T Cell-based RAS Activity and Insulin Levels in Obese Subjects with Low Grade Inflammation.

BACKGROUND: Obesity is a major contributor to inflammation and oxidative stress that are key underlying causes for insulin resistance (IR) and diabetes. Accumulated evidence suggest that RAS may serve as a strong link between IR and obesity. We investigated RAS activity in circulating T cells by obese subjects with and without angiotensin (Ang) II stimulation in presence or not of IR and of low-grade inflammation. METHODS: We studied 29 obese and 10 healthy subjects. After T-lymphocytes isolation, mRNAs for angiotensin converting enzyme (ACE) and angiotensin 1-receptor (AT1-R) were quantified by reverse transcription polymerase chain reaction (RT-PCR). High-sensitivity C-reactive protein (hs-CRP), insulin and inflammatory cytokines serum levels, plasma renin activity (PRA) and ACE activity in cell pellet and supernatant, and angiotensin (Ang) II T cell content were also measured. RESULTS: Under baseline conditions, RAS gene expressions, ACE activity and Ang II levels in T cells, but not PRA, of obese subjects with or without IR and with or without hs-CRP ≥3mg/dl were higher than in controls (p < 0.05). The increase in all parameters induced by Ang II was significantly higher in T cells from the obese subjects with hs-CRP ≥3 mg/dl than in controls or in the obese subjects with hs-CRP <3 mg/dl. In the obese subjects with low grade inflammation and IR, the cytokine serum levels and T cells RAS gene expression was inversely correlated with insulin serum concentration. CONCLUSIONS: Low grade inflammation amplifies the T cell RAS response to Ang II stimulation. T cell RAS gene expressions and serum levels of inflammatory cytokines were inversely related with insulin serum concentration. A protective role of insulin towards the development of inflammatory events can be hypothesized.

Zr and U determination at trace level in simulated deep groundwater by Q ICP-MS using extraction chromatography.

In the framework of trace element analysis by Q ICP-MS in a simulated deep Callovo-Oxfordian groundwater, separation procedures based on extraction chromatography were developed to eliminate the high salt contents and to concentrate Zr and U simultaneously. Theoretical and experimental speciation studies showed the importance of adjusting the medium to HNO3/HF (0.5 M/0.005 M) to guarantee the stability over time of the analytes before removal of the matrix. Two preconcentration methods based on TRU and TODGA resins were optimized for the simultaneous isolation of Zr and U prior to Q ICP-MS measurements. Using TRU resin, alkali and alkali earth metals contained in the deep groundwater were removed with 2 M HNO3 whereas Zr and U were recovered with a HNO3/NH4HC2O4 (0.02 M/0.05 M) medium. For the separation protocol based on TODGA resin, alkali and alkali earth metals were eliminated with 3 M and 11 M HNO3 while Zr and U were simultaneously stripped with a HNO3/HF (0.5 M/0.2 M) medium. The procedure optimized on TODGA resin was validated with the French AFNOR NF T90-210 standard by studying linearity, limits of quantification (LOQ) and separation yields. The LOQ was determined at 0.008 µg L(-1) for Zr and U after the separation. Both analytes were recovered quantitatively. Compared to a sample dilution implemented to reduce the matrix effects, the developed preconcentration method allowed improving the sensitivity up to a 20 fold factor for Zr and U measurements at trace level by Q ICP-MS.

Increased cardiac sympathetic activity and insulin-like growth factor-I formation are associated with physiological hypertrophy in athletes.

Physiological hypertrophy represents the adaptive changes of the heart required for supporting the increased hemodynamic load in regularly trained healthy subjects. Mechanisms responsible for the athlete's hypertrophy still remain unknown. In 15 trained competitive soccer players and in 15 healthy men not engaged in sporting activities (sedentary control subjects) of equivalent age, we investigated the relationship among cardiac growth factor formation, cardiac sympathetic activity, and left ventricular morphology and function. Cardiac formation of insulin-like growth factor (IGF)-I, endothelin (ET)-1, big ET-1, and angiotensin (Ang) II was investigated at rest by measuring artery-coronary sinus concentration gradients. Cardiac sympathetic activity was studied by [(3)H]norepinephrine (NE) kinetics. Cardiac IGF-I, but not ET-1, big ET-1, and Ang II, formation was higher in athletes than in control subjects (P<0.01). NE levels in arterial and peripheral venous blood did not differ between groups. In contrast, coronary sinus NE concentration was higher in athletes than in control subjects (P<0.01). Cardiac, but not total systemic, NE spillover was also increased in athletes (P<0.01), whereas cardiac [(3)H]NE reuptake and clearance were not different. Echocardiographic modifications indicated a volume overload-induced hypertrophy associated with increased myocardial contractility. Multivariate stepwise analysis selected left ventricular mass index as the most predictive independent variable for cardiac IGF-I formation and velocity of circumferential fiber shortening for cardiac NE spillover. In conclusion, increased cardiac IGF-I formation and enhanced sympathetic activity selectively confined to the heart appear to be responsible for the physiological hypertrophy in athletes performing predominantly isotonic exercise.

Activation of cardiac renin-angiotensin system in unstable angina.

OBJECTIVES: The aim of this study was to investigate the activity of the cardiac renin-angiotensin system (RAS) in unstable angina (UA). BACKGROUND: Angiotensin (Ang) II locally produced by continuously operating cardiac RAS may affect the pathophysiology of UA. METHODS: In 35 patients with UA, 32 with stable effort angina (SA) and 21 with atypical chest pain (controls), cardiac RAS was investigated during coronary angiography after five days of Holter monitoring by combining the measurement of aorta-coronary sinus gradient for Ang I and Ang II with the kinetics study of 125I-Ang I. Messenger RNAs (mRNA) for all the components of RAS were also quantified with the reverse transcriptase-polymerase chain reaction (RT-PCR) and localized by in situ hybridization in myocardial biopsy specimens from patients who underwent aorta-coronary bypass surgery. RESULTS: Cardiac Ang II generation was higher in patients with UA than it was in patients with SA or in controls (p < 0.001) due to increased de novo cardiac Ang I formation and its enhanced fractional conversion rate to Ang II. Messenger RNA levels for angiotensinogen (AGTN), angiotensin-converting enzyme (ACE) and Ang II type 1 (AT1) subtype receptors were higher in patients with UA (p < 0.01) than they were in patients with SA or in control hearts. Messenger RNAs for AGTN and ACE were almost exclusively expressed on endothelial and interstitial cells. Angiotensin II formation was correlated with ischemia burden (p < 0.001). However, the amount of Ang II formed and the expression levels of mRNAs for AGTN, ACE and AT1 were not related to the time that had elapsed since the last anginal attack. CONCLUSIONS: In patients with UA, cardiac RAS is activated, resulting in increased Ang II formation. Myocardial ischemia is essential for RAS activation, but it is unlikely to be a direct and immediate cause of RAS activation.

Cardiac angiotensin II formation in the clinical course of heart failure and its relationship with left ventricular function.

In 76 patients with heart failure (HF) (New York Heart Association [NYHA] classes I through IV) and in 15 control subjects, cardiac angiotensin II (Ang II) generation and its relationship with left ventricular function were investigated by measuring aorta-coronary sinus concentration gradients of endogenous angiotensins and in a part of patients by studying (125)I-labeled Ang I kinetics. Gene expression and cellular localization of the cardiac renin-angiotensin system components, the density of AT(1) and AT(2) on membranes and isolated myocytes, and the capacity of isolated myocytes for synthesizing the hypertrophying growth factors insulin-like growth factor-I (IGF-I) and endothelin (ET)-1 were also investigated on 22 HF explanted hearts (NYHA classes III and IV) and 7 nonfailing (NF) donor hearts. Ang II generation increased with progression of HF, and end-systolic wall stress was the only independent predictor of Ang II formation. Angiotensinogen and angiotensin-converting enzyme mRNA levels were elevated in HF hearts, whereas chymase levels were not, and mRNAs were almost exclusively expressed on nonmyocyte cells. Ang II was immunohistochemically detectable both on myocytes and interstitial cells. Binding studies showed that AT(1) density on failing myocytes did not differ from that of NF myocytes, with preserved AT(1)/AT(2) ratio. Conversely, AT(1) density was lower in failing membranes than in NF ones. Ang II induced IGF-I and ET-1 synthesis by isolated NF myocytes, whereas failing myocytes were unable to respond to Ang II stimulation. This study demonstrates that (1) the clinical course of HF is associated with progressive increase in cardiac Ang II formation, (2) AT(1) density does not change on failing myocytes, and (3) failing myocytes are unable to synthesize IGF-I and ET-1 in response to Ang II stimulation.

Cardiac growth factors in human hypertrophy. Relations with myocardial contractility and wall stress.

The aim of the present study was to investigate whether and which cardiac growth factors are involved in human hypertrophy, whether growth factor synthesis is influenced by overload type and/or by the adequacy of the hypertrophy, and the relationships between cardiac growth factor formation and ventricular function. Cardiac growth factor formation was assessed by measuring aorta-coronary sinus concentration gradient in patients with isolated aortic stenosis (n=26) or regurgitation (n=15) and controls (n=12). Gene expression and cellular localization was investigated in ventricular biopsies using reverse transcriptase-polymerase chain reaction and in situ hybridization. Cardiac hypertrophy with end-systolic wall stress <90 kdyne/cm2 was associated with a selective increased formation of insulin-like growth factor (IGF)-I in aortic regurgitation and of IGF-I and endothelin (ET)-1 in aortic stenosis. mRNA levels for IGF-I and preproET-1 were elevated and mainly expressed in cardiomyocytes. At stepwise analysis, IGF-I formation was correlated to the mean velocity of circumferential fiber shortening (r=0.86, P<0.001) and ET-1 formation to relative wall thickness (r=0.82, P<0. 001). When end-systolic wall stress was >90 kdyne/cm2, IGF-I and ET-1 synthesis by cardiomyocytes was no longer detectable, and only angiotensin (Ang) II was generated, regardless of the type of overload. The mRNA level for angiotensinogen was high, and the mRNA was exclusively expressed in the interstitial cells. Ang II formation was positively correlated to end-systolic stress (r=0.89, P<0.001) and end-diastolic stress (r=0.84, P<0.001). Multivariate stepwise analysis selected end-systolic stress as the most predictive variable and left ventricular end-diastolic pressure as the independent variable for Ang II formation (r=0.93, P<0.001). In conclusion, the present results indicate that the course of human left ventricular hypertrophy is characterized by the participation of different cardiac growth factors that are selectively related both to the type of hemodynamic overload and to ventricular function.

Excessive vasoconstriction after stress by the aging kidney: inadequate prostaglandin modulation of increased endothelin activity.

The adaptive capacity of the aging kidney to stimulation of the sympathetic nervous system, as induced by a 30-minute mental stress (MS), was assessed in 8 elderly healthy women (68 to 82 years of age) and compared with that of 8 younger women (24 to 40 years of age). The study encompassed 4 consecutive 30-minute periods (baseline, mental stress, recovery 1, and recovery 2). In the elderly subjects, baseline effective renal plasma flow (ERPF)(iodine 131-labeled hippurate clearance) was lower and glomerular filtration rate (GFR)(iodine 125-labeled iothalamate clearance) was proportionally less reduced than in the younger group; the filtration fraction (FF) was higher. The elderly group excreted more endothelin 1 (ET-1) (P < .05), prostaglandin E2 (PGE2), and 6-keto-prostaglandin F1alpha (6-keto PGF1alpha)(P < .001 for both)(radioimmunoassay). Mental stress induced similar increases in blood pressure, heart rate, and plasma catecholamines in the 2 age groups, limited to the stimulation period. In the elderly group, mental stress caused a prolonged decrease in ERPF that reached its maximum 60 minutes after mental stress (-33%, P < .05), while GFR remained constant during the whole experiment, so that FF increased. In the younger subjects, renal hemodynamic changes were limited to the mental stress period. ET-1 increased during mental stress and the first recovery period in the elderly group (+50% and +25%, P < .05) as it did in the younger group, but the elderly group differed from the younger in that vasodilating prostaglandins increased only during mental stress. In conclusion, the aging kidney reacts to adrenergic stimulation with more-pronounced and -prolonged vasoconstriction that is probably caused by a defect in prostaglandin modulation of endothelin activity. Autoregulation of GFR is maintained at the expense of increased intraglomerular pressure.

Human vascular renin-angiotensin system and its functional changes in relation to different sodium intakes.

A growing body of evidence supports the existence of a tissue-based renin-angiotensin system (RAS) in the vasculature, but the functional capacity of vascular RAS was not investigated in humans. In 28 normotensive healthy control subjects, the metabolism of angiotensins through vascular tissue was investigated in normal, low, and high sodium diets by the measurement of arterial-venous gradient of endogenous angiotensin (Ang) I and Ang II in two different vascular beds (forearm and leg), combined with the study of 125I-Ang I and 125I-Ang II kinetics. In normal sodium diet subjects, forearm vascular tissue extracted 36+/-6% of 125I-Ang I and 30+/-5% of 125I-Ang II and added 14.9+/-5.1 fmol x 100 mL(-1) x min(-1) of de novo formed Ang I and 6.2+/-2.8 fmol x 100 mL(-1) x min(-1) of Ang II to antecubital venous blood. Fractional conversion of 125I-Ang I through forearm vascular tissue was about 12%. Low sodium diet increased (P<.01) plasma renin activity, whereas de novo Ang I and Ang II formation by forearm vascular tissue became undetectable. Angiotensin degradation (33+/-7% for Ang I and 30+/-7% for Ang II) was unchanged, and vascular fractional conversion of 125I-Ang I decreased from 12% to 6% (P<.01). In high sodium diet subjects, plasma renin activity decreased, and de novo Ang I and Ang II formation by forearm vascular tissue increased to 22 and 14 fmol x 100 mL(-1) x min(-1), respectively (P<.01). Angiotensin degradation did not significantly change, whereas fractional conversion of 125I-Ang I increased from 12% to 20% (P<.01). Leg vascular tissue functional activities of RAS paralleled those of forearm vascular tissue both at baseline and during different sodium intake. These results provide consistent evidence for the existence of a functional tissue-based RAS in vascular tissue of humans. The opposite changes of plasma renin activity and vascular angiotensin formation indicate that vascular RAS is independent from but related to circulating RAS.

Relationship between depressed baroreflex function and exaggerated sympathetic response to exercise in patients with heart failure.

The relationship between impaired baroreflex sensitivity (BS) and the degree of sympathetic activation during exercise in patients with heart failure (HF) has not been studied in detail. For this purpose, we studied BS and measured plasma norepinephrine (NE) at rest, and during and after treadmill exercise in 15 patients and 10 controls. HF patients showed lower BS in comparison to controls (3. 51 +/- 3.62 vs. 9.74 +/- 4.56 ms/mm Hg; p < 0.001), and higher levels of plasma NE at rest (449.3 +/- 147.1 vs. 261.1 +/- 82.48 pg/ml; p < 0.001) and during exercise (1,542 +/- 361.2 vs. 524.6 +/- 92.61 pg/ml; p < 0.001). BS was directly related to pVO2 (r = 0.62; p = 0.0008) and inversely related to NE at peak exercise and to the increase in NE during exercise (r = 0.59, p = 0.005, and r = 0.53; p = 0.0058). Thus, during exercise, a marked sympathetic activation exists in patients with moderate HF. The relationship between increased plasma NE during exercise and decreased BS suggests that impaired baroreceptor function may be present in sympathetic activation in HF patients.

Renal adaptation to stress: a possible role of endothelin release and prostaglandin modulation in the human subject.

The aim of this study was to define the neurohumoral response associated with the renal hemodynamic perturbations induced by mental stress acting as an adrenergic stimulus. In 8 healthy women, the effects of mental stress were studied during four consecutive 30-minute periods (baseline, mental stress, recovery I, recovery II). Mental stress induced sympathetic activation as evidenced by increases in blood pressure, heart rate, and plasma norepinephrine level. Effective renal plasma flow (iodine 131-labeled hippurate clearance) decreased only during mental stress (-22%, p < 0.05 vs baseline); glomerular filtration rate (iodine 125-labeled iotalamate clearance) remained constant during the entire experiment; the filtration fraction increased significantly during mental stress and recovery I (+30% and +22%, respectively, p < 0.02 for both). Complex neuroendocrine responses were associated with the hemodynamic changes. Urinary excretion of endothelin-1 and 6-keto-PGF(1alpha) increased during mental stress (+53%, p < 0.01, and +20%, p < 0.01, respectively) and recovery I (+49% and +29%, respectively, p < 0.01 for both). Urinary cyclic guanosine monophosphate rose only during mental stress (+77%, p < 0.05), whereas excretion of PGE2 showed a stepwise increase throughout recovery I and II (+292%, p < 0.01, and +360%, p < 0.001, respectively). In conclusion, the present experiments demonstrate that renal hemodynamic response induced by mental stress is a complex reaction in which endothelin-1, prostaglandins, and presumably nitric oxide take part.

Evidence for the existence of a functional cardiac renin-angiotensin system in humans.

BACKGROUND: The presence of mRNA for the essential components of the renin-angiotensin system (RAS) has been found in animal and human hearts. The present study was designed to provide evidence for the existence of a (functional) cardiac RAS. METHODS AND RESULTS: Twenty-four patients with atypical chest pain undergoing coronary angiography for diagnostic purposes were investigated. The cardiac production rate of angiotensins was estimated by measurement of the cardiac extraction of 125I-angiotensin I and 125I-angiotensin II associated with the determination of endogenous angiotensins in aortic and coronary sinus blood in normal, low, or high sodium diets. In a normal sodium diet, angiotensin I and II aorta-coronary sinus gradients were tendentially negative (-1.8 +/- 2.5 and -0.9 +/- 1.7 pg/mL, respectively), and the amounts of angiotensin I and II added by cardiac tissues were 6.5 +/- 3.1 and 2.7 +/- 1.3 pg/mL, respectively. The low sodium diet caused a significant increase in both plasma renin activity (PRA) and angiotensin I concentration in aortic but not in coronary sinus blood, resulting in a more negative aorta-coronary sinus gradient (-9.7 +/- 3.1 pg/mL, P < .01). Angiotensin formation by PRA in blood during transcardiac passage increased (P < .001), whereas angiotensin I formed by cardiac tissues decreased dramatically. Accordingly, in the low sodium diet, 125I-angiotensin II extraction did not change, the cardiac fractional conversion rate of 125I-angiotensin I to 125I-angiotensin II notably decreased (P < .01), and angiotensin II formation by cardiac tissues was undetectable. The high sodium diet caused a decrease in PRA and no changes in cardiac extraction of radiolabeled angiotensins; conversely, angiotensin I formed by cardiac tissues, cardiac Ang I fractional conversion rate, and angiotensin II formed during transcardiac passage significantly (P < .01 for all) increased. CONCLUSIONS: These results provide evidence for the existence of a functional cardiac RAS independent of but related to the circulating RAS.

Platelet activation and platelet lipid composition in pulmonary cancer.

In order to investigate the possible mechanisms underlying platelet functional changes in patients affected by neoplasms, platelet lipid composition, plasma beta-thromboglobulin (Beta-TG) and serum thromboxane B2 (TXB2) were investigated in 16 male patients affected by pulmonary carcinoma and in 16 comparable control subjects. In patients high levels of plasma Beta-TG (67 +/- 9 versus controls 14 +/- 4 ng/ml, p < 0.001) and serum TXB2 (434 +/- 56 versus 223 +/- 48 ng/ml, p < 0.001) were observed. Also platelet lipid composition was found altered in patients with respect to controls (lower percent levels in n-3 fatty acids and in linoleic acid esterified in the main platelet phospholipid fractions: at least p < 0.05). These results indicate that in vivo platelet activation is detectable in neoplastic patients and it is associated with alterations in platelet lipid composition. In the light of the important role played by membrane lipids in platelet functions related to thrombosis and haemostasis we conclude that platelet lipid changes could cooperate in platelet activation and increased thrombotic risk so frequently observed in neoplastic disease.

Effect of low-dose heparin treatment on fibrinolysis in patients with previous myocardial infarction.

The present study was designed to investigate whether medium-term, low-dose heparin treatment is able to affect the fibrinolytic system. In a randomized cross-over study 10 asymptomatic patients with previous (1-6 years) myocardial infarction underwent two sequential 15-day treatments, respectively, on heparin and on placebo (saline solution), preceded and separated by 10-day wash-out periods. Heparin (as calcium heparin, 12,500 IU in 0.5 ml) and saline (0.5 ml) were subcutaneously administered once a day at 8 a.m. Blood samples for fibrinolysis studies were withdrawn on the first and 15th day of each period immediately before and 4 h after heparin or saline administration before and after 10 min venous occlusion (VO) respectively. Four hours after the first heparin administration tissue plasminogen activator antigen (t-PA ag) levels significantly increased with respect to saline administration (p < 0.01 and p < 0.05, respectively). After 15-day heparin treatment a decrease in euglobulin lysis time (p < 0.05) and an increase in t-PA activity (act) (p < 0.05) and in t-PA ag (p < 0.01) in comparison with placebo were observed before VO. No statistically significant changes in plasminogen activator inhibitor-1 (PAI-1) levels were found. The variations of fibrinolytic system activity induced by heparin treatment were more marked when evaluated after VO. These results indicate that medium-term low-dose heparin treatment increases t-PA ag formation and/or release with consequent t-PA act increase.

[The tissue renin-angiotensin systems]. / I sistemi renina-angiotensina tessutali.

Over the past 10 years, our understanding of the renin-angiotensin system has changed greatly, reflecting significant advances in this field. Studies in cell biology, the application of molecular biological techniques, and studies of angiotensin kinetics have provided evidence for the existence in various sites (cardiac, cerebral, renal, vascular and gonadal) of tissue-based renin-angiotensin system that function independently of the plasma renin-angiotensin system. These local systems contribute to the functional activity of their plasma counterpart through the local production of angiotensins I and II; at the same time, they are autonomous autocrine-paracrine systems with their own regulatory mechanisms. This multicentric organization of the renin-angiotensin system has physiologic and pathophysiologic implications. However, it also has important therapeutic consequences, since it follows that inhibition of the plasma renin-angiotensin system alone is probably not sufficient for therapeutic purposes.

Silent ischemia in unstable angina is related to an altered cardiac norepinephrine handling.

BACKGROUND: Inferential evidence suggests that silent ischemia might be related to sympathetic activity. Study of [3H]norepinephrine kinetics is a suitable tool to assess the regional sympathetic activity. This method was applied to investigate whether silent myocardial ischemia in unstable angina is related to and depends on cardiac sympathetic overactivity. METHODS AND RESULTS: Patients with active unstable angina were compared with patients with inactive unstable angina, stable effort angina, and controls. Silent myocardial ischemia was evaluated by three 24-hour Holter monitoring periods on alternate days, and [3H]norepinephrine kinetics was assessed under rest conditions and following the cold pressor test. Simultaneously, catecholamine concentrations were measured in the aortic, coronary sinus, and peripheral venous blood. Different than the other groups (p = 0.0013), in patients with active unstable angina, the majority of silent ischemic episodes occurred without increase in heart rate. These patients had a positive coronary sinus-aorta norepinephrine gradient, both at rest and following the cold pressor test. [3H]Norepinephrine kinetics demonstrated an increased selective cardiac spillover, both at rest and, even more, after the cold pressor test. Reduced cardiac [3H]norepinephrine extraction also was found. A significant relation was found between the number of ischemic episodes or the overall duration of silent ischemia and norepinephrine spillover, both at rest and following cold application. CONCLUSIONS: During the acute phase of unstable angina (but not in the quiescent phase or in stable effort angina), a disorder in cardiac norepinephrine handling occurs. This results in a reflex cardiac sympathetic overactivity that plays a major role in the occurrence of silent myocardial ischemia.

Changes in thromboxane A2 generation and plasma lipid pattern in pseudomenopause induced by gonadotropin releasing hormone (GnRH) analogue buserelin.

An increased risk of cardiovascular disease has been found in postmenopausal women in comparison to premenopausal women. The aim of this study was to investigate platelet function, blood clotting and plasma lipid levels in 12 women with a condition of hypoestrogenism, similar to the postmenopausal status induced by treatment with the GnRH analogue buserelin for uterine leiomyoma. Platelet aggregation in whole blood and platelet-rich plasma (PRP), serum thromboxane (TX) B2 production, fibrinopeptide A (FPA) plasma levels and plasma lipid pattern were measured before and after 13 weeks of buserelin treatment. No changes of platelet aggregability were found either in whole blood or PRP. Serum TXB2 generation increased significantly after 13 weeks of therapy (p less than 0.001). No signs of increased thrombin generation were found, as indicated by unchanged FPA plasma levels. Total cholesterol plasma levels were found increased after 13 weeks, LDL cholesterol levels showed a tendency to increase although not significantly. HDL cholesterol and triglyceride concentrations were unaffected. The changes of arachidonic acid metabolism and lipid pattern suggest that buserelin treatment may induce a condition of increased thrombotic risk even if the lack of enhanced thrombin generation and increased platelet aggregability indicates that no blood clotting activation occurs.