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ABSTRACT: Plasminogen Activator Inhibitor-1 (PAI-1) is a member of the SERine Protease INhibitor (SERPIN) superfamily and is the main physiological inhibitor of urokinase (uPA) and tissue-type plasminogen activator (tPA) [1]. Studies using PAI-1 deficient (PAI-1(-/-) ) mice, have demonstrated that PAI-1 not only regulates the fibrinolytic system but also modulates other physiological and pathophysiological processes, including inflammation, angiogenesis, tumor growth, and cardiovascular disease [2-6]. Regions of human PAI-1 that are critical for inhibition of plasminogen activation, binding to vitronectin (VN), and binding to Low-density lipoprotein Receptor-related Protein (LRP) have been identified [7-9]. © 2012 International Society on Thrombosis and Haemostasis.
Journal of Thrombosis and Haemostasis 10/2012; · 5.73 Impact Factor
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ABSTRACT: Conantokins are naturally-occurring small peptide antagonists of ion flow through NMDA/glycine activated-N-methyl-d-aspartate receptor (NMDAR) ion channels. One member of the conantokin family, conantokin (con)-G, a 17-residue peptide, is selective for NMDARs containing the N-methyl-d-aspartate receptor subunit 2 B (NR2B), whereas the homologous peptides, con-T and con-R, show broader selectivity for NR2 subunits. In this study, con-G, con-R, and con-T variants were chemically synthesized and employed to investigate their subunit selectivities as inhibitors of agonist-evoked ion currents in human embryonic kidney-293 (HEK-293) cells expressing various combinations of NMDAR subunits that contain NR1a or NR1b combined with NR2A or NR2B. Using truncation and point mutants, as well as chimeric conantokins, we determined that the N-terminus of con-G contains all the determinants for NR2B selectivity. With this information, a large number of (con) variants were synthesized and used to establish minimal sequence determinants for selectivity. Tyr at position 5 broadens the NR2 selectivity, and recovery of NR2B selectivity in Tyr5 peptides was achieved by incorporating Ala or Gly at position 8. NR2B selectivity in con-R can be conferred through deletion of the Ala at position 10, thereby shifting the γ-carboxyglutamate (Gla) from position 11 to position 10, where a Gla naturally occurs in con-G and con-T. The nature of the amino acid at position 6 is also linked to subunit selectivity. Our studies suggest that the molecular determinants of conantokins that dictate NMDAR subunit selectivity are housed in specific residues of the N-termini of these peptides. Thus, it is possible to engineer desired NMDAR functional properties into conantokin-based peptides.
Neuroscience 10/2010; 170(3):703-10. · 3.38 Impact Factor
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Journal of Thrombosis and Haemostasis 10/2010; 8(10):2341-4. · 5.73 Impact Factor
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ABSTRACT: Alterations in expression of protein C (PC) pathway components have been identified in patients with active inflammatory disease states. While the PC pathway plays a pivotal role in regulating coagulation and fibrinolysis, activated PC (aPC) also exhibits cytoprotective properties. For example, PC-deficient mice challenged in septic/endotoxemic models exhibit phenotypes that include hypotension, disseminated intravascular coagulation, elevated inflammatory mediators, neutrophil adhesion to the microvascular endothelium, and loss of protective endothelial and epithelial cell barriers. Further, inflammatory bowel disease has been correlated with diminished endothelial PC receptor and thrombomodulin levels in the intestinal mucosa. Downregulated expression of the cofactor, protein S, as well as PC, is also associated with ischemic stroke. Studies to elucidate further the structural elements that differentiate the various functions of PC will serve to identify novel therapeutic approaches toward regulating these and other diseases.
Journal of Thrombosis and Haemostasis 08/2009; 7 Suppl 1:140-5. · 5.73 Impact Factor
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Journal of Thrombosis and Haemostasis 09/2008; 6(11):1993-5. · 5.73 Impact Factor
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ABSTRACT: The interaction of urokinase-type plasminogen activator (uPA) and its receptor, uPAR, on cell surfaces facilitates the generation of cell-bound plasmin, thus allowing cells to establish a proteolytic front that enables their migration through protein barriers. This complex also activates cell signalling pathways that influence cell functions. Clinical studies have identified uPA as an indicator of poor overall survival in patients with colorectal cancer. In the current study, a mouse model of colon cancer, Apc(Min/+), with an additional deficiency of uPA (Apc(Min/+)/Plau-/-) was used to determine the effects of uPA on tumour initiation and growth. Utilizing this model, it was found that the number of tumours was diminished in these mice relative to Apc(Min/+) mice, which correlated with the decreased leukocyte infiltration in the tumours. However, tumour growth was not impeded in Apc(Min/+)/Plau-/- mice, and proliferation and tumour vascularization were, in fact, enhanced in Apc(Min/+)/Plau-/- mice. These latter effects are consistent with a mechanism involving up-regulation of COX-2 expression and Akt pathway activation in Apc(Min/+)/Plau-/- mice. The results from this study suggest that uPA plays dual and opposing roles in regulating lesion development: one early, during the transition from normal epithelia to dysplastic lesions, and another later during tumour growth.
The Journal of Pathology 12/2007; 213(3):266-74. · 6.32 Impact Factor
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ABSTRACT: Upregulation of the activated Factor VII (FVIIa)/Tissue Factor complex, downregulation of natural anticoagulation pathways, and inhibition of fibrinolysis, are major contributors to coagulopathies associated with acute inflammation. Provision of FVIIa, and consequent downstream coagulation-related proteases, also stimulates further inflammatory changes, which can result in disseminated intravascular coagulation. Thus, the potential protective effects in vivo of a genetic-based reduction in FVII levels have been investigated in a murine model of acute inflammation, namely lipopolysaccharide (LPS)-induced lethal endotoxaemia. Mice with a total FVII deficiency do not survive the neonatal period. Therefore mice expressing low levels of FVII (FVII(tTA/tTA)), producing sufficient amounts of FVII for survival (approximately 5% of wild-type (WT) FVII), were employed to investigate in vivo pathways involved in the crosstalk between coagulation, inflammation, and survival, consequent to administration of a lethal dose of LPS. The FVII(tTA/tTA) mice presented with reduced mortality, coagulation, and inflammatory responses in comparison with similarly treated WT mice after administration of LPS. The attenuated inflammatory responses in FVII(tTA/tTA) mice were associated with downregulation of Egr-1 signalling. Administration, in vivo, of specific inhibitors of FXa and thrombin demonstrated that the inflammatory responses were unaltered in WT mice, but further reduced in FVII(tTA/tTA) mice. Therefore, a FVII deficiency enhances survival from lethal endotoxaemia both through attenuation of inflammatory responses that result directly from reduced FVIIa levels, and, indirectly, from downregulation of coagulation proteases downstream of the FVII-dependent cascade.
The Journal of Pathology 01/2007; 210(4):488-96. · 6.32 Impact Factor
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ABSTRACT: Severe inflammation leads to haemostatic abnormalities, such as the development of microvascular thrombi. As a result, ischaemia-related downstream organ damage can occur. The present study demonstrates that mice with a total deficiency of fibrinogen (Fg(-/-)) present with altered responses to challenge with Gram-negative lipopolysaccharide (LPS). Early survival in response to continuous LPS challenge was increased in Fg(-/-) mice and histological findings indicated that this improvement correlated with a lack of fibrin deposition in organs. Neutrophils appeared early in the lungs of challenged wild-type (WT) mice, but occurred in Fg(-/-) mice at later times. This delayed response in Fg(-/-) mice was confirmed by studies that showed a strong dependence on Fg of binding of neutrophils to endothelial cells in the presence of LPS. While cytokines were also elevated in both WT and Fg(-/-) mice, their levels were generally lower at early times in this latter group. The time course of MIP-2 expression correlated with the occurrence of pulmonary leakage after LPS challenge, which was delayed in Fg(-/-) mice. These results suggest that fibrin(ogen) plays a role as an early mediator in the cross-talk between coagulation and inflammation.
The Journal of Pathology 12/2006; 210(3):325-33. · 6.32 Impact Factor
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ABSTRACT: The inflammatory response to implanted biomaterials severely limits their deployment in patients. Plasminogen has been shown to play a central role in cell migration, and therefore could regulate this inflammatory response. We sought to determine if plasminogen influences recruitment of inflammatory cells to a biomaterial implanted into plasminogen-deficient (Plg(-/-)) mice. Small disks of polyethylene terephthalate, a material used in vascular grafts, were surgically implanted into the peritoneum of wild-type and Plg(-/-) mice. Recruitment of neutrophils and monocytes/macrophages into the peritoneum and onto the disks was measured, primarily at 18 h. Monocyte/macrophage recruitment was markedly blunted in Plg(-/-) mice compared with wild-type mice. Unexpectedly, neutrophil recruitment was also markedly decreased in the Plg(-/-) mice. While recruitment of leukocytes into the peritoneum was plasminogen-dependent, the adhesion of the emigrating cells to the implants was not. In contrast, adhesion but not recruitment was reduced in fibrinogen-deficient mice. Reconstitution of Plg(-/-) mice with intravenous or intraperitoneal plasminogen differentially restored monocyte/macrophage and neutrophil recruitment. Tranexamic acid, an inhibitor of the lysine binding sites of plasminogen, suppressed leukocyte recruitment in wild-type mice, but aprotinin, a plasmin inhibitor, did not. Plasminogen exerts a marked influence on both neutrophil and monocyte/macrophage recruitment to implanted biomaterials. This role is distinct from that of fibrinogen, and the two inflammatory cell types use plasminogen in different ways. Plasminogen represents a therapeutic target for controlling the inflammatory response to implanted materials.
Journal of Thrombosis and Haemostasis 11/2004; 2(10):1798-805. · 5.73 Impact Factor
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ABSTRACT: Aromatic amino acid residues within kringle domains play important roles in the structural stability and ligand-binding properties of these protein modules. In previous investigations, it has been demonstrated that the rigidly conserved Trp25 is primarily involved in stabilizing the conformation of the kringle-2 domain of tissue-type plasminogen activator (K2tPA), whereas Trp63, Trp74, and Tyr76 function in ω-amino acid ligand binding, and, to varying extents, in stabilizing the native folding of this kringle module. In the current study, the remaining aromatic residues of K2tPA, viz., Tyr2, Phe3, Tyr9, Tyr35, Tyr52, have been subjected to structure–function analysis via site-directed mutagenesis studies. Ligand binding was not significantly influenced by conservative amino acid mutations at these residues, but a radical mutation at Tyr35 destabilized the interaction of the ligand with the variant kringle. In addition, as reflected in the values of the melting tempe0ratures, changes at Tyr9 and Tyr52 generally destabilized the native structure of K2tPA to a greater extent than changes at Tyr2, Phe3, and Tyr35. Taken together, results to date show that, in concert with predictions from the crystal structure of K2tPA, ligand binding appears to rely most on the integrity of Trp63 and Trp74, and aromaticity at Tyr76. With regard to aromatic amino acids, kringle folding is most dependent on Tyr9, Trp25, Tyr52, Trp63, and Tyr76. As yet, no obvious major roles have been uncovered for Tyr2, Phe3, or Tyr35 in K2tPA.
European Journal of Allergy and Clinical Immunology 12/2003; 53(6):656 - 664. · 1.30 Impact Factor
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ABSTRACT: Conantokin-G (con-G) is a small, gamma-carboxyglutamic acid (Gla)-containing peptide that functions neurophysiologically by inhibiting the N-methyl-d-aspartate receptor (NMDAR). In the current study, the receptor binding properties of an alanine-rich, Gla-deficient con-G variant, Ala-con-G, were assessed following tracer radioiodination with 125I. Direct binding experiments with [125I]Ala-con-G yielded a single site defined by a Kd value of 516 +/- 120 nm. Displacement of [125I]Ala-con-G binding by Ala-con-G resulted in 100% displacement with an IC50 value of 564 +/- 33 nm, while heterologous displacement by con-G[S16Y], con-G, con-T, and con-R[1-17] yielded IC50 values in the range of 15-45 microm. No displacement was observed with d-gamma-con-G or con-G[L5A], analogs that are inactive at NMDARs. Specific [125I]Ala-con-G binding was displaced by NMDA and 2-amino-5-phosphopentanoic acid in a dose-dependent manner, suggesting an interaction at the glutamate binding site. The direct binding of [125I]Ala-con-G to adult rat brain sections revealed an anatomical distribution of binding sites in all regions known to contain the NR2B subunit of the NMDAR. These results constitute the only known demonstration of the direct binding of a radiolabeled conantokin to the NMDARs present in rat brain membrane preparations and rat brain sections, and suggest that radiolabeled Ala-con-G, and similar conantokin derivatives, may find utility as probes of NMDARs in a variety of systems.
European Journal of Allergy and Clinical Immunology 07/2003; 61(6):307-17. · 1.30 Impact Factor
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ABSTRACT: Conantokin-G (con-G) is a small, γ-carboxyglutamic acid (Gla)-containing peptide that functions neurophysiologically by inhibiting the N-methyl-d-aspartate receptor (NMDAR). In the current study, the receptor binding properties of an alanine-rich, Gla-deficient con-G variant, Ala-con-G, were assessed following tracer radioiodination with 125I. Direct binding experiments with [125I]Ala-con-G yielded a single site defined by a Kd value of 516 ± 120 nm. Displacement of [125I]Ala-con-G binding by Ala-con-G resulted in 100% displacement with an IC50 value of 564 ± 33 nm, while heterologous displacement by con-G[S16Y], con-G, con-T, and con-R[1–17] yielded IC50 values in the range of 15–45 μm. No displacement was observed with d-γ-con-G or con-G[L5A], analogs that are inactive at NMDARs. Specific [125I]Ala-con-G binding was displaced by NMDA and 2-amino-5-phosphopentanoic acid in a dose-dependent manner, suggesting an interaction at the glutamate binding site. The direct binding of [125I]Ala-con-G to adult rat brain sections revealed an anatomical distribution of binding sites in all regions known to contain the NR2B subunit of the NMDAR. These results constitute the only known demonstration of the direct binding of a radiolabeled conantokin to the NMDARs present in rat brain membrane preparations and rat brain sections, and suggest that radiolabeled Ala-con-G, and similar conantokin derivatives, may find utility as probes of NMDARs in a variety of systems.
European Journal of Allergy and Clinical Immunology 05/2003; 61(6):307 - 317. · 1.30 Impact Factor
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ABSTRACT: Factor X (FX)-deficient embryos suffer partial embryonic lethality with approximately 30% of the embryos arresting at midgestation. The remaining animals survive to term but die perinatally mainly from abdominal or intracranial hemorrhage. We have rescued FX-deficient mice by transplanting fetal liver cells from FX+/+, Rosa26 fetuses into midgestation embryos derived from FX+/- heterozygous crosses. FX-/- embryos were born at the expected frequency and approximately 50% of the FX-/- neonates survived longer than 4 months. FX-/- embryos receiving saline injections that survived to term died perinatally similar to untreated FX-deficient mice. The plasma levels of FX in the rescued 16-week-old FX-/- mice were approximately 1-6% of wild-type levels. beta-Galactosidase-staining cells derived from the donor Rosa26 fetal liver cells were detected in 47% of the livers of adult mice. In addition, donor-derived cells were also recovered in the bone marrow, spleen, lung, and occasionally in the brain and testis. These results suggest that in utero cell transplantation could be an effective therapeutic strategy to treat pathologies resulting from the deficiency of hepatic-expressed factors.
Journal of Thrombosis and Haemostasis 02/2003; 1(1):19-27. · 5.73 Impact Factor
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ABSTRACT: The characterization of conantokin-T (con-T), conantokin-R (con-R), and variants thereof, using the whole-cell patch clamp technique, was undertaken to evaluate the contribution of various residues towards the onset and recovery of N-methyl-D-aspartate (NMDA) receptor inhibition in cultured embryonic murine hippocampal neurons. The results obtained indicate that the two most C-terminal gamma-carboxyglutamic acid (Gla) residues of the conantokins, while not essential for activity, provided for more tenacious binding to the receptor. Specifically, con-T[gamma10K/gamma14K] and con-R[gamma11A/gamma15A] displayed 5.6- and 8.4-fold decreases in tau(off), respectively, compared to the parent peptides. For the truncated con-T variants, con-T[1-9/Q6G], and a sarcosine (Src)-containing species, con-T[1-9/G1Src/Q6G], the tau(off) was over 80- and 40-fold faster, respectively, compared to con-T. For the latter peptide, the coapplication of 300 microM spermine enhanced the onset rate constant from 3.1x10(3)M(-1) x s(-1) to 12.6x10(3)M(-1) x s(-1). From analysis of equilibrium dose-inhibition curves using the Cheng-Prusoff equation, a K(i) value of 1.1 microM for the peptide was obtained. Con-T[1-9/G1Src/Q6G] demonstrated an apparent competitive mode of inhibition relative to NMDA. Schild analysis of the data yielded an equilibrium dissociation constant of 2.4 microM for the interaction of con-T[1-9/G1Src/Q6G] with the receptor.
Neuropharmacology 01/2002; 41(7):801-10. · 4.81 Impact Factor
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ABSTRACT: Components of the fibrinolytic system have been implicated in cell migratory events associated with tissue remodeling. Studies in plasminogen-deficient mice (PG(-/-)) indicated that skin wound healing is impaired, but is resolved with an additional fibrinogen deficiency. Plasminogen activator inhibitor-1 (PAI-1) expression by keratinocytes has been identified shortly after wound injury. PAI-1 expression could affect wound healing by regulating the fibrinolytic environment of the wounded area, as well as influencing events associated with cell attachment and detachment through interactions with matrix proteins. The present study directly assesses PAI-1 involvement in skin wound healing through analyses of a dermal biopsy punch model in PAI-1-deficient (PAI-1(-/-) mice. While the cellular events associated with the healing process are similar between wild-type (WT) and PAI-1(-/-) mice, the rate of wound closure is significantly accelerated in PAI-1(-/-) mice.
American Journal Of Pathology 12/2001; 159(5):1681-8. · 4.89 Impact Factor
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ABSTRACT: The three members of the conantokin peptide family identified to date are conantokin(con)-G, -T and -R. Their defining attributes include a high relative content of gamma-carboxyglutamic acid (Gla), N-terminal sequence identity, as well as considerable overall sequence homology, and antagonism of the N-methyl-D-aspartate receptor (NMDAR). As promising templates for the design of neuroprotective agents, a thorough evaluation of structure-function relationships in these peptides will be invaluable in aiding rational drug modeling. To this end, a comprehensive assessment of the contributions of individual residues to conantokin structure and function is required. The current review summarizes recent efforts in this area, and also includes the effects of peptide length, as well as structural-stabilization and -destabilization on the structural and inhibitory profiles of an extensive panel ofconantokin derivatives.
Current Drug Targets 10/2001; 2(3):313-22. · 3.55 Impact Factor
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ABSTRACT: The involvement of the glutamate-glycine activated ion channels of the NMDA receptor in various neurophysiological processes has made this ion channel the focus of intense research. The excessive release of glutamate in a variety of neuronal hypoxic conditions implicates the NMDA receptor in a number of neuropatholological states, such as stroke, chronic pain, Parkinson's disease, Alzheimer's disease, ALS, and epilepsy, among others, thus making this receptor a prime drug target candidate. A variety of agents are known to be effective in opening and closing of the ion channels of this receptor, among the latter group of agents is the peptidic conantokins. Through the use of electrophysiological measurements with a number of cell types containing natural and recombinant subunits of the NMDA receptor, much knowledge is evolving regarding the mechanism of action of activators and inhibitors of the NMDA receptor ion channels. In addition, structure-function studies of the conantokins in these systems have been revealing in terms of their complimentary sites on the NMDA receptor. These relationships serve as the main focus of this review.
Current Drug Targets 10/2001; 2(3):323-9. · 3.55 Impact Factor
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F J Castellino
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ABSTRACT: Protein C (PC) is the zymogen form of a serine protease, activated protein C (APC), a naturally occurring anticoagulant. In control of the coagulation of blood, APC functions by attenuating thrombin formation. It serves this role through inactivation, by limited proteolysis, of two important cofactors for overall clot formation, one of which, Factor Va (FVa), stimulates prothrombin activation, and another, Factor VIIIa (FVIIIa), enhances activation of coagulation Factor X (FX). In maintaining the fluidity of blood, APC also indirectly functions in fibrinolysis, in one manner by directly inactivating an inhibitor of plasminogen activation, plasminogen activation inhibitor-1 (PAI-1), and in another manner via its role in attenuating thrombin production, with the resulting effect of limiting production of another thrombin-dependent fibrinolytic inhibitor, thrombin activatable fibrinolysis inhibitor (TAFI). PC, and other components of the PC anticoagulant pathway, e.g., protein S (PS), thrombomodulin (Tm), and endothelial cell protein C receptor (EPCR), also can serve as anti-inflammatory mediators, through a number of different thrombin-dependent and thrombin-independent mechanisms. A large number of symptomatic and asymptomatic mutations occur in PC in humans, which express a variety of phenotypes. Generation and characterization of a murine model of a total PC gene inactivation has demonstrated that while an untreated total PC deficiency results in neonatal death through DIC-related abnormalities, a valuable resource is now available to study phenotypes of less severe deficiencies of this protein. Such studies will lead to advances in an understanding of the relative role of this protein system in the various pathways in which it has an influence.
Frontiers in Bioscience 08/2001; 6:D807-19. · 3.52 Impact Factor
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ABSTRACT: Whole cell voltage clamp recordings were performed to assess the ability of conantokin-G (con-G), conantokin-T (con-T), and a 17-residue truncated form of conantokin-R (con-R[1-17]) to inhibit N-methyl-d-aspartate (NMDA)-evoked currents in human embryonic kidney 293 cells transiently expressing various combinations of NR1a, NR1b, NR2A, and NR2B receptor subunits. Con-T and con-R[1-17] attenuated ion currents in cells expressing NR1a/NR2A or NR1a/NR2B. Con-G did not affect NMDA-evoked ionic currents in cells expressing NR1a/NR2A, but it showed inhibitory activity in cells expressing NR1a/NR2B receptors and the triheteromeric combination of NR1a/NR2A/NR2B. An Ala-rich con-G analog, con-G[Q6G/gamma7K/N8A/gamma10A/gamma14A/K15A/S16A/N17A] (Ala/con-G, where gamma is Gla), in which all nonessential amino acids were altered to Ala residues, manifested subunit specificity similar to that of con-G, suggesting that the replaced residues are not responsible for selectivity in the con-G framework. A sarcosine-containing con-T truncation analog, con-T[1-9/G1Src/Q6G], inhibited currents in NR1a/NR2A and NR1a/NR2B receptors, eliminating residues 10-21 as mediators of the broad subunit selectivity of con-T. In contrast to the null effects of con-G and Ala/con-G at a NR1a/NR2A-containing receptor, some inhibition ( approximately 40%) of NMDA-evoked currents was effected by these peptides in cells expressing NR1b/NR2A. This finding suggests that the presence of exon 5 in NR1b plays a role in the activity of the conantokins. Analysis of various conantokin analogs demonstrated that Leu(5) of con-G is an important determinant of conantokin selectivity. Taken as a whole, these results suggest that the important molecular determinants on conantokins responsible for NMDA receptor activity and specificity are discretely housed in specific residues of these peptides, thus allowing molecular manipulation of the NMDA receptor inhibitory properties of the conantokins.
Journal of Biological Chemistry 08/2001; 276(29):26860-7. · 4.77 Impact Factor
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ABSTRACT: A minimally invasive laser-induced injury model is described to study thrombus development in mice in vivo. The protocol involves focusing the beam of an argon-ion laser through a compound microscope on the vasculature of a mouse ear that is sufficiently thin such that blood flow can be visualized by intravital microscopy. Two distinct injury models have been established. The first involves direct laser illumination with a short, high-intensity pulse. In this case, thrombus formation is inhibited by the GPIIb/IIIa antagonist, G4120. However, the anticoagulants, hirulog, PPACK, and NapC2 have minimal effect. This indicates that thrombus development induced by this model mainly involves platelet interactions. The second model involves low-intensity laser illumination of mice injected with Rose Bengal dye to induce photochemical injury in the region of laser illumination. Thrombi generated by this latter procedure have a slower development and are inhibited by both anticoagulant and anti-platelet compounds.
American Journal Of Pathology 06/2001; 158(5):1613-22. · 4.89 Impact Factor
