Plasma concentrations of Gas6 (growth arrest specific protein 6) and its soluble tyrosine kinase receptor sAxl in sepsis and systemic inflammatory response syndromes

Blood samples for the analyses of plasma proteins and lactate were collected from subjects at enrollment in the study in 5 ml plastic vacutainer tubes containing 0.5 ml 0.129 mol/l sodium citrate, as previously described [16]. The Gas6 [17] and sAxl [11] ELISAs have been described earlier. In short, maxisorb (Nunc) plates were coated with a catching polyclonal antibody before blocking with 3% fish gelatin in 50 mM Tris-HCL, 150 mM NaCl, pH 7.4 with 0.1% Tween 20. The samples were diluted in the blocking buffer and incubated overnight before washing and detection with a biotinylated secondary antibody. The signal was amplified using ABC/HRP (Dako, Glostrup, Denmark) and visualized with 1,2-phenylenediamine dihydrochloride and hydrogen peroxide. Sulphuric acid was added to stop the reaction before measuring the absorbance at 490 nm. The absorbance of the samples was compared to a standard curve prepared by a dilution series with known amounts of the respective protein.

The study cohort has previously been described in detail [16]. Briefly, 232 patients were enrolled in a prospective study at the Clinic for Infectious Diseases, University Hospital, Lund, Sweden. The inclusion criteria were fever (≥38°C) and a suspected infection. Only adults (≥18 years of age) were included. The blood sampling was performed within 12 hours after admission to the hospital. The ethics committee of Lund University approved the project protocol, and informed consent was obtained from all patients or their close relatives.

Based on the presence of SIRS criteria (body temperature ≥38°C, WBC >12 × 10⁹/l or <4 × 10⁹/l, pulse rate >90/minute and respiratory rate >20/minute [13]), or a significant hypotension (a systolic blood pressure of <90 mmHg or a fall of >40 mmHg from baseline), presence or absence of organ failure, and the final diagnosis, the patients were categorized into various groups. The criteria were those proposed by the American College of Chest Physicians/Society of Critical Care Medicine [13].

The patients were divided into the following groups: Severe Sepsis, Sepsis, Infection and SIRS. Severe sepsis was defined as an infectious disease, at least two SIRS criteria, and the presence or development of hypotension and/or organ failure within 24 h of the collection of the blood samples. Sepsis was defined as an infectious disease, at least two SIRS criteria, but no presence or development of organ failure. Infection was defined as an infectious disease without SIRS. SIRS was defined as a non-infectious disease with at least two SIRS criteria. Renal failure was defined using the RIFLE criteria [18].

Two hundred and thirty-two patients were included. Seventy patients were diagnosed with severe sepsis, 99 patients with sepsis, 43 patients with infection without SIRS, and 20 patients with SIRS without infection. Detailed patient demographic data and diagnoses have been presented elsewhere [16]. Pneumonia and urinary tract infections were common and also overrepresented in the severe sepsis and the sepsis groups. Infected patients without SIRS suffered mostly from upper respiratory infections. The 20 patients with non-infectious SIRS suffered from various diseases such as vasculitis, cardiac failure, gastrointestinal bleeding, pulmonary embolism and pancreatitis. The over-all mortality rate was 3.4%. In the severe sepsis group, the mortality rate was 10%, and out of the 26 patients with septic shock, 19% died.

The plasma concentrations of Gas6 and sAxl were determined in the acutely ill patients who were found to suffer from severe sepsis, sepsis, infections without SIRS, or SIRS without infection (Figure 1). When compared to the controls, all patient groups had significantly increased plasma concentrations of Gas6, the median Gas6 concentration being 0.58, 0.50, 0.48 and 0.52 nM for the patient groups and 0.25 nM for the controls. The patients with severe sepsis had significantly increased Gas6 concentrations when compared to the sepsis group (Figure 1a). The median plasma sAxl concentrations were 1.19, 1.00, 1.14, 1.29 and 0.99 nM, respectively. There were also statistical differences in sAxl between the controls and the patient groups, but they were not close to the significance levels observed for Gas6. There were several individuals with very high sAxl concentrations in the patient groups, but the differences between the groups were less pronounced than what was found for Gas6 (Figure 1b).

Both Gas6 and sAxl concentrations correlated with previously measured analytes [16]. When combining all the 232 samples in one group, the concentration of Gas6 correlated with those of IL-6, procalcitonin and number of failing organs. Also sAxl concentrations correlated to Gas6, procalcitonin and number of failing organs (Table 1).

When evaluating the severe sepsis and sepsis groups separately, Gas6 correlated to IL-6, bilirubin, INR, procalcitonin and number of failing organs. sAxl correlated to bilirubin, Gas6, number of organs in failure and inversely to C-reactive protein (Table 2).

Gas6 was higher in patients with organ failure, kidney failure and in patients receiving intensive care, whereas sAxl was higher in patients with organ failure. There was a non-significant trend towards higher Gas6 in patients that did not survive (Table 3).