M. Margaglione

Plasma Thrombin-Activatable Fibrinolysis Inhibitor Antigen Concentration and Genotype in Relation to Myocardial Infarction in the North and South of Europe

The thrombin-activatable fibrinolysis inhibitor (TAFI) is a recently described inhibitor of fibrinolysis that decreases plasminogen binding to the fibrin surface. The plasma TAFI concentration is almost entirely genetically determined. We investigated whether plasma TAFI levels and polymorphisms located in the TAFI gene could constitute risk markers of myocardial infarction (MI). Plasma TAFI antigen (Ag) levels were assayed by ELISA and 2 TAFI gene polymorphisms (Ala147Thr and C+1542G in the 3′ untranslated region) were determined in a large European case-control study. This study compared 598 men recruited 3 to 6 months after MI with 653 age-matched controls from North Europe (Stockholm, Sweden, and London, England) and South Europe (Marseilles, France, and San Giovanni Rotondo, Italy). A TAFI Ag value above the 90th percentile was associated with a significantly lower risk of MI (odds ratio 0.55, P <0.02), indicating that elevated TAFI may be protective against MI. As previously shown, the 2 TAFI gene polymorphisms were in strong linkage disequilibrium and were associated with the TAFI Ag concentration, with carriers of the Thr147 and 1542C alleles having higher levels (P <0.0005). These effects were similar in controls and cases and in each center. There was a difference in allele frequency between cases and controls for the Ala147Thr polymorphism, with Thr147 allele carriers being more frequent in controls than in cases in 2 centers, Stockholm (P =0.03) and San Giovanni Rotondo (P =0.03); the odds ratio for the entire cohort was 0.78 (P <0.05). In conclusion, patients with a recent MI presented lower values of TAFI Ag and higher frequencies of the “TAFI-decreasing” alleles. The geographical differences observed do not contribute to explaining the North-South gradient in MI risk in Europe.

Abnormally high thromboxane biosynthesis in homozygous homocystinuria. Evidence for platelet involvement and probucol-sensitive mechanism.

Homocystinuria due to homozygous cystathionine beta-synthase deficiency is an inborn error of metabolism characterized by a high incidence of thrombosis and premature atherosclerosis. We evaluated TXA2 biosynthesis in vivo and several in vitro tests of platelet function in 11 homocystinuric patients and 12 healthy controls. In vitro, patients platelet aggregation was within control values as were TXB2 formation, fibrinogen binding, and ATP secretion in response to thrombin. In contrast, the urinary excretion of 11-dehydro-TXB2, a major enzymatic derivative of TXA2, was > 2 SD of controls in all patients (1,724 +/- 828 pg/mg creatinine, mean +/- SD, in patients vs. 345 +/- 136 in controls, P < 0.001). The administration to four patients of low-dose aspirin (50 mg/d for 1 wk) reduced metabolite excretion by > 80%. The recovery of 11-dehydro-TXB2 excretion over the 10 d that followed aspirin cessation occurred with a pattern consistent with the entry into the circulation of platelets with intact cyclooxygenase activity. Prolonged partial reduction in the abnormally high excretion of both 11-dehydro-TXB2 and 2,3-dinor-TXB2, was also observed in seven patients who ingested 500 mg daily for 3 wk of the antioxidant drug probucol. These results provide evidence for enhanced thromboxane biosynthesis in homocystinuria and for its partial dependence on probucol-sensitive mechanisms. Furthermore, the elevated TXA2 formation in homocystinuria is likely to reflect, at least in part, in vivo platelet activation.

Abnormally high circulation levels of tissue plasminogen activator and plasminogen activator inhibitor-1 in patients with a history of ischemic stroke.

We evaluated 106 subjects with and 109 subjects without a history of ischemic stroke. All were attending a metabolic ward. The two groups were compared for major risk factors for ischemic events. A positive family history for ischemic complications of atherosclerosis was more common in subjects with a history of stroke than in those without; moreover, plasma levels of plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (TPA) were higher in patients with documented previous events. A strong positive significant correlation was found between TPA and PAI-1 levels, and an interaction between age and TPA was observed when the sample was stratified according to ages being above or below 70 years. When the patient population was analyzed according to the number of ischemic events, it was found that 62 of the 106 subjects with a history of stroke had experienced more than one ischemic event. Under these conditions, the levels of TPA and PAI-1 still correlated with the occurrence of previous ischemic episodes. As in the whole patient sample, TPA was the strongest discriminator. We conclude that in subjects attending a metabolic ward, TPA and PAI-1 levels consistently help identify subjects with a history of cerebral ischemic episodes and that TPA is the strongest discriminator.

Anticardiolipin antibodies in patients with liver disease.

Objective:Our aim was to test the hypothesis that anticardiolipin antibodies (aCL) may cause an antiphospholipid syndrome and thrombotic events in patients with liver disease.Methods:aCL were measured in 116 healthy controls and 372 patients with liver disease of different stage and etiology: 136 cases secondary to hepatitis C virus (HCV) infection, 139 due to hepatitis B virus (HBV) infection, 69 with alcoholic liver damage, and 28 cryptogenic in origin. Prior thrombotic events were recorded. The results were related to age, gender, stage, severity, and etiology of the liver disease, as well as to the occurrence of organ- and nonorgan-specific autoantibodies.Results:aCL were positive in 4.4% of controls and in 18.8% of patients (p < 0.0002). Patients with aCL were more frequently men with an advanced cirrhosis and simultaneous occurrence of anti–smooth-muscle antibodies (ASMA) in serum (p < 0.0006); their liver damage was often secondary to HBV (37.3%) or alcohol abuse (18.5%). At conditional logistic regression analysis, only the presence of ASMA (odds ratio [OR] = 3.02, 95% confidence interval [CI] 1.7–5.5, p= 0.0003), HBV (OR = 3.4, 95% CI 1.6–7.2, p= 0.0013), or alcoholic liver disease (OR = 5.3, 95% CI 2.3–12.2, p= 0.0001) were independently associated with aCL. Thrombosis was encountered in 24 patients (6.4%). At conditional logistic regression analysis, thrombosis was significantly associated with advanced age (OR = 1.07, 95% CI 1.0–1.1, p= 0.0094), development of hepatocellular carcinoma (OR = 17.8, 95% CI 1.6–196.0, p= 0.01), HBV etiology (OR = 6.3, 95% CI, 1.6–24.6, p= 0.0076), or cryptogenic liver disease (OR = 54.8, 95% CI 5–599.9, p= 0.001). Of the five patients with newly documented portal thrombosis during the follow-up, only one tested positive for aCL.Conclusion:In patients with nonautoimmune liver disease, aCL production is an epiphenomenon of the liver damage and is not associated with thrombotic complications. These data do not support the hypothesis that HCV is a cause of the antiphospholipid syndrome.

Association of Plasminogen Activator Inhibitor (PAI)-1 (SERPINE1) SNPs With Myocardial Infarction, Plasma PAI-1, and Metabolic Parameters The HIFMECH Study

Objective— The purpose of this study was to investigate the effects of plasminogen activator inhibitor-1 (PAI-1) gene (SERPINE1) single nucleotide polymorphisms (SNPs) on the risk of myocardial infarction (MI), on PAI-1 levels, and factors related to the metabolic syndrome. Methods and Results— Eleven SNPs capturing the common genetic variation of the SERPINE1 gene were genotyped in the HIFMECH study. In the 510 male cases and their 543 age-matched controls, a significant gene-smoking interaction was observed. In nonsmokers, the rs7242-G allele was more frequent in cases than in controls (0.486 versus 0.382, P=0.013) whereas the haplotype derived from the rs2227631 (−844A>G)-G and rs2227683-A alleles was ≈3-fold lower in cases than in controls (0.042 versus 0.115, P=0.006). SERPINE1 haplotypes explained 3.5% (P=0.007) of the variability of PAI-1 levels, which was attributable to the combined effects of 3 SNPs, −844A>G, rs2227666, and rs2227694. The rs6092 (Ala15Thr) and rs7242 SNPs acted additively to explain 4.4% of the variability of plasma insulin levels and 1.6% of the variability of BMI (P<10−3 and P=0.023, respectively). Conclusions— SERPINE1 haplotypes are mildly associated with plasma levels of PAI-1 and with the risk of MI in nonsmokers. They are also associated with insulin levels and BMI.

Protein Z gene polymorphisms are associated with protein Z plasma levels

studied endothelial activation markers. Therefore, acquired free PS deficiency may be associated with lipid profile levels and/or another variable common to lipids and PS, conferring an increased potential thrombotic risk on HIV-infected patients naive of treatment. Additionally, this free PS deficiency may have some role in the development of avascular necrosis seen in HIV-infected patients naive of any antiretroviral treatment.

Hemostatic variables in homozygous familial hypercholesterolemia. Effect of regular plasma cholesterol removal by low density lipoprotein apheresis.

Plasma levels of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) and the in vitro ability of platelets to aggregate and of monocytes to express procoagulant (tissue factor) activity (PCA) were evaluated in five patients who are homozygous for familial hypercholesterolemia (FH) before and after a single and a regular 5-month cholesterol removal by low density lipoprotein (LDL) apheresis. The biweekly procedure resulted in a 25% to 30% reduction (approximately 150 mg/dl) in total and LDL cholesterol (both were greater than 550 mg/dl at the beginning of the study). The basal levels of t-PA antigen and fibrinolytic activity before and after 10 minutes of venous stasis, basal PAI activity, and PAI-1 antigen were comparable to controls and were not affected by LDL apheresis. Likewise, regardless of the cholesterol removal, the PCA of freshly isolated monocytes and that of monocytes incubated with lipopolysaccharide did not differ from control values. Finally, the pre-apheresis sensitivity of platelets to adenosine diphosphate, arachidonic acid, and collagen was 1.5 to 2 times the normal value. This ratio was unchanged throughout the 5-month procedure. We conclude that fibrinolysis and monocyte PCA are normal in FH patients, whereas platelet aggregation is abnormally high, and none of these parameters is significantly affected by a 25% to 30% reduction in total and LDL cholesterol by LDL apheresis. Furthermore, our data suggest that removal of cholesterol from plasma by LDL apheresis is important for gaining insight into the mechanisms involved in the ischemic complications of arteriosclerosis in FH patients.

A role for platelets and thrombin in the juvenile stroke of two siblings with defective thrombin-adsorbing capacity of fibrin(ogen).

Binding of iodine-125-labeled thrombin to fibrin clots from two siblings with juvenile stroke was 30% of normal, and abnormally high amounts of the radioligand (not adsorbed by fibrin) were found in the supernatant. In concordance with this finding, supernatants from the patients fibrin clots caused abnormal enhancement of platelet aggregation, ATP secretion, and binding of 125I-fibrinogen to platelets exposed to subthreshold concentrations of ADP or epinephrine. Hirudin suppressed the enhancing effect of the patients supernatants, and substitution of gamma-thrombin for alpha-thrombin led to normalization of platelet responses. Under some experimental conditions, degradation of the patients fibrinogen by plasmin was impaired. However, the euglobulin lysis time, the rate of fibrin degradation by plasmin, and the lysis of the patients plasma clots by human melanoma tissue-type plasminogen activator were normal. Patients plasmas, as well as purified fibrinogen, showed a prolonged thrombin time (partially corrected by 10 mM CaCl2) and an impaired release of fibrinopeptide A in response to thrombin. However, the release in response to reptilase was normal, and the reptilase, ancrod, and thrombin coagulase times were within control (normal) values. In addition, the patients fibrinogen showed normal polymerization of preformed fibrin monomers, normal sialic acid content, and normal binding to ADP or epinephrine-stimulated platelets. Our studies support the concept that thrombin and platelets play an important role in the occurrence of stroke in these patients and suggest a direction to be followed to identify the mechanism(s) contributing to thrombosis in subjects with abnormal fibrinopeptide release.

Fibrinogen and mechanisms of thrombosis. A difficult link

Epidemiological observations indicate that high plasma fibrinogen levels are strongly correlated to the frequency of two major thrombotic complications of atherosclerosis: stroke and myocardial infarction. Thrombosis is increasingly recognized as a central mechanism in stroke and myocardial infartion, and fibrinogen is involved in events thought to play a major role in thrombosis. Therefore, elucidation of the relationship between fibrinogen and thrombosis may strengthen the predictive value of this protein and define new interventions against stroke and myocardial infarction. In addition, advances in the understanding of the atherogenic potential of several risk factors of coronary heart disease took advantage of information emerging from the measurement of the factor in population-based studies. Thus, it is conceivable that measuring plasma fibrinogen to predict stroke and myocardial infarction is a major direction to be followed to gain insight into the thrombogenic potential on this protein and inspire new strategies against thrombotic complications of atherosclerosis.

Plasma predictors of ischemic complications of atherosclerosis: open issues

In western countries, acute myocardial infarction is the commonest cause of morbidity and mortality [19]. An occlusive coronary thrombus on an ulcerated atherosclerotic plaque in the coronary arteries is the etiological event in more than 90% of patients with Q-wave myocardial infarction [38]. The underlying abnormality in non-Q-wave myocardial infarction is often a ruptured atherosclerotic plaque, which acts as a nidus for the deposition and activation of platelets. In this case, thrombosis occurs, but may not be totally occlusive, or an early spontaneous recanalization may occur. On the other hand, some clinical trials showed that a prolonged treatment with antiplatelet drugs significantly reduces the recurrence of coronary ischemia. Thus, atherosclerosis is a necessary condition for myocardial infarction, but it is not sufficient in that it usually needs the occurrence of thrombosis. However, only 25-30% of these thrombotic events are prevented by the administration of antiplatelets drugs. In recent years, epidemiological studies identified some hemostatic parameters whose abnormalities may help predict the risk of ischemic events: fibrinogen [14], plasminogen activator inhibitor-1 (PAI-1) [3], lipoprotein(a) [46], anticardiolipin antibodies (ACA) and lupus anticoagulant (LA) [10], leukocyte count [34], blood viscosity [34]. Some of these, such as fibronogen and PAI-1 are acute-phase proteins.(ABSTRACT TRUNCATED AT 250 WORDS)