Abstract
Abstract
Background:
Platelets are the key factor in primary hemostasis. It has been shown that chronic altitude exposure increases platelets' aggregability. Nevertheless, data about acute effects and the underlying mechanisms are sparse.
Methods:
Sixteen healthy volunteers were examined in our hospital (519 m alt.) and 30 minutes after arrival in the environmental research station on the Zugspitze Mountain (2656 m alt.). Serum levels of soluble p-selectin were examined to elucidate platelet activation. In addition, serum levels of chromogranin A (CGA) as a measure of adrenergic activation, endothelin 1 (ET-1) representing pulmonary vascular tone and monocyte chemoattractant protein-1 (MCP-1) as a measure of inflammatory response were examined.
Results:
Acute altitude exposure induced a significant increase of p-selectin (116 ± 4.8 pg/mL vs. 132 ± 6.2 pg/mL; p < 0.01). Whereas MCP-1 was significantly lowered (538 ± 50.6 pg/mL to 470 ± 41.1 pg/mL; p = 0.02) and CGA was not altered significantly (88 ± 47.4 ng/mL vs. 79 ± 44 ng/mL; p = 0.22), ET1 was increased significantly from 0.8 ± 0.07 pg/mL to 1.15 ± 0.09 pg/mL (p < 0.01).
Conclusion:
Our study could demonstrate relevant platelet activation that was accompanied by a 44% increase of ET-1. This activation might obtain clinical relevance in patients with pre-existing cardiovascular disease as a trigger for acute events.
Background
Platelet aggregation plays a crucial role in primary hemostasis. Nevertheless, a large variety of influences can cause platelet activation also in the absence of required hemostasis. Platelet activation can be caused not only by metabolic changes or inflammation, but also by rheological changes or increased vascular resistance. Among others, different kinds of stress are able to induce platelet activation. Stress reaction is made by two hormonal axes. The acute stress reaction is implemented by the hypothalamic–adrenal medulla axis. The key effectors of this axis are the catecholamines adrenaline and noradrenalin. The more chronic reaction follows the hypothalamic–hypophysis adrenal cortex route. Key effectors of stress are corticotropin-releasing hormone and cortisol. Especially activation of the acute stress system causes a relevant activation of platelet function (Nickel et al., 2016).
Stress cannot be objectified directly, but several substances in the blood (besides the actors of the earlier described stress axes) that have shown to be altered in the context of acute stress can serve as surrogate parameters for stress and have been described previously (Wilbert-Lampen et al., 2010).
p-Selectin is an integrin stored in vesicles of platelets. After platelet activation p-selectin becomes membrane-bound and promotes platelet aggregation to endothelium through platelet–fibrin and platelet–platelet binding. Besides, p-selectin is of importance for leucocyte activation at the site of vascular injury. After platelet activation p-selectin is rapidly shed into the blood (Au and Josefsson, 2017). Therefore, soluble p-selectin serves as a marker of platelet activation (Andre, 2004).
Acute exposure to high altitude represents a form of environmental stress and might cause platelet activation. Thus, in this pilot study we intended to elucidate the effect of acute altitude exposure on platelet activation in young and healthy subjects with respect to a potential underlying pathophysiological role of the three following stress surrogate markers:
Endothelin-1 (ET-1) is produced by endothelial cells, smooth muscle cells, monocytes, and macrophages after stimulation by catecholamines, cortisol, hypoxia, or interleukins. The effect of ET-1 on the pulmonary vessels is vasoconstriction not only in pulmonary disease, but also in healthy subjects as a result of acute hypoxia (Kylhammar and Radegran, 2017). We hypothesize that this vasoconstriction could cause an activation of platelets due to the concomitant rheological changes. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine synthesized mainly in monocytes and macrophages. Several factors affect synthesis of MCP-1, for example, tumor necrosis factor α, interferon-γ, and angiotensin that are inducing synthesis of MCP-1 through nuclear factor kappa-light-chain-enhancer of activated B cells. Thereby, MCP-1 may serve as a marker for inflammatory stress reaction (Melgarejo et al., 2009). In our survey MCP-1 is used as an inaccurate surrogate for acute inflammatory response. Chromogranin A (CGA) is an essential part of secretory vesicle in endocrine cells, neurons, and neuroendocrine cells. As a part of secretory vesicles CGA is released with the content of the vesicle. There is evidence that increased levels of CGA are associated with physical stress (Wilbert-Lampen et al., 2010; Nickel et al., 2016) and may serve as a marker for catecholaminergic activation within the scope of acute stress reaction.
Methods
Study population
Sixteen healthy volunteers with age >18 years and without any relevant (in particular cardiac or pulmonary) disease were examined. Written informed consent was obtained from all volunteers before enrolment. The study protocol was approved by the local ethics committee.
Blood was taken from an antecubital vein in our hospital (519 m alt.). Afterward all individuals were transferred to Garmisch-Partenkirchen by car and to the Environmental Research Station Schneefernerhaus (UFS) at the Zugspitze Mountain (2650 m alt.) by cable car. Blood was taken 30 minutes after arrival by a new puncture of an antecubital vein. Immediate centrifugation and freezing of serum specimens was performed on site. In addition, peripheral transcutaneous oxygen saturation was measured in our hospital as well as at the UFS.
Measurement of platelet activation and stress parameters
Serum levels of soluble p-selectin, CGA (as a measure of adrenergic activation), ET-1 (representing pulmonary vascular tone), and MCP-1 (as a measure of inflammatory response) were measured with a standard ELISA kit according to the manufacturer's instruction after the transfer of the frozen specimens from the UFS (CGA; Antikoerper Online, Germany; p-selectin, ET-1 and MCP-1; Biocat, Germany).
Statistical analysis
Results and patient characteristics are expressed in mean ± standard error of mean. Data were evaluated for normal distribution by the Anderson–Darling test. For data with normal distribution paired Student's t-test was used for statistical testing. Values of p < 0.05 were considered statistically significant.
Results
Baseline characteristics and oxygen saturation are depicted in Table 1. Participants had a mean age of 37.2 years. Acute exposure to altitude led to a significant decrease of oxygen saturation compared with baseline measurement in our hospital (91.4 ± 0.6% vs. 97.0 ± 0.2%; p < 0.05).
Characteristics
SpO2, oxygen saturation as measured by pulse oximetry.
This decrease of oxygen saturation was accompanied by a significant activation of platelets indicated by an increase of soluble p-selectin (116 ± 4.8 ng/mL vs. 132 ± 6.2 ng/mL; p < 0.01; Fig. 1A). CGA did not show a significant alteration (88 ± 47.4 ng/mL vs. 79 ± 44 ng/mL; p = 0.22; Fig. 1B). MCP-1 decreased from 538 ± 50.6 pg/mL to 470 ± 41.1 pg/mL (p = 0.02, Fig. 1C), whereas ET-1 significantly increased from 0.8 ± 0.07 pg/mL to 1.15 ± 0.09 pg/mL (p < 0.01, Fig. 1D).
Levels of soluble p-selectin
Discussion
To the best of our knowledge this is the first study to elucidate the effect of acute altitude exposure on platelet activation with respect to the underlying pathophysiological mechanisms in young healthy individuals.
Our major finding was a significant increase of soluble p-selectin as a marker of platelet activation. This activation was accompanied by an increase of ET-1, whereas levels of MCP-1 and CGA did not show an increase.
The effect of altitude exposure on platelet function has been investigated previously. Chronic altitude exposure can result in an increase of platelet counts as well as platelet reactivity leading to a prothrombotic phenotype after several days (Rocke et al., 2018). In accordance Tyagi et al. (2014) found a decrease in bleeding time and increase of platelet aggregability after incubating rats for a much shorter time of only 6 hours in a decompression chamber with conditions equivalent to an altitude of 7620 m. In contrast, Toff et al. (2006) did not see any changes of platelet activation (soluble p-selectin) after a hypobaric chamber simulation of an 8 hour long-haul air travel in 73 healthy volunteers.
Nevertheless, a prothrombotic phenotype after long-term altitude exposure can be of clinical relevance as Isik et al. (2013) could demonstrate that patients after ST-elevating myocardial infarction living at an altitude of 1960 m had a higher rate of reinfarction compared with patients living at sea level.
The instant effects of acute altitude exposure, for example, as part of civil aviation, affect a large population. Unfortunately, no comparable data about these instant effects of altitude exposure on platelet function, such as our 14% increase of soluble p-selectin after 30 minute exposure, are available.
Our current study also tried to give a hint for possible mechanisms for platelet activation after acute altitude exposure. We hypothesized that acute altitude exposure will cause an acute physiological stress reaction, mediated predominantly by catecholamine liberation. As a surrogate for catecholamine liberation CGA was found without significant alteration. This could be explained by the fact that acute altitude exposure did not cause a relevant acute stress reaction in our young and healthy volunteers.
As a second possible mechanism of platelet activation MCP-1 was examined as a measure of inflammatory stress reaction. Hartmann et al. (2000) described pronounced alteration of inflammatory mediators after high altitude exposure of healthy volunteers for several days. Nevertheless, the lack of an increase of MCP-1 in our study suggests inflammatory mediators of platelet activation are not involved, at least after this very short exposure (Hartmann et al., 2000).
In our current survey, platelet activation was accompanied by a 44% increase of ET1. Speculating about the way how ET-1 activates platelets two possible mechanisms are thinkable. Our finding could be explained by platelet-activating properties of ET-1 as described by Jagroop and Mikhailidis (2000). Besides, also rheological changes as a consequence of hypoxic vasoconstriction could cause platelet activation. Hypoxic pulmonary vasoconstriction (HPV) serves to optimize ventilation–perfusion matching in focal hypoxia and may improve pulmonary gas exchange (Kylhammar and Radegran, 2017). During global hypoxia as given after altitude exposure, HPV induces general pulmonary vasoconstriction, which may raise pulmonary total vascular resistance and impair exercise capacity (Kylhammar and Radegran, 2017). ET-1 is a key player of hypoxic vasoconstriction and, therefore, gives room for speculation regarding platelet activation due to rheological mechanisms, of which ET-1 might be a marker.
Limitations and future perspectives
Our study cohort is small and contains only young and healthy subjects, so we cannot generalize our findings, especially not to subjects with pre-existing cardiovascular disease. Methodology of evaluation of platelet activation was limited to measurement of one single serum parameter and no diagnostic procedure was performed to evaluate pulmonary pressure or pulmonary vascular resistance (e.g., echocardiography). Our future study will have to deal with these three major limitations and prove the current theories in a well-founded setting before therapeutic intervention (e.g., antagonism of ET-1) will come in the scope of future studies.
Conclusion
Our current study on young healthy subjects could demonstrate platelet activation after an acute 30 minute high altitude exposure, which was accompanied by a 44% increase of ET-1. A causal relation could rest on direct platelet-activating properties of ET-1 or by concomitant rheological chances, of which ET-1 might be a marker. The shown platelet activation might obtain clinical relevance in patients with pre-existing cardiovascular disease as a trigger for acute cardiovascular events.
Author Contributions
K.L. was in charge of the study design, conduction of experiments, and draft of the article. Conduction of experiments and revision of the article for relevant intellectual content were done by C.G.S., P.M., J.G., and K.M. S.B. performed the study design, conduction of experiments, and revision of the article for relevant intellectual content.
Footnotes
Acknowledgment
This study was supported by the young investigator grant of Ludwig Maximilians University—“LMUexcellent.”
Author Disclosure Statement
No competing financial interests exist.