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OBJECTIVES: To evaluate the safety and potential efficacy of antithrombin III (AT III) in reducing mortality in patients with severe sepsis. DESIGN: Prospective, randomized, placebo-controlled, double-blind, phase II, multicenter, multinational clinical trial. SETTING: Seven academic medical center intensive care units (ICU) in Belgium, Denmark, the Netherlands, Norway and Sweden. PATIENTS: 42 patients with severe sepsis who received standard supportive care and antimicrobial therapy, in addition to the administration of AT III or placebo. INTERVENTIONS: Patients received either an intravenous loading dose of 3000 IU AT III followed by a maintenance dose of 1500 IU every 12 h for 5 days or equivalent amounts of placebo. MEASUREMENTS AND RESULTS: All patients were evaluated for safety and for 30-day all-cause mortality. CONCLUSIONS: 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.

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We evaluated the effect of C1 inhibitor (C1-inh), an inhibitor of the classical pathway of complement and the contact system, on the physiologic and inflammatory response in baboons suffering from lethal Escherichia coli sepsis. Five animals pretreated with 500 U/kg C1-inh (treatment group; n = 5), followed by a 9-h continuous infusion of 200 U/kg C1-inh subsequent to bacterial challenge, were compared with five controls receiving E. coli alone. Of the treatment group, one animal survived and another lived beyond 48 h, whereas all control animals died within 27 h. In four of five treated animals, less severe pathology was observed in various target organs. C1-inh administration did not prevent the hemodynamic or hematologic changes observed upon E. coli infusion. The activation of fibrinolysis and the development of disseminated intravascular coagulation were essentially unaffected by C1-inh. However, C1-inh supplementation significantly reduced decreases in plasma levels of factor XII and prekallikrein and abrogated the systemic appearance of C4b/c, indicating substantial inhibition of activation of the contact system and the classical complement pathway, respectively. Furthermore, treated animals displayed a reduced elaboration of various cytokines including TNF, IL-10, IL-6, and IL-8. Thus, the administration of C1-inh may have a beneficial but modest effect on the clinical course and outcome of severe sepsis in nonhuman primates. We suggest that activated complement and/or contact system proteases may, at least in part, contribute to the attendant manifestations of septic shock through an augmentation of the cytokine response.

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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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We assessed the pharmacokinetic characteristics of a new high-purity pasteurized FVIII concentrate in comparison with an intermediate purity pasteurized concentrate, produced by the same manufacturer. The study was designed as a cross-over single-dose pharmacokinetic investigation in 8 non-bleeding patients with severe hemophilia A. All patients were given 25 IU/kg of each of the two concentrates, with an interval of at least one week between the two administrations. Decay curves were assessed by collecting 10 serial blood samples over 36 hours following the end of infusion. The concentration of Factor VIII in blood samples was determined in triplicate in three different laboratories using each of the following assay methods: a one-stage clotting assay, a two-stage clotting assay, and a two-stage chromogenic-peptide substrate assay. All pharmacokinetic parameters were calculated by model-independent methods. The two products were found to differ significantly both in the clearance, which was on average 13.8% lower for Haemate P, and in the in-vivo recovery, which was 11.7% lower for Factor VIII:C P on the average. In comparison with previous pharmacokinetic data obtained from other heated Factor VIII concentrates, the clearance of Haemate P was found to be significantly slower, while the half-life of both products was longer. No differences were observed in the Vd-area. These findings indicate that the purification procedures to which both products are subjected do not increase the in-vivo rate of plasma disappearance of Factor VIII.

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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)

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BACKGROUND AND METHODS: Seventeen adult patients with acute lymphoblastic leukemia (ALL) treated with L-asparaginase (20,000 IU/m2 on six alternate days) were infused with antithrombin III (AT III) concentrates (Kybernin P, Behring). Substitution therapy was aimed at increasing the reduced AT III concentration usually found in these patients, since AT III deficiency is thought to be associated with an increased risk of thrombosis. Two schedules of AT III administration, different in dosage, timing and duration were evaluated. The first 7 patients (group A) received a fixed dose of 2,000 U every day for 6 times, starting with the second L-asparaginase (L-ase) infusion, independently of their plasma AT III levels. In the following 10 patients (group B), 20-25 U/Kg b.w. were administered daily for 7 times only when the plasma AT III level was lower than 60% with plasma fibrinogen higher than 100 mg/dl and platelet count higher than 50 x 10(9)/l, or when AT III was below 40%. Thirteen patients who received L-ase without AT III substitution served as controls. RESULTS AND CONCLUSIONS: Both substitution regimens resulted in mean plasma AT III nadir values significantly (p less than 00.1) higher than in the controls. Our data suggest that, in ALL patients receiving L-ase according to the L20 protocol, satisfactory plasma AT III levels may be assured with infusions of 20-25 U/Kg b.w./day for 7-10 days, starting by day 2 of L-ase treatment.

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This study was aimed at assessing the reproducibility of Factor VIII assays between different laboratories using the same reagents. A total of 176 post-dose plasma samples were obtained from 8 Italian subjects with hemophilia-A treated with a single dose of Factor VIII concentrates. Three laboratories (in FRG, Italy, and Sweden) participated in the study. Frozen aliquots of each sample were dispatched to each of the laboratories, where the aliquots were assayed using the same one-stage, two-stage and chromogenic methods. The one-stage and the chromogenic methods were well reproducible between the three centers: pairwise correlation analyses yielded r-values ranging from 0.88 to 0.91 for the one-stage method and from 0.91 to 0.96 for the chromogenic method. The agreement between these two assays was less evident in samples with activity below 200 IU/L in which the one-stage gave, on average, higher Factor VIII concentrations than those provided by the chromogenic method. The two-stage method was not well reproducible, and the pairwise r-values ranged from 0.48 to 0.73. Our study emphasises the need to develop multi-center quality control programs to verify the reproducibility of Factor VIII assays.

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The effect of purified fibrin(ogen) degradation product fragment D on cultured bovine aortic endothelial cells has been investigated. Binding to the cells was studied using purified radiolabelled fragment D and assessment of its recently postulated deleterious action was made by three different approaches: release of 51Cr from prelabelled cell layers, uptake of 3H-adenine by the cultivated cells as well as determination of residual cell number after exposure to various concentrations of fragment D were carried out. None of these methods applied was able to detect any harmful effect of the fragment on endothelial cell integrity and function in vitro. Neither could a specific interaction of the protein with the cultured cells be established. Even huge amounts of fragment D (1 mg/ml) exposed to the cells over 20 h did not reveal any alterations in the typical monolayer morphology. Thus, fragment D, which is generated in large amounts during systemic fibrinolytic treatment, does not exert any perturbation on cultured vascular endothelial cells as had previously been proposed.

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A method for long-term cultivation of large amounts of human microvascular endothelial cells from the omental tissue (human omental tissue microvascular endothelial cells, HOTMECs) was devised. The method originally described by Kern, Knedler, and Eckel was modified: HOTMECs were isolated by enzymatic dissociation with collagenase. For primary cultivation and passages, HOTMECs were plated either onto fibronectin-coated petri dishes or onto a human fibroblast extracellular matrix (HFB-ECM) prepared from the same tissue. Omental tissue (10-15 g) yielded 4-8 X 10(5) HOTMECs; more than 90% of the cells adhered to precoated dishes and grew in Waymouth's culture medium supplemented with 20% heat-inactivated fetal calf serum. Confluence was reached 3-5 days after seeding with an average of 1-2 X 10(6) cells/dish. Confluent HOTMEC layers were subcultured at a split ratio of 1:3 up to 11 passages by plating the cells onto dishes coated with HFB-ECM and maintained in long-term culture for up to 3 months. The endothelial origin of these cells was demonstrated as follows. The cells in culture showed the typical "cobblestone" growth pattern and synthesized von Willebrand factor (vWF) as determined by metabolic labeling. Using an indirect immunostaining technique, the cytoplasm of the HOTMECs stained for vWF. A monoclonal antibody specific for human endothelial cells bound exclusively to the cultured cells. The expression of thrombomodulin on the surface of the cultured cells was demonstrated by the activation of protein C by thrombin. In control experiments, these features could be detected on neither fibroblasts nor mesothelial cells.

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Exposure to lipopolysaccharides (LPS; 10 micrograms/ml derived from either S. enteritidis or E. coli or to their lipid A moiety alone induced procoagulant activity in cultured human endothelial cells. This exclusively cell-associated activity was identified as tissue factor activity by two criteria: Firstly, the presence of Factor VII was required for its expression and, secondly, clotting was abolished by the addition of the IgG fraction of anti-human tissue factor antibodies. Concomitant analysis of prostacyclin (PGl2) formation by the cells showed a substantial increase in the production of this potent platelet inhibiting substance during exposure to endotoxin. LPS-induced release of PGl2 did not result in refractoriness of the cells to generate new PGl2 as indicated by the retained response to stimulation with 20 microM arachidonic acid. While the release of PGl2 could be inhibited by pretreatment of the cells with 100 microM acetylsalicylic acid (ASA), the induction of tissue factor activity remained unaffected by ASA. In contrast to LPS-free control cultures, ASA did not completely prevent PGl2 formation by human endothelial cells after exposure to LPS suggesting the induction of a cyclooxygenase-independent pathway by LPS.

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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)

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Intact 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 (ATIII) 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 ATIII 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 microM) 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, 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 microM) 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.(ABSTRACT TRUNCATED AT 250 WORDS)

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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 (Mr 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.

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The important role of protein C (PC) in the regulation of hemostasis has been appreciated since the description of patients who were deficient in PC and presented with severe thromboembolic events. The potentially fatal complications associated with PC-deficiency require an early and reliable identification of those patients affected with this inherited disorder. The present study introduces a test procedure for the functional assessment of PC in plasma samples. The test utilizes the thrombin/thrombomodulin complex to achieve complete and rapid formation of activated PC whose proteolytic capacity is subsequently determined with a chromogenic substrate. Homogenate obtained from rabbit lung effectively substituted the purified component thrombomodulin in the assay system. This new approach simplifies the test procedure without losing specificity and accuracy. Proteases, such as plasmin, streptokinase and urokinase did not influence the assay and the inhibitory effect of heparin on the PC-activation could easily be overcome by the addition of protamine sulphate. The PC-activity in a group of unselected patients (n = 50), who did not reveal any abnormalities in global coagulation tests, amounted to 100 +/- 12% (mean +/- SD) with a range from 54 to 143% when analyzed in comparison to a plasma pool constituted from healthy volunteers. Since the synthesis of PC depends on the availability of vitamin K, patients receiving phenprocoumon have also been analyzed. These patients (n = 103) presented 40 +/- 11% residual PC-activity accompanied by a concomitant decrease in PC-antigen levels to 43 +/- 10% (mean +/- SD). The test described is specific, sensitive, less time-consuming and can be performed on a routine basis.

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The endothelial lining contributes in many respects to the patency of the vasculature. The production of heparan sulphate, release of prostacyclin and expression of the membrane cofactor thrombomodulin that is essentially required for the activation of protein C represent important mechanisms that warrant thromboresistance. If the integrity of the vessel wall is lost, the exposed subendothelium that has been built up by the endothelial cells serves as a highly reactive surface for platelets whose adherence is facilitated by another endothelial cell product, the von Willebrand Factor. Induction of tissue factor production after exposure to endotoxin also emphasizes an important role für the endothelium in the pathogenesis of disseminated intravascular coagulation. Once thrombosis has occurred the release of plasminogen activator of tissue-type from the endothelium leads to dissolution of the clot and a functional restoration of the blood vessel.

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