Subjects and ethical approval

Twenty patients recently diagnosed with lacunar stroke (53–84 years) and fifteen healthy age-matched controls (55–77 years) were included in the study (Table 1). However, conventional markers of coagulation (fibrinogen, thrombocytes, APTT, and coagulation factors) were only assessed in eight stroke patients and fifteen control participants.

Table 1 Subject characteristics

The clinical criteria for inclusion of the patients was a recent incidence of ischemic small vessel occlusion stroke (lacunar stroke). The stroke patients were recruited at the Copenhagen University Hospital, Herlev, Stroke Unit, Department of Neurology, Copenhagen, Denmark, and the age-matched controls were recruited through local advertisement and through online portals. Pre-determined inclusion criteria for the stroke patients were: >18 years of age, clinical symptoms of small vessel occlusion stroke and concomitant brain imaging (computed tomography scan (CT)/magnetic resonance imaging (MR)-scan with signs of a relevant ischemic small vessel occlusion lesion within the last 5 years), MR and/or CT verified acute lacunar supratentorial or infratentorial infarct (< 2 cm in diameter in acute phase, 1.5 cm in chronic phase), and no clinically significant carotid stenosis or cardioembolic cause of infarct. None of the participants were diagnosed with thrombocythemia, but the stroke patients received single antiplatelet-treatment (three patients received dual antiplatelet-treatment), as part of a standard treatment procedure for lacunar stroke [21, 22]. Seventeen of the stroke patients received single antiplatelet therapy with 75 mg clopidogrel and three stroke patient received 75 mg clopidogrel and 75 mg aspirin (Table 1). CYP2C19 mutations were measured by a gene test to assess the intra-individual ability for activating a clopidogrel antiplatelet response. Only one of the stroke patients was homozygote for this gene, with possible reduced clopidogrel antiplatelet activity. Pre-determined inclusion criteria for the controls were: men and women between 50 to 80 years of age, BMI < 30 kg∙m−2 and physically inactive lifestyle (< 2 h of physical exercise per week). Pre-determined exclusion criteria for both groups included: other chronic diseases (e.g., cancer, heart disease, immune diseases), drug or alcohol abuse, and smoking. The median duration from the clinical diagnosis of stroke onset to patient assessment was 24 days, with a range from 6 to 756 days.

The study was approved by the ethical committee of Copenhagen, Region H (H-16048498), conducted in accordance with the guidelines of the Declaration of Helsinki and was registered at ClinicalTrials.gov (NCT03635177). The participants were informed about any risk and discomfort related to participating in the study before they gave their written consent to participate.

Overall study design

Prior to inclusion in the study, participants went through a medical examination carried out by a medical doctor. For stroke patients the health examination took place at Herlev Hospital, and for the control subjects at the Department of Nutrition, Exercise and Sports, University of Copenhagen. The medical examination included a medical history by the doctor identifying the general state of health and former diseases, a resting ECG measurement, resting blood pressure, and a blood sample for screening of health-related factors, such as anemia, infection, coagulation profile, cholesterol, kidney function, fasting blood sugar and plasma glucose. The recruitment and medical examination of the stroke patients were performed in close connection with a stroke neurologist involved in their stroke treatment.

Before the experimental day, participants were not allowed to drink any beverage containing caffeine or take medicine for 24 h prior (except for the antiplatelet therapy for stroke patients) and participants avoided strenuous exercise for 48 h prior. On the experimental day, participants arrived at the laboratory at least 2 h post-prandial (light meal). Participants rested in a supine position for ~ 30 min and a peripheral venous catheter (18-gauge, 32 mm; 393224; BD Venflon Pro Safety) was placed in the antecubital vein for blood sampling performed before, immediately after, and 1 h after a graded cycle test with a “Talk Test” (see below), for determination of clot microstructure, assessed as fractal dimension (df), conventional markers of coagulation and full blood count.

Cycling exercise protocol

The exercise protocol was a graded cycle test with a “Talk Test”, which has been verified as being appropriate for patients with lacunar strokes [23]. In brief, the participants performed a brief warm-up at 15 watts for 2 min at 60 rpm on a Monark cycle ergometer (Monark Ergomedic 928E, Vansbro, Sweden), then the resistance was increased 15 watts every minute. During the last 10 s of each stage of the cycling test, the participants were required to recite a simple paragraph in Danish out loud to the researchers. The test was terminated when the participant could no longer repeat the paragraph comfortably, which was based on bystander evaluation and self-assessment. This should result in a similar relative workload. The participants wore continuous heart rate monitors (POLAR Team Pro (Polar Electro Oy), Kempele, Finland) during the test.

Measurements of biomarkers indicating the susceptibility of thrombosisBlood clot microstructure

To determine the blood clot microstructure, the gel point of an incipient blood clot was determined. The measurement of the gel point was conducted by rheometric analysis on untreated blood, immediately after the blood drawn, as described elsewhere [15, 24,25,26]. Gel point is defined as the transition from a viscoelastic fluid to a viscoelastic solid [15]. From the analysis of the gel point measurement, fractal dimension (df) of the incipient blood clot can be determined [15]. Df analysis provides information about the fibrin network of the developing ex-vivo clot, and thereby provides a functional measure of clot microstructure [15, 27]. In brief, blood was drawn from the antecubital vein into a 9 mL blood collection tube without additive (455001; Greiner Bio-One, Austria). 3.5 mL of fresh unaltered blood was added to two opposite sides of a double concentric geometry of a controlled stress rheometer (AR-G2; TA Instruments, New Castle, USA) together with 2 drops of low-viscosity oil, at the constant temperature of 37\(^\circ\)C. Sequential frequency measurements (2, 0.93, 0.43 and 0.2 Hz) were immediately performed after loading of the sample, thereby over time obtaining gel point [15]. From the obtained gel point (\(\alpha\)) measurement, the clot microstructure was quantified by calculating the corresponding fractal dimension (df) using the following equation [28]:

$$d_f=\left(10\cdot\left(\alpha/90\right)-15\right)\;/\left(2\cdot\left(\alpha/90\right)-6\right)$$

Blood biomarkers of coagulation

After appropriate filling of vacutainers (3.5 mL, 3.2% Sodium Citrate, BD Vacutainer, Becton Dickinson, USA), fibrinogen, activated partial thromboplastin time (APTT), coagulation factors II + VII + X and the International Normalized Ratio (INR) were analyzed at either the Department of Clinical Biochemistry at Rigshospitalet, using an ACL TOP 550 CTS analyzer (Werfen, Warrington, UK) or the Department of Clinical Biochemistry at Herlev Hospital using a Sysmex CS5100 (Siemens Healthcare, Germany). In addition, after gently inverting the vacutainer, platelet count was measured in citrated blood using either a Sysmex XN-450 (Sysmex, Kobe, Japan) or ADVIA 2120i 3M (Siemens Healthcare, Erlagen, Germany).

Measurement of a biomarker of fibrinolysisPlasminogen activator inhibitor-1 (PAI-1)

PAI-1 protein levels were analyzed in EDTA plasma by use of ELISA according to the manufacturer’s protocol (ab269373, Abcam, Cambridge, UK).

Statistical analysis

The calculation of sample size was based on previous data from our laboratory on the primary outcome; fractal dimension after acute exercise with a power value for the analyses of > 0.8. The required sample size was estimated to at least 15 subjects in each group [10]. The statistical analyses were performed using Rstudio (Version 4.1.2, R Foundation for Statistical Computing, Vienna, Austria). Graphs were made using GraphPad Prism (GraphPad Software for Windows, Version 9.3.1., San Diego, CA, USA). Data are reported as mean \(\pm\) standard deviation (SD) unless otherwise stated. Differences in baseline subject characteristics (Table 1) and delta changes between post exercise and rest between groups were detected using a two-tailed unpaired t-test. A linear mixed-model approach was used to investigate differences within and between groups for the following parameters: fractal dimension, thrombocytes, fibrinogen, APTT, coagulation factors, and PAI-1. Fixed factors were ‘group’ (Control subjects and Stroke patients) and ‘Intervention’ (rest, immediately post exercise, and 1 h post exercise). Subjects were specified as a repeated factor and identifier of random variation. Differences were identified using the ‘emmeans’ package and Sidak adjusted p-values are presented. Model reduction was not performed to avoid potential selective inference, meaning that post hoc tests were performed despite non-significant interactions [29]. Normal distribution was confirmed using Q-Q plots. Twenty stroke patients and fifteen control subjects were included, however, conventional markers of coagulation (fibrinogen, thrombocytes, APTT, and coagulation factors) were assessed in eight stroke patients and fifteen control participants (See figure legends). Missing data points are described below. For the cardiovascular risk factor parameters (Table 1) two data points were missing in the stroke group due to difficulties drawing blood (n = 18), and for hematocrit and erythrocytes additional two data points were missing (n = 16) due to a mistake running the analysis (n = 2). For the measurement of fractal dimension, in the stroke group, one data point was missing at rest and post-exercise, and three data points in total were missing at 1 h post-exercise due to difficulties drawing blood. In the control group, two data points were missing 1 h post-exercise due to difficulties drawing blood (n = 1) and due to a system error during analysis (n = 1). For thrombocyte count, one data point was missing from the stroke patients at rest and post-exercise, due to difficulties drawing blood and due to machine error when running the analysis, and one data point was missing from the control subjects 1 h post-exercise due to difficulties drawing blood. For the measurement of fibrinogen and APTT, one data point was missing for stroke patients 1 h post-exercise and from the control subjects pre-exercise, in both cases due to difficulties drawing blood. For APTT, an additional data point was missing in the control subjects post-exercise. For coagulation factors and INR, one data point was missing from the control subjects pre-exercise difficulties drawing blood. For measurements of PAI-1, two data points were missing from the stroke subjects 1 h post-exercise due to insufficient material. The significance level was set at p < 0.05, and trends were reported if p < 0.07.