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Edward F Plow

Publications and source records attributed to Edward F Plow.

34 records · Page 2Linked to original sources
PubMed2004

Integrin alphaMbeta2 orchestrates and accelerates plasminogen activation and fibrinolysis by neutrophils.

Plasmin, the pivotal thrombolytic enzyme, is generated on the surface of many cell types, where urokinase receptor (uPAR)-bound urokinase (uPA) activates cell-bound plasminogen (Plg). It has been reported that neutrophils mediate endogenous thrombolysis involving a uPA-dependent mechanism, and we previously demonstrated that both uPAR and integrin alpha(M)beta(2) recognize uPA to control cell migration and adhesion. In the present study, we report that the alpha(M)beta(2) regulates neutrophil-dependent fibrinolysis. Phorbol 12-myristate 13-acetate (PMA)-stimulated but not resting neutrophils dissolved fibrin clots, and this activity was not only uPA- and Plg-dependent but also alpha(M)beta(2)-dependent. Purified alpha(M)beta(2) directly bound uPA (K(d) = 40 nm) and Plg (K(d) = 1 microm) in a dose-dependent and saturable manner. In Plg activation assays, addition of purified alpha(M)beta(2), but not a control protein, to a single chain uPA (sc-uPA)/Plg mixture, decreased the K(m) from 2 to 0.1 microm, thereby augmenting the overall reaction efficiency by 50-fold. The binding of sc-uPA to alpha(M)beta(2) was critical for the alpha(M)beta(2)-mediated enhancement of plasmin (Plm) generation, because this effect was lost when WT-sc-uPA was replaced with a kringle-less mutant (DeltaK-sc-uPA), which does not bind to alpha(M)beta(2). Plm inactivation by alpha(2)-antiplasmin was significantly delayed when Plm was preincubated with purified, soluble alpha(M)beta(2). When Plg was added to PMA-stimulated neutrophils, both uPA and Plg were co-immunoprecipitated with alpha(M)beta(2.) Thus, assembly of Plg and uPA on integrin alpha(M)beta(2) regulates Plm activity and, thereby, plays a crucial role in neutrophil-mediated thrombolysis.

Cell Adhesion↗
PubMed2004

Coronary artery disease and the thrombospondin single nucleotide polymorphisms.

GeneQuest was a high throughput, large-scale analysis of single nucleotide polymorphisms (SNPs) to identify gene associated with familial, premature coronary artery disease and myocardial infarction. The three SNPs showing the highest and most significant associations with disease were all members of the thrombospondin gene family, thrombospondin-1, thrombospondin-2 and thrombospondin-4. These unanticipated associations have kindled efforts to understand how the three SNPs influence the structures and functions of the thrombospondins. The SNP in thrombospondin-1 and thrombospondin-4 reside in their coding regions and result in single amino acid changes: in thrombospondin-1, the predominant asparagine at position 700 is changed to a serine while, in thrombospondin-4, it is a change of an alanine to a proline at position 387. The SNP in thrombospondin-2 is a base change in the 3'-untranslated region of the mRNA. At this early stage of investigation, predictive analyses suggest that the substitutions in thrombospondin-2 and thrombospondin-4 should alter structure, and there is direct evidence to indicate that the thrombospondin-1 SNP alters conformational stability. In addition, profound differences in the function of the thrombospondin-4 SNP variants have been identified with respect to their capacity to support endothelial cell adhesion and proliferation. While substantial additional information is needed to understand if and how the polymorphic forms of the thrombospondins affect coronary artery disease, the data assembled to date suggest marked effects of these SNPs on the structures and functions of the thrombospondins, which are consistent with induction of a proatherogenic and prothrombotic phenotype.

Binding Sites↗
PubMed2004

The functions of plasminogen in cardiovascular disease.

Plasminogen (Plg) and its derivative serine protease, plasmin, together with the activators, inhibitors, modulators, and substrates of the Plg network, are postulated to regulate a wide variety of biologic responses that could influence cardiovascular disease. The development of Plg-deficient mice has provided an incisive approach to test these proposed functions in vivo. Several different models of atherosclerosis, restenosis, aneurysm, and thrombosis have been analyzed in these mice and have demonstrated profound effects of Plg on these events as well as on the inflammatory response, which contributes to these cardiovascular diseases. Plasmin (ogen) may influence the progression of cardiovascular diseases through its degradation of matrix proteins, including fibrin; its activation of matrix metalloproteinases; its regulation of growth factor and chemokine pathways; or its influence on directed cell migration. Dissection of these mechanisms represents a future challenge toward understanding the roles of Plg in vivo.

Animals↗
PubMed2004

Leukocyte engagement of fibrin(ogen) via the integrin receptor alphaMbeta2/Mac-1 is critical for host inflammatory response in vivo.

The leukocyte integrin alpha(M)beta(2)/Mac-1 appears to support the inflammatory response through multiple ligands, but local engagement of fibrin(ogen) may be particularly important for leukocyte function. To define the biological significance of fibrin(ogen)-alpha(M)beta(2) interaction in vivo, gene-targeted mice were generated in which the alpha(M)beta(2)-binding motif within the fibrinogen gamma chain (N(390)RLSIGE(396)) was converted to a series of alanine residues. Mice carrying the Fibgamma(390-396A) allele maintained normal levels of fibrinogen, retained normal clotting function, supported platelet aggregation, and never developed spontaneous hemorrhagic events. However, the mutant fibrinogen failed to support alpha(M)beta(2)-mediated adhesion of primary neutrophils, macrophages, and alpha(M)beta(2)-expressing cell lines. The elimination of the alpha(M)beta(2)-binding motif on fibrin(ogen) severely compromised the inflammatory response in vivo as evidenced by a dramatic impediment in leukocyte clearance of Staphylococcus aureus inoculated into the peritoneal cavity. This defect in bacterial clearance was due not to diminished leukocyte trafficking but rather to a failure to fully implement antimicrobial functions. These studies definitively demonstrate that fibrin(ogen) is a physiologically relevant ligand for alpha(M)beta(2), integrin engagement of fibrin(ogen) is critical to leukocyte function and innate immunity in vivo, and the biological importance of fibrinogen in regulating the inflammatory response can be appreciated outside of any alteration in clotting function.

Animals↗
PubMed2003

Thrombospondin-4 and its variants: expression and differential effects on endothelial cells.

BACKGROUND: In a recent large-scale genetic association study, a single nucleotide polymorphism in the thrombospondin-4 (TSP-4) gene, resulting in a proline-for-alanine substitution at position 387, was associated with a significantly increased risk for premature atherosclerosis. TSP-4 had not previously been implicated in vascular pathology, and very little information is available on its expression and functions. METHODS AND RESULTS: The goal of this study was to assess TSP-4 expression in vessel wall and to identify differences in functions of TSP-4 variants that could account for the proatherogenic effects of the (P387)TSP-4 variant. TSP-4 expression was demonstrated in human endothelial cells (ECs) and vascular smooth muscle cells from brain blood vessels and coronary arteries. (P387)TSP-4 and its fragment (residues 326 to 722), but not the A(387) forms, suppressed EC adhesion and proliferation. The (P387)TSP-4 was more active in inducing the phosphorylation of focal adhesion kinase, consistent with inhibition of proliferation. Both variant fragments increased the proliferation of human aortic smooth muscle cells. CONCLUSIONS: TSP-4 is expressed by vascular cells and influences the vessel wall by modulating the proliferation of ECs and smooth muscle cells. The A387P substitution is a "gain-of-function" mutation, favoring a form of TSP-4 that interferes with EC adhesion and proliferation and may thereby be proatherogenic.

Arteriosclerosis↗
PubMed2003

Increased expression of thrombospondin-1 in vessel wall of diabetic Zucker rat.

BACKGROUND: Thrombospondin-1 (TSP-1) expression in the vascular wall has been related to the development of atherosclerotic lesions and restenosis. TSP-1 promotes the development of neointima and has recently been associated with atherogenesis at a genetic level. Because TSP-1 expression is responsive to glucose stimulation in mesangial cells, we hypothesized that glucose may stimulate its production by vascular cells. Thus, TSP-1 expression in the blood vessel wall may increase, providing a molecular link between diabetes and accelerated vascular lesion development. METHODS AND RESULTS: To determine whether the expression level of TSP-1 in vessel wall is increased in diabetes, aorta and carotid arteries of Zucker rats were used for immunostaining, Western blotting, and in situ RNA hybridization. A significant increase in TSP-1 expression was found in the adventitia of blood vessels from diabetic rats. Consistent with the well-known antiangiogenic effect of TSP-1, the number of vasa vasorum was reduced in aortas from diabetic rats. In cultured endothelial cells, vascular smooth muscle cells, and fibroblasts, TSP-1 expression increased in response to glucose stimulation (>30-fold). After balloon catheter injury to carotid arteries, expression of TSP-1 protein and mRNA was higher at all time points in the vessels of diabetic rats. CONCLUSIONS: Increased expression of TSP-1 in blood vessels in diabetes may represent a new link between diabetes, atherogenesis, and accelerated restenosis. This increase in TSP-1 production may be a direct response of vascular cells to glucose.

Animals↗
PubMed2003

Identification of a novel binding site for platelet integrins alpha IIb beta 3 (GPIIbIIIa) and alpha 5 beta 1 in the gamma C-domain of fibrinogen.

The interactions of platelets with fibrinogen mediate a variety of responses including adhesion, platelet aggregation, and fibrin clot retraction. Whereas it was assumed that interactions of the platelet integrin alpha IIb beta 3 with the AGDV sequence in the gamma C-domain of fibrinogen and/or RGD sites in the A alpha chains are involved in clot retraction and adhesion, recent data demonstrated that fibrinogen lacking these sites still supported clot retraction. These findings suggested that an unknown site in fibrinogen and/or other integrins participate in clot retraction. Here we have identified a sequence within gamma C that mediates binding of fibrinogen to platelets. Synthetic peptide duplicating the 365-383 sequence in gamma C, designated P3, efficiently inhibited clot retraction in a dose-dependent manner. Furthermore, P3 supported platelet adhesion and was an effective inhibitor of platelet adhesion to fibrinogen fragments. Analysis of overlapping peptides spanning P3 and mutant recombinant gamma C-domains demonstrated that the P3 activity is contained primarily within gamma 370-383. Integrins alpha IIb beta 3 and alpha 5 beta 1 were implicated in recognition of P3, since platelet adhesion to the peptide was blocked by function-blocking monoclonal antibodies against these receptors. Direct evidence that alpha IIb beta 3 and alpha 5 beta 1 bind P3 was obtained by selective capture of these integrins from platelet lysates using a P3 affinity matrix. Thus, these data suggest that the P3 sequence in the gamma C-domain of fibrinogen defines a previously unknown recognition specificity of alpha IIb beta 3 and alpha 5 beta 1 and may function as a binding site for these integrins.

Amino Acid Sequence↗
PubMed2003

Integrin alphaIIbbeta3 and its antagonism.

AlphaIIbbeta3, the major membrane protein on the surface of platelets, is a member of the integrin family of heterodimeric adhesion receptors. The alphaIIb and beta3 subunits are each composed of a short cytoplasmic tail, a single transmembrane domain, and a large, extracellular region that consists of a series of linked domains. Recent structural analyses have provided insights into the organization of this and other integrins and how a signal is initiated at its cytoplasmic tail to transform the extracellular domain of alphaIIbbeta3 into a functional receptor for fibrinogen or von Willebrand factor to support platelet aggregation and thrombus formation. These functions of alphaIIbbeta3 have been targeted for antithrombotic therapy, and intravenous alphaIIbbeta3 antagonists have been remarkably effective in the setting of percutaneous coronary interventions, showing both short-term and long-term mortality benefits. However, the development of oral antagonists has been abandoned on the basis of excess of mortality in clinical trials, and the extension of therapy with existing alphaIIbbeta3 antagonists to broadly treat acute coronary syndromes has not fully met expectations. An in-depth understanding of how antagonists engage and influence the function of alphaIIbbeta3 and platelets in the context of the new structural insights may explain its salutary and potential deleterious effects.

Angioplasty, Balloon, Coronary↗
PubMed2003

Regulation of plasminogen receptors.

Many eukaryotic and prokaryotic cells bind plasminogen in a specific and saturable manner. When plasminogen is bound to cell-surface proteins with C-terminal lysines via its lysine binding sites, its activation to plasmin is accelerated, and cell-bound plasmin is protected from inactivation by natural inhibitors. Plasmin mediates direct or indirect degradation of the extracellular matrix, and bound plasmin is used by cells to facilitate migration through extracellular matrices. Since cell migration and tissue remodelling are the underpinnings of many physiological and pathological responses, the modulation of plasminogen receptors may serve as a primary regulatory mechanism for control of many cellular responses. Specific examples of cell types on which plasminogen receptors undergo modulation include: fibroblasts, where modulation may contribute to cartilage and bone destruction in rheumatoid arthritis; leukemic cells, where enhanced plasminogen binding may contribute to the heightened fibrinolytic state in the patients; other tumor cells, where up-regulation may support invasion and metastasis; bacteria, where enhanced plasminogen binding may facilitate tissue destruction and invasion; platelets, where up-regulation of plasminogen binding may play a role in regulating clot lysis; and adipocytes, where the modulation of plasminogen receptor expression may regulate cell differentiation and fat accumulation. Two pathways for modulation of plasminogen receptors have been characterized: A protease-dependent pathway can either up-regulate or down-regulate plasminogen binding to cells by changing the availability of plasminogen-binding proteins with C-terminal lysines. New receptors may be generated by trypsin-like proteases, including plasmin, which create new C-terminal lysines; other enzymes may expose existing membrane proteins by altering the cell surface; or receptor function may be lost by removal of C-terminal lysines. The basic carboxypeptidases of blood carboxypeptidase N and plasma carboxypeptidase B (TAFI) mediate such down-regulation. A non-protease dependent pathway for modulation of plasminogen receptors may be initiated by growth factors, chemokines or cytokines that alter the cell membrane and/or cytoskeleton architectures to expose plasminogen binding sites. Many examples of the modulation of plasminogen receptors have been demonstrated in vitro, and the development of knock-out mice may soon lead to incisive evaluations of the significance of the regulation of plasminogen receptors in vivo.

Animals↗
PubMed2002

Convergence of the adhesive and fibrinolytic systems: recognition of urokinase by integrin alpha Mbeta 2 as well as by the urokinase receptor regulates cell adhesion and migration.

Previous studies demonstrated that integrin alpha(M)beta(2) (CD11b/18, Mac-1) forms a physical complex with the urokinase-type plasminogen activator receptor (uPAR/CD87) on leukocytes. In this study, we used human peripheral blood neutrophils and transfected cells expressing alpha(M)beta(2), uPAR, or both receptors to show that the integrin can directly interact with urokinase (uPA). We demonstrate that alpha(M)beta(2) supported adhesion and migration of these cells to uPA, and, in each case, blockade of alpha(M)beta(2) suppressed the response. Within uPA, both the kringle and proteolytic domains are recognized by alpha(M)beta(2), which are distinct from the growth factor domain that binds to uPAR. Within the alpha(M) subunit of the integrin, the I domain interacts with uPA, which is distinct from the region that interacts with uPAR. On cells expressing uPAR and alpha(M)beta(2), both receptors mediated adhesion and migration. This cooperation was particularly apparent in the responses of neutrophils to uPA, where blockade of alpha(M)beta(2) reduced uPAR-mediated responses and engagement of uPAR enhanced recognition of uPA by alpha(M)beta(2). Thus, recognition of uPA by alpha(M)beta(2) allows for formation of a multicontact trimolecular complex, in which a single uPA ligand may bind simultaneously to both uPAR and alpha(M)beta(2). This complex may play an important role in the control of inflammatory cell migration and vascular homeostasis.

Cell Adhesion↗
PubMed2002

Delineation of the key amino acids involved in neutrophil inhibitory factor binding to the I-domain supports a mosaic model for the capacity of integrin alphaMbeta 2 to recognize multiple ligands.

To gain insight into the mechanism by which the alpha(M)I-domain of integrin alpha(M)beta(2) interacts with multiple and unrelated ligands, the identity of the neutrophil inhibitory factor (NIF) recognition site was sought. A systematic strategy in which individual amino acid residues within three previously implicated segments were changed to those in the alpha(L)I-domain, which is structurally very similar but does not bind NIF, was implemented. The capacity of the resulting mutants, expressed as glutathione S-transferase fusion proteins, to recognize NIF was assessed. These analyses ultimately identified Asp(149), Arg(151), Gly(207), Tyr(252), and Glu(258) as critical for NIF binding. Cation binding, a function of the metal ion-dependent adhesion site (MIDAS) motif, was assessed by terbium luminescence to evaluate conformational perturbations induced by the mutations. All five mutants bound terbium with unaltered affinities. When the five residues were inserted into the alpha(L)I-domain, the chimera bound NIF with high affinity. Another ligand of alpha(M)beta(2), C3bi, which is known to use the same segments of the alpha(M)I-domain in engaging the receptor, failed to bind to the chimeric alpha(L)I-domain. Thus, the alpha(M)I-domain appears to present a mosaic of exposed amino acids within surface loops on its MIDAS face, and different ligands interact with different residues to attain high affinity binding.

Amino Acid Sequence↗
PubMed2002

Identification and characterization of two cation binding sites in the integrin beta 3 subunit.

The midsegment of the beta(3) subunit has been implicated in the ligand and cation binding functions of the beta(3) integrins. This region may contain a metal ion-dependent adhesion site (MIDAS) and fold into an I domain-like structure. Two recombinant fragments, beta(3)-(95-373) and beta(3)-(95-301), were expressed and found to bind fibrinogen. Whereas 0.1 mm Ca(2+) supported ligand binding to both recombinant fragments, 1.0 mm Ca(2+) suppressed binding to the longer but not the shorter fragment. These properties suggest that beta(3)-(95-373) contains both the ligand-competent (LC) and inhibitory (I) cation binding sites, and beta(3)-(95-301) lacks the I site. In equilibrium dialysis experiments, beta(3)-(95-373) contained two divalent cation binding sites, one reactive with either Mg(2+) or Ca(2+) and one Ca(2+)-specific, whereas beta(3)-(95-301) lacked the Ca(2+)-specific site. Mutant forms of beta(3)-(95-373) suggested that the LC site is a MIDAS motif involving Asp(119), Ser(121), Ser(123), Asp(217), and/or Glu(220) as coordination sites, and the I site was dependent upon residues within beta(3)-(301-323). In a molecular model of beta(3)-(95-373), a second Ca(2+) could be docked onto a flexible loop in close proximity to the MIDAS. These results indicate that the ligand competent and Ca(2+)-specific inhibitory cation binding sites are distinct and reside in beta(3)-(95-373).

Amino Acid Sequence↗
PubMed2002

In vivo regulation of plasminogen function by plasma carboxypeptidase B.

The major functions of plasminogen (Plg) in fibrinolysis and cell migration depend on its binding to carboxy-terminal lysyl residues. The ability of plasma carboxypeptidase B (pCPB) to remove these residues suggests that it may act as a suppressor of these Plg functions. To evaluate this role of pCPB in vivo, homozygote pCPB-deficient mice were generated by homologous recombination, and the resulting pCPB(-/-) mice, which were viable and healthy, were mated to Plg(-/-) mice. Plg(+/-) mice show intermediate levels of fibrinolysis and cell migration compared with Plg wild-type and deficient mice, reflecting the intermediate levels of the Plg antigen in their plasma. Differences in Plg-dependent functions between pCPB(+/+), pCPB(+/-), and pCPB(-/-) mice were then analyzed in a Plg(+/-) background. In a pulmonary clot lysis model, fibrinolysis was significantly increased in mice with partial (pCPB(+/-)) or total absence (pCPB(-/-)) of pCPB compared with their wild-type counterparts (pCPB(+/+)). In a thioglycollate model of peritoneal inflammation, leukocyte migration at 72 hours increased significantly in Plg(+/-)/pCPB(+/-) and Plg(+/-)/pCPB(-/-) compared with their wild-type counterparts. These studies demonstrate a definitive role of pCPB as a modulator of the pivotal functions of Plg in fibrinolysis and cell migration in vivo.

Animals↗
PubMedDate not supplied

Phase II trial of GM-CSF + thalidomide in patients with androgen-independent metastatic prostate cancer.

BACKGROUND: Androgen-independent metastatic prostate cancer is a major therapeutic dilemma. Granulocyte-macrophage colony stimulating factor (GM-CSF) and thalidomide have some biologic activity as single agents in this disease subset. We performed a Phase II trial of this combination to assess its toxicity and potential utility as an immunomodulatory approach to management of advanced prostate cancer. METHODS: Twenty-two patients were treated with GM-CSF 250 microg administered SC on Monday, Wednesday and Friday of each week. Thalidomide was escalated to reach the study dose of 200 mg/day. Patients were assessed every 4 weeks with therapy continuing to a maximum of 6 months. RESULTS: All 22 patients had a decrement in PSA at 2 weeks postinitiation of therapy. Five patients had a > or =50% decline (56, 64,66,66, and 94%, respectively) from baseline verified at 4 weeks post best response. This corresponds to an observed response rate of 23% (95% confidence interval 8-45%). Therapy was well tolerated with the majority of patients experiencing only one event. CONCLUSIONS: The combination of GM-CSF + thalidomide is relatively well tolerated and has the potential to produce antitumor activity in a population of patients with metastatic, androgen independent prostate cancer. This nonchemotherapy combination should be explored in a subset of patients with less advanced disease.

Aged↗
PubMedDate not supplied

In vivo comparison of the inflammatory response induced by different vascular biomaterials.

Biomaterial implants induce a local inflammatory response. A comparison of the inflammatory cell response was made between several biomaterials commonly used as vascular prostheses. Disks of polyethylene terephthalate (PET), polytetrafluoroethylene (PTFE), aluminum, titanium, copper, and stainless steel were surgically placed into the peritoneum of mice. Recruited macrophage and neutrophil populations were measured after recovery from the disk surface and peritoneal lavage. Following peritoneal biomaterial implants, there was no difference in total neutrophil or macrophage recruitment between mice implanted with PET, PTFE, aluminum, or titanium disks. However, there was significant attenuation of total neutrophil and macrophage recruitment to stainless steel compared with the other implants. Similarly, there was no significant difference in the percentage of leukocytes adherent to the PET, aluminum, or titanium disks. Macrophage adherence to the stainless steel disks was attenuated by 19.1%, and the number of neutrophils was attenuated by 69.1% when compared with PET implant mice. Mice implanted with copper disks universally expired. Leukocyte recruitment did not differ between PET, PTFE, aluminum, or titanium disks, suggesting that these materials stimulate similar inflammatory responses. Stainless steel disks recruited both fewer neutrophils and fewer macrophages and support lower adherence of these cells than the other biomaterials. Copper incited an overwhelming and fatal response.

Animals↗