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(1/9) Protein kinase C- and calcium-regulated pathways independently synergize with Gi pathways in agonist-induced fibrinogen receptor activation.

Platelet fibrinogen receptor activation is a critical step in platelet plug formation. The fibrinogen receptor (integrin alphaIIbbeta3) is activated by agonist-mediated G(q) stimulation and resultant phospholipase C activation. We investigated the role of downstream signalling events from phospholipase C, namely the activation of protein kinase C (PKC) and rise in intracellular calcium, in agonist-induced fibrinogen receptor activation using Ro 31-8220 (a PKC inhibitor) or dimethyl BAPTA [5,5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N', N'-tetra-acetic acid], a high-affinity calcium chelator. All the experiments were performed with human platelets treated with aspirin, to avoid positive feedback from thromboxane A2. In the presence of Ro 31-8220, platelet aggregation caused by U46619 was completely inhibited while no effect or partial inhibition was seen with ADP and the thrombin-receptor-activating peptide SFLLRN, respectively. In the presence of intracellular dimethyl BAPTA, ADP- and U46619-induced aggregation and anti-alphaIIbbeta3 antibody PAC-1 binding were completely abolished. However, similar to the effects of Ro 31-8220, dimethyl BAPTA only partially inhibited SFLLRN-induced aggregation, and was accompanied by diminished dense-granule secretion. When either PKC activation or intracellular calcium release was abrogated, aggregation and fibrinogen receptor activation with U46619 or SFLLRN was partially restored by additional selective activation of the G(i) signalling pathway. In contrast, when both PKC activity and intracellular calcium increase were simultaneously inhibited, the complete inhibition of aggregation that occurred in response to either U46619 or SFLLRN could not be restored with concomitant G(i) signalling. We conclude that, while the PKC- and calcium-regulated signalling pathways are capable of inducing activating fibrinogen receptor independently and that each can synergize with G(i) signalling to cause irreversible fibrinogen receptor activation, both pathways act synergistically to effect irreversible fibrinogen receptor activation.  (+info)

(2/9) Significance of platelet activation in vascular access survival of haemodialysis patients.

BACKGROUND: Vascular access failure is the most common cause of morbidity and hospitalization in haemodialysis (HD) patients. Although there are reports that anti-platelet agents can prevent vascular access thrombosis, the relationship between platelet activation and vascular access failure is not clear. The aim of this study was to investigate the role of platelet activation in recurrent vascular access failure. METHODS: The studied subjects were divided into three groups: group I included 23 HD patients with recurrent vascular access failure (native arteriovenous fistula <2 year survival or synthetic arteriovenous graft <1 year survival), group II included 15 HD patients with longer vascular access survival (>5 year survival) and group III included 10 healthy volunteers as controls. The expression of platelet activation markers (CD62P and fibrinogen receptor) and the numbers of platelet-derived microparticles were measured and compared between groups. RESULTS: CD62P-positive platelets were significantly higher in group I than in both group II (7.3+/-3.7 vs 3.5+/-1.3%; P<0.0005) and group III (2.9+/-0.9%; P<0.00005). Fibrinogen receptor-positive (PAC-1-positive) platelets were also significantly higher in group I than in group II (2.2+/-2.1 vs 0.9+/-0.7%; P<0.01) and group III (0.8+/-0.6%; P<0.01). CONCLUSIONS: A higher level of circulating activated platelets is associated with shorter survival of vascular access in HD patients. The higher level of circulating activated platelets may be a predictor of recurrent vascular access failure. The potential advantageous effects of anti-platelet therapy on this patient population warrant further investigation.  (+info)

(3/9) Fibrinogen receptor antagonists induce conformational changes of the human platelet glycoprotein IIb.

BACKGROUND: Controversial results have been reported concerning the ability of fibrinogen receptor antagonists (fibans) to induce conformational changes in the fibrinogen receptor after binding to it as the initial step of fibrinogen binding and platelet activation. METHODS: Platelets in citrated whole blood were stained with several pairs of anti-glycoprotein (anti-GP) IIb-directed monoclonal antibodies conjugated to phycoerythrin (PE) or indirectly labeled with Cy5. Pairs of monoclonal antibodies that induced a high-fluorescence resonance energy transfer (FRET) efficiency served as tools to detect activation-dependent changes of GP IIb after addition of adenosine diphosphate and several fibans. RESULTS: Using the combination of the clones 5B12-PE and P2-biotin/SA-Cy5, a concentration-dependent alteration of the GP IIb conformation was observed after addition of tirofiban, eptifibatide, and lotrafiban. Magnitude and kinetics differed among the investigated substances. CONCLUSION: The newly developed FRET assay allows the direct investigation of conformational changes of GP IIb after addition of platelet agonists or receptor ligands, as shown for three fibans.  (+info)

(4/9) The fibrinogen receptor FbsA promotes adherence of Streptococcus agalactiae to human epithelial cells.

Streptococcus agalactiae is a major cause of bacterial pneumonia, sepsis, and meningitis in human neonates. During the course of infection, S. agalactiae adheres to a variety of epithelial cells but the underlying mechanisms are only poorly understood. The present report demonstrates the importance of the fibrinogen receptor FbsA for the streptococcal adherence and invasion of epithelial cells. Deletion of the fbsA gene in various S. agalactiae strains substantially reduced their binding of soluble fibrinogen and their adherence to and invasion of epithelial cells, indicating a role of FbsA in these different processes. The adherence and invasiveness of an fbsA deletion mutant were partially restored by reintroducing the fbsA gene on an expression vector. Heterologous expression of fbsA in Lactococcus lactis enabled this bacterium to adhere to but not to invade epithelial cells, suggesting that FbsA is a streptococcal adhesin. Flow cytometry experiments revealed a dose-dependent binding of FbsA to the surface of epithelial cells. Furthermore, tissue culture experiments exhibited an intimate contact of FbsA-coated latex beads with the surfaces of human epithelial cells. Finally, host cell adherence and invasion were significantly blocked in competition experiments with either purified FbsA protein or a monoclonal antibody directed against the fibrinogen-binding epitope of FbsA. Taken together, our studies demonstrate that FbsA promotes the adherence of S. agalactiae to epithelial cells but that FbsA does not mediate the bacterial invasion into host cells. Our results also indicate that fibrinogen-binding epitopes within FbsA are involved in the adherence of S. agalactiae to epithelial cells.  (+info)

(5/9) Cranial neural crest recycle surface integrins in a substratum-dependent manner to promote rapid motility.

Cell migration is essential for proper development of numerous structures derived from embryonic neural crest cells (NCCs). Although the migratory pathways of NCCs have been determined, the molecular mechanisms regulating NCC motility remain unclear. NCC migration is integrin dependent, and recent work has shown that surface expression levels of particular integrin alpha subunits are important determinants of NCC motility in vitro. Here, we provide evidence that rapid cranial NCC motility on laminin requires integrin recycling. NCCs showed both ligand- and receptor-specific integrin regulation in vitro. On laminin, NCCs accumulated internalized laminin but not fibronectin receptors over 20 min, whereas on fibronectin neither type of receptor accumulated internally beyond 2 min. Internalized laminin receptors colocalized with receptor recycling vesicles and were subsequently recycled back to the cell surface. Blocking receptor recycling with bafilomycin A inhibited NCC motility on laminin, indicating that substratum-dependent integrin recycling is essential for rapid cranial neural crest migration.  (+info)

(6/9) Src family kinase-mediated and Erk-mediated thromboxane A2 generation are essential for VWF/GPIb-induced fibrinogen receptor activation in human platelets.

The binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein Ib-IX (GPIb-IX) results in platelet activation. In this study, we sought to clarify previous conflicting reports and to elucidate the mechanism of activation and the precise role of extracellular signal-regulated kinase (Erk) in VWF-induced platelet activation. Erk2 is activated in platelets on stimulation with VWF/ristocetin in a time-dependent manner. VWF-induced Erk2 phosphorylation and thromboxane A2 (TXA2) release were completely blocked by PP2, an Src family kinase inhibitor, suggesting that Erk is downstream of Src family kinases. U73122, a phospholipase C inhibitor, also abolished TXA2 generation and Erk phosphorylation. Although VWF fostered the agglutination of platelets regardless of any additional treatment, the inhibition of mitogen-activated protein kinase kinase (MEK) with U0126 abolished VWF-induced platelet aggregation and thromboxane production in non-aspirin-treated washed platelets. However, in platelets treated with aspirin, VWF failed to cause any aggregation. Thus, we conclude that VWF stimulation of platelets results in phospholipase A2 activation through Erk stimulation and that Src family kinases and phospholipase C play essential roles in this event. We further conclude that VWF-induced platelet aggregation does not directly depend on Erk activation but has an absolute requirement for Src/Erk-mediated TXA2 generation.  (+info)

(7/9) Endothelial cell activation by IL-1beta in the presence of fibrinogen requires alphavbeta3.

OBJECTIVE: The purpose of this study was to investigate the receptor requirements for enhanced IL-1beta-induced secretion of nitric oxide (NO) by endothelial cells (ECs) in the presence of fibrinogen. METHODS AND RESULTS: ECs were exposed to IL-1beta with or without fibrinogen and NO was measured as nitrite. NO production by EC exposed to fibrinogen (0.3+/-0.1 micromol/L) was comparable concentration to control (0.2+/-0.1 micromol/L), but IL-1beta significantly increased NO production (0.8+/-0.1 micromol/L), and the combination of both fibrinogen and IL-1beta resulted in a further increase to 2.2+/-0.2 micromol/L (P<0.002). 7E3 or LM609, antibodies to alphavbeta3, inhibited NO production stimulated by fibrinogen-bound IL-1beta to 0.2+/-0.1 micromol/L (P<0.001) or 0.2+/-0.03 micromol/L (P<0.0001), respectively. These levels were comparable to control and significantly less than with IL-1beta (P<0.002). EC or fibroblasts exposed to both fibrinogen and IL-1beta, but not vitronectin and IL-1beta, demonstrated positive Western blotting for alphavbeta3 after immunopurification with anti- IL-1R, indicating specific association between alphavbeta3 and IL-1R. Dual immunofluorescence also revealed colocalization of alphavbeta3 and IL-1R only when the cells were exposed to both fibrinogen and IL-1beta. CONCLUSIONS: The enhanced NO production by ECs in the presence of fibrinogen-bound IL-1beta requires the coordinated effects of colocalized alphavbeta3 and IL-1R.  (+info)

(8/9) Human platelets express functional alpha7-nicotinic acetylcholine receptors.

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