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E F Plow

Publications and source records attributed to E F Plow.

At least 37 records · Page 2Linked to original sources
PubMed1999

Peptide ligands can bind to distinct sites in integrin alphaIIbbeta3 and elicit different functional responses.

The spatial relationship between the binding sites for two cyclic peptides, cyclo(S,S)KYGCRGDWPC (cRGD) and cyclo(S,S)KYGCHarGDWPC (cHarGD), high affinity analogs for the RGD and HLGGAKQAGDV peptide ligands, in integrin alphaIIbbeta3 (GPIIb-IIIa) has been characterized. For this purpose, cRGD and cHarGD were labeled with fluorescein isothiocyanate and tetramethylrhodamine 5-isothiocyanate, respectively. Both cyclic peptides were potent inhibitors of fibrinogen binding to alphaIIbbeta3, particularly in the presence of Mn2+; IC50 values for cRGD and cHarGD were 1 and <0.1 nM in the presence of Mn2+. Direct binding experiments and fluorescence resonance energy transfer analysis using the purified receptor showed that both peptides interacted simultaneously with distinct sites in alphaIIbbeta3. The distance between these sites was estimated to be 6.1 +/- 0.5 nm. Although cRGD bound preferentially to one site and cHarGD to the other, the sites were not fully specific, and each cyclic peptide or its linear counterpart could displace the other to some extent. The binding affinity of the cHarGD site was dramatically affected by Mn2+. cRGD, but not cHarGD, bound to recombinant beta3-(95-373) in a cation-dependent manner, indicating that the cRGD site is located entirely within this fragment. With intact platelets, binding of c-RGD and cHarGD to alphaIIbbeta3 resulted in distinct conformational alterations in the receptor as indicated by the differential exposure of ligand-induced binding site epitopes and also induced the opposite on membrane fluidity as shown by electron paramagnetic resonance analyses using 5-doxylstearic acid as a spin probe. These data support the concept the two peptide ligands bind to distinct sites in alphaIIbbeta3 and initiate different functional consequences within the receptor itself and within platelets.

Binding Sites↗
PubMed1999

Platelet GPIIb-IIIa blockers.

Regardless of the event that stimulates the aggregation of platelets, the receptor alpha(IIb)beta3--one of a family of adhesion receptors known as integrins--has a key role in the process. The past decade has seen the publication of 10 phase III (randomised) clinical trials of four members of a new class of antiplatelet drugs, the GPIIb-IIIa blockers, targeted at this important receptor. Three (abciximab, eptifibatide, and tirofiban) are licensed for human use. 10 other GbIIb-IIIa blockers are in phase II or III human studies. In all 10 placebo-controlled trials, done in the clinical settings of percutaneous coronary intervention or acute coronary syndrome in patients on aspirin, the endpoints favoured the active drug, with a risk reduction for death or non-fatal myocardial infarction of about 21% overall. With attention to heparin dose the risk of bleeding is not a major concern with these agents. The GPIIb-IIIa blockers are taking the clinician and patient out of the era of aspirin monotherapy when platelet inhibition is required.

Humans↗
PubMed1999

A role of plasminogen in atherosclerosis and restenosis models in mice.

In addition to its preeminent role in fibrinolysis, the plasminogen system is believed to play a key role in mediating cell migration. Leukocyte migration into the vessel wall is a key and early event in the development of the lesions of atherosclerosis and restenosis, pathologies which may be viewed as specific examples of vascular inflammatory responses. The development of mice in which the plasminogen gene has been inactivated affords an opportunity to test the contribution of plasminogen in leukocyte migration during in vivo. This article summarizes recent studies conducted in murine models of the inflammatory response, restenosis and atherosclerosis in which leukocyte migration, and in particular monocyte/macrophage migration, has been evaluated in plasminogen-deficient mice. Recruitment of these cells through the vessel wall in inflammatory response models and into the vessel wall in restenosis and transplant atherosclerosis models is substantially blunted. These data implicate plasminogen in the migration of leukocytes in these murine models. With the numerous correlations between components and/or activation of the plasminogen system in restenosis and atherosclerosis, these results also support a role of plasminogen in the corresponding human pathologies.

Animals↗
PubMed1998

Activation of alphaVbeta3 on vascular cells controls recognition of prothrombin.

Regulation of vascular homeostasis depends upon collaboration between cells of the vessel wall and blood coagulation system. A direct interaction between integrin alphaVbeta3 on endothelial cells and smooth muscle cells and prothrombin, the pivotal proenzyme of the blood coagulation system, is demonstrated and activation of the integrin is required for receptor engagement. Evidence that prothrombin is a ligand for alphaVbeta3 on these cells include: (a) prothrombin binds to purified alphaVbeta3 via a RGD recognition specificity; (b) prothrombin supports alphaVbeta3-mediated adhesion of stimulated endothelial cells and smooth muscle cells; and (c) endothelial cells, either in suspension and in a monolayer, recognize soluble prothrombin via alphaVbeta3. alphaVbeta3-mediated cell adhesion to prothrombin, but not to fibrinogen, required activation of the receptor. Thus, the functionality of the alphaVbeta3 receptor is ligand defined, and prothrombin and fibrinogen represent activation- dependent and activation-independent ligands. Activation of alphaVbeta3 could be induced not only by model agonists, PMA and Mn2+, but also by a physiologically relevant agonist, ADP. Inhibition of protein kinase C and calpain prevented activation of alphaVbeta3 on vascular cells, suggesting that these molecules are involved in the inside-out signaling events that activate the integrin. The capacity of alphaVbeta3 to interact with prothrombin may play a significant role in the maintenance of hemostasis; and, at a general level, ligand selection by alphaVbeta3 may be controlled by the activation state of this integrin.

Adenosine Diphosphate↗
PubMed1998

Interaction of the fungal pathogen Candida albicans with integrin CD11b/CD18: recognition by the I domain is modulated by the lectin-like domain and the CD18 subunit.

Interactions of microorganisms with integrins are central to the host defense mechanisms. The leukocyte integrin CD11b/CD18 is the principal adhesion receptor on leukocytes for Candida albicans, a major opportunistic pathogen. In this study we have investigated the roles of three regions within the receptor, the inserted (I) and lectin-like domains within the CD11b subunit, and the CD18 subunit, in CD11b/CD18-C. albicans interactions. We report four major findings. 1) A mutation in CD18 exerts a dominant negative effect on the function of the CD11b/CD18 complex. This interpretation is based on the observation that in the absence of CD18, the CD11b subunit alone binds C. albicans well, but a single point mutation at Ser138 of CD18 abolishes CD11b/CD18 binding of the fungus. 2) The lectin-like domain is not sufficient for CD11b/CD18-C. albicans interactions. Rather, the lectin-like domain appears to influence CD11b/CD18 binding activity by modulating the function of the I domain. 3) The I domain is the primary binding site for C. albicans in the receptor and is sufficient to support an efficient interaction. 4) We have identified specific amino acid sequences within the I domain that engage the microorganism. Compared with other ligands of CD11b/CD18, C. albicans has some unique as well as common contact sites within the I domain of the receptor. Such unique contact sites may underlie the ability of C. albicans to modulate CD11b/CD18 function and raise the possibility for selective interference of the microorganism-host leukocyte interactions.

CD18 Antigens↗
PubMed1998

Identification of a novel recognition sequence for integrin alphaM beta2 within the gamma-chain of fibrinogen.

The interaction of leukocyte integrin alphaMbeta2 (CD11b/CD18, Mac-1) with fibrinogen has been implicated in the inflammatory response by contributing to leukocyte adhesion to the endothelium and subsequent transmigration. Previously, it has been demonstrated that a peptide, P1, corresponding to residues 190-202 in the gamma-chain of fibrinogen, binds to alphaM beta2 and blocks the interaction of fibrinogen with the receptor and that Asp199 within P1 is important to activity. We have demonstrated, however, that a double mutation of Asp199-Gly200 to Gly-Ala in the recombinant gamma-module of fibrinogen, spanning region 148-411, did not abrogate alphaM beta2 recognition and considered that other binding sites in the gamma-module may participate in the receptor recognition. We have found that synthetic peptide P2, duplicating gamma377-395, inhibited adhesion of alphaM beta2-transfected cells to immobilized D100 fragment of fibrinogen in a dose-dependent manner. In addition, immobilized P2 directly supported efficient adhesion of the alphaM beta2-expressing cells, including activated and non-activated monocytoid cells. The I domain of alphaM beta2 was implicated in recognition of P2, as the biotinylated recombinant alphaMI domain specifically bound to both P2 and P1 peptides. Analysis of overlapping peptides spanning P2 demonstrated that it may contain two functional sequences: gamma377-386 (P2-N) and gamma383-395 (P2-C), with the latter sequence being more active. In the three-dimensional structure of the gamma-module, gamma190-202 and gamma377-395 reside in close proximity, forming two antiparallel beta strands. The juxtapositioning of these two sequences may form an unique and complex binding site for alphaM beta2.

Amino Acid Sequence↗
PubMed1998

Plasminogen deficiency differentially affects recruitment of inflammatory cell populations in mice.

It is widely held that the plasminogen (Plg) system plays a role in inflammation through plasmin-mediated directional cell migration. However, substantial evidence for its involvement in the inflammatory response has been obtained from indirect studies and lacks firm biological confirmation. To directly characterize plasminogen's involvement in the inflammatory response, we used thioglycollate to induce a peritoneal inflammatory reaction in Plg(+/+), Plg(+/-), and Plg(-/-) mice. At 6 hours poststimulation, neutrophil recruitment into the peritoneum was maximal and similar between Plg(+/+), Plg(+/-), and Plg(-/-) mice. In contrast, monocyte recruitment was significantly diminished after 24 hours poststimulation in Plg(-/-) mice relative to Plg(+/+) mice. Lymphocyte recruitment also was blunted. Blood monocyte levels in these mice indicated that diminished recruitment into the peritoneum was not the result of a diminished source of cells in the circulation. Macrophage phagocytic function was similar between Plg(+/+) and Plg(-/-) mice. This study establishes a direct involvement of plasminogen in monocyte recruitment during a representative inflammatory response.

Animals↗
PubMed1997

Networking in the hemostatic system. Integrin alphaiibbeta3 binds prothrombin and influences its activation.

Prothrombin activation is a pivotal event in thrombosis and hemostasis because thrombin can mediate fibrin formation and can activate and aggregate platelets. Platelet aggregation depends upon the binding of adhesive proteins to integrin alphaIIbbeta3 on the platelet surface. In the present study, a novel interface between the blood coagulation system and platelets is demonstrated by showing that 1) prothrombin binds to alphaIIbbeta3 and 2) this interaction accelerates prothrombin activation. Prothrombin bound to purified alphaIIbbeta3 in a specific, saturable, and divalent cation-dependent manner. This interaction was inhibited by certain monoclonal antibodies to alphaIIbbeta3, by the alphaIIbbeta3 ligands fibrinogen and RGD peptides, but not by thrombin or unrelated proteins. Prothrombin also interacted with alphaIIbbeta3 on resting and stimulated platelets as demonstrated by soluble ligand binding and platelet adhesion assays. Activation of prothrombin by Factor Xa alone or Factor Xa-Va was accelerated by alphaIIbbeta3, and this enhancement was blocked by a monoclonal antibody that inhibited prothrombin binding to the receptor. Taken together, these data identify a previously unrecognized linkage between platelets and the blood coagulation system that may have a significant regulatory consequence.

Amino Acid Sequence↗
PubMed1997

Identification and reconstruction of the binding site within alphaMbeta2 for a specific and high affinity ligand, NIF.

Engagement of the alphaMbeta2 (CD11b/CD18, Mac-1) integrin on neutrophils supports adhesion and induces various cellular responses. These responses can be blocked by a specific ligand of alphaMbeta2, neutrophil inhibitory factor (NIF). The molecular basis of alphaMbeta2-NIF interactions was studied. The single chain alphaM subunit, expressed on the surface of human 293 cells, bound NIF with an affinity equivalent to that of alphaMbeta2 heterodimer. This observation, coupled with previous data showing that the alphaMI domain alone supported high affinity NIF binding, indicated that the binding site for NIF is restricted to the I domain. Guided by the crystal structure of the alphaMI domain, 16 segments corresponding to the entire outer hydrated surface of alphaMI domain were switched to their counterparts sequences in alphaL, which does not bind NIF. Surface expression and heterodimer formation were achieved for all mutants, and correct folding was confirmed. Of the 16 switches, only 5 affected NIF binding substantially, reducing affinity by 8-300-fold. These data confined the NIF-binding site to a narrow region composed of Pro147-Arg152, Pro201-Lys217, and Asp248-Arg261 of alphaM. Verifying this localization, when these segments were introduced into the alphaXI-domain, the resulting chimeric receptor was converted into a high affinity NIF-binding protein.

Binding Sites↗
PubMed1997

On the mechanism of the antifibrinolytic activity of plasma carboxypeptidase B.

The precursor of plasma carboxypeptidase B (pCPB) also known as thrombin-activable fibrinolysis inhibitor can be converted by thrombin to an active enzyme capable of eliminating C-terminal Lys- and Arg-residues from proteins. The activation is about 1000-fold more efficient in the presence of thrombomodulin (TM). We investigated the antifibrinolytic potency of maximally activated pCPB in plasma and explored the antifibrinolytic mechanism of pCPB. During clotting of plasma in the presence of 3.3 NIH units/ml thrombin and 1 microg/ml soluble TM, more than 80% pro-pCPB was converted into the active form causing an increase of plasma carboxypeptidase activity from 100 units/liter (constitutive activity ascribed to plasma carboxypeptidase N) to 430 units/liter as measured with furoylacroleyl-alanyl-arginine substrate. Under these conditions, lysis of a plasma clot induced by a range of tissue-type plasminogen activator (t-PA) concentrations (0.2-2 microg/ml) was retarded more than 4-fold. A considerable retardation of fibrinolysis was observed upon addition of as little as 12 ng/ml soluble TM, a concentration comparable with physiological concentrations of soluble TM in human plasma. The presence of Ca2+ appeared to be a critical requirement for effective activation of pro-pCPB by thrombin-TM in plasma. Plasminogen-binding sites (C-terminal lysines) on the surface of a plasmin-treated fibrin clot were eliminated within 1-3 min by plasma with maximally activated pCPB, as studied in a recently described model involving fluorescence microscopy. Confocal fluorescence microscopy showed that in the absence of TM plasminogen strongly accumulated on fibrin fibers during t-PA-induced lysis of a plasma clot. In the presence of TM (and a concomitant pro-pCPB activation), lysis was slow and was not accompanied by accumulation of plasminogen on the fibers. In conclusion, generation of active pCPB during clotting of plasma in the presence of Ca2+ and TM leads to a retardation of plasma clot lysis in a wide range of t-PA concentrations, from low to therapeutic, and to a fast elimination of plasminogen-binding sites on partially degraded fibrin. This is a likely mechanism for the antifibrinolytic effect of active pCPB.

Antifibrinolytic Agents↗
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

Fibrinogen is a ligand for integrin alpha5beta1 on endothelial cells.

Previous studies have shown that fibrinogen can associate with endothelial cells via an Arg-Gly-Asp (RGD) recognition specificity. In the present study, we have characterized the specificity of fibrinogen binding to endothelial cells under different cation conditions. Fibrinogen binding to suspended endothelial cells was selectively supported by Mn2+ and was suppressed by Ca2+. The Mn2+-supported interaction was completely inhibited by RGD peptides but not by alphavbeta3 blocking monoclonal antibodies. In contrast, the interaction was completely blocked by two alpha5beta1 monoclonal antibodies. This interaction was not mediated by fibronectin bound to the integrin; could be demonstrated with purified alpha5beta1; and also was observed with a second alpha5beta1-bearing cell type, platelets. The binding of fibrinogen to alpha5beta1 on endothelial cells in the presence of Mn2+ was time-dependent, specific, saturable, and of high affinity (Kd = 65 nM). By employing anti-peptide monoclonal antibodies, the carboxyl-terminal RGD sequence at Aalpha 572-574 was implicated in fibrinogen recognition by alpha5beta1. Two circumstances were identified in which alpha5beta1 interacted with fibrinogen in the presence of Ca2+: when the receptor was activated with monoclonal antibody (8A2) or when the fibrinogen was presented as an immobilized substratum. These results identify fibrinogen as a ligand for alpha5beta1 on endothelial and other cells, an interaction which may have broad biological implications.

Cells, Cultured↗