Gabriele Himmelreich

Pathophysiologic role of contact activation in bleeding followed by thromboembolic complications after implantation of a ventricular assist device

The time period after implantation of a ventricular assist device in patients with end-stage heart disease is complicated by hemorrhage in the early postoperative period and by thromboembolism in the later course. To investigate the pathophysiologic role of contact activation in 12 bridging patients (10 patients with a paracorporeal Berlin Heart [Berlin Heart GmbH, Berlin, Germany], 2 patients with an intracorporeal Novacor system [Novacor N100; Baxter, Oakland, CA]), hemostatic parameters were determined until heart transplantation or at least up to the 51st postoperative day. The following were observed: 1) In the early postoperative period, until day 15, levels of contact factors XI, XII, and prekallikrein were below normal, whereas levels of plasmin-a2-antiplasmin (PAP) complexes were elevated. Thrombin-antithrombin III (TAT) complexes, as well as platelet factor 4 and beta-thromboglobulin, significantly increased immediately after surgery. 2) In the later postoperative period, starting with the third postoperative week, an increase of factors XI, XII, and prekallikrein was observed. PAP and TAT complexes, as well as platelet factor 4 and beta-thromboglobulin, remained elevated. It is concluded that, in the early postoperative period, hemostasis is influenced mainly by an activation of the intrinsic contact system dependent fibrinolytic system with consumption of contact factors and increased levels of PAP complexes, whereas later system dependent fibrinolysis becomes less important, leading to a shift of the balance toward coagulation, with sustained prothrombin and platelet activation. This is in accord with the observed clinical complications (e.g., early postoperative bleeding, and thromboembolic events later on).

Evidence that intraoperative prostaglandin E1 infusion reduces impaired platelet aggregation after reperfusion in orthotopic liver transplantation.

Recent investigations measuring platelet aggregation during 10 orthotopic liver transplantations showed a significant decrease in platelet aggregation immediately after reperfusion and in the perfusate. As prostaglandin E1 has been shown to exhibit a beneficial effect in the treatment of ischemic injury of the liver, we investigated in a prospective, randomized, and open study the effect of PGE1 infusion during OLT on platelet function. Ten patients were randomized to receive a continuous PGE1 infusion (PG group) and another ten patients served as controls. Platelet function was determined ex vivo by measuring the adenosine diphosphate-, collagen-, and ristocetin-induced aggregation in platelet-rich plasma. A significantly higher platelet aggregability was measured in the PG group throughout the whole operation for ADP (1 and 2 mumol/L) and collagen (0.5 micrograms/ml). The same was true for collagen (1 microgram/ml) and ristocetin (1.2 mg/ml) after reperfusion. Not only the postreperfusional decrease in platelet aggregation but also the decline in platelet count that occurred in the control group could be prevented greatly by PGE1 infusion. In the perfusate, released from the liver graft vein by flushing with arterial blood, a significantly lower platelet aggregability was seen in comparison with the systemic circulation before reperfusion in the control group, a difference that was not found when PGE1 infusion was given intraoperatively. However, blood product requirements during OLT were comparable in both groups. In conclusion, PGE1 therapy during OLT preserves platelet function and prevents the drop in platelet count observed in the control group after revascularization.

Different aprotinin applications influencing hemostatic changes in orthotopic liver transplantation

The effect of different aprotinin applications on hemmtatic changes and blood product requirements in orthotopic liver transplantation was investigated in a prospective, open, and randomized study. From November 1989 to June 1990, 13 patients received aprotinin as a bolus of 0.5 Mill, kallikrein inac-tivator units (KIU) on three occasions in the course of an OLT, whereas 10 other patients were treated with continuous aprotinin infusion of 0.1–0.4 Mill. KIU/hr. Before and after reperfusion of the graft liver, signs of hyperfibrinolysis, measured by thrombelastography, were significantly lower in the infusion group. Tissue-type plasminogen activator (t-PA) activity increased during the anhepatic phase but to a significantly lesser extent in the infusion group. Blood product requirements during OLT were tendentiously higher in the bolus group but not significantly so. However, the use of packed red blood cells was significantly lower in the postoperative period, whereas there was no significant difference in fresh frozen plasma requirements between the two groups. All 23 patients have survived, and only one woman of each group required retransplantation due to severe host-versus-graft reactions. Furthermore, we investigated the perfusate of the graft liver in both groups and detected signs of a decreased t-PA release in the infusion group. Our results demonstrate an advantage of aprotinin given as continuous infusion over bolus application in OLT.

Decreased platelet aggregation after reperfusion in orthotopic liver transplantation

Increased blood loss is a major contributory factor to postoperative short- and longterm outcome in orthotopic liver transplantation (OLT). Hyperfibrinolysis, occurring mainly in the anhepatic phase, and disseminated intravascular coagulation (DIC), starting immediately after reperfusion, have been observed to parallel increased bleeding tendency. However, other factors may also contribute. For the first time we investigated platelet aggregation during the course of 10 OLTs and found a marked reduction of adenosine diphosphate-, collagen-, and ristocetin-induced platelet aggregation immediately after reperfusion. Since this is the point at which increased bleeding tendency is known to occur, it would seem that, apart from DIC and hyperfibrinolysis, an intermediate dysfunction of platelet aggregation may be a major cause of intraoperative bleeding.

Mediators of leukocyte activation play a role in disseminated intravascular coagulation during orthotopic liver transplantation.

Leukocytes play an important role in the development of disseminated intravascular coagulation (DIC). In the reperfusion phase of OLT a DIC-like situation has been described and has been held responsible for the high blood loss during this phase. We investigated the role of leukocytes in the pathogenesis of DIC in OLT by measuring the leukocytic mediators released upon activation (cathepsin B, elastase, TNF, neopterin) and the levels of thrombin-antithrombin III (TAT) complexes, seen as markers of prothrombin activation. Arterial blood samples were taken at 10 different time points during and after OLT. Samples were also taken of the perfusate released from the liver graft vein during the flushing procedure before the reperfusion phase. Aprotinin was given as a continuous infusion (0.2-0.4 Mill. KIU/hr) and its plasma levels were determined. Significantly elevated levels of neopterin (15-fold; P < 0.01), cathepsin B (440-fold; P < 0.01) in the perfusate, as compared with the systemic circulation, as well as their significant increases in the early reperfusion phase suggested that they were released by the graft liver. This was paralleled by elevated levels of elastase (1.3-fold, P < 0.05), TNF (1.5-fold, P = NS), and TAT complexes (1.4-fold; P < 0.1) in the perfusate. Significant correlations could be identified between the parameters of leukocyte activation and TAT complexes, whereas no correlation was observed between any of the parameters investigated and the aprotinin levels. Our results strongly indicate a release of leukocytic mediators from the graft liver during its reperfusion which seems to be related to the parallely increased prothrombin activation. No correlation could be seen between levels of aprotinin and levels of leukocytic mediators.

Possible role of extracellularly released phagocyte proteinases in coagulation disorder during liver transplantation.

Orthotopic liver transplantation is frequently associated with a complex coagulation disorder, influencing the outcome of the procedure. In this respect, disseminated intravascular coagulation (DIC) had been suggested to be of causative importance for bleeding complications after reperfusion of the liver graft. In 10 consecutive patients undergoing orthotopic liver transplantations, we studied the occurrence of two phagocyte proteinases of different origin in the graft liver perfusate and in systemic blood during the operation, as well as their effects on hemostasis. As compared with plasma samples taken at the end of the anhepatic phase, highly significant increases of cathepsin B and thrombin-anti-thrombin III complexes (TAT), as well as highly significant decreases in antithrombin III, protein C, and C1-inhibitor were observed in graft liver perfusate. Von Willebrand factor and fibrinogen were slightly decreased, whereas the elastase-alpha 1 proteinase inhibitor complexes (EPI) were elevated. In plasma the activity of cathepsin B remained unchanged during the prereperfusion phases, but immediately after revascularization of the graft this cysteine proteinase increased. The EPI showed a gradual increase in plasma during the preanhepatic and anhepatic phases but a more pronounced increase in the reperfusion phase. In parallel with the rise in these two proteinases TAT increased and the activities of antithrombin III and C1-inhibitor in plasma decreased after reperfusion. At 12 hr after revascularization plasma levels of TAT, antithrombin III, and C1-inhibitor had returned to the prereperfusion ranges, whereas cathepsin B and EPI were significantly above the baseline levels. These observations are consistent with the hypothesis that extracellularly released lysosomal proteinases may play a role in the development of a DIC-like constellation, including thrombin formation after revascularization of the liver graft. For the first time we could prove the occurrence of phagocyte proteinases in graft liver perfusate and evaluate the importance of these proteinases for the understanding of the pathophysiology leading to bleeding complications in patients undergoing orthotopic liver transplantation.

In vitro inhibition of platelet aggregation by the liver with UW solution as the preservation fluid.

The influence of UW preservation fluid in comparison with that of Euro-Collins and Bretschneider solutions on collagen, adenosine diphosphate and ristocetin-induced platelet aggregation was investigated in vitro using platelet-rich plasma of 4 healthy volunteers. The concentrations of the solutions tested were comparable to those that may be used in the transplant situation. 2UW solution inhibited ADP and collagen-induced platelet aggregation—an effect that could be attributed mainly to adenosine and secondarily to penicillin in UW solution—whereas ristocetin-induced aggregation was not affected. Euro-Collins and Bretschneider solutions did not alter platelet aggregation. The inhibition of platelet aggregation by UW may, on the one hand, contribute to bleeding complications after reperfusion and, on the other hand, be responsible for the observed lower incidence of hepatic artery thrombosis.

Protein S in end stage liver disease and in the course of liver transplantation

Protein S (PS) is mainly synthesized in the liver and in endothelial cells. Determination of PS levels is complicated by the fact that total PS antigen (total PS Ag) exists in plasma as free and functionally active protein (free PS Ag) and in a reversible inactive complex with C4b-binding protein. In blood samples from patients with end stage liver diseases we studied PS levels before, during and after orthotopic liver transplantation (OLT). Median levels of total and free PS Ag were below and within the lower part of the reference interval, respectively. Two different groups in PS distribution could be distinguished. In 70% of the patients with preoperative levels of total and free PS Ag within the reference interval showed an impaired free/total PS Ag ratio towards higher unbound and free PS Ag levels. With reperfusion of the graft liver total and free PS Ag decreased significantly and the free/total PS Ag ratio normalized. In 30% of the patients reduced PS Ag levels together with a normal free/total PS Ag ratio were seen. In the reperfusion phase no further reduction of total and free PS Ag could be observed.