Release of pro-coagulant microparticles after moderate endurance exercise

To the editor,

Elevated plasma concentrations of cell-membrane-derived microparticles (MP) are found in many diseases such as sepsis, cancer, and diabetes mellitus [1], [2]. Various cell types are known as sources of MP, which play a crucial role in angiogenesis, endothelial cell function as well as coagulation and inflammation. A uniform characteristic of the various cell-specific MP is the surface exposure of negatively charged phospholipids. Due to these phospholipids and also due to the exposure of tissue factor (TF) and/or TF pathway inhibitor, MP are believed to be involved in the regulation of coagulation [3], [4]. Furthermore, MP bear cell-specific antigens enabling them to interact with a variety of target cells. Thus, MP can be considered as intercellular signal transferring entities [5], [6]. Low levels of circulating MP, mainly derived from platelets, are also present in healthy individuals but their functional relevance in haemostasis and inflammation is not well understood [7].

Increased levels of circulating MP are known to be associated with an increased risk of cardiovascular disease and atherothrombotic events [1], [8]. Since moderate physical exercise is known to lower the risk of cardiovascular diseases [9], [10], we were interested to study the effect of moderate physical exercise on both concentration and pro-coagulant activity of circulating MP.

After approval by the local Ethics Committee and obtaining written informed consent, 16 healthy male volunteers (age 25 ± 3 years; mean ± SD) underwent a singular 90 min exercise test on a bicycle ergometer with a constant power of 80% of individual anaerobic threshold (IAT). IAT was determined one week prior to the test according to Stegmann et al. [11]. Blood samples were taken before (rest), immediately post (post) and 45 min (45 min post) as well as 2 hours (2 h post) after exercise.

MP were identified by flow cytometry according to their size and the binding of FITC-labelled annexin V as well as by the binding of PE-labelled antibodies against cell-specific antigens: platelet-derived MP (PMP) by anti-CD42a, monocyte-derived MP (MMP) by anti-CD14 and endothelial cell-derived MP (EMP) by anti-CD62E. To estimate pro-coagulant activities of MP two different tests were performed: The ACTICHROME® test (American Diagnostica), which measures the pro-thrombinase activity of FXa/Va of MP bound to an annexin V-coated surface in absence of any other plasma constituents and a one-stage clotting test, in which the formation of a fibrin clot was monitored by measuring the optical density in MP-containing plasma samples after re-calcification and addition of a small amount of TF.

The 90 min exercise at 80% IAT (mean power 173 ± 73 W) increased heart rate to 156 ± 16 beats/min, the systolic blood pressure to 156 ± 25 mm Hg and plasma lactate concentration to 2.17 ± 0.81 mmoles/l. As shown in Table I, the exercise resulted in a significant increase in the numbers of white blood cells and platelets. It has been shown elsewhere that the increase in platelet and leukocyte counts is not only due to a reduction in plasma volume but also due to a release of platelets and leukocytes from various storage sites (spleen, lung, bone marrow etc.) [9]. With respect to MP, we also observed an increase in the number of all annexin-positive MP as well as PMP, whereas EMP and MMP remained unchanged. Maximum values in platelet number were found immediately post exercise and returned to basal level at least after 45 min post exercise. In contrast, white blood cells and MP counts peaked at 45 min post-exercise and did not return to basal levels within 2 h post exercise (Table I).

Table I.  Time-dependent changes in the numbers of blood cells and circulating MP after physical exercise. Data are given as mean ± SD. ANOVA for repeated measurements was used to test for significant time-dependent changes; t-test for paired samples was applied to test for significant effects of exercise in comparison to basal values (rest). ns, not significant; § p ≤ 0.05.

Abosolute counts/µl	Rest	Post	45 min post	2 h post	ANOVA
White blood cells (*10^3)	5 ± 1	9 ± 3 §	11 ± 3 §	11 ± 2 §	p < 0.01
Platelets (*10^3)	238 ± 44	288 ± 51 §	244 ± 44	242 ± 40	p < 0.01
Annexin positive particles	3290 ± 1085	5990 ± 2334 §	8394 ± 3849 §	7045 ± 2976 §	p < 0.01
PMP	970 ± 473	1596 ± 779 §	2671 ± 1314 §	2184 ± 1592 §	p < 0.01
EMP	394 ± 212	440 ± 246	440 ± 231	440 ± 242	ns
MMP	33 ± 23	44 ± 26	45 ± 29	42 ± 27	ns

The procoagulant activity of annexin V-bound MP (Actichrome test) was nearly two-fold increased immediately post-exercise when compared to rest and remained elevated at least up to 2 h after exercise (Figure 1A). A similar time course was observed for the TF-initiated fibrin formation in the one-stage clotting test with MP-rich plasma (Figure 1B).

Figure 1. Functional activity tests at rest, immediately post-, 45 min post-, 2 h post exercise. A: MP pro-coagulant activity given as concentration of negatively charged phospholipids (nM). B: Maximum velocity (value*10⁻²) of fibrin formation (Δ optical density/time); § p ≤ 0.05 for differences to rest (t-test).

We could show in the present study that moderate exercise in young healthy subjects increased the number of circulating pro-coagulant MP. The increase was mainly due to a release of CD42a-positive PMP with a maximum at 45 min post-exercise, whereas the numbers of MMP and EMP remained unchanged. As PMP and platelet count had different time-course, the increase in PMP is probably caused by exercise-induced platelet activation rather then a simple consequence of the increase in platelet count. Exercise-induced platelet activation was recently reported [12], [13].

Interestingly, pro-coagulant activity of annexin V-positive MP and their absolute number in plasma have different time-course. Maximum of activity was found immediately post-exercise whereas maximum number was found at 45 min post-exercise. Thus one could speculate that the specific activity of the MP decreases with time whereas the number of MP still increases.

Two mechanisms might contribute to the release of PMP after physical exercise. Platelets that are present in the circulation at the beginning of exercise become stimulated by agonists and/or shear stress, release MP and afterwards will be removed by phagocytes. As another consequence of exercise, juvenile platelets from spleen, bone marrow and the intravascular pool of the lungs enter the circulation. These platelets have a high sensitivity to agonists such as epinephrine and nor-epinephrine and may release MP with other procoagulant activity then elderly platelets. Furthermore, juvenile platelets have high energetic capacity enabling them to reorganise their cytoskeleton after MP release, and continue to circulate and function [14], [15].

Moderate exercise in young as well as middle-aged, healthy subjects has been shown to activate coagulation [16], [17]. Our data on MP and their procoagulant activity after moderate exercise may support the phenomenon of an exercise-induced increase in the haemostatic potential. In a previous study with coronary artery diseased patients we observed positive correlations between the number of circulating PMP and thrombin generation [18].

Numerous epidemiological and prospective cohort studies confirm an important beneficial role of moderate endurance exercise in the prevention and treatment of cardiovascular diseases [9]. Thus, our data presented may support the hypothesis that MP are not only ‘bad’ but–at least in some conditions–also ‘good’ [1].