AGGREGATION OF HUMAN PLATELETS INDUCED BY PORCINE ENDOTHELIAL-CELLS IS DEPENDENT UPON BOTH ACTIVATION OF COMPLEMENT AND THROMBIN GENERATION

The binding of human xenoreactive antibody (XNA) to porcine endotheliu m with complement (C) activation via the classical pathways is conside red the major event triggering hyperacute rejection (NAR) with microva scular thrombosis in vivo. As C components are linked to key events in blood coagulation, we have examined pathways whereby activation of co mplement by endothelial cells results in xenogeneic platelet activatio n in vitro. Methods: Cultured porcine aortic endothelial cells (pEC), human aortic endothelial cells (HAEC) or human umbilical vein endothel ial cells (HUVEC) were prepared in suspension (5 x 10(6)/ml) using EDT A-collagenase, Human platelet rich plasma (PRP) and washed platelets w ith platelet poor plasma (PPP) were prepared from control, drug free, volunteer donors, All aggregation tests used a two-sample, four-channe l, model 560 Ca Lumi-Aggregometer (Chronolog Corporation, Havertown, P A), Selected assays for complement (C3a; C5b-C9; CH50: and AP50) were then performed on supernatant fluids. To test the effects of complemen t inhibition and thrombin antagonists, the following agents were pre-i ncubated with PRP (or PPP) in various titrations or 10 min at 37 degre es C prior to combination with pEC in the aggregometer: soluble comple ment receptor type 1 (sCR1); Cobra Venom Factor (CVF), heparin, hirudi n, and anti-CD31 (anti-PECAM). In addition, pEC, HAEC, and HUVEC were incubated with 10-20% human PPP; supernatant fluids were harvested al various time points and used for platelet activation assays and for fu nctional tests of thrombin or levels of thrombin-antithrombin complexe s (TAT). Results: pEC but not HAEC/HUVEC resulted in activation of PRP or washed platelets only in the presence of supplemental PPP. Platele t 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 sCR1 (1-2 mg/ml). Complement assays confirmed activation of C3 by the classical pathway with reduction of the CH50 while C6 deficient s amples also supported platelet activation. Aggregation of platelets wa s 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 fo und to have increased levels of thrombin and thrombin-antithrombin com plexes 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 indep endent, at least in part, of the assembly of terminal complement compo nents. Consequent PEC generation of thrombin appeared adequate to trig ger platelet aggregation which could in turn be inhibited by hirudin o r heparin in vitro.