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D Collen

Publications and source records attributed to D Collen.

At least 271 records · Page 15Linked to original sources
PubMed1994

Functional properties of recombinant staphylokinase variants obtained by site-specific mutagenesis of methionine-26.

Variants of recombinant staphylokinase (Sak) were produced by site-specific mutagenesis of the unique Met-26 residue and purified to homogeneity from the cell extract of transformed E. coli. The desired mutations were confirmed by cDNA and amino-acid sequence analysis. Sak-M26L, Sak-M26C, Sak-M26R, Sak-M26V and Sak-M26A were selected for further analysis on the basis of their plasminogen activating activity. The specific fibrinolytic activities of Sak-M26L, Sak-M26C and Sak were comparable (76,000 +/- 10,000, 75,000 +/- 2400 and 78,000 +/- 9700 HU/mg, respectively; mean +/- S.E., n = 3 or 4). Active site exposure in equimolar (4.5 microM) mixtures plasminogen at room temperature was more rapid with Sak-M26L than with Sak (quantitative exposure within 4 min and 8 min, respectively). Activation of 1 microM plasminogen by catalytic amounts (5 nM) of Sak-M26L initially appeared to be somewhat faster, but comparable 50 to 60% activation was obtained within 30 min. In contrast, Sak-M26R and Sak-M26V were virtually inactive, did not form active complexes with plasminogen and did not activate plasminogen. The catalytic efficiencies for plasminogen activation were comparable for plasmin-Sak-M26L, plasmin-Sak-M26C and plasmin-Sak (0.14 microM-1 s-1, 0.16 microM-1 s-1 or 0.12 microM-1 s-1, respectively). Comparable dose-dependent lysis of 0.06 ml 125I-fibrin labeled human plasma clots submerged in 0.3 ml human plasma was obtained with Sak-M26L, Sak-M26C and Sak (concentration required for 50% lysis in 2 h, EC50, of 17 +/- 1.6 nM, 19 +/- 1.4 nM and 14 +/- 2.5 nM, respectively), whereas Sak-M26R or Sak-M26V were inactive. Sak-M26A did not form a stable complex with plasminogen, as shown by gel filtration. These data establish that substitution of the unique Met residue in position 26 of the Sak sequence with Leu or Cys has little or no influence on its plasminogen activating or fibrinolytic potential. In contrast, substitution of Met-26 with either Arg or Val results in total loss of the functional activity. Thus, the amino acid in position 26 of Sak appears to be of crucial importance for the activation of plasminogen by staphylokinase.

Amino Acid Sequence↗
PubMed1994

High yield production and purification of recombinant staphylokinase for thrombolytic therapy.

Recombinant plasmids were constructed in which the signal sequence of the sak42D and the sakSTAR staphylokinase genes were replaced by an ATG start codon and which express staphylokinase under the control of a tac promoter and two Shine-Dalgarno sequences in tandem. Induction of transfected E. coli TGl cells in a bacterial fermentor produced intracellular staphylokinase representing 10 to 15% of total cell protein. Gram quantities of highly purified recombinant staphylokinase were obtained from cytosol fractions by chromatography, at room temperature, on SP-Sepharose and on phenyl-Sepharose columns, with yields of 50 to 70 percent. The material, at a dose of 4 mg/kg, did not produce acute reactions or affect body weight in mice. Intravenous administration of 10 mg SakSTAR over 30 minutes in five patients with acute myocardial infarction induced complete coronary artery recanalization, without associated fibrinogen degradation. However, neutralizing antibodies appeared in the plasma of all patients within 12 to 20 days. Thus, the present expression and purification method for recombinant staphylokinase yields large amounts of highly purified mature protein (approximately 200 mg per liter fermentation broth) suitable for a more detailed clinical investigation of its potential as a thrombolytic agent.

Base Sequence↗
PubMed1994

Oxidized lipoproteins in atherosclerosis and thrombosis.

Oxidized low density lipoproteins (LDL) and autoimmune antibodies against oxidized LDL have been demonstrated in human atherosclerotic lesions; they may enhance the progression of these lesions by 1) enhancing monocyte adhesion and macrophage foam cell generation; 2) inducing smooth muscle cell migration, proliferation, and foam cell generation; 3) enhancing platelet adhesion and aggregation, which may stimulate macrophage foam cell generation and smooth muscle cell proliferation; 4) triggering thrombosis; and 5) impairing vasodilation, which results in increased shear stress. The oxidation of LDL probably occurs in the arterial wall, where it is sequestered from circulating antioxidants. Atherosclerotic arterial walls contain increased levels of redox-active metal ions, and the LDL of patients with atherosclerotic cardiovascular disease are more susceptible to oxidation, possibly as a result of reduced endogenous antioxidants such as vitamin E. Dietary supplementation with vitamin E (up to 1,000 IU/day) or administration of probucol reduce the oxidation rate of LDL and may significantly decrease the risk of coronary heart disease.

Animals↗
PubMed1994

Time course of the effects of a single bolus injection of F(ab')2 fragments of the antiplatelet GPIIb/IIIa antibody 7E3 on arterial eversion graft occlusion, platelet aggregation, and bleeding time in dogs.

The time course of the effects of a single intravenous bolus injection of 10 mg/kg aspirin or 0.8 mg/kg F(ab')2 fragments of the monoclonal antiplatelet glycoprotein IIb/IIIa receptor antibody 7E3 [7E3-F(ab')2] on arterial occlusion, platelet aggregation, and bleeding time was studied in 30 dogs with an everted (inside out) carotid arterial segment inserted into the femoral artery. In the absence of an antiplatelet agent, the eversion grafts occluded spontaneously with platelet-rich thrombus within 30 minutes. With aspirin, arterial occlusion persisting for 2 hours occurred in 5 of 10 dogs and cyclic occlusion and reflow in 4 animals; arterial occlusion was observed in all dogs at 24 hours. With 7E3-F(ab')2, arterial patency persisted throughout a 2-hour observation period in all of 10 dogs and for 24 hours in 4 of the 10 dogs. Contralateral eversion grafting 24 hours after aspirin or 7E3-F(ab')2 injection was associated with graft patency for 2 hours in 1 of 5 aspirin dogs and in 3 of 5 7E3-F(ab')2 dogs; patency persisted for 24 hours. In dogs grafted 48 hours after aspirin or 7E3-F(ab')2 injection, patency at 24 hours was seen in 0 of 5 dogs given aspirin and 3 of 5 dogs given 7E3-F(ab')2. The overall frequencies of arterial graft patency at 2, 24, 48, and 72 hours after study drug injection were significantly higher in the 7E3-F(ab')2 groups than in the aspirin groups (P < .0005, n = 10 in each group; P < .05, n = 15; P < .005, n = 15; and P = .05, n = 5, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Contribution of platelets and the vessel wall to the antithrombotic effects of a single bolus injection of Fab fragments of the antiplatelet GPIIb/IIIa antibody 7E3 in a canine arterial eversion graft preparation.

The contribution of platelets and the vessel wall to the antithrombotic effects of a single intravenous bolus injection of 0.8 mg/kg Fab fragments of the monoclonal antiplatelet glycoprotein IIb/IIIa receptor antibody 7E3 (7E3-Fab), combined with continuous heparin anticoagulation (100 U/kg bolus and 50 U/kg per hour), was studied in a canine preparation consisting of an everted (inside out) carotid arterial segment that had been inserted into a transected femoral artery. In all 6 control dogs without antibody, persistent or transient eversion graft occlusion occurred during an initial 2-hour observation period, and 5 of the 6 grafts were occluded at 24 hours. In 6 dogs given 7E3-Fab 24 hours before receiving an everted carotid artery segment from a donor dog, cyclic occlusion and reflow occurred in all dogs, whereas the grafts were patent at the end of a 2-hour observation period in 5 of the 6 dogs (P = .056 versus control). When transferred back to the donor dogs, the patient eversion segments showed brief periods of cyclic occlusion and reflow within 2 hours in 3 of 5 dogs (P = .034 versus control), whereas all of the 5 eversion segments were patent at 24 hours (P < .005 versus control).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Stimulation with a monoclonal antibody (mAb4E4) of scavenger receptor-mediated uptake of chemically modified low density lipoproteins by THP-1-derived macrophages enhances foam cell generation.

mAb4E4, a murine monoclonal antibody that is specific for acetylated LDL and malondialdehyde-treated LDL, binds specifically to modified LDL present in human atherosclerotic lesions. It is directed against an epitope that is poorly exposed in delipidated and solubilized apolipoprotein B-100 from modified LDL. mAb4E4, as well as its F(ab')2 and Fab fragments, enhanced the uptake of both acetylated LDL and malondialdehyde-treated LDL by THP-1-derived macrophages resulting in a sixfold increase of cytoplasmic cholesteryl ester levels. The increased uptake of modified LDL/mAb4E4 complexes did not occur via the Fc receptor and did not depend on aggregation of modified LDL particles. However, their uptake was inhibited by blocking the scavenger receptors with fucoidin or by downregulation of receptor expression with endotoxins or interferon-gamma, indicating that their uptake is mediated via these receptors. Thus, generation of autoimmune antibodies against modified LDL and subsequent endocytosis of soluble modified LDL/antibody complexes via scavenger receptors may enhance foam cell generation. This mechanism may contribute to the progression of atherosclerotic lesions.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

On the immunogenicity of recombinant staphylokinase in patients and in animal models.

Streptokinase and staphylokinase, the presently available thrombolytic agents of bacterial origin, are immunogenic in man; their use may cause allergic reactions and/or refractoriness to renewed administration. Infusion of 2 to 10 mg of recombinant staphylokinase (STAR) in 20 patients with acute myocardial infarction or peripheral arterial occlusion induced IgG-related neutralizing activity in plasma with a lag phase of 10 to 12 days, from a baseline of 0.2 +/- 0.06 microgram STAR neutralized per ml plasma (mean +/- SEM) to a maximum of 30 +/- 6.2 micrograms/ml after 3 to 9 weeks, which persisted at a level of 14 +/- 5.8 micrograms/ml after 18 months (n = 4). In 4 baboons with a 125I-fibrin labeled clot in an extracorporeal arteriovenous loop, i.v. administration of 63 micrograms/kg STAR over 1 h, repeated at weekly intervals, induced a progressive increase of STAR-neutralizing activity (from 0.05 +/- 0.1 microgram/ml at baseline to 4.8 +/- 1.5 micrograms/ml at week 6), which was paralleled by a reduction of in vivo clot lysis (from 60 +/- 7% to 8 +/- 3%). After temporary discontinuation of STAR-administration, neutralizing activity reverted to baseline within 7 weeks, whereafter the sensitivity of in vivo clot lysis to STAR was restored. In rabbits, i.v. administration of 250 micrograms/kg STAR over 1 h, repeated at weekly intervals, also induced a progressive increase of STAR-neutralizing activity (from 0.5 +/- 0.2 microgram/ml at baseline to 6.4 +/- 1.1 micrograms/ml at week 6), which was paralleled by a reduction of in vivo clot lysis (from 68 +/- 3% to 31 +/- 7%).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Comparative thrombolytic properties of tissue-type plasminogen activator and of a plasminogen activator inhibitor-1-resistant glycosylation variant, in a combined arterial and venous thrombosis model in the dog.

rt-PA-K, a variant of recombinant tissue-type plasminogen activator (rt-PA) with substitution of amino acids 296 to 299 with alanine (KHRR296-299AAAA) has increased fibrin-specificity and reduced sensitivity to plasminogen activator inhibitor-1; rt-PA-T, with threonine 103 replaced by asparagine has an additional glycosylation site and a reduced clearance; and rt-PA-N, with asparagine 117 mutagenized to glutamine lacks the high mannose carbohydrate side chain. We have investigated whether combination of these properties in a single molecule might yield an improved thrombolytic agent. The thrombolytic potency and fibrin-specificity of the combination mutant rt-PA-TNK was compared with that of rt-PA in a combined venous whole blood clot model and platelet-rich arterial eversion graft thrombosis model in dogs given intravenous heparin and aspirin. Infusion of 0.125 to 1.0 mg/kg over 60 min in groups of 4 to 5 dogs produced dose-dependent fibrin-specific venous clot lysis. The thrombolytic potency (percent lysis per mg compound administered per kg body weight) of rt-PA-TNK was significantly higher than that of rt-PA as evidenced by a higher maximal rate of lysis of 480 +/- 100% versus 140 +/- 40% within the 2 h observation period per mg of compound administered per kg body weight (mean +/- SEM, p = 0.004) and a significantly lower dose of 0.08 +/- 0.01 versus 0.21 +/- 0.04 mg/kg body weight at which the maximal rate of lysis was obtained (p = 0.004).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Prevalence and induction of circulating antibodies against recombinant staphylokinase.

Streptokinase (SK) is a routinely used thrombolytic agent but it is immunogenic and allergenic; staphylokinase (STA) is a potential alternative agent which is under early clinical evaluation. The comparative prevalence of antibodies against recombinant STA (STAR) and against SK was studied in healthy subjects and their induction with intravenous administration in small groups of patients. Enzyme-linked immunosorbent assays, using microtiter plates coated with STAR or SK and calibration with affinospecific human antibodies, revealed 2.1 to 65 micrograms/ml (median 11 micrograms/ml) anti-STAR antibodies and 0.9 to 370 micrograms/ml (median 18 micrograms/ml) anti-SK antibodies (p < 0.001 vs anti-STAR antibodies) in plasma from 100 blood donors, with corresponding values of 0.6 to 100 micrograms/ml (median 7.1 micrograms/ml) and 0.4 to 120 micrograms/ml (median 7.3 micrograms/ml), respectively, in 104 patients with angina pectoris. Three out of 17 patients with Staphylococcus aureus bacteremia had significantly increased anti-STAR antibody levels (150, 75 and 75 micrograms/ml), and STAR neutralizing activities (2.2, 3.6 and 4.1 micrograms STAR neutralized per ml plasma, respectively). In 6 patients with acute myocardial infarction, given 10 mg STAR intravenously over 30 min, median anti-STAR antibody levels were 3.5 micrograms/ml at baseline, 2.9 micrograms/ml at 6 to 8 days and 1.2 mg/ml at 2 to 9 weeks, with median corresponding titers of STAR neutralizing activity at 2 to 9 weeks of 42 micrograms/ml plasma. Conversely, in 5 patients treated with 1,500,000 units SK over 60 min, median anti-SK antibodies increased from 2.9 micrograms/ml at baseline to 360 micrograms/ml at 5 to 10 days, with corresponding median SK neutralizing activities of 13 micrograms/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Angina Pectoris↗
PubMed1994

Expression and characterization of clustered charge-to-alanine mutants of low M(r) single-chain urokinase-type plasminogen activator.

In an effort to modify the fibrinolytic and/or pharmacokinetic properties of recombinant low M(r) single-chain urokinase-type plasminogen activator (rscu-PA-32k), mutants were prepared by site-directed mutagenesis of clusters of charged amino acids with the highest solvent accessibility. The following mutants of rscu-PA-32k were prepared: LUK-2 (Lys 212, Glu 213 and Asp 214 to Ala), LUK-3 (Lys 243 and Asp 244 to Ala), LUK-4 (Arg 262, Lys 264, Glu 265 and Arg 267 to Ala), LUK-5 (Lys 300, Glu 301 and Asp 305 to Ala) and LUK-6 (Arg 400, Lys 404, Glu 405 and Glu 406 to Ala). The rscu-PA-32k moieties were expressed in High Five Trichoplasiani cells, and purified to homogeneity from the conditioned cell culture medium, with recoveries of 0.8 to 3.7 mg/l. The specific fibrinolytic activities (220,000 to 300,000 IU/mg), the rates of plasminogen activation by the single-chain moieties and the rates of conversion to two-chain moieties by plasmin were comparable for mutant and wild-type rscu-PA-32k moieties, with the exception of LUK-5 which was virtually inactive. Equi-effective lysis (50% in 2 h) of 60 microliters 125I-fibrin labeled plasma clots submerged in 0.5 ml normal human plasma was obtained with 0.7 to 0.8 microgram/ml of wild-type or mutant rscu-PA-32k, except with LUK-5 (no significant lysis with 16 micrograms/ml). Following bolus injection in hamsters, all rscu-PA-32k moieties had a comparably rapid plasma clearance (1.3 to 2.7 ml/min), as a result of a short initial half-life (1.4 to 2.5 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Antithrombotic effects and bleeding time prolongation with synthetic platelet GPIIb/IIIa inhibitors in animal models of platelet-mediated thrombosis.

Cyclic Arg-Gly-Asp (RGD) containing synthetic peptides such as L-cysteine, N-(mercaptoacetyl)-D-tyrosyl-L-arginylglycyl-L-alpha-aspartyl- cyclic (1-->5)-sulfide, 5-oxide (G4120) and acetyl-L-cysteinyl-L-asparaginyl-L-propyl-L-arginyl-glycyl-L-alpha- aspartyl-[0-methyltyrosyl]-L-arginyl-L-cysteinamide, cyclic 1-->9-sulfide (TP9201) bind with high affinity to the platelet GPIIb/IIIa receptor. The relationship between antithrombotic effect, ex vivo platelet aggregation and bleeding time prolongation with both agents was studied in hamsters with a standardized femoral vein endothelial cell injury predisposing to platelet-rich mural thrombosis, and in dogs with a carotid arterial eversion graft inserted in the femoral artery. Intravenous administration of G4120 in hamsters inhibited in vivo thrombus formation with a 50% inhibitory bolus dose (ID50) of approximately 20 micrograms/kg, ex vivo ADP-induced platelet aggregation with ID50 of 10 micrograms/kg, and bolus injection of 1 mg/kg prolonged the bleeding time from 38 +/- 9 to 1,100 +/- 330 s. Administration of TP9201 in hamsters inhibited in vivo thrombus formation with ID50 of 30 micrograms/kg, ex vivo platelet aggregation with an ID50 of 50 micrograms/kg and bolus injection of 1 mg/kg did not prolong the template bleeding time. In the dog eversion graft model, infusion of 100 micrograms/kg of G4120 over 60 min did not fully inhibit platelet-mediated thrombotic occlusion but was associated with inhibition of ADP-induced ex vivo platelet aggregation and with prolongation of the template bleeding time from 1.3 +/- 0.4 to 12 +/- 2 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

On the future of thrombolytic therapy for acute myocardial infarction.

The beneficial effects of thrombolytic therapy in acute ischemic coronary syndromes, and particularly in acute myocardial infarction, are now well established. The limited efficacy and potentially life-threatening side effects of currently available thrombolytic agents, however, remain a problem. Available evidence suggests that the efficacy of coronary thrombolysis could be augmented either by shortening the time to treatment, by improvement of the potency and specificity of fibrin-dissolving agents, by optimized conjunctive treatment with anticoagulant and antiplatelet agents, or by a combination of all 3. These objectives can be pursued by intensified education of the public, paramedical personnel, and physicians; development of novel thrombolytic agents; and further elucidation of pathophysiologic determinants of recanalization and its maintenance. Although it is clear that compounds with antithrombin or antiplatelet properties may enhance and sustain the action of thrombolytic agents, their optimal use and potential hemorrhagic side effects remain to be further explored. Optimized thrombolytic therapy eventually will most likely consist of administration of potent specific plasminogen activators in combination with conjunctive targeted anticoagulant and/or antiplatelet agents.

Humans↗