PubMed Health⌕ Search

Biomedical subjects

D C Rijken

Publications and source records attributed to D C Rijken.

At least 91 records · Page 5Linked to original sources
PubMed1987

alpha 2-Antiplasmin Enschede: dysfunctional alpha 2-antiplasmin molecule associated with an autosomal recessive hemorrhagic disorder.

alpha 2-Antiplasmin (alpha 2-AP) is a major fibrinolysis inhibitor, whose complete, congenital absence has been found to be associated with a distinct hemorrhagic diathesis. We studied a 15-yr-old male with a hemorrhagic diathesis after trauma from early childhood on. This bleeding tendency was associated with a minimal alpha 2-AP level recorded functionally in the immediate plasmin inhibition test: less than or equal to 4% of normal. However, a normal plasma concentration of alpha 2-AP antigen (83%) was found. His sister (5 yr old) showed similar results (2 and 92%). In their family, eight heterozygotes could be identified by half-normal activity results and normal antigen concentrations. The inheritance pattern is autosomal recessive. On analysis, the alpha 2-AP of the propositus was homogeneous in all respects tested, suggesting a homozygous defect. We designated the abnormal alpha 2-AP as alpha 2-AP Enschede. alpha 2-AP Enschede showed the following characteristics: (a) complete immunological identity with normal alpha 2-AP; (b) normal molecular weight (sodium dodecyl sulfate-polyacrylamide gel electrophoresis); (c) normal alpha-electrophoretic mobility; (d) presence in plasma of both molecular forms excluding an excessive conversion to the less reactive non-plasminogen-binding form; (e) quantitatively normal binding to lys-plasminogen and to immobilized plasminogen kringle 1-3; and (f) normal Factor XIII-mediated binding to fibrin. Functional abnormalities were found in: (i) no inhibition of amidolytic activities of plasmin and trypsin, even on prolonged incubation; (ii) no formation of plasmin-antiplasmin complexes in plasma with plasmin added in excess; and (iii) no inhibition of fibrinolysis by fibrin-bound alpha 2-AP. In the heterozygotes, the presence of abnormal alpha 2-AP did not interfere with several functions of the residual normal alpha 2-AP. One-dimensional peptide mapping showed an abnormal pattern of papain digestion. We conclude that in this family, abnormal antiplasmin molecules, defective in plasmin inhibition but with normal plasminogen-binding properties, have been inherited. The residual plasminogen-binding properties do not protect against a hemorrhagic diathesis.

Adolescent↗
PubMed1986

Clearance of the heavy and light polypeptide chains of human tissue-type plasminogen activator in rats.

In order to assess which part of the tissue-type plasminogen activator (t-PA) molecule should be (genetically) modified to obtain more-slowly-clearing mutants, two-chain t-PA and its isolated heavy and light chains were radiolabelled and injected into rats. The vast majority of t-PA and the heavy chain disappeared from the blood circulation with half-lives of 2.3 and 1.0 min respectively. The clearance of the light chain was biphasic, owing to complex-formation with plasma proteinase inhibitors. The disappearance of di-isopropylphospho-light chain, which has a blocked active site, was nearly monophasic, with a half-life of 5.7 min. Organ distribution studies showed that hepatic clearance constituted the major pathway in all cases. These results strongly suggest that t-PA is recognized by the liver primarily through the heavy chain.

Animals↗
PubMed1986

Characterization and fibrin-binding properties of different molecular forms of pro-urokinase from a monkey kidney cell culture.

Culture fluid of a monkey kidney cell culture was harvested every two days, for a two week period, in order to obtain urokinase in the zymogen form. Pro-urokinase was isolated by immunoadsorption chromatography and gel filtered on Sephadex G-150, which resulted in three peaks with pro-urokinase activity. SDS-polyacrylamide gel electrophoresis showed that the first peak contained 55 kd pro-urokinase, aggregated with high molecular weight contaminants, whereas the second and third peaks consisted of almost pure 55 kd and 30 kd pro-urokinase, respectively. The latter form represented a relatively unknown and inactive precursor of low molecular weight urokinase, which was, like 55 kd pro-urokinase, activatable with plasmin. In comparison with tissue-type plasminogen activator, 55 kd and 30 kd pro-urokinase only bound weakly to purified fibrin clots and fibrin-sepharose columns. The extent of binding of the two pro-urokinases and their plasmin-activated forms to fibrin-sepharose decreased in the following order: 55 kd pro-urokinase 30 kd pro-urokinase 55 kd urokinase 30 kd urokinase. These results indicate that the two precursors exhibited stronger binding to fibrin-sepharose than the corresponding active enzymes, and the two 55 kd forms exhibited stronger binding than the corresponding 30 kd forms. This indicates the importance of both the zymogen nature and an intact NH2-terminal part of the molecules for binding to fibrin.

Animals↗
PubMed1986

Specific fibrinolytic properties of different molecular forms of pro-urokinase from a monkey kidney cell culture.

The specific fibrinolytic properties of both high molecular weight (55 kd) and low molecular weight (30 kd) pro-urokinase from a monkey kidney cell culture were evaluated in a plasma clot lysis system and compared with those of human urokinase. The system was composed of a radiolabelled plasma clot immersed in plasma containing the fibrinolytic agent. On unit base, 55 kd pro-urokinase was approximately 1.5 times more effective in lysing the clot than 30 kd pro-urokinase and equally effective as urokinase. In contrast to urokinase, both pro-urokinase forms induced clot lysis without degrading fibrinogen in the surrounding plasma. However, a considerable activation of the fibrinolytic system in the plasma occurred as a large amount of alpha 2-antiplasmin was consumed, indicating that pro-urokinase was not fully fibrin-specific. Quenching antibodies against tissue-type plasminogen activator (t-PA) added to the plasma clot lysis system retarded but did not prevent pro-urokinase-induced clot lysis. This indicated that not only was t-PA in plasma involved in the activation of pro-urokinase (probably via plasmin), but that an additional mechanism also existed.

Animals↗
PubMed1986

Isolation and functional characterization of the heavy and light chains of human tissue-type plasminogen activator.

Two-chain tissue-type plasminogen activator (t-PA), which consists of a heavy chain (Mr congruent to 38,000) and a light chain (Mr congruent to 31,000) connected by a disulfide bridge, was reduced with 2-mercaptoethanol and then air-reoxidized at a low protein concentration and carboxamidomethylated. The two chains were separated by means of zinc chelate-agarose, which was found to bind the light chain selectively. The light chain was fully active on the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine p-nitroanilide (S-2288) and partially active on plasminogen. The plasminogen activator activity of the light chain was, in contrast to that of two-chain t-PA, not stimulated by fibrin or fibrinogen fragments. Fibrin-agarose chromatography of radiolabeled chains showed that only the heavy chain bound to fibrin. These results indicate that the active site-containing light chain in t-PA needs the heavy chain for fibrin stimulation of its plasminogen activator activity.

Amino Acids↗
PubMed1985

On the composition and function of the carbohydrate moiety of tissue-type plasminogen activator from human melanoma cells.

Two variants (I and II) of tissue-type plasminogen activator (t-PA) from human melanoma cells were separated by Lysine Sepharose chromatography. The carbohydrate compositions of the forms were determined by gas-liquid chromatography. Variant I contained 12.8 g and variant II 7.1 g of carbohydrate per 100 g protein. Both variants contained N-acetylgalactosamine, suggesting O-glycosylation in addition to N-glycosylation. The possible role of N-linked oligosaccharides for the biological activity of t-PA was studied using t-PA secreted by melanoma cells in the presence of tunicamycin, an inhibitor of N-glycosylation. The latter t-PA showed the same plasminogen activating and fibrin binding properties as normally glycosylated t-PA, indicating that N-linked carbohydrate is not involved in the fibrinolytic activity of t-PA.

Carbohydrates↗
PubMed1985

Fibrinolytic shut-down after surgery: impairment of the balance between tissue-type plasminogen activator and its specific inhibitor.

In nine patients with non-malignant diseases undergoing major upper abdominal surgery, the mechanism of the postoperative fibrinolytic shut-down was investigated because of its potential significance for postoperative deep vein thrombosis by employing new and specific methods for assessing and stimulating the fibrinolytic system. The shut-down was found to result from an impairment of the balance between tissue-type plasminogen activator, t-PA, and its recently discovered fast-acting inhibitor. In this balance, the t-PA antigen concentrations both in resting conditions and after stimulation evoked by desamino-D-arginine vasopressin (DDAVP) were found to be unchanged by surgery. However, there was a significant postoperative increase in t-PA inhibitor levels. The release of t-PA under the stimulus of DDAVP infusion overcame the postoperative shut-down of t-PA activity. However, DDAVP infusion was associated with potentially unfavourable increases in the Factor VIII/von Willebrand factor complex. The discovery of increased t-PA inhibitor in the postoperative period opens new possibilities for a rational approach to reduce or abolish the postoperative fibrinolytic shut-down.

Adult↗
PubMed1985

The postoperative fibrinolytic shutdown: a rapidly reverting acute phase pattern for the fast-acting inhibitor of tissue-type plasminogen activator after trauma.

The plasma activity level of the recently discovered fast-acting inhibitor of tissue-type plasminogen activator (t-PA) was found to be temporarily increased after surgery, myocardial infarction and severe trauma. Detailed analysis of the postoperative period revealed simultaneously increased t-PA antigen and inhibition and decreased t-PA activity only on the first postoperative day. These changes were more rapid than those in fibrinogen and C-reactive protein. It is concluded that t-PA inhibition shows the most rapidly changing pattern observed so far in response to trauma. The postoperative fibrinolytic shutdown in blood fibrinolytic activity can be ascribed to a primary increase in t-PA inhibitor levels.

Antigens↗
PubMed1984

Fibrinolytic activators and inhibitors in terminal renal insufficiency and in anephric patients.

Fibrinolytic factors were measured before and after DDAVP-infusion in 18 patients on chronic, regular haemodialysis, 11 of whom underwent bilateral nephrectomy, and in 7 patients in whom non-functioning kidneys were still present. Baseline fibrinolytic activity was normal or high in all but two cases. Before haemodialysis, the response to DDAVP-infusion was greatly reduced in the majority of patients as compared with healthy controls, irrespective of the baseline level. This was in accordance with mean t-PA-antigen levels which increased only slightly after DDAVP. When DDAVP was given after haemodialysis, previous non-responders showed a normal increase in fibrinolytic activity. The level of free t-PA-inhibitors was normal in most cases as were levels of alpha 2-antiplasmin and alpha 2-macroglobulin.

Antigens↗
PubMed1984

Masking of fibrinolytic response to stimulation by an inhibitor of tissue-type plasminogen activator in plasma.

Patients with hyperlipoproteinaemia or spontaneous thromboembolism, known to be poor responders to DDAVP or to venous occlusion with regard to the rise in fibrinolytic activity of the blood, appeared to show a normal increase in t-PA-antigen after the same procedure. In their plasma a higher than normal level of free, fast-acting t-PA-inhibitor was found as measured by titration with purified t-PA. This free t-PA-inhibitor level only decreased after the test, in contrast to its complete disappearance in normal responders. The same happened in a healthy volunteer who failed to exhibit a rise in fibrinolytic activity after exhaustive exercise. We suppose that the lack of response of the fibrinolytic activity in these cases is due to a high inhibitor level and not to impaired release of t-PA into the blood. In contrast, patients with terminal renal insufficiency showed only a slight increase in t-PA-antigen after DDAVP. The level of free fast-acting inhibitor was normal in most cases and did not change appreciably during DDAVP-infusion. In these patients, a true impairment of the release of t-PA appears to exist.

Antigens↗
PubMed1984

Modulation of rapid plasminogen activator inhibitor in plasma by stanozolol.

Fibrinolytic activity in plasma euglobulin fractions can be increased by oral administration of stanozolol. This increase is not caused by increased synthesis or release of tissue-type plasminogen activator. A decreased level of fast acting t-PA inhibition is very probably the cause of the higher activity. These results suggest that this inhibition has a regulatory role on fibrinolysis in vivo.

Adult↗
PubMed1984

Quantitation of tissue-type plasminogen activator in human endothelial cell cultures by use of an enzyme immunoassay.

Endothelial cells from human umbilical cord were cultured to study plasminogen activator synthesis and secretion. Since simultaneous production of plasminogen activator inhibitor(s) prevented detection of plasminogen activators by use of fibrinolytic assays, an enzyme immunoassay for tissue-type plasminogen activator (t-PA) was developed. In this assay, t-PA in test samples was adsorbed onto microtiter plates coated with rabbit antibody against t-PA and then quantitated by successive incubation with goat antibody against t-PA and enzyme labeled rabbit antibody against goat IgG. The sensitivity of the assay was found to be 1 ng t-PA/ml. In the absence of serum, arterial and venous endothelial cells continuously produce t-PA antigen during a 24 h period, reaching a level of 5.1 +/- 2.5 ng t-PA/ml (n = 8). In serum containing (20%) medium, 9.3 +/- 6.0 ng t-PA/ml (n = 17) was produced during this period (0.1 - 0.2 ml medium per cm2 confluent cells). It is concluded that the enzyme immunoassay is a useful method for quantitating t-PA secretion by endothelial cells in the presence of proteinase inhibitors.

Cells, Cultured↗
PubMed1983

Measurement of human tissue-type plasminogen activator by a two-site immunoradiometric assay.

A two-site immunoradiometric assay for human extrinsic (tissue-type) plasminogen activator was developed by using rabbit antibodies raised against plasminogen activator purified from human melanoma cell culture fluid. Samples of 100 microliters containing 1 to 100 ng/ml plasminogen activator were incubated in the wells of polyvinyl chloride microtiter plates coated with antibody. The amount of bound extrinsic plasminogen activator was quantitated by the subsequent binding of 125I-labeled affinospecific antibody. The mean level of plasma samples taken at rest was 6.6 +/- 2.9 ng/ml (n = 54). This level increased approximately threefold by exhaustive physical exercise, venous occlusion, or infusion of DDAVP. Extrinsic plasminogen activator in plasma is composed of a fibrin-adsorbable and active component (1.9 +/- 1.1 ng/ml, n = 54, in resting conditions) and an inactive component that does not bind to a fibrin clot (probably extrinsic plasminogen activator-proteinase inhibitor complexes). The fibrin-adsorbable fraction increased approximately fivefold to eightfold after physical exercise, venous occlusion, or DDAVP injections. Potential applications of the immunoradiometric assay are illustrated by the measurement of extrinsic plasminogen activator in different tissue extracts, body fluids, and cell culture fluids and in oocyte translation products after injection with mRNA for plasminogen activator.

Culture Techniques↗
PubMed1983

Complexes between tissue-type plasminogen activator and proteinase inhibitors in human plasma, identified with an immunoradiometric assay.

Extrinsic (tissue-type) plasminogen activator antigen in human plasma, as measured by a two-site immunoradiometric assay, is composed of a fibrin-adsorbable and a nonadsorbable fraction. Gel filtration on Ultrogel AcA 44 in 1.6M KSCN of the fibrin-adsorbable fraction showed a peak with Mr congruent to 70,000, which contained plasminogen activator activity and was assumed to represent free extrinsic plasminogen activator. The nonadsorbable fraction showed a broad peak with Mr congruent to 140,000 without plasminogen activator activity. Overnight incubation at 37 degrees C of postexercise plasma revealed a shift of the Mr congruent to 70,000 peak to the Mr congruent to 140,000 position, suggesting that the Mr congruent to 140,000 peak consists of extrinsic plasminogen activator-protease inhibitor complex(es). alpha 2-Antiplasmin is the main inhibitor of extrinsic plasminogen activator in plasma 13 and is therefore most probably at least in part responsible for the generation of the Mr congruent to 140,000 component. A possible involvement of other plasma proteinase inhibitors was explored by incubation of 125I-labeled extrinsic plasminogen activator in alpha 2-antiplasmin-depleted plasma. A complex was formed with a t1/2 of about 1 hr, which was identified by immunoprecipitation as extrinsic plasminogen activator-alpha 1-antitrypsin complex. Additional evidence for the presence of extrinsic plasminogen activator complexes with alpha 2-antiplasmin and alpha 1-antitrypsin in plasma was obtained from two-site immunoradiometric assays, in which solid-phase anti-inhibitor antibody bound the corresponding complex, which was then detected with radiolabeled, affinospecific antibody against extrinsic plasminogen activator. It was concluded that plasma contains both free extrinsic plasminogen activator and plasminogen activator complexes with alpha 2-antiplasmin and alpha 2-antitrypsin. These complexes are also present in plasma collected on the active site inhibitor, D-Phe-Pro-Arg-CH 2Cl, at rest and after exercise and are therefore assumed to circulate in vivo.

Chromatography, Gel↗
PubMed1982

Purification of human tissue-type plasminogen activator in centigram quantities from human melanoma cell culture fluid and its conditioning for use in vivo.

Human tissue-type plasminogen activator was produced in centigram quantities from 50-60 1 batches of conditioned medium of a human melanoma cell culture. The yields of the procedure remained essentially unchanged during 50 subsequent preparations. The final products were of high purity as assessed by SDS gel electrophoresis. These materials, after filtration on 0.22 microM Milliporefilters were sterile and free of viruses and pyrogens. Their stability was very good in the fluid form at temperatures up to 56 degrees C. Material obtained by the present procedure has been used to investigate the biological and thrombolytic properties of tissue-type plasminogen activator.

Cell Line↗
PubMed1982

Characterization of a plasminogen activator secreted by cultured bovine aortic endothelial cells.

A plasminogen activator was purified from the serum-free conditioned medium of bovine aortic endothelial cell cultures by chromatography on zinc chelate-agarose and benzamidine-CH-Sepharose. The final material consisted of a main fibrinolytically active component with Mr 30,000 and a minor component with Mr 41,000. It was obtained with a yield of 60%, a purification factor of 35 and a purity of 25-50%. The activity of this plasminogen activator was completely neutralized by antibodies to human urokinase but not by antibodies against human tissue plasminogen activator. Purified tissue plasminogen activator from bovine heart, however, was completely neutralized by antibodies against human tissue plasminogen activator but unaffected by antibodies to human urokinase. These findings indicate that bovine aortic endothelial cells in culture secrete mainly a urokinase-like. These findings indicate that bovine aortic endothelial cells in culture secrete mainly a urokinase-like plasminogen activator, and not a tissue-type plasminogen activator as was generally assumed.

Animals↗
PubMed1982

Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin.

The kinetics of the activation of Glu-plasminogen and Lys-plasminogen (P) by a two-chain form of human tissue plasminogen activator (A) were studied in purified systems, and in the presence of fibrinogen (f) and of fibrin films (F) of increasing size and surface density. The activation in the purified systems followed Michaelis-Menten kinetics with a Michaelis constant of 65 microM and a catalytic rate constant of 0.06 s-1 for Glu-plasminogen as compared to 19 microM 0.2 s-1 for Lys-plasminogen. In the presence of fibrinogen plots of 1/v versus 1/[P] or 1/v versus 1/[f] yielded straight lines with an apparent Michaelis constant at infinite [f] of 28 microM and a catalytic rate constant of 0.3 s-1 for Glu-plasminogen as compared to 1.8 microM and 0.3 s-1 for Lys-plasminogen. In the systems with fibrin, plasmin was estimated from the rate of release of 125I from 125I-labeled fibrin films. The initial rate of activation (v) was calculated and Lineweaver-Burk plots of 1/v versus 1/[P] or 1/v versus 1/[F] yielded straight lines. Activation occurred with an intrinsic Michaelis constant of 0.16 microM and a catalytic rate constant of 0.1 s-1 for Glu-plasminogen as compared to 0.02 microM and 0.2 s-1 for Lys-plasminogen. The kinetic analysis suggested that the activation in the presence of fibrin occurs through binding of an activator molecule to the clot surface and subsequent addition of plasminogen (sequential ordered mechanism) to form a cyclic ternary complex. The Low Michaelis constant in the presence of fibrin allows efficient plasminogen activation on a fibrin clot, while its high value in the absence of fibrin prevents efficient activation in plasma.

Enzyme Activation↗