Immobi lized RTL551 supported mouse platelet surface adhesion and

Immobi lized RTL551 supported mouse platelet surface adhesion and lamellipodia formation. Consistent with previous reports, mouse selleck chemical platelets exhibit only partial spreading on a fibrinogen surface. RTL supports platelet actin cytoskeletal reorganization and Ca2 Inhibitors,Modulators,Libraries mobilization Upon activation, platelets rapidly mobilize intracellular stores of calcium and assemble actin rich structures such as filopodia, lamellipodia and stress fibers. Fluorescent labeling of the actin cytoskeleton showed the formation of stress fibers in the human platelets on immobilized RTL1000. Real time imaging of platelets loaded with the calcium reporter dye, Oregon Green BAPTA 1 AM, revealed that platelets generated a rapid burst of oscillating intracellular Ca2 following binding to RTL1000, which subsequently declined over a period of 3 10 min.

In contrast, a rhythmic series of Ca2 spikes were observed on fibrinogen. Characterization of the platelet signaling cascade downstream Inhibitors,Modulators,Libraries of RTL We next aimed to determine the intracellular Inhibitors,Modulators,Libraries mechan isms that mediate human platelet spreading on RTL1000. Addition of the ADP scavenger apyrase in combination with the cyclooxygenase inhibitor indomethacin had no effect on platelet spreading on RTL1000. In contrast, treatment of platelets with inhibitors to Src kinases, Syk kinase, PI3 kinases, protein kinase C or an intracellular Ca2 chelator abrogated platelet lamellipodia formation Inhibitors,Modulators,Libraries on RTL1000 coated surfaces.

Platelet lamellipodia formation on RTL1000 was also inhibited in the presence of the aIIbb3 receptor Inhibitors,Modulators,Libraries antago nist, eptifibatide, whereas adhesion to fibrinogen was eliminated in the presence of eptifibatide We next determined whether the interaction of RTL1000 with human platelets led to the activation of signal transduction pathways with known roles in plate let adhesion and spreading. Platelet binding to RTL1000 was associated with an increase in total tyrosine phos phorylation levels as compared to platelets under con trol conditions. The addition of the ADP scavenger apyrase reduced the degree of tyrosine our website phosphorylation in whole cell lysates as well as the phosphorylation of Akt and of GSK3b. The Src family kinase inhibitor, PP2, the PI3 kinase inhibitor, wortmannin, or the Syk kinase inhibitor, BAY 61 3606, abrogated phos phorylation of Akt and GSK3b. In contrast, phosphorylation of Akt and GSK3b was not affected by the protein kinase C inhibitor, R0 31 8220. Effect of RTL on platelet aggregation, coagulation and occlusive thrombus formation Next we determined the effect of RTL on the initiation of coagulation. RTL1000 did not affect the activated par tial prothrombin time or the clotting time of platelet poor plasma initiated by the addition of excess molar Ca2.

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