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D C Rijken

Publications and source records attributed to D C Rijken.

At least 73 records · Page 4Linked to original sources
PubMed1991

Substrate specificity of tissue-type and urokinase-type plasminogen activators.

Recent studies suggest that plasminogen activators not only hydrolyse a specific arginine-valine bond in plasminogen, but may also cleave other proteins such as fibronectin. We studied the substrate specificity, particularly the preference for arginyl over lysyl peptide bonds, of tissue-type plasminogen activator (t-PA) as well as of two-chain urokinase-type plasminogen activator (u-PA). The arginine/lysine preference was determined with three pairs of tripeptidyl-p-nitroanilide substrates having either arginine or lysine in the P1 position and varied from 5.2 to 14.1 for u-PA and from 55.6 to 99.8 for t-PA. It was concluded that both t-PA and u-PA preferred arginyl to lysyl peptide bonds. However, u-PA had a significantly lower arginine/lysine preference than t-PA, indicating that u-PA represents a less specific proteinase. This may point to functions of u-PA other than plasminogen activation, which involve cleavage of lysyl bonds.

Amino Acid Sequence↗
PubMed1991

Acceleration of the thrombin inactivation of single chain urokinase-type plasminogen activator (pro-urokinase) by thrombomodulin.

The in vitro effects of thrombomodulin on the inactivation of single chain urokinase-type plasminogen activator (scu-PA) by thrombin were investigated by incubating scu-PA with varying concentrations of human thrombin, in both the absence and presence of soluble rabbit thrombomodulin. 50% inactivation of scu-PA occurred in 45 min at 160 ng/ml thrombin in the absence of thrombomodulin and at 4.6 ng/ml thrombin in the presence of thrombomodulin. No difference was found in either the absence or the presence of thrombomodulin between the inactivation rates of high molecular weight scu-PA, and a low molecular weight scu-PA which lacked the growth factor and kringle domains. Enzyme kinetic experiments with varying concentrations of scu-PA showed that thrombomodulin decreased the Km of thrombin for scu-PA from 7.8 to 0.43 microM and increased the kcat from 0.30 to 1.2 s-1, corresponding to a 70-fold increase in the second-order rate constant kcat/Km. SDS-polyacrylamide gel electrophoresis showed that scu-PA was cleaved into two chains upon inactivation by thrombin, and confirmed the acceleration effect of thrombomodulin on inactivation of scu-PA. Thrombomodulin thus not only has anticoagulant properties but is also antifibrinolytic. The acceleration may imply a new mechanism for the regulation of local plasminogen activator activity on the cell surface.

Calcium↗
PubMed1990

Plasminogen activation at low temperatures in plasma samples containing therapeutic concentrations of tissue-type plasminogen activator or other thrombolytic agents.

It is known that in vitro plasminogen activation in blood samples taken during thrombolytic therapy with tissue-type plasminogen activator (t-PA) may lead to artefactually low fibrinogen and alpha 2-antiplasmin values. To mimic this phenomenon, pooled normal plasma was supplemented with 2.5 micrograms/ml t-PA and incubated at various temperatures. The rates of fibrinogen degradation and alpha 2-antiplasmin consumption were most pronounced at 37 degrees C, were less pronounced at 25 degrees C, but surprisingly, did not further decrease at 10 degrees C, 0 degrees C or -8 degrees C. In contrast, when plasma was supplemented with 160 IU/ml urokinase or 30 IU/ml streptokinase, the rates of fibrinogen degradation and alpha 2-antiplasmin consumption gradually decreased with incubation temperature and were negligible at 10 degrees C and lower temperatures. The rate of plasminogen activation also decreased gradually with temperature in mixtures of purified fibrinogen, plasminogen, alpha 2-antiplasmin and t-PA. These results imply that, in a plasma milieu, additional factors with a stimulatory activity are involved in t-PA-induced plasminogen activation at around 0 degrees C. The abnormally high reaction rate at low temperatures explains in vitro plasminogen activation observed during the processing of t-PA-containing blood samples. In contrast to the activation of plasminogen by t-PA, the slow inhibition of t-PA (2.5 micrograms/ml) by proteinase inhibitors in plasma could be minimized to a negligible level by keeping the plasma samples at 0 degrees C. This makes it possible to reliably monitor t-PA activity during thrombolytic therapy.

Chromogenic Compounds↗
PubMed1990

Receptor-mediated endocytosis of tissue-type plasminogen activator (t-PA) by liver cells.

Tissue-type plasminogen activator (t-PA) has a short half-life in the circulation because the enzyme is rapidly cleared by the liver. This short review summarizes recent literature concerning mechanisms of uptake and degradation of t-PA in the liver. In vivo studies in rats show that degradation takes place via a lysosomal pathway. Saturation of the uptake system at high t-PA concentrations suggests a receptor-mediated mechanism. Competition experiments with various glycoproteins indicate that the asialoglycoprotein receptor is not involved, but they point to a role for the mannose receptor, which recognizes t-PA via its high mannose-type oligosaccharide on the first kringle domain. Both in vivo and in vitro studies with isolated liver cells demonstrate that parenchymal cells, as well as liver endothelial cells, are involved in the clearance of t-PA. Parenchymal cells, as the hepatoma cell line Hep G2, endocytose t-PA via a still unknown, possibly t-PA specific receptor, while liver endothelial cells catabolize t-PA via the mannose receptor.

Amino Acid Sequence↗
PubMed1989

Binding and degradation of tissue-type plasminogen activator by the human hepatoma cell line Hep G2.

In this study, binding and degradation of tissue-type plasminogen activator (t-PA) by the human hepatoma cell line Hep G2 was investigated. Binding at 4 degrees C was time-dependent and reached a maximum after ca. 2 hours. Scatchard analysis of saturation experiments showed about 170,000 high affinity binding sites for t-PA per cell with an apparent Kd of 90 nM. These binding sites were calcium-dependent. Part of the binding to the hepatoma cells was non-saturable, owing to a large amount of low affinity binding sites which are at least partially located on the extracellular matrix of the cells. Competition with mannose- and galactose-terminated glycoproteins had no effect on total binding of 125I-t-PA. Degradation products of 125I-t-PA were found in the supernatant after a short lag phase and then increased linearly for at least 5 hours at 37 degrees C. Degradation could be inhibited by chloroquine, NH4Cl and NaN3. We conclude that the human hepatoma cell line Hep G2 has a specific binding mechanism for t-PA which is not mediated by known carbohydrate receptor systems. Binding is followed by cellular uptake and degradation in the lysosomes.

Binding, Competitive↗
PubMed1989

Pharmacokinetics and systemic effects of tissue-type plasminogen activator in normal subjects.

Pharmacokinetics and systemic effects of recombinant tissue-type plasminogen activator (rt-PA) were studied in 18 healthy male volunteers after 30-minute intravenous infusions of placebo, 0.25 mg/kg rt-PA, and 0.5 mg/kg rt-PA. Highly comparable pharmacokinetic parameters were obtained after analysis of rt-PA as both an antigen and an activity. Mean clearance (antigen) was 620 +/- 70 (SD) ml/min, volume of distribution at steady state was 8.1 +/- 0.8 L, initial volume of distribution was 4.4 +/- 0.6 L, and dominant half-life was 4.4 +/- 0.3 minutes. The pharmacokinetics of rt-PA were linear, showed low interindividual variation, and are compatible with rapid hepatic elimination of the protein. Systemic plasminogen activation was minimal as assessed by hemostatic assays of plasma samples treated with anti-rt-PA Immunoglobulin G (IgG) to inhibit in vitro fibrinogenolysis. Circulating fibrinogen levels, clotting times, and coagulation factors were unchanged; plasminogen and alpha 2-antiplasmin decreased maximally to 85% and 65% of baseline values, respectively. The data are consistent with the fibrin specificity of t-PA, which is derived from its role in physiologic fibrinolysis.

Adult↗
PubMed1988

Characterization of the interaction in vivo of tissue-type plasminogen activator with liver cells.

The interaction in vivo of 125I-labeled tissue-type plasminogen activator (t-PA) with the rat liver and the various liver cell types was characterized. Intravenously injected 125I-t-PA was rapidly cleared from the plasma (t1/2 = 1 min), and 80% of the injected dose associated with the liver. After uptake, t-PA was rapidly degraded in the lysosomes. The interaction of 125I-t-PA with the liver could be inhibited by preinjection of the rats with ovalbumin or unlabeled t-PA. The intrahepatic recognition site(s) for t-PA were determined by subfractionation of the liver in parenchymal, endothelial, and Kupffer cells. It can be calculated that parenchymal cells are responsible for 54.5% of the interaction of t-PA with the liver, endothelial cells for 39.5%, and Kupffer cells for only 6%. The association of t-PA with parenchymal cells was not mediated by a carbohydrate-specific receptor and could only be inhibited by an excess of unlabeled t-PA, indicating involvement of a specific t-PA recognition site. The association of t-PA with endothelial cells could be inhibited 80% by the mannose-terminated glycoprotein ovalbumin, suggesting that the mannose receptor plays a major role in the recognition of t-PA by endothelial liver cells. An excess of unlabeled t-PA inhibited the association of 125I-t-PA to endothelial liver cells 95%, indicating that an additional specific t-PA recognition site may be responsible for 15% of the high affinity interaction of t-PA with this liver cell type. It is concluded that the uptake of t-PA by the liver is mainly mediated by two recognition systems: a specific t-PA site on parenchymal cells and the mannose receptor on endothelial liver cells. It is suggested that for the development of strategies to prolong the half-life of t-PA in the blood, the presence of both types of recognition systems has to be taken into account.

Animals↗
PubMed1988

Alpha 2-antiplasmin Enschede is not an inhibitor, but a substrate, of plasmin.

alpha 2-Antiplasmin Enschede is a variant of alpha 2-antiplasmin which has lost its ability to inhibit plasmin irreversibly and which is associated with a haemorrhagic disorder [Kluft et al. (1987) J. Clin. Invest. 80, 1391-1400]. The abnormal protein was purified from the plasma of a homozygous patient and subjected to one-dimensional peptide mapping using papain for digestion. A slightly abnormally migrating polypeptide (Mr 17,000) was found which represented the C-terminal part of the molecule (the N-terminus of the polypeptide corresponded to Gly-338 in normal alpha 2-antiplasmin) and which contained the reactive centre. The interaction of plasmin with alpha 2-antiplasmin Enschede was studied by adding plasmin to plasma of the homozygous patient. SDS/polyacrylamide-gel electrophoresis and immunoblotting showed that no complex persisted, but that the abnormal alpha 2-antiplasmin was cleaved into two fragments of Mr 56,000 and 14,000 respectively. The latter fragment co-migrated with the post-complex peptide, which is cleaved from normal alpha 2-antiplasmin during complex-formation with plasmin. In a purified system, catalytic amounts of plasmin rapidly cleaved alpha 2-antiplasmin Enschede into the aforementioned fragments. In kinetic studies alpha 2-antiplasmin Enschede reversibly and temporarily inhibited the plasmin-catalysed hydrolysis of D-valyl-L-leucyl-L-lysine p-nitroanilide ('S-2251') as a competitive inhibitor (Ki,app. 35 nM). It was concluded that alpha 2-antiplasmin Enschede apparently forms a normal complex with plasmin. The complex is, however, not stable, but disintegrates rapidly to a cleaved form of alpha 2-antiplasmin Enschede and active plasmin. The abnormal protein thus behaves like a substrate, instead of an inhibitor, of plasmin.

Amino Acid Sequence↗
PubMed1988

Daytime fluctuations in blood of tissue-type plasminogen activator (t-PA) and its fast-acting inhibitor (PAI-1).

Circadian fluctuation in blood fibrinolytic activity was studied in 10 volunteers in the day-time period at 09.00, 12.00 and 15.00 h. Activity of tissue-type plasminogen activator (t-PA) was found to increase from 09.00 to 15.00 h in accordance with known results with global assays of blood activity. Also, activity and antigen of plasminogen activator inhibitor 1 (PAI-1) and antigen of t-PA showed fluctuations but with an opposite pattern to that of t-PA activity, with highest levels in the morning and lowest ones in the afternoon. Activity of a reversible t-PA inhibitor was constant. The discordance between fluctuations in antigen and in activity of t-PA is attributed to the inhibitory effect of PAI-1 on t-PA activity. It is concluded that the appearance into the blood of t-PA and PAI-1 shows a daytime fluctuation with a similar pattern, suggesting a co-ordinated circadian fluctuation in production of both proteins by endothelial cells.

Adult↗
PubMed1988

Binding of tissue-type plasminogen activator to fibrinogen fragments.

In order to localize the binding site(s) for tissue-type plasminogen activator (t-PA) in the fibrin(ogen) molecule, the following binding assay was developed. Two-chain t-PA was immobilized onto microtitration plates. The t-PA-coated plates were then incubated with fibrinogen and various fibrinogen fragments. The extent of binding was quantified with enzyme-labelled antibodies against fibrin(ogen) and its fragments. Hardly any binding to t-PA was observed with fibrinogen or fragments X, Y and E; a moderate binding was observed with fragments Dcate and DEGTA and a strong binding with the cyanogen bromide fragment FCB-2 (Kd apparent = 140 nM). The binding of fibrinogen and its fragments to immobilized Lys-plasminogen was measured by the same method as a control for the binding assay. Results were in line with literature data: virtually no binding to Lys-plasminogen with fibrinogen or fragments X and Y, a moderate binding with fragments Dcate, DEGTA and E and a strong binding with FCB-2 (Kd apparent = 70 nM). The stimulatory capacity of the various fragments on the Lys-plasminogen activation by t-PA, as studied in a spectrophotometric assay, was found to be absent for fragment E, low for fibrinogen, fragments X, Y, Dcate and DEGTA, and high for FCB-2. It is concluded that a t-PA-binding site resides in the C-terminal globular domains of fibrinogen from which fragments D and FCB-2 originate. The site is hidden in the native fibrinogen molecule and in early fibrinogen degradation products. Binding of both Lys-plasminogen and t-PA appears to be required for a stimulator of the plasminogen activation, as illustrated by fragment E which only binds Lys-plasminogen and has no stimulatory capacity.

Binding Sites↗
PubMed1988

[Recombinant tissue-type plasminogen activator: pharmacokinetics and effect on the hemostasis system of the human].

The in vitro interaction of rt-PA with the coagulation and fibrinolytic system was investigated. After addition of rt-PA to citrated plasma prolonged clotting times for prothrombin time, partial thromboplastin time and thrombin time, decreased activities of individual clotting factors and decreases of alpha 2-antiplasmin, plasminogen and fibrinogen were measured. The alterations were dependent on incubation times and rt-PA concentrations and were mainly observed at rt-PA concentrations exceeding therapeutic levels. These in vitro phenomena could be prevented to different degrees using various inhibitors. Because of interferences with some fibrinolytic and coagulation assays aprotinin was only of limited utility as an inhibitor. PPACK lengthened clotting time assays based on generation of endogenous thrombin, but did not affect fibrinolytic assays. The most versatile inhibitor was a specific anti-rt-PA antibody which enabled correct measurements of all fibrinolytic and most coagulation assays. It is concluded that high rt-PA levels in samples taken from patients during fibrinolytic therapy induce in vitro artefacts which can be prevented by the use of suitable inhibitors. In 8 healthy male volunteers aged 32 +/- 7 years pharmacokinetic investigations were done. After infusion of 0.25 mg rt-PA/kg bw mean maximal plasma levels were 970 +/- 130 ng/ml. The elimination of rt-PA from plasma was fitted to a two compartment model and was characterized by two half lives of t1/2 alpha = 3.3 and t1/2 beta = 26 minutes.

Adult↗
PubMed1988

Relationships between structure and function of tissue-type plasminogen activator.

This mini-review deals with structure-function relationships of human tissue-type plasminogen activator. The enzyme consists of a single polypeptide chain of 527 amino acids. A two-chain form is produced by proteolytic cleavage of the Arg 275-Ile 276 peptide bond. The aminoterminal heavy or A-chain consists of a finger domain, a growth factor domain and two kringle domains. The carboxyterminal light or B-chain contains the active site and is homologous to the catalytic chains of other serine proteases. The light chain is able to activate plasminogen, but requires the heavy chain for fibrin-binding and fibrin-stimulation. Particularly, the finger domain and kringle 2 of the heavy chain are involved in the interaction with fibrin. Other specific properties of the plasminogen activator, such as its rapid hepatic clearance and its inhibition by plasminogen activator inhibitors have not yet been related to specific domains in the protein structure.

Chemical Phenomena↗
PubMed1988

Pharmacokinetics of antigen and activity of recombinant tissue-type plasminogen activator after infusion in healthy volunteers.

Pharmacokinetics of recombinant tissue plasminogen activator (rt-PA, large-scale process) were determined based on antigen and activity after infusion of 0.25 mg/kg in 8 healthy volunteers. Plasma antigen was measured using enzyme-linked immunosorbent assay (ELISA) with and without treatment of blood at collection with D-Phe-Pro-Arg-CH2Cl (P PACK); activity was quantified in acidified plasma both on fibrin plates and in a chromogenic assay. Highest rt-PA concentrations were measured in ELISA with P PACK-treated samples, yielding the following pharmacokinetic parameters (2-compartment model, mean +/- S.D.): Cmax = 973 +/- 133 ng/ml, CL = 687 ml/min, dominant half-life t1/2 alpha = 3.3 +/- 0.4 min, t1/2 beta = 26 +/- 12 min, V1 = 3.9 +/- 0.6 l and Vss = 7.2 +/- 1.0 l. The other assays yielded lower rt-PA concentrations, which affected clearance and volume parameters but not t1/2 beta and t1/2 beta. Linear regressions of the fibrin plate and chromogenic assay results vs. ELISA yielded excellent correlations (R greater than 0.96, n = 55-57) but slopes of 0.76 and 0.64, respectively. This indicates that about 25-35% of rt-PA antigen in thawed plasma samples are not detected in activity assays, due at least partially to in vitro binding of rt-PA by proteinase inhibitors.

Chromogenic Compounds↗
PubMed1987

Butyrate stimulates tissue-type plasminogen-activator synthesis in cultured human endothelial cells.

Incubation of cultured human endothelial cells with 5 mM-dibutyryl cyclic AMP led to an approx. 2-fold increase in tissue-type plasminogen-activator (t-PA) production over a 24 h incubation period. The stimulating effect of dibutyryl cyclic AMP could be explained by the slow liberation of butyrate, as the effect could be reproduced by addition of free butyrate to the medium, but not by addition of 8-bromo cyclic AMP or forskolin, agents known to raise intracellular cyclic AMP levels. With butyrate, an accelerated accumulation of t-PA antigen in the conditioned medium (CM) was observed after a lag period of about 6 h. Increasing amounts of butyrate caused an increasingly stimulatory effect, reaching a plateau at 5 mM-butyrate. The relative enhancement of t-PA production in the presence of 5 mM-butyrate varied among different endothelial cell cultures from 6- to 25-fold in 24 h CM. Such an increase in t-PA production was observed with both arterial and venous endothelial cells. The butyrate-induced increases in t-PA production were accompanied by increased t-PA mRNA levels. Analysis of radiolabelled CM and cell extracts by SDS/polyacrylamide-gel electrophoresis indicated that the potent action of butyrate is probably restricted to a small number of proteins. The accumulation of plasminogen activator inhibitor type 1 (PAI-1) in CM from butyrate-treated cells varied only moderately. In our study of the relationship between structure and stimulatory activity, we found that a straight-chain C4 monocarboxylate structure with a methyl group at one end and a carboxy moiety at the other seems to be required for the optimal induction of t-PA in cultured endothelial cells.

Butyrates↗
PubMed1987

Alpha 2-antiplasmin Enschede: alanine insertion and abolition of plasmin inhibitory activity.

An abnormal alpha 2-antiplasmin that is associated with a serious bleeding tendency has been found in a Dutch family and is referred to as alpha 2-antiplasmin Enschede. This abnormal alpha 2-antiplasmin is converted from an inhibitor of plasmin to a substrate. The molecular defect of alpha 2-antiplasmin Enschede, as revealed by sequencing of cloned genomic DNA fragments, consists of an alanine insertion near the active site region of the molecule. Substitution of this fragment into complementary DNA for a wild-type alpha 2-antiplasmin yields a translation product with physical and functional properties typical of the abnormal alpha 2-antiplasmin Enschede. The naturally occurring mutant may serve as a model for investigating the structures that determine the properties of an inhibitor versus those of a substrate in serine protease inhibitors.

Amino Acid Sequence↗
PubMed1987

Increased plasminogen activator inhibition levels in malignancy.

Plasminogen activator(PA)-tissue and urokinase type-and PA-inhibition in plasma were investigated in 52 consecutive cancer patients with a variety of tumors. At first patients were analyzed as one group. Secondly patients were subdivided into two groups, one with (n = 42) and one without (n = 10) metastasis. Our results show that tissue-type-PA antigen (t-PA-antigen) and PA-inhibition were both significantly increased irrespective of the presence or absence of tumor metastasis (p less than 0.001 compared to age matched healthy controls. In the group without metastasis a significantly decreased level of t-PA activity was found (p less than 0.001) but in the group with metastasis t-PA activity was normal. These data seem to reject the hypothesis that decreased plasma fibrinolytic activity is one of the prerequisites for tumor metastasis.

Antigens↗
PubMed1987

On the usefulness of fibrinolysis variables in the characterization of a risk group for myocardial reinfarction.

In a prospective study selected fibrinolysis variables were assessed in plasma samples from 29 consecutive patients recovering a first instance of acute myocardial infarction and the results were correlated with reinfarction during the next four years. Nine patients suffered a reinfarction leaving a group of 20 patients without evidence of relapse. The reinfarction group was characterized by lower tissue plasminogen activator activities in plasma euglobulins (p less than 0.05), significantly higher plasma concentrations of tissue plasminogen activator antigen (p less than 0.002) and a tendency to a higher plasma level of plasminogen activator inhibition capacity. There were no significant differences between the groups in plasma concentrations of plasminogen, histidine-rich glycoprotein, plasminogen kringle-4-binding-protein, and alpha 2-antiplasmin.

Fibrinolysis↗