Marco Morale

Microalbuminuria and endothelial dysfunction in essential hypertension

Microalbuminuria (urinary albumin excretion between 20 and 200 micrograms/min) and endothelial dysfunction coexist in patients with essential hypertension. To evaluate whether the two phenomena are related and the determinants of that association, we recruited 10 untreated males with essential hypertension and microalbuminuria without diabetes to be compared with an equal number of matched patients with essential hypertension excreting albumin in normal amounts and 10 normal controls. The status of endothelial function was inferred from circulating von Willebrand Factor antigen (vWF), a glycoprotein secreted in greater amounts when the vascular endothelium is damaged. vWF concentrations were higher in hypertensive patients with microalbuminuria than in hypertensive patients without and controls. Individual vWF and urine albumin-excretion values were correlated (r = 0.55, p < 0.002). Blood pressure correlated with both urinary albumin excretion and vWF. Left ventricular mass index and minimal forearm vascular resistances were comparable in patients with hypertension and higher than in controls; total and low-density lipoprotein cholesterol, triglycerides, lipoprotein-a, Factor VII, and plasminogen activator inhibitor-1 did not differ. Fibrinogen was higher and creatinine clearance lower in microalbuminurics. Albuminuria in essential hypertension may reflect systemic dysfunction of the vascular endothelium, a structure intimately involved in permeability, haemostasis, fibrinolysis, and blood pressure control. This abnormality may have important physiopathological implications and expose these patients to increased cardiovascular risk.

Soluble Vascular Cell Adhesion Molecule-1 as a Biohumoral Correlate of Atherosclerosis

Vascular cell adhesion molecule-1 (VCAM-1) is a protein expressed on the surface of activated endothelial cells and expressed in early atherosclerosis. Because part of the protein is shed in the circulation and can be detected in peripheral plasma [soluble (s) VCAM-1], we hypothesized that sVCAM-1 may be a circulating marker of the presence and severity of atherosclerosis in humans. We selected 11 patients with essential hypertension plus peripheral vascular disease (PVD) and matched them for age, gender, body mass index, and smoking habits with 11 patients with uncomplicated essential hypertension (UH) and 11 healthy controls. We evaluated plasma concentrations of sVCAM-1 along with those of the soluble form of two other endothelial leukocyte adhesion molecules [sE-selectin and s-intercellular adhesion molecule-1 (sICAM-1)] and other markers of endothelial dysfunction/ damage [s-thrombomodulin, plasminogen activator inhibitor type I, and von Willebrand factor (vWF)]. We also measured insulin, glucose, fibrinogen, total and HDL cholesterol, and the urinary albumin excretion (UAE), which may also be related to atherosclerosis. Results of these assays were related to the echographic assessment of the maximum intima-media thickness (IMTmax) at the carotid bifurcation, as an index of atherosclerosis in the carotids. PVD patients had a clearly elevated IMTmax [2.7 (1.1-3.1) mm, median (range)] compared with both UH patients [1.2 (0.8-2.4) mm] and controls [1 (0.6-2) mm]. sVCAM-1 was clearly higher in PVD patients [990 (273-1808) ng/mL, median (range)] versus 340 (236-975) ng/mL in UH and 386 (204-835) ng/mL in controls, and it separated clinical categories better than sICAM-1, vWF, glucose, insulin, UAE, triglycerides, or total, LDL or HDL cholesterol, sVCAM-1 was also the best biohumoral correlate of IMTmax (R = .59; P < .001) in univariate analysis. Because many of the biohumoral variables assessed were mutually intercorrelated, they were entered in a multivariate analysis to assess their contribution in explaining IMTmax variability. sVCAM-1 remained the only independent predictor of IMTmax and totally abolished the contribution of other variables to IMTmax variability. Thus, sVCAM-1 is a good biohumoral correlate of overt atherosclerosis, independent of underlying hypertension, and may be an in vivo marker of endothelial activation. Its potential value as a surrogate for global risk assessment and its behavior in intervention studies remain to be determined.

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.

Modulation of hemostatic balance with antithrombin III replacement therapy in a case of liver cirrhosis associated with recurrent venous thrombosis

Patients with liver failure can present both thrombotic and hemorragic complications because of the deficiency in coagulation factors and inhibitors (protein C and S, antithrombin III) and impairment of fibrinolytic balance. Here we report the case of a 63-year-old man with liver cirrhosis, recurrent thrombosis, and features of low-grade consumption coagulopathy, showing severe antithrombin III deficiency (about 30% of normal values). Treatment with antithrombin III (2000 U/day) and low doses of heparin (5000 U b.i.d.) was successful in modulating the coagulation system toward an antithrombotic effect. After discharge from hospital the ambulatory treatment with antithrombin III concentrates (2000 U twice a week) allowed the attainment of antithrombin III activity of about 60% and prevented the patient from recurrence of venous thrombosis.

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.

Assessment of coagulation and fibrinolysis in synovial fluid of rheumatoid arthritis patients

Summary In order to understand the mechanism of fibrin deposition in the synovia during the progression of rheumatoid arthritis (RA), activities of coagulation factors, fibrinolytic enzymes, and corresponding inhibitors were measured in synovial fluid of 12 RA patients. Almost all of the synovial fluid (SF) fibrinogen molecules were found to be fragmented and some portions of the molecule that were slightly affected were found to be in a complex with albumin molecules. Immunohistochemical analysis showed deposits of insoluble fibrin in synovial membranes and pannus, in levels related to the progression of disease. Coagulation factor VII, VIII, X, XII, and XIII activities in SF were found to be 30 to 50% of plasma levels but factors II and V activities were less than 25% of plasma levels. Protein C and antithrombin III activities in SF were 30% of plasma levels. However, the two orders of magnitude, higher level of thrombin-antithrombin III (TAT) complex in SF than in plasma, suggests a steady activation of coagulation cascade in synovia. The observed increased levels of fibrinogen, TAT complexes, Bβ 15–42 peptide and plasminogen activator inhibitor-1 (PAI-1) in plasma are consistent with the systemic inflammatory state of RA patients. Although a 30% decrease in plasma levels of α2-plasmin inhibitor activity, and a 2-fold higher level of PAI-1 activity in SF were found, the presence of D-dimer and Bβ 15–42 peptide in SF suggests an active participation of plasmin in the fibrino(geno)lysis. In addition, elevated levels of elastase-α1-proteinase inhibitor complexes and of thrombin-increasable fibrinopeptide A in SF suggest the participation of leukocyte elastase in fibrin(ogen)olysis as well.

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.

Activation of coagulation in smoking and non-smoking women using a third-generation oral contraceptive containing desogestrel

The concurret use of smoking and oral contraceptives affects the hemostatic balance, thereby inducing a thrombophilic state. In order to clarify the effects of this association on the hemostatic system, the possible changes in the markers of activation of coagulation (thrombin-antithrombin III complexes and prothrombin fragment F1+2) were evaluated in 35 women given a third-generation oral contraceptive for 6 months; 13 of these women (37.1%) were mild or moderate smokers. No differences were found in basal levels of the coagulation and fibrinolytic parameters between smokers and non-smokers. During oral contraceptive administration, both F1+2 fragment and thrombin-antithrombin III complex concentrations significantly increased both in smokers and in non-smokers (p < 0.01). Fibrinogen plasma levels increased in both groups (p < 0.01). Antithrombin III activity was reduced in both groups during treatment, but the difference was significant only in smokers (p < 0.05). Although the sample size of smokers was too small to draw definitive conclusions, present results appeared to confirm previous data about the effect of the concurrent use of smoking and oral contraceptives on antithrombin III levels, but did not demonstrate any additional effect of moderate smoking on the activation of the clotting system induced by this oral contraceptive preparation.