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.

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.

Different aprotinin applications influencing hemostatic changes in orthotopic liver transplantation.

The effect of different aprotinin applications on hemostatic 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 inactivator 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.

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.

Increased urokinase-type plasminogen activator (u-PA) levels in graft liver perfusate and decreased single chain u-PA activation with higher levels of aprotinin.

In orthotopic liver transplantation (OLT) the graft liver is perfused with arterial blood prior to the opening of the hepatocaval anastomosis. In the present investigation we focused on the reperfusion of the graft liver in order to study the hepatic influence in the regulation of urokinase-type plasminogen activator (u-PA levels). Two different aprotinin schedules were used in 43 patients. We measured u-PA levels in the perfusate and in the corresponding systemic circulation. u-PA levels were higher in the perfusate as compared to systemic blood samples despite the dilution of the perfusate sample by the preservation fluid. This suggests u-PA secretion by the graft liver. In the presence of lower aprotinin levels signs of single-chain u-PA (scu-PA) activation was in the perfusate more prominent than systemically--a difference which was not seen in the presence of higher aprotinin levels. This seems to be an argument for the effectiveness of higher dosed aprotinin application in preventing scu-PA activation.

Evolution of urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) in orthotopic liver transplantation (OLT).

In orthotopic liver transplantation (OLT) hyperfibrinolysis seems to be of causative importance for intra- and postoperative bleeding. Although recently hyperfibrinolysis has been successfully reduced by intraoperative aprotinin treatment, small increases of fibrinolysis still remain during OLT. Originally, tissue-type plasminogen activator (t-PA) was considered to be responsible for the increases, but the efficacy of aprotinin which inhibits besides plasmin also kallikrein and urokinase-type plasminogen activator (u-PA) suggested also a role for the intrinsic and contact system-dependent plasminogen activators. We investigated the role of u-PA. From 29 patients undergoing OLT with intraoperative aprotinin infusion arterial blood samples were taken at 7 different time points. The preoperative median values for u-PA antigen (u-PA Ag) and plasmin-activatable single-chain u-PA (scu-PA) levels, which were more than 2-fold above normal (both: p < 0.01), decreased slightly during the preanhepatic phase and remained unchanged during the anhepatic phase. With reperfusion of the graft liver the two levels decreased significantly (p = 0.0003 and p = 0.006, respectively) to almost normal values, probably due to clearance by the graft liver. Active two-chain u-PA (tcu-PA) was preoperatively 2-fold above the detection limit, remained stable during the preanhepatic phase and increased 2-fold in the anhepatic phase (p = 0.0018). As expected tcu-PA also relapsed upon reperfusion, but to the preoperatively enhanced level, possibly caused by sustained activation of scu-PA by cathepsin B. t-PA activity levels were at the upper end of the normal range preoperatively, slightly increased during preanhepatic and anhepatic phases and decreased significantly with reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Coagulation changes and the influence of the early perfusate in the course of orthotopic liver transplantation (OLT) when aprotinin is used intra-operatively.

The changes in relevant haemostatic parameters during the course of ten orthotopic liver transplantation were studied when aprotinin was given intra-operatively. Increases of tissue-type (P = 0.008) and urokinase-type (P = 0.009) plasminogen activators during the anhepatic phase could be correlated with hyperfibrinolysis. Thrombin-antithrombin III complexes (TAT) increased after revascularization of the liver graft (P = 0.003). Parallel studies in the perfusate showed that TAT concentrations were 350% and protease inhibitor activities (antithrombin III, protein C) only 52% of the systemic circulation before reperfusion, suggesting that thrombin activation together with protease inhibitor consumption occurs during graft liver reperfusion. The relatively smaller increases in profibrinolytic parameters and a lower blood loss when compared with other groups may be explained by aprotinin administration in our patients.

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).

[Recurrent thromboembolism due to increased acetylsalicylic acid-resistant platelet aggregation]. / Rezidivierende Thromboembolien infolge gesteigerter, gegen Acetylsalicylsäure resistenter Thrombozytenaggregation.

A 49-year-old man had for 30 years suffered from severe recurrent thromboembolism with leg-vein thrombosis, pulmonary emboli, mesenteric infarction, cerebrovascular accident, cerebral vein thrombosis, portal vein thrombosis and femoral artery occlusion requiring leg amputation. In addition to moderately increased clotting activation with single-fold positive demonstration of fibrin monomers and a D-dimer concentration of 1 mg/l platelet aggregation was increased and could not be influenced by aspirin, 300 mg daily. Despite aspirin there were recurrent transitory attacks of cerebral ischaemia. Fibrin monomers were threefold positive and D-dimer concentration was increased to 4 mg/l (elevated clotting activation). Ticlopidine administration (250 mg daily) reduced adenosine diphosphate-induced platelet aggregation by 30% without effect on collagen-induced platelet aggregation. In parallel to these changes the patient's general condition clearly improved: fibrin monomers were no longer demonstrated and the D-dimer level fell to 0.5 mg/l.

[Reiter's disease in a patient with chronic recurring parotitis]. / Morbus reiter bei einem Patienten mit chronisch-rezidivierender Parotitis.

We report a 30-year-old patient who suffered from an acute exacerbation of chronic parotitis followed by manifestation of Reiter's syndrome one month later. Clinical course of both symptoms were in favour of a connection between them.

[Secondary osteoporosis induced by anticoagulants?]. / Sekundäre Osteoporose durch Antikoagulantien?

Generalized osteoporosis is a result of different causes and pathogenic mechanisms, which often combine forces to become clinically relevant. Among the different exogenic factors, drugs play an important role, frequently in connection with other factors such as immobilization or pregnancy. It has been suggested that anticoagulation therapy with heparins or coumarins may induce osteoporotic changes or enhance the development of osteoporosis for other reasons. According to in vitro experiments, preclinical trials, and clinical investigations, it seems reasonable to assume that heparins induce increased bone loss in a time- and dose-related manner. Low-molecular-weight heparins most likely have less effect on bone turnover when compared to unfractionated heparin. Oral anticoagulation therapy with vitamin K-antagonists is believed to have a weak effect on induction of osteoporosis, but clinical studies are contradictory. In spite of the fact that a relevant effect of these drugs on the induction of osteoporosis is questionable, it must be taken into consideration that anticoagulant drugs may enhance the negative effects on bone density of other risk factors capable of inducing osteoporosis such as immobilization, pregnancy, or endocrinological disorders.

[Non-tropical sprue and chronic inflammatory rectal stenosis in a patient with abuse of ergotamine-containing suppositories]. / Nicht-tropische Sprue und chronisch-entzündliche Rektumstenose bei einer Patientin mit Abusus von ergotaminhaltigen Suppositorien.

A 51 year old Yugoslavian patient was admitted to our hospital in reduced general condition with distinct hypocalcemia, osteomalacia, and with rectum stenosis. Our investigations led to the diagnosis of a malabsorption syndrome due to non-tropical sprue. The most likely cause of the rectum stenosis is an abuse of ergotamine-containing suppositories for several years. A gluten-free diet and the interruption of the use of the suppositories improved her general condition remarkably.

Influence of prostaglandin E1 infusion on hemostasis in orthotopic liver transplantation.

In the control group, a significant decrease in platelet aggregability could be demonstrated after reperfusion. This was paralleled by a decrease in platelet counts. When PGE1 was infused during OLT, the post-reperfusional decreases in platelet aggregability and platelet counts in the control group could be prevented. Furthermore, our investigation demonstrated that PGE1 infusion led to higher t-PA activity during the anhepatic phase. This was paralleled and followed by lower alpha 2AP levels at the end of the anhepatic phase and after reperfusion. The higher t-PA levels in the PG group did not result in clinical signs of hyperfibrinolysis during OLT. The aprotinin administration in both groups is most certainly responsible for the absence of hyperfibrinolytic signs in the TEG and the low overall requirement for transfusions, explaining the comparable transfusion rate in the two groups (Fig. 5). Further investigations involving more patients are required to evaluate the clinical effect of PGE1 therapy.

Thrombomodulin: a marker for endothelial damage during orthotopic liver transplantation.

Thrombomodulin is a surface protein of vascular endothelial cells. A smaller form of thrombomodulin in blood and urine, the soluble (s)thrombomodulin, appears to be derived from injured endothelial cells or to be proteolytically cleaved from thrombomodulin by proteases. Several in vitro and in vivo studies have suggested (s)thrombomodulin as a marker of endothelial damage. In orthotopic liver transplantation, platelet and leukocyte activation as well as prothrombin activation are suspected of being caused by damaged endothelial cells in the graft liver. We determined (s)thrombomodulin antigen as well as thrombin-antithrombin III complexes, protein C, and antithrombin III activities in the course of 23 orthotopic liver transplantations. Samples were taken at 7 different time-points intraoperatively, as well as out of the perfusate released from the graft liver vein during the flushing procedure with arterial blood prior to the opening of the hepatocaval anastomosis. Levels of (s)thrombomodulin antigen and thrombin-antithrombin III complexes showed a significant increase with reperfusion of the graft liver and levels in the perfusate were higher (both: P = 0.0001) than the corresponding systemic levels. In parallel, antithrombin III decreased significantly with reperfusion and perfusate levels of antithrombin III and protein C activities were lower in the systemic circulation (both: P = 0.0001). In conclusion, high levels of (s)thrombomodulin antigen in the early reperfusion phase and in the perfusate strongly indicate endothelial damage to the graft liver vascular bed, paralleled and followed by signs of prothrombin activation.