Arterial hypoxemia and its impact on coagulation; significance of

altered redox homeostasis.

Lewis Fall, Karl J New, Kevin A Evans and Damian M Bailey

Neurovascular Research Laboratory, Research Institute of Science & Health Faculty of Life

Sciences & Education, University of South Wales, UK

Key Words: Oxidative Stress, Blood Coagulation; Hypoxia

Correspondence:

Dr Lewis Fall,

Neurovascular Research Laboratory,

Faculty of Life Sciences and Education,

University of South Wales,

UK.

Tel: +44-1443-4821066/Fax: +44-1443-482285

email: lewis.fall@southwales.ac.uk

Word Count: 1224

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Abstract

Aims: Arterial hypoxemia stimulates free radical formation. Cellular studies suggest this may

be implicated in coagulation activation, though human evidence is lacking. To examine this,

an observational study was designed to explore relationships between systemic oxidative

stress and haemostasic responses in healthy participants exposed to inspiratory hypoxia.

Results: Activated partial thromboplastin time and International Normalised Ratio were

measured as routine, clinical biomarkers of coagulation and ascorbate free radical (A •-) as a

direct, global biomarker of free radical flux. Six hours of hypoxia activated coagulation and

increased formation of A•-, with inverse correlations observed against oxyhaemoglobin

saturation.

Conclusions: This is the first study to address the link between free radical formation and

coagulation in-vivo. This “proof-of-concept” study demonstrated functional associations

between hypoxaemia and coagulation that may be subject to redox activation of the intrinsic

pathway. Further studies are required to identify precisely which intrinsic factors are subject

to redox activation.

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Introduction

Hypoxia increases the reduction state of mitochondrial electron transport carriers, favouring

leakage of the superoxide anion . Recently, it has been established in-vitro that an increase in

reactive oxygen species (ROS) formation activates coagulation . Though the underlying

mechanisms remain unclear, it has been suggested that NADPH oxidase-mediated ROS

formation within the vessel wall where tissue factor resides may be implicated, given that

tissue factor expression per se is sensitive to ROS . Equally, it has also been suggested that

coagulation factor VIII bioactivity may be involved given that it too is subject to redox

regulation .

Arterial hypoxaemia is a hallmark feature of patients with congestive heart failure, peripheral

arterial occlusive disease, chronic obstructive lung disease and obstructive sleep apnoea .

These conditions are associated with increased all-cause cerebrovascular and cardiovascular

mortality . Traditionally, activated coagulation has been attributed to systemic inflammation

with no consideration for alternative, arguably “upstream”, redox-active pathways.

Given this lack of information, we designed a “proof-of-concept” study in healthy

participants in order to isolate the independent metabolic impact of hypoxia and identify to

what extent redox function and haemostasis are altered and potentially related. We utilised

inspiratory hypoxia to reduce the healthy participants’ arterial oxygen tension to a level

similar to that of the circulatory-compromised patient population for six hours, as a simulated

model of disease without any confounding comorbidities. We hypothesised that (a)

inspiratory hypoxia would increase systemic free radical formation and activate coagulation,

confirmed by an elevation in A•- and shortened activated partial thromboplastin time (aPTT)

and (b) that both oxidative and haemostatic stress would be inversely related to the degree of

arterial hypoxaemia reflected by the reduction in arterial oxyhaemoglobin saturation (SaO2).

Methodology

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Subjects: Based on a simple 2-sided t-test comparing means, and assuming a 5% significance

level, 90% power and (average) effect size of 1.15 on primary outcome variables (aPTT and

A•-) we required 16 participants in each arm (normoxia and hypoxia) for analysis (calculated

using nQuery v4.0). Due to incomplete data sets and dropout, data are presented on thirteen

healthy men aged 25 ± 1 (mean ± S.E.M). A retrospective power analysis (SPSS statistics 21,

IBM, Edinburgh, UK) revealed that the primary outcome variables (aPTT and A•-) were

powered at 0.875 and 1.00 respectively when computed using an α-level of 0.05.

Participants with a known pre-existing coagulopathy or with pre-diagnosed risk factors for

vascular disease were excluded. Volunteers who were taking any regular medication known

to affect the oxidative, inflammatory or coagulation systems were excluded and also those

who habitually ingested dietary vitamin supplements or who refused to cease

supplementation 8 weeks prior to testing.

Experimental design: Baseline measurements were obtained at rest following a 12 hour

overnight fast both in normoxia (FIO2 of 21%) and following 6 hours exposure to normobaric

hypoxia (FIO2 of 12%) in a thermo-neutral environmental chamber (Weiss Technik, Ebbw

Vale, UK).

Haemostasis: Blood was obtained by single venipuncture from a cephalic vein, mixed with

sodium citrate, and centrifuged at 600 g (4°C) for 10 min. The plasma concentration of

fibrinogen (FB) and the corresponding prothrombin time (PT), and activated partial

thromboplastin time (aPTT) were determined using a Futura Pluss automated coagulometer

(Instrumentation Laboratory, Warrington, UK). The intra-assay and inter-assay coefficients

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of variation (CV) for all parameters were both <5%. The International Normalised Ratio

(INR) was calculated as:

where ISI is the international sensitivity index and is provided by the reagent

manufacturer, distinct to the batch.

Oxidative stress: Electron paramagnetic resonance (EPR) spectroscopy was used to directly

measure ascorbate free radical (A•-) as a biomarker of global free radical formation . Exactly

1 mL of K-EDTA plasma was injected into a high-sensitivity multiple-bore sample cell

(AquaX, Bruker Daltonics Inc., Billerica, MA, USA) housed within a TM110 cavity of an

electron paramagnetic resonance (EPR) spectrometer operating at X-band (9.87 GHz).

Samples were analysed using a modulation frequency of 100 kilohertz (kHz), modulation

amplitude of 0.65 gauss (G), microwave power of 10 milliwatt (mW), receiver gain of 2×105

AU, time constant of 41 ms, magnetic field centre of 3477 Gand scan width of ± 50 gauss (G)

for three incremental scans. After identical baseline correction and filtering, each of the

spectra were subject to double integration using graphical analysis software (OriginLab Pro,

version 8.5, OriginLab Corporation, MA, USA). The intra and inter-assay CV’s were both <5

%.

Cardiovascular: Arterial haemoglobin oxygen saturation (SaO2) was determined using

finger-tip pulse oximetry (Novametrix 515C, Philips Healthcare, Guilford, UK).

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Statistical analysis: Following confirmation of distribution normality using Shapiro-Wilks W

tests, data were analysed by paired samples t-tests. Relationships were assessed via Pearson

Product Moment Correlations. The α-level for all two-tailed tests was established at P < 0.05

and values are reported as mean ± SEM.

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Results

As anticipated, SaO2 decreased in hypoxia (P < 0.05 vs. normoxia). Hypoxia was also shown

to increase A•- and decrease aPTT (P < 0.05 vs. normoxia), changes that correlated inversely

against SaO2 (r = -0.34 and -0.40 respectively, P < 0.05).

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Discussion

This is the first study that has investigated the potential relationship between systemic

hypoxaemia, oxidative stress and activation of coagulation in humans. Normobaric hypoxia

was employed to simulate an arterial oxygen tension equivalent to that experienced by

patients with vascular disease in order to isolate the metabolic impact of hypoxia without the

potential confounds associated with related comorbidities. INR and aPTT were utilised as

markers of coagulation routinely employed in a clinical environment.

Given that the concentration of ascorbate in human plasma is orders of magnitude greater

than any oxidising free radical, combined with the low one-electron reduction potential (E ΄ =

282 mV) associated with the A•-/ascorbate monanion (AH-) couple , any radical generated

within the systemic circulation has the potential to react endogenously with this terminal

small-molecule antioxidant to form the distinctive A•- EPR doublet [(R• + AH- A•- + R-H] .

Thus, the elevation in A•- combined with the inverse relationship with SaO2 provides direct

evidence that hypoxaemia increased free radical-mediated oxidative stress.

The shortening of aPTT (reference range = 35.2 to 24.6 s) during hypoxia and the inverse

correlation with SaO2 suggests that hypoxia is associated with activated coagulation. From a

clinical perspective, a shortened aPTT would traditionally be disregarded as clinically benign.

However, recent research suggests that a decrease in aPTT predisposes to an increased risk

of thromboembolic events and myocardial infarction . In contrast, the elongation of PT as

reflected by the increase in INR is not clinically significant since the reference range lies

between 0.9 to 1.2 AU. The association of increased contact factor (intrinsic) coagulation

pathway activity (as shown by shortened aPTT) and increased oxidative stress tentatively

suggests that hypoxia may indeed predispose to thrombophilia. In short, these data provide

preliminary evidence that hypoxaemia per se initiates both oxidative stress and coagulation

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in-vivo and that these two processes may be functionally linked. A potential mechanism could

be via a free radical-mediated increase in the bioactivity of coagulation factor VIII and von

Willebrand factor , since these are two markers associated exclusively with the contact factor

pathway. Factor analysis, which was beyond the scope of this initial study, will be required to

ascertain if this is the case and a long-term antioxidant intervention is needed to establish

whether oxidative stress is indeed the primary causative factor.

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Licence for Publication

The Corresponding Author has the right to grant on behalf of all authors and does grant on

behalf of all authors, an exclusive licence (or non-exclusive for government employees) on a

worldwide basis to the BMJ Publishing Group Ltd to permit this article to be published in

JCP and any other BMJPGL products and sublicences such use and exploit all subsidiary

rights.

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Competing Interests

None declared.

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Acknowledgments

We thank the participants for their cheerful participation and our faculties for their continuing

support.

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Author Contributors

LF, KE and DMB designed the study. LF and KE performed the research. LF and KN

analysed the data, LF, KN and DMB wrote the paper.

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Key Messages

Arterial hypoxaemia is associated with increased all-cause cardiovascular mortality.

Hypoxaemia per se initiates both oxidative stress and coagulation in-vivo.

These two processes may be functionally linked.

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Table 1

Normoxia Hypoxia

(%)

aPTT (s)

INR (AU)

(AU)

98 ± 0.3

27.0 ± 0.6

0.98 ± 0.02

296 ± 188

85 ± 1.4 *

24.6 ± 0.8 *

1.03 ± 0.03 *

3656 ± 242 *

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Tables legend

Table 1: Changes in activated partial thromboplastin time (aPTT), international normalised ratio (INR), arterial haemoglobin oxygen saturation ( ) and plasma ascorbate free radical ( ). Values are expressed as means ± standard error; * different from normoxia (P < 0.05).

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