The administration of AT III was safe and well-tolerated. It was followed by a 39 % reduction in 30-day all-cause mortality (NS). The reduction in mortality was accompanied by a considerably shorter stay in the ICU. Patients treated with AT III exhibited a better performance in overall severity of illness and organ failure scores (Acute Physiology and Chronic Health Evaluation II, multiple organ failure, organ system failure), which was noticeable soon after initiation of treatment. Patients treated with AT III demonstrated a better resolution of pre-existing organ failures and a lower incidence of new organ failures during the observation period. A meta-analysis comprising this and two other double-blind, placebo-controlled trials with AT III with a total of 122 patients suffering from severe sepsis confirms the positive trend. The results of the meta-analysis demonstrate a 22.9 % reduction in 30-day all-cause mortality in patients treated with AT III. Although still too small to be confirmative, the meta-analysis clearly points to the fact that a sufficiently powered phase III trial is warranted to prove whether AT III has a beneficial role in the treatment of severe sepsis.

The endothelial cell surface provides a receptor for thrombin-designated thrombomodulin (TM) which regulates thrombin formation and the activity of the enzyme at the vessel wall surface by serving as a potent cofactor for the activation of protein C by thrombin. Heparin-like structures of the vessel wall have been proposed as another regulatory mechanism catalyzing the inhibition of thrombin by antithrombin III. In the present study, the interaction of antithrombin III with the thrombin-TM complex and its interference with heparin and polycations were investigated by using human components and TM isolated from the microvasculature of rabbit lung. Purified TM bound thrombin and acted as a cofactor for protein C activation. The addition of heparin (0.5 unit/mL) to the reaction mixture interfered neither with the binding of thrombin to TM nor with the activation of protein C. However, the polycations protamine (1 unit/mL) as well as polybrene (0.1 mg/mL) affected the thrombin-TM interaction. This was documented by an increase in the Michaelis constant from 8.3 microM for thrombin alone to 19.5 microM for thrombin-TM with the chromogenic substrate compound S-2238 in the presence of 1 unit/mL protamine. When the inhibition of thrombin by antithrombin III was determined, the second-order rate constant k2 = 8.4 X 10(3) M-1 s-1 increased about 8-fold in the presence of TM, implying an accelerative function of TM in this reaction. Although purified TM did not bind to antithrombin III-Sepharose, suggesting the absence of heparin-like structures within the receptor molecule, protamine reversed the accelerative effect of TM in the inhibition reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular-leak syndrome (VLS) is a common complication in the first 3 weeks after bone marrow transplantation (BMT). The patients present with weight gain, generalized edema, ascites, pericardial or pleural effusions, tachycardia, arterial hypotonia, and/or pre-renal failure. The aim of our study was to investigate the role of the complement system in VLS. The protein concentrations of C3 and C4 were studied by immunodiffusion, and total hemolytic complement activity was studied by assessment of CH50. C1 esterase inhibitor (C1 Inh), the major inhibitor of the classical pathway of complement, was assessed by a functional test. Activation of complement was assessed by C4d (a C4 activation product). Twelve patients were followed prospectively from start of conditioning therapy to day +21 after bone marrow transplantation. Eight of 12 patients did not develop VLS. These patients had an increase of C3 between day +9 and day +13 (range: 1.3- to 1.5-fold, median: 1.4-fold), C4 (range: 1.3- to 1.9-fold, median: 1.4-fold), CH50 (range: 1.3- to 1.6-fold, median: 1.4-fold), and C1 Inh (range: 1.2- to 1.5-fold, median: 1.3-fold). Four of 12 patients developed VLS. C1 Inh activity was decreased to 0.60- to 0.80-fold. This decrease began 2-6 days prior to clinical diagnosis of VLS (n = 3), or at onset of VLS (n = 1). Patients with VLS showed elevated C4d concentrations (up to 2.4 mg/dl, upper normal threshold value: 0.9 mg/dl). Patients with VLS reveal an activated state of the complement system which is accompanied by a reduced activity of C1 Inh. Insufficient control of complement activation may contribute to VLS in patients after BMT.

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SummaryIntact vascular endothelium provides several anticoagulant mechanisms for the maintenance of blood fluidity and the prevention of thrombosis. High-affinity binding of proteolytic active thrombin to thrombomodulin at the cell surface effectively facilitates the activation of the potent anticoagulant protein C (PC). Rapid inactivation of cell-bound thrombin by antithrombin III (AT III) accelerated by heparin-like structures represents another anticoagulant mechanism. In the present investigation the interference of these two events has been studied. Inhibition of thrombin bound to cultured bovine aortic endothelial cells (BAEC) by AT III and the effect of the inhibitor on the activation of PC has been studied using purified components of bovine origin. Exposure of thrombin (45 nM) with prewashed confluent BAEC-monolayers for 10 min resulted in the binding of 12% thrombin. The subsequent incubation with various concentrations (0.3-2.4 μM) of ATIII revealed no acceleration of the inhibition of thrombin by ATIII at the endothelial cell surface when compared with the uncatalyzed fluid phase reaction. However, heparin added to the reaction mixture substantially increased the inactivation of cell-bound thrombin. Modified ATIII that did not possess heparin cofactor activity presented a comparable inactivation pattern for endothelial cell bound-thrombin as native ATIII indicating that heparin-like structures did not accelerate the interaction. When PC (32 nM) and ATIII (1.8 μM) competed for thrombin bound to BAEC, activation of PC was demonstrated within the initial 6 min of the incubation amounting to 62% of the activated PC formation in the absence of ATIII. Preincubation of BAEC with blocking antibodies against ATIII excluded a possible influence of BAEC-bound ATIII on the capacity of cell-bound thrombin to activate PC. The results suggest that an enhancing mechanism for the inactivation of thrombin by ATIII was not operative at the surfaces of cultured bovine aortic endothelial cells, but instead, appreciable amounts of activated PC become generated in the presence of an excess of ATIII.

Deposition of polymerizing fibrin on the vascular endothelium is the final event in intravascular coagulation. Exposure of fibrin clots to confluent monolayers of cultured human endothelial cells for 4 to 24 hours resulted in the disappearance of their normal cobblestone morphology and in the formation of endothelial cell aggregates. The present study was designed to evaluate the conditions and structural requirements of the fibrin clot for the induction of disorganization. Even after harsh treatment with denaturing agents or loading with large amounts of fibrinogen antibodies, polymerized fibrin always induced disorganization of the monolayers. In contrast, soluble fibrin that was kept in solution by either fibrinogen, fragment D-cate, or the tetrapeptide Gly-Pro-Arg-Pro did not cause any alteration of the monolayers. The fibrinogen degradation product D-cate (M r 94,000) itself had no microscopically detectable influence on the monolayer structure. In the absence of fibrin, the effect of thrombin on endothelial cells was found to be distinct from that induced by fibrin; however, the exposure of pieces of glass coverslips caused alterations in morphology indistinguishable from the fibrin-induced disorganization of the monolayer. Experiments using protein-coated polyester films indicated that the ability of the endothelial cells to attach to the overlying material, independent of its chemical structure, is the prerequisite for the induction of disorganization, but not a defined component of the fibrin molecule. Disorganization of vascular endothelium in vivo might be important for the organization and revascularization of an occluding thrombus. (Arteriosclerosis 6:139-145, March/April 1986) T he vascular endothelium contributes anticoagulant as well as procoagulant activities to the regulation of the coagulation system. A nonthrombogenic surface and the expression of fibrinolytic activities by the endothelial cells guarantee the patency of the vascular tree. Surface-bound heparan sulphate, which accelerates the inactivation of thrombin by enhancing the formation of the antithrombin Ill/thrombin complex, 1 and the membrane cofactor thrombomodulin, which facilitates the activation of protein C in conjunction with thrombin, 2 are important surface-mediated mechanisms of the vessel wall that are anticoagulant in nature. In addition, the synthesis and release of prostacyclin 3 and of tissue plasminogen activators 4 account for the inhibition of platelet activation and the continuous lysis of small amounts of fibrin. On the other hand, the synthesis of facator VIIIR:Ag, 56 specific binding of factors IX/IXa and X/Xa, 7 ' 8 and the induction of tissue factor production in the This study was supported by the Stiftung Volkswagenwerk, Hannover, West Germany.Part of this work was presented at the 10th International Congress on Thrombosis and Haemostasis, San Diego, California, in 1985 and is published in abstract form (Thromb Haemostas 1985;54:194).Address for reprints: Dr. Ulrich Delvos, Clinical Research Unit for Blood ...

Previous studies have shown that antithrombin III levels are low in fulminant hepatic failure, and heparin kinetics are abnormal, making control of heparinization difficult during hemodialysis of these patients who are at risk of bleeding. In this study, we have performed a controlled, randomized trial of antithrombin III supplementation on heparin activity, occurrence of bleeding and the platelet count and activation during hemodialysis in 24 patients with fulminant hepatic failure. The treated group of 12 patients was given 3,000 units of antithrombin III before hemodialysis. Antithrombin III supplementation was shown to normalize antithrombin III levels during hemodialysis (prelevels: 0.22 +/- 0.03 U/ml S.E.; at 1 hr 0.99 +/- 0.06 U/ml; p less than 0.001; control prelevels: 0.24 +/- 0.03 U/ml; at 1 hr 0.23 +/- 0.04 U/ml). Total heparin usage was significantly decreased by antithrombin III supplementation (median 5,200 U; range = 2,000 to 13,000) as compared with the control group (median 10,200 U; range = 5,000 to 16,500; p less than 0.005). Blood heparin level (antifactor Xa activity) after the initial bolus was significantly greater in the antithrombin III-supplemented subjects (0.40 +/- 0.07 U/ml compared with 0.22 +/- 0.05 U/ml in the control group; p less than 0.05). The significant reduction in platelet count observed in the control patients (18% +/- 6% at 1 hr; p less than 0.05) did not occur in antithrombin III patients (6% +/- 4% at 1 hr), which was reflected by a lower release of the platelet-specific protein beta-thromboglobulin. Two of 12 patients in both groups showed minor bleeding around vascular access sites during the first hemodialysis.(ABSTRACT TRUNCATED AT 250 WORDS)