Figure 3.

Typical Western blotting analyses of plasma samples to evaluate the time-dependent changes in extracellular processed VS-I (top panel), and simultaneous hemodynamic profile (bottom panel) during a human septic shock. Time (Day X) runs over 3 days from admission (Day 1, hour 10:00 pm (H22)) to the end of the third day (Day 3, 02.00 pm (H14) and is represented on the X-axis (top and bottom lines). Plasma samples harvested by 8 h, from admission until Day 3, were immediately centrifugated (4000 rounds/min at 4 °C), and SDS-PAGE electrophoresis followed by electrophoretic blotting with immunological detection of VS-I was performed in standard conditions. The anti-VS-I antibodies were a generous gift of Pr A. Corti, Milan, Italy. Periods of norepinephrine and dobutamine infusion are represented as bold arrows from admission until weaning. Please note that the processing of chromogranin A is notably changing around Day 2 at 2:00 (H14) when the patient’s circulatory status no longer requires the two vasopressors. The global immunoblots are decreasing in intensity, and both small and large molecule multimerization is decaying (top panel). This phenomenon is contemporary to the decrease in doses of vasopressors (norepinephrine and dobutamine) and corresponds to circulatory failure recovery. Altogether, these data explain (i) why dosages of any of these proteins must be performed at a similar time window of the disease if a proper interpretation of their role is to be considered; (ii) that a pharmacological intervention must be scheduled at a moment when it can be efficient. Thus, our data on 4% albumin-impact on multimerization show that such an intervention must start as early as possible after admission, and it has no sense after the 5th day of disease onset [16]. Please remember that the apparent molecular weight (MW) of full-length CGA is around 70–75 kDa, and that of VS-I is approximately 18 kDa, which explains the immunoblotting of multimers on the top panel. Boxes (solid line, dashed line…) focus on processed molecules of interest, tagged with VS-I-antibodies: a careful identification must specify whether some multimers are not just large monomeric, full-length CGA molecules containing the VS-I domain.