Tissue Factor Pathway Inhibitor and Anitithrombin Trial Results

Crit Care Clin 21 (2005) 433 – 448

Tissue Factor Pathway Inhibitor and Anitithrombin Trial Results Steven P. LaRosa, MDa,*, Steven M. Opal, MDb a

Infectious Disease Division, Rhode Island Hospital, Gerry House 113, 593 Eddy Street, Providence, RI 02903, USA b Infectious Disease Division, Memorial Hospital, 111 Brewster Street, Pawtucket, RI 02860, USA

Severe bacterial and fungal infections often lead to the development of severe sepsis, that is, systemic inflammation with accompanying end organ failure. It has been established that outer membrane components of infectious organisms can trigger the release of pro-inflammatory cytokines, TNF-a and IL-1. These cytokines in turn can damage the endothelial lining of blood vessels leading to activation of the extrinsic pathway of coagulation and the deposition of fibrin clots in end organs. Naturally occurring anticoagulant molecules including tissue factor pathway inhibitor (TFPI), activated Protein C, and antithrombin (AT), are available to abrogate the coagulation response but these molecules are thought to both be consumed and the their production diminished by the septic process [1,2]. A large phase 3 trial of a recombinant version of rTFPI (OPTIMIST Trial) was recently shown to be ineffective in a randomized, placebo-controlled trial in patients with severe sepsis [3]. This brief review will evaluate the results of this trial in detail, and attempt to explain the results in the context of what has been learned about the molecule’s mechanisms of action, pharmacology, and from prior animal and human studies. The plan for the future development of the molecule will then be discussed.

Drs. LaRosa and Opal receive investigator grants and consulting fees from Chiron Corporation, the manufacturer of rTFPI. * Corresponding author. Infectious Disease Division, Memorial Hospital, 593 Eddy Street, Providence, RI 02903. E-mail address: [email protected] (S.P. LaRosa). 0749-0704/05/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ccc.2005.02.002 criticalcare.theclinics.com

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Clinical trial results A large, randomized, double blind, placebo controlled trial, multicenter phase III trial (OPTIMIST Trial) comparing recombinant tissue factor pathway inhibitor (rTPFI) with placebo in patients with severe sepsis was completed on September 27, 2001. Patients were enrolled in this study in two stages. The first stage enrolled a primary efficacy population composed of patients with two signs of the systemic inflammatory response syndrome: infection, the presence of two signs of organ dysfunction and or hypotension, and an international normalized ratio (INR) of 1.2 (high INR group). This population was based on an analysis from an earlier phase II study that demonstrated a statistically significant treatment by INR interaction (P = 0.037); increased efficacy was seen in the rTFPI group with increasing INR. The second stage of the study was composed of a safety population with the same criteria except the INR was b1.2 (low INR group). In both stages of the trial, patients were randomized in 1:1 ratio to receive either a 96-hour continuous infusion of rTPFI at a dose of 0.025 mg/kg/hr or placebo. The primary efficacy end point was 28-day all-cause mortality. Additional analyses examined the effects of rTFPI on the evolution of organ dysfunction, biomarkers of inflammation and coagulation, and safety parameters. A total of 1,754 patients were enrolled in the high INR population. The difference in mortality in the rTFPI arm (34.2%) was not statistically significant when compared with the placebo arm (33.9%), P = 0.75. Increasing INR was not predictive of a treatment benefit. The TFPI patients in the low INR population had a statistically significant lower mortality rate compared with placebo patients (12% versus 22.9%), P = 0.03. Subgroup analyses revealed a very clear drug– drug interaction between heparin and rTFPI. In the primary efficacy population a large mortality benefit was seen in the population that had not received heparin (rTFPI 34.6% versus placebo 42.7%, P = 0.05). A trend toward harm was seen in the rTFPI arm when heparin was co-administered (rTFPI 34% versus placebo 29.8%, P = 0.12) [3]. The presence of a documented infection was found to be important to the apparent efficacy of rTFPI. Mortality benefits with rTFPI were greatest in patients with documented bloodstream infections, and in documented cases of pneumonia especially in the absence of heparin. A trend toward harm with rTFPI was seen in patients without documented infection, especially those that had received heparin [4] (Table 1). A discussion of the results of the OPTIMIST trial would not be complete without commenting on the curious changes in the mortality rates observed during the course of the trial. At the beginning of the trial, a strong benefit with rTFPI was observed, and the time of the second interim analysis, the mortality in the rTFPI arm was 29.1% compared with a mortality of 38.9% in the placebo arm (P = 0.006). In the latter part of the study a complete reversal was seen with a dramatic decrease in the placebo mortality, and a sharp increase in the rTFPI arm mortality, which led to the overall neutral trial result. Although an in-depth investigation was performed by the study’s pharmaceutical sponsor in concert with outside academicians, a systemic lesion that could be attributed to the re-

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Table 1 rTFPI phase III trial results Group

28-Day mortality rTFPI

28-Day mortality placebo

P value

ITT population INR  1.2 (n = 1754) INR b 1.2 (n = 201) Heparin, INR  1.2) (n = 1200) No heparin, INR  1.2) (n = 554) Documented bloodstream infection (n = 535) Documented pneumonia (n = 504) No documented infection (n = 462) No documented infection, heparin (n = 326) Documented pneumonia, no heparin (n = 157)

34.2% 12% 34% 34.6% 27.7% 31.3% 40% 39.6% 31%

33.9% 22.9% 29.8% 42.7% 35.4% 39.8% 30% 25.7% 55%

0.75 0.03 0.12 0.05 0.053 0.05 0.02 0.01 0.002

Summary of results of the rTFPI phase III trial and major subgroups (references [3,4]).

versal of this trial could not be found. Potential causes that were ruled out included: errors in the randomization codes, changes in the pharmacodynamic potency of the drug, changes in the practice of critical care throughout the study, and changes in the severity of illness and baseline characteristics of the population in the two halves of the trial [3]. Animal work performed by Opal and colleagues using newer rTFPI at the beginning of the trial, compared with the ‘‘aged’’ material used at the end of the trial, did suggest decreased rTFPI efficacy with the aged material, despite similar effects of the two drug lots on clotting parameters (S. Opal, personal communication, 2004). The disparate results in the two halves of the trial are either due to ‘‘the play of chance’’ or some as yet undetermined etiology. The remainder of this review tries to explain the trial results based upon what is known about rTFPI. Deciphering tissue factor pathway inhibitor’s mechanism TFPI is a three-Kunitz-domain protease inhibitor. TFPI inhibits factor Xa by binding it at the second Kunitz domain. In a factor Xa-dependent process, TFPI is able to limit the production of factor Xa further, by binding factor VIIa / tissue factor (TF) complex at the first Kunitz domain [5] (Fig. 1). This anticoagulant function is the best known role for TFPI, and it is the one most easily measured by the prolongation of the prothrombin time. Additional mechanisms have been recently described for TFPI. For example, factor Xa, in concert with factor VIIa and tissue factor, can signal protease–activated receptors (PARS 1 and 2) on the cell membrane leading to downstream effects [6] including: production of proinflammatory cytokines [7], display of adhesion molecules [7], and endothelial vasorelaxation [8]. TFPI would, by virtue of Xa inhibition and TF/VIIa binding, inhibit PAR activation. In a model of lipopolysaccharide (LPS) challenge using rats, TFPI decreased lung injury, TNF levels, and myleloperoxidase levels. This study demonstrated that these effects were not directly related to anticoagulant effects, because similar effects were not achieved with site-inactivated factor Xa or factor VII [9].

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Systemic Inflammation IL-6 Contact Factor Pathway Amplification Loop

Tissue Factor Pathway

TF F XIa

F VII TF:F VIIa FX

F XII

F IXa

TNFα

Antithrombin

Fibrinolytic System

+F VIIIa

F Xa TFPI

+F Va Prothrombin

Thrombin

Fibrinogen Common pathway

Plasminogen PAI-1 u-PA Plasmin t-PA

Fibrin monomer F XIII

Fibrin polymer

Fig. 1. Pathways of coagulation activation in sepsis and the major inhibitory actions of TFPI and AT. Major activation steps (thick solid arrows); minor activation steps (thin solid arrows); inhibited steps (open arrows); IL, interleukin; F, factor; TF, tissue factor; TNFa, tumor necrosis factor alpha; u-PA, urokinase type plasminogen activator; t-PA, tissue type plasminogen activator; PAI-1, plasminogen activator inhibitor-1.

In a lethal baboon model by Hinshaw [10], a survival benefit with TFPI was seen and was also associated with decreased IL-6 levels and decreased end-organ injury. Similar protective effects were not seen with active–site inhibited Xa. In a rabbit model of Escherichia coli peritonitis, Fink and colleagues were able to demonstrate protective efficacy of TFPI with dosages much lower than those required to prolong coagulation tests by inhibiting factors VIIa/TF and Xa [11]. In an in vitro model, TFPI was able to bind LPS and prevent its transfer via lipopolysaccharide binging protein to the CD14 receptor suggesting a regulatory role in innate immunity [12]. An additional model of E. coli sepsis using baboons, demonstrated decreased levels of Fas—a marker of apoptosis— in TFPI-treated survivors, which suggests a beneficial effect on endothelial cell function [13]. What becomes clear through all of these studies, is that it is unclear which one or group of TFPI’s protective functions is being targeted in sepsis.

Tissue factor pathway inhibitor levels and target dosing The desired mechanistic target for TFPI in sepsis is tied to a discussion of TFPI levels and dosing. TFPI exists in the body in three distinct pools: ~3% is contained within platelets; ~10% is associated with plasma lipoproteins; and the remaining 80%–85% is bound to endothelial surface glycosaminoglycans (GAGs). It is the GAG-associated TFPI that is considered responsible for the mechanisms mentioned above. Levels of TFPI measured in healthy subjects are

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usually on the order of 100ng/mL [14]. Currently available rTFPI assays do not indicate the amount of TFPI available at its critical location on the endothelium. Studies performed measuring TF antigen levels and TFPI levels in the plasma of patients with acute respiratory distress syndrome [15] and with sepsis [16] found that tissue factor levels, but not TFPI levels, were significantly higher in severe disease and were associated with a poor outcome. TFPI levels in both studies were slightly higher than in normal controls. The authors conclude that TF not balanced by TFPI leads to a worse outcome. They also comment that human neutrophil elastase levels rise in severe sepsis and may lead to cleavage and inactivation of TFPI. In a study of TF and TFPI levels in children with meningococcal disease, plasma TFPI levels were decreased while urine TFPI levels increased, which suggests increased destruction and clearance of TFPI [17]. Finally, in a murine model of intraperitoneal LPS injection, decreased TFPI mRNA expression was found in lung tissue, as well as in cultured endothelial cells [18]. All these studies suggest an inadequate endothelial pool of TFPI in severe infection, but also the inadequacy of plasma levels to guide potential studies. Given the absence of data on what the intended mechanism of TFPI is, and what the levels of TFPI at the endothelial surface need to be, dosing decisions for TFPI have been quite difficult. Initial clinical studies in patients with sepsis were performed with dosages of TFPI equivalent to those found to be protective in the lethal baboon model of E. coli sepsis. Dosages of 0.33 mg/kg/hr and 0.66 mg/kg/hr were found to excessively prolong prothrombin time and increase bleeding events. Subsequent clinical trials in patients with sepsis used doses 13-fold lower, based on the results of Fink’s rabbit model previously mentioned [14]. Ultimately, a phase II dose escalation study was performed; plans were to study doses up to 0.10 mg/kg/hr for 96 hours. However, when a trend toward an increased number of serious adverse bleeding events was seen in the 0.05 mg/kg/ hr treatment group, the decision was made by a data safety monitoring board to not test higher doses. A relatively small population was studied in this trial, in which 69 patients received placebo, 80 patients received 0.025 mg/kg/hr, and 61 received 0.05 mg/kg/hr. The observed mortality was better in the ‘‘all-TFPI dose group’’ compared with placebo (30% versus 38%, P = 0.30). Although the observed mortality was lower in the 0.025 mg/kg/hr group than in the 0.05 mg/kg/hr group, the sample size was far too small to draw a conclusion regarding better efficacy at the lower dose. Of note, the ‘‘all-TFPI’’ treated patients demonstrated lower thrombin–AT complexes and prothrombin fragment (PF)1.2—markers of thrombin generation— over time compared with placebo, but by no means did these doses completely block the coagulation response seen in these patients. Effects on these markers were not presented by dose, so it is unclear if a dose-response was present [19]. This is important as a human endotoxin challenge model of the 0.05 mg/kg/hr dose was able to demonstrate statistically significant lower PF1.2 levels compared with placebo, but was not able to blunt the coagulation response like the 0.2 mg/kg/hr dose that was also studied in the model [20]. This finding was confirmed in the large

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phase III trial where the 0.025 mg/kg/hr dose tested, attenuated the rise in PF1.2 levels seen in the placebo group, but did not decrease the PF1.2 levels from baseline. The levels of PF1.2 in the rTFPI group, as a percentage of the baseline, were still quite high at 24 hours (101%) and at 96 hours (126%) [3]. It appears, based on all the accumulated data, that the 0.025 mg/kg/hr dose is likely not adequate to sufficiently block the coagulopathic response in severe sepsis. Dosing TFPI to achieve the potential anti-inflammatory responses appears even less straightforward. In the phase II trial, the combined data from the 0.025 mg/kg/hr and 0.05 mg/kg/hr dosed patients demonstrated statistically significant lower IL-6 levels over time compared with the placebo-treated patients [19]. IL-6 data from the Phase III trial using the 0.025 mg/kg/hr dose is not published. The relatively high dose of 0.2 mg/kg/hr tested in the human endotoxin challenge model did not significantly affect markers of neutrophil activation, endothelial cell response, systemic cytokines, chemokines or acute phase reactants compared with a placebo group [21]. It is possibile that this dose is truly not high enough to impact the inflammatory pathways that are not triggered by thrombin, or that this model is either not severe enough, or does not generate enough thrombin to demonstrate TFPI’s ability to decrease endothelial pro-inflammatory cytokine production. If the predominant anti-inflammatory mechanism of TFPI is its ability to bind LPS and prevent its transfer to the CD14 via LBP, then finding the correct dose and level to accomplish this function becomes important. Studies done by Park et al [12] showed that the doses and levels of TFPI achieved in the lethal baboon challenge models (~50nmol) would likely be sufficient to prevent LBP-mediated LPS transfer to CD14. The results of all these studies indicate that the dosage of TFPI tested to date may not be sufficient to maximize the molecule’s anti-inflammatory properties. Heparin– tissue factor pathway inhibitor interaction With all the questions that surround the findings of the phase III trial, the heparin –TFPI interaction is probably the easiest to explain. The third Kunitz domain of TFPI has a heparin-binding domain. Studies have demonstrated that the binding of heparin to TFPI leads to its release from endothelium, where it may be active in sepsis [22]. This finding is true of both low-molecular-weight heparin and unfractionated heparin [23], and appears to occur even with low dosages of heparin [24]. The trend toward harm in the TFPI-heparin treated arm in the OPTIMIST trial is harder to explain than is the decrease in efficacy. Unlike the results in the AT trial, a higher rate of bleeding events and serious adverse events in the heparin–TFPI arm does not explain this result. An intriguing possibility for this apparent negative interaction relates back to the third Kunitz region of TFPI. This region of TFPI has been found to bind both heparin and LPS. Also, heparin is able to inhibit the binding of TFPI to LPS [12]. Is it possible that this drug interaction is caused by heparin’s interference with TFPI’s regulation of innate immunity? Given the large percentage of severe sepsis patients who receive heparin for deep vein thrombosis prophylaxis or for renal

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replacement therapy, it is clear that alternative methods would need to be used to accomplish such goals in any further trials of TFPI in sepsis. Documented infection and pneumonia as a therapeutic target The increased efficacy of TFPI in the patient subgroups with documented infection, and documented pneumonia in particular, was an unanticipated but fascinating finding from the OPTIMIST trial. It may be possible that TFPI’s role in blocking LPS signaling is a paramount mechanism and explains the importance of having a demonstrable pathogen. If this were the case, one would think that the treatment benefit might be greater with TFPI; maybe greater in gramnegative infections than gram-positive infections, an observation not found in the OPTIMIST trial data. Opal and colleagues [25] have, however, demonstrated endotoxemia, presumably from a splanchnic source, in patients with grampositive sepsis. Baseline data from the PROWESS trial involving recombinant human activated protein C revealed that markers of inflammation and coagulation were highest in those patients with documented infections, especially those with bloodstream infections [26]. Thus, the presence of a documented infection may simply represent a patient with greater perturbations of the coagulation system and a better candidate for this type of agent. The impressive reduction of TFPI in patients with documented pneumonia is also fascinating especially given that a similar finding was observed with recombinant human activated protein C [4,27]. Many studies have pointed to the extensive activation of the extrinsic pathway of coagulation in severe pneumonia. Studies by both Gross and McGhee [28,29] demonstrated tissue factor expression in both rat pulmonary macrophages and alveolar epithelial cells. Additional studies by Gunther, Levi, and Schultz [30–32] have demonstrated activation of coagulation and high levels of tissue factor in acute bronchopneumonia and acute lung injury. The preponderance of data would suggest that a patient population with severe acute bronchopneumonia would serve as the best therapeutic target for TFPI. Summary A great deal has been learned about TFPI from in vitro, animal, and human trials but multiple questions abound. The most important questions are: what is or are the protective mechanism(s) of TFPI, and what is the dose and level necessary to achieve these effects? It seems clear that doses of TFPI 0.05 mg/mg/hr need to be tested in a large patient population to see if enhanced anti-inflammatory and anti-coagulant effects, and hopefully enhanced efficacy, can be observed with a sufficient degree of safety. It also appears that a homogenous population with severe pneumonia is the best biologic target for TFPI. Finally, any future trials of TFPI should be done in the absence of concomitant heparin therapy. Fortunately, such a trial is underway in patients with severe community acquired pneumonia. We eagerly await the results and hope the trial answers the questions raised here.

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Clinical trials with antithrombin in sepsis Early clinical investigations AT therapy for sepsis-induced coagulation abnormalities is certainly not a new idea. Schipper and coworkers first reported the use of AT transfusion for disseminated intravascular coagulation (DIC) back in 1978 [33]. Heparin had been attempted as a treatment strategy for the consumptive coagulopathy associated with severe infection for many years without any convincing evidence of efficacy [34,35]. Since AT levels were noted to decrease in the presence of systemic infection, it provided a therapeutic rationale for the replacement of this naturally occurring circulating anticoagulant in septic patients [36] Efforts to determine the actual clinical benefit of AT in sepsis-induced DIC have yielded some tantalizing insights into possible improvements in outcome, but variable and generally inconsistent results have punctuated these clinical investigations for the last 25 years [1,37] A brief summary of these clinical studies is provided in the following paragraphs. After Schipper’s initial report, several case reports and case series were published [36]. The first phase II randomized study of AT versus heparin in patients with shock and DIC was reported in 1985 [37]. Duration of DIC appeared to be shortest in the group receiving AT alone, while the group given a combination of AT and heparin or heparin alone had less favorable responses. Studies in primate sepsis models then appeared, which indicated that AT not only prevented DIC, it improved overall survival rates [38]. Extensive studies then followed in multiple animal models with a variety of septic stimuli (endotoxin, gram-negative pathogens, gram-positive pathogens, polymicrobial infections) verifying the survival benefits that accrue from the administration of AT in experimental sepsis models [39–43]. Many small clinical studies ensued and generally supported the hypothesis that AT supplementation may provide a survival benefit for severely septic patients with or without sepsis-associated DIC [44]. Coagulation activation was shown to be an early and omnipresent feature of sepsis, even in the absence of any clinical evidence of coagulopathy [45]. The extensive interactions between the innate immune response to systemic inflammation, invasive microbial infection, and the coagulation system have become increasing appreciated [1,46,47]. It has subsequently become evident that AT and other endogenous anticoagulants have extensive immuno-modulatory effects separate and distinguishable from their anti-thrombotic properties. These anti-inflammatory actions may be essential to their therapeutic role in sepsis [46]. In 1993, Fourrier et al [48] reported a 44% relative risk reduction in sepsis mortality with high-dose AT treatment in a 35-patient, randomized, placebocontrolled, double-blind study in the presence of septic shock and DIC. A subsequent study in 45 patients with sepsis was reported in which AT treatment was co-administered with 6 IU of heparin/kg/h, and a 14% relative risk reduction in mortality was observed [49]. In this trial, there was a suggestion of excess

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bleeding risk with co-administration of heparin and AT. Another European study randomized 42 patients with severe sepsis and reported a 39% relative risk reduction in mortality in the AT group when compared with the placebo group [50]. The meta-analysis of all three of these randomized trials estimated an overall a 22.9% reduction in 30-day all-cause mortality with AT treatment in severe sepsis [51]. Baudo and colleagues [52] performed a larger (n = 120) controlled, randomized, double blind, multi-center study with AT therapy in severely septic patients with or without post-operative surgical complications in 1998. They found no overall difference in outcome with AT versus the placebo control group, but did observe a significant survival benefit for the most severely ill subgroup of patients with septic shock. A subsequent study by Balk and coworkers [53] found that in less severely ill, septic patients (n = 34) the AT treatment group actually experiencing a trend toward worsening outcome compared with a placebo group. These clinical studies served as the basis for the design and implementation of the large phase III study for high-dose AT in severe sepsis or septic shock [54]. A pharmacokinetic study was first undertaken to determine the optimal dosing strategy to achieve supraphysiological levels of AT in severely septic patients [55]. Detailed animal experimentation [41–44] indicated the need to attain high levels of AT (N 250% of normal plasma concentrations) for optimal antiinflammatory effects of AT (see following section on mechanism of AT). It was observed that a loading dose followed by a continuous infusion of 6,000 IU of AT maintained a blood level of N 200% of normal plasma levels for more than 4 days in septic patients. The phase III clinical trial of high dose antithrombin in severe sepsis This pharmacokinetic study was then followed by a large, phase III multinational sepsis study involving more than 2,314 patients worldwide (the KyberSept trial) [54]. The study failed to meet its primary endpoint of an intent to treat, significant improvement in the 28 day mortality rate by the administration of AT versus placebo in this overall septic patient population (Table 2). As in a number of other clinical sepsis studies, careful review of the data revealed potentially favorable effects of the study treatment in clinically relevant subgroups within the trial. Table 2 KyberSept trial results with high dose antithrombin 28-day mortality

90-day mortality

Group

AT

Placebo

AT

Placebo

P value

All patients (n = 2314) No heparin subgroup (n = 698) SAPS II (30%-60%) (n = 1008)

38.9% 37.8% 36.9%

38.7% 43.6% 40.7%

48.5% 44.9%* 45.5%*

50.0 52.5% 51.6%

NS *P b.05 *P b 0.05

Summary of results of the antithrombin phase III trial and major subgroups (reference [54]). * Statistically significant.

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The KyberSept study was a double-blind, placebo-controlled, multi-center phase III clinical trial in patients with severe sepsis conducted in 211 study sites in 19 countries worldwide. The study enrolled 2,314 patients in the intentionto-treat population who were randomized into two equal groups of 1,157 patients, to receive either intravenous high-dose AT (30,000 IU in total over 4 days) or placebo (1% human albumin). Low doses of heparin (up to 10,000 units/d) were allowed at the discretion of the investigator during or after the study drug infusion to allow for prophylaxis against deep venous thrombosis or other indications, if clinically warranted. It was recognized that heparin administration with AT could potentially increase bleeding risk and might alter the pharmacokinetics of AT, interfering with its local binding sites within the microcirculation [36,41,44]. It was hypothesized that low doses of heparin could be given with high doses of AT without excess bleeding risk and without major interference with AT’s postulated beneficial effects on microcirculatory events. For this reason, a pre-specified analysis was planned to examine the group of patients with concomitant heparin administration as a separate subgroup. There was no formal randomization to heparin or no heparin in the KyberSept trial. The overall results were disappointing for many reasons. Not only did the primary efficacy end point fail to be reached, some of the trial assumptions were not confirmed in the actual clinical trial. The overall, intent to treat, allcause 28-day mortality rate did not differ between the AT and placebo groups (38.9 versus 38.7%, P = ns). It was anticipated that the stringent entry criteria would identify a severely septic patient population that would have a moderate to high risk of death due to severe sepsis/septic shock. In the final trial results, less than half (43.6%) of the overall study population had a predicted mortality by SAPS II scores of 30%-60% (the initial target population). As shown in Table 2, a 90-day survival time analysis of this subgroup actually reached nominal statistical significance with a mortality rate of 45.5% in the AT group and 51.6% in the placebo group [56]. Perhaps the use of a clinical coordinating center tasked with the review of all possible study patients before study randomization, could have increased the number of patients considered to be the optimal cohort for a pivotal clinical trial. Another disappointing finding was the failure to achieve the target blood level of AT in the treated arm of the KyberSept trial. A blood level N 200% normal AT activity was anticipated, based on the dosing schedule, but the mean blood level in the treatment arm was only 180%. The pre-clinical data clearly indicate the need to give supraphysiological doses to maximize the therapeutic potential of AT [41–43]. The outcome of those patients randomized to the AT treatment arm, who attained a blood level of 200% AT levels after 24 hours, was significantly better in the AT group whose blood levels were less than 200% (30.7% versus 36.2%) [57]. The reason(s) for the inability to achieve the target blood level of AT in the treated group are not known, but it should be noted that the 30,000 IU treatment was a fixed dose in this study, and not based on body weight or rate of AT consumption as measured by AT blood levels. Some of these study related issues have been addressed by the authors in an earlier publication [58].

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Another major disappointment with this study was the extent to which even relatively low doses of heparin with high doses of AT exacerbated bleeding risk and appeared to limit the efficacy of the AT infusion. The excess bleeding risk seen in the KyberSept trial attributable to the AT treatment (22% AT versus 12.8% placebo; P b 0.001), was largely confined to the subgroup who received concomitant heparin treatment during the study drug infusion. The bleeding risk was 23.8% in the heparin + AT group, and 17.8% in the AT only group. There was no association between the AT blood levels observed after 24 hours of AT infusion and bleeding risk [54]. The drug interaction between AT and heparin was primarily responsible for the excess bleeding risk in this study population. It is indeed unfortunate that the use of concomitant heparin use during the study drug infusion actually increased over the course of the clinical trial [57]. A further complicating factor with heparin use in combination with high dose AT was the loss of possible efficacy attributable to AT itself. The AT subgroup that received AT without concomitant heparin had a nominal survival benefit at 90 days compared with patients randomized to the placebo group without heparin treatment (44.9 versus 52.5%; P b 0.05) [54]. This favorable trend was observed in the 30%-60% SAPS II subgroup and was also seen in a secondary analysis of the long-term morbidity and quality of life measures in this study [59]. Some recent laboratory findings and experimental studies into the detailed molecular interactions with AT and heparin and heparin sulfate may help account for these pronounced AT–heparin interactions. It could be argued that heparin in the absence of AT may have benefited the patients enrolled into this trial. The differential effect seen with heparin is related to the lower mortality in the placebo group that received heparin. Patients enrolled in the KyberSept trial were not randomized into heparin or no heparin therapy groups, received different dosages and formulations of heparin, and upon review of the baseline characteristics have not been randomly distributed by disease severity. The administration of heparin in this study was often a postrandomization event that unavoidably introduces a bias favoring heparin in this critically ill patient population. Patients who die early from sepsis are unable to receive heparin while surviving patients may receive heparin at anytime during the 28-day follow up used in each study. This introduces a selection bias and an allocation bias into the interpretation since the sickest patients are more likely to end up in the no heparin group. A well-performed randomized, placebocontrolled trial with heparin alone will be necessary determine if heparin itself has any salutary effects in management of severe sepsis [60]. Mechanisms of antithrombin and interactions with heparin and related heparan sulfate glycoaminoglycans AT is a serine protease inhibitor that inactivates multiple clotting enzymes (Xa, IXa, XIa, tissue factor: VIIa complex) and, in particular, thrombin by forming thrombin-AT complexes (TAT) (Fig. 1). Sepsis generates a procoagulant state by multiple mechanisms including IL-6 induced (and other cytokines and chemo-

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kines) tissue factor expression. TNF is principally responsible for activation of the fibrinolytic pathways in systemic inflammatory states. TAT complex formation is accelerated by an endogenous heparin-like molecule, heparan sulfate, a glycosaminoglycan (GAG) found upon the endothelial surface [1,36,41]. Specific heparin-like GAGs bearing repeating units of sulfated pentasaccharides activate AT over 1,000-fold by the process of allosteric activation with conformational changes in the active site of AT [61]. Through its interaction with these GAGs AT may stimulate the production of prostacyclin (PGI2) by endothelial cells. Prostacyclin has anti-inflammatory properties, including diminishing TNF synthesis from monocytes, inflammatory mediator release from neutrophils, and neutrophil adhesion to endothelial cells [62]. Recent evidence indicates that the anti-inflammatory activity attributable to AT may have a more direct mechanism underlying its immune modulating properties. AT directly binds to neutrophil and monocyte membranes via highly specific and saturable binding to sulfated pentasaccharide structures found on a glycoprotein known as Syndecan-4 [63]. This interaction between AT and its cellular receptor limits signaling pathways such as NFkB nuclear translocation [64], attenuates cytokine production and chemotactic activity [65], and limits white cell-endothelial cell adherence [66]. These anti-inflammatory effects are markedly impaired in the presence of therapeutic or even prophylactic doses of heparin [63,66]. These interactions may explain the loss of activity observed with AT therapy in the KyberSept trial [54]. Future directions for antithrombin use in severe sepsis The ultimate clinical utility of AT therapy in severe sepsis is presently uncertain. The general strategy taken in the KyberSept trial is clearly not sufficient to define a patient population likely to benefit from AT therapy, and minimize bleeding risk at acceptably low levels in these critically ill patients. Heparin should be avoided during the administration of high-dose AT treatment. The use of continuous infusion strategies dosed in accordance to individual patient needs determined by AT blood levels is a reasonable approach. Hoffman and coworkers [67] recently published a randomized study comparing a 14-day AT infusion study versus standard care where they sought to maintain AT levels about a threshold value of 120% in severely septic patients. They showed significant improvements in a number of coagulation parameters in this study of 40 severely septic patients. A comparative study with AT versus heparin alone or AT versus activated protein C would also be a possible means to further investigate AT efficacy in a large clinical trial. Clinical trial design should incorporate methods to limit the study to those patients most likely to benefit (severely ill but not moribund patients, no concomitant heparin use, evidence of DIC or other measure of coagulation abnormalities). Another potential strategy is the use of recombinant human AT rather than plasma-derived AT. Recombinant human AT from transgenic animals has been developed and has similar activities to plasma-derived AT

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despite differences in glycosylation patterns and pharmacokinetic properties [68,69]. AT is likely to be used more intelligently in the future for selected patients with significant deficiencies in endogenous AT activity. In the future, the use of AT, or any other anticoagulant agent for sepsis, will need to be carefully considered in light of significant patient differences, and in terms of individual timing, dosing, treatment duration, and safety issues. Going Forward It is unclear from the current phase III studies whether AT and rTFPI actually provide clinical survival benefits. Both rTFPI and AT should be subjected to additional efficacy trials. In designing these trials careful attention must be focused on the dosing regimen. Safeguards must be put in place to prevent deterioration of study drug during the trial. Heparin use must be prohibited during the period of study drug administration. Continuous use of pneumatic compression stockings for deep venous thrombosis prophylaxis, and regional citrate for continuous renal replacement therapy, could be used in lieu of systemic heparin. Furthermore, a welldone, randomized, placebo-controlled study of heparin for severe sepsis should take place. A homogenous population of patients with severe sepsis due to community acquired pneumonia may provide the best target population for these drugs. The use of a clinical trial coordinating center that works cohesively with study sites and sponsor research physicians should be used if at all possible. A coordinating center provides the critical oversight to facilitate every opportunity for a successful study in this exceedingly difficult field of clinical investigation. In conclusion, the story of anticoagulant therapy for severe sepsis is not over. To paraphrase Winston Churchill, the current status of these anticoagulants for sepsis, ‘‘is not at the end, it’s not even the beginning of the end, but it’s the end of the beginning.’’

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