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D Collen

Publications and source records attributed to D Collen.

At least 199 records · Page 11Linked to original sources
PubMed1996

Recombinant staphylokinase variants with altered immunoreactivity. II: Thrombolytic properties and antibody induction.

BACKGROUND: The substitution variants K35A,E38A,K74A, E75A,R77A (SakSTAR.M38) and K74A,E75A,R77A,E80A, D82A (SakSTAR.M89) of recombinant staphylokinase (SakSTAR) with reduced antibody reactivity were assayed for thrombolytic potency and antibody induction in animal models and in patients. METHODS AND RESULTS: In a 125I-fibrin-labeled pulmonary embolism model in the hamster, the doses giving 50% clot lysis in 90 minutes were 25 micrograms/kg for SakSTAR, 85 micrograms/kg for SakSTAR.M38, and 90 micrograms/kg for SakSTAR.M89. In rabbits with 125I-fibrin-labeled plasma clots incorporated into extracorporeal arteriovenous loops, lysis within 2 hours was 76 +/- 18% (mean +/- SD, n = 28) with 400 micrograms/kg SakSTAR, 53 +/- 13% (n = 8) with 1000 micrograms/kg SakSTAR.M38, and 39 +/- 13% (n = 6) with 800 micrograms/kg SakSTAR.M89. When groups of eight rabbits were immunized by intravenous administration of 0.2 to 1.0 micrograms/kg compound followed by subcutaneous injection of 0.4 mg in Freund's adjuvant at 2, 3, and 5 weeks, SakSTAR.M38 and SakSTAR.M89 elicited markedly less circulating neutralizing activity, compared with SakSTAR, when determined at 6 weeks (neutralizing 6.1 +/- 3.0 and 4.9 +/- 1.3 micrograms compound/mL plasma, respectively, versus 20 +/- 17 micrograms/mL; P = .02 and P = .01, respectively) and induced significantly less resistance to thrombolysis (residual thrombolytic potency producing 59 +/- 25% and 39 +/- 12% lysis, respectively, versus 8.5 +/- 5.7%; P = .008 and P = .006, respectively). In patients with peripheral arterial occlusion, intra-arterial administration of SakSTAR.M38 (n = 4) or SakSTAR.M89 (n = 4) induced significantly fewer circulating neutralizing antibodies (P = .03) and specific IgG (P = .01) at 2 to 3 weeks than SakSTAR (n = 8). CONCLUSIONS: SakSTAR.M38 and SakSTAR.M89 induce less antibody formation and might constitute preferred agents for thrombolytic therapy in humans.

Animals↗
PubMed1996

Adenoviral-mediated transfer of the human endothelial nitric oxide synthase gene reduces acute hypoxic pulmonary vasoconstriction in rats.

Nitric oxide (NO), a vasodilator involved in the regulation of pulmonary vascular tone, is synthesized by a family of enzymes, nitric oxide synthases (NOS). To investigate whether adenoviral-mediated overexpression of constitutive endothelial NOS (ceNOS) would attenuate hypoxic pulmonary vasoconstriction, we aerosolized 3 X 10(9) plaque forming units of a recombinant adenovirus containing the ceNOS gene (AdCMVceNOS) into rat lungs. Four days after infection, transgene expression was confirmed using immunoblot techniques. Diffuse ceNOS immunostaining was detected in alveoli and medium-sized and small pulmonary vessels of AdCMVceNOS-transduced lungs. AdCMVceNOS-transduction was associated with an 86% increase in [3H]arginine to [3H]citrulline conversion and a rise in pulmonary cGMP levels from 7 +/- 1 to 59 +/- 9 pmol/mg protein in lungs from AdCMVceNOS versus control rats, (P < 0.05). During acute hypoxia (FIO2 = 0.10) for 25 min, mean pulmonary artery pressure (PAP) increased significantly from 17 +/- 1 to 27 +/- 1 mmHg in rats aerosolized with saline (n = 4) and from 18 +/- 1 to 28 +/- 1 mmHg in rats given an adenoviral vector expressing a nuclear-targeted beta-galactosidase gene (AdCMV beta gal, n = 8). In contrast, in AdCMVceNOS-transduced rats (n = 8) the hypoxia-induced increase in PAP was significantly attenuated (18 +/- 1 to 23 +/- 2 mmHg). Systemic blood pressure was not affected by aerosol gene transfer. Thus, adenoviral-mediated ceNOS gene transfer to rat lungs increases ceNOS expression and activity, and reduces acute hypoxic pulmonary vasoconstriction. Aerosolized recombinant adenovirus overexpressing vasodilatory proteins can act as a selective pulmonary vasodilator and may hold promise as a future therapeutic strategy for pulmonary hypertension.

Adenoviridae↗
PubMed1996

Urokinase-type plasminogen activator is effective in fibrin clearance in the absence of its receptor or tissue-type plasminogen activator.

The availability of gene-targeted mice deficient in the urokinase-type plasminogen activator (uPA), urokinase receptor (uPAR), tissue-type plasminogen activator (tPA), and plasminogen permits a critical, genetic-based analysis of the physiological and pathological roles of the two mammalian plasminogen activators. We report a comparative study of animals with individual and combined deficits in uPAR and tPA and show that these proteins are complementary fibrinolytic factors in mice. Sinusoidal fibrin deposits are found within the livers of nearly all adult mice examined with a dual deficiency in uPAR and tPA, whereas fibrin deposits are never found in livers collected from animals lacking uPAR and rarely detected in animals lacking tPA alone. This is the first demonstration that uPAR has a physiological role in fibrinolysis. However, uPAR-/-/tPA-/- mice do not develop the pervasive, multi-organ fibrin deposits, severe tissue damage, reduced fertility, and high morbidity and mortality observed in mice with a combined deficiency in tPA and the uPAR ligand, uPA. Furthermore, uPAR-/-/tPA-/- mice do not exhibit the profound impairment in wound repair seen in uPA-/-/tPA-/- mice when they are challenged with a full-thickness skin incision. These results indicate that plasminogen activation focused at the cell surface by uPAR is important in fibrin surveillance in the liver, but that uPA supplies sufficient fibrinolytic potential to clear fibrin deposits from most tissues and support wound healing without the benefit of either uPAR or tPA.

Animals↗
PubMed1996

Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele.

The endothelial cell-specific vascular endothelial growth factor (VEGF) and its cellular receptors Flt-1 and Flk-1 have been implicated in the formation of the embryonic vasculature. This is suggested by their colocalized expression during embryogenesis and the impaired vessel formation in Flk-1 and Flt-1 deficient embryos. However, because Flt-1 also binds placental growth factor, a VEGF homologue, the precise role of VEGF was unknown. Here we report that formation of blood vessels was abnormal, but not abolished, in heterozygous VEGF-deficient (VEGF+/-) embryos, generated by aggregation of embryonic stem (ES) cells with tetraploid embryos (T-ES) and even more impaired in homozygous VEGF-deficient (VEGF-/-) T-ES embryos, resulting in death at mid-gestation. Similar phenotypes were observed in F1-VEGF+/- embryos, generated by germline transmission. We believe that this heterozygous lethal phenotype, which differs from the homozygous lethality in VEGF-receptor-deficient embryos, is unprecedented for a targeted autosomal gene inactivation, and is indicative of a tight dose-dependent regulation of embryonic vessel development by VEGF.

Alleles↗
PubMed1996

Generation and characterization of urokinase receptor-deficient mice.

Mice homozygously deficient for the urokinase-type plasminogen activator (u-PA) receptor (u-PAR-1-) were generated by homologous recombination in D3, embryonic stem cells. The genomic sequences comprising exon 2 through 5 of the u-PAR gene were replaced by the neomycin resistance gene, resulting in inactivation of both u-PAR splice variants. The inactivated u-PAR allele was transmitted via mendelian inheritance, and fertility. Inactivation of u-PAR was confirmed by the absence of binding of rabbit anti-murine u-PAR or of an aminoterminal fragment of murine u-PA (mu-PA.1-48) to u-PAR-1- embryonic fibroblasts and macrophages. u-PAR-1- mice displayed normal lysis of a murine plasma clot injected via the jugular vein. Invasion of macrophages into the peritoneal cavity after thioglycollate stimulation was similar in u-PAR-1- and u-PAR-1- mice. u-PAR-1- peritoneal macrophages had a threefold decreased initial rate of u-PA-mediated plasminogen activation in vitro but degraded extracellular matrix proteins in vitro as efficiently as u-PAR-1- macrophages.

Animals↗
PubMed1996

Pluripotential rabbit embryonic stem (ES) cells are capable of forming overt coat color chimeras following injection into blastocysts.

The isolation of pluripotent embryonic stem (ES) cell lines from preimplantation rabbit embryos and their in vitro properties have been previously described. In the present investigation, these ES cell lines were further characterized and their capacity to contribute to formation of adult, fertile animals upon injection into recipient New Zealand White blastocysts demonstrated. The efficiency of chimera formation was low (5% of live born), but the degree of chimerism, as assessed by coat color contribution from the Dutch belted strain, was high (10-50%). Thus a significant step is taken toward the development of gene-targeting technology in the rabbit, an animal whose physiology and size lend itself to unique applications in biomedical research.

Animals↗
PubMed1996

Gene manipulation and transfer of the plasminogen and coagulation system in mice.

The blood coagulation and the fibrinolytic (or plasminogen/plasmin) systems determine the balance between the formation and dissolution of blood clots, but in addition contribute to the pathogenesis of various cardiovascular disorders such as thrombosis, atherosclerosis, and restenosis. Furthermore, they participate in a variety of other (patho)biological processes such as embryonic development, reproduction, wound healing, cancer, and brain function. Two recently developed technologies, gene targeting and gene transfer, that allow manipulation of the genetic balance of these proteinase systems in a controllable manner have allowed a more definitive elucidation of the biological role of these systems. This review summarizes the insights that have been obtained from the gene targeting studies and discusses the use of adenovirus-mediated transfer of fibrinolytic genes to study and possibly to develop novel strategies for the treatment of restenosis and thrombosis.

Animals↗
PubMed1996

Characterization of a helicase-like transcription factor involved in the expression of the human plasminogen activator inhibitor-1 gene.

A 5.4-kb cDNA encoding the protein that binds to the B Box of the plasminogen activator inhibitor-1 (PAI-1) gene was isolated and sequenced. The protein, named helicase-like transcription factor (HLTF), contains a DNA-binding domain, a RING finger domain, and seven helicase domains and is homologous to SWI/SNF proteins. Two HLTF mRNAs of 5.5 and 4.5 kb were detected in most human tissues, a single gene was located on chromosome 3q24-25, and the protein was located in the nucleoplasm. Two HLTF proteins differing in translation start site (Met-1 or Met-123) were obtained by in vitro translation in reticulocyte lysate or by immunoprecipitation from HeLa cell nuclear extracts. In vitro transcription from the PAI-1 promoter in HeLa cell extracts was inhibited by HLTF antibodies and by the HLTF DNA binding domain. Over-expression of HLTF or HLTFMet123 produced a three-fold induction of PAI-1-LUC transient expression in HeLa cells. Mutation of the PAI-1 B Box led to an eight-fold reduction of basal PAI-1-LUC expression in these cell lines, but did not affect the four- to six-fold induction by phorbol esters.

Amino Acid Sequence↗
PubMed1996

Comparative antigenicity of recombinant wild-type staphylokinase (SakSTAR) and a selected mutant (SakSTAR.M38) in a baboon thrombolysis model.

Staphylokinase, a bacterial plasminogen activator, is a potent, highly fibrin-specific but antigenic thrombolytic agent in humans. In an effort to attenuate the antigenicity of wild-type staphylokinase (SakSTAR variant), 2 of its 3 immunodominant epitopes were altered by substituting clusters of 2 or 3 charged amino acids with alanine, yielding the mutant SakSTAR.M38 (K35A, E38A, K74A, E75A, R77A), which was less antigenic in inbred New Zealand White rabbits. In the present study, groups of 6 baboons (Papio hamadryas) were randomized to SakSTAR (group 1) or SakSTAR.M38 (group 2). The thrombolytic potencies of 50 micrograms/kg compound at baseline, assessed in an extracorporeal thrombosis model, were similar: 77 +/- 2.9% (mean +/- SEM) clot lysis in group 1 and 83 +/- 3.6% in group 2. Groups 1 and 2 were immunized subcutaneously at 2, 3, and 5 weeks with 500 micrograms SakSTAR or SakSTAR.M38, respectively. From 6 weeks, group 1 developed significantly more antibody-related neutralizing activity than group 2 (maximal titer at 8 weeks of 100 +/- 23 micrograms SakSTAR and of 22 +/- 7.1 micrograms SakSTAR.M38 neutralized per milliliter of plasma, respectively). Neutralizing activities subsequently decreased gradually to 10-20% of peak values at 18 weeks. At 6 weeks, both groups were resistant to thrombolysis with 50 micrograms/kg of either compound. Rechallenge at 18 weeks with 250 micrograms/kg of the immunizing compound showed a significantly better recovery of the thrombolytic potency of SakSTAR.M38 (68 +/- 4.5% clot lysis) than of SakSTAR (39 +/- 5.3% clot lysis). Neither agent degraded fibrinogen or depleted alpha 2-antiplasmin. Therefore, SakSTAR.M38 is comparably active and fibrin-specific but less antigenic than wild-type SakSTAR. These findings in outbred primates confirm and extend earlier observations in inbred rabbits and provide a basis for the further development of staphylokinase variants with reduced antigenicity in humans.

Animals↗
PubMed1996

Genetic analysis of the plasminogen and coagulation system in mice.

The blood coagulation and the fibrinolytic (or plasminogen/plasmin) systems determine the balance between the formation and dissolution of blood clots, but in addition contribute to the pathogenesis of various cardiovascular disorders such as thrombosis, atherosclerosis and restenosis. Furthermore, they participate in a variety of other (patho)biological processes such as embryonic development, reproduction, wound healing, cancer and brain function. Two recently developed technologies, gene targeting and gene transfer, that allow to manipulate the genetic balance of these proteinase systems in a controllable manner have allowed to more definitively elucidate the biological role of these systems. This review summarizes the insights that have been obtained from the gene targeting studies and discusses the use of adenovirus-mediated transfer of fibrinolytic genes to study and possibly to develop novel strategies for the treatment of restenosis and thrombosis.

Animals↗
PubMed1996

Optimising thrombolytic therapy in elderly patients with acute myocardial infarction.

The incidence of myocardial infarction (MI) and its subsequent mortality increases with age. However, elderly patients are less likely to receive thrombolytic therapy. Among other reasons, this may be because elderly patients with evolving MI often have atypical signs and symptoms and delay their presentation for medical treatment by 2 hours compared with younger patients. The risk of (most often fatal) cerebral bleeding in elderly patients treated with aspirin, heparin and thrombolytic drugs is about 1 to 2 per 1000 patients, and is 2 times higher than in younger patients. In order to decrease the incidence of intracranial haemorrhage in elderly patients with acute MI, thrombolytic treatment should not be recommended in patients at increased risk of cerebral bleeding. Otherwise, the benefit of thrombolytic treatment outweigh the risk; for every 1000 elderly patients treated with thrombolytic drugs, approximately 36 are saved compared with those receiving control treatment.

Aged↗
PubMed1996

Characterization of a cDNA encoding murine coagulation factor VII.

The cDNA encoding murine coagulation factor VII (mfVII) was isolated and reconstructed from a lambda Zap cDNA library generated from murine liver mRNA. The cDNA contains 1903 nucleotides spanning 15 bases upstream of the 5'-translation initiation codon, an open reading frame of 1338 nucleotides, 550 nucleotides downstream of the first stop codon and a 3' poly(A) tail. The translation product is composed of a 41-amino acid signal/propeptide region followed by a 405-residue mature protein. The latter is highly homologous to that of human, rabbit, bovine, Rhesus monkey, and canine fVII. All protein domains of hfVII are strictly conserved in mfVII.

Amino Acid Sequence↗
PubMed1996

Comparative effects of staphylokinase and alteplase in rabbit bleeding time models.

BACKGROUND: The pathogenesis of bleeding associated with thrombolytic therapy remains largely unknown, although spontaneous bleeding appears to correlate with bleeding time prolongation. Here, the comparative effects on cuticle bleeding times (CBT) and ear puncture bleeding times (EBT) of recombinant staphylokinase (Sak) and alteplase (recombinant tissue-type plasminogen activator, rt-PA) at equivalent doses, alone and in combination with aspirin and heparin, were studied in rabbits. METHODS AND RESULTS: Groups of 4 to 9 rabbits were allocated to one of the 8 following intravenous infusions: saline; aspirin 15 mg/kg and heparin - 100 IU/kg bolus and 10 IU/kg infusion over one hour; 1.5 mg/kg rt-PA; 1.5 mg/kg rt-PA plus aspirin and heparin; 4.5 mg/kg rt-PA; 0.5 mg/kg Sak; 0.5 mg/kg Sak plus aspirin and heparin and 1.5 mg/kg Sak. Bleeding times were determined 30 and 15 min before and 5, 15, 30 and 60 min after the administration over one min of saline, rt-PA or Sak, by simultaneously severing a nail cuticle (CBT) and by puncturing the ear (EBT). Bleeding times were unaffected by saline and by both doses of Sak in monotherapy. Heparin-aspirin and low dose rt-PA significantly lengthened EBT but not CBT. Both CBT and EBT were significantly prolonged (to a mean of > 4 times pretreatment at 5 min) after high-dose rt-PA and after the combined administration of heparin and aspirin with either Sak or tr-PA. rt-PA provoked significantly longer bleeding than Sak in the CBT (p = 0.001; mean estimated difference = 23 min), but not in the EBT. rt-PA but not Sak degraded plasma fibrinogen dose-dependently. CBT correlated inversely with fibrinogen (r= -0.66, p=0.001) but EBT did not. CONCLUSION: At equivalent doses Sak displays a significantly higher fibrin specificity and prolongs bleeding time less than rt-PA, particularly in the nail cuticle bleeding time model in which larger vessels are injured that require fibrinogen for hemostasis.

Animals↗
PubMed1996

A pilot study on bolus administration of recombinant staphylokinase for coronary artery thrombolysis.

Recombinant staphylokinase (Sak) is a highly fibrin-specific thrombolytic agent but the optimal dose and mode of administration remain to be defined. Intravenous (i.v.) infusion over 5 min of 20 mg Sak in 12 patients with acute myocardial infarction induced complete coronary patency (TIMI perfusion grade 3) in 7 patients (58%) within 60 min. In 3 of the 5 patients with no or suboptimal flow (TIMI grade 0, 1 or 2) at 60 min, an additional 10 mg i.v. bolus of Sak resulted in TIMI grade 3 flow at 90 min. No major treatment-related complication occurred. Residual fibrinogen and alpha 2-antiplasmin levels at 90 min were 110 +/- 6.0% and 98 +/- 4.1% (mean +/- SEM) of baseline, respectively. Median antibody-related Sak-neutralizing activity was low at baseline (0.0 microgram/ml) and after 1 week (0.5 microgram/ml) but increased from day 10 on (to 4.0 micrograms/ml). Thus, bolus thrombolysis with Sak may induce efficient coronary artery recanalization while preserving circulating fibrinogen and alpha 2-antiplasmin. Comparative trials of coronary thrombolysis with double-bolus Sak appear to be warranted.

Aged↗
PubMed1996

Correlation between oxidized low density lipoproteins and von Willebrand factor in chronic renal failure.

An ELISA specific for a wide spectrum of oxidized apo B-100 in OxLDL was developed and applied to blood samples from 27 control subjects, 20 mild chronic renal failure (MCRF) patients, 21 severe chronic renal failure patients on conservative treatment (SCRF) and 56 severe chronic renal failure patients on maintenance hemodialysis (HEMO). Mean levels of OxLDL were 0.59 mg/dl in controls (95% CI, 0.52-0.66 mg/dl), and were 2.7-fold (p < 0.01), 3.1-fold (p < 0.001) and 5.4-fold (p < 0.001) higher in MCRF, SCRF and HEMO patients, respectively. Levels of von Willebrand factor, a marker of endothelial injury, were 100 percent in controls (95% CI, 90-110 percent), and were 1.5-fold (p = NS), 1.6-fold (p < 0.01) and 2.1-fold (p < 0.001) higher in MCRF, SCRF and HEMO patients, respectively. Multiple regression analysis revealed that the extent of renal failure (F = 14; p = 0.0004) accounted for a significant fraction of the variation in OxLDL levels, also after exclusion of patients with evidence of ischemic atherosclerotic disease (F = 21; p = 0.0001). After adjustment for the extent of renal failure, hemodialysis (F = 5.6; p = 0.021) and LDL cholesterol levels (F = 7.1, p = 0.0095) contributed significantly to the variation in OxLDL levels. Whereas the extent of renal failure contributed only marginally to the individual variations in vWF levels (F = 4.1; p = 0.048), these levels correlated significantly with plasma levels of OxLDL (F = 26; p = 0.0001). In conclusion, OxLDL increase progressively during the development of renal failure suggesting that the oxidation of LDL may be associated with endothelial injury and atherogenesis in these patients.

Aged↗