Aggregation of human platelets induced by porcine endothelial cells is dependent upon both activation of complement and thrombin generation

Abstract

Abstract: The binding of human xenoreactive antibody (XNA) to porcine endothelium with complement (C) activation via the classical pathways is considered the major event triggering hyperacute rejection (HAR) with microvascular thrombosis in vivo. As C components are linked to key events in blood coagulation, we have examined pathways whereby activation of complement by endothelial cells results in xenogeneic platelet activation in vitro.Methods: Cultured porcine aortic endothelial cells (pEC), human aortic endothelial cells (HAEC) or human umbilical vein endothelial cells (HUVEC) were prepared in suspension (5 × 106/ml) using EDTA‐collagenase. Human platelet rich plasma (PRP) and washed platelets with platelet poor plasma (PPP) were prepared from control, drug free, volunteer donors. All aggregation tests used a two‐sample, four‐channel, model 560 Ca Lumi‐Aggregometer (Chronolog Corporation, Havertown, PA). Selected assays for complement (C3a; C5b‐C9; CH50; and AP50) were then performed on supernatant fluids. To test the effects of complement inhibition and thrombin antagonists, the following agents were pre‐incubated with PRP (or PPP) in various titrations for 10 min at 37°C prior to combination with pEC in the aggregometer: soluble complement receptor typel (sCR1); Cobra Venom Factor (CVF), heparin, hirudin, and anti‐CD31 (anti‐PECAM). In addition, pEC, HAEC, and HUVEC were incubated with 10–20% human PPP; supernatant fluids were harvested at various time points and used for platelet activation assays and for functional tests of thrombin or levels of thrombin‐antithrombin complexes (TAT).Results: pEC but not HAEC/HUVEC resulted in activation of PRP or washed platelets only in the presence of supplemental PPP. Platelet activation could be inhibited by pre‐incubation of samples with CVF (2–5 IU/ml to deplete complement components) and to a variable extent with sCRl (1–2 mg/ml). Complement assays confirmed activation of C3 by the classical pathway with reduction of the CH50 while C6 deficient samples also supported platelet activation. Aggregation of platelets was inhibited by preincubation of PRP with concentrations of hirudin (1 unit/ml) and heparin (5–10 units/ml) sufficient to neutralize thrombin generated. Supernatant fluids containing PPP removed from pEC were found to have increased levels of thrombin and thrombin‐antithrombin complexes and could also activate platelets in a hirudin sensitive manner.Discussion: Platelet and pEC combinations underwent aggregation only in the presence of complement components. This process appeared independent, at least in part, of the assembly of terminal complement components. Consequent pEC generation of thrombin appeared adequate to trigger platelet aggregation which could in turn be inhibited by hirudin or heparin in vitro.