 28 ng/mL rtcu-PA was detected but no fibrinogen breakdown had occurred. Fifty percent fibrinogen breakdown did occur only when rtcu-PA had reached a level of 1,000 +/- 270 ng/mL (at 150 +/- 21 minutes). rscu-PA, 2 micrograms/mL, induced 50% clot lysis in 160 +/- 41 minutes (n = 6); no fibrinogen degradation occurred within 4 hours and rtcu-PA levels did not exceed 80 ng/mL. In the absence of a fibrin clot, 5 micrograms/mL rscu-PA added to human plasma did not result in significant generation of rtcu-PA (less than 50 ng/mL after 4 hours) and no fibrinogen degradation was observed. These results indicate that clot lysis with rscu-PA in a plasma milieu does not require extensive systemic conversion of rscu-PA to rtcu-PA, and that fibrinogen degradation occurs secondarily to systemic conversion of rscu-PA to rtcu-PA.

The relative contribution of several mechanisms to plasminogen activation and fibrin dissolution by urokinase-type plasminogen activator (u-PA) in vitro was quantitated. The activation of plasminogen by recombinant single chain u-PA (rscu-PA), by its two chain derivative (rtcu-PA) and by a plasmin-resistant mutant, rscu-PA- Glu158, obeys Michaelis-Menten kinetics with catalytic efficiencies of 0.00064, 0.046, and 0.00005 L/mumol.s for native plasminogen (Glu- plasminogen) and of 0.0061, 1.21, and 0.0004 L/mumol.s for partially degraded plasminogen (Lys-plasminogen). In a purified system consisting of a fibrin clot submerged in a plasminogen solution, the equi- effective doses (50% lysis in one hour) for rscu-PA, rtcu-PA, and rscu- PA-Glu158 were 16, 6.5, and 32,000 ng/mL for Glu-plasminogen and two- to fourfold lower for Lys-plasminogen. In a plasma milieu, 50% lysis in two hours was obtained for a plasma clot with 2.1 micrograms/mL rscu- PA, 0.5 micrograms/mL rtcu-PA, and greater than 200 micrograms/mL rscu- PA-Glu158 and for a purified fibrin clot with 1.3 micrograms/mL rscu-PA and 0.27 microgram/mL rtcu-PA. After predigestion of a purified fibrin clot with plasmin, the apparent potency of rscu-PA and rtcu-PA increased by 40% and 20%, respectively. In conclusion, rscu-PA has an intrinsic plasminogen activating potential that is only about 1% of that of rtcu-PA and that is 13 times higher than that of rscu-PA- Glu158. Conformational transition of Glu-plasminogen to Lys-plasminogen enhances its sensitivity to activation by all u-PA moieties ten- to 20- fold. Predigestion of fibrin clots with associated increased binding of plasminogen results in a minor apparent increase of the fibrinolytic potency of rscu-PA and rtcu-PA. The relative fibrinolytic potency of rtcu-PA is two to three orders of magnitude higher than that of rscu-PA- Glu158 but only two- to five-fold higher than that of rscu-PA, both in purified systems and in a plasma milieu. These results indicate that conversion of rscu-PA to rtcu-PA constitutes the primary mechanism of fibrin dissolution.

Endothelial cells are centrally involved in regulation of fibrinolysis, and receptors for plasminogen and urokinase provide a mechanism by which cells can regulate their fibrinolytic function. Therefore, the existence and characteristics of receptors for these fibrinolytic components on cultured human umbilical vein endothelial cells were examined. We verified the presence of plasminogen receptors on these cells (Kd = 2.1 +/- 1.3 mumol/L, and 1.8 +/- 1.3 x 10(7) binding sites/cell). These binding parameters and other characteristics indicate that these receptors are closely related to the plasminogen receptors on many circulating and adherent cells. Specific binding sites that interact with two-chain urokinase of mol wt 55,000 with a dissociation constant of 2.1 +/- 1.7 nmol/L, with 2.9 +/- 2.9 x 10(5) sites/cell were also identified. Single-chain urokinase of mol wt 55,000, but not the two-chain degradation product of mol wt 33,000 bound to the cells, implicating the amino-terminal aspects of the ligand in receptor recognition. When endothelial cells were stimulated with thrombin, an agent that modulates their fibrinolytic potential, both receptor types were modestly affected; urokinase binding increased 17%, whereas plasminogen binding decreased 19%. The presence and modulation of plasminogen and urokinase receptors provide a potentially important additional mechanism by which endothelial cells may regulate fibrinolysis.

An antigen immunochemically indistinguishable from plasma alpha 2- antiplasmin, the primary plasmin inhibitor, was detected in human platelets. By radioimmunoassay, 33–114 ng alpha 2-antiplasmin antigen was quantitated in the detergent-soluble extract of 10(9) washed human platelets from 10 normal donors with a mean level of 62 +/- 24 ng/10(9) platelets. Plasma alpha 2-antiplasmin, either in the platelet suspending medium or on the surface of the platelets, could account for less than 8% of the antigen present in the platelet extracts. When stimulated with thrombin, the platelets released alpha 2-antiplasmin antigen without cell lysis, and greater than 85% of the alpha 2- antiplasmin antigen was released at a high thrombin dose. At a lower dose of thrombin, alpha 2-antiplasmin and platelet factor 4 were partially released without concomitant secretion of serotonin. No alpha 2-antiplasmin antigen was detected in extracts or red blood cells, polymorphonuclear leukocytes, and adherent and nonadherent mononuclear cells. Thus, the platelet is the only peripheral blood cell containing significant amounts of alpha 2-antiplasmin.

A potential physiological role of stromelysin-1 (MMP-3) in the expression or activation of gelatinase A (MMP-2) or gelatinase B (MMP-9) in the wall of injured arteries was studied with the use of homozygous MMP-3–deficient (MMP-3-/-) mice. One week after perivascular electric injury of the carotid or femoral artery in wild-type (MMP-3+/+) or MMP-3-/- mice, 70 kD and 65 kD proMMP-2 levels were enhanced by twofold to fourfold, with corresponding increases of 20- to 40-fold for active 61 kD and 58 kD MMP-2, and of 10- to 80-fold for 94 kD proMMP-9. Active MMP-2 species represented approximately one third of the total MMP-2 concentration for both MMP-3+/+ and MMP-3-/- mice. Active 83 kD MMP-9 was not detected in noninjured carotid or femoral arteries, whereas one week after injury its contribution to the total MMP-9 level was 11% to 18% for MMP-3+/+ and MMP-3-/- mice. Immunostaining of arterial sections confirmed enhanced expression of both MMP-2 and MMP-9 after vascular injury. Double immunostaining showed colocalization of MMP-9 with macrophages in the adventitia, whereas MMP-2 was also detected mainly in the adventitia but failed to colocalize with smooth muscle cells. Cell culture experiments confirmed comparable ratios of active versus latent MMP-2 in skin fibroblasts and smooth muscle cells derived from MMP-3+/+ and MMP-3-/- mice. Addition of plasmin(ogen) did not significantly affect activation of proMMP-2. In MMP-3+/+ and MMP-3-/- macrophages, comparable levels of 94 kD proMMP-9 were detected, and plasmin(ogen)-mediated conversion to 83 kD MMP-9 was obtained in both genotypes. These data thus indicate that proMMP-2 activation may occur via a plasmin- and MMP-3–independent mechanism, whereas plasmin can directly activate proMMP-9 via a MMP-3–independent mechanism.

Homozygous plasminogen-deficient (Plg-/-) mice had a significantly reduced thrombolytic capacity toward intravenously injected 125I-fibrin labeled plasma clots prepared from Plg-/- murine plasma (9% +/- 3% lysis after 8 hours; (mean +/- SEM, n = 6), as compared with 82% +/- 8% in wild-type mice; P < .0001). Bolus injection of 1 mg purified murine plasminogen in 10- to 17-week-old Plg-/- mice increased the plasminogen antigen and activity levels at 8 hours to normal levels (130 +/- 5 micrograms/mL). Plasminogen administration was associated with significant restoration of thrombolytic potential (64% +/- 7% spontaneous clot lysis; P < .0001 versus lysis without plasminogen injection). Bolus injection of 1 mg plasminogen in homozygous tissue- type plasminogen activator-deficient (t-PA-/-) mice doubled the plasminogen antigen and activity levels after 8 hours and increased 125I-fibrin clot lysis at 8 hours from 13% +/- 3% to 34% +/- 5% (P = .008). Fibrinogen, t-PA antigen and alpha 2-antiplasmin activity levels after 8 hours were not significantly different in the groups with or without plasminogen injection. Injection of plasminogen induced a variable increase (on average 7- to 10-fold) of PAI-1, but no correlation with the extent of spontaneous clot lysis was observed. Histopathologic examination at the end of the experiments revealed that fibrin deposition in the liver of Plg-/- mice was slightly reduced 8 hours after bolus plasminogen injection (P = .007) and markedly reduced after 24 hours (P < .0001). Plasminogen antigen levels in liver extracts were comparable with those found in wild-type mice at 8 hours (130 +/- 20 versus 110 +/- 15 ng/mg protein) and decreased to 25 +/- 3.2 ng/mg protein at 24 hours. Thus, restoration of normal plasminogen levels in Plg-/- mice normalized the thrombolytic potential toward experimentally induced pulmonary emboli, and resulted in removal of endogenous fibrin deposits within 24 hours.

Platelet aggregation plays an important role in the pathogenesis in arterial thrombotic disorders. The binding of fibrinogen via the Arg- Gly-Asp (RGD) recognition sequence to the platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor is an essential step of platelet aggregation induced by various physiologic agonists, and RGD-containing peptides that bind to the GPIIb/IIIa receptor inhibit thrombus formation in vivo. L-cysteine, N-(mercaptoacetyl)D-tyrosyl-L- arginylglycyl-L alpha-aspartyl-cyclic (1----5)-sulfide, 5-oxide (G4120), a cyclic RGD-containing synthetic pentapeptide, inhibits adenosine diphosphate (ADP)-induced platelet aggregation with 50% inhibition (IC50) at a concentration of 0.05 microgram/mL in human plasma, 0.12 microgram/mL in hamster plasma, and 11 micrograms/mL in rat plasma. Corresponding values for the linear tetrapeptide Arg-Gly- Asp-Phe (RGDF) were 7 and 100 micrograms/mL in human and hamster plasma. The antithrombotic effects of G4120 and RGDF were evaluated in a hamster model consisting of a mural platelet-rich femoral vein thrombus induced by standardized endothelial cell damage. Bolus intravenous injection of G4120 was followed by a biphasic disappearance of G4120 from plasma with t1/2 alpha of 3.7 minutes and t1/2 beta of 63 minutes, corresponding to a plasma clearance of 5.2 +/- 0.68 mL/min. Bolus intravenous injection of G4120 inhibited ex vivo platelet aggregation with 0.5 mumol/L ADP and in vivo thrombus formation in a dose-dependent manner, with ID50 of 11 and 11 micrograms/kg, respectively. Bolus injection of RGDF inhibited in vivo thrombus formation; 43% inhibition was obtained at a dose of 30 mg/kg. Thus, this hamster platelet-rich femoral vein thrombosis model may be useful for the investigation of the antithrombotic properties of platelet GPIIb/IIIa antagonistic peptides. The cyclic synthetic peptide G4120 appears to have a very potent antithrombotic activity in vivo.

Targeting of plasminogen activators to the fibrin component of a thrombus with the use of monoclonal antibodies (MA) directed against human fibrin may enhance their thrombolytic potency and fibrin- specificity. The thrombolytic and pharmacokinetic properties of rscu- PA/MA-FU1–74, an immunoconjugate of recombinant single-chain urokinase- type plasminogen activator (rscu-PA) and a bispecific MA directed against u-PA and against the beta-chain of human fibrin (MA-FU1–74), were investigated in baboons with a [125I]fibrin-labeled autologous blood clot in the femoral vein. Continuous intravenous infusion of rscu- PA/MA-FU1–74 (1:1.2 molar ratio) over 2 hours showed a fivefold increased thrombolytic potency (lysis per unit dose) over that of unconjugated rscu-PA, as evidenced both by a higher maximal rate of lysis (380% +/- 68% v 78% +/- 25% lysis per mg u-PA equivalent of compound administered per kg body weight, P less than .001), and by a lower dose at which the maximal rate of lysis occurs (0.19 +/- 0.03 v 0.82 +/- 0.10 mg compound per kg body weight, P less than .001). The specific thrombolytic activity (percent lysis per unit steady-state plasma u-PA antigen level) was lower for rscu-PA/MA-FU1–74 than for rscu-PA, as shown by both a lower maximal rate of lysis (60% +/- 13% v 220% +/- 22% lysis per microgram/mL u-PA antigen level in plasma, P less than .001) and a higher plasma antigen level at which maximal lysis is achieved (1.2 +/- 0.17 v 0.20 +/- 0.01 microgram/mL, P less than .001). The thrombolytic potency of rscu-PA/MA-UK1–3, an immunoconjugate of rscu-PA with the parental anti-u-PA antibody was similar to that of unconjugated rscu-PA. Clot lysis was achieved without systemic fibrinogen or alpha 2-antiplasmin consumption, and with a minor transient prolongation of the bleeding time. After the end of the infusions, u-PA-related antigen disappeared from plasma in a biphasic manner, with an initial half-life of 3.3 +/- 0.4 minutes for rscu-PA, 13 +/- 1 minutes for rscu-PA/MA-FU1–74, and 13 +/- 1 minutes for rscu-PA/MA-UK1–3, with corresponding plasma clearances of 340 +/- 28, 10 +/- 1, and 37 +/- 4 mL/min, respectively (mean +/- SEM). rscu- PA/MA-FU1–74 has a fivefold higher thrombolytic potency than unconjugated rscu-PA, as a result both of fibrin targeting by the specific idiotype of the antibody and of a slower plasma clearance.