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PubMed1996

Human trophoblast cell adhesion to extracellular matrix protein, entactin.

PROBLEM: Trophoblast interaction with endometrial extracellular matrix (ECM) is crucial during human embryo implantation and placentation. Entactin, a ubiquitous basement membrane glycoprotein, plays a central role in ECM assembly, cell attachment, and chemotaxis. The present study was conducted to examine the possible role of entactin in promoting human trophoblast adhesion. METHODS: Using an extended life span first trimester trophoblast cell line HTR-8/SVneo (HTR) and a cell adhesion assay, we measured the adherence of human first trimester trophoblasts to recombinant entactin and its domains. Also, we used flow cytometry and indirect immunofluorescence to detect the presence of integrins that may be involved in human trophoblast-entactin interaction; these methods were used to analyze HTR cells, as well as tissue sections and freshly isolated human trophoblasts from first trimester and term placenta. RESULTS: We found that first trimester trophoblast cells were highly adherent to entactin and its E and G2 domains but not to G1 or G3 domains. Using indirect immunofluorescence and flow cytometry, we found that both beta 1 and beta 3 integrin subunits were expressed on the surface of HTR trophoblast cells adhering to entactin; in contrast, beta 2 and beta 4 integrin subunits were not detected. In addition, we found that alpha v beta 3 was expressed on freshly isolated villous cytotrophoblasts and cytotrophoblast and syncytiotrophoblasts in tissue sections from term placenta. The beta 3 integrin subunit was expressed in cytotrophoblasts and syncytiotrophoblasts in villi of first trimester placental tissue sections. CONCLUSION: Recombinant entactin promotes human trophoblast cell adhesion through both its E and G2 domains and these specific adhesive interactions may be mediated by beta 1 and/or beta 3 class integrins.

Antigens, CD↗
PubMed2002

Candida albicans expresses a focal adhesion kinase-like protein that undergoes increased tyrosine phosphorylation upon yeast cell adhesion to vitronectin and the EA.hy 926 human endothelial cell line.

The signaling pathways triggered by adherence of Candida albicans to the host cells or extracellular matrix are poorly understood. We provide here evidence in C. albicans yeasts of a p105 focal adhesion kinase (Fak)-like protein (that we termed CaFak), antigenically related to the vertebrate p125Fak, and its involvement in integrin-like-mediated fungus adhesion to vitronectin (VN) and EA.hy 926 human endothelial cell line. Biochemical analysis with different anti-chicken Fak antibodies identified CaFak as a 105-kDa protein and immunofluorescence and cytofluorimetric analysis on permeabilized cells specifically stain C. albicans yeasts; moreover, confocal microscopy evidences CaFak as a cytosolic protein that colocalizes on the membrane with the integrin-like VN receptors upon yeast adhesion to VN. The protein tyrosine kinase (PTK) inhibitors genistein and herbimycin A strongly inhibited C. albicans yeast adhesion to VN and EA.hy 926 endothelial cells. Moreover, engagement of alpha v beta 3 and alpha v beta 5 integrin-like on C. albicans either by specific monoclonal antibodies or upon adhesion to VN or EA.hy 926 endothelial cells stimulates CaFak tyrosine phosphorylation that is blocked by PTK inhibitor. A role for CaFak in C. albicans yeast adhesion was also supported by the failure of VN to stimulate its tyrosine phosphorylation in a C. albicans mutant showing normal levels of CaFak and VNR-like integrins but displaying reduced adhesiveness to VN and EA.hy 926 endothelial cells. Our results suggest that C. albicans Fak-like protein is involved in the control of yeast cell adhesion to VN and endothelial cells.

Antigens, Bacterial↗
PubMed2001

Anti-integrin therapy.

The glycoprotein IIb/IIIa integrin receptor binds fibrinogen and is therefore a final common pathway responsible for platelet aggregation. One antibody (abciximab) and two synthetic compounds (tirofiban and eptifibatide) are clinically available to antagonize the function of this receptor. Several large-scale studies have documented the benefit of these compounds in acute coronary syndromes and during percutaneous interventions. Current data suggest that abciximab is the preferred drug in the catheterization laboratory, whereas the other compounds reduce risk for patients with unstable angina before coronary interventions are performed. The highest benefit is achieved in diabetic patients and in patients with elevated troponins. Adverse reactions are rare, and bleeding complications are minor when weight-adjusted heparin is given. Oral compounds have been associated with excess mortality, precluding their clinical use.

Angioplasty, Balloon, Coronary↗
PubMed2006

Roles of phosphatidylinositol 3-kinase and osteopontin in steatosis and aminotransferase release by hepatocytes treated with methionine-choline-deficient medium.

Feeding mice a methionine and choline-deficient (MCD) diet serves as an experimental animal model for nonalcoholic steatohepatitis (NASH). In the present study we examined the effect of exposing AML-12 hepatocytes to MCD culture medium in regard to mechanisms of steatosis and alanine amino-transferase (ALT) release. Cells exposed to MCD medium developed significant and progressive steatosis from 6 to 24 h and also had significantly increased loss of ALT into the medium at 18 and 24 hours of incubation. No increased oxidative injury or cell death was observed. Osteopontin (OPN) mRNA in cells and protein expression in medium were significantly increased during 6-24 hours of incubation. MCD medium treatment also resulted in activation of PI3-kinase by 30 minutes and its downstream target p-Akt within 1hour of incubation. Steatosis was associated with increased expression of microsomal triglyceride transfer protein (MTTP) mRNA and increased ALT release with over expression of ALT mRNA, all of which were completely prevented by inhibition of PI3-kinase (LY294002). Blocking OPN signaling by treating with anti-OPN or anti-beta3-integrin antibody prevented the increased ALT release while only partially prevented the increased ALT mRNA expression, but had no effect on either steatosis or MTTP expression. In conclusion, incubation of cultured hepatocytes with MCD medium results in cellular steatosis and OPN dependent ALT release. PI3-kinase plays a central role in signaling the MCD medium-induced steatosis and increased OPN expression, whereas OPN appears to play a role in signaling hepatocyte ALT release but not steatosis.

Animals↗
PubMed2005

Matrix metalloproteinase-9 gene deletion facilitates angiogenesis after myocardial infarction.

Matrix metalloproteinases (MMPs) are postulated to be necessary for neovascularization during wound healing. MMP-9 deletion alters remodeling postmyocardial infarction (post-MI), but whether and to what degree MMP-9 affects neovascularization post-MI is unknown. Neovascularization was evaluated in wild-type (WT; n = 63) and MMP-9 null (n = 55) mice at 7-days post-MI. Despite similar infarct sizes, MMP-9 deletion improved left ventricular function as evaluated by hemodynamic analysis. Blood vessel quantity and quality were evaluated by three independent studies. First, vessel density was increased in the infarct of MMP-9 null mice compared with WT, as quantified by Griffonia (Bandeiraea) simplicifolia lectin I (GSL-I) immunohistochemistry. Second, preexisting vessels, stained in vivo with FITC-labeled GSL-I pre-MI, were present in the viable but not MI region. Third, a technetium-99m-labeled peptide (NC100692), which selectively binds to activated alpha(v)beta3-integrin in angiogenic vessels, was injected into post-MI mice. Relative NC100692 activity in myocardial segments with diminished perfusion (0-40% nonischemic) was higher in MMP-9 null than in WT mice (383 +/- 162% vs. 250 +/- 118%, respectively; P = 0.002). The unique finding of this study was that MMP-9 deletion stimulated, rather than impaired, neovascularization in remodeling myocardium. Thus targeted strategies to inhibit MMP-9 early post-MI will likely not impair the angiogenic response.

Animals↗
PubMed1997

An Arg-Gly-Asp peptide stimulates constriction in rat afferent arteriole.

The potential role of integrins in the myogenic mechanism was studied in the rat afferent arteriole (AA) by fluorescence immunolocalization and microperfusion of isolated AA. Confocal fluorescence images were acquired from frozen sections of rat kidney after indirect immunostaining for various integrin beta- and alpha-subunits. The beta 1-, beta 3-, alpha 3-, alpha 5-, and alpha V-integrins were found on the plasma membrane in smooth muscle of AA, providing the morphological basis for participation of integrins in mechanotransduction. With 1 mM nitro-L-arginine methyl ester (L-NAME) in the luminal perfusate to inhibit endogenous nitric oxide (NO) production from AA, the hexapeptide GRGDSP (10(-7)-10(-3)M) induced immediate vasoconstriction. The constriction was dose dependent and specific or peptides with arginine-glycine-aspartic acid (RGD) motifs, commonly found on the binding sites of extracellular matrix to integrins. In controls, the hexapeptide GRGESP induced no constriction. GRGDSP, 1 mM, induced a 21.6 +/- 2.6% decrease (P < 0.05, n = 6) in lumen diameter for 30 s and an 18.3 +/- 4.1% increase (P < 0.05, n = 6) in smooth muscle intracellular calcium concentration for 18 s, as measured by the emission ratio of Fluo-3/Fura Red. Binding of exogenous RGD motifs with exposed integrins on AA smooth muscle therefore triggers calcium-dependent vasoconstriction. However, the dose response to RGD was not sensitive to the myogenic tone of the vessel, which suggests that the integrin-mediated vasoconstriction is different from myogenic constriction.

Amino Acid Sequence↗
PubMed2002

Engagement of glycoprotein IIb/IIIa (alpha(IIb)beta3) on platelets upregulates CD40L and triggers CD40L-dependent matrix degradation by endothelial cells.

BACKGROUND: CD40L-CD40 interactions induce inflammatory signals in cells of the vascular wall. We evaluated the effects of glycoprotein (GP) IIb/IIIa (alpha(IIb)beta3) engagement that occurs during platelet-endothelium interactions on CD40L surface exposure on platelets and initiation of proteolytic activity in human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: Transient (60-minute) adhesion of thrombin-prestimulated platelets enhanced HUVEC expression of urokinase-type plasminogen activator receptor and membrane type-1 matrix metalloproteinase (MT1-MMP) (reverse transcriptase-polymerase chain reaction, flow cytometry) and secretion of urokinase-type plasminogen activator, tissue-type plasminogen activator, and MMP-1 (ELISA) and induced proteolytic activity via MMP-2 and MMP-9 (gelatin zymography). These effects were abrogated by hindrance of physical platelet-endothelial contacts using transwell systems or inhibited by GRGDSP, mAbs anti-GP IIb/IIIa (7E3), anti-alpha(v)beta3 (LM609), or anti-CD40L (TRAP1). In addition, MMP-2 and MMP-9 were inhibited by specific GP IIb/IIIa antagonists tirofiban, lamifiban, or integrelin. On endothelial cells, induction of proteolytic activity by activated platelets was mimicked by CD40 engagement using soluble CD40L but not affected by antibody clustering of alpha(v)beta3. On platelets, CD40L and CD62P exposure was enhanced on adhesion to HUVECs or immobilized fibrinogen and was abrogated by GRGDSP or LM609. In suspension, cross-linking of GP IIb/IIIa by fibrinogen plus secondary mAb upregulated CD40L surface exposure. Consistently, bivalent mAb 7E3 upregulated CD40L, whereas ligation of GP IIb/IIIa by soluble fibrinogen alone or monovalent Fab-fragment c7E3 had no effect. CONCLUSIONS: Platelet adhesion via GP IIb/IIIa upregulates CD40L and CD62P surface exposure. Proteolytic activity of HUVEC is induced by the concerted action of beta3-integrin-mediated platelet adhesion and subsequent CD40L-induced signals in HUVECs. Effective anti-GP IIb/IIIa or anti-CD40L strategies might, therefore, contribute to plaque stabilization.

Blood Platelets↗
PubMed2002

Plasminogen activator inhibitor-1 promotes formation of endothelial microparticles with procoagulant potential.

BACKGROUND: Endothelial dysfunction is emerging as a common denominator for diverse and highly prevalent cardiovascular diseases. Increased level of plasminogen activator inhibitor-1 (PAI-1) and procoagulant activity have been recognized as hallmarks of endothelial dysfunction. This study was aimed at investigating cellular actions of PAI-1 and a potential link between PAI-1 and procoagulant state. METHODS AND RESULTS: Human umbilical vein endothelial cells treated with PAI-1 were subjected to laser confocal fluorescence microscopy, immunoprecipitation and Western blotting, and FACS analysis for isolation and identification of endothelial microparticles. PAI-1 treatment resulted in a reduced expression of uPAR, its colocalization with caveolin, and the concomitant increase of uPAR abundance in the culture medium. FACS analysis revealed that PAI-1 rapidly and dose-dependently increased the number of endothelial microparticles expressing uPAR and alpha(V)beta3 integrin. This process was attenuated by pretreatment with neutralizing anti-uPAR antibodies. PAI-1 knockout mice showed a significantly decreased number of circulating endothelial microparticles than wild-type mice; however, PAI-1-deficient animals responded to infusion of PAI-1 with a more pronounced rise in the number of microparticles. PAI-1 treatment increased the number of microparticles stained with Annexin V, evidence for the expression of anionic phospholipids. This was accompanied by the accelerated generation of thrombin. CONCLUSIONS: The data disclose a novel effect of PAI-1 to dose-dependently promote formation of endothelial microparticles with the reduced transmembrane asymmetry of phospholipids. This phenomenon may be responsible for the observed increase in in vitro thrombin generation. These findings could potentially link these hallmarks of endothelial dysfunction-elevated levels of PAI-1 and propensity toward thrombosis.

Animals↗
PubMed2000

Angiotensin II enhances integrin and alpha-actinin expression in adult rat cardiac fibroblasts.

Angiotensin II (Ang II) plays an important role in cardiac remodeling through stimulation of proliferation and extracellular matrix (ECM) production in cardiac fibroblasts. Integrins are a family of transmembrane receptors that mediate the attachment of cells to ECM. We hypothesized that Ang II regulation of integrins further contributes to its role in cardiac remodeling. We cultured adult rat cardiac fibroblasts with and without Ang II (100 nmol/L) to determine the effects on mRNA and protein levels of integrins, as well as alpha-actinin and other cytoskeletal proteins that link to integrins at the site of focal adhesions. Ang II was also added in the presence of irbesartan (10 micromol/L), a specific Ang II type 1 (AT(1)) receptor antagonist, or PD 123319 (10 micromol/L), a specific Ang II type 2 receptor antagonist. To investigate the function of these integrins, we determined the effects of blocking antibodies on Ang II-induced adhesion to ECM. We also treated spontaneously hypertensive rats (SHR) with an AT(1) receptor blocker, losartan, or with hydralazine to investigate integrin and alpha-actinin expression in treated and untreated SHR. Ang II enhanced alpha(v), beta(1), beta(3), and beta(5) integrins; osteopontin; and alpha-actinin mRNA and protein levels in cardiac fibroblasts. All of these effects were inhibited by irbesartan but not by PD 123319. Pretreatment of cardiac fibroblasts with Ang II enhanced cell attachment to ECM proteins and induced focal adhesion kinase phosphorylation. Blocking antibodies to beta(3) and alpha(v)beta(5) attenuated Ang II-induced adhesion. In SHR, ventricular alpha(v) and beta(5) integrin expression and alpha-actinin were increased compared with those in Wistar-Kyoto rats. Although both losartan and hydralazine lowered mean arterial pressure and decreased peripheral vascular resistance, only losartan attenuated the increased integrin, alpha-actinin, fibronectin laminin, and osteopontin expression and the increased left ventricular mass (as determined with echocardiography). Hydralzine had none of these effects. Although both agents attenuated beta-myosin heavy chain expression, a marker of hypertrophy, losartan had a greater effect. These results suggest that integrins and alpha-actinin are upregulated by Ang II and in left ventricular hypertrophy and that the block of expression of these proteins through inhibition of the AT(1) receptor is associated with attenuation of the hypertrophic response. Ang II induces integrin and alpha-actinin expression in cardiac fibroblasts that is associated with adhesion and left ventricular hypertrophy and blocked through inhibition of the AT(1) receptor.

Actinin↗
PubMed2004

Thromboxane A2 receptor signaling inhibits vascular endothelial growth factor-induced endothelial cell differentiation and migration.

Vascular endothelial growth factor (VEGF) is an important patho-physiological mediator of angiogenesis. VEGF-induced endothelial cell (EC) migration and angiogenesis often occur in complicated environments containing multiple agents capable of modifying the response. Thromboxane (TX) A2 is released from multiple cell types and is a prime mediator of pathogenesis of many vascular diseases. Human EC express both TXA2 receptor (TP) isoforms; however, the effects of individual TP isoforms on VEGF-induced EC migration and angogenesis are unknown. We report here that the TXA2 mimetic [1S-(1alpha, 2beta(5Z), 3alpha(1E, 3R), 4alpha]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxab icyclo-[2.2.1]heptan-2yl]-5'-heptenoic acid (IBOP) (100 nmol/L) is a potent antagonist (IC50 30 nmol/L) of VEGF-induced EC migration and differentiation. TPbeta, but not TPalpha, expression is required for the inhibition of VEGF-induced migration and angiogenesis. IBOP costimulation suppressed nitric oxide (NO) release from VEGF-treated EC through decreased activation of Akt, eNOS, and PDK1. TPbeta costimulation also ablated the increase in focal adhesion formation in response to VEGF. This mechanism was characterized by decreased recruitment of focal adhesion kinase (FAK) and vinculin to the alpha(v)beta3 integrin and reduced FAK and Src activation in response to VEGF. Addition of NO donors together with transfection of a constitutively active Src construct could circumvent the blockade of VEGF-induced migration by TP; however, neither intervention alone was sufficient. Thus, TP stimulation appears to limit angiogenesis, at least in part, by inhibiting the pro-angiogenic cytokine VEGF. These data further support a role for antagonism of TP activation in enhancing the angiogenic response in tissues exposed to elevated TXA2 levels in which revascularization is important.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2005

Distinct pathways regulate expression of cardiac electrical and mechanical junction proteins in response to stretch.

To define mechanisms regulating expression of cell-cell junction proteins, we have developed an in vitro system in which neonatal rat ventricular myocytes were subjected to pulsatile stretch. Previously, we showed that expression of the gap junction protein, connexin (Cx) 43, is increased by approximately 2-fold after 1 hour of stretch, and this response is mediated by stretch-induced secretion of vascular endothelial growth factor (VEGF). Here, we report that the mechanical junction proteins plakoglobin, desmoplakin, and N-cadherin are also upregulated by pulsatile stretch but by a mechanism independent of VEGF or other secreted chemical signals. Stretch-induced upregulation of mechanical junction proteins was blocked by anti-beta1 and anti-beta3 integrin antibodies. Transfection of cells with adenovirus expressing GFP-FRNK, a dominant-negative inhibitor of focal adhesion kinase (FAK)-dependent signaling, blocked stretch-induced upregulation of Cx43 and mechanical junction proteins but did not block the ability of exogenous VEGF to upregulate Cx43 expression. Conditioned medium removed from uninfected cells after stretch increased Cx43 expression when added to nonstretched cells, and this effect was blocked by anti-VEGF antibodies, but stretch-conditioned medium from GFP-FRNK cells had no effect on Cx43 expression. The src kinase inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine blocked stretch-induced upregulation of mechanical junction proteins but not Cx43. Thus, stretch upregulates expression of both electrical and mechanical junction proteins via integrin-dependent activation of FAK. Stretch-induced upregulation of Cx43 expression is mediated by FAK-dependent secretion of VEGF. In contrast, stretch-induced upregulation of adhesion junction proteins involves intracellular mechanotransduction pathways initiated via integrin signaling and acting downstream of src kinase.

Animals↗
PubMed2005

Cellular pathology of atherosclerosis: smooth muscle cells promote adhesion of platelets to cocultured endothelial cells.

Although platelets do not ordinarily bind to endothelial cells (EC), pathological interactions between platelets and arterial EC may contribute to the propagation of atheroma. Previously, in an in vitro model of atherogenesis, where leukocyte adhesion to EC cocultured with smooth muscle cells was greatly enhanced, we also observed attachment of platelets to the EC layer. Developing this system to specifically model platelet adhesion, we show that EC cocultured with smooth muscle cells can bind platelets in a process that is dependent on EC activation by tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1. Recapitulating the model using EC alone, we found that a combination of TGF-beta1 and TNF-alpha promoted high levels of platelet adhesion compared with either agent used in isolation. Platelet adhesion was inhibited by antibodies against GPIb-IX-V or alpha(IIb)beta3 integrin, indicating that both receptors are required for stable adhesion. Platelet activation during interaction with the EC was also essential, as treatment with prostacyclin or theophylline abolished stable adhesion. Confocal microscopy of the surface of EC activated with TNF-alpha and TGF-beta1 revealed an extensive matrix of von Willebrand factor that was able to support the adhesion of flowing platelets at wall shear rates below 400 s(-1). Thus, we have demonstrated a novel route of EC activation which is relevant to the atherosclerotic microenvironment. EC activated in this manner would therefore be capable of recruiting platelets in the low-shear environments that commonly exist at points of atheroma formation.

Atherosclerosis↗
PubMed1997

Integrin signaling transduces shear stress--dependent vasodilation of coronary arterioles.

A direct relationship exists between shear stress and endothelium-dependent NO-mediated vasodilation of blood vessels. The transduction of shear stress to the biochemical signals resulting in the production of NO is, however, unknown. We tested the hypothesis that integrin binding to Arg-Gly-Asp(RGD) peptide sequences in extracellular matrix proteins is a critical step in initiation of the signaling sequence whereby shear stress activates endothelial tyrosine kinase(s) and induces vasodilation of isolated arterioles. Isolated coronary arterioles were exposed to increasing shear stress under control conditions and in the presence of a synthetic peptide, GRGDNP, to competitively inhibit integrin binding to extracellular matrix proteins containing RGD peptide sequences. Intraluminal GRGDNP (0.1, 0.5, and 1.0 mmol/L) inhibited shear stress-induced vasodilation in a concentration-dependent manner. Application of GRGDNP had no effect on endothelium-dependent relaxation to substance P (10(-12) to 10(-8) mol/L). An inactive structural analogue, GRGESP, did not alter shear stress-induced vasodilation. To further elucidate the integrin involved in shear stress-induced vasodilation, we administered a blocking antibody to the integrin beta 3 chain (F11) and observed significant attenuation of the vasodilation. Shear stress was also associated with an increase in tyrosine kinase activity, as assessed by anti-phosphotyrosine binding. Application of GRGDNP significantly decreased anti-phosphotyrosine binding during shear stress, suggesting a link between tyrosine kinase activation and integrin signaling during this vasodilatory response. Taken together, these results indicate that integrin-matrix interactions, possibly at focal adhesions, are of cardinal importance in the signaling pathway of shear stress-induced vasodilation.

Animals↗
PubMed2003

Resident murine alveolar and peritoneal macrophages differ in adhesion of apoptotic thymocytes.

Apoptotic cells must be cleared efficiently by macrophages (Mø) to prevent autoimmunity, yet their ingestion impairs Mø microbicidal function. The principal murine resident lung phagocyte, the alveolar Mø (AMø), is specifically deficient at apoptotic cell ingestion, both in vitro and in vivo, compared with resident peritoneal Mø (PMø). To further characterize this deficiency, we assayed static adhesion in vitro using apoptotic thymocytes and resident AMø and PMø from normal C57BL/6 mice. Adhesion of apoptotic thymocytes by both types of Mø was rapid, specific, and cold-sensitive. Antibody against the receptor tyrosine kinase MerTK (Tyro12) blocked phagocytosis but not adhesion in both types of Mø. Surfactant protein A increased adhesion and phagocytosis by AMø, but not to the levels seen using PMø. Adhesion was largely cation-independent for PMø and calcium-dependent for AMø. Adhesion was not inhibited in either Mø type by mAbs against beta1 or beta3 integrins or scavenger receptor I/II (CD204), but AMø adhesion was inhibited by specific mAbs against CD11c/CD18. Thus, resident murine tissue Mø from different tissues depend on qualitatively disparate receptor systems to bind apoptotic cells. The decreased capacity of murine AMø to ingest apoptotic cells is only partially explained by reduced initial adhesion.

Animals↗
PubMed1994

Fibrinogen mediates platelet-polymorphonuclear leukocyte cooperation during immune-complex glomerulonephritis in rats.

The metabolic and functional alterations which occur during the acute phase of nephrotoxic nephritis (NTN) in rats, a model of immune-mediated glomerulonephritis, result from a cooperative interaction between PMNs and platelets (PLTs). In consequence, we hypothesized that fibrinogen (Fg) might play a critical role in this process and, accordingly, we found that defibrination of animals decreased both the acute phase proteinuria in NTN (approximately 70%) as well as the influx of PLTs and PMNs into the glomerulus (approximately 40-50%). In contrast, blockade of the PLT Fg receptor, alpha IIb beta 3, with the RGD peptidomimetic SC-49992 decreased proteinuria (approximately 90%) without substantially altering the influx of PMNs or PLTs. Immunocytochemistry showed a marked increase in beta 3 integrin expression in inflamed glomeruli which was prevented either by PMN or PLT depletion before disease induction. FACS and immunocytochemical analysis of glomerular cell dissociates demonstrated that beta 3 integrin expression was predominantly on intraglomerular PLTs. In vitro, activated PLTs stimulated the PMN respiratory burst, an interaction which could be inhibited by Fg receptor blockade. In sum, acute NTN is accompanied by a marked increase in glomerular beta 3 integrin expression predominantly due to the influx of PLTs which localize to the glomerulus in a PMN-dependent fashion. Fg appears to serve a major role as a coactivating stimulus for PLT-PMNs in situ via alpha IIb beta 3, potentially mediating the PMN respiratory burst which contributes to proteinuria. Fg may also play a subsidiary role in PMN/PLT comigration.

Animals↗
PubMed2001

A Glanzmann's mutation in beta 3 integrin specifically impairs osteoclast function.

Osteoclastic bone resorption requires cell-matrix contact, an event mediated by the alpha v beta 3 integrin. The structural components of the integrin that mediate osteoclast function are, however, not in hand. To address this issue, we generated mice lacking the beta 3 integrin gene, which have dysfunctional osteoclasts. Here, we show the full rescue of beta 3(-/-) osteoclast function following expression of a full-length beta 3 integrin. In contrast, truncated beta 3, lacking a cytoplasmic domain (h beta 3c), is completely ineffective in restoring function to beta 3(-/-) osteoclasts. To identify the components of the beta 3 cytoplasmic domain regulating osteoclast function, we generated six point mutants known, in other circumstances, to mediate beta integrin signaling. Of the six, only the S(752)P substitution, which also characterizes a form of the human bleeding disorder Glanzmann's thrombasthenia, fails to rescue beta 3(-/-) osteoclasts or restore ligand-activated signaling in the form of c-src activation. Interestingly, the double mutation Y(747)F/Y(759)F, which disrupts platelet function, does not affect the osteoclast. Thus similarities and distinctions exist in the mechanisms by which the beta 3 integrin regulates platelets and osteoclasts.

Amino Acid Sequence↗
PubMed2005

Increased tyrosine phosphorylation of platelet proteins including pp125(FAK) suggests endogenous activation and aggregation in pulmonary hypertension.

Despite of several lines of evidence indicating a pathophysiologic role of platelets in pulmonary hypertension, the occurrence of chronic endogenous platelet activation has been a matter of debate. It was hypothesized that the pattern of tyrosine phosphorylation of platelet proteins examined ex vivo could provide information on the state of platelet activation. This was examined in 10 patients with pulmonary arterial hypertension aged 18 to 53 years. Phosphotyrosine density and the amounts of specific proteins were analyzed in resting platelets after reaction with anti-phosphotyrosine, anti-pp60(s-src), anti-pp125(FAK), and anti-alphaIIbbeta3 antibodies. There was a 79% increase in protein-associated phosphotyrosine in patients in comparison to levels in controls (p<0.05). In particular, phosphorylation on tyrosine residues of pp120 and pp125(FAK) increased 24% and 57%, respectively (p<0.05). Although pp60(s-src)-associated phosphotyrosine was not altered in the patient group as a whole, it was clearly decreased in three subjects. Platelet content of beta3 integrin, pp60(s-src), and pp125(FAK), was not altered. This pattern of phosphorylation suggests an ongoing process of platelet activation. Because phosphorylation of pp125(FAK) is a late, integrin-dependent event, results suggest that platelet activation and aggregation occur in vivo in these patients.

Adolescent↗
PubMed1999

Cytoskeletal organization and incorporation of beta 3 integrin in thrombin-stimulated platelets: effect of acetylsalicylic acid.

Platelet stimulation by agonists is followed by changes in cytoskeletal organization that includes actin polymerization and association of the membrane skeleton (which is connected with the integrin alpha IIb beta 3) with the underlying cytoplasmic actin filaments. The effect of orally administered acetylsalicylic acid to healthy volunteers on incorporation of contractile protein and beta 3 integrin into the cytoskeletal core of thrombin-stimulated platelets was studied. Stimulation was followed by increased contractile protein and beta 3 incorporation into the cytoskeleton. Acetylsalicylic acid intake resulted in decreased incorporation of myosin and actin (32% and 20%, respectively), and a decrease (36%) in the association of beta 3 integrin with the cytoskeletal elements was evident. In conclusion, we have shown that acetylsalicylic acid, besides the known inhibitory effect on thromboxane synthesis, promotes changes in the cytoskeletal organization of thrombin-stimulated platelets that could limit thrombus formation.

Actinin↗