B.R. Binder

Circadian fluctuations in plasma levels of tissue plasminogen activator antigen and plasminogen activator inhibitor activity

Abstract Daily fluctuations of t-PA antigen and PAM activity were measured in plasma samples of physically active young healthy volunteers (group 1, n = 11; age range 20–38 years) and compared to data obtained from resting patients (group 11, n = 23; age range 44–67 years) suffering from moderate valvular disease without evidence for inflammatory, neoplastic, or thrombosis-related diseases (e.g. deep vein thrombosis, coronary artery disease). t-PA antigen concentration showed a similar diurnal pattern in both study groups with the peak value at 06:00 but was significantly increased in the higher aged group at all collection times. PAM activity had its acrophase in both groups at or around 03:00 but no age-dependent differences could be demonstrated. t-PA antigen as well as PAI-1 activity fluctuations conserved their typical pattern despite differences in physical activity in the study groups.

Soluble M6P/IGF2R Released by TACE Controls Angiogenesis via Blocking Plasminogen Activation

Rationale: The urokinase plasminogen activator (uPA) system is among the most crucial pericellular proteolytic systems associated with the processes of angiogenesis. We previously identified an important regulator of the uPA system in the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R). Objective: Here, we wanted to clarify whether and how did the soluble form of M6P/IGF2R (sM6P/IGF2R) contribute to modulation of the uPA system. Methods and Results: By using specific inhibitors and RNA interference, we show that the tumor necrosis factor &agr; convertase (TACE, ADAM-17) mediates the release of the ectodomain of M6P/IGF2R from human endothelial cells. We demonstrate further that sM6P/IGF2R binds plasminogen (Plg) and thereby prevents Plg from binding to the cell surface and uPA, ultimately inhibiting in this manner Plg activation. Furthermore, peptide 18-36 derived from the Plg-binding site of M6P/IGF2R mimics sM6P/IGF2R in the inhibition of Plg activation and blocks cancer cell invasion in vitro, endothelial cell invasion in vivo, and tumor growth in vivo. Conclusions: The interaction of sM6P/IGF2R with Plg may be an important regulatory mechanism to inhibit migration of cells using the uPA/uPAR system.

Dissecting Mannose 6-Phosphate-Insulin-like Growth Factor 2 Receptor Complexes That Control Activation and Uptake of Plasminogen in Cells

Background: The plasminogen system is central in cell migration and is thus involved in many patho/physiological processes. Results: M6P-IGF2R is a regulatory factor in plasminogen-associated complexes and mediates plasminogen internalization. Conclusion: The uptake of plasminogen by M6P-IGF2R might be an important pathway to control plasminogen activation in cells. Significance: M6P-IGF2R restricts plasmin activity and its loss might lead to rampant fibrinolysis. The plasminogen (Plg) activation cascade on the cell surface plays a central role in cell migration and is involved in a plethora of physiological and pathological processes. Its regulation is coordinated by many receptors, in particular the urokinase-type plasminogen activator receptor (uPAR, CD87), receptors that physically interact and functionally cooperate with uPAR, and Plg binding molecules. Here we studied the impact of one of the Plg binding molecules, the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P-IGF2R, CD222), on cellular Plg activation. By developing both in vitro and in vivo Plg activation assays on size-fractionated lysates of M6P-IGF2R-silenced cells, we identified Plg-associated complexes with M6P-IGF2R as the regulatory factor. Using lipid raft preserving versus dissolving detergents, we found lipid dependence of the Plg regulatory function of these complexes. Furthermore, M6P-IGF2R-silencing in uPAR-positive human cell lines reduced internalization of Plg, resulting in elevated Plg activation. In contrast, the expression of human M6P-IGF2R in mouse embryonic fibroblasts derived from M6P-IGF2R knock-out mice enhanced Plg internalization. Finally, peptide 18–36 derived from the Plg-binding site within M6P-IGF2R enhanced Plg uptake. Thus, by targeting Plg to endocytic pathways, M6P-IGF2R appears to control Plg activation within cells that might be important to restrict plasmin activity to specific sites and substrates.

Effect of notoginsenoside R1 on the synthesis of components of the fibrinolytic system in cultured human pulmonary artery endothelial cells and human skin microvascular endothelial cells

Summary We have previously reported that Notoginsenoside R1 (NR1) increases the synthesis of tissue-type plasminogen activator (t-PA) and decreases plasminogen activator inhibitor-1 (PAI-1) activity in cultured human umbilical vein endothelial cells (HUVECs). It was the aim of this study to investigate whether the effect of NR1 on the synthesis of components of the fibrinolytic system is also operative in endothelial cells from other vascular beds. Therefore cultured human pulmonary artery endothelial cells (HPAECs) and human skin microvascular endothelial cells (HSMECs) were treated with NR1. When cultured HPAECs and HSMECs (passage 2–3) were conditioned with NR1, a dose (0.01–100 g NR1/ml for 24 h) dependent increase in t-PA and u-PA synthesis was observed, which was significant from 0.1 g NR1/ml on. t-PA antigen increased from 5.5 ± 0.2 ng/10 5 cells/24 h to 9.8 ± 0.6 ng/10 5 cells/24 h in HPAECs, and from 1.9 ± 0.1 ng/10 5 cells/24 h to 3.0 ± 0.3 ng/10 5 cells/24 h in HSMECs; u-PA antigen increased from 0.8 ± 0.1 ng/10 5 cells/24 h to 1.8 ± 0.4 ng/10 5 cells/24 h in HPAECs, and from 3.0 ± 0.1 ng/10 5 cells/24 h to 5.7 ± 0.7 ng/10 5 cells/24 h in HSMECs on addition of 100 g NR1/ml. In contrast, no change in PAI-1 antigen synthesis in both HPAECs and HSMECs was seen. As judged from fibrin autography (FA) and reverse fibrin autography (RFA), t-PA activity increased 3.5-fold whereas PAI-1 activity decreased to approximately 50% in conditioned media of HPAECs treated with 100 g/ml NR1 as compared to control cells. On Northern blot analysis of mRNA obtained from NR1-stimulated and control HPAECs and HSMECs, NR1 induced a significant increase in specific mRNA levels of t-PA and u-PA (t-PA mRNA: 180% and 160% of control value in HPAECs and HSMECs, respectively, at 100 g NR1/ml; u-PA mRNA: 140% of control value in HSMECs at 100 g NR1/ml), while PAI-1 mRNA remained unchanged. In conclusion, our data give evidence that NR1 can increase the fibrinolytic and proteolytic potential of endothelial cells from different vascular sources in vitro by increasing the production of t-PA and u-PA.

Comparative cross-over study of the effects of lisinopril and doxazosin on insulin, glucose and lipoprotein metabolism and the endogenous fibrinolytic system

Summary Objective: The present study was performed to compare the effects of the alpha-1-blocker doxazosin (4 mg/d) with the ACE-inhibitor lisinopril (10 mg/d) in a cross-over study on plasma levels of metabolic and fibrinolytic parameters. Patients, methods: In 10 male patients with upper body obesity, proven stable coronary artery disease and hypertension, measurements included baseline determination of lipoproteins and fibrinolytic parameters and determinations of glucose and insulin during intravenous glucose tolerance tests after two 12-week treatment periods. Results: Basal insulin levels were significantly decreased by both doxazosin and lisinopril (from 15.5±3.5 μU/ml to 11.6±2 μU/ml, P≤0.05 and from 16.5±2.8 μU/ml to 11.2±2.4 μU/ml, P≤0.001; respectively). Lisinopril decreased the area under the insulin-concentration time curve by 31.9% (P≤0.007) as compared to 23.6% (n.s.) after doxazosin treatment. HDL-cholesterol was increased by lisinopril from 38±3.5 to 43.5±4.4 mg/dl (P≤0.05), t-PA antigen was increased by doxazosin from 8.3±0.7 to 11.4±1.6 ng/ml (P≤0.05) and PAI-1 was not affected by either therapy. Conclusion: These potentially favorable effects on insulin and lipid metabolism and the endogenous fibrinolytic system might contribute to a higher efficacy of antihypertensive treatment in patients with coronary artery disease and accompanying metabolic cardiovascular risk factors.

Fibrinolysis in patients with the 1691 G→A mutation in factor V gene and history of deep vein thrombosis

Summary It was observed that cosegregation of two inherited risk factors for deep vein thrombosis (DVT), e.g. resistance to activated protein C (APC) with protein C or protein S deficiency increases clinical manifestation of DVT. The aim was to establish if there exists cosegregation of the 1691 G→A mutation in factor V gene, the major cause of resistance to APC and impairment of fibrinolysis, and if possible cosegregation of both defects effects severity of the disease. Eighty-eight consecutive patients (37 females, 51 males, aged 19–60, mean 42 years) were investigated 18±10 months after acute DVT. In 15 patients (16%), the 1691 G→A mutation in factor V gene was observed. These patients did not differ from other DVT patients by clinical characteristics, age, body mass index, fasting glucose and cholesterol, or by resting levels of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1) and euglobulin activity (t-PA antigen: 7.5 vs 7.1 ng/mL; t-PA activity: 0.9 vs 1.2 IU/mL; PAI-1 antigen: 18.1 vs 13.6 ng/mL; PAI activity 13.8 vs 13.9 IU/mL, all values medians; euglobulin clot lysis time: 308±32 vs 298±78 min, mean ±SD). Moreover, frequencies of abnormal levels of t-PA, PAI-1 (t-PA antigen >13 ng/mL, t-PA activity 37 ng/mL, PAI activity >35 IU/mL) and euglobulin activity (euglobulin clot lysis time >410 min) were similar in both subgroups of patients. Recurrent DVT was not more frequent in patients with both defects: the 1691 G→A mutation in factor V gene and impaired fibrinolysis. It was concluded that altered fibrinolysis is not an aggrevating factor for DVT in patients with the 1691 G→A mutation in factor V gene.

Serine protease inhibitors (serpins) in human seminal plasma: Concentrations and inhibition of acrosin

Abstract Acrosin is a trypsin-like serine protease present in its zymogen form in the acrosome of spermatozoa. The activated enzyme is thought to digest a pathway for the sperm through the zona pellucida of the ovum during the process of fertilisation. We have shown that in a purified system boar acrosin was inhibited by human protein C inhibitor with an apparent second order rate constant (k app ) of 3.7×10 4 M −1 s −1 1. Protein C inhibitor is present in high concentrations in seminal plasma and endogenous protein C inhibitor was found in the immediate vicinity of disrupted acrosomal membranes of washed human spermatozoa. This serpin could therefore function as a scavenger of prematurely activated acrosin in the male reproductive tract. Since little is known about the interaction of acrosin with other serpin type inhibitors, we analysed in this study the interaction of boar acrosin with other purified human serpins. Antithrombin III, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, and α 1 -antitrypsin inhibited acrosin activity. The following apparent k app s were calculated: Antithrombin III: 19.5×10 4 M −1 s −1 , plasminogen activator inhibitor-1: 21.5×10 4 M −1 s −1 , plasminogen activator inhibitor-2: 3.3×10 4 M −1 s 1 , α 1 -antitrypsin: 0.09×10 4 M −1 s 1 . α 2 -antiplasmin and heparin cofactor II did not inhibit acrosin. SDS-stable acrosin/serpin complexes were only seen with antithrombin III; all other acrosin inhibitors were cleaved by the enzyme. As determined by enzyme-linked immunosorbent assays, the concentrations of protein C inhibitor, plasminogen activator inhibitor-1, and plasminogen activator inhibitor-2 in individual seminal plasma samples from healthy donors were 5.33±0.47 μM, 88±24 pM and 163±17 pM (means±SD), respectively. The concentrations of antithrombin III in seminal plasma were ⩽50 nM (semiquantitative immunoblotting). Therefore considering both, the k app calculated for the inhibition of boar acrosin in a purified system and the concentration of each serpin in seminal plasma, protein C inhibitor seems to be the best candidate to function as a physiological acrosin inhibitor in the male reproductive tract.

Effect of caffeine intake on platelet antigen levels of plasminogen activator inhibitor-1 (PAI-1).

Plasminogen activator inhibitor 1 (PAI-1) antigen levels which could be released by addition of thrombin from isolated platelets as well as the total amount of platelet PAI-1 antigen were determined in healthy young volunteers after one week of caffeine ingestion (3×100mg caffeine in addition to caffeine-containing beverages ad libitum) and one week of total avoidance of caffeine intake. The total PAI-1 antigen contained in platelets ranged between 0.36 and 1.6fg/platelet (0.80 ± 0.33fg/platelet) before initiation of the study. After one week of caffeine intake the respective mean values were 0.8 ± 0.26fg/platelet and after one week of caffeine avoidance 1.14 ± 0.48fg/platelet which was significant (P<0.005; paired t-test). Furthermore, the mean amount of PAI-1 released from platelets was also significantly different: 0.52 ± 0.19fg/platelet with and 0.76 ± 0.28fg/platelet without caffeine intake (P<0.005). These data suggest that caffeine intake in fact can alter PAI-1 levels.

Interactions of uPAR: impact on receptor regulation and signal transduction

Summary This review focuses on signal transduction via the urokinase receptor, a very controversial issue. It describes data on membrane and extracellular matrix proteins interacting with this receptor, phosphorylation events induced by activation of the receptor, and influence of receptor activation on gene regulation and biologic functions of various cells. Due to the use of many different cells and culture conditions by different authors, and their focus on specific events within signaling cascades, a conclusive pathway of signal transduction via the urokinase receptor is still lacking. Actually this receptor seems to modify different signaling pathways used by integrins or cytokines rather than activate a signaling pathway of its own.

Effect of endothelin on the regulation of the fibrinolytic system of cultured human vascular smooth muscle cells

Summary The effect of endothelin-1 (ET-1) and ET-3 on the fibrinolytic system of cultured human vascular smooth muscle cells (SMCs) was investigated. When confluent cultures of human aortic SMCs (HASMCs) and human basilaris artery SMCs (HBASMCs) were conditioned with ET-1 or ET-3 (0.1 to 100 nM) plasminogen activator inhibitor-1 (PAI-1) synthesis increased up to 135% (HASMCs) or 179% (HBASMCs) of control whereas tissue-type plasmingen activator (t-PA) synthesis decreased down to 70% of control in both types of SMCs. PAI-1 mRNA levels were upregulated to 200% of control levels and t-PA mRNA decreased by 50% when the cells were treated with 100 nM ET-1 or ET-3. We provide evidence for a new function of endothelins towards vascular smooth muscle cells. By decreasing the fibrinolytic potential of SMCs, endothelin might contribute to the development of thrombosis seen in cardiovascular diseases associated with vascular injury and/or smooth muscle cell activation such as atherosclerosis and myocardial infarction.