The thrombin antagonist hirudin fails to inhibit endotoxin-induced leukocyte/endothelial cell interaction and microvascular perfusion failure

Besides its central role in coagulatory pathways, thrombin is known to be a key mediator of macrophage and granulocyte activation in vitro. During recent years the concept of thrombin inhibition by the specific thrombin inhibitor, hirudin, has been established to treat septic disorders. Since basic mechanisms of sepsis include leukocyte/endothelial cell interaction and deterioration of capillary perfusion, we hypothesized that hirudin modulates leukocyte activation and microvascular injury. Severe endotoxemia was induced in Syrian hamsters by intravenous administration of endotoxin (lipopolysaccharide [LPS], E. coli, 2mg/kg) at 0 h. Hirudin (0.25 mg/kg/h) was substituted intravenously during the 4 h after the induction of endotoxemia (n = 7, hirudin). In control animals (n = 6, control) LPS was given without hirudin substitution. In skinfold chamber preparations leukocyte/endothelial cell interaction and functional capillary density (FCD, measure of capillary perfusion) were analyzed during a 24-h period after LPS injection using intravital fluorescence microscopy. Hirudin effectively normalized thromboplastin time and antithrombin activity when compared to controls (P < 0.05, ANOVA). However, hirudin did not attenuate LPS-induced arteriolar and venular leukocyte adherence, and even tended to increase leukocyte adherence after 24 h (P > 0.05, MANOVA). In parallel, addition of hirudin led to a significant deterioration of FCD over time when compared to controls (hirudin: baseline = 171 +/- 19 cm(-1) versus 16 +/- 9 at 24 h; control: baseline = 150 +/- 20 cm(-1) versus 62 +/- 18 at 24 h; P < 0.05). The fall in FCD in hirudin animals was associated with a significant increase of wet-to-dry weight ratios in lung, kidney, muscle, and small intestine (P < 0.05 versus control, ANOVA). Thus our study does not indicate a protective effect of hirudin on microcirculation during endotoxemia, despite an improvement of coagulatory parameters. This result may at least in part explain the lack of efficacy of hirudin on lethality during endotoxemia and sepsis.     

凝血酶对脑微血管内皮细胞的损害及水蛭素的保护作用

目的：探讨凝血酶对大鼠脑微血管内皮细胞（BMVEC）的损伤以及凝血酶抑制剂水蛭素的保护作用。方法：分离新生大鼠脑微血管内皮细胞并进行培养,取第二或第三代内皮细胞进行实验。实验分成3组：对照组、凝血酶组和水蛭素组。通过透射电镜和流式细胞术检测3组细胞凋亡的情况。利用TRITC-鬼笔环肽对细胞进行染色观察3组细胞骨架的形态变化。结果：凝血酶可以增加血管内皮细胞凋亡并导致细胞骨架重排、紊乱,而水蛭素可以减轻凝血酶的这种作用。结论：凝血酶可以损害血管内皮细胞骨架,引起细胞收缩、变形,同时增加内皮细胞凋亡,破坏血脑屏障。水蛭素可以减轻这种改变。     

Secretion of tissue-type plasminogen activator and plasminogen activator inhibitor by cultured human endothelial cells: modulation by thrombin, endotoxin, and histamine

Primary cultures of pooled endothelial cells obtained from four to six human umbilical cord veins were grown to confluency in M 199 cell culture medium containing 20% human serum. The secretion of tissue-type plasminogen activator antigen (t-PA Ag) and fast-acting plasminogen activator inhibitor activity (PA-I activity) was measured in the conditioned medium after incubation of confluent cultures with serum-free medium for 24 hours (1.5 ml/25 cm2 cell surface). The baseline production of t-PA Ag was 2.7 +/- 1.4 ng/ml (mean +/- SD) and of PA-I activity 36 +/- 18 IU/ml. Stimulation with thrombin resulted in a dose-dependent and time-dependent increase of the secretion of both components. At 1 NIH U thrombin per milliliter, a fourfold increase of t-PA Ag and a twofold increase of PA-I activity were observed. This effect was dependent on a free active site in thrombin and specific protein synthesis (inhibited by cycloheximide and dactinomycin) but unrelated to prostacyclin synthesis (no effect of aspirin or indomethacin). No free t-PA activity could be demonstrated on fibrin plates in conditioned medium from either stimulated or nonstimulated endothelial cell cultures. Endotoxin stimulation resulted in a specific secretion of PA-I activity but not of t-PA Ag, whereas histamine stimulation at concentrations of 0.1 to 1 mumol/L caused a specific secretion of t-PA Ag but not of PA-I. These findings indicate that the secretion of t-PA Ag and PA-I activity are not necessarily coupled.(ABSTRACT TRUNCATED AT 250 WORDS)     

水蛭素融合蛋白研究进展

水蛭素是一种高效特异的凝血酶抑制剂，临床上用于血栓病的预防和治疗，但它半衰期短、出血副作用强和功能单一。近年来，通过把水蛭素与某些功能蛋白融合表达来解决这些问题，得到的融合蛋白或在体内延长水蛭素的半衰期，或降低其出血副作用，或带来新的功能，如：溶栓、抑制血小板凝集、特异寻靶等。本文对近年来国内外水蛭素融合蛋白研究状况进行综述。     

Thrombin upregulates production of plasminogen activator inhibitor type 1 in human peritoneal mesothelial cells.

OBJECTIVE: To determine the influence of thrombin, which is generated intraperitoneally during peritoneal dialysis, on the synthesis of fibrinolytic system components in human peritoneal mesothelial cells (HMC). METHODS: Confluently grown HMC, isolated from the omental tissue, were used in the experiments. Conditioned media were obtained by incubating cells with serum-free M199 containing the appropriate concentration of the test compound. Tissue type plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1) antigen concentrations were measured by ELISA. Northern blot analysis was conducted for mRNA expression experiments. To test thrombin specificity, we used the thrombin inhibitor hirudin. The protein kinase C (PKC) inhibitor Ro 31-8220 was inserted to examine whether the effect of thrombin depends on PKC activity. RESULTS: Thrombin increased PAI-1 antigen in the conditioned media of HMC in a time- and concentration-dependent manner. After 24 hours incubation, PAI-1 levels increased from 350+/-30 ng/10(5) cells in control conditions to 620+/-30 ng/10(5) cells in HMC exposed to 5 U/mL thrombin (n = 8, p < 0.05). In contrast, there was no effect of thrombin on tPA antigen levels. An increase of PAI-1 mRNA expression was also observed by Northern blot hybridization. Hirudin (10 U/mL) inhibited the thrombin-induced increase in PAI-1 synthesis. In addition, a complete inhibition of the stimulating effect of thrombin on PAI-1 synthesis was obtained by blocking PKC activity with Ro 31-8220 (3 micromol/L). CONCLUSIONS: Thrombin increases PAI-1 synthesis in HMC via a PKC-dependent mechanism.Thereby the synthesis of tPA is not affected. Thus, thrombin may not only promote fibrin formation in the peritoneal cavity, but may also inhibit fibrin degradation by release of free PAI-1 from HMC.     

Bradykinin and its metabolite bradykinin 1-5 inhibit thrombin-induced platelet aggregation in humans

Bradykinin 1-5 is a major stable metabolite of bradykinin, formed by the proteolytic action of angiotensin-converting enzyme. In vitro and animal studies suggest that bradykinin 1-5 possesses biological activity. This study tests the hypothesis that bradykinin 1-5 affects vasodilation, fibrinolysis, or platelet aggregation in humans. Graded doses of bradykinin (47-377 pmol/min) and bradykinin 1-5 (47-18,850 pmol/min) were infused in the brachial artery in random order in 36 healthy subjects. Forearm blood flow (FBF) was measured, and simultaneously obtained venous and arterial plasma samples were analyzed for tissue plasminogen activator (t-PA) antigen. In seven subjects each, alpha- and gamma-thrombin-induced platelet aggregation was measured in platelet-rich plasma obtained from antecubital venous blood at baseline and during peptide infusions. Bradykinin caused dose-dependent increases in FBF and net t-PA release (P < 0.001 for both). Bradykinin 1-5 did not affect FBF (P = 0.13) or net t-PA release (P = 0.46) at concentrations >1500 times physiologic. In contrast, both bradykinin and bradykinin 1-5 inhibited alpha-and gamma-thrombin-induced platelet aggregation (P < 0.01 versus baseline). Bradykinin 1-5 inhibited gamma-thrombin-induced platelet aggregation 50% at a calculated dose of 183 +/- 3 pmol/min. Neither bradykinin nor bradykinin 1-5 affected thrombin receptor-activating peptide-induced platelet aggregation, consistent with the hypothesis that bradykinin and bradykinin 1-5 inhibit thrombin-induced platelet aggregation by preventing cleavage of the thrombin receptor and liberation of thrombin receptor-activating peptide. This study is the first to demonstrate biological activity of bradykinin 1-5 following in vivo administration to humans. By inhibiting thrombin-induced platelet aggregation without causing vasodilation, bradykinin 1-5 may provide a model for small molecule substrate-selective thrombin inhibitors.     

Thrombin suppresses matrix metalloproteinase 2 activity and increases tissue inhibitor of metalloproteinase 1 synthesis in cultured human peritoneal mesothelial cells.

OBJECTIVE: Recently, high levels of intraperitoneally generated thrombin were found in the effluent of patients treated with continuous ambulatory peritoneal dialysis (CAPD). The aim of the present study was to investigate in human peritoneal mesothelial cells (HMCs) the effect of thrombin on the activity and synthesis of matrix metalloproteinases (MMPs), which regulate the degradation of basement membrane collagen. METHODS: Cultured HMCs were isolated from omental tissue and used at confluence for the experiments. Conditioned media were obtained by incubating cells with serum-free M199 containing the relevant doses of thrombin. Activity of MMP-2 and MMP-9 were determined by an activity assay system. The antigen levels of MMPs and of the specific tissue inhibitor of metalloproteinases 1 (TIMP-1) were measured by ELISA. Northern blot analysis was applied to analyze mRNA expression of MMP-2 and TIMP-1. RESULTS: Incubation of HMCs with increasing doses of thrombin resulted in a concentration- and time-dependent suppression of MMP-2 activity. No changes in MMP-9 activity were seen. After a 48-hour stimulation period with thrombin (5 U/mL), MMP-2 activity decreased to 53% of that seen in control conditions. Antigen measurements revealed that this decrease was paralleled by a slight reduction in MMP-2 levels, which became significant at a thrombin dose of 5 U/mL [50.65 +/- 7.5 ng/10(5) cells (48 hours, 5 U/mL) vs 64.6 +/- 10.1 ng/10(5) cells (control)]. Under the same conditions, TIMP-1 levels were considerably increased [3.9 +/- 0.46 microg/10(5) cells (48 hours, 5 U/mL) vs 1.2 +/- 0.14 microg/10(5) cells (control)]. Hirudin (10 U/mL) completely inhibited the thrombin-induced effects on MMP-2 and TIMP-1 synthesis. These results were also reflected by Northern blot hybridization, where a slight decrease in MMP-2 and an increase in TIMP-1 mRNA expression were observed in response to thrombin. CONCLUSIONS: Our results suggest that high thrombin levels suppress MMP-2 activity through decreased MMP-2 and increased TIMP-1 synthesis. Thus, thrombin may promote the accumulation of basement membrane collagen. In addition to fibrin formation, this mechanism may represent a further contribution by thrombin to peritoneal thickening during CAPD.     

Protease‐activated receptor‐1 cleaved at R46 mediates cytoprotective effects

Summary. Background: Activated protein C (aPC) mediates powerful cytoprotective effects through the protease‐activated receptor‐1 (PAR1) that translate into reduced harm in mouse injury models. However, it remains elusive how aPC‐activated PAR1 can mediate cytoprotective effects whereas thrombin activation does the opposite. Objectives: We hypothesized that aPC and thrombin might induce distinct active conformations in PAR1 causing opposing effects. Methods: We analyzed antibody binding to, and cleavage and signalling of PAR1 in either endogenously expressing endothelial or overexpressing 293T cells. Results: In thrombin‐cleaved PAR1 neither the tethered ligand nor the hirudin‐like domain were available for anti‐PAR1 ATAP2 and WEDE15 binding unless the tethered ligand was quenched. In contrast, aPC irreversibly prevented ATAP2 binding while not affecting WEDE15 binding. Reporter constructs with selective glutamine substitutions confirmed R41 as the only thrombin cleavage site in PAR1, whereas aPC preferentially cleaved at R46. Similarly, we report distinct cleavage sites on PAR3, K38 for thrombin and R41 for aPC. A soluble peptide corresponding to R46‐cleaved PAR1 enhanced the endothelial barrier function and reduced staurosporine toxicity in endothelial as well as in 293T cells if PAR1 was expressed. Overexpression of PAR1 variants demonstrated that cleavage at R46 but not R41 is required for cytoprotective aPC signaling. Conclusions: We provide a novel concept on how aPC and thrombin mediate distinct effects. We propose that the enzyme‐specific cleavage sites induce specific conformations which mediate divergent downstream effects. This unexpected model of PAR1 signaling might lead to novel therapeutic options for the treatment of inflammatory diseases.     

Protease-activated receptor 1 mediates thrombin-dependent, cell-mediated renal inflammation in crescentic glomerulonephritis

Protease-activated receptor (PAR)-1 is a cellular receptor for thrombin that is activated after proteolytic cleavage. The contribution of PAR-1 to inflammatory cell-mediated renal injury was assessed in murine crescentic glomerulonephritis (GN). A pivotal role for thrombin in this model was demonstrated by the capacity of hirudin, a selective thrombin antagonist, to attenuate renal injury. Compared with control treatment, hirudin significantly reduced glomerular crescent formation, T cell and macrophage infiltration, fibrin deposition, and elevated serum creatinine, which are prominent features of GN. PAR-1-deficient (PAR-1(-/-)) mice, which have normal coagulation, also showed significant protection from crescentic GN compared with wild-type mice. The reductions in crescent formation, inflammatory cell infiltration, and serum creatinine were similar in PAR-1(-/-) and hirudin-treated mice, but hirudin afforded significantly greater protection from fibrin deposition. Treatment of wild-type mice with a selective PAR-1-activating peptide (TRAP) augmented histological and functional indices of GN, but TRAP treatment did not alter the severity of GN in PAR(-/-) mice. These results indicate that activation of PAR-1 by thrombin or TRAP amplifies crescentic GN. Thus, in addition to its procoagulant role, thrombin has proinflammatory, PAR-1-dependent effects that augment inflammatory renal injury.     

Inhibition by human thrombomodulin of factor Xa-mediated cleavage of prothrombin.

Human thrombomodulin significantly inhibited the rate of prothrombin conversion to thrombin by Factor Xa in the presence of phospholipid or platelets, calcium, and Factor Va. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 125I-prothrombin activation revealed that thrombomodulin reduced the rate of prothrombin activation but did not alter the cleavage pattern. The inhibition was reversed by the inclusion of a highly specific rabbit antithrombomodulin antibody. If thrombomodulin was replaced by hirudin, the rate of thrombin generation was not decreased excluding the possibility that the inhibition by thrombomodulin was secondary to the binding of small amounts of thrombin formed early in the reaction and the prevention of feedback breakdown of prothrombin by thrombin. The inhibitory activity of thrombomodulin was overcome by increasing the concentration of Factor Xa and specific, saturable binding of thrombomodulin to Factor Xa was demonstrated. These results indicate that thrombomodulin binds to Factor Xa and thereby inhibits the activity of the prothrombinase complex.     

Fibrinogen γ' chain binds thrombin exosite II

Summary.  A high-affinity thrombin-binding site in an alternately processed fibrinogen variant, the γA/γ' isoform, is characterized in this report. The binding site has been shown to be situated between γ' 414 and 427, and Tyr418 and 422 in this part of the γ' chain are known to be sulfated. A synthetic peptide corresponding to the γ' chain carboxyl terminus is shown to bind thrombin with a K _d = 0.63 ± 0.16 µmol L⁻¹. Maximum binding of this peptide requires negative charges on Tyr418 and 422. Competitive binding studies with hirudin peptides, heparin and DNA aptamers specific for thrombin exosites I or II indicate thrombin binds to the γ' peptide via exosite II. Thus, thrombin binding to the γ' chain leaves exosite I and the active site accessible to substrates. This may explain why fibrin-bound thrombin can retain enzymatic activity, and why fibrin-bound thrombin is heparin-resistant.     

Factor XII-dependent increases in thrombin activity induce carboxypeptidase-mediated attenuation of pharmacological fibrinolysis

Summary.  Activation of the contact system in patients treated with fibrinolytic agents may be an important source of thrombin that activates thrombin-activated fibrinolysis inhibitor (TAFI) and attenuates fibrinolysis. Factor (F)XIIa in plasma increased 2-fold over 60 min in patients given either tissue plasminogen activator (t-PA) or streptokinase (SK). To determine whether FXIIa-mediated generation of thrombin and activated TAFI (TAFIa) attenuates fibrinolysis in vitro , plasma clots were incubated with SK (250 U mL⁻¹) or t-PA (2.5 g mL⁻¹) and the rate of lysis was measured. Plasma FXIIa impaired lysis judging from marked acceleration when 2.5 µ m corn trypsin inhibitor were added (lysis increased by 172 ± 144% for SK and 40 ± 31% for t-PA vs. no inhibitor, n  = 16, P  < 0.01). Moreover, inhibition of thrombin with hirudin and TAFIa with carboxypeptidase inhibitor accelerated lysis. We conclude that activation of FXII increases thrombin generation, which promotes TAFIa-mediated attenuation of fibrinolysis.     

重组水蛭素克隆的表达及其产物的分离纯化

目的：在原核细胞中表达具有生物活性的重组水蛭素变异物１（ｒＨＶ１）并进行重组产物的分离纯化研究。方法：以质粒ｐＢＶ２２０为载体，在大肠杆菌ＤＨ５α中表达ｒＨＶ１，发色底物法测定其抗凝血酶生物活性；利用超滤、ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０以及凝血酶亲合层析进行重组产物的分离纯化。结果：在大肠杆菌中获得了ｒＨＶ１的活性表达，表达量约占总蛋白的１６．９％，活力达到２０～３０ＡＴＵ／ｍｌ培养液；初步的纯化研究得到了具抗凝生物活性的ｒＨＶ１纯品，ＳＤＳ聚丙烯酰胺凝胶电泳呈单一条带。结论：重组水蛭素变异物１的活性表达及其分离纯化研究填补了国内空白，为研制国产高效重组抗凝药物带来希望。     

The relative kinetics of clotting and lysis provide a biochemical rationale for the correlation between elevated fibrinogen and cardiovascular disease

BACKGROUND: Elevated plasma fibrinogen is a well known risk factor for cardiovascular disease. The mechanistic rationale for this is not known. OBJECTIVES: These studies were carried out to determine the fibrinogen concentration dependencies of clotting and lysis times and thereby determine whether these times rationalize the correlation between an increased risk of cardiovascular disease and elevated plasma fibrinogen. METHODS: The time courses of clot formation and lysis were measured by turbidity in systems comprising a) fibrinogen, thrombin and plasmin, or b) fibrinogen, thrombin, plasminogen and t-PA, or c) plasma, thrombin and t-PA. From the lysis times, k(cat) and K(m) values for plasmin action on fibrin were determined. RESULTS: The time to clot increased linearly from 2.9 to 5.6 minutes as the fibrinogen concentration increased from 1 to 9 microM and did not increase further as the fibrinogen concentration was raised to 20 microM. In contrast, the clot lysis time increased linearly over the input fibrinogen concentration range of 2 to 20 microM. A similar linear trend was found in the two systems with t-PA and plasminogen. Apparent K(m) and k(cat) values for plasmin were 1.1 +/- 0.6 microM and 28 +/- 2 min(-1), respectively. K(m) values for plasmin in experiments initiated with t-PA and plasminogen were 1.6 +/- 0.2 microM in the purified system and 2.1 +/- 0.9 microM in plasma. CONCLUSION: As the concentration of fibrinogen increases, especially above physiologic level, the balance between fibrinolysis and clotting shifts toward the latter, providing a rationale for the increased risk of cardiovascular disease associated with elevated fibrinogen.     

The use of hirudin as universal anticoagulant in haematology, clinical chemistry and blood grouping.

Undesirable interactions between anticoagulants and diagnostic test kit procedures so far have prevented the development of a single uniform blood sampling tube. Contrary to K2-EDTA, heparin and other anticoagulants, hirudin only minimally alters blood cells and dissolved blood constituents, thus qualifying as a universal anticoagulant for diagnostic purposes. Automated complete blood counts, automated analyses of clinical chemistry analytes and immunohaematology were performed from hirudinised and routinely processed blood obtained from healthy volunteers (n=35) and hospitalised patients (n=45). Hirudin (400 ATU/ml blood) sufficiently anticoagulated blood for diagnostic purposes. The measurements of automated complete blood counts obtained from K2-EDTA-anticoagulated and hirudinised blood correlated significantly as did the measurements of 24 clinical chemistry analytes from hirudinised plasma and serum. Regression analysis revealed that the results of complete blood counts and clinical chemistry tests were predictable from the respective measurements from hirudinised blood (p=0.001). Immunohaematological tests and cross-matching from hirudinised and native blood of the same donors gave identical results. Single clotting factors, but not global coagulation analytes, could be measured from hirudinised blood. Therefore, a universal hirudin-containing blood sampling tube could be designed for automated analysis of haematological, serological and clinical chemistry analytes.     

Preparation, characterization and in vivo observation of phospholipid-based gas-filled microbubbles containing hirudin

The objective of this work was to prepare echogenic phospholipid-based gas-filled microbubbles (PGM) and investigate their physical characteristics, echogenicity and loading ability of hirudin under various NaCl concentrations. PGM were prepared by a sonication-lyophilization method. Hirudin was used as a model drug to evaluate the drug encapsulation efficiency of the PGM. PGM loaded with hirudin were prepared by dissolving lyophilized powder with hirudin solution. The morphology, particle size and microbubble concentration of PGM were measured. The hirudin encapsulation efficiency as a function of NaCl concentration was determined. The mean particle size and microbubble concentration of PGM were unchanged by the presence of hirudin for at least 60 min after preparation. Hirudin encapsulation quantity was proportional to the hirudin concentration until saturation occurred at high concentration, and the encapsulation efficiency had an inverse relationship. Hirudin encapsulation efficiency was affected by NaCl concentration. When NaCl concentration was increased from 10 mg mL(-1) to 20 mg mL(-1) in PGM solution, hirudin encapsulation efficiency decreased from 35.8 to 26.7%, and microbubble concentration decreased from 2.7 x 10(8) to 1.7 x 10(8) microbubbles per mL. The PGM were shown easily to be visible in in vivo rabbit liver. There was no difference in echogenicity between the loaded and unloaded bubbles. PGM prepared by the sonication-lyophilization method exhibited satisfactory physical characteristics and loading ability and are suitable for use in imaging and ultrasound-triggered delivery.     

Thrombomodulin modulates growth of tumor cells independent of its anticoagulant activity.

Thrombomodulin (TM), recognized as an essential vessel wall cofactor of the antithrombotic mechanism, is also expressed by a wide range of tumor cells. Tumor cell lines subcloned from four patients with malignant melanoma displayed a negative correlation between TM expression and cell proliferation in vitro and in vivo. Overexpression of wild-type TM decreased cell proliferation in vitro and tumor growth in vivo. TM mutants with altered protein C activation capacity lead to a similar effect. In contrast, transfection of melanoma cells with mutant TM constructs, in which a portion of the cytoplasmic or lectin domain was deleted, abrogated the antiproliferative effect associated with overexpression of wild-type TM. Experiments performed with either peptide agonists/antagonists of the thrombin receptor, with hirudin, or with inhibitors of thrombin-TM interaction did not alter the growth inhibitory effect of TM overexpression. These data suggest that TM exerts an effect on cell proliferation independent of thrombin and the thrombin receptor, possibly related to the binding of novel ligands to determinants in the lectin domain which might trigger signal transduction pathways dependent on the cytoplasmic domain.     

Thrombin induction of plasminogen activator-inhibitor in cultured human endothelial cells.

We have examined the effect of thrombin on the activity of plasminogen activator (PA) and plasminogen activator-inhibitor (PA-I) in medium conditioned by primary cultures of human umbilical vein endothelial cells. PA activity was measured by fibrinolytic and esterolytic assays, and total tissue-type PA (tPA) antigen by radioimmunoassay. Net PA-I activity was assayed by titration of human urokinase esterolytic activity. Incubation of confluent endothelial cell cultures with thrombin for 24 h caused a sixfold increase in PA-I activity. The effect of thrombin was half-maximal at approximately 0.4 U/ml (less than 4 nM), and required concomitant RNA and protein synthesis. The stimulation of PA-I activity required active alpha-thrombin and was not obtained with gamma-thrombin nor with thrombin catalytically inactivated with hirudin. Because of the excess of PA-I, PA activity was not measurable in either control or thrombin-treated cells. Thrombin did, however, increase medium concentration of tPA antigen by approximately fourfold. The thrombin-induced PA-I inhibited both tPA and urokinase, did not lose activity upon acidification, and was stable to sodium dodecyl sulfate and thiol reduction. We conclude that physiologic concentrations of thrombin increase both PA-I activity and tPA antigen in medium conditioned by human umbilical vein endothelial cells. Because there was always a several-fold increase in the net activity of free PA-I, these observations suggest that the net effect of thrombin is to decrease fibrinolytic activity in human endothelial cells. Thus, thrombin, in addition to its role in coagulation, may protect clots from premature lysis by increasing the amount of a specific fibrinolytic inhibitor.     

Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition

In human gene therapy applications, lentiviral vectors may have advantages over gamma-retroviral vectors in several areas, including the ability to transduce nondividing cells, resistance to gene silencing and a potentially safer integration site profile. However, unlike gamma-retroviral vectors it has been problematic to drive the expression of multiple genes efficiently and coordinately with approaches such as internal ribosome entry sites or dual promoters. Using different 2A peptides, lentiviral vectors expressing two-gene T-cell receptors directed against the melanoma differentiation antigens gp100 and MART-1 were constructed. We demonstrated that addition of amino-acid spacer sequences (GSG or SGSG) before the 2A sequence is a prerequisite for efficient synthesis of biologically active T-cell receptors and that addition of a furin cleavage site followed by a V5 peptide tag yielded optimal T-cell receptor gene expression. Furthermore, we determined that the furin cleavage site was recognized in lymphocytes and accounted for removal of residual 2A peptides at the post-translational level with an efficiency of 20-30%, which could not be increased by addition of multiple furin cleavage sites. The novel bicistronic lentiviral vector developed herein afforded robust anti-melanoma activities to engineered peripheral blood lymphocytes, including cytokine secretion, cell proliferation and lytic activity. Such optimal vectors may have immediate applications in cancer gene therapy.     

Effect of thrombin on the fibrinolytic activity of cultured bovine endothelial cells.

The vascular endothelium is a rich source of plasminogen activator (PA) and thus of blood vessel-associated fibrinolytic activity. Cultured bovine aortic endothelial cells were employed to determine if components of the coagulation system interact with the endothelium to modify expression of this activity. The addition of thrombin to these cultures led to a rapid decline in intracellular PA activity, with as little as 3 ng/ml, or 0.1 nM thrombin causing a 50% decrease within 30 min. Thrombin inactivated with diisopropylflurophosphate or hirudin did not elicit the response. Although control cultures secreted high levels of PA, no PA activity could be detected in the media surrounding the thrombin-treated cells. This loss of activity did not appear to result from direct inactivation of PA by thrombin. These observations indicate that the fibrinolytic potential of cultured endothelial cells is rapidly suppressed by trace amounts of thrombin. The generation of thrombin at sites of vascular injury may have a similar effect on the endothelium.