Biomedical subjects

D Collen

Publications and source records attributed to D Collen.

At least 145 records · Page 8Linked to original sources

PubMed1998

Function of the plasminogen/plasmin and matrix metalloproteinase systems after vascular injury in mice with targeted inactivation of fibrinolytic system genes.

The matrix metalloproteinase (MMP) system, which may be activated via the plasminogen (Plg)/plasmin system, is claimed to play a role in matrix degradation and smooth muscle cell migration. To test the role of both systems, expression of fibrinolytic and gelatinolytic activity was quantified after vascular injury in mice with targeted inactivation of tissue-type Plg activator (tPA-/-), urokinase-type Plg activator (uPA-/-), or Plg (Plg-/-). Neointima formation 1 week after vascular injury was impaired in uPA-/- and Plg-/- mice compared with wild-type (WT) mice or tPA-/- mice (reduction of neointimal area to 30% and 10% of WT, respectively). Cell accumulation at the borders of the injury was significantly (P<0.01) impaired compared with that in WT mice. One week after injury of the femoral artery, tPA-mediated fibrinolytic activity in arterial sections or extracts of WT, uPA-/-, or Plg-/- mice was not altered, whereas uPA activity levels in tPA-/- and Plg-/- mice were 2- to 3-fold higher than in uninjured controls. Total levels (latent plus active) of MMP-2 (gelatinase A) were increased by 2- to 4-fold, whereas the contribution of active MMP-2 represented 38% to 63% of the total in the different genotypes. MMP-9 (gelatinase B) was not detectable in the majority of control arteries, whereas total MMP-9 levels after injury were dramatically increased (up to 50-fold above the detection limit). Active MMP-9 represented 20% to 46% of total MMP-9 in WT, tPA-/-, and uPA-/- mice but was not consistently detectable in Plg-/- mice. Similar results were obtained in carotid arteries. Thus, the unaltered ratios of active and latent MMP-2 suggest that proMMP-2 activation may occur in the absence of tPA, uPA, or Plg, whereas no active MMP-9 was detected in the absence of Plg. The data of this study confirm a role for uPA and Plg but not for tPA in smooth muscle cell migration and neointima formation after vascular injury and indicate that impairment of these phenomena may occur despite the observed increases in MMP-2 or MMP-9 levels after vascular injury.

Animals↗

PubMed1998

Nucleotide structure and characterization of the murine gene encoding anticoagulant protein C.

The 15,160 bp murine gene encoding anticoagulation protein C (PC) was cloned and sequenced, including 414 bp upstream of exon 1 and 80 bp downstream of the translation stop codon. Nine exons and eight introns were identified. The first exon was untranslated and contained the major transcriptional start site, the surrounding nucleotide sequence of which matched reasonably well with the consensus eukaryotic Cap element sequence. The translational initiator methionine residue was located in exon 2. The other introns were positioned as splices between the major domain units of the protein. The 5' untranslated region contained two possible CCAAT sequences and GC boxes, but no TATA box was obvious within the optimal range of distances from the transcription start site. The 3'-flanking nucleotides included a probable polyadenylation site (ATTAAA), beginning 80 nucleotides downstream of the translation stop codon, and a downstream consensus sequence (AGTGTTTC) required for the efficient formation of a 3' terminus of mRNA. Several high probability transcription factor recognition sequences, including proteins that are enriched in, or specific to, the liver, such as C/EBP alpha, C/EBP beta, HNF1, and HNF3 beta, have been located in the 5' region of the gene. These results indicate that all elements are present for liver-based transcription of the gene for murine PC.

Amino Acid Sequence↗

PubMed1998

Feasibility study of catheter-directed thrombolysis with recombinant staphylokinase in deep venous thrombosis.

The feasibility of catheter-directed thrombolysis with recombinant staphylokinase was evaluated in six selected patients with deep vein thrombosis. The patients underwent intrathrombus infusion of recombinant staphylokinase (2 mg bolus followed by a continuous infusion of 1 mg/h). Heparin was given via the catheter as a bolus (5000 U) and as a continuous infusion (1000 U/h). Complete lysis was obtained in five patients and partial lysis in one patient. Complications consisted of minor bleeding in four subjects. Symptomatic reocclusion occurred in one. Debulking of the thrombus mass by a high speed rotating impeller (n = 1) and stenting (n = 3) were used as additional interventions. An underlying anatomical abnormality was present in two patients. Long term follow up revealed normal patency in all patients and normal valve function in four patients. Symptomatic venous insufficiency with valve dysfunction was present in the two with a second thrombotic episode. Thus catheter-directed infusion of recombinant staphylokinase in patients with deep vein thrombosis appears feasible and may be associated with a high frequency of thrombolysis. Larger studies to define the clinical benefit of this treatment appear to be warranted.

Adolescent↗

PubMed1998

Regulation of gelatinase activity in mice with targeted inactivation of components of the plasminogen/plasmin system.

To investigate a potential physiological role of the plasminogen/plasmin system in activation of the matrix metalloproteinase (MMP) system, the distribution of latent and active MMP-2 (gelatinase A) or MMP-9 (gelatinase B) was monitored in aorta extracts and in serum-free conditioned cell culture medium obtained from wild-type (WT) mice and from mice with deficiency of tissue-type plasminogen activator (t-PA(-/-)), urokinase-type plasminogen activator (u-PA(-/-)), plasminogen activator inhibitor-1 (PAI-1(-/-)) or plasminogen (Plg(-/-)). In aorta extracts, the contribution of active MMP-2 to the total MMP-2 level ranged between 7 and 16% for the different genotypes, whereas active MMP-9 was not detected. The contribution of active 58 kDa MMP-2 to the total MMP-2 level (active plus latent) ranged between 14 and 29% (mean of 3 experiments) for fibroblasts of the different genotypes, and between 18 and 32% for smooth muscle cells, and was relatively constant in time (7-72 h). The contribution of active 83 kDa MMP-9 to the total MMP-9 level ranged between 15 and 29% for fibroblasts of the different genotypes and was relatively constant in time (24-72 h); corresponding values were 17 to 57% for smooth muscle cells, with the exception of Plg(-/-) smooth muscle cells which had undetectable levels of active MMP-9. Addition of plasmin(ogen) to the cell culture medium of fibroblasts did not significantly affect the distribution of active and latent MMP-2, but resulted in an approximately two-fold enhancement of the contribution of active MMP-9. In macrophages of Plg(-/-) mice, active MMP-9 was detected only when the cells were cultured in the presence of plasminogen. These data indicate that activation of proMMP-2 occurs independently of the physiological plasminogen activators and of plasmin(ogen) in all the cell types evaluated. Activation of proMMP-9 was enhanced in the presence of plasmin(ogen), but active MMP-9 was also detected in fibroblasts of Plg(-/-) mice, indicating that in vivo activation may occur via plasmin(ogen)-independent mechanisms.

Animals↗

PubMed1998

Cloning and characterization of a cDNA encoding murine coagulation factor X.

The cDNA encoding murine coagulation factor X (fX) was isolated and reconstructed from a lambdaZap cDNA library generated from murine liver mRNA. The cDNA contains 1486 bases starting at the 5'-translation initiation codon. It includes an open reading frame of 1443 nucleotides, followed by an 18 residue 3' nontranslated sequence downstream of the first stop codon. and a 3' poly(A) tail. The translation product is composed of a 40-amino acid signal/propeptide region followed by a 441-residue mature protein. The latter is highly homologous to that of human and rat fX. All protein domains of human and rat fX are strictly conserved in mouse fX. The cDNA coding for mouse fX has been expressed in human embryonic kidney 293 cells and generates fX activity measured in a clotting assay using human fX-deficient plasma.

Amino Acid Sequence↗

PubMed1997

Effects of adenovirus-mediated human apo A-I gene transfer on neointima formation after endothelial denudation in apo E-deficient mice.

BACKGROUND: Inactivation of apolipoprotein (apo) E genes in mice markedly increases beta-VLDL levels and accelerates progression of complex atherosclerotic lesions. The present study investigated (1) the effect of apo E deficiency (apo E-/-) on neointima formation after endothelial denudation; and (2) the effect of increased HDL, induced by adenovirus-mediated transfer of a human apo A-I gene, on neointima formation. METHODS AND RESULTS: Guidewire-induced abrasion of the endothelium of the common carotid artery did not produce neointima formation within 18 days after injury in C57BL/6J mice (n=12) but was associated with an intima/media ratio of 0.82+/-0.25 in age-matched C57BL/6J apo E-/- mice (n=12). Neointima consisted primarily of smooth muscle alpha-actin positive cells. Injection in C57BL/6J apo E-/- mice of 2x10(9) (n=5) or 4x10(9) (n=7) plaque forming units (p.f.u.) of a recombinant human apo A-I adenovirus 3 days before injury resulted in an increase of HDL cholesterol from 36+/-5 to 75+/-3 mg/dL (P<.05) and to 96+/-13 mg/dL (P<.05), respectively, and of the HDL cholesterol/non-HDL cholesterol ratio from 0.063+/-0.003 to 0.15+/-0.01 (P<.05) and to 0.16+/-0.015 (P<.05), respectively. Intima/media ratio decreased to 0.28+/-0.06 (P=NS versus C57BL/6J apo E-/- mice) with 2x10(9) p.f.u. of apo A-I virus and to 0.03+/-0.01 with 4x10(9) p.f.u. (P<.01 versus C57BL/6J apo E-/- mice). Injection of 4x10(9) p.f.u. of RR5 (n=7) or tissue plasminogen activator (t-PA) control virus (n=6) did not result in a significant alteration of HDL cholesterol (44+/-11 and 26+/-4 mg/dL, respectively) nor in a reduction of intima/media ratio (0.81+/-0.35 and 0.86+/-0.23, respectively). CONCLUSIONS: Apo E deficiency is associated with increased neointima formation after endothelial denudation. Gene transfer of apo A-I increases HDL cholesterol and significantly reduces neointima formation, which suggests a direct vascular protective effect of HDL.

Adenoviridae↗

PubMed1997

Mice lacking factor VII develop normally but suffer fatal perinatal bleeding.

Blood coagulation in vivo is initiated by factor VII (FVII) binding to its cellular receptor tissue factor (TF). FVII is the only known ligand for TF, so it was expected that FVII-deficient embryos would have a similar phenotype to TF-deficient embryos, which have defective vitello-embryonic circulation and die around 9.5 days of gestation. Surprisingly, we find that FVII-deficient (FVII-/-) embryos developed normally. FVII-/- mice succumbed perinatally because of fatal haemorrhaging from normal blood vessels. At embryonic day 9.5, maternal-fetal transfer of FVII was undetectable and survival of embryos did not depend on TF-FVII-initiated fibrin formation. Thus, the TF-/- embryonic lethal and the FVII-/- survival-phenotypes suggest a role for TF during embryogenesis beyond fibrin formation.

Animals↗

PubMed1997

Inhibitory role of plasminogen activator inhibitor-1 in arterial wound healing and neointima formation: a gene targeting and gene transfer study in mice.

BACKGROUND: Plasminogen-deficient mice display impaired vascular wound healing and reduced arterial neointima formation after arterial injury, suggesting that inhibition of plasmin generation might reduce arterial neointima formation. Therefore, we studied the consequences of plasminogen activator inhibitor-1 (PAI-1) gene inactivation and adenoviral PAI-1 gene transfer on arterial neointima formation. METHODS AND RESULTS: Neointima formation was evaluated in PAI-1-deficient (PAI-1(-/-)) mice with perivascular electric or transluminal mechanical injury. PAI-1 deficiency improved vascular wound healing in both models: the cross-sectional neointimal area was 0.001+/-0.001 mm2 in PAI-1(+/+) and 0.016+/-0.008 mm2 in PAI-1(-/-) mice within 1 week after electric injury (P<.02) and 0.055+/-0.008 mm2 in PAI-1(+/+) and 0.126+/-0.006 mm2 in PAI-1(-/-) mice within 3 weeks after mechanical injury (P<.001). Proliferation of smooth muscle cells was not affected by PAI-1 deficiency. Topographic analysis of arterial wound healing after electric injury revealed that PAI-1(-/-) smooth muscle cells, originating from the uninjured borders, more rapidly migrated into the necrotic center of the arterial wound than wild-type smooth muscle cells. On the basis of immunostaining, PAI-1 expression was markedly upregulated during vascular wound healing. There were no genotypic differences in reendothelialization of the vascular wound. When PAI-1(-/-) mice were intravenously injected with replication-defective adenovirus expressing human PAI-1 (AdCMVPAI-1), plasma PAI-1 antigen levels increased in a dose-dependent fashion up to to 61+/-8 microg/mL with 2x10(9) plaque-forming units (pfu) virus. Luminal stenosis was 35+/-13% in control AdRR5-treated (2x10(9) pfu) and suppressed to 5+/-5% in AdCMVPAI-1-treated (6x10(8) pfu) PAI-1(-/-) mice (P<.002). CONCLUSIONS: By affecting cellular migration, PAI-1 plays an inhibitory role in vascular wound healing and arterial neointima formation after injury, and adenoviral PAI-1 gene transfer reduces arterial neointima formation in mice.

Adenoviridae↗

PubMed1997

Adenovirus-mediated transfer of tissue-type plasminogen activator augments thrombolysis in tissue-type plasminogen activator-deficient and plasminogen activator inhibitor-1-overexpressing mice.

Impaired fibrinolysis, resulting from increased plasminogen activator inhibitor-1 (PAI-1) or reduced tissue-type plasminogen activator (t-PA) plasma levels, may predispose the individual to subacute thrombosis in sepsis and inflammation. The objective of these studies was to show that adenovirus-mediated gene transfer could increase systemic plasma t-PA levels and thrombolytic capacity in animal model systems. Recombinant adenovirus vectors were constructed that express either human wild type or PAI-1-resistant t-PA from the cytomegalovirus (CMV) promoter. Both t-PA-deficient (t-PA(-/-)) and PAI-1-overexpressing transgenic mice were infected by intravenous injection of these viruses. Intravenous injection of recombinant adenovirus resulted in liver gene transfer, t-PA synthesis, and secretion into the plasma. Virus dose, human t-PA antigen, and activity concentrations in plasma and extent of lysis of a 125I-fibrin-labeled pulmonary embolism were all closely correlated. Plasma t-PA antigen and activity were increased approximately 1,000-fold above normal levels. Clot lysis was significantly increased in mice injected with a t-PA-expressing virus, but not in mice injected with saline or an irrelevant adenovirus. Comparable levels of enzyme activity and clot lysis were obtained with wild type and inhibitor-resistant t-PA viruses. Adenovirus-mediated t-PA gene transfer was found to augment clot lysis as early as 4 hours after infection, but expression levels subsided within 7 days. Adenovirus-mediated transfer of a t-PA gene can effectively increase plasma fibrinolytic activity and either restore (in t-PA-deficient mice) or augment (in PAI-1-overexpressing mice) the thrombolytic capacity in simple animal models of defective fibrinolysis.

Adenoviridae↗

PubMed1997

Nonisotopic quantitative analysis of protein-DNA interactions at equilibrium.

Two versions of an enzyme-linked immunosorbent assay-type method to quantify protein-DNA interactions at equilibrium were developed. The first variant comprised immobilization of DNA-binding protein on microtiter plates, incubation with biotinylated DNA, and tagging of bound DNA with streptavidin- and biotin-substituted horseradish peroxidase. In the second version, biotinylated DNA was immobilized on streptavidin-substituted microtiter plates, incubated with DNA-binding protein, and bound protein was quantified with specific antibodies. To illustrate the method, the interaction of a fusion protein between glutathione-S-transferase and the DNA-binding domain of the helicase-like transcription factor with its cis-element (the B box of the plasminogen activator inhibitor-1 promoter) was determined with both versions: a 1:1 stoichiometric interaction with an equilibrium dissociation constant (Kd) of 1 nM was found, which is similar to the value determined by electrophoretic mobility shift assay, demonstrating the validity of the assays.

Bacterial Proteins↗

PubMed1997

Epitope mapping by negative selection of randomized antigen libraries displayed on filamentous phage.

Since most antibodies directed against protein antigens recognize epitopes composed of several discontinuous segments of the polypeptide chain, attempts to delineate the amino acids constituting these epitopes with the use of linear peptides have generally been unsuccessful. Here, a method is described based on error-prone PCR, phage display and negative selection, whereby amino acid residues constituting the functional epitope are identified in the context of the native protein. First a library of randomized antigen variants containing most single, double and triple amino acid mutants generated by single nucleotide substitutions is produced by error-prone PCR amplification of the DNA sequence encoding the protein antigen. The phage-displayed library is then negatively selected for epitope loss mutants by passing through an affinity matrix derivatized with a specific antibody and positively selected for retention of function. This method was applied to the mapping of the epitopes of two murine monoclonal antibodies (MA-7H11 and MA-3G10) on staphylokinase, a 136 amino acid plasminogen activator secreted by some strains of Staphylococcus aureus. After two negative/positive selection cycles, DNA sequencing of several clones revealed preferential amino acid mutations at positions 35 and 130 (with MA-7H11), and at positions 62, 66 and 136 (with MA-3G10). Affinity measurements of staphylokinase variants carrying single amino acid mutations at these positions confirmed their contribution to the free energy of binding to MA-7H11 and MA-3G10. This approach may be useful for isolating mutants with altered antigenic or functional properties and in general to map critical regions for protein-protein interactions.

Amino Acid Sequence↗

PubMed1997

Role of the Arg123-Tyr166 paired helix of apolipoprotein A-I in lecithin:cholesterol acyltransferase activation.

The Arg123-Tyr166 central and Ala190-Gln243 carboxyl-terminal pairs of helices of apoA-I were substituted with the pair of helices of apoA-II, resulting in the apoA-I(Delta(Arg123-Tyr166), nablaA-II(Ser12-Ala75)) and apoA-I(Delta(Ala190-Gln243), nablaA-II(Ser12-Gln77)) chimeras, respectively. The structures of these chimeras in aqueous solution and in reconstituted high density lipoproteins (rHDL) and the lecithin:cholesterol acyltransferase (LCAT) activation properties of the rHDL were studied. Recombinant human apoA-I and the chimeras were expressed in Escherichia coli and purified from the periplasmic space. Binding of the apolipoproteins with palmitoyloleoylphosphatidylcholine was associated with a similar shift of Trp fluorescence maxima from 337 to 332 nm, from 339 to 334 nm, and from 337 to 333 nm, respectively. All rHDL had a Stokes radius of 4.8 nm and contained 2 apolipoprotein molecules/particle. Circular dichroism measurements revealed eight alpha-helices per apoA-I and per chimera molecule. The catalytic efficiencies of LCAT activation were 1.5 +/- 0.33 (mean +/- S.D.; n = 3), 0.054 +/- 0.009 (p < 0.001 versus apoA-I), and 1.3 +/- 0.32 (p = not significant versus apoA-I) nmol of cholesteryl ester/h/microM, respectively. The lower LCAT activity of the central domain chimera was due to a 27-fold reduced Vmax with unaltered Km. Binding of radiolabeled LCAT to rHDL of apoA-I and apoA-I(Delta(Arg123-Tyr166), nablaA-II(Ser12-Ala75)) was very similar. In conclusion, although substitution of the Arg123-Tyr166 central or Ala190-Gln243 carboxyl-terminal pair of helices of apoA-I with the pair of helices of apoA-II yields chimeras with structure similar to that of native apoA-I, exchange of the central domain (but not the carboxyl-terminal domain) of apoA-I reduces the rate of LCAT activity that is independent of binding to rHDL.

Apolipoprotein A-I↗

PubMed1997

Screening panels of monoclonal antibodies using phage-displayed antigen.

A procedure is described to screen panels of hybridomas or purified monoclonal antibodies using antigen displayed on the surface of filamentous bacteriophage. In this system, samples containing murine monoclonal antibodies are incubated with phage-displayed antigen in microtiter plates coated with rabbit anti-mouse IgG, and bound antibody-phage complex is detected with horseradish peroxidase-sheep anti-phage M13 conjugate. The assay has been validated with a panel of 16 monoclonal antibodies directed against human plasminogen, using phage-displayed miniplasmin-(ogen) (amino acids Ala444 through Asn791 comprising kringle 5 and the proteinase domain of plasminogen) or microplasminogen (amino acids Ala543 through Asn791 comprising the proteinase domain). Six monoclonal antibodies were identified directed against miniplasminogen and miniplasmin; this was confirmed using a microtiter plate coated with antigens. One of these monoclonal antibodies (MA-42B12) did not react with microplasminogen, suggesting that its epitope is comprised within the kringle 5 domain. This test is rapid and sensitive (detecting 10-20 ng/ml of monoclonal antibody), and screening can be performed using phage-displayed zymogens or active enzymes or selected domains thereof. The procedure eliminates the need for large amounts of purified antigen for screening. Furthermore, immunization can be performed with partially purified antigen because only antibodies raised against the antigen of interest will be identified with the use of phage-displayed antigen. Therefore, this test may offer distinct advantages over the classical one-site enzyme-linked immunosorbent assay using antigen-coated microtiter plates.

Animals↗

PubMed1997

Role of the carboxy-terminal domain of human apolipoprotein AI in high-density-lipoprotein metabolism--a study based on deletion and substitution variants in transgenic mice.

Cholesterol levels in high-density lipoprotein (HDL) of transgenic mice overexpressing human apolipoprotein AI (apoAI), a des-(190-243)-apoAI deletion mutant or an apoAI-(1-189)-apoAII-(12-77) chimera were 2.8-fold (P<0.001), 1.3-fold (P<0.05) and 2.2-fold (P<0.001) higher than in control mice, respectively. Human apolipoprotein levels in apoAI and in apoAI-(1-189)-apoAII-(12-77) transgenic mice were 5.2-fold and 3.5-fold higher than in des-(190-243)-apoAI transgenic mice, whereas their HDL cholesterol levels were 2.1-fold and 1.6-fold higher. PAGE of HDL isolated by ultracentrifugation revealed that murine HDL migrated as 9.6-nm and 7.2-nm particles. Overexpression of human apoAI and apoAI-(1-189)-apoAII-(12-77) resulted in the production of polydisperse HDL (9.6, 9.2, 8.4 and 7.2 nm) particles, whereas overexpression of des-(190-243)-apoAI primarily resulted in an increase of 7.2-nm particles. The fractional catabolic rates of human apoAI and apoAI-(1-189)-apoAII-(12-77) were very similar, whereas that of des-(190-243)-apoAI was 4.9-fold higher. The endogenous production rates of human apoAI, des-(190-243)-apoAI and apoAI-(1-189)-apoAII-(12-77) in transgenic mice were very similar. It is concluded that deletion of the carboxy-terminal domain of apoAI reduces its lipoprotein association, resulting in the production of small, phospholipid-rich HDL particles that are cleared more rapidly. Substitution of the carboxy-terminal helices of apoAI with helices of apoAII restores lipoprotein association, resulting in the production of HDL, which migrates as human HDL3 and HDL2. Although the carboxy-terminal domain of the chimera contained more than 80% of the amino acid sequence of apoAII, its HDL-distribution profile in transgenic mice was very similar to that of human apoAI. This study demonstrates the importance of the helical structure of apoAI of the carboxy-terminal domain of apoAI, rather than of its exclusive amino acid sequence, in HDL metabolism.

Animals↗

PubMed1997

Insights in vessel development and vascular disorders using targeted inactivation and transfer of vascular endothelial growth factor, the tissue factor receptor, and the plasminogen system.

VEGF has been proposed to participate in normal and pathological vessel formation. Surprisingly, lack of only a single VEGF allele resulted in embryonic lethality due to abnormal formation of intra- and extra-embryonic vessels. Homozygous VEGF-deficient embryos, generated by tetraploid aggregation, revealed an even more severe defect in vessel formation. These results (1) suggest a tight regulation of early vessel development by VEGF and, indirectly, the presence of other VEGF-like molecules; (2) reveal an unprecedented lethal phenotype associated with heterozygous deficiency of an autosomal gene, and (3) demonstrate that tetraploid aggregation was a valid and the only method to study the phenotype of the homozyogous VEGF-deficient embryos. The dominant and strict dose-dependent role of VEGF in vivo renders this molecule a desirable therapeutic target for promoting or preventing angiogenesis. Tissue factor (TF) is the principal cellular initiator of coagulation and its deregulated expression has been related to thrombogenesis in sepsis, cancer, and inflammation. However, TF appears to be also involved in a variety of non-hemostatic functions including inflammation, cancer, brain function, immune response, and tumor-associated angiogenesis. Surprisingly, TF deficiency resulted in embryonic lethality due to abnormal extra-embryonic vessel development and defective vitelloembryonic circulation. The abnormal yolk sac vasculature is reminiscent of that observed in embryos lacking VEGF, possibly suggesting that both gene functions are interconnected. These targeting studies extend the recently documented role of TF in tumor-associated angiogenesis and warrant further study of its role in angiogenesis during other pathological disorders. The plasminogen system, via its triggers, tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), has been implicated in thrombosis, arterial neointima formation, and atherosclerosis. Studies in mice with targeted gene inactivation of t-PA, u-PA, PAI-1, the urokinase receptor (u-PAR), and plasminogen (Plg) revealed (1) that deficiency of t-PA or u-PA increase the susceptibility to thrombosis associated with inflammation and that combined deficiency of t-PA:u-PA or deficiency of Plg induces severe spontaneous thrombosis; (2) that vascular injury-induced neointima formation is reduced in mice lacking u-PA-mediated plasmin proteolysis, unaltered in t-PA- or u-PAR-deficient mice and accelerated in PAI-1-deficient mice, but that it can be reverted by adenoviral PAI-1 gene transfer; and (3) that atherosclerosis in mice doubly deficient in apolipoprotein E (apoE) and PAI-1 is reduced after 10 weeks of cholesterol-rich diet. Thus, the plasminogen system significantly affects thrombosis, restenosis, and atherosclerosis.

Animals↗

PubMed1997

Enzymatic properties of phage-displayed fragments of human plasminogen.

Two low-molecular-mass forms of human plasminogen, plasminogen-(543-791)-peptide (micro-plasminogen), comprising the serine protease domain, and plasminogen-(444-791)-peptide (mini-plasminogen), which in addition contains kringle 5, were displayed on filamentous phage by fusion to the N-terminus of the minor coat protein pIII, to levels of 0.5 molecules micro-plasminogen-pIII/phage particle and 0.1 molecules mini-plasminogen-pIII/phage particle. The proenzymes, quantitatively activated by urokinase, showed catalytic efficiencies that were virtually identical to their soluble counterparts, and activity remained associated with the phage as demonstrated by phage ELISA and biopanning with human alpha2-antiplasmin or the inhibitor Phe-Pro-Arg-CH2Cl. Micro-plasminogen-pIII was activated by streptokinase and staphylokinase, two non-enzymatic plasminogen activators, to the same extent as by urokinase. Activated forms of mini-plasminogen-pIII micro-plasminogen-pIII and mini-plasminogen dissolved 125I-labelled fibrin films in a dose-dependent time-dependent manner, with 50% lysis in 20 h requiring 0.52, 3.2 and 0.46 nM active plasmin, respectively. Thus, proenzyme moieties derived from plasminogen can be successfully displayed on phage with maintenance of their enzymatic properties. The micro-plasminogen and mini-plasminogen phage-display systems may be useful to study mechanisms of plasminogen activation.

Amino Acid Sequence↗

PubMed1997

Plasminogen and plasminogen activators protect against renal injury in crescentic glomerulonephritis.

The plasminogen/plasmin system has the potential to affect the outcome of inflammatory diseases by regulating accumulation of fibrin and other matrix proteins. In human and experimental crescentic glomerulonephritis (GN), fibrin is an important mediator of glomerular injury and renal impairment. Glomerular deposition of matrix proteins is a feature of progressive disease. To study the role of plasminogen and plasminogen activators in the development of inflammatory glomerular injury, GN was induced in mice in which the genes for these proteins had been disrupted by homologous recombination. Deficiency of plasminogen or combined deficiency of tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA) was associated with severe functional and histological exacerbation of glomerular injury. Deficiency of tPA, the predominant plasminogen activator expressed in glomeruli, also exacerbated disease. uPA deficiency reduced glomerular macrophage infiltration and did not significantly exacerbate disease. uPA receptor deficiency did not effect the expression of GN. These studies demonstrate that plasminogen plays an important role in protecting the glomerulus from acute inflammatory injury and that tPA is the major protective plasminogen activator.

Animals↗

PubMed1997

Staphylokinase requires NH2-terminal proteolysis for plasminogen activation.

Staphylokinase (Sak), a single-chain protein comprising 136 amino acids with NH2-terminal sequence,SSSFDKGKYKKGDDA forms a complex with plasmin, that is endowed with plasminogen activating properties. Plasmin is presumed to process mature (high molecular weight, HMW) Sak to low molecular weight derivatives (LMW-Sak), primarily by hydrolyzing the Lys10-Lys11 peptide bond, but the kinetics of plasminogen activation by HMW-Sak and LMW-Sak are very similar. Here, the requirement of NH2-terminal proteolysis of Sak for the induction of plasminogen activating potential was studied by mutagenesis of Lys10 and Lys11 in combination with NH2-terminal microsequence analysis of equimolar mixtures of Sak and plasminogen and determination of kinetic parameters of plasminogen activation by catalytic amounts of Sak. Substitution of Lys10 with Arg did not affect processing of the Arg10-Lys11 site nor plasminogen activation, whereas substitution with His resulted in cleavage of the Lys11-Gly12 peptide bond and abolished plasminogen activation. Substitution of Lys11 with Arg did not affect Lys10-Arg11 processing or plasminogen activation, whereas replacement with His did not prevent Lys10-His11 hydrolysis but abolished plasminogen activation. Substitution of Lys11 with Cys yielded an inactive processed derivative which was fully activated by aminoethylation. Deletion of the 10 NH2-terminal amino acids did not affect plasminogen activation, but additional deletion of Lys11 eliminated plasminogen activation. Thus generation of plasminogen activator potential in Sak proceeds via plasmin-mediated removal of the 10 NH2-terminal amino acids with exposure of Lys11 as the new NH2 terminus. This provides a structural basis for the hypothesis, derived from kinetic measurements, that plasminogen activation by Sak needs to be primed by plasmin and a mechanism for the high fibrin selectivity of Sak in a plasma milieu.

Amino Acid Sequence↗