Cécile H. Kicken

The flavanol (-)-epicatechin and its metabolites protect against oxidative stress in primary endothelial cells via a direct antioxidant effect

Accumulating evidence suggests that foods rich in flavanols decrease the risk of developing cardiovascular diseases. Attenuation of oxidative stress was suggested to contribute to the cardiovascular benefit of flavanols. Up to now it was unclear whether flavanol metabolites can also protect cells from oxidative stress. The aim of the present study was to determine the potential contribution of several glucuronidated, methylated and sulfated metabolites of (-)-epicatechin (EC) and (+)-catechin (Cat) to the protection of human vascular endothelial cells (HUVECs) against oxidative stress. The relative potency of the tested compounds to scavenge superoxide anion radicals showed that a free catechol moiety in the molecule is important for the direct antioxidant activity. EC and Cat (0.5, 1, 10µM) were potent radical scavengers and provided protection against intracellular oxidative stress induced by hydrogen peroxide. Although the metabolites provided less intracellular protection compared to EC and Cat, the tested methylated and glucuronidated metabolites reduced oxidative stress significantly in HUVECs. Our results indicate that the metabolites have a relevant contribution in the intracellular protection of EC and Cat against oxidative stress. Also, the direct antioxidant activity plays an important role in this protection.

Response of platelet concentrates to pressure and temperature changes without impairment of the in vitro function

BACKGROUND Rapid platelet concentrate (PC) transfusion is crucial for hemostatic resuscitation. Pressure-aided and warmed transfusion combined with pneumatic tube system (PTS) transport from the laboratory to the operating theatre offers a potentially rapid delivery technique. The aim of this study is a quantitative assessment of in vitro platelet function after PTS transport followed by warmed and/or pressure-aided mock transfusions. METHODS Ten PC samples entered a single PTS run and were subsequently aliquoted for testing. PCs were warmed in a blood warmer and/or subjected to pressure-aided mock transfusion at 300 mm Hg on day 2 or day 7 after collection. Platelet function was assessed using light-transmission aggregometry and multiple-electrode aggregometry to measure the response to ADP, arachidonic acid, collagen, and thrombin-receptor activating peptide. Data were analyzed with non-parametric testing; P<0.05 was considered statistically significant. RESULTS Single PTS transport markedly reduced ADP response in fresh PCs. Seven-day storage had a pronounced effect on both ADP and collagen response. All other tested platelet agonists revealed preserved function. Subsequent warming and/or application of pressure did not significantly compromise platelet function. CONCLUSIONS Pressure-aided plus warmed transfusion and PTS transport was not found to be detrimental to the PC. Further clinical studies are required to determine safety and efficacy of the product.

Hypoxia Induces a Prothrombotic State Independently of the Physical Activity

Hypoxia (oxygen deprivation) is known to be associated with deep vein thrombosis and venous thromboembolism. We attempted to get a better comprehension of its mechanism by going to high altitude, thereby including the potential contributing role of physical activity. Two groups of 15 healthy individuals were exposed to hypoxia by going to an altitude of 3900 meters, either by climbing actively (active group) or transported passively by cable car (passive group). Both groups were tested for plasma fibrinogen, von Willebrand factor and factor VIII levels, fibrinolysis, thrombin generating capacity, heart rate, oxygen saturation levels and blood pressure. As a control for the passive group, 7 healthy volunteers stayed immobile in bed for 7 days at normoxic conditions. The heart rate increased and oxygen saturation levels decreased with increasing altitude. Fibrinolysis and fibrinogen levels were not affected. Factor VIII and von Willebrand factor levels levels increased significantly in the active group, but not in the passive group. Plasma thrombin generation remained unchanged in both the active and passive group with increasing altitude and during 7 days of immobility in healthy subjects. However, by applying whole blood thrombin generation, we found an increased peak height and endogenous thrombin potential, and a decreased lagtime and time-to-peak with increasing levels of hypoxia in both groups. In conclusion, by applying whole blood thrombin generation we demonstrated that hypoxia causes a prothrombotic state. As thrombin generation in plasma did not increase, our results suggest that the cellular part of the blood is involved in the prothrombotic phenotype induced by hypoxia.

Application of an optimized flow cytometry-based quantification of Platelet Activation (PACT) : Monitoring platelet activation in platelet concentrates

Background Previous studies have shown that flow cytometry is a reliable test to quantify platelet function in stored platelet concentrates (PC). It is thought that flow cytometry is laborious and hence expensive. We have optimized the flow cytometry-based quantification of agonist induced platelet activation (PACT) to a labor, time and more cost-efficient test. Currently the quality of PCs is only monitored by visual inspection, because available assays are unreliable or too laborious for use in a clinical transfusion laboratory. Therefore, the PACT was applied to monitor PC activation during storage. Study design and methods The optimized PACT was used to monitor 5 PCs during 10 days of storage. In brief, optimized PACT uses a ready-to-use reaction mix, which is stable at -20°C. When needed, a test strip is thawed and platelet activation is initiated by mixing PC with PACT. PACT was based on the following agonists: adenosine diphosphate (ADP), collagen-related peptide (CRP) and thrombin receptor-activating peptide (TRAP-6). Platelet activation was measured as P-selectin expression. Light transmission aggregometry (LTA) was performed as a reference. Results Both PACT and LTA showed platelet function decline during 10-day storage after stimulation with ADP and collagen/CRP; furthermore, PACT showed decreasing TRAP-induced activation. Major differences between the two tests are that PACT is able to measure the status of platelets in the absence of agonists, and it can differentiate between the number of activated platelets and the amount of activation, whereas LTA only measures aggregation in response to an agonist. Also, PACT is more time-efficient compared to LTA and allows high-throughput analysis. Conclusion PACT is an optimized platelet function test that can be used to monitor the activation of PCs. PACT has the same accuracy as LTA with regard to monitoring PCs, but it is superior to both LTA and conventional flow cytometry based tests with regard to labor-, time- and cost efficiency.

Early platelet recovery following cardiac surgery with cardiopulmonary bypass

Abstract Coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB) is frequently associated with low platelet count (PC) and disturbed platelet function (PF). While PC is easy to measure, PF is more difficult to assess. Moreover, the time-related platelet dysfunction and recovery after CPB is not fully elucidated. Platelet dysfunction could lead to bleeding but also to coronary graft failure. Laboratory tests could provide more insights into PF after CABG. The aim of the current study was to investigate the time-related PF induced by CPB. Blood samples of 20 patients with a preoperative PC of more than 250 × 109/L were collected before incision, after weaning from CPB, and 24 h postoperative. Platelet contribution to coagulation was quantified by PLTEM (calculated by means of EXTEM and FIBTEM results). PF was assessed by multiple electrode impedance aggregometry (MEIA) in whole blood and by light transmission aggregometry (LTA) in platelet-rich plasma after stimulation with arachidonic acid (AA), adenosine diphosphate, collagen, and thrombin-receptor-activating peptide. LTA and MEIA analysis demonstrated significant platelet dysfunction after CPB, with partial recovery within 24 h after surgery. AA-induced platelet aggregation increased to higher levels within 24 h after surgery compared to baseline values as measured by LTA. PLTEM maximum clot firmness remained unchanged throughout the study. Correlation analyses revealed that MEIA and rotational thromboelastometry (ROTEM), but not LTA, were dependent on PC and hematocrit. No correlations were found between LTA, MEIA, ROTEM, PC, and clinical outcome parameters. Our results demonstrate a reversible platelet dysfunction recovering within 24 h after CPB. Interestingly, AA-induced platelet aggregation increases to higher levels during the first 24 h postoperatively, which might be important for early initiation of antiplatelet therapy after CABG. MEIA as POC test is able to detect platelet dysfunction during cardiac surgery with a PC of ≥150 × 109/L.

Platelet Function During Hypothermia in Experimental Mock Circulation.

Alterations in platelet function are a common finding in surgical procedures involving cardiopulmonary bypass and hypothermia. Although the combined impact of hypothermia and artificial circulation on platelets has been studied before, the ultimate strategy to safely minimize the risk for bleeding and thrombosis is yet unknown. The aim of this study was to evaluate the use of a mock circulation loop to study the impact of hypothermia for platelet-related hemostatic changes. Venous blood was collected from healthy adult humans (n = 3). Closed mock circulation loops were assembled, each consisting of a centrifugal pump, an oxygenator with integrated heat exchanger, and a hardshell venous reservoir. The experiment started with the mock circulation temperature set at 37°C (T0 [0 h]). Cooling was then initiated at T1 (+2 h), where temperature was adjusted from 37°C to 32°C. Hypothermia was maintained from T2 (+4 h) to T3 (+28 h). From that point in time, rewarming from 32°C to 37°C was initiated with similar speed as cooling. From time point T4 (+30 h), normothermia (37°C) was maintained until the experiment ended at T5 (+32 h). Blood samples were analyzed in standard hematological tests: light transmission aggregometry (LTA) (arachidonic acid [AA], adenosine diphosphate [ADP], collagen [COL], thrombin-receptor-activating-peptide-14 [TRAP]), multiple electrode aggregometry (MEA) (AA, ADP, COL, TRAP), and rotational thromboelastometry (ROTEM) (EXTEM, FIBTEM, PLTEM). Hemoglobin, hematocrit, and platelet count decrease more substantially during temperature drop (37-32°C) than during hypothermia maintenance. Hb and Hct continue to follow this trend during active rewarming (32-37°C). PC increase from the moment active rewarming was initiated. None of the values return to the initial values. LTA values demonstrate a similar decrease in aggregation after stimulation with the platelet agonists between the start of the mock circulation and the start of cooling. Except for platelet stimulation using COL, this trend continues during temperature drop from 37°C to 32°C. LTA values using AA and TRAP demonstrate a considerable decline in platelet function throughout the experiment that was most pronounced after 24 h of circulation at 32°C. LTA values using ADP and COL further decline after rewarming. MEA ADP, ASPI, and COL identify platelet dysfunction patterns analogous with LTA, between the start of the mock circulation and the start of cooling. Except for MEA TRAP, this trend continues during temperature drop from 37°C to 32°C. MEA ASPI and ADP demonstrate a considerable decline in platelet function throughout the experiment, which was most pronounced after 24 h of circulation at 32°C. For MEA COL and TRAP, further decline in platelet function is observed after rewarming. This study quantitatively assessed the effect of temperature changes on platelet function during experimental mock circulation demonstrating a considerable decline in platelet function during hypothermia without uniform recovery of platelet function observed after rewarming.

Hypobaric Hypoxia Causes Elevated Thrombin Generation Mediated by FVIII that is Balanced by Decreased Platelet Activation

INTRODUCTION Epidemiological studies suggest that hypobaric hypoxia at high altitude poses a risk for developing venous thromboembolism. The cause of this observed hypercoagulability remains unclear. Therefore, this study aimed to investigate the effect of hypobaric hypoxia at 3,883 m above sea level on thrombin generation and platelet activation. METHODS After complying with medical ethical procedures, 18 participants were recruited, of whom 1 had to leave the study prematurely due to mild acute mountain sickness. Blood was drawn first at 50 m above sea level and second at 3,883 m altitude after gradual acclimatization for 6 days. Thrombin generation was measured in whole blood, platelet-rich plasma and platelet-poor plasma. Platelet activation was assessed using a whole blood flow-cytometric assay. Coagulation factor levels, D-dimer levels and markers of dehydration and inflammation were measured. RESULTS Hypobaric hypoxia at 3,883 m altitude caused increased thrombin generation, measured as peak height and endogenous thrombin potential, in whole blood, platelet-rich and platelet-poor plasma without or at low tissue factor concentration. The elevated thrombin generation was mediated by increased factor VIII levels and not caused by dehydration or inflammation. In contrast, spontaneous and agonist-induced platelet activation was decreased at high altitude. CONCLUSION Hypobaric hypoxia causes increased factor VIII-mediated thrombin generation. The hypercoagulability was balanced by decreased platelet activation. These findings may explain why venous, and not arterial thrombotic events occur more frequently at high altitude.

Effects of Repeated Bouts of Exercise on the Hemostatic System

Abstract Physical activity is beneficial for health, for example, by lowering the risk of cardiovascular events. However, vigorous exercise is associated with the occurrence of thromboembolic events and sudden cardiac death, in particular in untrained individuals. Whereas acute exercise is known to cause a hypercoagulable state, repeated exposure to (strenuous) exercise by means of training may actually condition the hemostatic response to exercise. To date, the effects of exercise training on blood coagulability and the underlying mechanisms have yet to be fully discerned. In this review, the authors provide an overview of existing literature on how training programs and training status influence hemostasis in healthy individuals. Furthermore, they present data of a pilot study in which we studied the effects of repetitive submaximal intensity cycling on procoagulant and anticoagulant processes. It is known that factor VIII (FVIII) and von Willebrand factor (VWF) increase after exercise, but we found that this increase in FVIII and VWF (antigen, propeptide, and VWF in active conformation) was smaller on each of three subsequent days, suggesting either adaptation of endothelial activation or exhaustion of endothelial VWF supplies. With respect to thrombin generation, elevated FVIII significantly increased the thrombin generation peak but not the endogenous thrombin potential. In contrast, platelet activation in terms of P‐selectin expression after stimulation with protease‐activated receptor‐1 and glycoprotein VI agonists decreased after exercise and did not recover, indicating exhaustion of the platelet response to repetitive exercise.

Hemostasis during Extreme Exertion

Abstract Exercise is protective against cardiovascular disease, but can also provoke sudden cardiac death, a phenomenon referred to as “the exercise paradox.” Extreme exertion is known to induce a rebalanced hemostatic state by causing hypercoagulability and concomitantly enhanced fibrinolysis. Over the past decade, novel techniques for quantifying hemostasis have been introduced, which may provide new insights into this process. This review summarizes recent literature on the effect of extreme exertion of both short and long duration on coagulation, fibrinolysis, and recovery of hemostatic balance. Extreme exertion introduces a temporary hypercoagulable state, mainly through upregulation of the contact pathway by increased FVIII levels. Additionally, von Willebrand factor levels and platelet activation are increased. Simultaneously, increased fibrinolysis results from increased tissue‐type plasminogen activator levels, suggesting a rebalanced hemostasis. The vascular endothelium appears to play a pivotal role in both processes. Data on the effect of exercise on endogenous anticoagulants are scarce. Hypercoagulability persists for hours to a day after prolonged extreme exertion, while fibrinolytic parameters appear to return to baseline levels more quickly. Hence, the balance in the rebalanced hemostatic state may be lost during recovery. Training induces lower amplitude of the hypercoagulable response, and quicker recuperation toward baseline. Repetitive exercise exhausts the endothelium, attenuating procoagulant changes. Additional research is needed to identify if the hemostatic balance is lost during recovery, and if so, when the shift toward thrombosis appears. Moreover, differences between sexes need to be addressed, since women are known to have a different pathophysiological mechanism behind cardiovascular events, but are underrepresented in recent literature.