Activated Protein C has No Effect on Pulmonary Capillary Endothelial Function in Septic Patients with Acute Respiratory Distress Syndrome: Association of Endothelial Dysfunction with Mortality

The study was approved by the Evangelismos Hospital Research Ethics Committee (94/9-9-2003). All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964, also as revised in 2013. Ιnformed written consent was obtained from all patients’ next-of-kin prior to any study procedure. Our study was conducted and completed in 2004, before the International Committee of Medical Journal Editors initiated the policy requiring investigators to deposit information about trial designs. Consequently, our study was not registered.

In 2004, 19 critically-ill septic patients with ARDS treated with rhAPC were enrolled in the study. All subjects were hospitalized in a general intensive care unit (ICU), were mechanically ventilated, and had at least two organ dysfunctions consequent to infection, one of which was sepsis-associated respiratory dysfunction. All patients suffered from severe sepsis or septic shock and, furthermore, all patients had ARDS except three who had acute lung injury, according to the respective definitions at the time of the study [10, 11]; based on the current definitions, they are all considered to have suffered from sepsis or septic shock and ARDS [12, 13]. The decision to treat with rhAPC was taken by the patients’ attending physicians, in compliance with the administration criteria valid at that time [8]. They all received rhAPC at 24 μg/kg/h for 96 h in a continuous IV drip. All the precautions were taken regarding the risk of bleeding before and after major or minor surgical procedures. Major bleeding events were not noticed. After the completion of the study, patients were followed up and complete clinical and laboratory data were recorded for 28 days or until their discharge from the ICU. At the end of the study, the subjects were grouped according to their outcome (28 days survival or prior discharge from the ICU) into survivors and non-survivors.

PCEB-ACE activity was measured prior to initiation of rhAPC treatment (measurement #1 at baseline), during the 96-h infusion (measurement #2 at 24 h; measurement #3 at 48 h; measurement #4 at 96 h) and 3 days after the end of the rhAPC infusion (measurement #5, day 7). No patient was under ACE inhibitors. Each patient’s baseline measurement served as control of him/herself.

A detailed description of the methodology used is given elsewhere [2]. Trace amounts of the radiolabelled synthetic ACE substrate ³[H] benzoyl-Phe-Ala-Pro (BPAP) were injected as a rapid bolus into a (jugular or subclavian) central vein, and arterial blood was withdrawn into a fraction collector for analysis.

Single transpulmonary pass substrate utilization is expressed as percent metabolism (%M) and transpulmonary hydrolysis (v = [E] × t_c × k_cat/K_m), where [E] equals the enzyme concentration available for reaction, t_c the capillary transit time, k_cat the catalytic rate constant, and K_m the Michaelis–Menten constant. Functional capillary surface area (FCSA) was additionally calculated as hydrolysis × pulmonary plasma flow and depends on the total enzyme mass that is available for reaction, and the constants k_cat and K_m. Both v and %M are indices that reflect ACE activity per capillary, while FCSA is a reflection of ACE activity per vascular bed [1, 2, 14].

Data are presented as mean ± SE, or median (interquartile range) when data distribution was skewed. Comparisons between groups were made by Student’s t test or Mann–Whitney test, when appropriate, for continuous data, and Chi square test for qualitative data. Differences within time were examined by the Friedman test. Mixed effect models were fitted to examine the effect of time, outcome and their interaction. The Tukey–Kramer test was used for multiple comparisons adjustment. All p values are two-sided. Differences were considered significant at p < 0.05.

Patient baseline demographic and clinical data are shown in Table 1. Patients were grouped according to their clinical outcome (28 days survival) as survivors (n = 9) and non-survivors (n = 10). There were no statistically significant differences between the two groups in baseline characteristics.

In all patients, BPAP  %M did not significantly change during the study period: from 26.4 ± 2.6% (baseline) to 24.4 ± 2.9% at the end of the 96-h rhAPC infusion, and to 22.5 ± 2.8 at the end of the 7 days study (Fig. 1a; p = 0.541). In a similar pattern a non-significant fluctuation of hydrolysis (v) was observed in all patients from 0.32 ± 0.04 at baseline to 0.29 ± 0.04 at day 4 and to 0.27 ± 0.04 at day 7 (Fig. 1b; p = 0.641). FCSA at baseline was 1550 ± 212 ml/min, while similar values were obtained at day 4 (1549 ± 276 ml/min) and at day 7 (1561 ± 292 ml/min) (Fig. 1c; p = 0.652).

Neither %M nor hydrolysis changed significantly with time in either group. In survivors, BPAP %M tended to decrease from 32.7 ± 3.4% at baseline to 25.6 ± 2.9% at day 7, and in non-survivors from 20.8 ± 2.8% at baseline to 15.5 ± 5% at day 7. Likewise, v tended to decrease from 0.41 ± 0.06 at baseline to 0.30 ± 0.04 at day 7 in survivors, and 0.24 ± 0.04 to 0.18 ± 0.06 in non-survivors (Fig. 2a, b; p = 0.489 and p = 0.499, respectively). Nonetheless, ACE substrate utilization was significantly lower in non-survivors as compared to survivors (Fig. 2a, b; p = 0.044 and p = 0.049).

FCSA increased slightly, from 1760 ± 327 ml/min at baseline to 1890 ± 338 ml/min at day 7 in survivors, while in the non-survivors FCSA dropped from 1362 ± 276 to 821 ± 402 ml/min. It tended to be higher in survivors, without, however, reaching significance (Fig. 2c, p = 0.075). It should be noted that the obtained values might be influenced by differences in cardiac output per patient, observed during the study (data not shown), since they affect FCSA measurements (see “Methods”).

Patients’ PO₂/FiO₂ did not significantly change with time (Fig. 3a; p = 0.458). However, when patients were divided into survivors and non-survivors, the former revealed significantly higher values than the latter (Fig. 3b; p = 0.032). Similar patterns were observed for lung injury scores (LIS) (Fig. 3c; p = 0.726). Again, survivors had significantly lower LIS than non-survivors (Fig. 3d; p = 0.025). SOFA scores decreased with time in all patients (Fig. 4a; p = 0.007). The same pattern (i.e. decrease with time) was observed in both survivors and non-survivors, and were lower in survivors than in non-survivors (Fig. 4b; p = 0.001 and p = 0.04, respectively). Patients’ APACHE II scores also decreased with time in all patients (Fig. 4c; p = 0.033). The same pattern (i.e. decrease with time) was observed in both survivors and non-survivors (Fig. 4d; p = 0.045). However no differences were noted between the two groups (Fig. 4d; p = 0.189).

It is worth mentioning that PCEB-ACE activity indices were the only significantly different parameters between survivors and non-survivors at baseline; no other disease or lung injury severity score was significantly different at baseline (data not shown). More specifically, in survivors, BPAP %M at baseline was 32.7 ± 3.4% compared to 20.8 ± 2.8% in non-survivors (Mann–Whitney, p = 0.017). Baseline hydrolysis (v) was 0.41 ± 0.06 in survivors and 0.24 ± 0.04 in non-survivors (Mann–Whitney, p = 0.017).