Antioxidant and antiplatelet effects of the alpha-tocopherol-aspirin combination in type 1-like diabetic rats

We analyze the effect of the combination of acetylsalicylic acid (2 mg/kg/day p.o.) and alpha-tocopherol (25 mg/kg/day p.o.) in a type-1-like experimental model of diabetes mellitus on platelet factors, endothelial antithrombotic factors and tissue oxidative stress. In diabetic rats, the combination of drugs had a greater inhibitory effect on platelet aggregation than in untreated control animals with diabetes (88.87%). The combination of drugs had little effect on the inhibition of thromboxane production (-90.81%) in comparison to acetylsalicylic acid alone (-84.66%), potentiated prostacyclin production (+162%) in comparison to alpha-tocopherol alone (+30.55%), and potentiated nitric oxide production (+241%) in comparison to either drug alone (acetylsalicylic acid +125%, alpha-tocopherol +142%). The combination of the two drugs improved the thromboxane/prostacyclin balance (0.145+/-0.009) in comparison to untreated diabetic animals (4.221+/-0.264) and in untreated healthy animals (0.651+/-0.045). It did not potentiate the antioxidant effect of either drug alone, but did increase tissue concentrations of reduced glutathione, especially in vascular tissue (+90.09% in comparison to untreated animals). In conclusion, in the experimental model of diabetes tested here, the combination of acetylsalicylic acid and alpha-tocopherol led to beneficial changes that can help protect tissues from thrombotic and ischemic phenomena.     

Identification of a sequence of human activated protein C (residues 390-404) essential for its anticoagulant activity.

Activated protein C (APC) exerts its physiologic anticoagulant role by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. To identify the regions on the surface that mediate anticoagulant activity, 26 synthetic peptides were prepared representing 90% of the human protein C heavy chain primary structure and tested for their ability to inhibit APC anticoagulant activity. Peptide-(390-404) specifically inhibited APC activity in activated partial thromboplastin time and Xa-1-stage coagulation assays in normal, in protein S-depleted and Factor VIII-deficient plasma with 50% inhibition at 5 microM peptide. Polyclonal antibodies raised against this peptide and immunoaffinity-purified on a protein C-Sepharose column inhibited APC anticoagulant activity in activated partial thromboplastin time and Xa-1-stage assays in normal, protein S-depleted, and Factor VIII-deficient plasma with half-maximal inhibition at 30 nM anti-(390-404) antibody. Neither the peptide-(390-404) nor the anti-(390-404) antibodies inhibited APC amidolytic activity or the reaction of APC with recombinant [Arg358] alpha 1-antitrypsin. Furthermore, in a purified system, peptide-(390-404) inhibited APC-catalyzed inactivation of Factor Va in the presence as well as in the absence of phospholipids with 50% inhibition at 4 microM peptide. These data suggest that the region containing residues 390-404 in APC is essential for anticoagulant activity and is available to interact with antibodies or with other proteins such as the macromolecular substrates Factors Va or VIIIa.     

Eicosanoids in preeclampsia

Preeclampsia is characterized by an imbalance between two cyclooxygenase metabolites of arachidonic acid, thromboxane and prostacyclin, that favors thromboxane. Because of the biologic actions of these two eicosanoids, this imbalance might explain major clinical symptoms of preeclampsia, such as hypertension, platelet aggregation and reduced uteroplacental blood flow. In the maternal circulation, this imbalance is primarily manifested by decreased production of prostacyclin by endothelial cells. Platelet thromboxane synthesis is only increased in severe preeclampsia. In the placenta and in leukocytes, the imbalance is exacerbated by increased production of thromboxane coupled with decreased production of prostacyclin in both mild and severe preeclampsia. Longitudinal measurements of urinary metabolites of thromboxane and prostacyclin reveal that the thromboxane/prostacyclin imbalance predates the onset of clinical symptoms of preeclampsia. The imbalance between thromboxane and prostacyclin is most likely caused by oxidative stress, which is manifest in preeclampsia by increased lipid peroxidation and decreased antioxidant protection. Oxidative stress may drive this imbalance because lipid peroxides activate the cyclooxygenase enzyme to increase thromboxane synthesis, but at the same time they inhibit prostacyclin synthase to decrease prostacyclin synthesis. Low-dose aspirin therapy (50-150 mg/day) has been considered for the prevention of preeclampsia because it selectively inhibits thromboxane synthesis. Several studies reported dramatic decreases in the incidence of preeclampsia with low-dose aspirin therapy. However, two large multicenter studies reported only modest decreases, which dampened enthusiasm. The two large studies were "intent to treat" studies which included patients who were noncompliant and who discontinued the use of aspirin. In one of the studies for which compliance statistics were available only 53% of the aspirin group had a compliance rate greater than 75%, which raises a question as to whether the effectiveness of aspirin was being tested. Low-dose aspirin therapy should not yet be dismissed for the prevention of preeclampsia, but be reconsidered with emphasis on compliance using doses of aspirin in the range of 100-150 mg/day combined with antioxidants.     

Therapeutic control of anticoagulant treatment.

Control of anticoagulant treatment was studied in 250 patients. One hundred and fifty patients receiving long-term anticoagulant treatment (group 1) were studied for 52 weeks and the remaining 100 (group 2) for 12 weeks after discharge from hospital. The desired British correlated ratio range was 2.5-3.3, and a range of 2.3-3.5 was classified as satisfactory. In group 1 a satisfactory ratio was obtained for 70% of the study period and 120 of the 150 patients were maintained within this range for over 60% of the time. In group 2 only half of the patients were maintained within the satisfactory range and for 50% of the study period or less. The time and effort expended in therapeutic control were more than most clinics could afford, and the results for group 2 were disappointing. The standard of long-term anticoagulant treatment should be improved by continuous review of control and by "therapeutic quality control."     

Antibody profile of pregnant women with antiphospholipid syndrome and pregnancy outcome after treatment with low dose aspirin and low-weight-molecular heparin

The aim of the research was to show our diagnostic and therapeutic experience with antiphospholipid syndrome (APS) in pregnant women. 36 pregnant women suspect on APS were included in the study: 32 with primary antiphospholipd syndrome (PAPS) and 4 with secondary antiphospholipid syndrome (SAPS). All pregnant women received low-molecular-weight-heparin (LMWH) and low dose aspirin (LDA) therapy. Control group represented 26 women with SAPS and previous bad reproductive anamnesis. Average pregnancy lasted 37.06 +/- 0.707 weeks. LMWH and LDA therapy was successful in 97.22%. Lupus anticoagulant (LA) was found to be more frequent in PAPS group (71.87%). Anticardiolipin antibodies (aCL) were found to be more frequent in SAPS (26.66%). For three patients (3.37%), PAPS was diagnosed due to a fact that they had positive antibeta2-glycoproteinl (antibeta-GP1). To make APS diagnosis, it is of great importance to search for all antiphospholipid antibodies. LMWH and low dose of acetylsalicylic acid should be the first choice therapy.     

Anticoagulant activities of oleanolic acid via inhibition of tissue factor expressions

Oleanolic acid (OA), a triterpenoid known for its anti-inflammatory and anti-cancer properties, is commonly present in several medicinal plants but its anticoagulant activities have not been studied. Here, the anticoagulant properties of OA were determined by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrin polymerization as well as cell-based thrombin and activated factor X (FXa) generation activities. Data showed OA prolonged aPTT and PT significantly and inhibited thrombin catalyzed fibrin polymerization. In addition, OA inhibited the activities of thrombin and FXa and inhibited the generation of thrombin or FXa in human endothelial cells. OA also inhibited TNF-α-induced tissue factor expression on human endothelial cells. In accordance with these anticoagulant activities, OA showed an anticoagulant effect in vivo. These results indicate that OA possesses antithrombotic activities and suggest that daily consumption of a herb containing OA may be preventing thrombosis in pathological states.     

The “therapeutic range” of the one-stage prothrombin time in the control of anticoagulant therapy: The effect of different thromboplastin preparations

Commercially available thromboplastin reagents and two human brain preparations have been compared using the one-stage prothrombin time and plasma samples from patients receiving long-term oral anticoagulant therapy. Considerable variation is noted between various thromboplastins using the same plasma sample. The commercially available thromboplastins give shorter prothrombin times than do human brain preparations. With the latter, the “therapeutic range” is represented by a prothrombin time of about 1.8 to 3.0 times the normal control value, whereas with commercial preparations the “therapeutic range” is about 1.25 to 1.75 times normal. The implications of these observations are discussed; the desirability of standardization of the one-stage prothrombin time is emphasized.     

An Investigation of the Hemorrhagic Diathesis in Patients Receiving Coumarin and Indanedione Anticoagulants

Coagulation studies were carried out on 10 patients who bled during anticoagulant therapy, in whom no other underlying cause for bleeding could be demonstrated, and 10 patients with similar degrees of hypoprothrombinemia who were not bleeding. The average age and sex distribution of the two groups was similar, and no association was noted between the occurrence of hemorrhage and the type of anticoagulant used, the duration of treatment or the nature of the underlying disease. Comparison of the results revealed no differences in the levels of factors II, VII, IX and X or in the glass and silicone (Siliclad) clotting time, the thromboplastin generation test and Thrombotest. It was concluded that all patients on anticoagulant drugs whose prothrombin time is in the therapeutic range or longer are potential bleeders and that one cannot necessarily predict those who will bleed on the basis of coagulation studies.     

Study on a new chromogenic substrate for the prothrombin time determination

The aim of our study was to evaluate the possibility of using a chromogenic substrate for the prothrombin time determination. The reagent used by us (Chromoquick) is composed of a human placenta thromboplastin and chromogenic substrate (Tos-Gly-Pro-Arg-5-amino-2-nitrobenzoic acid-isopropylamide), calcium chloride and a buffer. Normal subjects, patients with liver disease, patients on oral anticoagulant therapy, patients on heparin therapy, heterozygous and homozygous patients for prothrombin complex defects and other miscellaneous conditions have been investigated. The results of chromoquick have been related with standard prothrombin time obtained using a human placenta thromboplastin (Thromborel) and rabbit brain and lung thromboplastin (Simplastin). The normal range was 18-23 s for chromoquick and 13.5-15.5 s for the standard prothrombin times using Thromborel and Simplastin. In all groups of patients examined we noticed a significant correlation between the chromogenic and the classic prothrombin times with r values varying between +0.505 and +0.947. The statistical significance resulted from p values varying between less than 0.05 and less than 0.001. Only in the case of some heterozygotes for prothrombin complex factor defects the values obtained have not been unequivocal in the sense that in a few instances the heterozygotes seemed to escape detection. Therefore, it seems that the introduction of chromogenic substrates in laboratory practice for the prothrombin time determination is possible and can offer considerable advantages like standardization and automation. The only disadvantage may be caused by costs involved.     

How cardiologists manage antithrombotic treatment of patients with atrial fibrillation undergoing percutaneous coronary stenting: the WOEST survey 2018

BackgroundAntithrombotic treatment choices are complicated when patients have both atrial fibrillation (AF) and acute coronary syndrome and/or undergo percutaneous coronary intervention (PCI). In this study, we aimed to gain insight into antithrombotic management strategies in daily clinical practice.MethodsWe invited interventional cardiologists to complete the WOEST (What is the Optimal antiplatElet & Anticoagulant Therapy in Patients With Oral Anticoagulation and Coronary StenTing) survey 2018. In this questionnaire, we presented a patient with a non-ST-elevation myocardial infarction (NSTEMI) and an elective PCI case.ResultsThe results were based on 118 completed questionnaires (response rate 69.4%). In the case of the AF patient with NSTEMI, most cardiologists indicated they would initiate dual antiplatelet therapy (acetylsalicylic acid and clopidogrel) and continue non-vitamin K antagonist oral anticoagulant (NOAC) therapy at admission and during coronary angiography/PCI. At discharge, 70.3% would prescribe triple antithrombotic therapy (oral anticoagulation, acetylsalicylic acid and clopidogrel), mostly for 1 month. One year after NSTEMI, 83.1% would cancel the antiplatelet therapy and prescribe NOAC monotherapy. For the AF patient undergoing elective PCI, 51.7% would start dual antiplatelet therapy prior to the procedure and 52.5% would discontinue NOAC therapy prior to the PCI. At discharge, 55.1% would start triple antithrombotic therapy. Furthermore, 25.4% responded they routinely prescribe a reduced dose of NOAC after discharge. One year after PCI, 89.0% would continue NOAC monotherapy.ConclusionThe WOEST survey demonstrated heterogeneity in antithrombotic management strategies among interventional cardiologists. This observed variety mirrors the heterogeneity of the many guidelines and consensus documents. Further research is needed to guide patient-tailored medicine for AF patients undergoing PCI.Electronic supplementary materialThe online version of this article (10.1007/s12471-020-01500-3) contains supplementary material, which is available to authorized users.     

Increased tissue-plasminogen activator (t-PA) levels in patients under oral anticoagulant therapy

The fibrinolytic system was investigated in 30 patients under oral anticoagulant therapy, and in 23 control patients not receiving oral anticoagulants. Patients under oral anticoagulant therapy had significantly higher tissue-plasminogen activator (t-PA) antigen levels than patients in the control group. Mean t-PA levels before venous occlusion were 18.4 ng/ml in the anticoagulated patients vs. 7.9 ng/ml in the control patients (p less than 0.001). After venous occlusion for 10 minutes, t-PA levels were 45.0 ng/ml in the anticoagulated patients and 24.2 ng/ml in the control patients (p less than 0.01). Plasminogen activator inhibitor (PAI) capacity was not significantly different in the two groups before venous occlusion (VO) but differed slightly (p less than 0.05) after VO. The net decrease in euglobulin lysis time (ELT) after venous occlusion (= ELT before VO - ELT after VO), indicating the relative potency of the fibrinolytic activity in blood, was also significantly higher in the anticoagulated patients (median 240 min vs. 125 min, p less than 0.001). These data indicate that oral anticoagulant therapy increases the fibrinolytic activity in blood, and thus may have an additional therapeutic effect in addition to anticoagulation.     

Recovery of megakaryocyte thromboxane production in vitro after aspirin inhibition

Megakaryocytes isolated in high purity from guinea pigs produced thromboxane B₂ in response to exogenously provided arachidonic acid. This production was inhibited by in vitro treatment with acetylsalicylic acid with a concentration response relationship similar to that seen in platelets. During in vitro culture, the aspirin-treated megakaryocytes recovered thromboxane synthetic ability. Following a lag of 12 hours, recovery of megakaryocyte thromboxane production resumed at a rate of 16% of control per day. This recovery was inhibited by the addition of cycloheximide to the culture medium.     

Bleeding complications after pacemaker or cardioverter-defibrillator implantation in patients receiving dual antiplatelet therapy

Introduction. The aim of the study was to define the prevalence of bleeding events in patients treated with dual antiplatelet therapy (DAT) in comparison with patients receiving only acetylsalicylic acid (ASA). Methods. Prospective two-centre registry of all first implantations of pacemakers, cardioverter-defibrillators and cardiac resynchronisation therapy units in patients receiving ASA (n=194) or DAT (n=53). Results. Bleeding complications were detected in 27 (16.2%) patients in the ASA group and in 13 (24.5%) in the DAT group. There was no significant difference in the overall number of complications between the patients receiving ASA or DAT, although there was a trend towards a higher incidence of overall complication rates in the DAT group (p=0.0637). The incidence of major complications (requiring blood transfusion or surgical intervention or prolonging hospital stay) was low (3.6%), and similar in both groups (3.6 and 3.8% respectively, ns). The rate of minor complications (subcutaneous haematomas) was greater in the DAT group (p=0.015). Conclusions. Treatment with DAT does not increase the risk of major bleeding complications as a result of device implantation; however, minor complications are significantly more frequent. Our results suggest that DAT could be continued in patients undergoing device implantation with a moderate risk of bleeding complications. (Neth Heart J 2010;18:230-5.)     

Pharmacological modulation of prostacyclin and thromboxane production of rat and cat venous tissue slices.

To reveal a potential modulating effect of vasoactive pharmacological agents on the prostanoid production of the venous wall, prostacyclin and thromboxane release from venous tissue slices was studied. Aortic and caval vein samples from 20 rats as well as from 21 cats were studied. Prostacyclin and thromboxane productions were determined by radioimmunoassay as 6-keto-PGF1 alpha and TxB2 released into the incubation medium. Venous tissue produced significantly less prostacyclin per unit weight than arterial tissue in rats (30.7 +/- 4.6 vs. 52.1 +/- 8.2 pg/mg/min), while in cats an opposite situation was found (16.6 +/- 3.2 vs. 7.06 +/- 1.9 pg/mg/min). Thromboxane production of venous tissue was consequently higher than corresponding values for aortic tissue (3.72 +/- 0.46 vs. 1.54 +/- 0.14 in rats and 3.4 +/- 0.6 vs. 1.33 +/- 0.19 in cats, all values in pg/mg/min). Norepinephrine and dopamine significantly increased both the prostacyclin and the thromboxane release from venous tissue, while isoproterenol had no effect. Vasopressin significantly increased thromboxane release and decreased the ratio of prostacyclin vs. thromboxane production (from 10.4 +/- 1.6 to 7.5 +/- 1.6, in acetylsalicylic acid pretreated cats). Angiotensin and thrombin had no significant effects. Bradykinin (0.5 microgram/ml) significantly augmented prostacyclin release from venous tissue (14.4 +/- 2.6 from 10.9 +/- 2.4 pg/mg/min) and decreased thromboxane release (0.65 +/- 0.18 from 1.35 +/- 0.22 pg/mg/min). Methionine-enkephalin (5 micrograms/ml) significantly reduced the thromboxane release from venous tissue slices. The presented material demonstrates that several vasoactive agents modulate the vasoactive prostanoid release of the venous wall. In some cases, the prostacyclin and the thromboxane productions are influenced separately, which in turn will have its impact on smooth muscle activity and thrombocyte aggregation.     

Anticoagulant properties of a sulfated galactan preparation from a marine green alga, Codium cylindricum

An anticoagulant was isolated from a marine green alga, Codium cylindricum. The anticoagulant was composed mainly of galactose with a small amount of glucose, and was highly sulfated (13.1% as SO3Na). The anticoagulant properties of the purified anticoagulant were compared with that of heparin by assays of activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using normal human plasma. The anticoagulant showed similar activities with heparin, however, weaker than heparin. On the other hand, the anticoagulant did not affect PT even at the concentration at which APTT and TT were strongly prolonged. The anticoagulant did not potentiate antithrombin III (AT III) and heparin cofactor II (HC II), thus the anticoagulant mechanism would be different from that of other anticoagulants isolated so far from the genus Codium.     

Anticoagulant activities of curcumin and its derivative

Curcumin, a polyphenol responsible for the yellow color of the curry spice turmeric, possesses antiinflammatory, antiproliferative and antiangiogenic activities. However, anticoagulant activities of curcumin have not been studied. Here, the anticoagulant properties of curcumin and its derivative (bisdemethoxycurcumin, BDMC) were determined by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT) as well as cell-based thrombin and activated factor X (FXa) generation activities. Data showed that curcumin and BDMC prolonged aPTT and PT significantly and inhibited thrombin and FXa activities. They inhibited the generation of thrombin or FXa. In accordance with these anticoagulant activities, curcumin and BDMC showed anticoagulant effect in vivo. Surprisingly, these anticoagulant effects of curcumin were better than those of BDMC indicating that methoxy group in curcumin positively regulated anticoagulant function of curcumin. Therefore, these results suggest that curcumin and BDMC possess antithrombotic activities and daily consumption of the curry spice turmeric might help maintain anticoagulant status.     

Sulfonation and anticoagulant activity of botryosphaeran from Botryosphaeria rhodina MAMB-05 grown on fructose

Botryosphaeran (EPS_FRU), an exopolysaccharide of the β-(1→3,1→6)-d-glucan type with 31% branching at C-6, is produced by the fungus Botryosphaeria rhodina MAMB-05 when grown on fructose as carbon source. Botryosphaeran was derivatized by sulfonation to induce anticoagulant activity. The effectiveness of the sulfonation reaction by chlorosulfonic acid in pyridine was monitored by the degree of substitution and FT-IR analysis of the sulfonated EPS_FRU (once sulfonated, EPS_FRU SULF; and re-sulfonated, EPS_FRU RESULF). Activated partial thromboplastin time (APTT) and thrombin time (TT) tests of EPS_FRU RESULF indicated significant in vitro anticoagulant activity that was dose-dependent. EPS_FRU did not inhibit any of the coagulation tests.     

Anticoagulant properties of a sulfated galactan preparation from a marine green alga, Codium cylindricum

An anticoagulant was isolated from a marine green alga, Codium cylindricum. The anticoagulant was composed mainly of galactose with a small amount of glucose, and was highly sulfated (13.1% as SO Na). The anticoagulant properties of the purified anticoagulant were compared with that of heparin by assays of activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using normal human plasma. The anticoagulant showed similar activities with heparin, however, weaker than heparin. On the other hand, the anticoagulant did not affect PT even at the concentration at which APTT and TT were strongly prolonged. The anticoagulant did not potentiate antithrombin III (AT III) and heparin cofactor II (HC II), thus the anticoagulant mechanism would be different from that of other anticoagulants isolated so far from the genus Codium.