Binding of tissue plasminogen activator to cultured human endothelial cells.

Tissue plasminogen activator (t-PA) and urokinase (u-PA), the major activators of plasminogen, are synthesized and released from endothelial cells. We previously demonstrated specific and functional binding of plasminogen to cultured human umbilical vein endothelial cells (HUVEC). In the present study we found that t-PA could bind to HUVEC. Binding of t-PA to HUVEC was specific, saturable, plasminogen-independent, and did not require lysine binding sites. The t-PA bound in a rapid and reversible manner, involving binding sites of both high (Kd, 28.7 +/- 10.8 pM; Bmax, 3,700 +/- 300) and low (Kd, 18.1 +/- 3.8 nM; Bmax 815,000 +/- 146,000) affinity. t-PA binding was 70% inhibited by a 100-fold molar excess of u-PA. When t-PA was bound to HUVEC, its apparent catalytic efficiency increased by three- or fourfold as measured by plasminogen activation. HUVEC-bound t-PA was active site-protected from its rapidly acting inhibitor: plasminogen activator inhibitor. These results demonstrate that t-PA specifically binds to HUVEC and that such binding preserves catalytic efficiency with respect to plasminogen activation. Therefore, endothelial cells can modulate hemostatic and thrombotic events at the cell surface by providing specific binding sites for activation of plasminogen.     

Acute endothelial tissue plasminogen activator release in pregnancy

Summary. Objective: Pregnancy is associated with marked changes in vascular physiology and an increased risk of thrombosis. The aim of the study was to assess the effect of pregnancy on the acute release of tissue plasminogen activator (t‐PA) from the endothelium. Methods and results: Ten primigravida pregnant women were recruited in the third trimester of pregnancy (week 36 ± 1) and compared with 20 age‐matched non‐pregnant women (day 9.8 ± 0.3 of menstrual cycle). Blood flow and plasma fibrinolytic factors were measured in both forearms by venous occlusion plethysmography and blood sampling, respectively, during unilateral brachial artery infusions of bradykinin (100–1000 pmol min^?1). Pregnant women had higher plasma plasminogen activator inhibitor type 1 (PAI‐1) antigen concentrations (77.1 ± 12.4 vs. 21.5 ± 9.8 ng mL^?1; P = 0.004) that resulted in lower basal t‐PA/PAI‐1 ratios (0.2 ± 0.1 vs. 0.6 ± 0.1; P = 0.02) and plasma t‐PA activity concentrations (0.17 ± 0.02 vs. 0.58 ± 0.06 IU mL^?1; P < 0.0004). In both groups, bradykinin caused dose‐dependent increases in blood flow and local release of plasma t‐PA antigen and activity (P < 0.005 for all). Both the plasma t‐PA/PAI‐1 ratios and the net release of active t‐PA were markedly reduced in pregnant women (P < 0.05 for both). Area under the curve for net active t‐PA release was reduced by 36%. Conclusions: Pregnancy is associated with major perturbations of endogenous fibrinolytic capacity with an overwhelming increase in plasma PAI‐1 concentrations and an inadequate release of active t‐PA. These prothrombotic effects may, in part, explain the increased risk of arterial and venous thrombosis in pregnant women.     

Anaphylactoid reaction to recombinant tissue plasminogen activator.

Anaphylactoid reaction to recombinant tissue plasminogen activator for the thrombolytic treatment of acute ischemic stroke is an uncommon complication. An increased risk of anaphylaxis may be found in patients concomitantly being treated with angiotensin-converting enzyme inhibitors, as illustrated by this case report describing a patient who experienced an urticaric rash, hypotension, tachycardia, orolingual angioedema, and airway obstruction following intravenous administration of alteplase. Possible pharmacologic interactions resulting in excessive serum bradykinin and subsequent systemic hypersensitivity responses are discussed.     

组织型纤溶酶原激活因子构建及其在人脐静脉内皮细胞的表达活性

背景：组织型纤溶酶原激活因子被认为是血管系统内初期血栓清除的关键酶。目的：构建pcDNA3．1（＋）组织型纤溶酶原激活因子真核表达载体转染人脐静脉内皮细胞，观察外源性组织型纤溶酶原激活因子的表达情况。设计：随机对照实验。单位：中南大学湘雅二医院及中国医学遗传学国家重点实验室。材料：实验于2002-09／2003-06在中国医学遗传学国家重点实验室完成。脐静脉内皮细胞取自中南大学湘雅二医院健康产妇分娩后胎盘脐带，pcDNA3．1为Smith Kline Beecham公司赠送。方法：构建真核表达载体pcDNA3．1（＋）组织型纤溶酶原激活因子，并将pcDNA3．1（＋）组织型纤溶酶原激活因子转入人脐静脉内皮细胞，用酶联免疫吸附法定量检测转基因后组织型纤溶酶原激活因子蛋白表达情况、发色底物法检测外源性组织型纤溶酶原激活因子活性。以转pcD-NA3．1（＋）组织型纤溶酶原激活因子基因人脐静脉内皮细胞为观察对象，未转pcDNA3．1（＋）组织型纤溶酶原激活因子基因人脐静脉内皮细胞作为对照。主要观察指标：组织型纤溶酶原激活因子蛋白定量及活性测定结果。结果：①组织型纤溶酶原激活因子蛋白定量表达结果为568．6ng／10。细胞／24h，未转pcDNA3．1（＋）组织型纤溶酶原激活因子的人脐静脉内皮细胞测得为17．8ng／10。细胞／24h。②组织型纤溶酶原激活因子活性为108．8IU／10。细胞／24h，未转pcDNA3．1（＋）组织型纤溶酶原激活因子的人脐静脉内皮细胞测得为5．6IU／105细胞／24h。结论：pcDNA3．1（＋）组织型纤溶酶原激活因子转染人脐静脉内皮细胞后，外源性组织型纤溶酶原激活因子基因获有效表达。为pcDNA3．1（＋）组织型纤溶酶原激活因子转入人体细胞确立了有效依据。     

组织型纤溶酶原激活因子构建及其在人脐静脉内皮细胞的表达活性

背景组织型纤溶酶原激活因子被认为是血管系统内初期血栓清除的关键酶.目的构建pcDNA3.1(+)组织型纤溶酶原激活因子真核表达载体转染人脐静脉内皮细胞,观察外源性组织型纤溶酶原激活因子的表达情况.设计随机对照实验.单位中南大学湘雅二医院及中国医学遗传学国家重点实验室.材料实验于2002-09/2003-06在中国医学遗传学国家重点实验室完成.脐静脉内皮细胞取自中南大学湘雅二医院健康产妇分娩后胎盘脐带,pcDNA3.1为SmithKline Beecham公司赠送.方法构建真核表达载体pcDNA3.1(+)组织型纤溶酶原激活因子,并将pcDNA3.1(+)组织型纤溶酶原激活因子转入人脐静脉内皮细胞,用酶联免疫吸附法定量检测转基因后组织型纤溶酶原激活因子蛋白表达情况、发色底物法检测外源性组织型纤溶酶原激活因子活性.以转pcDNA3.1(+)组织型纤溶酶原激活因子基因人脐静脉内皮细胞为观察对象,未转pcDNA3.1(+)组织型纤溶酶原激活因子基因人脐静脉内皮细胞作为对照.主要观察指标组织型纤溶酶原激活因子蛋白定量及活性测定结果.结果①组织型纤溶酶原激活因子蛋白定量表达结果为568.6 ng/106细胞/24 h,未转pcDNA3.1(+)组织型纤溶酶原激活因子的人脐静脉内皮细胞测得为17.8 ng/106细胞/24 h.②组织型纤溶酶原激活因子活性为108.8 IU/106细胞/24 h,未转pcDNA3.1(+)组织型纤溶酶原激活因子的人脐静脉内皮细胞测得为5.6IU/106细胞/24 h.结论pcDNA3.1(+)组织型纤溶酶原激活因子转染人脐静脉内皮细胞后,外源性组织型纤溶酶原激活因子基因获有效表达,为pcDNA3.1(+)组织型纤溶酶原激活因子转入人体细胞确立了有效依据.     

Interleukin 1 induces endothelial cell synthesis of plasminogen activator inhibitor

Human endothelial cells activated with IL-1 express a surface membrane-oriented procoagulant generating system characterized by increased tissue factor synthesis and decreased thrombomodulin activity. We now report that IL-1 also stimulates endothelial cell synthesis of plasminogen activator inhibitor. This array of IL-1-induced activities shifts the balance at the endothelial cell surface to a prothrombotic influence and may reflect an early response of the blood vessel wall to injury.     

Thrombin stimulates tissue plasminogen activator release from cultured human endothelial cells

The effect of thrombin on the release of tissue plasminogen activator from endothelial cells was studied in primary cultures of human umbilical vein endothelial cells. Tissue plasminogen activator concentration in conditioned medium was measured by a two-site radioimmunometric assay. The addition of increasing concentrations (0.01 to 10 U/ml) of thrombin to confluent cultures produced a saturable, dose-dependent increase in the rate of release of tissue plasminogen activator. A sixfold increase in tissue plasminogen activator concentration (from 2 to 12 ng/ml) occurred after the addition of 1 U/ml thrombin (8 X 10(-9) M) to cultures containing 5 X 10(4) cells/cm2. Enhanced release was not observed until 6 h after thrombin addition, reached a maximum rate of 1.3 ng/ml per h between 8 and 16 h, and then declined to 0.52 ng/ml per h after 16 h. The 6-h lag period before increased tPA release was reproducible and independent of thrombin concentration. Thrombin inactivated with diisopropylfluorophosphate or hirudin did not induce an increase in tissue plasminogen activator levels. A 50-fold excess of diisopropylfluorophosphate-treated thrombin, which inhibits binding of active thrombin to endothelial cell high affinity binding sites, did not inhibit the thrombin-induced increase. It is concluded that proteolitically active thrombin causes an increase in the rate of release of tissue plasminogen activator from cultured human endothelial cells. The 6-h interval between thrombin treatment and enhanced tissue plasminogen activator release may reflect a delaying mechanism that transiently protects hemostatic plugs from the sudden increase in the local concentration of this fibrinolytic enzyme.     

Tissue-type plasminogen activator increases the binding of glu-plasminogen to clots.

Porcine tissue-type plasminogen activator (t-PA) increases the binding of 125I-glu-plasminogen to clots made from human plasma or purified fibrinogen in a time and t-PA concentration dependent fashion. The accumulation of plasminogen was faster and greater on noncrosslinked plasma clots than on clots which had been crosslinked by Factor XIIIa. Furthermore, the uptake of plasminogen to crosslinked fibrin clots occurred at a slower rate in the presence of alpha 2-plasmin inhibitor (alpha 2 PI) than in its absence. The kinetics of the uptake of 125I-plasminogen were analyzed using SDS-polyacrylamide gel electrophoresis and radioautography of solubilized plasma clots formed in the presence of t-PA. During the initial phase there was a decrease of clot-bound glu-plasminogen; simultaneously, there was a slight increase in clot-bound glu-plasmin and in plasmin complexed to alpha 2 PI that was crosslinked to alpha-chain polymers of fibrin. This was followed by a marked increase in clot-bound plasminogen having glutamic acid as NH2-terminal (glu-plasminogen) and gluplasmin. t-PA-induced enhancement of glu-plasminogen uptake appears to be mediated by plasmin but does not require the conversion of glu-plasminogen to plasminogen having lysine or methionine as NH2-terminal. The described mechanism assures an adequate supply of clot-bound plasmin, which is the enzyme ultimately involved in the degradation of fibrin.     

Localization of the binding site of tissue-type plasminogen activator to fibrin.

Functionally active A and B chains were separated from a two-chain form of recombinant tissue-type plasminogen activator after mild reduction and alkylation. The A chain was found to be responsible for the binding to lysine-Sepharose or fibrin and the B chain contained the catalytic activity of tissue-type plasminogen activator. An extensive reduction of two-chain tissue-type plasminogen activator, however, destroyed both the binding and catalytic activities. A thermolytic fragment, Fr. 1, of tissue-type plasminogen activator that contained a growth factor and two kringle segments retained its lysine binding activity. Additional thermolytic cleavages in the kringle-2 segment of Fr. 1 caused a total loss of the binding activity. These results indicated that the binding site of tissue-type plasminogen activator to fibrin was located in the kringle-2 segment.     

Autocrine angiotensin system regulation of bovine aortic endothelial cell migration and plasminogen activator involves modulation of proto-oncogene pp60c-src expression.

Rapid endothelial cell migration and inhibition of thrombosis are critical for the resolution of denudation injuries to the vessel wall. Inhibition of the endothelial cell autocrine angiotensin system, with either the angiotensin-converting enzyme inhibitor lisinopril or the angiotensin II receptor antagonist sar1, ile8-angiotensin II, leads to increased endothelial cell migration and urokinase-like plasminogen activator (u-PA) activity (Bell, L., and J. A. Madri. 1990. Am. J. Pathol. 137:7-12). Inhibition of the autocrine angiotensin system with the converting-enzyme inhibitor or the receptor antagonist also leads to increased expression of the proto-oncogene c-src: pp60c-src mRNA increased 7-11-fold, c-src protein 3-fold, and c-src kinase activity 2-3-fold. Endothelial cell expression of c-src was constitutively elevated after stable infection with a retroviral vector containing the c-src coding sequence. Constitutively increased c-src kinase activity reconstituted the increases in migration and u-PA observed with angiotensin system interruption. Antisera to bovine u-PA blocked the increase in migration associated with increased c-src expression. These data suggest that increases in endothelial cell migration and plasminogen activator after angiotensin system inhibition are at least partially pp60c-src mediated. Elevated c-src expression with angiotensin system inhibition may act to enhance intimal wound closure and to reduce luminal thrombogenicity in vivo.     

The addition of endothelial cell growth factor and heparin to human umbilical vein endothelial cell cultures decreases plasminogen activator inhibitor-1 expression.

Plasminogen activator inhibitor-1 (PAI-1) is a specific and rapid inhibitor of tissue plasminogen activator (tPA) and urokinase. Clinical studies suggest that PAI-1 may play a crucial role in the regulation of fibrinolysis. A number of factors modulate PAI-1 activity in endothelial cell culture, and the isolation of PAI-1 cDNA now allows study of PAI-1 regulation at the mRNA level. We examined the effect of endothelial cell growth factor (ECGF) and heparin on PAI-1 expression in human umbilical vein endothelial cell (HUVEC) culture. The addition of ECGF and heparin to HUVEC cultures results in a 3-10-fold decrease in the PAI-1 activity secreted into the conditioned media. This effect is mediated at the mRNA level. A decrease in PAI-1 is also seen with higher concentrations of ECGF alone, but is greatly enhanced by the addition of heparin. No significant change in tPA antigen or mRNA levels was observed.     

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)     

脓毒症大鼠肺组织中尿激酶型纤溶酶原激活剂受体的表达及其与肺损伤程度的相关性研究

目的探讨脓毒症大鼠肺组织中尿激酶型纤溶酶原激活剂受体（uPAR）表达变化及其与肺损伤的关系。 方法将48只雄性成年大鼠分为假手术组及脓毒症组,每组各24只大鼠。采用盲肠结扎穿孔术建立脓毒症损伤模型,假手术组大鼠只行开腹手术、不行结扎及穿孔,各组分别在术后6、12、24 h各取8只大鼠分批处死后收集肺泡灌洗液并留取肺脏。观察所有大鼠各时间点肺组织病理变化,测定肺组织干湿重比及肺泡灌洗液中蛋白变化,并采用免疫组织化学染色测定uPAR表达情况。 结果光镜下可见,假手术组大鼠肺组织肺泡结构完整,术后24 h只有少量炎症细胞浸润;脓毒症组大鼠肺泡腔内大量炎症细胞浸润,肺泡结构破坏,并以术后24 h最明显。两组大鼠各时间点肺组织干湿重比、肺泡灌洗液蛋白水平及uPAR表达比较,差异均有统计学意义（F = 32.000、97.770、72.780,P均< 0.001）。与假手术组相比,脓毒症组大鼠6、12、24 h肺组织干湿重比[（0.75 ± 0.03）vs.（0.78 ± 0.03）;（0.77 ± 0.04）vs.（0.83 ± 0.05）;（0.78 ± 0.04）vs.（0.89 ± 0.05）]、肺泡灌洗液蛋白水平[（79 ± 22）mg/L vs.（110 ± 40）mg/L;（79 ± 23）mg/L vs.（168 ± 36）mg/L;（94 ± 24）mg/L vs.（199 ± 56）mg/L]及uPAR表达[（2.2 ± 1.3）分vs.（4.6 ± 1.9）分;（2.3 ± 0.9）分vs.（6.4 ± 1.8）分;（2.4 ± 0.9）分vs.（7.0 ± 1.8）分]均明显升高（P均< 0.05）;且脓毒症组大鼠12、24 h亚组肺组织干湿重比、肺泡灌洗液蛋白水平均显著高于6 h亚组,而uPAR表达仅24 h亚组显著高于6 h亚组（P均< 0.05）。同时,肺组织中uPAR表达与肺组织干湿重比、肺泡灌洗液蛋白浓度均呈正相关（r = 0.618,P< 0.001;r = 0.652,P< 0.001）。 结论与假手术组相比,uPAR在脓毒症大鼠肺组织中表达明显增加,并且与肺损伤程度呈正相关。     

Renal synthesis of urokinase type-plasminogen activator, its receptor, and plasminogen activator inhibitor-1 in diabetic nephropathy in rats: modulation by angiotensin-converting-enzyme inhibitor

Plasmin is an important factor in the degradation of extracellular matrix. In the study reported here we examined the expression of plasminogen-activator inhibitor-1 (PAI-1), urokinase-type plasminogen activator (uPA), and uPA receptor (uPAR), as well as the relevance of such expression to the production of type IV collagen, a major component of extracellular matrix, in the renal tissue of rats with streptozotocin-induced diabetes. Because angiotensin II is involved in the synthesis of PAI-1 and uPA, we also examined the effect of benazepril, an angiotensin-converting-enzyme inhibitor, on the expression of PAI-1, uPA, and uPAR messenger RNAs (mRNAs) and type IV collagen protein. Rats with streptozocin-induced diabetes-some untreated and some treated with 30 mg/L benazepril-and nondiabetic control rats were sacrificed at 4, 12, or 24 weeks after induction of diabetes. We examined the expression of PAI-1, uPA, and uPAR mRNAs through the use of in situ hybridization and that of type IV collagen by means of immunohistochemical methods. In control rats, we detected weak signals for PAI-1, uPA, and uPAR mRNAs in glomeruli. Diabetic rats exhibited high levels of expression of PAI-1, uPA, and uPAR mRNAs and type IV collagen protein, mainly in mesangial cells. These mRNAs were synthesized in various renal cells (epithelial, mesangial, and endothelial cells and Bowman's capsule). Benazepril inhibited increases in all 3 mRNAs, especially in the mesangium; reduced type IV collagen expression; and attenuated mesangial expansion. Our results indicated that altered expression of PAI-1, uPA, and uPAR in diabetic nephropathy was associated with mesangial expansion and that the beneficial effects of ACE-I may be at least associated with such expression.     

Gender-specific correlations of plasminogen activator inhibitor-1 and tissue plasminogen activator levels with cardiovascular disease-related traits

Summary.  Background:  The purpose of this study was to examine the correlations between plasma levels of plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (t-PA) and cardiovascular disease-related traits in a general population and whether these correlations differed between females and males. Methods:  Plasma PAI-1 and t-PA antigen levels and C-reactive protein (CRP), HDL-cholesterol, triglycerides, total cholesterol, systolic blood pressure, diastolic blood pressure, urinary albumin excretion, and glucose were measured in the population-based PREVEND study in Groningen, the Netherlands ( n  = 2527). Results:  Except for CRP and total cholesterol levels, all traits were significantly different between gender ( P  < 0.001). PAI-1 levels were correlated with all measured cardiovascular disease-related traits ( P  < 0.01) in both females and males. Except for urinary albumin excretion, similar results, albeit less significant, were found for t-PA levels. Age-adjusted correlations between PAI-1 and CRP, triglycerides, total cholesterol, systolic blood pressure, and diastolic blood pressure differed significantly between females and males ( P  < 0.01). Many of the gender differences were predominantly present between premenopausal females and males. Conclusion:  PAI-1 and t-PA levels were correlated with cardiovascular disease-related traits in subjects obtained from the general population and several of these correlations differed across gender. The correlations found in the present study suggest the presence of coordinated patterns of cardiovascular risk factors and indicate which traits might influence PAI-1 and t-PA levels and thereby provide a framework and potential tool for therapeutic intervention to reduce thromboembolic events in the general population.     

Determination of tissue plasminogen activator and its "fast" inhibitor in plasma

We describe efficient, accurate methods for specific determination of tissue plasminogen activator (t-PA, EC 3.4.21.31) and its "fast" inhibitor in plasma. In this coupled assay, a sample containing t-PA is incubated with plasminogen, a plasmin (EC 3.4.21.7) substrate of low Km and high Kcat, and fibrin as a stimulator. The inhibitor of t-PA is determined by incubating the sample with a known amount of t-PA in excess, then determining the residual t-PA. Both t-PA and t-PA inhibitor can be determined in many samples simultaneously within a few hours. These assays are modifications of procedures described by us (Clin Chim Acta 1983;127:279-88 and Thromb Res 1983;31:427-36). Their accuracy as assessed by analytical recovery of pure t-PA added to blood samples (91 +/- 4%) or of partly purified inhibitor added to plasma samples (102 +/- 10%) is satisfactory, as is their precision. For the t-PA assay the CV was 1.6% (within run) or 4.1% (between run). The corresponding values for the inhibitor assay were 4.5% (within run) or 8.4% (between run) if the inhibitor concentration exceeded 3 arb. units/mL.     

The use of tissue plasminogen activator infusion to re-establish function of tunneled hemodialysis catheters.

PURPOSE: To evaluate the safety and efficacy of the recombinant tissue plasminogen activator alteplase in the clearance of poorly functioning tunneled hemodialysis catheters. METHODS: We retrospectively reviewed the outcomes of 25 patients who presented with poorly functioning hemodialysis catheters and were treated with alteplase. After confirming fluoroscopically the need for thrombolytic therapy, alteplase was administered over 2 hours as a 2.5-mg/hour/catheter lumen infusion (total 10 mg). Treatment was considered a clinical success if a flow rate of 250 mL or more per minute was established. RESULTS: Clinical success was achieved in each of 25 patients (100%). There were no thrombolytic-related complications. Catheter survival was extended 30 days in 54% of patients and 45 days in 33% of patients. CONCLUSION: Alteplase is a safe and effective means of producing clearance of blocked tunneled catheters.