Lysates were separated by SDS PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% BSA for 1 hour prior to incubation with primary antibodies overnight at 4 C. Following washing with TBS T, membranes were incubated with the appropriate secondary antibodies. selleck chemical Crenolanib Subsequent immunoblotting was carried out as previously described. Inhibitors,Modulators,Libraries Platelet aggregation and clotting time measurements Platelet aggregation was assayed using 300 ul of washed platelets in a Chrono Log aggregometer with continuous stirring at 1200 rpm at 37 C. Activated partial thromboplastin time of pooled human platelet poor plasma and recalcification time of PPP were measured with a KC4 Coagulation Analyzer. Samples were pre treated with either vehicle, RTL1000, an anti FXI mAb or tissue factor for 3 min prior to the addition of 16.
6 mM CaCl2 for recalcifica tion times or aPTT reagent followed by 6. 6 mM CaCl2 for aPTT tests. Inhibitors,Modulators,Libraries In each test, clotting time was measured following the addition of CaCl2 as previously described. Capillary occlusion assay Capillary tubes were coated with fibrillar collagen, aligned vertically Inhibitors,Modulators,Libraries and connected to a reservoir as pre viously described. Sodium citrate antic oagulated whole blood was sequentially supplemented with 7. 5 mM Ca2 and 3. 75 mM Mg2. Capillary flow was driven by the force of gravity, and the height of the sample reservoir was regulated in order to produce an initial shear rate of 300 s 1 as previously described. Analysis of data Data are shown as means SEM. Statistical significance of differences between means was determined by ANOVA.
If means were shown to be significantly differ ent, multiple comparisons were performed by the Tukey test. Probability values of P 0. 05 were selected to be statistically Inhibitors,Modulators,Libraries significant. Results Characterization of RTL as a ligand for platelets To determine the ability of human blood platelets to support RTL1000 binding, purified platelets were immo bilized on a surface of fibrinogen prior to exposure to Inhibitors,Modulators,Libraries fluorescently labeled RTL1000. RTL1000 bound to the human platelet surface, demonstrating that a receptor for RTL may be present on the surface of human platelets. In an attempt to identify a potential RTL receptor on platelets, washed human platelets were incubated over RTL coated glass coverslips, and platelet adhesion and spreading was monitored with Nomarski differential interference contrast microscopy.
Our data show that RTL1000 supported human platelet sur face adhesion and lamellipodia formation. prompt delivery The degree of adhesion observed was similar to that of platelet adhesion to an immobilized surface of fibrino gen, which supports platelet adhesion through the aIIbb3 integrin. Minimal platelet adhesion was observed on BSA coated coverslips. A parallel series of experiments was performed with purified mouse platelets. RTL551 contains the peptide derived from mouse myelin oligodendrocyte glycoprotein.