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From experiments with purified proteins, it has been concluded that factor XIa (FXIa) is inhibited in plasma mainly by alpha 1-antitrypsin (a1AT), followed by antithrombin III (ATIII), C1-inhibitor (C1Inh), and alpha 2-antiplasmin (a2AP). However, the validity of this concept has never been studied in plasma. We established the relative contribution of different inhibitors to the inactivation of FXIa in human plasma, using enzyme-linked immunosorbent assays (ELISAs) for the quantification of complexes of FXIa with a1AT, C1Inh, a2AP, and ATIII. We found that 47% of FXIa added to plasma formed complexes with C1Inh, 24.5% with a2AP, 23.5% with a1AT, and 5% with ATIII. The distribution of FXIa between these inhibitors in plasma was independent of whether FXIa was added to plasma, or was activated endogenously by kaolin, celite, or glass. However, in the presence of heparin (1 or 50 U/mL), C1Inh appeared to be the major inhibitor of FXIa, followed by ATIII. Furthermore, at lower temperatures, less FXIa-C1Inh and FXIa-a1AT complexes but more FXIa-a2AP complexes were formed. These data demonstrate that the contribution of the different inhibitors to inactivation of FXIa in plasma may vary, but C1Inh is the principal inhibitor under most conditions.

Gram-negative sepsis is oftentimes complicated by activation of coagulation with disseminated intravascular coagulation and microthrombosis. This may contribute to the associated morbidity, multiple organ failure and death. Recent studies have established that the tissue factor-dependent pathway of blood coagulation has a significant participatory role in the initial endotoxin-induced activation of coagulation. Tissue factor (TF), expressed on the surface of activated monocytes and endothelial cells forms cell surface complexes with free circulating factors VII and VIIa. The latter complex proteolytically activates factors X and IX. Recent in vivo experiments have shown that a rapidly neutralizing TF monoclonal antibody prevents and arrests the endotoxin-induced activation of coagulation and similar studies have shown to reduce mortality in baboons. In this study we describe the preparation of a factor VII/VIIa neutralizing monoclonal Fab fragment and characterize its effect on in vivo activation of coagulation during experimental endotoxemia in chimpanzees. Four chimpanzees received a bolus intravenous injection of 4 ng/kg endotoxin in combination with Fab fragments of a factor VII/VIIa neutralizing murine monoclonal antibody (12D10) at a dose of either 50 micrograms/kg (n = 2) or 100 micrograms/kg (n = 2). Four control animals received a bolus injection of endotoxin alone. Administration of the 12D10 Fab fragments, immediately preceding the endotoxin bolus injection, effectively blocked the endotoxin-induced activation of coagulation. Plasma levels of products of in vivo activation, namely F1 + 2, TAT complexes and FpA remained at baseline values. The administration of 12D10 resulted in a rapid decline in factor VII/VIIa antigen levels which remained below 5 ng/ml for 180-240 min, followed by a rapid return to baseline levels.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of tumor necrosis factor-alpha in the shedding of soluble tumor necrosis factor receptors in endotoxemia was investigated. The appearance of the soluble tumor necrosis factor receptors was assessed in four healthy volunteers following an intravenous injection of tumor necrosis factor-alpha and in eight chimpanzees after intravenous administration of endotoxin in the absence or presence of concurrent treatment with a neutralizing anti-tumor necrosis factor-alpha monoclonal antibody. Injection of tumor necrosis factor-alpha in humans elicited a significant, instantaneous (after 15 min) increase in the plasma concentrations of both types of soluble tumor necrosis factor receptors. In chimpanzees, treatment with the anti-tumor necrosis factor-alpha antibody completely neutralized endotoxin-induced tumor necrosis factor-alpha activity. The release of soluble tumor necrosis factor receptors was strongly (80-90%) inhibited in the presence of the neutralizing antibody. Our results indicate that tumor necrosis factor-alpha is a prime mediator of endotoxin-induced release of its own soluble receptors.

Interleukin 10 (IL-10) has been shown to inhibit endotoxin-induced tumor necrosis factor (TNF) production. To assess the role of TNF in the induction of IL-10 in endotoxemia, four healthy men were studied after a bolus intravenous injection of recombinant human TNF (50 micrograms/m2). In addition, 13 healthy chimpanzees were investigated after a bolus intravenous injection of Escherichia coli endotoxin (4 ng/kg), 6 animals received endotoxin only, 4 animals received a simultaneous intravenous injection of a monoclonal anti-TNF antibody, whereas 3 chimpanzees were treated with an anti-TNF F(ab')2 fragment 30 min after the administration of endotoxin. TNF induced a modest rise in IL-10 concentrations peaking after 45 min (47 +/- 32 pg/ml; p < 0.05). IL-10 peaked 2 h after injection of endotoxin (202 +/- 61 pg/ml; p < 0.005). In both anti-TNF-treated groups, the early endotoxin-induced TNF activity was completely neutralized. Simultaneous anti-TNF treatment attenuated endotoxin-induced IL-10 release (73 +/- 13 pg/ml; p < 0.01 versus endotoxin alone), whereas postponed anti-TNF treatment did not significantly affect this response (p = 0.21). These results indicate that TNF, in part, mediates the induction of IL-10 in endotoxemia, resulting in an autoregulatory feedback loop.

This paper reviews our recent studies of blood coagulation activation in the chimpanzee which were carried out employing sensitive immunoassays that measure activation markers of blood coagulation in plasma. Infused factor VIIa activated both factors IX and X in vivo; this reaction depended on the formation of the factor VIIa-tissue factor (TF) complex. The infusion of endotoxin also led to assembly of the factor VIIa-TF complex, enhancing fibrin formation. This process occurred through the intermediate action of specific cytokines.

The role of interleukin 6 (IL-6) in the toxic sequelae of sepsis is controversial. To assess the part of IL-6 in inflammatory responses to endotoxin, we investigated eight chimpanzees after either a bolus intravenous injection of Escherichia coli endotoxin (n = 4; 4 ng/kg) or after the same dose of endotoxin with a simultaneous bolus intravenous injection of an anti-IL-6 mAb (30 mg; n = 4). Anti-IL-6 did not affect the induction of the cytokine network (tumor necrosis factor [TNF], soluble TNF receptors types I and II, and IL-8) by endotoxin, nor did it influence the occurrence of a neutrophilic leukocytosis and neutrophil degranulation, as monitored by the measurement of elastase-alpha 1-antitrypsin complexes. In contrast, anti-IL-6 markedly attenuated endotoxin-induced activation of coagulation, monitored with the plasma levels of the prothrombin fragment F1+2 and thrombin-antithrombin III complexes, whereas activation of fibrinolysis, determined with the plasma concentrations of plasmin-alpha 2-antiplasmin complexes, remained unaltered. We conclude that IL-6 does not have a feedback effect on the release of other cytokines after injection of endotoxin, and that it is not involved in endotoxin-induced neutrophilia or neutrophil degranulation. IL-6 is, however, an important intermediate factor in activation of coagulation in low grade endotoxemia in chimpanzees.

Platelet-activating factor (PAF) has been postulated to play a role in the pathogenesis of sepsis. Additionally, in vitro studies have revealed tight interactions between PAF and the cytokine network, and PAF is considered to be an important stimulator of neutrophil functions. To assess the intermediate role of PAF in the induction of cytokines and neutrophil degranulation in endotoxemia in vivo, 12 healthy adult chimpanzees were i.v. injected with a bolus dose of Escherichia coli endotoxin (4 ng/kg); four animals received endotoxin alone, whereas the other chimpanzees were infused with the specific and potent PAF antagonist TCV-309 (bolus of 100 micrograms/kg, followed by either 100 micrograms/kg/h (n = 4) or 500 micrograms/kg/h (n = 4) for 5 h). At both doses TCV-309 significantly inhibited the endotoxin-induced rise in cytokine levels. Peak TNF concentrations after injection of endotoxin alone were 366 +/- 96 pg/ml, vs 105 +/- 47 and 115 +/- 56 pg/ml after administration of endotoxin together with the lower or higher dose of TCV-309, respectively (p < 0.05). TCV-309 also reduced the appearance of soluble TNFRs. Maximal levels of the type I soluble TNFR were diminished from 2.53 +/- 0.27 ng/ml (endotoxin alone) to 1.69 +/- 0.36 ng/ml (high dose TCV-309; p < 0.05); peak values of the type II soluble TNFR were diminished from 8.62 +/- 1.19 ng/ml to 5.76 +/- 0.92 ng/ml (p < 0.05). Furthermore, TCV-309 attenuated the endotoxin-induced release of IL-6 (160 +/- 82 pg/ml after endotoxin alone, vs 63 +/- 30 pg/ml in the low dose TCV-309 group (p < 0.05) and 65 +/- 29 pg/ml in the high dose group (p = 0.07) as well as that of IL-8 (279 +/- 168, vs 71 +/- 15 and 46 +/- 17 pg/ml, respectively; both p < 0.05). TCV-309 tended to reduce the endotoxin-provoked rise in serum IL-1R antagonist levels. In contrast, TCV-309 did not affect the neutrophilic leukocytosis elicited by endotoxin, nor did it inhibit endotoxin-induced neutrophil degranulation, as monitored by the plasma levels of elastase-alpha 1-antitrypsin complexes. We conclude that PAF plays a role, either directly or indirectly, in the stimulation of the cytokine network and in the shedding of soluble TNFR in endotoxemia. PAF does not seem to be an important intermediate factor in endotoxin-induced neutrophilia or neutrophil degranulation.

To assess the role of tumor necrosis factor (TNF) in the appearance of interleukin-1 receptor antagonist (IL-1RA) in endotoxemia, 4 healthy humans were studied after a bolus intravenous injection of recombinant human TNF (50 micrograms/m2). In addition, 8 healthy chimpanzees were investigated after a bolus intravenous injection of Escherichia coli endotoxin (4 ng/kg) with (n = 4) or without (n = 4) a simultaneous intravenous injection of a monoclonal anti-TNF antibody (15 mg/kg). TNF induced a pronounced rise in IL-1RA concentrations, becoming apparent after 1 h and peaking after 3 h (P < .05). The rise in IL-1RA after administration of endotoxin started 2 h later. Neutralization of the early endotoxin-induced TNF activity by anti-TNF caused a marked reduction in IL-1RA concentrations (P < .05). These results indicate that TNF is an intermediate factor in IL-1RA release in endotoxemia.

Recent studies suggest that increased activity of the coagulation system, measured with sensitive assays for activation markers, may be important in the pathogenesis of vascular occlusion in sickle cell disease (SCD). Since most of these studies were carried out in adult patients and SCD is an inherited disorder with severe morbidity even in childhood, we decided to determine the activity of the coagulation system in children with SCD. In a prospective study markers of thrombin generation as well as coagulation inhibitors were investigated in 16 homozygous SCD patients and 16 age-matched control children. Significantly increased plasma concentrations of the prothrombin fragment F1+2 and of thrombin-antithrombin III (TAT) complexes were found in SCD patients. The levels of protein C activity and total and free protein S were significantly reduced in SCD patients as compared with control values. Plasma AT III levels were not different in the two groups. We conclude that, in children with SCD, evidence of enhanced thrombin generation is present, which may in part be due to reduced levels of the inhibitors proteins C and S. The clinical relevance of this coagulation imbalance has to be demonstrated.

Tumor necrosis factor (TNF) is considered to be a pivotal mediator of endotoxin-induced lethality. To assess the intermediate role of TNF in specific systemic inflammatory responses known to contribute to tissue injury in endotoxemia, eight healthy adult chimpanzees were intravenously injected with Escherichia coli endotoxin (4 ng/kg). In four of these animals the administration of endotoxin was followed immediately by a bolus intravenous injection of an anti-TNF monoclonal antibody (15 mg/kg). Treatment with anti-TNF completely prevented the endotoxin-induced increase in serum TNF activity, and profoundly reduced the appearance of interleukin-6 and -8 (both P < .05). Neutrophilia and lymphopenia were not affected by anti-TNF, whereas neutrophil degranulation, as measured by the plasma concentrations of elastase-alpha 1-antitrypsin complexes, was only slightly reduced (peak levels after endotoxin alone 31.0 +/- 3.4 ng/mL, versus 25.5 +/- 3.4 ng/mL after endotoxin with anti-TNF; P < .05). Anti-TNF did not influence endotoxin-induced activation of the coagulation system, as reflected by unchanged increases in the plasma concentrations of the prothrombin fragment F1 + 2 and thrombin-antithrombin III complexes. In contrast, anti-TNF strongly attenuated the activation of the fibrinolytic system, ie, peak plasma levels of plasmin-alpha 2-antiplasmin were 33.8 +/- 11.1 nmol/L after endotoxin alone and 17.0 +/- 2.9 nmol/L after endotoxin with anti-TNF (P < .05). These results suggest that TNF is not the common mediator of systemic inflammatory changes in low-grade endotoxemia. Moreover, the finding that in this mild model anti-TNF specifically inhibited fibrinolysis suggests that treatment with anti-TNF potentially may enhance the tendency towards microvascular thrombosis in sepsis.

Knowledge of the pathogenetic mechanisms responsible for the activation of the coagulation system associated with endotoxemia is important for the development of improved modalities for prevention and treatment. We analyzed the appearance in plasma of TNF, IL-6, and indices of coagulation and fibrinolytic system activation in normal chimpanzees after intravenous infusion of endotoxin. Endotoxin infusion elicited reproducible and dose-dependent elevations in serum TNF and IL-6, as well as marked increases in thrombin generation in vivo as measured by immunoassays for prothrombin activation fragment F1 + 2, thrombin-antithrombin III complexes, and fibrinopeptide A. Activation of the fibrinolytic mechanism was monitored with assays for plasminogen activator activity and plasmin-alpha 2-antiplasmin complexes. To potentially intervene in the molecular pathways elicited by endotoxin, pentoxifylline, an agent that interrupts "immediate early" gene activation by monocytes, or a potent monoclonal antibody that neutralizes tissue factor-mediated initiation of coagulation, were infused shortly before endotoxin. Pentoxifylline markedly inhibited increases in the levels of TNF and IL-6, as well as the effects on coagulation and fibrinolysis. In contrast, the monoclonal antibody to tissue factor completely abrogated the augmentation in thrombin generation, but had no effect on cytokine levels or fibrinolysis. We conclude that the endotoxin-induced activation of coagulation appears to be mediated by the tissue factor-dependent pathway, the fibrinolytic response triggered by endotoxin is not dependent on the generation of thrombin, and that the release of cytokines may be important in mediating the activation of both the coagulation and the fibrinolytic mechanisms in vivo.

To assess the role of tumor necrosis factor (TNF) in the appearance of soluble TNF receptors (sTNFRs), 20 consecutive patients with a clinical diagnosis of sepsis were studied as were 7 chimpanzees after administration of endotoxin (4 ng/kg) with or without pentoxifylline. The patients had markedly elevated serum levels of sTNFR-p55 and sTNFR-p75 compared with healthy controls (P < .0001 for both receptors). The levels of both soluble receptors correlated with simultaneously measured immunoreactive TNF concentrations (p55: r = .63, P < .01; p75: r = .69, P < .001). In the chimpanzees, endotoxin induced subsequent rises in the serum concentrations of TNF and sTNFRs. Although pentoxifylline reduced the TNF response to intravenous endotoxin to 20% (P < .05), the appearance of sTNFRs was only moderately inhibited (sTNFR-p55 to 79% on average, P < .05; sTNFR-p75 to 77%, P = .12). These results indicate that TNF either does not play an important role in the appearance of sTNFRs in systemic infection or that a small amount of TNF remaining in the circulation after some bacterial challenges is sufficient to preserve the secretion of its soluble receptors.

The human coagulation system continuously generates very small quantities of Factor Xa and thrombin. Current evidence suggests that basal level activation of the hemostatic mechanism occurs via Factor VIIa-dependent activation of Factor X, but direct proof has not been available for the participation of tissue factor in this pathway. To examine this issue, we infused relatively high concentrations of recombinant Factor VIIa (approximately 50 micrograms/kg body wt) into normal chimpanzees and observed significant increases in the plasma levels of Factor IX activation peptide, Factor X activation peptide, and prothrombin activation fragment F1+2. Metabolic turnover studies with radiolabeled Factor IX activation peptide, Factor X activation peptide, and F1+2 indicate that elevated levels of the activation peptides are due to accelerated conversion of the three coagulation system zymogens into serine proteases. The administration of a potent monoclonal antibody to tissue factor, which immediately neutralizes function of the Factor VIIa-tissue factor complex in vitro, abolishes the activation of Factor X and prothrombin mediated by the infused recombinant protein, and also suppresses basal level activation of Factor IX and Factor X. The above results suggest that recombinant Factor VIIa functions as a prohemostatic agent by interacting with endogenous tissue factor sites, but definitive proof will require studies in hemophilic animals using relevant hemostatic endpoints.

Objective: To review new insights in the pathogenetic mechanisms involved in the development of disseminated intravascular coagulation (DIC) in septic patients, in order to develop new directions for therapeutic intervention., Data Sources: Articles and published peer-reviewed abstracts on the mechanism of the initiation of DIC in sepsis., Study Selection: Studies selected for detailed review were those reporting specifics about the mechanism of activation of coagulation and fibrinolysis in experimental human and animal models of sepsis. Data extraction guidelines for assessing data quality included validity of the model, quality of the laboratory assessment of activation of coagulation and fibrinolysis, and methodological considerations, such as the presence of control experiments and statistical analysis., Data Synthesis: After the presence of endotoxin in the circulation, significant coagulation activation can be detected. This activation is preceded by an increase in the serum levels of various cytokines, such as tumor necrosis factor and interleukins. Inhibition of the increase in tumor necrosis factor results in inhibition of coagulation activation. Measurement of molecular markers for the activation of coagulation proteins at various levels indicates that the activation of coagulation is mediated by the tissue factor-dependent pathway, which is further confirmed by experiments in which the inhibition of the tissue factor-dependent pathway resulted in complete inhibition of coagulation activation. The activation of coagulation seems to be amplified by impaired function of the protein C-protein S inhibitory pathway. An imbalance between coagulation and fibrinolysis, ultimately leading to plasminogen activator inhibitor type 1-mediated inhibition of fibrinolysis, may further promote the procoagulant state., Conclusion: The increased knowledge of the various pathogenetic mechanisms of coagulation activation and fibrinolysis in sepsis may have therapeutic implications; however, their efficacy needs to be assessed in appropriate clinical trials.

Costimulation of neutrophils and cytokines may play an important role in organ injury in sepsis. Pentoxifylline inhibits various neutrophil functions in vitro, and attenuates endotoxin-induced production of TNF in both in vitro and in vivo models. To assess the effect of pentoxifylline on neutrophil activation in endotoxemia, nine adult chimpanzees (Pan troglodytes) were i.v. injected with saline (n = 2), Escherichia coli endotoxin (4 ng/kg; n = 4), or E. coli endotoxin (4 ng/kg) in combination with pentoxifylline (500 mg/3 h, starting 30 min before the endotoxin injection; n = 3). Serial blood samples were obtained for measurements of leukocyte counts and the granulocytic proteinases elastase complexed with alpha 1-antitrypsin and lactoferrin, and cytokines during the next 5 h. No changes were observed in the saline-treated chimpanzees. Endotoxin induced a marked leukocytosis and neutrophilia, which were slightly reduced by pentoxifylline. In contrast, pentoxifylline almost completely prevented endotoxin-induced neutrophil degranulation: peak elastase-alpha 1-antitrypsin was 164 +/- 21 ng/ml (mean +/- SE) after endotoxin alone, vs 71 +/- 7 ng/ml after endotoxin with pentoxifylline (t = 3 h; p < 0.05); peak lactoferrin was 329 +/- 15 and 182 +/- 5 ng/ml, respectively (t = 5 h; p < 0.05). Pentoxifylline also inhibited the endotoxin-induced release of TNF (271 +/- 26 vs 55 +/- 23 pg/ml at t = 1.5 h; p < 0.05) and IL-6 (225 +/- 42 vs 73 +/- 25 pg/ml at t = 2 h; p < 0.05). IL-8 release was not significantly inhibited by pentoxifylline. In none of the animals activation of the C system could be detected. We conclude that pentoxifylline attenuates neutrophil activation in endotoxemia in chimpanzees, probably in part by inhibiting the release of TNF.

Disseminated intravascular coagulation is a frequent finding in critically ill patients, and may be diagnosed in the majority of patients with Gram-negative sepsis. Tissue damage may result from intravascular thrombosis, and disseminated intravascular coagulation is an underestimated causal factor in the pathogenesis of organ failure in sepsis. The diagnosis of disseminated intravascular coagulation is difficult, as the initial coagulation process that leads to thrombosis is counteracted by fibrinolytic responses, that in the context of ongoing consumption of clotting factors may result in an overwhelming bleeding tendency. Hence, depending on underlying disease, and the time at which the patient is evaluated, different laboratory results may be obtained. The treatment of disseminated intravascular coagulation is even more challenging than its diagnosis. No well-designed, controlled clinical trials exist that form a basis for rational treatment decisions. Treatment frequently needs to be individualized, and rapid adjustments may be necessitated by the course of the disease. Nonetheless, we believe that recent insights in the pathophysiology of disseminated intravascular coagulation, in particular concerning the role of the extrinsic coagulation pathway, provide ground for some optimism concerning future therapeutic options.

We investigated coagulation system activation following estrogen treatment in 29 healthy postmenopausal women. Study participants received conjugated estrogens at 0.625 and 1.25 mg per day, and placebo for 3-month periods in a randomized crossover protocol. Blood samples were obtained on two consecutive days at the end of each treatment period for immunoassays of F1+2 and fibrinopeptide A (FPA), markers of factor Xa action on prothrombin and thrombin action on fibrinogen in vivo, respectively. Treatment with estrogens at a dose of 0.625 or 1.25 mg resulted in significant increases in mean F1+2 levels of 40 and 98%, respectively, and in mean FPA levels of 37 and 71%, respectively. The measurements of F1+2 were significantly higher in women receiving 1.25 mg of estrogen than 0.625 mg. We also observed significant declines in the levels of antithrombin III and total protein S antigen. Immunologic levels of protein C increased modestly at only the 1.25 mg estrogen dose level. These data indicate that low doses of oral estrogens (< or = 1.25 mg per day) frequently increase the amount of thrombin generated in vivo. Our observations may help to explain the increased thrombotic risk that has been observed with higher doses of this medication (> or = 2.5 mg).

We have infused recombinant factor VIIa into patients with hereditary factor VII deficiency with marked reductions in plasma concentrations of factor IX activation peptide (FIXP), factor X activation peptide (FXP), and prothrombin activation fragment F1+2. These investigations show substantial elevations in these markers of coagulation activation and thereby demonstrate that the factor VII-tissue factor pathway is largely responsible for the activation of factor IX as well as factor X in the basal state (ie, the absence of thrombosis or provocative stimuli). We have administered a monoclonal antibody purified factor IX concentrate to individuals with hemophilia B. These studies show an increase in the plasma levels of FIXP that were initially greatly decreased, but no change in FXP or F1+2. We have also infused highly purified factor VIII concentrate into patients with hemophilia A. The data demonstrate no significant changes in the plasma concentrations of FXP and F1+2. The above observations indicate that factor IXa generated by the factor VII-tissue factor pathway is unable to activate factor X under basal conditions. Based upon the above findings, we outline a model of blood coagulation system function under basal conditions, and suggest a process by which the generation of factor Xa and thrombin might be accelerated during normal hemostasis and in the setting of thrombotic disorders.

We assessed the safety and efficacy of the novel low molecular weight heparinoid Lomoparan (Org 10172) for the prevention of deep-vein thrombosis in patients undergoing elective total hip replacement in a randomized, placebo-controlled, double-blind trial in 197 consecutive patients. The heparinoid (750 anti-factor Xa-units, s.c., b.i.d.) was administered to 97 patients and 99 patients received placebo. Study medication was started preoperatively and continued for 10 days. Efficacy was assessed by bilateral phlebography at day 10, postoperatively. The incidence of deep-vein thrombosis was 56.6% and 15.5% respectively in the placebo and heparinoid treated patients (incidence reduction: 74%; P less than 0.001). This reduction was observed both for proximal-vein thrombosis (25% to 8%; P less than 0.005) and isolated calf-vein thrombosis (31% to 7%; P less than 0.001). No major hemorrhage was observed. The number of red-cell units transfused and drain-fluid loss were comparable for the two study groups. Six patients in the heparinoid group and none in the control group developed minor wound hematomas (P less than 0.05). During an 8-week post-discharge follow-up period three patients with a normal venogram at day 10 developed clinically apparent venous thromboembolism, which was confirmed by objective testing. All three patients belonged to the heparinoid-treated group. We conclude that 750 anti-factor Xa units Org 10172 s.c. twice daily starting preoperatively is safe and effectively reduces early deep-vein thrombosis following elective total hip replacement. Further studies on the incidence of post-discharge thromboembolism are required.

In a cross-over study increasing doses of protamine hydrochloride (20-100 mg) or placebo were administered to six groups of four healthy male volunteers each, following a single intravenous dose of 3200 anti-Xa units of Org 10172. No neutralising effects were observed on the Org 10172 induced changes in the bleeding time, prothrombin time and thrombin time. A small and statistically not significant temporary decrease in anti-Xa activity was observed after doses of 80 and 100 mg protamine chloride. The anti-thrombin activity was dose-dependently and partly irreversibly neutralised by protamine chloride to a maximum of approximately 60%. This neutralisation correlated with the observed shorter prolongation of the thrombin time. The thrombin-generation inhibition activity was for approximately 35% neutralised by protamine chloride doses of 60-100 mg.

We performed a retrospective analysis on the influence of three types of anaesthesia on the incidence of deep vein thrombosis (DVT) following total hip replacement (THR) in consecutive patients randomized to either the low molecular weight heparinoid Org 10172 (97 patients), or placebo (99 patients). Ninety patients were operated under epidural anaesthesia, 77 patients under psoas compartment block with additional inhalation anaesthesia, and 29 patients under general anaesthesia. DVT assessment was performed by bilateral venography between days 8 and 12 postoperatively. The overall incidence of DVT in the 196 patients was 37% in the epidural anaesthesia group, 35% in the psoas compartment block group, and 36% in the general anaesthesia group. Although the incidence of DVT in patients randomized to placebo was similar in the two anaesthesia groups (53%), there was an important reduction of the occurrence of proximal DVT by the heparinoid in the psoas compartment block group (from 20 to 0%), compared to the epidural anaesthesia group (from 27 to 18%) (p less than 0.0061). Significantly more minor wound hematomas occurred in the psoas compartment block group as compared to the epidural anaesthesia group (p less than 0.05). Synergism of thrombin generation inhibition by the heparinoid and inhibition of platelet aggregation at the damaged vessel wall, by high local concentrations of bupivacaine in the psoas compartment block technique, is proposed as a possible mechanism behind this observation.

Despite significant progress in elucidating the biochemistry of the hemostatic mechanism, the process of blood coagulation in vivo remains poorly understood. Factor IX is a vitamin K-dependent glycoprotein that can be activated by factor XIa or the factor VII-tissue factor complex in vitro. To investigate the role of these two pathways in factor IX activation in humans, we have developed a sensitive procedure for quantifying the peptide that is liberated with the generation of factor IXa. The antibody population used for the immunoassay was raised in rabbits and chromatographed on a factor IX-agarose immunoadsorbent to obtain antibody populations with minimal intrinsic reactivity toward factor IX. We determined that the mean level of the factor IX activation peptide (FIXP) in normal individuals under the age of 40 years was 203 pmol/L and that levels increased significantly with advancing age. The mean concentration of FIXP was markedly reduced to 22.7 pmol/L in nine patients with hereditary factor VII deficiency (factor VII coagulant activity less than 7%) but was not significantly different from normal controls in nine subjects with factor XI deficiency (factor XI coagulant activity less than 8%). These data indicate that factor IXa generation in vivo results mainly from the activity of the tissue factor mechanism rather than the contact system (factor XII, prekallikrein, high molecular-weight kininogen, factor XI). Our results may also help to explain the absence of a bleeding diathesis in many patients with deficiencies of the contact factors of coagulation.

Tumor necrosis factor has been implicated in the activation of blood coagulation in septicemia, a condition commonly associated with intravascular coagulation and disturbances of hemostasis. To evaluate the early dynamics and the route of the in vivo coagulative response to tumor necrosis factor, we performed a controlled study in six healthy men, monitoring the activation of the common and intrinsic pathways of coagulation with highly sensitive and specific radioimmunoassays. Recombinant human tumor necrosis factor, administered as an intravenous bolus injection (50 micrograms per square meter of body-surface area), induced an early and short-lived rise in circulating levels of the activation peptide of factor X, reaching maximal values after 30 to 45 minutes (mean +/- SEM increase after 45 minutes, 34.2 +/- 18.2 percent; tumor necrosis factor vs. saline, P = 0.015). This was followed by a gradual and prolonged increase in the plasma concentration of the prothrombin fragment F1+2, peaking after four to five hours (mean increase after five hours, 348.0 +/- 144.8 percent; tumor necrosis factor vs. saline, P less than 0.0001). These findings signify the formation of factor Xa (activated factor X) and the activation of prothrombin. Activation of the intrinsic pathway could not be detected by a series of measurements of the plasma levels of factor XII, prekallikrein, factor XIIa-C1 inhibitor complexes, kallikrein-C1 inhibitor complexes, and the activation peptide of factor IX. The delay between the maximal activation of factor X and that of prothrombin amounted to several hours, indicating that neutralization of factor Xa activity was slow. We conclude that a single injection of tumor necrosis factor elicits a rapid and sustained activation of the common pathway of coagulation, probably induced through the extrinsic route. Our results suggest that tumor necrosis factor could play an important part in the early activation of the hemostatic mechanism in septicemia.

A new heparinoid, Org 10172, was given to five patients with hemorrhagic stroke with thromboembolic complications. Treatment did not cause progression of cerebral bleeding, and thrombotic processes were inhibited. No side effects were encountered.

Twenty consecutive patients undergoing elective transurethral resection of the prostate were allocated randomly to one of two groups. Group I (n = 10) received lumbar extradural analgesia with 0.5% bupivacaine. Group II (n = 10) received general anaesthesia with spontaneous respiration, using 60% nitrous oxide and 1-2% halothane in oxygen. There was a significant inhibitory effect on platelet aggregation in the extradural group. No such effect was observed in the general anaesthesia group. Measured indices of coagulation and fibrinolysis showed no abnormalities compared with control in either group except for a significant decrease in alpha 2-antiplasmin during surgery in group II. These results suggest that the possible thromboprophylatic effect of extradural analgesia with bupivacaine may result from an inhibitory effect on platelet aggregation which is in addition to the increase in lower limb blood flow.

Several investigators have reported that tumor necrosis factor (TNF) can alter the hemostatic properties of vascular endothelial cells in vitro. We have examined the in vivo effects on the hemostatic mechanism of recombinant human TNF administered as a continuous intravenous infusion to 23 cancer patients with active disease. A battery of sensitive and specific immunochemical techniques were used to monitor changes in blood coagulability. Serial determinations of F1 + 2, the protein C activation peptide (PCP), and fibrinopeptide A (FPA) were obtained prior to the initiation of the TNF infusions and at three and 24 hours after the start of therapy in 12 individuals who received greater than 3 x 10(5) U/m2/24h. The mean levels of F1 + 2, PCP, and FPA were significantly elevated at both time points as compared to the baseline values. The metabolic behavior of 125I-F1 + 2 in an animal model was not affected by infusions of the cytokine. We therefore conclude that the observed elevations in the concentration of this marker in humans receiving TNF result from hemostatic system hyperactivity. In 11 subjects infused with 1 x 10(5) to 2.4 x 10(5) U/m2/24 h of the cytokine, the mean levels of F1 + 2, PCP, and FPA were not significantly greater at 24 hours as compared with the baseline values, indicating that there is a threshold dose at which the cytokine can exert a biochemical effect on the coagulation system. Our studies demonstrate that TNF is able to provide a substantial net procoagulant stimulus to the hemostatic mechanism, and suggest that this cytokine may be a mediator of certain hypercoagulable states in humans.

A sensitive radioimmunoassay (RIA) for the fragment that is liberated from factor X when this zymogen is activated by factor VII/VIIa-tissue factor or factor IXa was developed. Antisera were raised in rabbits to a synthetic 15 amino acid peptide containing the COOH-terminal sequence of the activation fragment coupled to bovine serum albumin with glutaraldehyde. The reactivity of the antibody population obtained toward the factor X zymogen was negligible (less than 1/36,000 that of the activation peptide on a molar basis). However, because other plasma constituents contributed to a nonspecific basal signal in the RIA, a procedure by which the peptide could be reproducibly extracted from plasma was developed. The mean level of this species in normal individuals younger than the age of 40 was 66.4 pmol/L, and elevations up to 550 pmol/L were observed in patients with evidence of disseminated intravascular coagulation. The validity of these measurements of factor X activation is supported by the fact that the RIA signal migrates on reverse-phase high pressure liquid chromatography in a manner identical to that of the native peptide and can be quantitatively recovered. The mean concentration of the activation fragment was markedly decreased to 25.7 pmol/L in patients with hereditary factor VII deficiency (P = .0001 v normal controls), whereas the mean level in subjects with factor VIII deficiency was 61.1 pmol/L (P greater than .1 v normal controls). These data indicate that the basal (ie, in the absence of thrombosis or provocative stimuli) levels of FXP under in vivo conditions result mainly from the activity of the extrinsic pathway.

Org 10172, a low MW heparinoid derived from animal intestinal mucosal tissue, has a mean molecular weight of 6500 D and a specific activity of 8.0 anti-Xa U/mg. Its elimination half-life after i.v. administration is 18 hours. Six human volunteers received repeated single i.v. injections of 800 and 3200 anti-Xa units of Org 10172, 5000 IU heparin or placebo. Bleeding time, platelet count and plasma beta thromboglobulin were not affected by Org 10172 or heparin. Heparin stimulated ADP-induced platelet aggregation (0.2 uM; p less than 0.05) and inhibited thrombin induced aggregation (0.3 U/ml; p less than 0.05), while the heparinoid lacked these effects. Heparin increased plasma platelet factor 4, whereas Org 10172 had no effect. In contrast to heparin Org 10172 had only a minor effect on the activated partial thromboplastin time and thrombin time, while both compounds induced anti-Xa activity in plasma. In a crossover study in six haemodialysis patients, both heparin and Org 10172 (34.4 and 22.4 anti-Xa units/kg/body weight) successfully prevented clotting of the extracorporeal circuit. Microscopical analysis of the artificial kidney membranes showed that the 34.4 unit Org 10172 dosage was as effective as heparin in preventing fibrin deposition. The haemostatic and coagulation effects were as expected from those observed in the volunteers except that there was a slower elimination of the plasma anti-Xa response. In addition heparin and Org 10172 (34.4 anti-Xa units/kg) inhibited the Xa-induced platelet aggregation (0.5 U/ml; p less than 0.01 and p less than 0.001 respectively).

In this review an interim analysis was made of all clinical studies performed with low-molecular-weight heparin(oid)s (LMWH) up to January 1987. Thus far, many experimental studies on LMWH show these substances to have an increased benefit/risk ratio concerning efficacy and bleeding as compared to standard heparin. In man, this increased ratio was verified in some small open studies. However, until the present day, controversy still exists. Some of the difficulties in assessing the results of different investigations include the existence of various kinds of LMWH and the lack of a consensus as to the unitage in which they are expressed. Furthermore, no international agreement exists as to the test measuring dosage/effect and its standard. In the following review all published clinical studies were analyzed for patient groups. Important issues concerning controversy with regard to efficacy, bleeding risk, diagnosis, and testing are discussed. In general, it may be concluded that LMWH have an antithrombotic potential comparable to that of heparin, and that the benefit/risk ratio i.e., decreased bleeding potential when compared to standard heparin, as found in animal experiments, has not been clearly demonstrated in man.

In preparation for an efficacy study, the effect of the low molecular weight heparinoid Org 10172 on postoperative blood loss was assessed in a randomized double-blind, placebo controlled study in patients undergoing transurethral resection of the prostate (TURP). Org 10172 and placebo were given twice daily as i.v. injection for three postoperative days starting one hour preoperatively. Three doses of Org 10172 (800, 1600, and 2400 anti-Xa units b.d.) were evaluated against placebo in three consecutive patient blocks respectively. Each block consisted of 20 patients, 15 receiving Org 10172 and 5 patients placebo. The study was discontinued after 9 patients of the third block had completed the protocol because of excessive urinary blood loss. Data analysis showed a dose-dependent increase in postoperative haemoglobin loss, this was not significant for the 800 anti-Xa units b.d. dosage but was significant in those patients treated with 1600 (p less than 0.05) and 2400 anti-Xa units b.d. (p less than 0.01). It was concluded that the heparinoid Org 10172 caused a dose dependent increase in urinary blood loss following TURP.

Postoperative hemorrhage remains a serious complication in cardiopulmonary bypass (CPB) surgery. In our study, alternative anticoagulation with a new low molecular weight (LMW) heparinoid (Org 10172) was compared with a standardized heparin regimen. A preliminary dose-finding study indicated the minimal effective heparinoid dose to be 260 anti-Xa U/kg body weight, which was comparable to the standardized heparin regime, as revealed by similar plasma anti-Xa values. The following randomized open pilot study in 12 mongrel dogs undergoing CPB showed the heparinoid to be as effective as heparin, with an additional advantageous decrease in postoperative blood loss in the Org 10172 group. Our randomized blind study in 16 mongrel dogs undergoing CPB was performed to confirm previous results. Both antithrombotic agents were effective in the prevention of clot formation within the extracorporeal circuit. Hematocrit values and erythrocyte and platelet counts showed no significant intergroup differences. Post-CPB leukocyte counts revealed a significantly more rapid increase in the group given heparinoid (P less than 0.05). In the group given heparin, the expected prolongations of both the thrombin time (TT) and activated partial thromboplastin time (APTT) were noted, whereas in the group given heparinoid, only a transient peak prolongation of the TT after dose administration was revealed, and no significant prolongation of the APTT. Mean anti-Xa plasma levels were similar during CPB, showing a rapid decrease in the group given heparin on protamine administration, as did the APTT. Assessment of the operating field indicated an elevated intraoperative blood loss in the group given heparin. Postoperative blood loss measured over a period of 2.5 hours after closure of the thorax was significantly lower in the group given heparinoid than in the heparinized animals (625 +/- 100.0 ml, mean +/- SD, and 806 +/- 178.2 ml, respectively; P less than 0.05). Our observations suggest that the LMW heparinoid Org 10172 has an increased benefit/risk ratio over standard heparin and is effective in CPB in dogs. Additional investigations in humans should verify the possibility of use of this substance as an alternative means of anticoagulation during CPB in patients in whom heparin is relatively contraindicated.

In a prospective study, the clinical relevance of a quantitative chromogenic endotoxin assay in plasma (detection limit 10 ng/L, assay time 2.5 hours) versus blood cultures was evaluated in 51 critically ill patients with increased susceptibility for infectious complications. Of the 400 samples tested, the endotoxin assay and bacterial culture both were negative in 342 samples. In 21 samples from 15 patients, gram-negative aerobic microorganisms were cultured. Corresponding endotoxin assays were positive in 14 samples (mean 100 ng/L). Twenty-three samples grew gram-positive bacteria. The associated endotoxin assays all were negative. Twelve samples were found to be endotoxin-positive without a corresponding gram-negative bacterial culture. In 7 of these 12 positive endotoxin assays, a laboratory or clinical explanation for these positive tests could be provided. In view of the high sensitivity, specificity, and predictive values obtained, the authors conclude that the endotoxin assay used is a useful clinical adjunct for both the detection and exclusion of gram-negative septicemia.

In three groups of patients with advanced prostatic cancer the influence of three different forms of hormonal manipulation, i.e. estrogens, anti-androgens and bilateral orchidectomy, on the coagulation and fibrinolytic systems has been investigated. The groups, studied over a period of 35 days, were comparable as to age and stage of malignancy. A significant decrease in antithrombin III (AT-III) activity of 27% (range 7-46%) was found in patients on an initially high-dose estrogen (diethylstilbestrol) treatment regime. No changes in any of the monitored coagulation and fibrinolytic parameters were noted in the other treatment groups, including patients on maintenance estrogen therapy. The results of this study show that only high-dose estrogen therapy is accompanied by a selective decrease in AT-III activity. This may be an important etiological factor in the increased risk of thromboembolism in patients treated by this regime. The other means of hormonal manipulation studied, including low-dose estrogen treatment, did not influence the coagulation or fibrinolytic systems.

A new low molecular weight heparinoid, Org 10172 was compared to heparin in a randomized single blind cross-over study in 55 patients with end-stage renal failure undergoing chronic intermittent haemodialysis. The heparinoid administered as a single pre-dialysis i.v. injection of 34.4 anti-Xa units/kg body weight was compared to standard heparin (loading dose 2,500 IU + continuous infusion of 1,800 IU/hr). Mean anti-Xa plasma levels reached were 0.55 and 0.94 anti-Xa units/ml midway dialysis respectively. All 110 dialysis procedures were successfully performed without clotting or bleeding complications. Analysis of the number of clotted hollow-fibres within the dialysers showed a slight statistically calculated advantage in favour of heparin. Clinically no difference was detected. In conclusion, the heparinoid seems to be a good alternative means of anticoagulation in haemodialysis. As it is administered as a single i.v. predialysis injection it will simplify the dialysis procedure.

Veno-venous bypass without the use of systemic heparinization has recently become of increasing interest for application during liver transplantation and surgery on the large abdominal veins. However, possible adverse effects on blood components as demonstrated by means of hematologic and hemostatic parameters or on the occurrence of thromboembolic complications are until now not excluded. No consensus has been reached as to the efficacy of heparin coated circuits in those procedures. In the present study veno-venous bypass was performed for four hours in ten dogs using heparin coated and non coated circuits without further heparinization in a randomized blind fashion. No changes or significant intergroup differences were noted in the hematological and coagulation parameters. Macroscopic evaluation of the circuits revealed small strands of fibrin on all connector rims and clots in the center part of the pump head and at the cannula tips. The lungs showed two small emboli in large size pulmonary arteries and also two minor emboli in small size arteries. In four animals the emboli were equally divided between the two groups. As expected regarding the size of the clots no influences could be seen on hemodynamic or respiratory parameters. With Scanning Electronic Microscopy a monolayer of activated thrombocytes was observed on the surface of the bypass circuits in the coated as well as in the uncoated group. This study suggests that a veno-venous bypass without systemic heparinization is possible without serious damage to blood cellular elements or impressive activation of the coagulation system.(ABSTRACT TRUNCATED AT 250 WORDS)

Org 10172, a new, natural heparinoid, was used as the sole anticoagulant in twelve patients with acute or acute-on-chronic renal failure, who underwent haemodialysis 55 times. All patients had either intercurrent bleeding or a high risk of severe haemorrhagic complications if given standard heparin therapy. After a single loading dose of 300-600 mg of Org 10172, plasma anti-Xa levels in the range 0.42 - 0.93 U/ml were achieved. All haemodialysis runs were completed without adverse side-effects. There were no haemorrhagic complications and deposition of 125I-fibrinogen on the renal dialysis membrane was successfully inhibited in the 4 patients in whom this was studied. Org 10172 seems to prevent thrombosis during renal haemodialysis. It may have a lower risk/benefit ratio than other anticoagulants, such as heparin, in patients at high risk of haemorrhagic complications undergoing haemodialysis.

The solitary rectal ulcer and colitis cystica profunda are different manifestations of the solitary rectal ulcer syndrome. The cause of solitary rectal ulcer syndrome remains unknown. Since defecation disorders are common among patients with solitary rectal ulcer syndrome, defecography is indicated. Defecography was performed on 19 patients with solitary rectal ulcer syndrome. In five patients, the spastic pelvic floor syndrome had occurred. Twelve patients had internal intussusception of the rectum, and one patient had an anterior rectal wall prolapse. In one patient, no abnormalities could be detected. These abnormalities led to severe straining, which can damage the anterior rectal wall. Findings strongly support the hypothesis that solitary rectal ulcers are traumatic lesions caused by straining. Defecography is a suitable procedure for detecting the causative disorder of defecation and for selecting patients for treatment.

Using the chromogenic substrate S-2222, we have optimized and automated an amidolytic assay for heparin. The assay is based on the detection of anti-Xa activity generated by heparin in plasma. The method is reproducible (intra- and interassay CVs of 2.4 and 3.3%, respectively) and reliable in antithrombin III-deficient plasma. Results of this assay, obtained for plasma samples from patients and volunteers treated with heparin, correlate well (r = 0.899) with those of the test for activated partial thromboplastin time. Upon administration of a low-Mr heparinoid (Org 10172) and heparin fragment ( Kabi 2165), however, the activated partial thromboplastin time failed to detect anticoagulant activity, whereas the chromogenic heparin assay revealed anti-Xa activity. This automated amidolytic assay for heparin is therefore suitable not only for monitoring standard therapy with heparin but also for measuring the activity of recently developed heparin fractions.