PROTEIN C AND SOLUBLE THROMBOMODULIN IN RELATION TO TUMOR NECROSIS FACTOR a IN CRITICALLY ILL SEPTIC PATIENTS AND THEIR CORRELATION WITH SOFA SCORE
The objective of this study was to investigate the influence of sepsis on protein C (PC) activity and soluble thrombomodulin (s TM) in relation to tumor necrosis factor a (TNF a) Also, to correlate these parameters with the SOFA score and serum lactate concentration as predictors of morbidity and mortality in septic patients. Thirty two adult patients (17 with sepsis and 15 with severe sepsis) in the intensive care unit (ICU) as well as 10 healthy ageand sexmatched controls were accrued to the study. The results showed that the baseline values of PC activity were significantly lower in both groups of septic patients compared to the controls, whereas, the serum levels of s TM, TNF a and lactate were significantly higher in the former compared to the latter. Moreover, survivors in both patient groups had higher PC activity and lower serum levels of s TM, TNF a and lactate compared to non survivors, both on day (1) and day (3) of ICU admission with variable statistical significance reflecting the heterogeneity of sepsis and the well known individual patient variation. In addition, day (3) samples showed significant increase in PC activity and reduction in serum levels of s TM, TNF a, lactate and SOFA scores in survivors and the reverse in non survivors compared to baseline levels. Also, baseline PC activity was correlated negatively with SOFA score but not with serum TNF a in both groups of septic patients. It was also negatively correlated with serum TM and lactate in sepsis but not in severe sepsis. Conclusion: PC activity and serum levels of s TM, TNF a and lactate in addition to SOFA score are good predictors of clinical outcome in critically ill patients with sepsis. INTRODUCTION Sepsis is defined as systemic inflammatory response syndrome (SIRS) in the presence of documented or suspected infection. Severe sepsis is defined as sepsis associated with organ dysfunction. Septic shock is a subcategory of severe sepsis in which the cardiovascular system is dysfunctional and is defined as arterial hypotension despite adequate fluid resuscitation. The pathogenesis of sepsis involves the activation of a number of host defense mechanisms including the cytokine network, leukocytes, complement and coagulation systems. In addition to activation of coagulation in sepsis, there is an attenuation of natural anticoagulant responses and impaired fibrinolysis. The resulting shift toward a procoagulant state results in excessive thrombin generation, fibrin formation, and consumption of clotting factors and inhibitors such as antithrombin III and proteins C and S. The protein C (PC) pathway provides a useful model for the impact of inflammation on coagulation. The key features of this pathway is its ability to respond to the presence of thrombin. As the thrombin concentration rises, it binds to thrombomodulin (TM) an endothelial surface glycolprotein, leading to both rapid thrombin inactivation and change in the substrate preference of thrombin from procoagulants such as fibrinogen and factor V to anticoagulants such as PC especially in the capillaries. An endothelial protein C receptor (EPCR) augments PC activation by the thrombin TM complex more than 10fold in vivo. Conversely, once thrombin generation reverts to homostatic levels, the PC activation complex slows the rate of generation of activated protein C (APC) down to the physiologic levels. APC was originally recognized as an anticoagulant that works up by proteolytic inactivation of factors Va and VIIIa. In addition, APC is profibrinolytic by inactivation of plasminogen activator inhibitor 1 (PAI 1) APC also possesses anti inflammatory properties. In experimental animals challenged with endotoxin, APC inhibits the release of Alexandria Journal of Anaesthesia and Intensive Care AJAIC Vol. (8) No. 3 Sept. 2005 74 proinflammatory cytokines such as tumor necrosis factor a (TNF a) and limits leukocyte extravasation to the tissues. However, this pathway is not static and could be impaired in a variety of clinically relevant situations including sepsis in which there is systemic endothelial dysfunction. Recent trials have shown that mortality can be reduced among patients with severe sepsis through the use of a new therapy that inhibits both the procoagulant and the inflammatory cascades. Intravenous infusion of antithrombin, PC/APC, or tissue factor pathway inhibitor (TFPI) can reduce the frequency of lethal responses in non human primates and other animals. Recombinant APC is the first biologic agent shown to reduce significantly the mortality in patients with severe sepsis. The prognosis of septic patients is related to the severity of organ dysfunction. Scoring systems such as the Sequential Organ Failure Assessment (SOFA) score are useful tools for assessing and quantifying organ dysfunction and failure over time. The SOFA is used to evaluate morbidity and is a good indicator of mortality in critically ill patients. The aim of this work was to investigate the influence of sepsis on the PC activity and s TM in relation to serum TNF a. Also, to correlate these parameters with the SOFA score and serum lactate concentrations as predictors of morbidity and mortality in these patients. This could help to clarify the position of PC/APC and other novel therapeutic modalities in the intensive care armamentarium. PATIENTS AND METHODS This study was carried out on 42 individuals divided into 3 groups: Group I: 10 healthy age and sex matched adult individuals as a control group, Group II: 17 adult patients (11 males and 6 females) with sepsis (SIRS infection) systemic reaction to infection defined by two or more of the following: (a) Temperature 38degC or 36degC, (b) Heart rate 90 beats/min, (c) Respiration rate 20 breaths/ min or PaCO2 32mmHg, (d) Leukocytes 12 x 10/L, 4 x 10, or 10 immature (band) forms. (1) Their ages ranged between 22 and 59 years with a mean of 46.88+ 14.59 years. Group III: 15 adult patients (10 males and 5 females) with severe sepsis: sepsis with new signs of organ dysfunction or decrease in organ perfusion (lactate acidosis> 3mmol/L, oliguria 0.5 ml/kg body weight for at least 1 hour or 30 ml/hour, hypotension 90 mmHg or a decrease of 40 mmHg) mental alteration) Their ages ranged between 28 and 58 years with a mean of 49.33+ 11.94 years. The patients were selected from those admitted to the Intensive Care Unit, Main Alexandria University Hospital, during the period from April 2005 to September 2005. Exclusion criteria included age 16 years or 60 years, pregnancy, inherited or acquired bleeding or throbmotic disorders, hematological or non hematological malignancies, significant renal or hepatic dysfunction, medications altering bleeding or coagulation profiles, severe trauma and recent (within 24 hours) transfusion of fresh frozen plasma, blood coagulation products or platelets. All patients were subjected to: 1Full history taking with stress on the onset of fever, bleeding or thrombotic manifestations, and symptoms of infection. 2Thorough clinical examination with emphasis on detection of signs of inflammation and evidence of bleeding or thrombosis. 3Routine laboratory investigations were done daily including renal, liver function tests, complete blood count, prothrombin time (PT) and Activated partial thromboplastin time (A PTT) Also, arterial blood gases were done every 12 hours. 4Investigations of the septic patients on admission to the ICU including urine analysis, chest X ray, cultures for bacteria and fungi, CT scans and/or MRI, when indicated. 5Colorimetric assay for determination of serum lactate was done daily for the first 3 days after diagnosis using (Lactate PAP Fluid, Centronic GmbH. Germany) 6Upon diagnosis and after 48 hours the following tests were done:Alexandria Journal of Anaesthesia and Intensive Care AJAIC Vol. (8) No. 3 Sept. 2005 75 aMeasurement of protein C activity using (protein C reagent, Dade Behring/ Maburg. USA) bEstimation of serum thrombomodulin using (CD141 ELISA KIT, Diaclone. France) cEstimation of serum TNF a using (TNF a ELISA IM1121, IM1121, Beckman CoulterTM. France) 7Assessment of SOFA score was done daily for the first three days after diagnosis according to the criteria proposed by Ferreria et al. Samples collection and storage Plasma samples were obtained by mixing one part of sodium citrate (3.8% with 9 parts of venous blood and centrifuged immediately at approximately 3000 rpm for at least 10 minutes, the resulting plasma supernatent is used immediately for the estimation of PT and APTT while the remaining part stored in aliquots at 20degC until used for determineation of PC activity. Serum sample was obtained by centrifugation of clotted blood at approximately 3000 rpm for 15 minutes, then it was removed rapidly and stored in aliquots at 20degC until used for estimation of TNF a, s TM and lactate. STATISTICAL ANALYSIS Data were analyzed using SPSS V 10. RESULTS Table (1) illustrates the clinical data in the studied septic patients while Table (2) shows the statistical comparison between the studied parameters in controls and septic patients on day (1) The baseline values of PC activity were significantly lower in both groups of septic patients compared to control group. Whereas the serum levels of s TM, TNF a and lactate were significantly higher in the former compared to the latter. Moreover, higher PC activity and lower serum levels of s TM, TNF a and lactate were observed in sepsis compared to severe sepsis but the difference was significant for all parameters except TNF a. Table (3) demonstrates statistical comparison between survivors and non survivors in both groups of septic patients. On day (1) the mean values of PC activity were significantly higher in survivors than non survivors in sepsis (p 0.013) but not in severe sepsis. While on day (3) the difference was significant in both groups of patients (p 0.001 and 0.002 for sepsis and severe sepsis respectively