Activation of porcine endothelium in response to xenogeneic serum causes thrombosis independently of platelet activation.

BACKGROUND

Endothelial cell activation and microvascular thrombosis are hallmarks of hyperacute xenograft rejection. However, the molecular determinants of platelet-endothelial interaction and thrombus formation are poorly understood. This study investigated whether: (i) xenogeneic human serum (HS), as a source of xenoreactive antibodies and complement, activates porcine aortic endothelial cells (PAEC) to promote thrombus formation under high shear stress; (ii) the endothelial adhesive proteins vitronectin receptor and P-selectin are involved in the von Willebrand factor (VWF)-platelet interaction during the thrombotic process under flow; (iii) reactive oxygen species (ROS) are activated by complement and served as intracellular signals for adhesive protein up-regulation.

METHODS

The PAEC were pre-exposed for 90 min in static conditions to medium plus 10, 20, and 50% HS or 20% porcine serum (PS), as control, then cells were perfused at 50 dynes/cm2 in a parallel plate flow chamber with human blood and area occupied by thrombi was measured. The role of complement in HS-induced thrombus formation was assessed by incubating PAEC with 20% HS in the presence of soluble complement receptor type 1 (sCR1) before blood perfusion. The effect of platelet activation was assessed using human blood treated or not with ADP and then flowed over PAEC pre-exposed to 20% HS or 20% PS as control. To identify the endothelial adhesive proteins involved in thrombus formation PAEC treated with 20% HS were then incubated with anti-vitronectin receptor antibody, anti-P-selectin antibody or P-selectin glycoprotein ligand-1 (PSGL-1), the soluble ligand of P-selectin, before the adhesion assay. Confocal microscopy was used to detect changes in endothelial adhesive protein expression. VWF interaction with platelet receptors GPIb and alphaIIbbeta3 was assessed adding aurin tricarboxylic acid (ATA) and anti-alphaIIbbeta3 antibody to blood before perfusion. The ROS involvement in xenogeneic serum-induced thrombus formation was determined studying the intracellular production of hydrogen peroxide (H2O2). The effect of antioxidants and metal chelators on HS-induced thrombus formation was evaluated treating PAEC with pyrrolidine dithiocarbamate (PDTC) or 1,3-dimethyl-2-thiourea (DMTU) before and during incubation with 20% HS followed by blood perfusion. The effect of antioxidants and sCR1 on ROS generation was investigated treating PAEC with PDTC or DMTU before and during incubation with 20% HS. Intracellular ROS generation was measured by fluorescence spectroscopy using the probe dihydrorhodamine 123 (DHR-123).

RESULTS

Human serum but not PS caused thrombus formation on PAEC under high shear stress. Blockade of complement activation by sCR1 prevented xenogeneic serum-induced thrombus formation. Activated platelets did not promote thrombus formation on resting endothelium, and did not further increase platelet deposition on xenogeneic serum-treated PAEC. Vitronectin receptor and P-selectin were up-regulated on the endothelial surface by HS. Their functional blockade by specific antibodies prevented platelet deposition and thrombus formation. H2O2 production significantly increased when PAEC were exposed to the xenogeneic condition. Antioxidants and sCR1 completely prevented thrombus formation by reducing excessive ROS production and the expression of vitronectin receptor and P-selectin.

CONCLUSIONS

Xenogeneic complement induces endothelial cell activation and thrombosis which is independent of platelet activation. Complement deposition elicits a rapid generation of ROS that lead to overexpression of endothelial adhesive molecules instrumental for platelet deposition.