Gene polymorphisms of tissue plasminogen activator and plasminogen activator inhibitor-1 in patients with antiphospholipid antibodies

OBJECTIVE: Impaired fibrinolytical outcomes may be one of the pathogenic factors for thrombotic events in patients with antiphospholipid antibodies (aPL). We investigated the consequences of the gene polymorphisms of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) in patients positive for aPL. METHODS: Seventy-seven Japanese and 82 British patients with aPL were examined for Alu-repeat insertion (I)/deletion (D) polymorphism of the tPA gene by polymerase chain reaction (PCR), and 4G/5G polymorphism in the PAI-1 promoter gene by site-directed mutagenesis-PCR and restriction fragment length polymorphism analysis. Correlations between these polymorphisms and clinical symptoms of antiphospholipid syndrome (APS) (arterial thrombosis, venous thrombosis, miscarriage) were analyzed. RESULTS: Significant differences in the allele frequencies of these genes did not exist between patients and controls. There was no significant correlation between these gene polymorphisms and clinical symptoms of APS in patients with aPL. CONCLUSION: Polymorphisms of the tPA or PAI-1 genes probably do not significantly influence the risk of anerial thrombosis, venous thrombosis, or pregnancy morbidity in patients with aPL.     

Unique secretory dynamics of tissue plasminogen activator and its modulation by plasminogen activator inhibitor-1 in vascular endothelial cells

We analyzed the secretory dynamics of tissue plasminogen activator (tPA) in EA.hy926 cells, an established vascular endothelial cell (VEC) line producing GFP-tagged tPA, using total internal reflection-fluorescence (TIR-F) microscopy. tPA-GFP was detected in small granules in EA.hy926 cells, the distribution of which was indistinguishable from intrinsically expressed tPA. Its secretory dynamics were unique, with prolonged (> 5 minutes) retention of the tPA-GFP on the cell surface, appearing as fluorescent spots in two-thirds of the exocytosis events. The rapid disappearance (mostly by 250 ms) of a domain-deletion mutant of tPA-GFP possessing only the signal peptide and catalytic domain indicates that the amino-terminal heavy chain of tPA-GFP is essential for binding to the membrane surface. The addition of PAI-1 dose-dependently facilitated the dissociation of membrane-retained tPA and increased the amounts of tPA-PAI-1 high-molecular-weight complexes in the medium. Accordingly, suppression of PAI-1 synthesis in EA.hy926 cells by siRNA prolonged the dissociation of tPA-GFP, whereas a catalytically inactive mutant of tPA-GFP not forming complexes with PAI-1 remained on the membrane even after PAI-1 treatment. Our results provide new insights into the relationship between exocytosed, membrane-retained tPA and PAI-1, which would modulate cell surface-associated fibrinolytic potential.     

培哚普利对慢性心力衰竭患者血浆t-PA和PAI-1水平的影响

目的评价培哚普利对慢性心力衰竭(CHF)患者血浆组织型纤溶酶原激活物(t-PA)和纤溶酶原激活物抑制物-1(PAI-1)水平的影响。方法采用酶联免疫吸附法测定60例CHF患者(CHF组)及20例健康人(正常对照组)血浆t-PA、PAI-1水平。CHF组患者又随机均分为常规治疗亚组和培哚普利亚组。培哚普利亚组在常规治疗基础上加用培哚普利2~4mg,每日1次。所有CHF患者治疗2周后复测血浆t-PA、PAI-1水平。结果CHF患者血浆t-PA、PAI-1水平比正常对照组明显增高(P<0.01)。治疗后,培哚普利亚组血浆PAI-1水平比常规治疗亚组明显降低(P<0.01),血浆t-PA水平比常规治疗亚组明显升高(P<0.01)。结论培哚普利不仅可降低PAI-1水平,而且可升高t-PA水平,改善内源性纤溶功能。     

Adipose tissue secretion of plasminogen activator inhibitor-1 in non-obese and obese individuals

Summary High plasma plasminogen activator inhibitor-1 (PAI-1) activity is a frequent finding in obesity, and both PAI-1 and obesity are risk factors for cardiovascular disease. To study the mechanisms underlying increased PAI-1 levels in obese individuals, gene expression and secretion of PAI-1 were measured in human abdominal subcutaneous adipose tissue. A total of 32 obese, otherwise healthy subjects and 10 never-obese healthy subjects with a body mass index (BMI) of 42.6 ± 1.2 and 24.3 ± 1.9 kg/m² (mean ± SEM), respectively, were investigated. Plasma PAI-1 activity, adipose tissue PAI-1 secretion and adipocyte PAI-1 mRNA levels were increased sevenfold (p < 0.0001), sixfold (p < 0.0001) and twofold (p < 0.05), respectively, in the obese group. There were clear associations between adipose tissue secretion of PAI-1 and PAI-1 mRNA levels on the one hand and fat cell volume on the other (r = 0.68, p < 0.0001 and r = 0.51, p < 0.01, respectively, in the obese group). PAI-1 mRNA levels were also related to the amount of PAI-1 secreted among obese individuals (r = 0.31, p = 0.09). It is concluded that adipose tissue secretes significant amounts of PAI-1, that PAI-1 secretion from adipose tissue is increased in obesity, and that PAI-1 secretion is related to the lipid content and cell volume of fat cells. Plasma PAI-1 activity is elevated in obesity, at least in part due to increased gene expression in adipocytes, which, in turn, enhances PAI-1 secretion from adipose tissue. [Diabetologia (1998) 41: 65–71] Received: 26 May 1997 and in revised form: 12 August 1997     

Studies of recombinant plasminogen activator inhibitor-1 in rabbits. Pharmacokinetics and evidence for reactivation of latent plasminogen activator inhibitor-1 in vivo

The pharmacokinetics of human recombinant plasminogen activator inhibitor-1 (rPAI-1) was studied in rabbits. Latent rPAI-1 (0-2 units of tissue-type plasminogen activator neutralizing activity per microgram protein); reactivated rPAI-1 (approximately 150 units/micrograms); and chloramine T-oxidized, nonreactivatable rPAI-1 (approximately 0.7 units/microgram) were studied. The pharmacokinetic parameters for the disposition of rPAI-1 antigen after an intravenous bolus injection of 1.0 or 2.5 mg/kg rPAI-1 were very similar for all three forms: the initial volume of distribution was approximately 60 ml/kg, the initial half-life in plasma was 6 minutes, and the plasma clearance was approximately 4 ml/kg/min. The disposition of PAI activity after injection of reactivated rPAI-1 was similar to that of rPAI-1 antigen. Injection of latent rPAI-1 was associated with a nearly threefold increase in the specific activity of circulating PAI-1 from 2 units/micrograms to 5.0 +/- 1.1 units/micrograms (p less than 0.01) within 1 minute, followed by a cumulative 25-fold increase in specific activity over 1 hour (p = 0.01). In contrast, the specific activity of oxidized or reactivated preparations of rPAI-1 did not increase in the first several minutes after injection. These findings support the existence of a fast-acting but low-capacity mechanism for the reactivation of rPAI-1 in vivo.     

Regulation of plasminogen activator inhibitor-1 secretion by urokinase and tissue plasminogen activator in rat epithelioid-type smooth muscle cells

Tissue plasminogen activator (tPA) and urokinase (uPA) are targets of plasminogen activator inhibitor-1 (PAI-1) inhibition. We have previously shown that both proteases can also induce PAI-1 secretion in rat smooth muscle cells (SMCs). We now report that both proteases appear to use very similar cellular mechanisms for signal transduction. They induced PAI-1 secretion using a pathway(s) involving protein kinase C (PKC). They also activated the Raf/Mek/mitogen-activated protein kinase (MAPK) pathway, which lies downstream of PKC activation. Activation of protein kinase A (PKA), however, lowered PAI-1 secretion induced by uPA and tPA, as a result of an inhibition of the PKC pathway and inhibition of Raf, Mek and MAPK phosphorylations. Src and syk family non-receptor tyrosine kinases (TK) were also involved in PAI-1 induction. The mechanisms of interaction of these tyrosine kinases with other pathways appeared to be quite different: src appeared to act within the PKC and PKA pathways, while syk operated independently of these pathways. Furthermore, whereas src inhibition resulted in inhibition of Raf/Mek/Erk phosphorylations, syk inhibition could only inhibit Mek and Erk phosphorylations but not the phosphorylation of Raf. These multiple pathways utilized by uPA and tPA to modulate PAI-1 secretion might be involved in determining the proteolytic or antiproteolytic potential of the SMCs under different pathophysiological conditions.     

Expression of urokinase-type plasminogen activator receptor and plasminogen activator inhibitor-1 in gastric cancer

In gastric cancer, the urokinase-type plasminogen activator (uPA) system plays important roles in invasion and metastasis, processes which entail proteolysis and adhesion. Both the urokinasetype plasminogen activator receptor (uPAR) and the plasminogen activator inhibitor-1 (PAI-1) are thought to be important factors in this system. To clarify the relationship between these two factors and gastric cancer invasiveness, we evaluated the expression of uPAR and PAI-1 in 91 cases of gastric cancer by immunohistochemistry and in situ hybridization. Urokinase-type plasminogen activator receptor-mRNA, PAI-1-mRNA, uPAR and PAI-1 protein were diffusely distributed in the cytoplasm of the cancer cells and concentrated at invasive foci. Urokinase-type plasminogen activator receptor protein expression correlated with lymphatic, venous invasion (P<.01) and lymph node metastasis (P<0.05); uPAR-mRNA expression correlated with lymphatic, venous invasion and lymph node metastasis (P<0.05). Plasminogen activator inhibitor-1 protein expression correlated with lymphatic, venous invasion, lymph node metastasis and depth of invasion (P<0.01); PAI-1-mRNA expression was linked to lymphatic, venous invasion (P<0.01), lymph node metastasis and depth of invasion (P<0.05). This suggests that the proteolytic activity of uPAR and the cellular motility of PAI-1 in gastric cancer cells may determine penetration of lymphatic and blood vessels, whereby lymph node metastasis may be promoted and that the promotion of cellular motility by PAI-1 may influence the depth of cancer invasion.     

Impact of adipose tissue on plasma plasminogen activator inhibitor-1 in dieting obese women.

The increased incidence of cardiovascular diseases in obese subjects could be partially attributed to impaired fibrinolysis due to elevated plasma levels of tissue plasminogen activator inhibitor 1 (PAI-1). The associations between changes in plasma PAI-1, metabolic variables, and adipose tissue during weight loss and regain were studied in 52 healthy, premenopausal, obese women participating in a weight reduction program with a hypocaloric diet. PAI-1, insulin, triglyceride, leptin, and adipsin levels were determined at entry, after the first week, after completion of the program, and after 5 months of follow-up. In the 33 obese women who completed the program, decreases in PAI-1 antigen (-54%), PAI activity (-74%), and leptin (-51%), but not of adipsin, were observed. Changes in PAI-1 were associated with changes in body mass index (BMI), body fat, leptin, and insulin. The decreased level of PAI-1 remained low after follow-up in the 14 women who maintained their reduced weight but increased in the 16 women who regained weight. This increase in PAI-1 was correlated with an increase in body fat and leptin. On multivariate analysis, BMI was the major determinant of PAI-1 level. In conclusion, during weight reduction with a hypocaloric diet, the decrease in PAI-1 is more closely related to changes in adipose tissue than to changes in metabolic variables, suggesting a significant role for adipose tissue in regulating plasma levels of PAI-1.     

Protection by α2-macroglobulin of tissue plasminogen activator against inhibition by plasminogen activator inhibitor-1

Tissue plasminogen activator (tPA) is widely used in the treatment of acute myocardial infarction (MI). However, its thrombolytic efficacy does not correlate with the dose administered. The interactions between tPA, α2-macroglobulin (α2-M), and plasminogen activator inhibitor-1 (PAI-1) were investigated both in vitro and in patients undergoing tPA therapy for MI in an attempt to identify variables that might affect the clinical efficacy of tPA.
Purified α2-M (5.4 mg/ml) protected 16.0% or 22.4% of tPA (12.5 IU/ml) activity from inhibition by PAI-1 at 4 or 8 IU/ml in vitro . Of nine patients treated with 5–20 mega IU of tPA for MI, the plasma activity of tPA remained increased for 15–30 min after the cessation of infusion in eight; the patient who failed to exhibit a persistent increase in tPA activity had a low plasma concentration of α2-M. Total tPA activity, derived from the area under the activity-versus-time curve (AUC), showed a significant inverse correlation with the ratio of the plasma PAI-1 activity to the plasma α2-M concentration. Total tPA activity did not correlate with plasma PAI-1 activity or plasma α2-M concentration alone. Results suggest that α2-M, by binding to tPA, protects the latter against inhibition by PAI-1.     

Increased levels of tissue plasminogen activator with a low plasminogen activator inhibitor-1 in a patient with postoperative bleeding

Recurrent postoperative bleeding in a previously healthy man with a moderate prolongation of PT and PTT which did not correct with vigorous fresh-frozen plasma infusion was identified as a unique abnormality of the fibrinolytic system: a combination of an excess of tissue plasminogen activator (t-PA) at 300% of normal activity accompanied by a concurrent deficiency of plasminogen activator inhibitor-1 activity (PAI-1) of 10-20% of normal activity. This observation underscores the potentially important regulatory role of PAI-1 in the degree to which clinical manifestations of bleeding tendencies may occur in patients in whom an excess amount of t-PA is expressed.     

Testosterone does not adversely affect fibrinogen or tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) levels in 46 men with chronic stable angina

OBJECTIVE: In women, sex hormones cause increased morbidity and mortality in patients with coronary heart disease (CHD) and adversely affect the coagulation profile. We have studied the effect of physiological testosterone replacement therapy in men on coagulation factor expression, to determine if there is an increased risk of thrombosis. METHODS: Double-blind, randomized, placebo-controlled trial of testosterone in 46 men with chronic stable angina. Measurements of free, total and bioavailable testosterone, luteinising hormone (LH) and follicle-stimulating hormone (FSH), estradiol, plasminogen activator inhibitor-1 (PAI-1), fibrinogen, tissue plasminogen activator (tPA) and full blood count were made at 0, 6 and 14 weeks. RESULTS: Bioavailable testosterone levels were: 2.58 +/- 0.58 nmol/l at baseline, compared with 3.35 +/- 0.31 nmol/l at week 14 (P < 0.001) after treatment compared with 2.6 +/- 0.18 nmol/l and 2.44 +/- 0.18 nmol/l in the placebo group (P was not significant). There was no change in fibrinogen (3.03 +/- 0.18 g/l at baseline and 3.02 +/- 0.18 g/l at week 14, P = 0.24), tPA activity (26.77 +/- 4.9 Iu/ml and 25.67 +/- 4.4 Iu/ml, P = 0.88) or PAI-1 activity (0.49 +/- 0.85 Iu/ml and 0.36 +/- 0.06 Iu/ml, P = 0.16) with active treatment and no differences between the groups (at week 14, P value 0.98, 0.59 and 0.8 for fibrinogen, PAI-1 and tPA respectively). Haemoglobin concentration did not change over time, in the testosterone group (1.44 +/- 0.02 g/l and 1.45 +/- 0.02 g/l, P = 0.22). CONCLUSION: Physiological testosterone replacement does not adversely affect blood coagulation status.     

Plasma plasminogen activator inhibitor activity and tissue plasminogen activator levels in patients with unstable angina and those with coronary spastic angina.

Plasminogen activator inhibitor (PAI) activity and tissue plasminogen activator (TPA) antigen were measured in venous samples in 14 patients with unstable angina consisting of eight patients with organic stenosed coronary arteries and six patients with coronary spastic angina (unstable angina group); in 14 patients with stable exertional angina (stable exertional angina group); and in 14 patients with chest pain syndrome (chest pain syndrome group). The plasma levels of PAI activity were higher (p less than 0.01) in the unstable angina group than in the stable exertional angina group and the chest pain syndrome group (12.3 +/- 1.0 versus 5.1 +/- 0.7 and 4.8 +/- 0.6 IU/ml). The plasma levels of TPA antigen were also higher (p less than 0.05) in the unstable angina group than in the stable exertional angina group and the chest pain syndrome group (10.2 +/- 1.3 versus 6.5 +/- 0.8 and 6.0 +/- 0.7 ng/ml). There were no significant differences in PAI activity and TPA antigen levels between the stable exertional angina group and the chest pain syndrome group. Furthermore, both PAI activity and TPA antigen levels in the unstable angina group decreased to the levels in the stable exertional angina group and the chest pain syndrome group after treatment (p less than 0.01). In conclusion, the increased plasma PAI activity in patients with unstable angina and in those with coronary spastic angina indicates that the fibrinolytic system is impaired in these patients.     

Thrombin regulation of mRNA levels of tissue plasminogen activator and plasminogen activator inhibitor-1 in cultured human umbilical vein endothelial cells

Cultured human umbilical vein endothelial cells release tissue plasminogen activator (t-PA) and type 1 plasminogen activator inhibitor (PAI-1) in response to alpha thrombin stimulation. In order to study the mechanisms of thrombin stimulation, we measured changes in levels of mRNA for t-PA and PAI-1 following exposure of endothelial cells to 3 U/mL alpha thrombin. Alpha thrombin causes a significant and time- dependent increase in the mRNA levels of both t-PA and PAI-1. Catalytically inactivated diisofluorophosphate (DIP) treated thrombin and alpha thrombin pretreated with hirudin do not alter t-PA and PAI-1 mRNA levels. We conclude that the increased secretion of t-PA and PAI-1 by human umbilical vein endothelial cells in response to alpha thrombin is mediated at least partially through an increase in mRNA levels. In addition, an active thrombin catalytic site is required for these increases in mRNA to occur.     

介入封堵术对成人先天性心脏病患者血浆t-PA和PAI-1水平的影响

目的评价介入封堵术对成人先天性心脏病(CHD)患者血浆组织型纤溶酶原激活物(t-PA)和纤溶酶原激活物抑制物-1(PAI-1)水平的影响.方法采用酶联免疫吸附法检测70例CHD患者及30例健康人(正常对照组)血浆t-PA、PAI-1水平.所有CHD患者分别于介入封堵术前头天,术后24 h,1、3、6、12个月检测血浆t-PA、PAI-1水平.结果介入封堵术前,CHD患者血浆t-PA、PAI-1水平比正常对照组明显增高(P＜0.01).介入封堵术后1个月,CHD组血浆PAI-1水平开始逐渐下降,术后6个月恢复正常水平;血浆t-PA水平在术后1个月升高,术后3个月始下降,术后6个月恢复正常水平.结论介入封堵术可通过改变血浆t-PA、PAI-1水平而改善成人CHD患者的内源性纤溶功能.     

Local insulin infusion stimulates expression of plasminogen activator inhibitor-1 and tissue-type plasminogen activator in normal subjects.

PURPOSE: Plasma levels of plasminogen activator inhibitor-1 are increased in obesity, hypertension, and diabetes. Their correlation with insulin levels supports the hypothesis that hypofibrinolysis may affect the development of atherosclerotic complications in patients with insulin resistance. To investigate the effect of insulin on fibrinolysis, we evaluated levels of plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) antigens during insulin infusion in the forearm vascular beds of 8 healthy subjects. MATERIALS AND METHODS: Insulin was infused in the brachial artery of each subject to raise local venous concentrations to approximately 100 microU/mL. Blood samples were obtained from the brachial artery and vein at baseline, after 30, 60, 90, and 120 minutes of infusion, and 30 minutes after the end of the infusion. RESULTS: Following intra-arterial infusion of insulin, forearm blood flow (mean +/- SD) increased progressively from 2.7 +/- 0.6 to 4.0 +/- 0.6 mL/dL/min (P <0.01) and did not return to baseline after the end of the infusion. Plasminogen activator inhibitor-1 balance increased (345 +/- 160 versus 8 +/- 152 fmol/dL/min, P <0.02) at 60 minutes, reaching baseline levels after the end of the infusion. After 90 minutes, tPA balance increased (40 +/- 26 versus 7 +/- 29 fmol/dL/min, P <0.01) with a profile similar to forearm blood flow. CONCLUSIONS: Local hyperinsulinemia induces regional vasodilation and expression of PAI-1 and tPA antigens. An alteration of this physiological process could be involved in the development of hypofibrinolysis and atherosclerosis in states of insulin resistance.     

Tissue plasminogen activator,plasminogen activator inhibitor-1 and von willebrand factor in rheumatoid arthritis

Summary Tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor (vWF), all of endothelial origin and active in the haemostasis, were analysed in 74 patients with rheumatoid arthritis. The concentrations were related to extra-articular disease and to the incidence of thromboembolic events (TE) registered in a 2-year follow-up period. Patients with extra-articular disease had a significant increase in PAI-1 activity and reduced tPA release in the venous occlusion test. von Willebrand factor, PAI-1 and also haptoglobin and triglycerides were significantly increased in the group of patients who later suffered from TE. In a multiple regression model, in which cholesterol, triglycerides and lipoprotein (a) showed significant association with TE, vWF had the strongest additive explanatory value. No distinct acute phase pattern of PAI-1 was found in any patient subgroup.     

冠心病稳定型心绞痛患者基质金属蛋白酶-9、可溶性E选择素及组织型纤溶酶原激活物抑制剂-1表达变化

目的:探讨冠心病稳定型心绞痛(SAP)患者基质金属蛋白酶-9(MMP-9)、可溶性E选择素(sE-se-lectin)及组织型纤溶酶原激活物抑制剂-1(tPAI-1)表达变化。方法:采用液相芯片分析系统同步检测206例经冠状动脉造影证实的SAP患者及28例健康对照者血清MMP-9、sE-selectin及tPAI-1表达程度,并比较3者之间及与冠状动脉病变程度的相关性。结果:SAP患者血清MMP-9、tPAI-1及sE-selectin表达程度较对照者升高,其中前2项差异有统计学意义(P<0.01);3者变化呈显著正相关(P<0.01)。结论:SAP患者冠状动脉斑块具有一定程度炎性反应,血液呈现高凝状态,故具有一定不稳定性。炎性因子和纤溶系统标记物可能成为判定SAP患者危险分层的依据。