Physical Exertion Induces Thrombin Formation and Fibrin Degradation in Patients With Peripheral Atherosclerosis

From the Wihuri Research Institute (P.M., R.L.) and the Department of Surgery (M.L.), Helsinki University Central Hospital, Helsinki, Finland.

Correspondence to Riitta Lassila, MD, PhD, Wihuri Research Institute, Kalliolinnantie 4, 00140 Helsinki, Finland.

	Abstract

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Abstract—Sudden extreme physical stress is associated with an increased risk of myocardial infarction mainly in people with preexisting atherosclerosis. In this study we compared the effect of submaximal exercise on coagulation and fibrinolysis in patients with peripheral arterial occlusive disease (PAOD) with that in healthy control subjects. Fifteen PAOD patients with intermittent claudication and 15 healthy control subjects, matched for age, sex, medication use, smoking habit, and conditioning, were studied. Thrombin–antithrombin III complex (TAT), D-dimer, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI)-1 antigens (Ag), t-PA activity, and plasmin-₂–antiplasmin complex (PAP), as well as plasma catecholamines, were measured before and after a treadmill exercise test. At rest, fibrinogen (3.3±0.5 versus 2.9±0.5 g/L [mean±SD]; P<.05), D-dimer (392±128 versus 271±113 ng/mL; P<.05), t-PA Ag (9.1±5.1 versus 5.5±1.2 ng/mL; P<.02), and PAI-1 Ag (14.9±7.1 versus 7.6±3.8 ng/mL; P<.002) levels in plasma were markedly higher in the patient group than in the control group. In patients but not in control subjects, exercise of similar intensity elevated circulating concentrations of TAT (from 3.43±1.45 to 4.83±2.27 ng/mL; P<.05). Exercise caused a parallel increase in D-dimer, t-PA Ag, t-PA activity, PAP, and catecholamines in both groups, whereas PAI-1 Ag remained stable. Plasma lactic acid was significantly higher in patients after exercise and was associated with lower-limb ischemia. Compared with healthy control subjects, patients with PAOD showed higher t-PA Ag, PAI-1 Ag, and D-dimer levels both at rest and after exercise. Notably, submaximal exercise on a treadmill enhanced thrombin formation in patients with PAOD but not in the control subjects. Sudden catecholamine release and local ischemia during exercise may accelerate the preexisting prothrombotic potential of the atherosclerotic vessel wall.

Key Words: atherosclerosis • exercise • coagulation • fibrinolysis • catecholamines

	Introduction

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Sudden unusual physical or emotional exertion can trigger acute myocardial infarction.^{1 2} Strenuous exercise induces activation in both the coagulation cascade and in the fibrinolytic system, which counterbalance each other.³ Nevertheless, a period occurs after exercise during which this balance seems to shift slightly toward the coagulation because of the short half-lives of components in the fibrinolytic system. It seems, however, that this prothrombotic tendency that prevails after exertion leads to thrombotic complications mainly in individuals with preexisting significant atherosclerosis.

During strenuous exercise, plasma catecholamine levels increase up to 10-fold compared with those in the resting state.^{4 5} This rise in epinephrine and norepinephrine levels may underlie the changes in hemostasis and fibrinolysis associated with exercise.⁶ For instance, epinephrine is known to promote both hemostasis and fibrinolysis by stimulating endothelial cells to liberate vWF⁷ and t-PA.⁸ In addition, we have recent evidence that epinephrine, at physiological concentrations, enhances vWF-dependent platelet interaction with a collagen-coated surface under blood flow.⁹ The epinephrine-induced potentiation of vWF-dependent platelet functions has also been reported in the form of enhanced high shear–induced platelet aggregation.¹⁰ Thus, adrenergic stimulation related to sudden physical exertion may well accelerate thrombus formation in atherosclerotic arteries after exposure of subendothelial vascular wall.

We subjected two groups, patients with intermittent claudication as the manifestation of PAOD and risk factor–matched healthy control subjects, to adrenergic stimulation in the form of a treadmill test with controlled levels of exertion. Our aim was to assess whether the presence of atherosclerotic vessel wall would influence the activation of coagulation and fibrinolysis during submaximal exercise that elevated catecholamine levels.

	Methods

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Study Subjects
The study protocol was approved by the institutional review committee and ethics board, and a written consent was obtained from study participants. Fifteen patients with documented stable intermittent claudication (Fontaine stage II) were included in the study. For each patient, we selected a healthy risk factor–matched (sex, age, BMI, physical activity, smoking, and alcohol consumption) control subject (Table 1). In all patients, the presence of atherosclerosis was confirmed by angiography of the lower extremities, and the functional severity of PAOD was assessed by the ABI, both at rest and after the treadmill exercise. One patient had undergone an intrainguinal venous bypass operation and another patient a thromboendarterectomy more than 3 years before the study, 1 patient had a vascular repair with prosthesis 1 year before, and 7 patients had undergone percutaneous transluminal coronary angioplasty at least 3 months earlier. Because more than 3 months had passed after these revascularization procedures in every case, their short-term, confounding effects on coagulation and fibrinolysis were avoided. In control subjects the presence of atherosclerotic disease was ruled out by a treadmill exercise test with continuous ECG monitoring and ABI measurements at rest and after the exercise test. Eleven of 15 patients were receiving regular ASA treatment, and the corresponding control subjects took ASA daily for at least 2 weeks before the study. None of the study subjects used any other medication, except for 1 patient who was receiving regular omeprazole treatment. None of the study subjects had diagnosed hypertension, diabetes mellitus, or any other acute illness.

Testing Procedure
The exercise tests were performed in the morning between 9 AM and 10:30 AM after a light carbohydrate-containing breakfast. Subjects took bed rest for approximately 15 minutes while they responded to an oral questionnaire before a baseline blood sample (sample 1) was drawn and ABI was measured. Subjects then mounted the treadmill, and a multistage treadmill stress test with continuous ECG registration was performed to the point of unbearable claudication in the patients and to the corresponding heart rate in the control subjects. Exercise tolerance was tested on a treadmill at a constant speed of 3.2 km/h. In the beginning of the exercise period, the treadmill remained flat for 2 minutes; thereafter, the inclination angle was increased by 2° every 2 minutes. All patients except 1 reached maximum walking distance within 24 minutes; all but one of the control subjects also reached a comparable heart rate within the same time. One patient and one control subject were able to continue beyond the maximal angle (22°), and the intensity of their exercise was further increased by speeding up the treadmill. Immediately after the exercise test, the study subjects lay down, the second blood sample (sample 2) was drawn, and ABI was measured. Subjects then rested in a recumbent position for 30 minutes, after which the third blood sample (sample 3) was collected.

Blood Sampling
Blood samples were always obtained with a free-flowing technique after the first 3 mL was discarded. Blood was collected with subjects supine (1) at rest, (2) immediately after the exercise test, and (3) 30 minutes after the exercise test. Samples 1 and 2 were collected through venipuncture from cubital veins of separate arms via an inserted polytetrafluoroethylene cannula (Viggo). After sample 2 was collected, the cannula was left in place and filled with low-molecular-weight heparin solution (dalteparin 100 antiXa U/mL in NaCl 0.9%), and sample 3 was collected via this cannula 30 minutes later. The effect of an inserted heparin-filled cannula on TAT at rest was tested in one healthy volunteer. Compared with the initial sample, TAT increased 3.8-fold at 30 minutes after exercise; similar results have also been reported previously.¹¹ Therefore, despite the heparin, sample 3 was used neither for TAT nor for D-dimer analysis. The following hemostatic and fibrinolytic blood values were measured: fibrinogen, vWF, TAT, D-dimer, t-PA and PAI-1 Ag, t-PA activity, and PAP. Details of blood collection and handling have been reported previously.^{12 13} Specifically, for the assessment of fibrinolytic enzymes blood was collected and handled as recommended by the Leiden fibrinolysis working party.¹⁴

Assays for Coagulation and Fibrinolysis
Fibrinogen was assessed by the functional method of Clauss with bovine thrombin (Dade, Baxter Healthcare Co). The following commercial ELISAs were used in the corresponding determinations: for vWF, Asserachrom vWF (Diagnostica Stago); for TAT, Enzygnost TAT micro (Behringwerke); for D-dimer, Asserachrom D-Di (Diagnostica Stago); for t-PA Ag, TintElize t-PA (Biopool); and for PAI-1 Ag, TintElize PAI-1 (Biopool); for t-PA activity, Spectrolyse/fibrin (Biopool); and for PAP, Enzygnost PAP micro (Behringwerke). Our intra-assay and interassay variations have been reported previously.¹²

Catecholamines
Venous plasma catecholamine concentrations were determined with high-performance liquid chromatography by means of electrochemical detection.¹⁵

Other Laboratory Tests
Plasma lactate levels were determined by use of a Kone Progress analyzer (Kone Instruments Oy), with Boehringer-Mannheim UV method reagents (catalog No. 256773), without deproteinization. Serum osmolality was assessed by the Advanced Micro Osmometer (model 3 MO plus). Hematocrit and blood cell counts were determined by the Trombocounter Coulter T-540 (Coulter Electronics, Inc). Serum cholesterol and triglycerides were analyzed by enzymatic methods (Boehringer-Mannheim), and CRP was determined by an immunochemical method (Orion Diagnostica).

Statistical Analysis
Values measured before and after the exercise test were analyzed by use of ANOVA for repeated measurements or Student's t test for paired data. Data for the patients and control subjects were compared by use of the Student's t test for independent samples. Data are given as mean±SD.

	Results

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The criteria for selecting patients for the study were clinically manifest intermittent claudication, the ischemic origin of which was disclosed by low ABI values, and the absence of any other acute or chronic illness. The healthy control subjects were symptom-free and had normal ABI levels and ECG recording, which were maintained during the exercise test. The resting ABI was 0.81 (range, 0.54 to 1.14) in the patient group and 1.16 (range, 1.09 to 1.25) in the control subjects (Fig 1). The maximum walking distance on a treadmill was 567 m (range, 140 to 1500 m) in the patient group and 931 m (range, 270 to 1860 m) for control subjects. After the exercise test, ABI decreased 47% in the patient group (P<.0001) but remained normal (>0.96)¹⁶ in all control subjects. The severity of PAOD in the patient group was relatively mild; all had mild to moderate claudication.

View larger version (17K): [in this window] [in a new window]   Figure 1. ABI measured before exercise (REST) and immediately after exercise (EXERCISE). Black bars represent patients and white bars represent the control subjects. #P<.0001 for patients vs control subjects at rest, patients vs control subjects after exercise; and for rest vs exercise in the patient group.

Total cholesterol and triglyceride levels, CRP, and blood cell counts were measured before the exercise test (Tables 1 and 2). The number of white cells was markedly higher in the patient group, but there was no difference in platelet number, hematocrit, CRP, cholesterol, or triglycerides.

The patients exercised until the point of unbearable claudication and the control subjects until a heart rate identical to that of the patients was reached. At the end of the exercise test, the maximal heart rate (percent of the age-dependent maximum) was 76±10% of maximum in the patient group and 76±11% in the control group (Fig 2). Before the exercise test, systolic blood pressure was somewhat higher in the patient group; this difference persisted throughout the study procedure. Plasma osmolality remained stable during the exercise: before the exercise test, it was 290±11 mOsm/kg in patients versus 292±19 mOsm/kg in control subjects, and after the exercise test, it was 285±12 mOsm/kg in patients versus 286±20 mOsm/kg in control subjects. Plasma epinephrine and norepinephrine levels did not differ between the groups either before or after the exercise test (Table 3). On average, exercise induced a 1.5-fold increase in epinephrine and a 1.7-fold increase in norepinephrine levels. Despite the relative similarity in level of exertion, plasma lactic acid concentration increased more in the patient group (Fig 2). Significant increases in platelet number and hematocrit were measured in both groups after exercise, but between the groups, the levels of these variables did not differ (Table 2). The number of white blood cells increased in both groups but remained markedly higher among the patients after the exercise test.

View larger version (16K): [in this window] [in a new window]   Figure 2. Effect of exercise on heart rate (A), systolic blood pressure (B), and plasma lactic acid concentration (C). Values were obtained with subjects in the supine position before exercise (1), immediately after exercise (2), and 30 minutes later, after bed rest (3). In A and B, represents the patients and the control subjects; in C, the black bars represent the patients and the white bars the control subjects. In A and B, #P<.0001 in both groups for time periods 2 vs 1. In B, *P<.05 for patients vs control subjects in time periods 1 to 3. In C, #P<.0001 in the patient group for time periods 2 vs 1; *P<.05 in the control group for time periods 2 vs 1 and for patients vs control subjects in time periods 2 and 3.

At rest, there was a difference between patients and control subjects regarding the levels of several parameters of coagulation and fibrinolysis. Fibrinogen concentration was higher in the patients than in the control subjects (Table 1). The difference in TAT levels did not reach statistical significance, but D-dimer was higher in the patient group (392±128 ng/mL versus 271±113 ng/mL in control subjects; P=.01) (Figs 3 and 4). Concentrations of t-PA and PAI-1 Ag were also markedly higher in the patients. On the other hand, the t-PA to PAI-1 Ag ratio, t-PA activity, and PAP levels were similar in both groups, indicating that in patients, elevated concentrations of PAI-1 were balanced by correspondingly increased t-PA levels (Fig 5 and Table 4).

View larger version (23K): [in this window] [in a new window]   Figure 3. Exercise-induced changes in TAT levels for individual patients (A) and control subjects (B) and mean levels (C) shown before exercise (REST) and immediately after exercise (EXERCISE). Horizontal line: upper limit of normal values (4.1 ng/mL). In C, black bars represent patients and white bars the control subjects. In C, **P<.01 for patients vs control subjects during exercise; and *P<.05 in patient group for rest vs exercise.

View larger version (25K): [in this window] [in a new window]   Figure 4. Exercise-induced changes in D-dimer levels for individual patients (A) and control subjects (B), and mean levels (C) shown before exercise (REST) and immediately after exercise (EXERCISE). Horizontal line: upper limit of normal values (400 ng/mL). In C, black bars represent patients and white bars the control subjects. In C, **P<.01 for rest vs exercise in patients and *P<.05 for rest vs exercise in control subjects and for patients vs control subjects in rest and in exercise.

View larger version (20K): [in this window] [in a new window]   Figure 5. Exercise-induced changes in t-PA (A) and PAI-1 Ag (B), with mean results shown before exercise (1), immediately after exercise (2), and 30 minutes after exercise (3). Black bars represent patients and white bars the control subjects. In A, ***P<.001 for time periods 2 vs 1 in both patients and control subjects; **P<.01 for patients vs control subjects in time period 2; and *P<.05 for patients vs control subjects in time periods 1 and 3. In B, ***P<.001 in time period 2 for patients vs control subjects; **P<.01 in time period 1 for patients vs control subjects and in patients in time periods 2 vs 3; and *P<.05 in time period 3 for patients vs control subjects.

View this table: [in this window] [in a new window]   Table 4. T-PA to PAI-1 Ag Ratio, t-PA Activity, and Plasmin-2–Antiplasmin Complex (PAP) Before and After Exercise

Intriguingly, exercise induced changes in the TAT that reflected thrombin generation in the patient group. After exercise, half the patients but only one of the control subjects had TAT levels exceeding normal reference values. Patients with the highest TAT levels also had the highest concentrations of D-dimer. Exercise induced a small but consistent increase in D-dimer in both groups, but the mean level of D-dimer remained higher in the patients after exercise. In the patient group, D-dimer increased from 392±128 to 413±137 ng/mL (P<.003), and in the control group, it increased from 271±113 to 286±116 ng/mL (P<.02). After the exercise test, the level of D-dimer was above normal reference values in half of the patients, whereas only 3 of 15 control subjects had above-normal values. The circulating fibrinolytic enzyme t-PA increased significantly as a consequence of exercise in both groups (Fig 5) and returned to resting levels 30 minutes later, whereas the level of PAI-1 remained constant or even decreased slightly. Consequently, the t-PA to PAI-1 Ag ratio and both t-PA activity and PAP levels were significantly higher after exercise compared with the resting state in both groups (Table 4).

	Discussion

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In this study, we report that while at rest, patients with peripheral atherosclerosis already showed signs of abnormal upregulation of both coagulation and fibrinolysis in the form of elevated fibrinogen, t-PA, PAI-1, and D-dimer levels (Table 1 and Figs 4 and 5), as has been reported before.^{17 18 19} Increased PAI-1 is associated with all forms of atherothrombosis,^{20 21 22} and continuous fibrin deposition on the atherosclerotic vessel wall appears to stimulate endogenous fibrinolysis, reflected in elevated t-PA levels.¹⁷ We also found that there was no difference between the patients and healthy age-, sex-, medication-, and cardiovascular risk factor–matched control subjects in resting levels of t-PA to PAI-1 Ag ratio, t-PA-activity, or PAP. In spite of this similarity in fibrinolytic potential measured from plasma, D-dimer was significantly higher in the patients, probably because of the increased amount of preexisting fibrin.

In our study, submaximal physical exercise led to increased thrombin generation in the patient group, whereas similar exercise levels failed to affect the levels of TAT in the control subjects. The simultaneous rise in TAT and D-dimer in half the patients would argue for generation of new fibrin during exercise. However, the small and consistent exercise-induced increase in D-dimer, which was also observed in the control subjects, may equally reflect degradation of preexistent fibrin as a consequence of activated fibrinolysis (Figs 4 and 5 and Table 4). In our study, submaximal physical exertion augmented fibrinolytic activity to a similar extent in both healthy control subjects and patients with PAOD (as measured by t-PA to PAI-1 Ag ratio, t-PA activity, D-dimer, and PAP) despite the finding that TAT was elevated only in the patients (Fig 3). This finding may reflect the prothrombotic potential of the pathological arterial wall.²³

Differences observed between our patients and control subjects, groups that were well matched regarding other factors known to affect blood coagulation and fibrinolysis,^{24 25} thus seem to reflect the influence of atherosclerosis. Age and sex distribution and mean BMI were similar in both groups (Table 1). Patients and control subjects did not differ in smoking habits or alcohol consumption. Furthermore, the regular aerobic exercise taken by the members of both groups was of similar quantity, eliminating the disturbing effect of differences in conditioning.^{26 27} Most of the PAOD patients were permanently taking ASA medication, and because of the well-known effects of ASA on platelet function and hemostasis,²⁸ the respective control subjects were also told to take this same medication for 2 weeks before the study, a period representing the life span of platelets. To further minimize any interference by medication, individuals who used ß-blockers or other medications known to affect hemostasis or the metabolism of catecholamines were excluded from the study.²⁹

At rest, depending on the severity of PAOD in the patients involved, various degrees of activation of the coagulation cascade have been reported previously by our group and others.^{17 18} The effect of exercise on hemostasis in patients with peripheral arterial disease has also been studied previously.¹⁹ In contrast to our results, the PAOD patients in the study by Herren et al¹⁹ showed enhanced thrombin formation (TAT) at rest compared with the age- and sex-matched healthy control group. Differences between the study populations may explain this dissimilarity: PAOD in the patients in the study by Herren et al¹⁹ was more severe than in our patient group; at the same time, the share of current smokers and hypertensives among their patients was significantly higher than in their control group. Additionally, Herren et al¹⁹ did not see any elevation of D-dimer or TAT after exercise, a result that also differed from ours. Direct comparisons cannot be made, however, because the patient population differed somewhat from ours, and the extent of their tested exercise remained unreported.

It is known that the intensity of exercise has profound effects on the reactivity of the hemostatic system.^{2 30 31} To compare the patients and the control subjects in the present study, both groups exercised to the same relative level, ie, the patients to the level of unbearable claudication and the control subjects to the corresponding heart rate.^{5 27} The mean percentage of age-dependent maximal heart rate was 76%, ranging from 53% to 92%. The patients showed higher lactic acid concentrations after the exercise test despite similar exercise intensity, as also reported previously.³² This reflects the exercise-induced ischemia of the lower limbs.

Levels of plasma catecholamines in the present study, both at rest and after exercise, are in accordance with results reported previously (Table 3).^{4 33} The exercise-induced changes in catecholamine concentrations in the two groups were similar, which implies that the intensity of exercise was equal. Catecholamines are known to affect fibrinolysis by stimulating endothelial cells to release t-PA,⁸ as well as stimulating coagulation by liberating factor VIII and vWF into the bloodstream.^{7 34} During exercise, release of t-PA begins when 50% of maximal exercise intensity is reached, whereas factor VIII and vWF are not liberated until 95% to 100% of maximal oxygen consumption is achieved.² In the presence of atherosclerosis in our study, signs of enhanced coagulation were already observed as a consequence of exercise with mean maximal intensity of 76%. This might be related to platelet activation, because several studies have suggested an association between platelet activation and adrenergic stimulation during exercise both in healthy subjects^{6 26 29 35} and in patients with coronary artery disease.³⁶ Adrenergic stimulation has direct effects on platelets via ₂-adrenergic receptors³⁷ but at physiological concentrations more apparent under flow conditions afforded in atherosclerotic vessels.^{9 10} Notably, aspirin inefficiently counteracts the platelet activation induced by catecholamines in vivo.^{38 39}

The prothrombotic tendency that appeared in our PAOD patients after exercise can be assumed to result from several additive factors. Even at rest, atherosclerosis is associated with upregulation of both coagulation and fibrinolysis. Progressive local ischemia provoked by the treadmill test can further modify this upregulation. During exercise, circulating platelets that come in contact with atherosclerotic arterial wall under pathological shear forces may be activated further by elevated catecholamines. If the intensity of exercise, under these pathological conditions, exceeds the level achieved in the present study, the reported release of vWF and factor VIII⁴⁰ would be yet an additional pertinent prothrombogenic factor.^{41 42} Sudden strenuous exercise thus seems to increase the risk of thrombosis in atherosclerotic patients.

	Selected Abbreviations and Acronyms

 

ABI = ankle-brachial systolic blood pressure index Ag = antigen ASA = acetylsalicylic acid BMI = body mass index CRP = C-reactive protein PAI = plasminogen activator inhibitor PAOD = peripheral arterial occlusive disease PAP = plasmin-2–antiplasmin complex TAT = thrombin–antithrombin III complex t-PA = tissue-type plasminogen activator vWF = von Willebrand factor

	Acknowledgments

 
This study was financially supported by the Finnish Heart Research Foundation and the Aarne Koskelo Foundation. The excellent technical assistance of Marita von Bell, Tuula Järvenpää, and Päivikki Määttälä is acknowledged. Vesa Manninen is thanked for his constant encouragement.

Received June 26, 1997; accepted October 8, 1997.

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