Early occlusion of coronary by-pass associated with the presence of factor V Leiden and the prothrombin 20210A allele: case report.

The presence of factor V Leiden mutation and the variation of the prothrombin gene 20210GA have been described as additional risk factors for arterial thrombosis when other acquired or metabolic risk factors are present. We report here a 56-year-old man who developed coronary artery disease since 1980 without any known risk factor and underwent a cardiopulmonary by-pass in 1997. In the first month after surgery, he became symptomatic, and an angiography showed complete occlusion of the grafts and some native coronary arteries. Three months after the second cardiopulmonary by-pass, a thrombophilic state was searched, and plasma levels of lipoprotein (a) (LPa) were measured. The patient is heterozygous for factor V Leiden mutation and has the variation 20210GA of the prothrombin gene and high levels of LPa. These findings induced us to add oral anticoagulation to the aspirin treatment, and the patient is in a good condition 11 months later.

Anticuerpos anti-beta, glicoproteína I y niveles plasmáticos de la proteína de unión a C4b / Antibodies against beta glycoprotein I and C4b binding protein plasmatic levels

Autoantibodies directed to beta2 glycoprotein I (a beta 2 GPI) are frequently found in patients with antiphospholipid antibodies (aPL). They are more strongly associated with clinical manifestations of the antiphospholipid syndrome then a PL. It has been shown that beta2 GPI and C4b bibding protein (C4bBP) share certain homology. In a previous study we have shown that anticardiolipin antibodies were associated with a plasma decrease of C4bBP. The aim of the present study was to evaluate in 131 patients with a PL whether the decrease in C4bBP is related to the presence of abeta2 GPI. Lower C4bBP levels (mean +- SD) in the group of patients having abeta2 GPI (n=57) were observed when compared with the normal group (n=44), (74.3 porciento +-28.1 vs 94.6 porciento +-20.9,p<0.005).This difference was more significant consideing the IgG isotype. The group of patients with positive abeta2GPI-igG (n=41) had lower values of C4bBP (70.1 porciento +- 26.8) than both the normal group (p<0.005) and the group of patients with negative abeta2 IgG (n=90, 86.0 porciento +- 30.5 porciento, p<0.05). C4bBP deficiency (level <70 porciento) was also morefrequent in the group abeta 2GPI-IgG (+) (63.4 porciento) then in the group abeta2GPI-IgG 8-) (34.4 porciento, p<0.005). Moreover, patients with a PL and previews venous thrombosis (n=32) showed lower C4bBP values (75.1 porciento +- 27.9) compared with the normal group (p<0.05). As this time, the mechanisms responsibles for the C4bBP decrease are not known. Our findings on the close relationship between abnormalitiesin the C4bBP/protein S system and the presence of abeta2GPI could explain the major thrombotic risk in patients havingthese autoantibodies

Pathogenic role of antiprotein-phospholipid antibodies.

Antiphospholipid antibodies (aPL) are a heterogeneous family of antibodies, including those specific for a variety of phospholipid (PL)-binding proteins and also those reacting with PL molecules. The former seem to be associated with the antiphospholipid syndrome (APS). At present, the main proteins proposed as antigens are beta 2 glycoprotein I, prothrombin, protein C, protein S, kininogens and annexin V. Anionic PL might play a key role "in vivo" in the binding of aPL to PL-bound proteins. Different mechanisms may be involved in the pathogenesis of the APS, including effects of aPL on the protein C system and antithrombin III and also on platelets, endothelial cells and monocytes. Recent data on experimental animal models have provided support for a causative role of aPL in the clinical complications of the APS.

Results of EMATAP: a double-blind placebo-controlled multicentre trial of ticlopidine in patients with peripheral arterial disease.

EMATAP, a randomized stratified, placebo-controlled double-blind multicentre trial was performed in Argentina in order to confirm the effect of ticlopidine in the prevention of thrombotic events in patients with intermittent claudication. Twenty-one clinical centres enrolled 615 patients, 304 (88 diabetic and 216 non diabetic) were assigned to the ticlopidine group and 311 (95 diabetic and 216 non diabetic) to the placebo group. Treatments were given for 24 weeks. The baseline characteristics were identical in both groups. The compliance was good and only 34 patients (17 in each group) did not reach the last visit. Their status however was checked and known at that time and according to the protocol, there was no patient lost to follow-up. Twenty-five patients experienced a first event during the follow-up period and the results show a very dramatic reduction of events in the ticlopidine group (5 vs 20), the difference being highly significant (p = 0.002) in intention-to-treat analysis. If we consider the subgrouping of outcome events: sudden deaths, myocardial infarctions and strokes on the one hand, vascular surgery on the other hand, a significant reduction is found in the ticlopidine group. Taking into account the total deaths plus non-fatal events (9 vs 21), the results were also significant (p = 0.027). These above results therefore demonstrate a consistent reduction in all outcome events. As regard side effects there were fewer gastro-intestinal disturbances and skin reactions than seen in North American trials.(ABSTRACT TRUNCATED AT 250 WORDS)

Natural inhibitors of blood coagulation and fibrinolysis in patients with lupus anticoagulant.

We studied the natural inhibitors (NI) of blood coagulation and fibrinolysis in 50 patients with lupus anticoagulant (LA), in order to identify possible alterations of these NI, that could favour thrombotic manifestations. We found no statistically significant difference in antithrombin III, protein C and alpha 2-antiplasmin between controls and patients with LA, irrespective of their clinical manifestations. We found an increase of plasminogen activator inhibitor (PAI, P < 0.001) and a decrease of free protein S (PSf, P < 0.001) and total protein S (PSt, 0.01 < P < 0.05) in the patients with LA when compared with the control group. We found no difference in the levels of NI between patients with thrombosis (n = 19) and without thrombosis (n = 31) nor between patients with (n = 25) or without thrombosis and/or foetal loss (n = 25). In contrast, we observed a decrease of PSf in women with foetal loss (n = 10) as compared with women without foetal loss (n = 22, 0.01 < P < 0.05) and a decrease of PSf when comparing 19 patients with systemic lupus erythematosus (SLE) with 31 patients without SLE (0.01 < P < 0.05). These findings show that the patients with LA had several abnormalities in the NI system, but there was no significant association between levels of PAI, PSf, PSt and a history of thrombosis.

Neutralization of lupus anticoagulant activity by human immunoglobulin "in vitro".

We studied the in vitro effect of human intravenous immunoglobulin (IVIg) on the lupus anticoagulant (LA) activity present in sera of 11 patients. LA potency was determined in all the cases and a fixed dilution of each serum was chosen to perform the dose-dependent neutralization experiments. For each patient, the dilute serum was incubated for 3 h at 37 degrees C with phosphate buffer saline (PBS) alone or containing IVIg at final concentrations of 0 to 50 mg/ml. Aliquots of the incubation mixtures were added to equal volumes of normal plasma and APTTs were performed. IVIg partially neutralized the LA activity present in 10 out of 11 patients sera. These neutralizations showed an IVIg dose-dependent behaviour. Statistically significant neutralizations were observed at least at one molar ratio (MR = [IVIg]/patient's [IgG] or [IgM]). In every case, a particular MR was found in which the neutralization was maximal (N%max). The N%max ranged from 33.6% to 79.5%. Eight patients showed maximal LA neutralization at MR ranging from 8.9 to 56.8. In one patient with drug-induced LA and another exhibiting LA cofactor effect, MRs were more elevated. We found poor negative correlation between N%max and LA potency (r = -0.46) or N%max and MR of N%max (r = 0.47), although no statistic significance was reached. However, there was good agreement between LA potency and MR of N%max (r = 0.98, p less than 0.001). We have shown that IVIg may neutralize LA activity in vitro. In view of these results, we believe that IVIg should be considered as an alternative therapy in patients with LA-related clinical complications.

Melatonin effect on arachidonic acid metabolism to cyclooxygenase derivatives in human platelets.

The effect of melatonin on thrombin-induced [3H]-arachidonic acid (AA) metabolism to cyclooxygenase derivatives was determined in platelets obtained from normal volunteers at 0830 and 2030 h. Percent conversion of radioactive AA was generally greater at 2030 h than at 0830 h for every cyclooxygenase derivative analyzed. Micromolar or greater concentrations of melatonin decreased significantly the conversion of [3H]-AA to prostaglandin (PG) F2 and thromboxane (Tx) B2, and inhibited slightly the conversion to PGE2 and PGD2. After preincubation of platelets with 1 mM imidazole, the melatonin inhibitory effect was significant for PGF2 only. Melatonin (10(-6) M) showed a significant inhibitory influence on platelet ATP release induced by phorbol-12 myristate-13 acetate (PMA) at 2030 h, an effect inhibited by 1 mM aspirin. These results indicate that at pharmacological concentrations melatonin inhibits human platelet cyclooxygenase.

Diurnal variation in melatonin effect on adenosine triphosphate and serotonin release by human platelets.

The effect of the pineal hormone melatonin on adenosine diphosphate-induced human platelet aggregation and adenosine triphosphate release was assessed in platelet-rich plasma obtained from normal volunteers at 08.30 and 20.30 h. In 10(-7)-10(-5) mol/l concentrations melatonin inhibited ADP-induced platelet aggregation only in the evening (p less than 0.05). ADP-induced ATP release, an index of platelet secretory processes, showed a generally greater, dose-dependent inhibition after adding melatonin (10(-9)-10(-5)mol/l) at 20.30 h as compared with 08.30 h. The inhibitory activity of melatonin (10(-9)-10(-5) mol/l) on [3H]serotonin release elicited by thrombin in washed human platelets obtained from normal volunteers was dose-dependent; the effect was generally greater at 20.30 h. The activity of the potent platelet anti-aggregating agent prostacyclin did not exhibit diurnal differences with respect to impairing ADP-induced platelet-rich plasma aggregation. These results indicate the existence of a diurnal variation of sensitivity to melatonin in human platelets.

Partial neutralization of a lupus anticoagulant by human immunoglobulin.

In a patient with a Lupus Anticoagulant (LA) and recurrent fetal loss, we observed a significant shortening of the APTT after high-dose intravenous immunoglobulin infusion (IVIg). The LA activity present in patient's serum and purified IgG was partially neutralized by IVIg in a dose-dependent way. In addition, IgG purified from IVIg and its F(ab')2 fragment neutralized LA activity of the patient's IgG. In both cases, the neutralization was dose-dependent and it was obtained with similar molar ratios. The "in vitro" neutralization of LA activity and the immediate shortening of the APTT after IVIg infusion, might be mediated through idiotype/antiidiotype interactions. On the other hand, the long-lasting effect of IVIg in this patient indicates that it may induce specific inhibition of autoantibody synthesis. We believe that IVIg should be considered as a therapeutic alternative for LA-related clinical disorders.

Inhibition of human platelet aggregation and thromboxane-B2 production by melatonin: evidence for a diurnal variation.

The effects of melatonin on platelet aggregation and thromboxane-B2 (TxB2) production induced by 1-4 x 10(-6) M adenosine diphosphate (ADP) or 0.6 x 10(-3) M arachidonic acid (AA) were assessed in platelet-rich plasma (PRP). Micromolar concentrations of melatonin inhibited in a dose-dependent way ADP-induced platelet aggregation with individual inhibitions 40% or more at 10(-6)-10(-5) M. A significant depression of AA-induced platelet aggregation was observed only at 10(-5)-10(-4) M melatonin. Morning (0830 h)-evening (1800 h) studies of ADP-induced platelet aggregation in seven normal men showed a higher sensitivity at 1800 h when analyzed as a global inhibitory effect of melatonin (P less than 0.01). Moreover, only during the evening hours did melatonin induce reversible aggregation, an index of inhibition of the platelet secretory process elicited by ADP exposure. No diurnal variability in melatonin inhibition of AA-induced aggregation was detected. TxB2 production elicited by AA in the evening was inhibited significantly in a concentration-related manner by a 2-min preincubation with 10(-9)-10(-5) M melatonin, while during the morning hours the inhibition was significant only at 10(-6) M or higher melatonin concentrations. In the case of ADP, the inhibition of TxB2 release attained significance at 10(-5)-M (0830 h) or 10(-6)-M concentrations (1800 h). In the presence of either stimulatory agent, melatonin depression of TxB2 generation was about 2-fold greater at 1800 h than at 0830 h. The diurnal changes in melatonin effect on TxB2 production were also observed in thrombin-stimulated washed platelets. The present data indicate the existence of circadian variations in platelet responsiveness to melatonin in humans.

Familial bleeding tendency with partial platelet thromboxane synthetase deficiency: reorientation of cyclic endoperoxide metabolism.

Three family members from three successive generations presented with a moderate bleeding tendency and a functional platelet defect. They had absent aggregation with arachidonic acid (0.6--3 microM), reversible aggregation with ADP (4 microgram) and cyclic endoperoxide analogues, single wave aggregation only with adrenaline (5.4 microgram) and a prolonged template bleeding time (> min). Malondialdehyde formation was reduced after N-ethylmaleimide stimulation (2--6 nmol/10(9) platelets; control values 8--12 nmol) and serum thromboxane B2 values were reduced (33--101 ng/ml; control values 200--700 ng/ml). When the platelets were incubated with [3H]arachidonic acid the final metabolite of the lipoxygenase pathway (HETE) was produced in normal amounts but the production of thromboxane B2 and HHT was decreased whereas prostaglandin F2a, and E2 and probably D2 were increased. Evidence for enhanced production of prostaglandin D2 was also provided by the rise in the patient's platelet cyclic AMP levels following stimulation with arachidonic acid. The patient's washed platelets stimulated the production of 6-keto PGF 1a by aspirin-pretreated cultured bovine endothelial cells. The plasma levels of 6-keto PGF1a (439--703 pg/ml; normal 181 +/- 46 pg/ml) were raised. The decreased production of thromboxane B2, HHT and malondialdehyde and increased formation of prostaglandin F2a, E2, D2 and of 6-keto PGF1a are compatible with a partial platelet thromboxane synthetase deficiency and reorientation of cyclic endoperoxide metabolism. The markedly prolonged bleeding time would result not only from reduced formation of thromboxane A2 but also from increased production of the aggregation inhibiting prostaglandins PGI2 and PGD2.