Upregulated but insufficient generation of activated protein C is associated with development of multiorgan failure in severe acute pancreatitis

Acute pancreatitis (AP) is a common disease with widely variable clinical outcome. Twenty-five per cent of patients suffer from the severe form of the disease with local and/or systemic complications, resulting in a mortality rate ranging from 2 to 10% [1]. Increased morbidity and mortality are associated with organ failure in 50% of severe AP cases [2].

Systemic inflammatory reaction and the development of organ failure in AP share similarities with complicated courses of sepsis, major trauma, and burns [3]. In systemic inflammation, excessive proinflammatory burst is rapidly followed by an anti-inflammatory reaction that may result in immune suppression [4]. Likewise, rapid activation of coagulation may turn into global or selected exhaustion of physiological anticoagulant systems. In sepsis, for example, coagulation, inflammation, and apoptosis all contribute to organ dysfunction and permanent damage. The interactions between coagulation and inflammatory pathways are essential in the pathogenesis of disseminated intravascular coagulation. For example, the proinflammatory cytokines tumour necrosis factor alpha, IL-1, and IL-6 upregulate thrombin formation and downregulate physiological antithrombotic defence mechanisms, especially the protein C (PC) pathway [5].

The PC pathway is both a major physiological anticoagulant system and a central link between inflammation and coagulation. The zymogen PC is converted to an active serine protease activated protein C (APC) by thrombin bound to thrombomodulin on the endothelial surface [6]. This effect is enhanced by the endothelial PC receptor [7]. APC conveys its anticoagulant function mainly by proteolytic inactivation of coagulation activated factor V and activated factor VIII. APC also exhibits distinct anti-inflammatory and anti-apoptotic properties [8‐11]. While the underlying mechanism remains incompletely understood, recombinant APC decreased the levels of IL-6 and D-dimer and reduced mortality in severe sepsis patients [12].

Few studies have explored systematically haemostatic disturbances during AP [13‐15]. An increase in plasma-soluble thrombomodulin predicted a lethal course of AP [16]. No data on APC in AP patients have been published. Based on the central role of the PC pathway in the acute systemic inflammatory response and the availability of two therapeutic approaches, zymogen PC concentrate [17] and recombinant APC [12], we decided to evaluate how the PC pathway evolves during the course of severe AP. In the current study we tested the hypothesis that a failure of the PC pathway homeostasis might be involved in the development of organ failure in AP patients.

The current observational study of the course of PC and APC levels during AP in 31 patients with a 42% incidence of MOF was conducted to address how often and to what extent the PC pathway would be disturbed and whether such perturbations would be associated with the development of MOF. The study setting inevitably resulted in limitations in interpreting the results in terms of causality. For example, the patients entered the university hospital at various stages of AP, some having MOF developed prior to the admission. Also, despite our continuous efforts during the two years of patient recruitment, the sampling schedule in the original protocol was not completely fulfilled. The patient series was rather uniform in disease severity and the incidence of MOF approached 50%, however, giving us the opportunity to test the hypothesis of whether a defective PC pathway would be detrimental to patients with AP.

The minimum levels of APC in MOF patients were significantly lower than in controls. The PC deficiency was therefore logically associated with a decrease of APC levels in patients with MOF. A more significant finding, however, may be that the APC levels seemed not to be grossly elevated. In fact, only one sample in one patient showed an absolute APC level that exceeded the reported upper limit of normality (200%) [20]. Thrombin is the APC activator, and APC is a feedback inhibitor for thrombin generation [22, 23]. The complicated balance between thrombin, PC, and APC varies depending on the circumstances. In resting healthy adults, the PC level rather than the thrombin level may be the major determinant of the circulating APC level [20, 24, 25]. During cardiopulmonary bypass, upon rapid thrombin generation during reperfusion, the APC/PC correlation was lost and a significant positive correlation between fibrinopetide A, a thrombin marker, and APC developed [24].

The upper limit for APC formation may be estimated from APC determinations in various clinical settings where coagulation is known to be activated. In our previous studies the most pronounced and fast enhancements from normal resting APC levels to levels typically ranging from 250% to over 800% of the normal mean were observed during the first minutes of reperfusion in liver transplantation [26] and during an infusion of antithymocyte globulin, a strong proinflammatory stimulus, in renal transplantation [27]. Liaw and colleagues recently reported that in acute sepsis patients, despite ongoing activation of coagulation, 25% of patients failed to increase their APC levels above 250% (while 75% had levels ranging from approximately 250 to 800%) [28]. They concluded that the septic patients varied markedly in their ability to generate APC in response to the physiological thrombin stimulus, and attributed the phenomenon to endothelial dysfunction [28]. In children with meningococceal sepsis, on the other hand, patients' ability to generate APC in response to thrombin did not seem to be grossly affected as infusion of PC resulted in elevated levels of APC, and significant correlations were observed between APC and thrombin markers at baseline [17].

Even though D-dimer is not a direct thrombin marker, the highly elevated D-dimer levels in the current study indicated that ample thrombin formation occurred throughout the observation period. Therefore, in good accordance with the study by Liaw and colleagues in adult septic patients [28], we assume that the lack of elevated free APC levels in the presence of activated coagulation in patients with AP in most cases reflected dysfunctional PC activation on the endothelium, or possibly enhanced inhibition of APC by plasma protease inhibitors. This suggestion, however, does not exclude the possibility that significant PC deficiency could also be rate limiting for APC formation in patients with severe AP, as seems to be the case in septic children [17].

The PC levels were subnormal in 92% of patients with MOF and in 44% of controls. Low PC was found to precede MOF, and early APC/PC ratios were frequently high in MOF patients. In an animal model of AP, the PC level was found to decline already one hour after initiation of AP [29]. The PC levels often fall rapidly in sepsis patients [30, 31], and this may associate with the development of organ failure [32]. Thus, in logical accordance with animal AP data and human sepsis studies, early PC deficiency was a most frequent finding during severe AP. While the causal relationship between low PC levels and MOF development cannot be proven in an observational study setting, one probable bias could be excluded. Namely, there was no indication that PC deficiency would associate with MOF after its diagnosis and, further, there was a significant trend for improvement of PC homeostasis during the course of MOF. It thus seems that, in patients developing MOF, the PC activation system reached its limits prior to the diagnosis of MOF, resulting in secondary deficiency of PC concomitantly with a failure to maintain steady APC levels similar to those observed in AP patients without MOF.

APC and PC correlated significantly with monocyte HLA-DR expression but failed to associate with the level of D-dimer. The existence of anti-inflammatory and anti-apoptotic properties of APC is currently widely accepted [22, 33]. Compared with the accumulating in vitro and animal data, however, observational human clinical studies provide scarce data on the specific relationship between APC and inflammation. The obvious limitation is the fact that thrombin formation is also enhanced whenever APC generation is enhanced, and the question remains whether APC or other components of the activated coagulation pathways might be involved.

The concomitant decrease in D-dimer and IL-6 during APC infusion in the PROWESS trial has been taken as circumstantial evidence of specific anti-inflammatory action of APC [12]. The remaining human studies finding associations between APC and inflammatory phenomena have involved only hyperacute proinflammatory/procoagulant situations such as reperfusion after cardiopulmonary bypass [34], liver transplantation [26], and renal transplantation [27]. We chose to measure HLA-DR%, which reflects the recent history of activation/functional suppression of the circulating monocyte population [21]. APC acts as an anti-inflammatory agent in vitro, largely through modulating monocyte activation during inflammation [9‐11]. The current correlations between HLA-DR expression and the PC and APC levels may therefore support the concept of interaction between inflammation and the PC pathway. In severe AP, levels of PC and APC were moderately associated with monocyte activation status.

The current study suggests that testing the therapeutic use of APC or PC to improve patient outcome might be feasible in AP. The concurrent activation of inflammation and coagulation during AP, the frequent occurrence of PC pathway defects in AP patients, their association with a significant clinical endpoint (MOF), and the timing of major PC defects to the early phase of the disease all lend support to attempt APC or PC infusion in severe AP.

In summary, the current study demonstrated significant PC pathway pathology in severe AP. The PC pathway defects were more frequent in patients developing MOF.