In vitro norepinephrine significantly activates isolated platelets from healthy volunteers and critically ill patients following severe traumatic brain injury

Abstract

Introduction: Norepinephrine, regularly used to increase systemic arterial blood pressure and thus improve cerebral perfusion following severe traumatic brain injury (TBI), may activate platelets. This, in turn, could promote microthrombosis formation and induce additional brain damage.

Methods: The objective of this study was to investigate the influence of norepinephrine on platelets isolated from healthy volunteers and TBI patients during the first two post-traumatic weeks. A total of 18 female and 18 male healthy volunteers of different age groups were recruited, while 11 critically ill TBI patients admitted consecutively to our intensive care unit were studied. Arterial and jugular venous platelets were isolated from norepinephrine-receiving TBI patients; peripheral venous platelets were studied in healthy volunteers. Concentration-dependent functional alterations of isolated platelets were analyzed by flow cytometry, assessing changes in surface P-selectin expression and platelet-derived microparticles before and after in vitro stimulation with norepinephrine ranging from 10 nM to 100 microM. The thrombin receptor-activating peptide (TRAP) served as a positive control.

Results: During the first week following TBI, norepinephrine-mediated stimulation of isolated platelets was significantly reduced compared with volunteers (control). In the second week, the number of P-selectin- and microparticle-positive platelets was significantly decreased by 60% compared with the first week and compared with volunteers. This, however, was associated with a significantly increased susceptibility to norepinephrine-mediated stimulation, exceeding changes observed in volunteers and TBI patients during the first week. This pronounced norepinephrine-induced responsiveness coincided with increased arterio-jugular venous difference in platelets, reflecting intracerebral adherence and signs of cerebral deterioration reflected by elevated intracranial pressure and reduced jugular venous oxygen saturation.

Conclusion: Clinically infused norepinephrine might influence platelets, possibly promoting microthrombosis formation. In vitro stimulation revealed a concentration- and time-dependent differential level of norepinephrine-mediated platelet activation, possibly reflecting changes in receptor expression and function. Whether norepinephrine should be avoided in the second post-traumatic week and whether norepinephrine-stimulated platelets might induce additional brain damage warrant further investigations.

Figures

Figure 1

Effect of norepinephrine and thrombin receptor-activating peptide (TRAP) on surface expression of P-selectin in platelets isolated from healthy controls. Norepinephrine, in a concentration-dependent manner, increased the number of P-selectin-positive platelets, which was significant only at norepinephrine concentrations of greater than or equal to 10 μM. Maximal increase was induced with TRAP. +P <0.001 TRAP versus norepinephrine; * P <0.001 norepinephrine of 10 and 100 μM versus norepinephrine of less than 10 μM; analysis of variance on ranks.

Figure 2

Significant concentration-dependent influence of norepinephrine and thrombin receptor-activating peptide (TRAP) on platelet microparticles isolated from healthy controls. This effect was significant only at norepinephrine concentrations of greater than or equal to 10 μM with a maximal increase induced with TRAP. +P <0.001 TRAP versus norepinephrine; * P <0.001 norepinephrine of 10 and 100 μM versus norepinephrine of less than 10 μM; analysis of variance on ranks.

Figure 3

Changes in expression of surface P-selectin in platelets isolated from severe traumatic brain injury patients compared with healthy controls. The relative number of P-selectin-positive arterial and jugular venous platelets was significantly decreased during the second week. * P <0.05 versus controls and first week; analysis of variance on ranks.

Figure 4

Relative increases in norepinephrine-induced expression of P-selectin in arterial (black bars) and jugular venous (grey bars) platelets isolated from severe traumatic brain injury (TBI) patients and peripheral venous platelets taken from healthy controls (white bars) expressed as a percentage of baseline values. Baseline values were determined in platelets not stimulated in vitro with norepinephrine. During the first week, the norepinephrine-mediated increase in P-selectin-positive platelets was significantly reduced compared with controls. In the second week, the norepinephrine-mediated increase in P-selectin expression significantly exceeded changes seen in the first week and in healthy volunteers. Overall, there was no significant difference between arterial and jugular venous platelets. During the second week, the TRAP-mediated increase in P-selectin-positive platelets significantly exceeded the TRAP-induced activation observed during the first week. #P <0.001 second week versus first week; +P <0.01 patients versus controls; * P <0.01 norepinephrine of greater than 500 nM versus norepinephrine of less than 10 μM. TRAP, thrombin receptor-activating peptide.