Ferdinando De Negri

COAGULATION AND FIBRINOLYTIC SYSTEM IMPAIRMENT IN INSULIN DEPENDENT DIABETES MELLITUS

Selected coagulation and fibrinolytic parameters were assessed in 40 insulin dependent diabetes mellitus patients with varying degrees of metabolic control; 30 healthy subjects matched for age and sex formed the control group. Activated Partial Thromboplastin Time, Prothrombin Time, Fibrinogen, Factor VII, Antithrombin III, Protein C, Plasminogen, alpha 2-Plasmin Inhibitor, Plasminogen Activator Inhibitor-1, tissue-Plasminogen Activator were functionally evaluated. Antigenic levels of tissue-Plasminogen Activator, Thrombin-Antithrombin complexes and fibrinolytic specific product B beta 15-42 were also determined. Compared to the control group diabetic patients displayed significantly higher levels of Fibrinogen (p < 0.01), Factor VII (p < 0.01), Thrombin-Antithrombin complexes (p < 0.01) and Plasminogen Activator Inhibitor-1 activity (p < 0.01). Regardless of the normal level of the tissue-Plasminogen Activator-related antigen, diabetic patients had tissue-Plasminogen Activator activity lower than the control group (p < 0.05). Coagulation Factor VII and Thrombin-Antithrombin complexes were increased only in the patients with poor metabolic control (p < 0.01). Activated Partial Thromboplastin Time, Prothrombin Time, Antithrombin III, Protein C, Plasminogen, alpha 2-Plasmin Inhibitor, B beta 15-42 fibrin peptide were found to be in the normal range. Fibrinogen correlated positively with fasting blood glucose (p < 0.05) and Thrombin-Antithrombin complexes with glycosylated haemoglobin (p < 0.05), whereas Factor VII was positively correlated with glycemia (p < 0.01) and glycosylated haemoglobin (p < 0.05). Higher levels of Fibrinogen were found in patients affected by nephropathy (p < 0.005) or neuropathy (p < 0.05). These results demonstrate an impairment of the haemostatic balance in diabetic patients, that is a possible hypercoagulable state, which represents an important factor in the pathogenesis of atherosclerotic complications.

Local insulin infusion stimulates expression of plasminogen activator inhibitor-1 and tissue-type plasminogen activator in normal subjects

PURPOSE Plasma levels of plasminogen activator inhibitor-1 are increased in obesity, hypertension, and diabetes. Their correlation with insulin levels supports the hypothesis that hypofibrinolysis may affect the development of atherosclerotic complications in patients with insulin resistance. To investigate the effect of insulin on fibrinolysis, we evaluated levels of plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) antigens during insulin infusion in the forearm vascular beds of 8 healthy subjects. MATERIALS AND METHODS Insulin was infused in the brachial artery of each subject to raise local venous concentrations to approximately 100 microU/mL. Blood samples were obtained from the brachial artery and vein at baseline, after 30, 60, 90, and 120 minutes of infusion, and 30 minutes after the end of the infusion. RESULTS Following intra-arterial infusion of insulin, forearm blood flow (mean +/- SD) increased progressively from 2.7 +/- 0.6 to 4.0 +/- 0.6 mL/dL/min (P <0.01) and did not return to baseline after the end of the infusion. Plasminogen activator inhibitor-1 balance increased (345 +/- 160 versus 8 +/- 152 fmol/dL/min, P <0.02) at 60 minutes, reaching baseline levels after the end of the infusion. After 90 minutes, tPA balance increased (40 +/- 26 versus 7 +/- 29 fmol/dL/min, P <0.01) with a profile similar to forearm blood flow. CONCLUSIONS Local hyperinsulinemia induces regional vasodilation and expression of PAI-1 and tPA antigens. An alteration of this physiological process could be involved in the development of hypofibrinolysis and atherosclerosis in states of insulin resistance.

Effect of Sulfaphenazole on Tissue Plasminogen Activator Release in Normotensive Subjects and Hypertensive Patients

Background— A nitric oxide–independent response, possibly mediated by hyperpolarization, regulates vascular tone, acting as a compensatory mechanism in the presence of impaired nitric oxide availability. Cytochrome P450 2C9 (CYP 2C9) is a source of endothelium-derived hyperpolarizing factors and modulates tissue-type plasminogen activator (tPA) release from endothelial cells; however, no effect of hyperpolarization on fibrinolysis has been documented in humans. We aimed to assess the effect of sulfaphenazole, a specific CYP 2C9 inhibitor, on tPA release in normotensive subjects and patients with essential hypertension. Methods and Results— tPA release was measured in the forearm microcirculation of 56 normotensivesubjects and 57 patients with essential hypertension after bradykinin (0.015 &mgr;g · 100 mL−1 · min−1) and acetylcholine (1.5 &mgr;g · 100 mL−1 · min−1) infusions, with or without sulfaphenazole (0.03 &mgr;g · 100 mL−1 · min−1). Bradykinin and acetylcholine infusions were repeated with NG-monomethyl-l-arginine (L-NMMA; 100 &mgr;g · 100 mL−1 · min−1) and/or sulfaphenazole. tPA release by bradykinin and acetylcholine was higher in normotensive subjects than in patients with essential hypertension (P<0.01). Sulfaphenazole (P<0.01) blunted bradykinin-induced but not acetylcholine-induced tPA release in both groups. In normotensive subjects, L-NMMA infusion reduced tPA release (P<0.01). When L-NMMA was coinfused with sulfaphenazole, tPA release induced by bradykinin, but not by acetylcholine, was further reduced (P<0.01). In patients with essential hypertension, tPA release by both agonists was unaffected by L-NMMA, but only bradykinin-induced tPA release was blunted by sulfaphenazole, alone or with L-NMMA (P<001). Conclusions— Sulfaphenazole inhibits bradykinin-induced tPA release, which suggests a modulatory role of CYP 2C9–derived endothelium-derived hyperpolarizing factors in tPA release in humans. In patients with essential hypertension, tPA release depends exclusively on endothelium-derived hyperpolarizing factor, which is an ineffective compensatory mechanism in the presence of impaired nitric oxide availability.

Nitric Oxide Modulates Tissue Plasminogen Activator Release in Normotensive Subjects and Hypertensive Patients

We evaluated the possible role of NO in modulating tissue plasminogen activator (t-PA) release in the forearm microcirculation of normotensive subjects and hypertensive patients. Essential hypertensive patients are characterized by endothelial dysfunction because of a reduced NO availability and also show an impaired t-PA release. In healthy volunteers and essential hypertensive patients, we studied local t-PA release and forearm blood flow changes (strain-gauge plethysmography) induced by intrabrachial administration of acetylcholine (0.45 and 1.5 &mgr;g/100 mL/min) and of sodium nitroprusside (0.5 and 1.0 &mgr;g/100 mL/min), an endothelium-dependent and -independent agonist, respectively. Acetylcholine was also repeated in the presence of intra-arterial infusion of the NO synthase inhibitor NG-monomethyl-l-arginine (100 &mgr;g/100 mL/min). In normotensive subjects, vasodilation to acetylcholine was blunted by NG-monomethyl-l-arginine. In these subjects, acetylcholine infusion induced a significant, dose-dependent increase in net forearm t-PA release. NG-monomethyl-l-arginine significantly reduced basal t-PA release, as well as acetylcholine-induced t-PA release. In essential hypertensive patients, vasodilation to acetylcholine was reduced as compared with controls and resistant to NG-monomethyl-l-arginine. In contrast to what was observed in healthy control subjects, in hypertensive patients, acetylcholine had no effect on t-PA release. Similarly, NG-monomethyl-l-arginine failed to modify either the tonic or the agonist-induced t-PA release. Both tonic and agonist-induced release of NO are directly involved in t-PA release by endothelial cells. Essential hypertension, characterized by a reduction in tonic and stimulated NO availability, is also associated with impaired capacity of t-PA release, suggesting a major role of impaired NO availability in worsening both vasodilation and t-PA release.

A 12-month clinical investigation with a 24-day regimen containing 15 μg ethinylestradiol plus 60 μg gestodene with respect to hemostasis and cycle control

Abstract The effects of a 24-day regimen containing 15 μg ethinyl estradiol (EE) plus 60 μg gestodene on cycle control and on hemostasis, were evaluated in 58 healthy women (age 19–47 years). All women received the pill for 12 months. Withdrawal bleeding at every cycle during the tablet-free interval was experienced by 84.5% of the women. The overall incidence of irregular bleedings was 19.3%. Hemostasis was evaluated in 20 women. No changes in plasma fibrinogen concentrations, nor in prothrombin fragment F1+2 were observed. A slight increase in thrombin-antithrombin III complexes was observed after 6 and 12 months of oral contraceptive use. Antithrombin III activity significantly increased after one-year of pill intake. The concentrations of tissue plasminogen activator and plasminogen activator inhibitor, both antigen and activity, did not change. These results show that very low doses of EE, such as 15 μg, do not impair hemostasis in healthy females. However, the reduction for the EE dose is responsible of some of the effects on cycle control.

Antithrombotic and antifibrinolytic effects of antithrombin III replacement in liver cirrhosis

In liver failure, chronic disseminated intravascular coagulation (DIC) may occur as shown by increases in markers of activation of the coagulation cascade (prothrombin fragment F1+2, thrombin-antithrombin [TAT] complexes) and fibrinolysis (plasmin-antiplasmin [PAP] complexes, D-dimer), although the contribution of impaired hepatic clearance to the increases in these products cannot be ruled out. DIC rarely becomes life-threatening in liver cirrhosis, but chronic activation and consumption of clotting factors can contribute to haemorrhagic and thrombotic complications. Antithrombin III (AT-III) inhibits thrombin, other activated coagulation factors, and plasmin, and previous studies suggest the usefulness of AT-III replacement in selected patients with AT-III deficiency and DIC. We studied the effect of AT-III replacement therapy on coagulation and fibrinolysis in cirrhotic patients. Ten cirrhotic patients (Child B-C stages) with low plasma AT-III (<600 U/L) were treated for 7 days with AT-III doses calculated to raise plasma values above 800 U/L (range 2000–4000 U per day). AT-III concentrates (Immuno, Vienna) were administered intravenously between 0800 h and 1000 h. Blood samples were obtained before AT-III injection. The antigenic levels of PAP complexes, F1+2 fragment, and TAT complexes were assayed by ELISA methods. All patients showed high basal concentrations of TAT, F1+2 fragment, and PAP complexes. During therapy, a progressive decrease of TAT complexes, F1+2 fragment, and PAP complexes was observed (figure). Plasma values of TAT complexes and F1+2 fragment were strongly correlated (p<0·001). In liver cirrhosis, hyperfibrinolysis, reduced synthesis of coagulation factors and inhibitors, and their consumption, restrain the adaptability of the haemostatic balance. Thus, depending on the triggering stimulus, the patient can develop thrombosis and/or haemorrhage. Whereas clotting proteins are present in excess and deficiencies must be severe to produce haemorrhagic effects, a moderate reduction of AT-III can severely impair the antithrombotic potential of plasma. Since second-order kinetics regulate the reaction of AT-III with thrombin and factor Xa, the rate of clotting enzyme inhibition is likely to depend on AT-III concentration. Moderate AT-III deficiency, characterised by a large molar excess of AT-III over the clotting factors, can cause thrombophilia through a kinetic rather than a capacity defect in the interaction between clotting factors and inhibitor. The finding of increased levels of F1+2 fragment and TAT complexes and their decrease after AT-III replacement demonstrates the presence of increased thrombin generation in cirrhotic patients, and also that ATIII replacement is able to inhibit prothrombin activation by quenching the coagulation cascade. In liver cirrhosis, hyperfibrinolysis, either primary or secondary to DIC, could play an important role in bleeding complications. In our patients, the decrease of PAP complexes after AT-III replacement suggests that hyperfibrinolysis in liver cirrhosis can be reduced by improving the antithrombotic potential of plasma. A trial is now needed to evaluate efficacy and indications for AT-III replacement therapy in cirrhotic patients.

Acetylcholine-mediated vasodilatation and tissue-type plasminogen activator release in normal and hypertensive men.

Muscarinic agents release tissue plasminogen activator (t-PA) in the forearm circulation of normal subjects, but no information exists about their effect in those hypertensive patients in whom the response to endothelial-mediated vasodilators is blunted. Acetylcholine, an endothelium-dependent vasodilator and a muscarinic agonist that releases t-PA from in-vitro systems, and sodium nitroprusside, an endothelium-indepen dent vasodilator, were infused into the brachial artery at rates calculated to cause a similar degree of vasodilatation. The study was performed in five elderly, smoking hyper tensive patients in whom the clustering of detrimental factors for endothelial function permitted prediction of defective endothelial-mediated vasorelaxation, and five young, normotensive, nonsmoking male volunteers. Forearm blood flow was assessed by venous plethysmography; t-PA and plasminogen activator inhibitor 1 (PAI-1) antigen values were expressed as flow-dependent (net release, the product of venoarterial concentration gradient and forearm blood flow) or independent (absolute and fractional concentration gradients) indices. In patients, acetylcholine did not change flow and net release and concentration gradients of t-PA, suggesting that vasodilatation as such, possibly by increasing fluid shear stress, may induce t-PA release in human forearm. In normal subjects, acetylcholine and sodium nitroprusside increased t-PA antigen net release at the highest infusion rate, an effect attributable to forearm hyperperfusion, since absolute and fractional gradients did not change significantly. PAI-1 antigen did not change during either infusion in both controls and patients, indicating the absence of an endothelial pool to be mobilized acutely.

Tissue-Type Plasminogen Activator Release in Healthy Subjects and Hypertensive Patients. Relationship With β-Adrenergic Receptors and the Nitric Oxide Pathway

The relationship between adrenergic stimuli and NO in modulating tissue-type plasminogen activator (t-PA) release from endothelial cells was investigated in normotensive subjects and essential hypertensive patients. Sympathetic activation, a well-known stimulus for endogenous fibrinolysis, is also involved in the determination of cardiovascular risk in essential hypertension. However, the existence of cross-talk between adrenergic stimuli and NO availability in modulating t-PA release is not well established yet. We assessed the release of t-PA in the forearm microcirculation of 58 normotensive subjects (mean age: 47±9 years) and 44 essential hypertensive patients (mean age: 48±11 years) under specific intra-arterial adrenergic stimuli. Intrabrachial infusion of epinephrine (0.1 to 0.3 &mgr;g/100 mL per minute) induced greater t-PA release in normotensive subjects as compared with essential hypertensive patients (P<0.05). However, inhibition of NO synthase with NG-monomethyl-l-arginine (100 &mgr;g/100 mL per minute) infusion blunted epinephrine-induced t-PA release in normotensive subjects (P<0.05) but not in essential hypertensive patients. In normotensive subjects, t-PA release by epinephrine was not affected by phentolamine (8 &mgr;g/100 mL per minute) coinfusion and was abolished in the presence of propanolol (10 &mgr;g/100 mL per minute). Intrabrachial isoproterenol (0.03 &mgr;g/100 mL per minute) induced a significant increase in t-PA release (P<0.01), an effect blunted by NG-monomethyl-l-arginine (P<0.05). In essential hypertensive patients, the response to isoproterenol was impaired as compared with normotensive subjects and was unaffected by NG-monomethyl-l-arginine coinfusion. In conclusion, the results of the present study demonstrate that adrenergic-induced t-PA release is mediated by β-adrenoreceptors via a mechanism involving the NO pathway. Our results show an impaired adrenergic-stimulated t-PA release among essential hypertensive patients, probably mediated via a reduced NO availability. This impaired fibrinolytic activity might contribute to the increased cardiovascular risk associated with hypertension.

Thrombin-Antithrombin III Complexes as an Additional Diagnostic Aid in Pulmonary Embolism

Plasma levels of selected coagulation and fibrinolytic parameters (activated partial thromboplastin time, prothrombin time, fibrinogen, antithrombin III, protein C, thrombin-anti-thrombin III complexes (TAT), plasminogen activator inhibitor-1 (PAI-1), plasminogen, alpha 2-plasmin inhibitor) were evaluated in 90 patients with clinical suspicion of pulmonary embolism (PE). Plasma levels of fibrinogen, PAI-1 and TAT were significantly higher in patients than in controls (p < 0.01): evaluation of TAT displayed a sensitivity of 96.1% and specificity of 30.8%, and positive and negative predictive values of 64.5 and 85.7%, respectively. The number of nonperfused lung segments correlated directly with TAT levels (p < 0.01) and inversely with arterial pO2 values (p < 0.01). No significant difference was found in the other parameters between patients and controls. Our results suggest that the finding of normal TAT plasma levels can help to exclude PE in patients with clinically suspected PE.

Long-term intramuscular administration of thyrotropin-releasing hormone tartrate in patients with cerebrovascular disease: effects on the pituitary-thyroid axis.

We studied the effects of long-term (30 days) refracted daily intramuscular administration of 4 mg TRH tartrate (TRH-T) on the pituitary-thyroid axis in 20 euthyroid patients affected by cerebrovascular disease (CVD). All subjects were assayed for T4, T3, FT4, FT3, TSH and TBG plasma levels before treatment (D0), after 15 and 30 treatment days (D15, D30), and after a 15-day washout (D45). In addition, TSH response to 200 micrograms intravenous TRH was assessed at D0, D30 and D45. We observed a significant increase in T4, FT4 and FT3 levels in the face of decreased TSH concentrations. A blunted TSH response to TRH bolus persisted at D30. These data demonstrate that the down-regulation mechanism may be partially overcome in vivo when thyrotrophs are chronically exposed to pharmacological TRH-T doses and that TSH pattern is mainly due to the negative feedback of thyroid hormones, even though pituitary TSH reserves may become depleted. Furthermore, prolonged TRH-T administration does not produce hyperthyroidism in euthyroid CVD patients.