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Biomedical subjects

D Collen

Publications and source records attributed to D Collen.

At least 595 records · Page 33Linked to original sources
PubMed1983

Heparin binding properties of human histidine-rich glycoprotein. Mechanism and role in the neutralization of heparin in plasma.

Human histidine-rich glycoprotein was found to interact strongly with heparin both in purified systems and in plasma, resulting in neutralization of the anti-coagulant activity of heparin. In purified systems, histidine-rich glycoprotein and heparin react with apparent 1:1 stoichiometry to form a complex with a dissociation constant of 7 nM. Covalent heparin-antithrombin complex still reacts with histidine-rich glycoprotein to form a complex with a dissociation constant of 29 nM. The interaction between a Mr = 4300-heparin fragment and histidine-rich glycoprotein appeared to be more complex. The mechanism of the interaction between histidine-rich glycoprotein and heparin appeared to be different from that between antithrombin III and heparin, since the former is abolished by EDTA and occurs both with heparin molecules having a high affinity or a low affinity for antithrombin III. In plasma, histidine-rich glycoprotein efficiently counteracts the anticoagulant activity of heparin. Both the thrombin times and the activated factor X inhibition following addition of heparin are markedly prolonged in the absence of histidine-rich glycoprotein and shortened by addition of purified histidine-rich glycoprotein. Low affinity heparin was found to efficiently compete with high affinity heparin for binding to histidine-rich glycoprotein but not to antithrombin III. This results in an increased anticoagulant activity of high affinity heparin in the presence of low affinity heparin. Since the effect of histidine-rich glycoprotein on the anticoagulant properties of heparin is clearly demonstrated in normal plasma, it may be of clinical significance.

Blood Proteins↗
PubMed1983

Thrombolysis with human extrinsic (tissue-type) plasminogen activator in rabbits with experimental jugular vein thrombosis. Effect of molecular form and dose of activator, age of the thrombus, and route of administration.

A simple venous thrombosis model in rabbits was used for the quantitative evaluation of the thrombolytic effect of human extrinsic (tissue-type) plasminogen activator as compared with urokinase.A thrombus was formed in an isolated segment of the jugular vein from a mixture of (125)I-labeled fibrinogen, whole rabbit blood, and thrombin. In order to immobilize the thrombus during lysis, it was formed around a woolen thread introduced longitudinally in the lumen of the vein. Thrombotic extension of the clot was prevented by subcutaneous injection of heparin. The extent of thrombolysis was measured as the difference between the radioactivity introduced in the clot and that recovered in the vein segment at the end of the experiment. In control animals the extent of thrombolysis was 5.6+/-1.4% (n = 5) after 6 h, 14.5+/-1.7% (n = 10) after 30 h, 16.0+/-1.5% (n = 11) after 78 h, and 48.1+/-2.7% (n = 10) after 174 h (mean+/-SEM). Extrinsic (tissue-type) plasminogen activator, highly purified from the culture fluid of a human melanoma cell line, was administered systemically or locally over a time period of 4 h and the percent thrombolysis measured 2 h after the end of the infusion. One- and two-chain extrinsic plasminogen activator had very similar thrombolytic potency. Systemic infusion resulted in a dose-dependent degree of thrombolysis. The activator-induced thrombolysis, after infusion of 100,000 IU ( congruent with1 mg protein), was approximately 75% for fresh clots, 35% for 1-d-old clots, 30% for 3-d-old clots, and 50% for 7-d-old clots. The thrombolytic activity of urokinase was more than five times lower than that of extrinsic plasminogen activator: Infusion of 500,000 IU resulted in approximately 40% lysis of fresh clots and 25% of 1-3-d-old clots, while 7-d-old clots appeared to have become resistent to urokinase. Local infusion resulted in a 5-10 times higher thrombolytic effect of both extrinsic plasminogen activator and urokinase. Thrombolysis with extrinsic plasminogen activator was not associated with systemic activation of the fibrinolytic system as evidenced by unaltered plasma levels of fibrinogen, plasminogen, and alpha(2)-antiplasmin. Systemic infusion of urokinase resulted in significant thrombolysis only at doses that were associated with disseminated plasminogen activation. Local infusion of urokinase required a 5-10-fold higher dose than extrinsic plasminogen activator to obtain a similar degree of thrombolysis, which also occurred in the absence of systemic activation of the fibrinolytic system. It is concluded that the extent of thrombolysis by extrinsic plasminogen activator is mainly determined by the dose of activator and its delivery in the vicinity of the thrombus and much less by the age of the thrombus or the molecular form of the activator. Extrinsic plasminogen activator appears to be superior to urokinase because of its higher (5-10-fold) specific thrombolytic activity and the absence of systemic activation of the fibrinolytic system, which results in defibrinogenation and a bleeding tendency.

Animals↗
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

Kinetics of the inhibition of plasmin in acidified human plasma.

Acid-treated human plasma is a competitive inhibitor of the hydrolysis of D-Val-Leu-Lys-Nan (S-2251) by plasmin. The rate of hydrolysis is decreased to 50% by 750 fold diluted acidified normal plasma and by 60 fold diluted acidified alpha 2-antiplasmin depleted plasma (alpha 2-antiplasmin concentration less than 2%). These findings suggest that alpha 2-antiplasmin is a contributary but not the main competitive inhibitor of acidified plasma. This interpretation is supported by the finding that alpha 2-antiplasmin depleted plasma reconstituted with purified alpha 2-antiplasmin inhibits the hydrolysis of S-2251 by plasmin at a 125 fold dilution following acidification and by the finding that in a purified system acid inactivated alpha 2-antiplasmin inhibits the hydrolysis of S-2251 by plasmin with a Ki of 25 nM. Thus, besides alpha 2-antiplasmin, other plasma proteins which are at least in part eliminated by the removal of alpha 2-antiplasmin from plasma by immunoadsorption appear to be competitive inhibitors for plasmin in acidified plasma. It is suggested that several competitive inhibitors for plasmin are present and/or generated in acidified plasma and that these inhibitors may at least in part be responsible for the variability in the results of measurements of plasminogen and/or plasmin in plasma following acidification.

Fibrinolysin↗
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

Partial primary structure of human alpha 2-antiplasmin-homology with other plasma protease inhibitors.

Human alpha 2-antiplasmin was digested with trypsin and with chymotrypsin and about 70 percent of the amino acids were sequenced and aligned in peptides ranging from 2 to 33 residues. Here we report five sequences of 21 to 33 residues. When these were compared with the primary structures of antithrombin III, alpha 1-antitrypsin and ovalbumin, which belong to the same protein superfamily (Hunt and Dayhoff [1980] Biochem Biophys Res Commun 95: 864-871), three peptides showed clear homologies with these proteins, indicating that alpha 2-antiplasmin also belongs to that superfamily. In addition, alpha 2-antiplasmin appeared to contain at least one internal homology.

Amino Acid Sequence↗
PubMed1982

Purification and partial primary structure of cyanogen bromide fragments from human alpha 2-antiplasmin.

Human alpha 2-antiplasmin was digested with CNBr and the partial NH2-terminal amino acid sequences of nine purified fragments were determined. One of these sequences (12 residues) revealed homologies with that of residues 158 to 168 and with that of residues 316-327 in the antithrombin III sequence and with the corresponding sequences in alpha 1-antitrypsin and ovalbumin. One CNBr-fragment of 69 residues with blocked NH2-terminus was subdigested with trypsin and chymotrypsin and most of its sequence aligned. This peptide probably represents the major part of the previously reported COOH-terminal fragment of alpha 2-antiplasmin with Mr 8,000 (1). In this sequence no clear homology with antithrombin III, alpha 1-antitrypsin or ovalbumin could be detected.

Amino Acid Sequence↗
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↗