Several studies have reported that an orthostatic challenge activates the coagulation system. Both active and passive standing are associated with (i) pooling of blood in the legs, (ii) increased transmural pressure, (iii) increased shear stress, as well as (iv) endothelial activation in the lower extremities, associated with (v) release of procoagulant glycoproteins, e.g., Tissue Factor (TF) (Masoud et al., 2008; Cvirn et al., 2012; Haider et al., 2013). All these studies have been performed in healthy young controls. Despite coagulation activation, no cases of thrombosis have been reported in these persons. It, therefore, appears that an orthostatic challenge, induced by either active or passive standing, does not provoke thromboembolic events in healthy controls. However, orthostatic challenge-induced coagulation activation might constitute a problem for patients at an already elevated risk for vascular events, for example, ischemic stroke.

We, therefore, wanted to investigate the influence of orthostatic challenge on the coagulation system in patients with a history of ischemic stroke. In our study, 22 older patients, who had recovered from ischemic stroke, performed a sit-to-stand test, a suitable model for orthostatic challenge. Twenty-two healthy age-matched controls served as controls.

Hemostatic profiling was carried out in both whole blood and platelet poor plasma samples. The clot formation process was monitored using TF-triggered thrombelastometry. Thrombin generation was assessed by means of calibrated automated thrombography (CAT), and by the determination of plasma levels of prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin complexes (TAT). For further assessment of orthostatic challenge-induced coagulation activation, we measured prothrombin times (PT), activated partial thromboplastin times (APTT), plasma levels of FII, FVII, FVIII, protein C, and protein S. In order to evaluate orthostatic challenge-induced endothelial activation, we determined plasma levels of TF and of tissue-plasminogen activator as well as of nitric oxide. Since nitric oxide has a very short half-life, it is very difficult to determine nitric oxide content in whole blood samples. We therefore measured nitrite + nitrate levels, the major stable metabolites of nitric oxide, which can be quantitatively determined (Romitelli et al., 2007).

It has been shown that standing is associated with hemoconcentration due to the transfer of intravascular fluid from the blood to the surrounding tissue (Hinghofer-Szalkay and Moser, 1986; Jacob et al., 2005). We therefore determined hematocrit values in pre- and post-orthostatic challenge blood samples to compare the hemoconcentration effect of orthostatic loading (standing) in both groups. Plasma volume changes were calculated from hematocrit, as described by Masoud et al. (2010).

Moreover, copeptin levels have been shown to be sensitive surrogate markers for the release of arginine-vasopressin, which is associated with orthostatic challenge (Goldsmith, 1989; Choi et al., 2015). We therefore determined levels of copeptin in pre- and post-orthostatic challenge plasma samples of the two groups in order to evaluate the orthostatic challenge-response on the hormonal levels.

Detailed anthropometric measurements of all test subjects are provided in Table 1. Although, a significantly higher number of patients who had recovered from ischemic stroke were treated with antiplatelet (acetylsalicylic acid and/or clopidogrel) and hypotensive drugs, the basal levels of coagulation parameters were not significantly different between the two groups with the exception of tissue-plasminogen activator. The basal tissue-plasminogen activator levels were significantly higher in the recovered stroke-patients group (10.1 vs. 7.3 ng/mL, medians, p = 0.023). Unsurprisingly, the number of previous vascular events and of vascular risk factors was greater in patients when compared with controls.

In the present study we quantified coagulation changes in response to orthostatic challenge (a sit-to-stand test) in patients who had recovered from ischemic stroke as compared with healthy, age-matched controls. Although, significantly more stroke-patients were treated with antiplatelet drugs (acetylsalicylic acid and/or clopidogrel), approximately same basal levels of thrombelastometry-derived coagulation times in both groups were found in our study. While acetylsalicylic acid has been shown to possess only a limited capability to affect thrombelastometry values (Trentalange and Walts, 1991; Luddington, 2005), clopidogrel can effectively prolong platelet-fibrin clot formation (Gurbel et al., 2007). Marked effects were found in patients loaded with 300 to 600 mg. We assume that this effect could not be observed in our study since our post-stroke patients were receiving only 75 mg of clopidogrel.

Our data show that orthostatic challenge is associated with activation of the coagulation system. In both groups of test subjects, prothrombin fragment 1+2 as well as thrombin/antithrombin-complex were significantly increased and prothrombin times were significantly shortened in the post standing samples compared to baseline to approximately the same extent. Activated partial thromboplastin times (median) were also shortened due to the orthostatic challenge, reaching significance only in the healthy control group. These findings are in agreement with two other studies, which investigated the effects of still standing on the coagulation cascade (Masoud et al., 2008, 2010). While the results from those studies were based on stationary standing periods that varied from 15 to 60 min, we observed that a short 6 min standing period also leads to a significant activation of the coagulation system. Furthermore, we observed that this activation of the coagulation is significantly more pronounced in patients than in healthy controls. Clotting times, determined by means of thrombelastometry, were significantly shorter in the standing samples compared with baseline in patients recovered from stroke but not in healthy controls. This indicates that orthostatic challenge is associated with a shift toward hypercoagulability in patients who had recovered from ischemic stroke, but not in healthy controls. Accordingly, alpha angles were significantly increased (compared to baseline) by orthostatic challenge in patients but not in healthy controls, indicating that orthostatic challenge apparently leads to enhancement of fibrin built-up and cross-linking in post-stroke patients but not in healthy controls. The changes were approximately the same by comparing both groups.

Moreover, peak rate of thrombin generation values, determined by means of calibrated automated thrombography, were greater in the standing samples in patients but not in healthy controls. This also indicates a shift toward hypercoagulability in patients recovered from stroke: orthostatic challenge seems to be associated with accelerated thrombin formation. Again, the changes were approximately the same when both groups were compared.

Furthermore, we found that orthostatic challenge significantly elevates plasma levels of Tissue Factor (a marker of endothelial activation, Tilley and Mackman, 2006) in patients compared to baseline. This also indicates an orthostatic challenge-induced shift toward hypercoagulability in the stroke-patients group, as Tissue Factor is the most important trigger of the coagulation cascade (Manly et al., 2011). Again, the changes were approximately the same across the two groups.

Plasma levels of tissue-plasminogen activator (another marker of endothelial activation) in healthy controls increased due to orthostatic challenge compared to baseline, indicating increased fibrinolytic activity in the standing samples in this group. However, we found no increase of tissue-plasminogen activator in the patients group compared to baseline, indicating that orthostatic challenge fails to activate the fibrinolytic system in this group (Mazzolai et al., 2002). Again, the changes were approximately the same across both groups.

Levels of another marker of endothelial activation, nitric oxide (determined as nitrite + nitrate plasma levels), were apparently not affected by orthostatic challenge. An explanation might be that the elevated basal levels of nitrite + nitrate in plasma (approximately 60 μM) (Rashid et al., 2003) mask the relatively low amounts of nitric oxide (most likely in the nanomolar range) produced in response to shear stress (Tian et al., 2010).

Interestingly, orthostatic challenge was associated with significant hemoconcentration in healthy controls (~5%) but not in the patient group. Apparently, the microvascular permeability in the stroke-patients group is significantly lower than in the control group. An explanation might be that 73% of the patients, who had recovered from ischemic stroke, but only 32% of the healthy controls, were treated with hypotensive drugs (primarily angiotensin-converting enzyme inhibitors). Angiotensin-converting enzyme inhibitors could have led to the decreases in the microvascular permeability via suppression of angiotensin II formation (Pupilli et al., 1999; Newton et al., 2005). Hematocrit changes were approximately the same when comparing both groups.

Our results also indicate that recovered stroke-patients show more hormonal responses to orthostatic challenge than healthy controls: Plasma levels of copeptin were greater in the standing samples (in comparison to baseline) in patients but not in controls. Copeptin is a valid surrogate marker for arginine-vasopressin release. Due to the arginine-vasopressin being a synergistic component of the hypothalamo-pituitary-adrenal axis, levels of arginine-vasopressin also reflect the participants orthostatic stress response (Katan et al., 2009; Masoud et al., 2010). Copeptin changes were significantly higher in Group 1 compared with Group 2 of test subjects.

We have recently observed that there is some degree of autonomic dysfunction–as reflected in the heart rate variability measurements–during orthostatic challenge even after 1 year of recovery from stroke (Rodriguez et al., in press). As autonomic dysfunction changes have been associated with orthostatic intolerance and clot formation at the site of the vascular injury (Lopez-Vilchez et al., 2009), it is not surprising that a greater tendency toward clot formation was seen in our stroke patients. Our results are also in agreement with the work of Davidoff and colleagues (Davydov et al., 2015).

In conclusion, our data indicate that orthostatic challenge, i.e., a simple sit-to-stand test, leads to a shift toward hypercoagulability in healthy controls, and, to a significantly higher extent, in patients who had recovered from ischemic stroke. In addition to other studies showing coagulation activation after prolonged quiet standing (15 up to 60 min), we show herein that significant coagulation activation occurs even after 6 min of still standing. In our study, protein C plasma levels remained unchanged by orthostatic challenge; this is in agreement with the findings of Masoud et al. (2008, 2010). Thus, it appears that the anticoagulant protein C pathway fails to neutralize the shift toward hypercoagulability (Masoud et al., 2008, 2010).

On the assumption that this simple sit-to-stand test is a valid method that exposes subjects to a procoagulant challenge, our data suggest that the coagulation system of patients with a history of ischemic stroke can be more easily shifted toward a hypercoagulable state than that of healthy controls. Thus, our findings may explain the frequent recurrence of thrombotic events in recovered stroke-patients (Becattini et al., 2012). Our data emphasize the importance of accurate, regular and long-term anticoagulant treatment of patients with a history of thrombosis. It has to be stated that, by absolute numbers, the shift toward a hypercoagulable state in the stroke-patients group is, although statistically significant, relatively small. All coagulation values remained within their reference values, as reported in a previous study (Lamprecht et al., 2013).

Notably, we show herein that even a short-term orthostatic challenge leads to significantly shortened standard coagulation times, particularly Prothrombin Times. With specific regard to the coagulation system, we could not find any literature that deals with the amount of time a patient should remain seated before providing blood coagulation assessments. From previous studies it is known that orthostatic challenge induced fluid shifts need about 30 min to return to baseline supine values (Hagan et al., 1978; Hinghofer-Szalkay et al., 2008; Goswami et al., 2012). Furthermore, using head-up-tilt and lower-body-negative pressure (Cvirn et al., 2012), we observed that endothelial activation (as assessed by tissue factor levels) returns to baseline levels around 20 min after orthostatic challenge. As patients are often standing/walking before they reach the consultation room, we recommend that when carrying out assessment of coagulation related parameters, patients should be allowed to rest in a seated or supine position for at least 20–30 min. This will help eliminate the influence of changes in posture on such tests as Prothrombin time.

All authors have made significant contributions in data collection, analysis and interpretation of the findings. All co-authors have been involved in writing the final version of the manuscript.