Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q9UE34 (fibrinogen)
 30,244 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous reports have shown that interleukin-6 (IL-6) enhances the responsiveness of platelets to thrombin stimulation and has modest thrombocytopoietic effects in vivo. Thrombopoietin (TPO; mpl ligand) has been shown to have dramatic thrombocytopoietic effect in vivo, but little is known of its capacity to alter platelet function. In this study, a direct comparison of the effects of IL-6 and TPO on platelet function in dogs has been performed, with modest doses of TPO (1 microgram/kg/d) chosen to match or moderately exceed the platelet counts achieved with IL-6 (40 micrograms/kg/d) for 10 days. Platelet responsiveness to thrombin stimulation was assessed in TPO-treated, IL-6-treated, and control dogs by flow cytometric measurement of P-selectin expression. On day 5, the dose of thrombin promoting half maximal stimulation (EC50) of platelets was not significantly changed in TPO-treated dogs, whereas in IL-6-treated dogs the EC50 decreased to 73.1% +/- 6.1% (mean +/- 1 SD; n = 5) of control values (P < 0.01). These experiments were performed on both gel-filtered platelets and washed whole blood, indicating that the observed changes in EC50 were caused by cytokine-mediated alteration of platelets rather than plasma components. Because it has been shown that thiazole orange specifically labels a subpopulation of dog platelets that is less than 24 hours old, the thrombin responsiveness of these young, newly synthesized platelets was determined. The EC50 of thiazole orange-positive platelets from IL-6-treated dogs decreased dramatically by day 5 to 46.5% +/- 13.1% (n = 4) of control values (P < 0.001), whereas TPO-treated dogs did not significantly change. When TPO was directly incubated with platelets ex vivo, no effects on either thrombin-mediated P-selectin expression or adenosine diphosphate-induced fibrinogen binding were observed. These data show that IL-6 alters platelet function, as measured by reactivity to thrombin, whereas TPO does not. This divergence in function is observed even though TPO is equally, or more, effective at promoting platelet production under these experimental conditions.
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PMID:Relative reactivity of platelets from thrombopoietin- and interleukin-6-treated dogs. 863 74

Normal circulating platelets do not adhere to intact, undisturbed endothelium. Studies have shown, however, that platelets will adhere to virally infected or thrombin-stimulated human umbilical vein endothelial cells. Using a novel platelet/endothelial cell adhesion assay we studied the interaction of thrombin-activated platelets to human saphenous vein endothelial cells (HSVEC), and its mechanism(s). Biotinylated platelets were exposed to Hepes-Tyrode buffer, 10E5 or PAC-1 [monoclonal antibodies (Mabs) blocking GPIIb-IIIa], AK4 (Mab blocking P-selectin, 6D1 (Mab blocking vWf binding to GPIb), RGDS (small peptide blocking the fibrinogen binding site), or EDTA (dissociates GPIIb-IIIa complex) and then activated with thrombin. The platelets were subsequently exposed to thrombin-stimulated monolayer HSVEC. Phycoerythrin-streptavidin was added to the wells to fluorescently label the platelets, followed by formaldehyde fixation and washing to remove nonadherent platelets. Adhesion of platelets to HSVEC was assessed using a fluorescent multiwell plate reader. Antibodies which blocked the GPIIb-IIIa receptor and agents which competitively bound the receptor all significantly inhibited activated platelet adhesion to the activated HSVEC. We have found that thrombin significantly increases platelet/HSVEC adhesion, and this event is mediated via the integrin GPIIb-IIIa (fibrinogen receptor). These GPIIb-IIIa receptor blocking Mabs and RGDS may be useful adjuncts for improving patency following angiographic intervention and/or vein grafting in patients with high risk of thrombosis. The assay we have developed is a valuable and relatively simple method for assessing platelet/endothelial cell adhesion and activation.
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PMID:Adhesion of activated platelets to venous endothelial cells is mediated via GPIIb/IIIa. 865 40

The sequence beta (3)203-228 is involved, in a yet undetermined manner, in alpha IIb beta 3 function. We now show that murine monoclonal antibody (MoAb) AP6, specific for beta (3)211-221, binds to alpha IIb beta 3 on adenosine diphosphate (ADP)-activated platelets only when the receptor is occupied by intact fibrinogen. The ligand-induced binding-site reported by AP6 is unique in that it is not expressed following occupancy by either RGD peptides or the gamma-chain carboxy-terminal dodecapeptide. Binding of AP6 to platelets coincides temporally with the binding of the MoAb 9F9, specific for a receptor-induced binding site on fibrinogen. Thus, AP6 reports the binding of fibrinogen to the recognition pocket of alpha IIb beta 3. Its binding to thrombin-stimulated washed platelets correlates with secretion as determined using an MoAb to P-selectin. When ultrathin sections of nonactivated platelets were examined by immunogold staining and electron microscopy, AP6 identified a pool of alpha IIb beta 3 colocalizing with P-selectin and suggesting the presence of alpha IIb beta 3-ligand complexes in the alpha-granule membrane. There was little binding of AP6 to surface alpha IIb beta 3 of unstimulated platelets. After ADP-induced activation, AP6 was abundantly distributed over the entire platelet surface, including pseudopods, but only when fibrinogen was present in the medium. ADP had little effect on AP6 reactivity within platelets. This contrasted with washed platelets and thrombin, where extensive AP6 binding was observed within internal membrane pools as early as 10 to 15 seconds after stimulation. Surface labeling with AP6 followed slower kinetics. Flow cytometry on Triton X-100 permeabilized fixed platelets confirmed AP6 binding to alpha IIb beta 3 within the platelet. Thus, our results provide evidence of (1) a pool of alpha-granule alpha IIb beta 3 occupied by ligand in nonactivated platelets; (2) thrombin-induced activation of alpha IIb beta 3 within the platelet, and (3) thrombin-induced mobilization of ligand-bound alpha IIb beta 3 to the surface.
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PMID:Distribution of ligand-occupied alpha IIb beta 3 in resting and activated human platelets determined by expression of a novel class of ligand-induced binding site recognized by monoclonal antibody AP6. 870 46

To examine the hypothesis that surface P-selectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo. Washed platelets prepared from nonhuman primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and reinfused into the same baboons. Three-color whole blood flow cytometry was used to simultaneously (i) identify platelets with a mAb directed against glycoprotein (GP)IIb-IIIa (integrin alpha 11b beta 3), (ii) distinguish infused platelets by their PKH2 fluorescence, and (iii) analyze platelet function with mAbs. Two hours after infusion of autologous thrombin-activated platelets (P-selectin-positive, PKH2-labeled), 95 +/- 1% (mean +/- SEM, n = 5) of the circulating PKH2-labeled platelets had become P-selectin-negative. Compared with platelets not activated with thrombin preinfusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 h after infusion and only slightly less 48 h after infusion. The loss of platelet surface P-selectin was fully accounted for by a 67.1 +/- 16.7 ng/ml increase in the plasma concentration of soluble P-selectin. The circulating PKH2-labeled, P-selectin-negative platelets were still able to function in vivo, as determined by their (i) participation in platelet aggregates emerging from a bleeding time wound, (ii) binding to Dacron in an arteriovenous shunt, (iii) binding of mAb PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (iv) generation of procoagulant platelet-derived microparticles. In summary, (i) circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, but continue to circulate and function; and (ii) we have developed novel three-color whole blood flow cytometric methods for tracking of platelets and measurement of platelet function in vivo.
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PMID:In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function. 887 31

This report describes the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet production and platelet function in humans. Subjects with advanced solid tumors received PEG-rHuMGDF daily for up to 10 days. There was no increase in circulating platelet count at doses of 0.03 or 0.1 microgram/kg/d by day 12 of study. At doses of 0.3 and 1.0 microgram/kg/d there was a threefold median increase (maximum 10-fold) in platelet count by day 16. The platelets produced in vivo in response to PEG-rHuMGDF showed unchanged aggregation and adenosine triphosphate (ATP)-release responses in in vitro assays. Tests included aggregation and release of ATP in response to adenosine diphosphate (ADP) (10, 5, 2.5, and 1.25 mumol/L), collagen (2 micrograms/mL), thrombin-receptor agonist peptide (TRAP, 10 mumol/L) and ristocetin (1.5 mg/mL). Administration of aspirin to an individual with platelet count of 1,771 x 10(3)/L resulted in the typical aspirin-induced ablation of the normal aggregation and ATP-release response to stimulation with arachidonic acid (0.5 mg/mL), collagen, and ADP (2.5 and 1.25 mumol/L). There was no change in the expression of the platelet-surface activation marker CD62P (P-selectin) nor induction of the fibrinogen binding site on glycoprotein IIb/IIIa as reported by the monoclonal antibody, D3GP3. An elevation of reticulated platelets was evident after 3 days of treatment with PEG-rHuMGDF and preceded the increase in circulating platelet count by 5 to 8 days; this reflected the production of new platelets in response to PEG-rHuMGDF. At later time points, the mean platelet volume (MPV) decreased in a manner inversely proportional to the platelet count. Levels of plasma glycocalicin, a measure of platelet turnover, rose 3 days after the initial increase in the peripheral platelet count. The level of plasma glycocalicin was proportional to the total platelet mass, suggesting that platelets generated in response to PEG-rHuMGDF were not more actively destroyed. Thus, the administration of PEG-rHuMGDF, to humans, increased the circulating platelet count and resulted in fully functional platelets, which showed no detectable increase in reactivity nor alteration in activation status.
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PMID:Administration of pegylated recombinant human megakaryocyte growth and development factor to humans stimulates the production of functional platelets that show no evidence of in vivo activation. 889 92

We examined the effects of nitric oxide (NO)/endothelium-derived relaxing factor (EDRF) on platelet surface glycoproteins (GP). As determined by flow cytometry, in both a washed platelet system and platelet-rich plasma, the EDRF congener (S-nitroso-N-acetylcysteine) markedly inhibited both the thrombin-induced and the (stable thromboxane A2 analogue) U-46619-induced upregulation of P-selectin (alpha-granule protein), CD63 (lysosomal protein), and the GPIIb-IIIa complex (fibrinogen receptor) but minimally inhibited downregulation of the GPIb-IX complex (von Willebrand factor receptor). The inhibitory effects of EDRF were markedly reduced in whole blood or by the addition of washed erythrocytes. Platelets in whole blood were still responsive to guanosine 3',5'-cyclic monophosphate (cGMP), as shown by complete inhibition of P-selectin upregulation by the stable analogue N6,2'-O dibutyryl cGMP. These data suggests that 1) cGMP negatively regulates the platelet surface expression of P-selectin, CD63, and the GPIIb-IIIa complex but not the platelet surface expression of the GPIb-IX complex and 2) hemoglobin within erythrocytes inhibits the effects of EDRF/NO on platelet surface glycoproteins.
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PMID:Effects of nitric oxide/EDRF on platelet surface glycoproteins. 892 69

Protein adsorption from human plasma and platelet binding and activation were studied at short blood-titanium/gold contact times. The protein adsorption was studied by ellipsometry-antibody techniques in situ, and adhering platelets were visualized with fluorescein isothiocyanate-labelled anti-CD 61 antibodies. Adhering platelets were quantified by counting labelled cells in microscopic image fields. The spreading of platelets was studied by scanning electron microscopy. The results show that after 1 min of plasma exposure, fibrinogen, IgG and albumin were detectable with antibodies on both surfaces. The amount of deposited fibrinogen and complement decreased with time on titanium, and the amount of adsorbed anti-high molecular weight kininogen increased. No complement was detected on gold surfaces after plasma incubation, and the antibody binding pattern also remained unchanged after prolonged plasma exposure. The surface-bound platelets were found to spread on the gold but not on titanium surfaces. C1q has been shown to induce the expression of P-selectin, i.e. cause secretion reactions in platelets. In this study secreted platelet-microvesicles were found on gold, but not on the titanium surfaces that bound significant amounts of C1q. Thus, the results of the present study indicate that the mixture of fibrinogen, C1q and kininogens, whilst causing adhesion and aggregation, does not result in the activation and microvesicle secretion of platelets. Platelet activation on biomaterial surfaces thus seems to be governed by the mixture of proteins present on that surface, and no one particular protein need cause a known reaction in platelets as obtained when platelets are exposed only to that particular protein.
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PMID:Platelet binding and protein adsorption to titanium and gold after short time exposure to heparinized plasma and whole blood. 896 16

Platelet activation and subsequent intravascular thrombus formation play a central role in the pathophysiology of acute coronary syndromes. Extracellular magnesium has an antithrombotic action due mainly to its antiplatelet effect. High concentrations of magnesium inhibit platelet aggregation and adhesion in vitro and reduce the generation of prothrombotic eicosanoids. In addition, magnesium modulates the intracellular platelet activation cascade and decreases calcium influx. The function of major platelet membrane receptors, such as the fibrinogen receptor GP-IIb-IIIa and P-selectin are also inhibited by extracellular magnesium. Intravenous magnesium inhibits platelet function in vivo-additive to aspirin. The antiplatelet effect of intravenously administered magnesium might be of benefit to patients with acute coronary syndromes when given before the development of an occlusive thrombotic plug.
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PMID:[Antithrombocytic effectiveness of magnesium]. 899 5

The initial reactions of two TiO2 surfaces with blood were investigated by short-time exposure to capillary blood and analysis of surface-adsorbed plasma proteins and surface-adhering cells by using immunofluorescence techniques. Antibodies directed against platelet membrane antigen and P-selectin were used to visualize platelet adhesion and activation. Acridine orange and anti-CD11b were used to detect adhesion and activation of polymorphonuclear granulocytes (PMNs). Antibodies against thrombospondin were used as markers for platelet alpha-granules. The fluorescence intensity was quantitated by computer-aided image analysis. Commercially pure, polished sheet titanium was oxidized in two different ways: (1) the natural oxide was dissolved with hydrofluoric acid and a new oxide layer was grown by oxidation in nitric acid, or (2) annealing was performed at 700 degrees C in air. Auger electron spectroscopy and x-ray photoelectron spectroscopy showed that both surfaces had similar composition consisting of TiO2 covered by a carbonaceous surface contamination layer. The thickness of the oxide layer was 4 nm on the acid-oxidized surface and 39 nm on the annealed surface. Optical profilometry and scanning electron microscopy showed that the acid-oxidized surface was rough and the annealed surface was smooth. The fibrinogen/prothrombin-thrombin ratio in the initial protein film differed between the surfaces. The number of adhering platelets was larger at the surface with a high surface concentration of adsorbed fibrinogen. Platelet activation (CD62) and priming of PMNs (CD 11b) were also significantly higher on the acid-oxidized surface. The results indicate that non-self recognition of biomaterials is an array of transient reactions comprising protein-material, protein-cell, and cell-cell interactions.
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PMID:Adhesion and activation of platelets and polymorphonuclear granulocyte cells at TiO2 surfaces. 901 89

We recently described a Quebec family with an autosomal dominant bleeding disorder characterized by mildly reduced-low normal platelet counts, an epinephrine aggregation defect, multimerin deficiency, and proteolytic degradation of several, soluble alpha-granular proteins. Similar clinical features led us to investigate a second family with an unexplained, autosomal dominant bleeding disorder. The affected individuals had reduced to normal platelet counts, absent platelet aggregation with epinephrine, and multimerin deficiency. Their platelet alpha-granular proteins factor V, thrombospondin, von Willebrand factor, fibrinogen, fibronectin, osteonectin, and P-selectin were proteolyzed and comigrated with the degradation products found in patients from the other family. However, their platelet albumin, IgG, external membrane glycoproteins, CD63 (a lysosomal and dense granular protein), calpain, and plasma von Willebrand factor were normal, indicating restriction in the proteins proteolyzed. Electron microscopy studies indicated preserved alpha-granular ultrastructure, despite degradation of soluble and membrane alpha-granular proteins. Immunoelectron microscopy studies of the patients' platelets indicated that fibrinogen, von Willebrand factor, P-selectin, multimerin, and factor V were within alpha-granules, with normal to reduced labeling for these proteins. Pathologic proteolysis of alpha-granular contents, rather than a defect in targeting proteins to alpha-granules, may be the cause of the protein degradation in the Quebec platelet disorder.
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PMID:Studies of a second family with the Quebec platelet disorder: evidence that the degradation of the alpha-granule membrane and its soluble contents are not secondary to a defect in targeting proteins to alpha-granules. 902 47


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