Biomedical subjects

Daniel A Lawrence

Publications and source records attributed to Daniel A Lawrence.

33 records · Page 2Linked to original sources

PubMed2003

Old dogs and new tricks: proteases, inhibitors, and cell migration.

A new model for the actions of plasminogen activator inhibitors (PAIs) on cell migration may resolve the conflicting research data on these proteins in metastasis and angiogenesis. Results from two groups reveal a role for PAI-1 in promoting cycles of attachment and detachment of the cell from the extracellular matrix that is independent of its role as an enzymatic inhibitor of urokinase-type plasminogen activator (uPA). Through the formation of a complex of integrins, uPA and its receptor, and the clearance receptors of the low-density lipoprotein family, PAI-1 may promote endocytosis and recycling of these adhesion-controlling proteins, allowing cycling of cellular attachment and detachment.

Animals↗

PubMed2003

Impaired fibrinolysis in multiple sclerosis: a role for tissue plasminogen activator inhibitors.

Tissue plasminogen activator (tPA), a neuronal as well as the key fibrinolytic enzyme, is found concentrated on demyelinated axons in multiple sclerosis lesions together with fibrin(ogen) deposits. The decreased tPA activity in normal-appearing white and grey matter and lesions of multiple sclerosis is reflected in diminished fibrinolysis as measured by a clot lysis assay. Nonetheless, peptide products of fibrin, including D-dimer, accumulate on demyelinated axons-the result of fibrinogen entry through a compromised blood-brain barrier (BBB). Analysis of tissue samples on reducing and non-reducing polyacrylamide gels demonstrates complexes of tPA with plasminogen activator inhibitor-1 (PAI-1) but not with neuroserpin, a tPA-specific inhibitor concentrated in grey matter. As total tPA protein remains unchanged in acute lesions and the concentration of PAI-1 rises several fold, complex formation is a probable cause of the impaired fibrinolysis. Although the tPA-plasmin cascade promotes neurodegeneration in excitotoxin-induced neuronal death, in inflammatory conditions with BBB disruption it has been demonstrated to have a protective role in removing fibrin, which exacerbates axonal injury. The impaired fibrinolytic capacity resulting from increased PAI-1 synthesis and complex formation with tPA, which is detectable prior to lesion formation, therefore has the potential to contribute to axonal damage in multiple sclerosis.

Adult↗

PubMed2003

Sequences within domain II of the urokinase receptor critical for differential ligand recognition.

The receptor for urokinase-type plasminogen activator (uPAR) plays important roles in a number of physiological and pathological processes by virtue of its interactions with urokinase-type plasminogen activator (uPA), vitronectin (Vn), and several other proteins. The uPA binding site spans all three domains (D1 to D3) of uPAR. However, the nature of the Vn binding site within uPAR is still not clear. In this study, we conducted homolog-scanning mutagenesis on uPAR by switching 14 individual segments of 4-8 residues to their counterpart sequences of a uPAR homolog CD59. All 14 mutants were well expressed, reacted with a panel of monoclonal antibodies, and exhibited correct molecular weights. Of these 14 mutants, six mutants were defective in both uPA and Vn binding. Most importantly, we found two unique mutants uPAR(Asn172-Lys175) and uPAR(Glu183-Asn186) within the D2 domain, which displayed differential ligand binding activity: both had high affinity uPA binding, but completely lost Vn binding, indicating that these two sequences constitute a novel Vn binding site. Indeed, two peptides, P1 (153CPGSNGFHNNDTFHFLKC) and P2 (171CNTTKCNEGPILELENLPQ), derived from the sequences of the identified uPA and Vn binding pockets within D2, respectively, behaved like bona fide ligand binding sites: peptide P1 bound uPA but not Vn, whereas peptide P2 bound Vn and inhibited uPAR-mediated cell adhesion, but did not interact with uPA. Altogether, our data demonstrated that uPAR D2 contains two distinct ligand binding sites for uPA and Vn. Such information will help us better understand the complex roles of uPAR in cell adhesion, migration, and tumor metastasis.

Amino Acid Sequence↗

PubMed2003

Mapping of a conformational epitope on plasminogen activator inhibitor-1 by random mutagenesis. Implications for serpin function.

The mechanism for the conversion of plasminogen activator inhibitor-1 (PAI-1) from the active to the latent conformation is not well understood. Recently, a monoclonal antibody, 33B8, was described that rapidly converts PAI-1 to the latent conformation (Verhamme, I., Kvassman, J. O., Day, D., Debrock, S., Vleugels, N., Declerck, P. J., and Shore, J. D. (1999) J. Biol. Chem. 274, 17511-17517). In an attempt to understand this interaction, and more broadly to understand the mechanism of the natural transition of PAI-1 to the latent conformation, we have used random mutagenesis to identify the 33B8 epitope in PAI-1. This site involves at least 8 amino acids scattered over more than two-thirds of the linear sequence that form a compact epitope on the PAI-1 three-dimensional structure. Surface plasmon resonance studies indicate a high affinity interaction between latent PAI-1 and 33B8 that is approximately 100-fold higher than comparable binding to active PAI-1. Structural modeling results together with surface plasmon resonance analysis of parental and site-directed PAI-1 mutants with disrupted 33B8 binding suggest the existence of a specific PAI-1 intermediate structure that is stabilized by 33B8 binding. These analyses strongly suggest that this intermediate form of PAI-1 has a partial insertion of the reactive center loop into beta-sheet A, and together, these data have significant implications for the general serpin mechanism of proteinase inhibition.

Animals↗

PubMed2003

Progressive ankylosis (Ank) protein is expressed by neurons and Ank immunohistochemical reactivity is increased by limbic seizures.

Ank is a 492-amino acid multipass transmembrane protein involved in the regulation of extracellular inorganic pyrophosphate levels and the control of tissue calcification. Previous Northern blot hybridization experiments revealed that Ank mRNA was expressed in the brain, but there have been no reports describing the anatomical sites or specific cell types in the brain that express Ank protein. In this study, we demonstrate that Ank is expressed primarily in human brain neurons, with the highest levels of expression observed in the thalamus, the III and V cortical layers, the Purkinje cells of the cerebellum, clusters of cells in the dorsal portion of the pons and midbrain, and neurons of the anterior horn of the spinal cord. In primary mouse neuronal cell cultures, Ank is detected on both the cell body and on cell extensions, mainly dendrites. In the rat brain, Ank mRNA is expressed at relatively high levels in the thalamus, midbrain, and spinal cord, and the Ank protein expression pattern is similar to that observed in the human brain. Finally, we observed a significant increase in Ank immunoreactivity in the rat amygdala, the CA-2 and CA-3 layers of the hippocampus, and the cerebral cortex after the induction of seizure activity. Ank regulation of ATP and/or inorganic pyrophosphate release from neurons may function to modulate the membrane excitability and cell death associated with seizure activity.

Adult↗

PubMed2003

A mutant, noninhibitory plasminogen activator inhibitor type 1 decreases matrix accumulation in experimental glomerulonephritis.

In fibrotic renal disease, elevated TGF-beta and angiotensin II lead to increased plasminogen activator inhibitor type 1 (PAI-1). PAI-1 appears to reduce glomerular mesangial matrix turnover by inhibiting plasminogen activators, thereby decreasing plasmin generation and plasmin-mediated matrix degradation. We hypothesized that therapy with a mutant human PAI-1 (PAI-1R) that binds to matrix vitronectin but does not inhibit plasminogen activators, would enhance plasmin generation, increase matrix turnover, and decrease matrix accumulation in experimental glomerulonephritis. Three experimental groups included normal, untreated disease control, and PAI-1R-treated nephritic rats. Plasmin generation by isolated day 3 glomeruli was dramatically decreased by 69%, a decrease that was reversed 43% (P < 0.02) by in vivo PAI-1R treatment. At day 6, animals treated with PAI-1R showed significant reductions in proteinuria (48%, P < 0.02), glomerular staining for periodic acid-Schiff positive material (33%, P < 0.02), collagen I (28%, P < 0.01), collagen III (34%, P < 0.01), fibronectin (48%, P < 0.01), and laminin (41%, P < 0.01), and in collagen I (P < 0.01) and fibronectin mRNA levels (P < 0.02). Treatment did not alter overexpression of TGF-beta1 and PAI-1 mRNAs, although TGF-beta1 protein was significantly reduced. These observations strongly support our hypothesis that PAI-1R reduces glomerulosclerosis by competing with endogenous PAI-1, restoring plasmin generation, inhibiting inflammatory cell infiltration, decreasing local TGF-beta1 concentration, and reducing matrix accumulation.

Animals↗

PubMed2003

Tissue-type plasminogen activator induces opening of the blood-brain barrier via the LDL receptor-related protein.

The regulation of cerebrovascular permeability is critical for normal brain homeostasis, and the "breakdown" of the blood-brain barrier (BBB) is associated with the development of vasogenic edema and intracranial hypertension in a number of neurological disorders. In this study we demonstrate that an increase in endogenous tissue-type plasminogen activator (tPA) activity in the perivascular tissue following cerebral ischemia induces opening of the BBB via a mechanism that is independent of both plasminogen (Plg) and MMP-9. We also show that injection of tPA into the cerebrospinal fluid in the absence of ischemia results in a rapid dose-dependent increase in vascular permeability. This activity is not seen with urokinase-type Plg activator (uPA) but is induced in Plg-/- mice, confirming that the effect is Plg-independent. However, the activity is blocked by antibodies to the LDL receptor-related protein (LRP) and by the LRP antagonist, receptor-associated protein (RAP), suggesting a receptor-mediated process. Together these studies demonstrate that tPA is both necessary and sufficient to directly increase vascular permeability in the early stages of BBB opening, and suggest that this occurs through a receptor-mediated cell signaling event and not through generalized degradation of the vascular basement membrane.

Animals↗

PubMed2003

Plasminogen activator inhibitor-1 in tumor growth, angiogenesis and vascular remodeling.

Plasminogen activator inhibitor-1 (PAI-1) is the principal inhibitor of urokinase type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), and as such is thought to play an important role in the regulation of extracellular matrix remodeling. In blood, PAI-1 is bound to the adhesion protein vitronectin and is associated with vitronectin in fibrin clots and the provisional matrix. Elevated levels of PAI-1 are associated with atherosclerosis and an increased thrombotic tendency, while PAI-1 deficiency leads to increased fibrinolysis and bleeding. PAI-1 is also elevated in many solid tumors and is associated with a poor prognosis in cancer. PAI-1 has been shown to be a potent regulator of both vascular cell migration in vitro and of angiogenesis and tumor growth in vivo. PAI-1 can both promote and inhibit tumor growth and angiogenesis. Low concentrations of PAI-1 can stimulate tumor angiogenesis while treatment of animals with high doses of PAI-1 inhibits angiogenesis and tumor growth. Hence, PAI-1 appears to have a multifunctional role in regulating the migratory and fibrinolytic activity of vascular cells, and this, in turn, may help to explain the many varied actions of PAI-1.

Animals↗

PubMed2002

Plasminogen promotes sarcoma growth and suppresses the accumulation of tumor-infiltrating macrophages.

The specific functions of plasminogen, stromal plasminogen activator, stromal plasminogen activator receptor, and stromal plasminogen activator inhibitor in the progression of the murine soft tissue sarcoma, T241 were investigated. Negation of plasminogen to the tumor blunted the orthotopic growth of the sarcoma in syngeneic mice. The reduced tumor growth was associated with a dramatic increase in tumor-infiltrating F4/80-positive macrophages and a diminution of vessel density, but not with obvious differences in fibrin and collagen deposition, or invasiveness of the tumor. Ablation of plasminogen activation by the tumor stroma only modestly impaired the prolonged growth of the sarcoma, suggesting that tumor cell-produced plasminogen activator is sufficient to mediate productive plasminogen activation. Plasminogen facilitated sarcoma progression, angiogenesis, and suppression of macrophage infiltration in the absence of either stromal urokinase plasminogen activator receptor or stromal plasminogen activator inhibitor. These data demonstrate that tumor cell-produced plasminogen activator and host plasminogen cooperate to facilitate soft tissue sarcoma growth and suppress the accumulation of tumor-infiltrating macrophages.

Animals↗

PubMed2002

Acyl-enzyme complexes between tissue-type plasminogen activator and neuroserpin are short-lived in vitro.

The serine protease tissue-type plasminogen activator (t-PA) initiates the fibrinolytic protease cascade and plays a significant role in motor learning, memory, and neuronal cell death induced by excitotoxin and ischemia. In the fibrinolytic system, the serpin PAI-1 negatively regulates the enzymatic activity of both single-chain and two-chain t-PA (sct-PA and tct-PA). In the central nervous system, neuroserpin (NSP) is a serpin thought to regulate t-PA enzymatic activity. We report that although both sct-PA and tct-PA rapidly form acyl-enzyme complexes with NSP in vitro, the interactions are short-lived, rapidly progressing to complete cleavage of NSP and regeneration of fully active enzyme. All NSP molecules appear to transit through the detectable acyl-enzyme intermediate and progress to completion of cleavage; no subpopulation that functions as a pure substrate was detected. Likewise, all molecules were reactive, with no evidence of a latent subpopulation. The interactions between NSP and t-PA were distinct from those between plasmin and NSP, wherein the same peptide bond was cleaved but there was no evidence of a detectable plasmin-NSP acyl-enzyme complex. The interactions between t-PA and NSP contrast with the formation of long-lived, physiologically irreversible acyl-enzyme complexes between t-PA and PAI-1, suggesting that the physiologic effect of t-PA-NSP interactions may be more complex than previously thought.

Binding Sites↗

PubMed2002

Adjuvant treatment with neuroserpin increases the therapeutic window for tissue-type plasminogen activator administration in a rat model of embolic stroke.

BACKGROUND: After stroke, the thrombolytic effect of tissue-type plasminogen activator (tPA) in the intravascular space is beneficial, whereas its extravascular effect on ischemic neurons is deleterious. We tested the hypothesis that neuroserpin, a natural inhibitor of tPA, reduces tPA-induced neuronal toxicity and increases its therapeutic window for treatment of embolic stroke. METHODS AND RESULTS: Rats were subjected to embolic middle cerebral artery occlusion (MCAO). Ischemic brains were treated with neuroserpin in combination with recombinant human tPA (n=7), tPA alone (n=7), or saline (n=9). Neuroserpin (20 micro L of 16 micro mol/L active neuroserpin) was intracisternally injected 3 hours and tPA (10 mg/kg) was intravenously administered 4 hours after ischemia. MRI measurements were performed to study blood brain barrier (BBB) leakage and ischemic lesion volume. Administration of tPA alone 4 hours after ischemia significantly (P<0.05) increased BBB leakage in the ischemic core measured by Gd-DTPA-enhanced MRI compared with rats treated with saline. However, treatment with neuroserpin in combination with tPA significantly (P<0.05) reduced BBB leakage, brain edema, and ischemic lesion volume compared with rats treated with tPA alone, although ischemic lesion volumes were the same in both groups before the treatment. Immunostaining revealed that MCAO resulted in reduction of neuroserpin immunoreactivity in the ipsilateral hemisphere after 2 to 6 hours of ischemia. Zymographic assay showed increased plasminogen activity in areas with BBB leakage in rats treated with tPA. CONCLUSIONS: Administration of neuroserpin after stroke is neuroprotective, seemingly because it blocks the extravascular effect of tPA, leading to subsequent decrease in stroke volume and widening of the therapeutic window for the thrombolytic effect of tPA.

Adjuvants, Pharmaceutic↗

PubMed2002

Association between conformational mutations in neuroserpin and onset and severity of dementia.

BACKGROUND: The aggregation of specific proteins is a common feature of the familial dementias, but whether the formation of neuronal inclusion bodies is a causative or incidental factor in the disease is not known. To clarify this issue, we investigated five families with typical neuroserpin inclusion bodies but with various neurological manifestations. METHODS: Five families with neurodegenerative disease and typical neuronal inclusions had biopsy or autopsy material available for further examination. Immunostaining confirmed that the inclusions were formed of neuroserpin aggregates, and the responsible mutations in neuroserpin were identified by sequencing of the neuroserpin gene (SERPINI1) in DNA from blood samples or from extraction of histology specimens. Molecular modelling techniques were used to predict the effect of the gene mutations on three-dimensional protein structure. Brain sections were stained and the topographic distribution of the neuroserpin inclusions plotted. FINDINGS: Each of the families was heterozygous for an amino acid substitution that affected the conformational stability of neuroserpin. The least disruptive of these mutations (S49P), as predicted by molecular modelling, resulted in dementia after age 45 years, and presence of neuroserpin inclusions in only a few neurons. By contrast, the most severely disruptive mutation (G392E) resulted, at age 13 years, in progressive myoclonus epilepsy, with many inclusions present in almost all neurons. INTERPRETATION: The findings provide evidence that inclusion-body formation is in itself a sufficient cause of neurodegeneration, and that the onset and severity of the disease is associated with the rate and magnitude of neuronal protein aggregation.

Adolescent↗

PubMed2002

Platelet-derived growth factor (PDGF)-induced tyrosine phosphorylation of the low density lipoprotein receptor-related protein (LRP). Evidence for integrated co-receptor function betwenn LRP and the PDGF.

The low density lipoprotein receptor-related protein (LRP) functions in the catabolism of numerous ligands including proteinases, proteinase inhibitor complexes, and lipoproteins. In the current study we provide evidence indicating an expanded role for LRP in modulating cellular signaling events. Our results show that platelet-derived growth factor (PDGF) BB induces a transient tyrosine phosphorylation of the LRP cytoplasmic domain in a process dependent on PDGF receptor activation and c-Src family kinase activity. Other growth factors, including basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-1, were unable to mediate tyrosine phosphorylation of LRP. The basis for this selectivity may result from the ability of LRP to bind PDGFBB, because surface plasmon resonance experiments demonstrated that only PDGF, and not basic fibroblast growth factor, epidermal growth factor, or insulin-like growth factor-1, bound to purified LRP immobilized on a sensor chip. The use of LRP mini-receptor mutants as well as in vitro phosphorylation studies demonstrated that the tyrosine located within the second NPXY motif found in the LRP cytoplasmic domain is the primary site of tyrosine phosphorylation by Src and Src family kinases. Co-immunoprecipitation experiments revealed that PDGF-mediated tyrosine phosphorylation of LRPs cytoplasmic domain results in increased association of the adaptor protein Shc with LRP and that Shc recognizes the second NPXY motif within LRPs cytoplasmic domain. In the accompanying paper, Boucher et al. (Boucher, P., Liu, P. V., Gotthardt, M., Hiesberger, T., Anderson, R. G. W., and Herz, J. (2002) J. Biol. Chem. 275, 15507-15513) reveal that LRP is found in caveolae along with the PDGF receptor. Together, these studies suggest that LRP functions as a co-receptor that modulates signal transduction pathways initiated by the PDGF receptor.

Amino Acid Sequence↗

PubMed2002

Structural similarity of the covalent complexes formed between the serpin plasminogen activator inhibitor-1 and the arginine-specific proteinases trypsin, LMW u-PA, HMW u-PA, and t-PA: use of site-specific fluorescent probes of local environment.

We have used two fluorescent probes, NBD and dansyl, attached site-specifically to the serpin plasminogen activator inhibitor-1 (PAI-1) to address the question of whether a common mechanism of proteinase translocation and full insertion of the reactive center loop is used by PAI-1 when it forms covalent SDS-stable complexes with four arginine-specific proteinases, which differ markedly in size and domain composition. Single-cysteine residues were incorporated at position 119 or 302 as sites for specific reporter labeling. These are positions approximately 30 A apart that allow discrimination between different types of complex structure. Fluorescent derivatives were prepared for each of these variants using both NBD and dansyl as reporters of local perturbations. Spectra of native and cleaved forms also allowed discrimination between direct proteinase-induced changes and effects solely due to conformational change within the serpin. Covalent complexes of these derivatized PAI-1 species were made with the proteinases trypsin, LMW u-PA, HMW u-PA, and t-PA. Whereas only minor perturbations of either NBD and dansyl were found for almost all complexes when label was at position 119, major perturbations in both wavelength maximum (blue shifts) and quantum yield (both increases and decreases) were found for all complexes for both NBD and dansyl at position 302. This is consistent with all four complexes having similar location of the proteinase catalytic domain and hence with all four using the same mechanism of full-loop insertion with consequent distortion of the proteinase wedged in at the bottom of the serpin.

Endopeptidases↗

PubMed2002

Regulation of seizure spreading by neuroserpin and tissue-type plasminogen activator is plasminogen-independent.

Tissue-type plasminogen activator (tPA) is a highly specific serine proteinase expressed in the CNS during events that require neuronal plasticity. In this study we demonstrate that endogenous tPA mediates the progression of kainic acid-induced (KA-induced) seizures by promoting the synchronization of neuronal activity required for seizure spreading, and that, unlike KA-induced cell death, this activity is plasminogen-independent. Specifically, seizure induction by KA injection into the amygdala induces tPA activity and cell death in both hippocampi, and unilateral treatment of rats with neuroserpin, a natural inhibitor of tPA in the brain, enhances neuronal survival in both hippocampi. Inhibition of tPA within the hippocampus by neuroserpin treatment does not prevent seizure onset but instead markedly delays the progression of seizure activity in both rats and wild-type mice. In tPA-deficient mice, seizure progression is significantly delayed, and neuroserpin treatment does not further delay seizure spreading. In contrast, plasminogen-deficient mice show a pattern of seizure spreading and a response to neuroserpin that is similar to that of wild-type animals. These findings indicate that tPA acts on a substrate other than plasminogen and that the effects of neuroserpin on seizure progression and neuronal cell survival are mediated through the inhibition of tPA.

Amygdala↗