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D C Rijken

Publications and source records attributed to D C Rijken.

At least 55 records · Page 3Linked to original sources
PubMed1995

Superficial accumulation of plasminogen during plasma clot lysis.

BACKGROUND: Binding of plasminogen to partially degraded fibrin is an important step in fibrinolysis, influencing its rate and fibrin specificity. Little is known about the spatial distribution of plasminogen and of plasminogen-binding sites inside thrombi during lysis. In the present study, we investigated this problem, which is important for a better understanding of the local regulation of fibrinolysis and the rate-limiting factors of therapeutic thrombolysis. METHODS AND RESULTS: An experimental system was used that allowed continuous visualization and quantification by fluorescence microscopy of the spatial distribution of fluorescein-labeled plasminogen inside and outside model thrombi. Strong superficial accumulation of plasminogen was observed during lysis of a plasma clot induced by tissue-type or urokinase-type plasminogen activators in the surrounding plasma. A distinctly visible plasminogen-accumulating shell moved continuously with the reducing surface of the clot. The accumulation decreased in conditions of exhaustive activation of plasminogen in the outer plasma. It was found in a purified system that a thin superficial layer (approximately 50 microns wide) of a plasmin-treated fibrin clot exposes about 2.5 plasminogen-binding sites per fibrin monomer with a Kd of 2.2 mumol/L. At a physiological concentration of plasminogen (1.5 mumol/L) in the outer medium, plasminogen was concentrated about 10-fold in this layer. The binding was dose-dependently inhibited by epsilon-aminocaproic acid. CONCLUSIONS: We conclude that the generation of potent surface-associated plasminogen-binding sites during thrombolysis results in a strikingly high plasminogen concentration at the dynamically changing surface of a lysing clot. The necessity of a continuous plasminogen supply from the plasma supports the use of fibrin-specific and plasminogen-sparing agents for thrombolytic therapy.

Blood Coagulation↗
PubMed1995

Plasminogen activators and plasminogen activator inhibitors: biochemical aspects.

Although this chapter does not represent a historical review, it will be clear how the biochemistry of t-PA, u-PA, PAI-1 and PAI-2 has evolved and where we stand in 1994. While the functional activities of the proteins were recognized at least three to four decades ago, highly purified preparations became available around 1980. In the mid-eighties the cDNAs of the proteins were cloned, representing a major breakthrough in the biochemistry of the four proteins. Amino acid sequences were derived from the nucleotide sequences, homologies with other proteins were recognized and larger amounts of (recombinant) proteins became available for research. In addition, mutant proteins were prepared by recombinant DNA technology, enabling investigation of structure-function relationships. This report is mainly based on the latter studies. Detailed information about three-dimensional structures of the proteins and the mode of interaction with other macromolecules is still lacking. To obtain this information will be the goal for biochemists in the coming years.

Amino Acid Sequence↗
PubMed1995

Native and non-glycosylated recombinant single-chain urokinase-type plasminogen activator are recognized by different receptor systems on rat parenchymal liver cells.

The recognition systems mediating the clearance of glycosylated high molecular weight single-chain urokinase-type plasminogen activator (HMW-scu-PA, produced in human embryonic kidney cells) and recombinant non-glycosylated scu-PA (rscu-PA, produced in E. coli) were analyzed by studying their binding characteristics to freshly isolated rat parenchymal liver cells. The binding of 125I-HMW-scu-PA at 4 degrees C was calcium-dependent and of high affinity (Kd = 37.6 nM) and could be inhibited by low molecular weight two-chain u-PA (LMW-tcu-PA) and lactose, but not by the low density lipoprotein receptor-related protein (LRP)-associated 39-kDa protein (RAP), rscu-PA or a mutant form lacking amino acids 11-135 (Delta 125-rscu-PA). Removal of the carbohydrate side chain of HMW-scu-PA by treatment with N-glycosidase F, completely reduced the specific binding to the parenchymal cells and strongly reduced its competition with 125I-HMW-scu-PA in cell binding. Recombinant scu-PA also bound with high affinity (Kd = 38.7 nM) to the parenchymal liver cells. The binding of 125I-rscu-PA could be competed for by unlabeled rscu-PA while Delta 125-rscu-PA, LMW-tcu-PA or lactose were ineffective. In contrast to HMW-scu-PA, binding of 125I-rscu-PA could be effectively inhibited by RAP (Ki = 1.1 nM), while also its association and degradation, as determined at 37 degrees C, were inhibited by RAP. Pretreatment of the parenchymal cells with proteinase K supplied further evidence for the involvement of two different receptor systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Characterization of the interaction of a complex of tissue-type plasminogen activator and plasminogen activator inhibitor type 1 with rat liver cells.

The present study was undertaken in order to determine the recognition site for tissue-type plasminogen activator-plasminogen activator inhibitor type 1 [t-PA-PAI-1] complexes in rat liver in vivo and in vitro. After intravenous injection into rats t-PA-PAI-1 complexes were rapidly removed from the plasma and the liver took up 80% of the injected dose. Within the liver parenchymal and endothelial liver cells contributed mainly to the uptake of t-PA-PAI-1, and were responsible for 62% and 24% of the liver uptake, respectively. The interaction of t-PA-PAI-1 with isolated rat parenchymal liver cells was of high affinity (Kd 17 nM). A well-known antagonist of the alpha 2-macroglobulin receptor (alpha 2MR/low-density lipoprotein receptor-related protein (LRP), GST-39kDa protein (GST-39kDaP) efficiently inhibited the binding (IC50 0.7 nM) of t-PA-PAI-1 to rat parenchymal liver cells. The interaction of t-PA-PAI-1 with LRP on rat parenchymal liver cells was not Ca2(+)-dependent and is most probably mediated by a specific determinant on PAI-1, since an anti-PAI-1 monoclonal antibody inhibited the binding of t-PA-PAI-1, where as free t-PA did not. The binding of t-PA-PAI-1 to rat hepatocytes could not be inhibited by a complex of plasmin and alpha 2-antiplasmin nor by various other ligands of LRP like beta-VLDL and lactoferrin. Binding of t-PA-PAI-1 to rat parenchymal liver cells was followed by internalization and subsequent degradation in the lysosomal compartment. It is concluded that parenchymal and endothelial liver cells mediate the removal of t-PA-PAI-1 complexes from the circulation. LRP on rat parenchymal liver cells is responsible for the uptake and degradation of t-PA-PAI-1 and may therefore be important for the regulation of the t-PA levels in the circulation.

Animals↗
PubMed1994

Activation of thrombin-inactivated single-chain urokinase-type plasminogen activator by dipeptidyl peptidase I (cathepsin C).

Single-chain urokinase-type plasminogen activator (scu-PA) is inactivated by thrombin, which cleaves the peptide bond between Arg156 and Phe157. In a search for potential activators of thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T), we found that the lysosomal aminopeptidase dipeptidyl-peptidase I or cathepsin C efficiently activates tcu-PA/T. Cathepsin C was as active towards tcu-PA/T as the bacterial proteinase thermolysin and about 300-times more active than plasmin. The activation by cathepsin C proceeded in a concentration-dependent and time-dependent manner with a pH optimum between 5 and 7. Furthermore, the effect of cathepsin C was inhibited by cystatin and stimulated by cysteine, typical for the action of a thiol proteinase. As no degradation of the tcu-PA/T molecule by cathepsin C was visible on SDS/PAGE, we suggest that activation of tcu-PA/T occurs by cleavage between Lys158-Ile159 and removal of the two N-terminal amino acid residues (Phe157-Lys158) of the B chain of tcu-PA/T. We conclude that both thrombin and dipeptidyl-peptidases like cathepsin C might play a regulatory role in the plasminogen-plasmin system by inactivating scu-PA and activating tcu-PA/T, respectively.

Cathepsin C↗
PubMed1994

In vitro stability of a tissue-type plasminogen activator mutant, BM 06.022, in human plasma.

BM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated. Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 microgram/ml to 38 min at 10 micrograms/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, alpha 2-antiplasmin and alpha 1-antitrypsin. During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of alpha 2-antiplasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i.e. within 45 min) converted into its two-chain form at concentrations of 5 micrograms/ml BM 06.022 and higher. In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, alpha 2-antiplasmin and alpha 1-antitrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug Stability↗
PubMed1993

Interaction of plasminogen activators and plasminogen with heparin: effect of ionic strength.

In order to define the possible effects of heparin on the fibrinolytic system under physiological conditions, we studied the interactions of this drug with plasminogen and its activators at various ionic strengths. As reported in recent literature, heparin stimulated the activation of Lys-plasminogen by high molecular weight (HMW) and low molecular weight (LMW) two-chain urokinase-type plasminogen activator (u-PA) and two-chain tissue-type plasminogen activator (t-PA) 10- to 17-fold. Our results showed, however, that this stimulation only occurred at low ionic strength and was negligible at a physiological salt concentration. Direct binding studies were performed using heparin-agarose column chromatography. The interaction between heparin and Lys-plasminogen appeared to be salt sensitive, which explains at least in part why heparin did not stimulate plasminogen activation at 0.15 M NaCl. The binding of u-PA and t-PA to heparin-agarose was less salt sensitive. Results were consistent with heparin binding sites on both LMW u-PA and the amino-terminal part of HMW u-PA. Single-chain t-PA bound more avidly than two-chain t-PA. The interactions between heparin and plasminogen activators can occur under physiological conditions and may modulate the fibrinolytic system.

Amino Acid Sequence↗
PubMed1993

Comparison of the in vitro fibrinolytic activities of low and high molecular weight single-chain urokinase-type plasminogen activator.

The fibrinolytic activity of low molecular weight (LMW) single-chain urokinase-type plasminogen activator (scu-PA) lacking the epidermal growth factor domain and the kringle domain was compared with the activity of high molecular weight (HMW) scu-PA. LMW scu-PA was 1-5 times less active than HMW scu-PA in a fibrin plate method, in a purified fibrin clot lysis assay and in a plasma clot lysis assay. Time course experiments in a chromogenic plasminogen activator assay suggested that LMW scu-PA was less sensitive to activation by plasmin than HMW scu-PA. This was confirmed in a scu-PA activation test, which showed that at a concentration of 40 IU/ml LMW scu-PA required a three-fold higher plasmin concentration for 50% activation in 20 min than did HMW scu-PA. Kinetic experiments in the presence of 0.1 M NaCl showed non-standard Michaelis-Menten kinetics for the activation by plasmin of both HMW and LMW scu-PA. In contrast, standard kinetics was observed at 0.15 M NaCl, showing a 2.6-fold lower catalytic efficiency for LMW scu-PA than for HMW scu-PA. It is concluded that the plasmin activation of LMW scu-PA is about three times slower than the activation of HMW scu-PA. This explains, at least partially, the lower fibrinolytic activity of LMW scu-PA in comparison with HMW scu-PA.

Amino Acid Sequence↗
PubMed1993

Role of the glycosaminoglycan component of thrombomodulin in its acceleration of the inactivation of single-chain urokinase-type plasminogen activator by thrombin.

Thrombomodulin (TM), a membrane proteoglycan on endothelial cells, binds thrombin in a 1:1 complex, accelerates the protein C activation by thrombin, promotes the thrombin inactivation by antithrombin III and inhibits the procoagulant properties of thrombin. The inactivation of single-chain urokinase-type plasminogen activator (scu-PA) by thrombin is accelerated about 70-fold by TM [De Munk, Groeneveld and Rijken (1991) J. Clin. Invest. 88, 1680-1684]. The present study investigates the role of the O-linked glycosaminoglycan moiety of TM in the latter reaction. In the presence of an excess of a fully-glycosylated soluble recombinant human TM mutant (high-Mr rec-TM), 0.11 nM thrombin inactivated 50% of 4.4 nM scu-PA in 45 min at 37 degrees C. In the presence of a soluble recombinant TM mutant lacking the glycosaminoglycans (low-Mr rec-TM), 1.9 nM thrombin was needed to inactivate 50% scu-PA, as compared with 4.7 nM thrombin in the absence of TM. Using the scu-PA inactivation assay the dissociation constant for the thrombin-TM interaction was found to be 0.4 nM for high-Mr rec-TM and 14 nM for low-Mr rec-TM. Treatment of high-Mr rec-TM with chondroitinase ABC to digest the glycosaminoglycans decreased the accelerating effect to the level of low-Mr rec-TM. A similar decrease was observed after treatment of solubilized rabbit TM with chondroitinase ABC. As expected, chondroitinase ABC had no influence on the accelerating effect of low-Mr rec-TM. The free glycosaminoglycans obtained by alkaline treatment of TM or chondroitin sulphate A also accelerated the inactivation of scu-PA by thrombin, but about 1000-fold higher concentrations than with TM were needed to obtain the same acceleration. It is concluded that the major glycosaminoglycan of TM plays a pivotal role in the inactivation of scu-PA by the TM-thrombin complex, both in the formation and in the activity of the complex.

Chondroitin Lyases↗
PubMed1992

Characterization of the interaction both in vitro and in vivo of tissue-type plasminogen activator (t-PA) with rat liver cells. Effects of monoclonal antibodies to t-PA.

The interaction of 125I-labelled tissue-type plasminogen activator (125I-t-PA) with freshly isolated rat parenchymal and endothelial liver cells was studied. Binding experiments at 4 degrees C with parenchymal cells and endothelial liver cells indicated the presence of 68,000 and 44,000 high-affinity t-PA-binding sites, with an apparent Kd of 3.5 and 4 nM respectively. Association of 125I-t-PA with parenchymal cells was Ca(2+)-dependent and was not influenced by asialofetuin, a known ligand for the galactose receptor. Association of 125I-t-PA with liver endothelial cells was Ca(2+)-dependent and mannose-specific, since ovalbumin (a mannose-terminated glycoprotein) inhibited the cell association of t-PA. Association of 125I-t-PA with liver endothelial cells was inhibited by anti-(human mannose receptor) antiserum. Anti-(galactose receptor) IgG had no effect on 125I-t-PA association with either cell type. Degradation of 125I-t-PA at 37 degrees C by both cell types was inhibited by chloroquine or NH4Cl, indicating that t-PA is degraded lysosomally. in vitro experiments with three monoclonal antibodies (MAbs) demonstrated that anti-t-PA MAb 1-3-1 specifically decreased association of 125I-t-PA with the endothelial cells, and anti-t-PA Mab 7-8-4 inhibited association with the parenchymal cells. Results of competition experiments in rats in vivo with these antibodies were in agreement with findings in vitro. Both antibodies decreased the liver uptake of 125I-t-PA, while a combination of the two antibodies was even more effective in reducing the liver association of 125I-t-PA and increasing its plasma half-life. We conclude from these data that clearance of t-PA by the liver is regulated by at least two pathways, one on parenchymal cells (not galactose/mannose-mediated) and another on liver endothelial cells (mediated by a mannose receptor). Results with the MAbs imply that two distinct sites on the t-PA molecule are involved in binding to parenchymal cells and liver endothelial cells.

Animals↗
PubMed1992

In vivo and in vitro interaction of high and low molecular weight single-chain urokinase-type plasminogen activator with rat liver cells.

The plasma clearance and the interaction of high (HMW) and low (LMW) molecular weight single-chain urokinase-type plasminogen activator (scu-PA) with rat liver cells was determined. 125I-Labeled HMW- and LMW-scu-PA were rapidly cleared from plasma with a half-life of 0.45 min and a maximal liver uptake of 55% of the injected dose. Liver uptake of scu-PA was mediated by parenchymal cells. Excess of unlabeled HMW-scu-PA reduced the liver uptake of 125I-HMW-scu-PA strongly. In vivo liver degradation of scu-PA was reduced by inhibitors of the lysosomal pathway. A high affinity binding site (Kd 45 nM, Bmax 1.7 pmol/mg cell protein) for both HMW- and LMW-scu-PA was determined on isolated parenchymal liver cells. Cross-competition binding studies showed that LMW- and HMW-scu-PA bind to the same site. Tissue-type plasminogen activator, mannose- or galactose-terminated glycoproteins did not affect the scu-PA binding to parenchymal liver cells. It is concluded that LMW- and HMW-scu-PA are taken up in the liver by a common, newly identified recognition site on parenchymal liver cells and are subsequently degraded in the lysosomes. It is suggested that this site is important for the regulation of the turnover of scu-PA.

Animals↗
PubMed1992

The amount of plasminogen, tissue-type plasminogen activator and plasminogen activator inhibitor type 1 in human thrombi and the relation to ex-vivo lysibility.

Thrombolytic therapy successfully reopens obstructed blood vessels in the majority of cases. However, it is not known why a substantial amount of thrombi are resistant to lysis by a fibrinolytic agent. In vitro studies have demonstrated that tissue-type plasminogen activator (t-PA) and plasminogen incorporated in the clot (during formation) increase lysibility. To test whether lysibility of in vivo formed human thrombi is related to their composition, we studied 25 venous thrombi obtained at autopsy and 21 arterial thrombi obtained during embolectomy. Plasminogen activator inhibitor-1 (PAI-1) antigen was measured in a phosphate-buffered saline (PBS) extract of each thrombus; t-PA antigen and plasminogen antigen were determined in a 6 M urea extract of the thrombus, representing bound proteins. Lysibility was measured as weight reduction during 8 h of incubation in PBS containing streptokinase (SK) 100 U/ml, corrected for spontaneous lysis, reflected by weight loss in PBS without SK. In addition, lysibility in SK was compared with lysibility in urokinase (UK) 100 U/ml and in t-PA 200 U/ml. Spontaneous lysis amounted to 29 +/- 5% (mean +/- SEM) and 33 +/- 5% in venous and arterial thrombi, respectively, and inversely correlated with the PAI-1 content of thrombi (r = -0.43, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Humans↗
PubMed1992

Isolation and characterization of the mannose receptor from human liver potentially involved in the plasma clearance of tissue-type plasminogen activator.

Various studies have shown that mannose receptors rapidly eliminate glycoproteins and microorganisms bearing high mannose-type carbohydrate chains from the blood circulation. The purpose of this study was to characterize the mannose receptor in the liver, which in vivo is involved in the rapid clearance of tissue-type plasminogen activator from the circulation. Human liver membranes were solubilized in Triton X-100, and the solution was applied to a tissue-type plasminogen activator Sepharose column. Bound proteins were eluted with ethylenediaminetetraacetate (10 mmol/L). A second, similar purification step rendered a single liver protein of 175,000 daltons. A combination of ligand blotting and a chromogenic assay for tissue-type plasminogen activator demonstrated that the identified liver protein is a mannose receptor because it bound tissue-type plasminogen activator, this tissue-type plasminogen activator binding being fully inhibited by 0.2 mol/L D-mannose. Western-blot analysis revealed that the isolated liver protein is immunologically identical to the human mannose receptor from placenta. Treatment of the liver protein and the placenta mannose receptor with trypsin yielded the same pattern of proteolytic degradation products as identified on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the physiologically relevant mannose receptor for tissue-type plasminogen activator clearance isolated from human liver is immunologically and structurally similar to or identical with the human mannose receptor isolated from placenta.

Blotting, Western↗
PubMed1991

Binding of tissue-type plasminogen activator by the mannose receptor.

Previous studies have shown that tissue-type plasminogen activator (t-PA) in blood is cleared by the liver partially through a mannose-specific uptake system. The present study was undertaken to investigate, in a purified system, whether t-PA is recognized by the mannose receptor which is expressed on macrophages and liver sinusoidal cells. The mannose receptor was isolated and purified from bovine alveolar macrophages and migrated as a single protein band at Mr 175,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Ligand blotting revealed that this protein specifically bound t-PA. The t-PA-receptor interaction was further characterized in a binding assay, which showed saturable binding with an apparent dissociation constant of 1 nM. t-PA binding required calcium ions and was negligible in the presence of EDTA or at acid pH. Mannose-albumin was an effective inhibitor, whereas galactose-albumin did not have a significant effect. From a series of monosaccharides tested, D-mannose and L-fucose were the most potent inhibitors, N-acetyl-D-glucosamine was a moderate inhibitor, whereas D-galactose and N-acetyl-D-galactosamine were ineffective. t-PA, deglycosylated by endoglycosidase H, did not interact with the receptor. It is concluded that the mannose receptor specifically binds t-PA, probably through its high mannose-type oligosaccharide.

Animals↗