Doreen Gläser

Transcription in response to physical stress— clues to the molecular mechanisms of exercise-induced asthma

To clarify stress‐induced immunological reactions and molecular events during exercise and the potential relevance to exercise‐induced bronchoconstriction, transcriptional responses to standardized physical stress were determined. Six healthy, young volunteers underwent an endurance exercise of 90% of their individual anaerobic threshold for 90 min. Time‐dependent alterations in the expression pattern of leukocytes from healthy, trained subjects were analyzed by DNA microarrays before and 2 h and 6 h after exercise. Starting out from a large collection of cDNA library clones comprising more than 70,000 human expressed sequence tags, we selected, designed, and immobilized oligonucleotide probes (60–70mers) for transcripts of 5000 stress‐and inflammation‐relevant genes. Exercise‐induced stress provoked changes in the expression of 433 gene activities 2 h and/or 6 h after exercise, which could be grouped into six clusters. The most prominent feature was an enhanced transcription of two genes, coding for 5‐lipoxygenase (ALOX5) and ALOX5‐activating protein. Moreover, enhanced levels of leukotriene B4 (LTB4) and LTC4 (P<0.05) were detected in plasma after exercise. Our data demonstrate that exercise alters the activities of a distinct number of genes. In particular, they possibly provide novel insights into the molecular mechanisms of exercise‐induced bronchoconstriction and suggest that enhanced transcription of ALOX5 and its activating protein together with a present predisposition of the subject critically contribute to exercise‐induced asthma.

Blood coagulation and fibrinolysis after extreme short-term exercise

INTRODUCTION Maximal exercise may be a trigger for cardiovascular events. The aim of the study was to investigate changes in blood coagulation and fibrinolysis following maximal short-term exercises with different durations up to 90 s. METHODS A total of 15 healthy nonsmokers underwent three isokinetic maximal tests on an SRM cycle ergometry system with durations of 15, 45, and 90 s. Blood samples were taken after a 30-min rest, immediately before and after exercise, 15 min, and 1 h after completion of exercise. For the investigation of blood coagulation, prothrombin fragment 1+2 (F1+2), thrombin-antithrombin III complex (TAT), intrinsic and extrinsic total (TTPin+ex), and endogenous thrombin potential (ETPin+ex) were measured. For testing fibrinolysis, determinations of plasmin-alpha(2)-antiplasmin complex (PAP), tissue-type plasminogen activator (tPA)-antigen, plasminogen activator inhibitor (PAI)-1-antigen and D-dimer were used. RESULTS Immediately after the exercise tests, only F1+2 (15- and 90-s test) and TTPin (45 and 90 s) showed a moderate increase (p<0.05), while TAT and ETP was unchanged. In contrast, a clear increase in PAP and tPA-antigen already after 15 s maximal exercise in relation to the exercise duration time could be investigated. These effects were not totally reversed to baseline 15 min after exercise; D-dimer and PAI-1-antigen still remained unchanged after these types of exercise. CONCLUSIONS Maximal short-term exercise does not lead to a relevant activation of blood coagulation in healthy young subjects, it is only slightly altered within the normal range. In contrast, fibrinolysis is clearly activated, and the increase is directly dependent on exercise duration. Additionally, it could be shown for the first time that fibrinolysis is already activated after 15 s maximal exercise duration.

Paraformaldehyde fixation induces a systematic activation of platelets.

In whole blood flow cytometric platelet assays sample fixation using paraformaldehyde (PFA) is considered very advantageous to prevent spontaneous activation of platelets in vitro. However, fixation is an important variable in activation assays and its influence on platelets is poorly understood. Using a direct immunofluorescence labelling technique and whole blood flow cytometry, the effect of PFA fixation was investigated for 4 different epitopes on platelet surface each of which mirrors a different aspect of platelet activation, namely P-selectin (CD62P), GP IIbIIa complex (CD41), the fibrinogen binding site of the activated GP IIaIIIb complex (PAC-1) and GP Ib-V-IX complex (CD42b). Platelets fixed with PFA (0.5%) before antibody labelling showed significant (P < 0.01) increases in mean fluorescence intensity (MFI) of CD62P (1.10 ± 0.14 vs. 0.94 ± 0.12 arbitrary units of fluorescence), CD41 (27.3 ± 6.3 vs. 15.6 ± 2.1) and PAC-1 (6.21 ± 1.25 vs. 0.55 ± 0.12) when compared to unfixed samples. At the same time, MFI of CD42b was reduced from 28.2 ± 1.6 to 22.6 ± 2.3 (P < 0.01). When fixation was initiated after antibody labelling, we observed less prominent increases in MFI of CD41 (P < 0.05) and PAC-1 (P < 0.05) while there was no significant difference for CD62P and rather a moderate rise in CD42b than a decrease (P < 0.05). Because these alterations cannot be explained by unspecific effects only, it must be concluded that PFA induces a systematic stimulation of platelets. The lowest in vitro platelet activation was found when antibody labelling was started immediately after blood sampling and when samples were analysed within 10 minutes after being stored without fixation of 4 degrees C in the dark.

Short-term exercise and platelet activity, sensitivity to agonist, and platelet-leukocyte conjugate formation

Strenuous exercise may be partially responsible for cardio-vascular events. The aim was to investigate the platelet activity, reactivity and different platelet-leukocyte-conjugate formation following maximal short-term exercises. Fifteen healthy non-smokers underwent three isokinetic maximal tests on a SRM cycle ergometry system with durations of 15, 45 and 90 s. Blood samples were taken after a 30-min rest, immediately before and after exercise, and 15 min and 1 h after completion of exercise. Platelets were detected flow-cytometrically by CD41, and activated platelets by CD62P. In addition, stimulation of the platelets in vitro with 7.5 μM TRAP-6 was initiated. For testing platelet-leukocyte-conjugates, antibodies against CD45, CD14 and CD41 were used. After the exercise tests the percent of non-stimulated CD62P-positive platelets (%PC) was unchanged. In contrast, an increase in %PC (CD62P) TRAP-6 stimulated (15-s test: 37.2±10.3 to 46.2±12.3%, P < 0.05; 90-s test: 40.6±9.5 to 51.7±10.2%, P < 0.01) and in platelet-granulocyte, platelet-lymphocyte, and platelet-monocyte conjugate formation 15 min after exercise (45- and 90-s test; P < 0.05) were observed in comparison with the changes on the control day. The changes nearly reversed 1 h after exercise. Maximal short-term exercise only leads to a moderate increase of platelet reactivity and to an increase in the different platelet-leukocyte conjugates. The implications of the changes in platelet-leukocyte conjugate formation should be investigated in future studies.

The influence of bromelain on platelet count and platelet activity in vitro

Bromelain is a general name for a family of sulfhydryl-containing, proteolytic enzymes from the pineapple plant. The aim of the present study was to investigate the influence of bromelain on platelet count, platelet aggregation and platelet activity in vitro. Blood samples were taken from the antecubital vein of 10 healthy male non-smokers. Platelet count decreased after incubation with 2.5 and 5 mg bromelain/ml from 277 ± 17 platelets/nl before to 256 ± 21 and 247 ± 19 platelets/nl after the treatment. The ADP and TRAP-6 induced platelet aggregation led to a significant decrease after the incubation with 2.5 mg (ADP: 48.6 ± 25.7%; TRAP-6: 49.6 ± 28.9%) or 5 mg (ADP: 5.0 ± 4.6%; TRAP-6: 9.0 ± 4.9%) bromelain/ml in comparison to control (ADP: 81.4 ± 5.0%; TRAP-6: 77.4 ± 10.4%). The percentage of unstimulated CD62P positive platelets which were investigated by flow cytometry was minimally higher after incubation with 5 mg bromelain/ml (0.57 ± 0.48% PC) in comparison to control (0.22 ± 0.11% PC), but after TRAP-6 stimulation the incubation with 5 mg bromelain/ml led to a remarkable decrease in comparison to the untreated control (50.4 ± 20.2 to 0.9 ± 0.8% PC). The changes of CD62P (TRAP-stimulated) and the results of platelet aggregation after incubation with bromelain in vitro may demonstrate the potential of bromelain as a substance for platelet inhibition.

Blood coagulation and fibrinolysis before and after exhaustive exercise in patients with IDDM

Diabetes mellitus involves changes in haemostasis which leads to the opinion that diabetes mellitus is a hypercoagulable state. However, little is known about the relationship of exercise and haemostasis in diabetics. Therefore, first of all the aim was to investigate if differences in blood coagulation and fibrinolysis can be demonstrated in subjects with insulin-dependent diabetes mellitus (IDDM) compared to controls and secondly, if differences concerning exercise induced changes can be seen in diabetics. 16 moderately fit subjects with IDDM and 16 matched controls underwent a maximal step test. Blood samples were taken after a 30 min rest, immediately and 1h after exercise and in addition after 30 min rest 7 days later at the same time of day. The rest values (mean of the two rest samples) in extrinsic total thrombin potential (TTPex, P=0.049), tPA-activity (P=0.007) were significantly higher and in PAI-1-antigen (P=0.002) -activity (P=0.049) lower in the diabetic group. APTT, PT, TAT (only control), TTPin, tPA-activity and -antigen and PAP were increased immediately and D-dimer (only control) 1 h after exercise, whereas PAI-1-activity and -antigen (only control) decreased immediately or 1 h after exercise (all minimal P<0.05). The increase of tPA-antigen and decrease in PAI-1-antigen after exercise were both lower in the diabetics (P<0.05). IDDM led to higher extrinsic total thrombin and fibrinolytic potential at rest, and reducing the exercise provoked distribution of tPA-antigen and decrease of PAI-1-antigen. Nevertheless a higher thrombotic risk after maximal exercise has not been investigated in young IDDM patients without complications and in good metabolic control.

Platelet activity, reactivity and platelet-leukocyte conjugate formation before and after exhaustive or moderate exercise in patients with IDDM.

Diabetes mellitus alters blood coagulation and platelet function which supports the suggestion that diabetes mellitus is a hypercoagulable state. Firstly the aim of the study was to investigate if differences in platelet activity, reactivity and platelet-leukocyte conjugate (PLC) formation can be observed in subjects with IDDM; secondly, if differences can be seen between the diabetic and control group concerning exercise-induced changes in platelet activation and conjugate formation; and thirdly, if different types of exercise lead to different patterns in platelet activation. Sixteen subjects with IDDM and 16 controls underwent a maximal step test and an endurance test (90% IAT, 45 min). Blood samples were taken after 30 min rest, and immediately and 1 h after completion of exercise. CD62P expression and differentiated platelet-leukocyte conjugates (CD45, CD14, CD41) were detected flow-cytometrically with and without stimulation with TRAP-6. The rest values of the platelet-granulocyte (PGC) and platelet-lymphocyte conjugates (PLyC) were higher (P<0.05) in the diabetics. After exercise, platelet reactivity (CD62P-TRAP; P<0.05) but not the activity (CD62P-unstimulated), as well as all different conjugates with or without stimulation were increased (P<0.05) independently from the group. Differences according to the type of exercise were barely observable. IDDM without vascular complications leads to higher PCG and PLyC at rest and to identical increases in differentiated platelet-leukocyte formation after exercise in comparison with matched controls.