Anti-CD154 monoclonal antibody and thromboembolism revisited

In view of reports of thromboembolic complications in nonhuman primates (1) and humans (2) receiving anti-CD154 monoclonal antibody (mAb) therapy, we briefly reported our own experience in the pig-to-baboon model where we had not experienced any such complications (3). We had carried out 10 pig hematopoietic cell transplants (4) and 8 pig kidney transplants (5) in baboons using anti-CD154 mAb and had seen no thromboembolic complications, with follow-up in some cases extending for more than 1 year. We had also investigated the effect of anti-CD154 mAb on platelet aggregation in vitro and had seen no significant modification of the response of baboon platelets to any known agonists of platelet aggregation (6). Recently, we have, however, had significant complications of coagulation that we believe are related to anti-CD154 mAb therapy in the presence of therapeutic doses of calcium chloride. We have performed nine heterotopic heart transplants (in the abdomen) in baboons using hearts from pigs transgenic for human decay accelerating factor (C. Knosalla, manuscript in preparation). All pig donors were fully heparinized (300 units/kg i.v.) before heart excision, and all baboon recipients received heparin (100 units/kg i.v.) before cross-clamping of the abdominal aorta and inferior vena cava. After reperfusion of the heart, we documented a reduction in serum calcium level from 8.4 (±1.4) to 6.7 (±1.2) mg/dl (mean, SD). In three baboons, to ensure good myocardial contractility, calcium chloride was administered by slow i.v. infusion, and calcium levels immediately returned to the normal range. In one further case, calcium was infused prophylactically during the operative procedure to ensure that, on reperfusion, the calcium level would be normal. In the three cases in which calcium was infused after reperfusion, thrombosis in both atrial appendages soon developed, although myocardial contractions remained satisfactory. In the one animal in which prophylactic calcium was given, thrombosis in the atrial appendages was rapidly followed by complete coagulation of blood in both ventricles. The heart became distended, contractions almost ceased, and the graft was excised. There were no technical reasons in any case for these complications and, in the one graft excised, there were no histopathological features suggesting hyperacute rejection. Furthermore, all of these baboons had been pretreated by immunoadsorption of anti-Gal antibody and by cobra venom factor to deplete complement, and we have never seen hyperacute rejection in this model. In the five baboons in this study that received identical therapy but without calcium supplementation, thrombosis in the atria did not develop, and myocardial function remained good. Calcium levels spontaneously returned to normal within 24 hr. The exact cause of the fall in calcium at the time of reperfusion remains uncertain but may be partly related to simple hemodilution, as all baboons received significant infusions of saline and packed red blood cells during the operative procedure. We observed a significant difference in platelet counts between these two groups of baboons. In the group that received calcium therapy, platelet count fell by 175,000 (±77,000)/μL (mean, SD), which represented 65% of baseline pretransplant level, within 8 hr posttransplant, and then either slowly recovered to baseline pretransplant levels over the next 1–7 days or, if falling rapidly to levels <30,000/μl, required a platelet transfusion. In contrast, in those baboons that did not receive supplementary calcium, the mean fall in platelet count was only 62,000 (±41,400)/μl, (i.e., 27%, within 8 hr of reperfusion) and recovery to baseline levels took place within 2 days. We have not seen thrombosis as a complication of pig kidney transplantation in baboons, but, in these cases, no calcium was administered. In one baboon, however, we have experienced thrombosis of the main pig renal vein 9 days after the transplant, which was not associated with any technical problems (7). We believe that this late thrombosis may have been related to the immunosuppressive regimen administered to this baboon, in which cyclophosphamide was omitted. We have considerable in vitro data from platelet aggregation assays that cyclophosphamide markedly reduces platelet aggregation and that this is maximal some 6 days after its administration (8). We suggest that this effect of cyclophosphamide balances any pro-aggregatory effect of anti-CD154 mAb. Nevertheless, despite these complications, anti-CD154 mAb remains an important agent in xenotransplantation as it remains the only agent we have tested to date that consistently prevents an elicited antibody response to transplanted porcine hematopoietic cells (4), kidneys (5) and hearts (C. Knosalla, manuscript in preparation). We therefore strongly encourage and support its development for clinical use. Christoph Knosalla Bernd Gollackner David K. C. Cooper