An endothelial dependent blood clotting assay was performed. Lysates of poly
An endothelial dependent blood clotting assay was performed. Lysates of poly(dA:dT) transfected endothelial cells accelerated blood clotting time when compared with timematched control cells (Fig. c, left). Stimulation of complete blood with lysates of endothelial cells treated with poly(dA:dT) alone (i.e. without cationic lipids) had no impact on blood clotting time (Fig. c, appropriate). Similar to cells transfected with the synthetic dsDNA analogue poly(dA:dT), lysates of endothelial cells transfected with human genomic DNA from peripheral human leukocytes also induced a substantially accelerated blood clotting compared to control cells right after hours (Fig. e). The prothrombotic effect of poly(dA:dT) immediately after hours was partly reversed just after prior transfection of endothelial cells with siRNA silencing RIGI receptor (Fig. d). issue (vWF) surface SPDB supplier Expression and platelet adhesion have been analyzed in primary human umbilical vein endothelial cells (HUVEC). Transfection of endothelial cells with poly(dA:dT) considerably improved surface expression of vWF immediately after hours as assessed by flow cytometry (Fig. a). To investigate the functional relevance of this observation, interactions in between endothelial cells and platelets had been examined inside a model of static adhesion. Hence endothelial cells pretreated with poly(dA:dT) for hours have been then cocultivated with freshly isolated platelets from healthy volunteers for hours. Endothelial
cells transfected with dsDNA showed drastically elevated numbers of adherent platelets as in comparison with nonstimulated cells (Fig. b). In PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/12056292 order to confirm our findings inside a much more physiological setting, we established a flow primarily based assay of platelet endothelium interaction. Hence freshly isolated human platelets were labeled with Calcein and flushed more than cultured endothelial cells within a flow chamber simulating a vascular shear stress of dyncm and plateletendothelial cell interactions were analyzed by immunofluorescence microscopy (Fig. c). Poly(dA:dT) transfected endothelial cells showed drastically increased level of tethering platelets in comparison with non stimulated cells (Fig. d). Additionally the number of really slow rolling platelets was higher on poly(dA:dT) transfected endothelial cells, nonetheless (thinking of the high variety of overall transfused platelets) the median velocity of platelets was not unique in both groups (Fig. e).Double stranded DNA induced prothrombotic proteins and accelerates endothelial dependent blood clotting in vitro. Subsequent, upregulation of prothrombotic molecules tissue factor and PAI was assessedDouble stranded DNA induces vWF upregulation and platelet tethering in vitro. Von WillebrandDouble stranded DNA accelerates microvascular thrombosis in vivo. To investigate the effects of double stranded DNA on thrombus formation in vivo, g poly(dA:dT) complexed with l of Lipofectamine or transfection reagent alone (handle group) was injected into the scrotum of CBl mice. Intravital microscopy of cremaster muscle vessels was performed hours after injection and thrombus formation was induced by lightdyeinjury following injection of FITClabeled dextran. Time of stimulation (hours)Time of stimulation (hours)Figure . Doublestranded DNA induces expression of prothrombotic genes in vascular endothelial cells. (a,b) Expression in the prothrombotic molecules tissue aspect (a) and Plasminogen activator inhibitor (PAI, b) as assessed by RTPCR upon transfection of vascular endothelial cells with poly(dA:dT). (c,d) Expression on the a.