The TT genotype of the C677T polymorphism in the methylentetrahydrofolate reductase as a risk factor in thrombotic microangiopathies: results from a pilot study.

In this study, we assessed the potential role of the TT genotype of the gene of the methylenetetrahydrofolate reductase for the manifestation of thrombotic microangiopathies, enrolling 40 affected patients (mean age [+/- standard deviation] 35 +/- 11 years). As a result, the methylenetetrahydrofolate reductase 677TT genotype was more prevalent in patients with thrombotic microangiopathies compared with controls (adjusted odds ratio = 2.58, 95% confidence interval = 1.2-5.7, P = .018), particularly in those suffering from the hemolytic uremic syndrome. A hemolytic more severe clinical course of thrombotic microangiopathies in carriers of the methylenetetrahydrofolate reductase 677TT genotype was not observed. In summary, our findings suggest a significant influence of the methylenetetrahydrofolate reductase genotype on the manifestation of thrombotic microangiopathies. The 677 TT genotype of this polymorphism appears to be a risk factor for manifestation of these rare thrombotic disorders, possibly explained by endothelial activation and increased oxidative stress.

The G1691A mutation of the factor V gene (factor V Leiden) and the G20210A mutation of the prothrombin gene as risk factors in thrombotic microangiopathies.

Factor V Leiden (FVL) mutation and prothrombin G20210A mutation are common hereditary risk factors for venous thrombosis. In the current study, 40 patients (mean age +/- standard deviation, 35 +/- 11 years) and 764 healthy control subjects (mean age +/- standard deviation, 37 +/- 14 years) were enrolled to assess the potential role of these mutations in the manifestation of thrombotic microangiopathies. Compared with controls, neither the heterozygous FVL mutation (7.5% vs 8.5%; P = 1) nor the heterozygous prothrombin mutation (2.5% vs 2.8%; P = 1) was more prevalent in the patients. The findings do not support a significant role of FVL and prothrombin mutations as risk factors for the manifestation of thrombotic microangiopathies. Thus, screening for these mutations does not allow the identification of individuals at increased risk for these rare thrombotic disorders.

Fatal bleeding due to a heparin-like anticoagulant in a 37-year-old woman suffering from systemic mastocytosis.

A 37-year-old female patient with systemic mastocytosis who was admitted with severe unexplained bleeding symptoms is studied. Laboratory procedures established the diagnosis of a patient-derived-heparin-like anticoagulant as a very rare hemostatic abnormality predisposing to bleeding. The patient died from refractory disease despite therapy with protamine, initiation of chemotherapy, and supportive measures. The case illustrates the clinical presentation and diagnosis of heparin-like anticoagulants. Etiology, pathophysiology, and therapeutic options are discussed.

Platelet adhesion onto immobilized fibrinogen under arterial and venous in-vitro flow conditions does not significantly differ between men and women.

BACKGROUND: Gender-related differences in incidence of arterial thrombosis have been a focus of interest for years. The platelet integrin alphaIIbbeta3 is primarily responsible for the interaction between platelets and fibrinogen and consecutive thrombus growth. In this study, we evaluated platelet adhesion onto immobilized fibrinogen under venous and arterial flow conditions in men and women. METHODS: Platelets in whole anticoagulated blood were labelled with the fluorescence dye Mepacrine and perfused through the rectangular flow chamber over glass cover slips coated with fibrinogen (shear rates of 50 s-1, 500 s-1 and 1500 s-1). A fluorescence laser-scan microscope was used for visualisation and quantification of platelet adhesion at 15 seconds, 1 and 5 minutes after the start of perfusion. RESULTS: During perfusion, the platelet adhesion linearly increased in regard to exposition time and shear rate. After five minutes of perfusion the platelet adhesion onto immobilized fibrinogen showed no significant gender related difference, neither at 50 s-1 nor at 500 s-1 and 1500 s-1 (p > 0.05), respectively. No significant difference in platelet adhesion onto immobilized fibrinogen, in regard to the menopausal status, was either observed (p > 0.05). CONCLUSION: In our in vitro experimental system, hormonal differences between men and women did not influence platelet adhesion onto immobilized fibrinogen, neither under venous nor under arterial rheological conditions.

HPA-1 polymorphism of alphaIIbbeta3 modulates platelet adhesion onto immobilized fibrinogen in an in-vitro flow system.

BACKGROUND: Platelet adhesion and subsequent thrombus formation on a subendothelial matrix at the site of vascular damage play a crucial role in the arrest of posttraumatic bleeding but also in different pathological thrombotic events, such as acute coronary syndrome and stroke. Recently published studies have clearly demonstrated that platelet integri alphaIIbbeta3 is intimately involved in the occlusive thrombus formation at the site of endothelial damage. Therefore, any genetic variation in the expression of this receptor may lead to an excessive bleeding or excessive thrombus formation. In this study, we evaluated the influence of HPA-1 polymorphism of integrin alphaIIbbeta3 on platelet adhesion onto immobilized fibrinogen using an in vitro system simulating blood flow. METHODS: Platelets in anticoagulated whole blood [49 healthy previously genotyped blood donors) were labelled with fluorescence dye and perfused through a rectangular flow chamber (shear rates of 50 s(-1), 500 s(-1) and 1500 s(-1)). A fluorescence laser-scan microscope was used for visualisation and quantification of platelet adhesion at 15 sec, 1 and 5 minutes after start of perfusion. RESULTS: During perfusion, the platelet adhesion linearly increased with regard to exposition time and shear rate. Perfusion of blood preincubated with Abciximab over fibrinogen-coated cover-slips showed reduced platelet adherence (absolute fluorescence: 168 +/- 35 U vs. 53000 +/- 19000 at control experiments, p < 0.05), as well as by perfusion over BSA-coated glass coverslips. Platelet with HPA-1a/1a genotype exhibited initial better adhesion but they also exhibited higher detachment under arterial flow conditions compared to the HPA-1b/1b platelets. Analysis of stable adhesion rate indicate that the platelets carrying the HPA-1b/1b genotype have a higher reactivity threshold for initial interaction with fibrinogen but under the higher shear rate (in regard to time of perfusion) also realize more stable bonds with fibrinogen than platelets with the HPA-1a/1a genotype. CONCLUSION: Our data support the contention that genetically determined variants of platelet integrins alphaIIbbeta3 could play a role in arterial thrombogenesis and thus confirm the hypothesis derived from epidemiological studies.

Antithrombin significantly influences platelet adhesion onto immobilized fibrinogen in an in-vitro system simulating low flow.

BACKGROUND: Adhesion of platelets onto immobilized fibrinogen is of importance in initiation and development of thrombosis. According to a recent increase in evidence of a multiple biological property of antithrombin, we evaluated the influence of antithrombin on platelet adhesion onto immobilized fibrinogen using an in-vitro flow system. METHODS: Platelets in anticoagulated whole blood (29 healthy blood donors) were labelled with fluorescence dye and perfused through a rectangular flow chamber (shear rates of 13 s-1 to 1500 s-1). Platelet adhesion onto fibrinogen-coated slips was assessed using a fluorescence laser-scan microscope and compared to the plasma antithrombin activity. Additionally the effect of supraphysiological AT supplementation on platelets adhesion rate was evaluated. RESULTS: Within a first minute of perfusion, an inverse correlation between platelet adhesion and plasma antithrombin were observed at 13 s-1 and 50 s-1 (r = -0.48 and r = -0.7, p < 0.05, respectively). Significant differences in platelet adhesion related to low (92 +/- 3.3%) and high (117 +/- 4.1%) antithrombin activity (1786 +/- 516 U vs. 823 +/- 331 U, p < 0.05) at low flow rate (13 s-1, within first minute) have been found. An in-vitro supplementation of whole blood with antithrombin increased the antithrombin activity up to 280% and platelet adhesion rate reached about 65% related to the adhesion rate in a non-supplemented blood (1.25 +/- 0.17 vs. 1.95 +/- 0.4 p = 0.008, respectively). CONCLUSION: It appears that antithrombin in a low flow system suppresses platelet adhesion onto immobilized fibrinogen independently from its antithrombin activity. A supraphysiological substitution of blood with antithrombin significantly reduces platelet adhesion rate. This inhibitory effect might be of clinical relevance.

The screening power of methylenetetrahydrofolate reductase C677T polymorphism versus plasma homocysteine concentration in patients with stenosis of the internal carotid artery.

BACKGROUND: Hyperhomocysteinemia is an important and independent risk factor for vascular disease. About 35% of patients with stroke and 47% of patients with peripheral arterial disease have elevated plasma homocysteine (HCY) concentrations. The relationship between plasma HCY and the methylentetrahydrofolate reductase (MTHFR) C677T polymorphism is still unclear, especially in regard to screening/diagnostic power. METHODS: This case-control study was performed on 96 patients, who underwent surgery due to asymptomatic or symptomatic high grade stenosis of the internal carotid artery (ICA), and 96 healthy age and sex-matched, controls. Plasma HCY concentration was determined using a commercial kit for fully automated analysis (AxSYM, Abbott). The C677T polymorphism of the MTHFR-gene was assessed by PCR. RESULTS: The mean plasma HCY concentration was significantly higher in the group with stenosis of ICA compared to the controls, 12.43 +/- 6.96 microM and 10.16 +/- 3.16 microM, respectively, (p < 0.05). An HCY plasma concentration of 1.5 SD above the mean value of the control group, was defined as cut-off for a pathological versus physiological plasma concentration. The sensitivity and specificity of HCY was 0.27 and 0.94, respectively. The positive predictive value was 0.82. There was no significant difference in the frequency of the MTHFR 677 CT and TT genotype between patients and controls (47% vs. 47% and 8.3% vs. 11.4%, respectively). Carriers of the T-allele (CT and TT genotypes) have significantly higher plasma HCY concentrations than CC patients, 14.1 +/- 7.6 microM and 10.29 +/- 5.2 microM, respectively, p < 0.05. Sensitivity and specificity of the MTHFR C677T polymorphism (T-allele) were 0.56 and 0.40, respectively. The positive predictive value was 0.48. There was no significant difference in plasma HCY or genotype frequency of the MTHFR C677T polymorphism between asymptomatic and symptomatic patients. CONCLUSION: Our study shows that in a population with a given pretest disease probability of 50%, the determination of plasma HCY concentration, with a positive predictive value of 0.82, is more suitable for screening of patients at risk than analysis of the MTHFR C677T polymorphism.

Effects of homocysteine on vascular and tissue adenosine: a stake in homocysteine pathogenicity?

Homocysteine may have deleterious effects on the cardiovascular system. It has been hypothesized that these effects may be brought about by a decrease in the adenosine concentration via the S-adenosylhomocysteine hydrolase reaction. A requirement for this causal relationship is proof of a reduction in vascular adenosine concentration during conditions of elevated homocysteine concentrations. In the present communication we summarize published data obtained during systematic variation of the arterial homocysteine concentration. Most of the results reported show that an increase in homocysteine concentration to 100 microM is associated with a 20-50% decrease in vascular adenosine concentration and an increase in tissue S-adenosylhomocysteine level. Homocysteine effects on the adenosine concentration seem to be more pronounced under conditions of impaired oxygenation. Further experiments, in particular on organs and tissue that release high amounts of homocysteine, i.e., the liver, are warranted to study the potential effects of homocysteine on vascular and tissue adenosine concentrations and consequent effects on organ function. The evidence obtained may be relevant for future assessment of risk indicators in conjunction with homocysteine pathogenicity, which might potentially be extended to measurements of adenosine or S-adenosylhomocysteine levels.

5, 10-Methylenetetrahydrofolate reductase (MTHFR) 677 C --> T genetic polymorphism in 228 Croatian volunteers.

5, 10-Methylenetetrahydrofolate Reductase (MTHFR) is one of the key enzymes in the metabolism of homocysteine, where it catalyses its remethylation. The autosomal recessive bp 677 C --> T mutation in the MTHFR gene leads to the substitution of valine for alanine. Individuals who are homozygous for this C677T mutation exhibit a decreased specific activity and increased thermolability of this enzyme. This leads to increased plasma levels of homocysteine, which is a known risk factor for atherosclerosis and various manifestations of the atherosclerotic disease. The aim of this study was to find out the distribution and frequency of this mutation in the general Croatian population. A group of 228 volunteers (175 males and 53 females) has been analyzed for the MTHFR polymorphism, which revealed the following distribution: 105 (46.05%) individuals were without mutation (C/C), 102 (44.74%) were heterozygous (C/T) and 21 (9.21%) homozygous (T/T). These findings are within the results of studies on other European populations.

Tracer adenosine: a novel myocardial flow marker.

UNLABELLED: Local myocardial blood flow measurements are of great importance in experimental and clinical settings. However, a lack of ideal markers is evident. Adenosine is suggested to be a potential candidate because of its high uptake and rapid intracellular sequestration. We specifically tested the hypothesis that the local deposition density of labeled adenosine within the heart reflects local myocardial blood flow. METHODS: Tracer microspheres, the recognized standard for local blood flow measurements, were injected and compared with simultaneously injected labeled adenosine ((3)H/(14)C) in tracer concentration into the left atrium of anesthetized Beagle dogs (n = 7). Myocardial deposition densities were assessed through beta-scintillation and gamma-counting measurements in samples (100-128 per heart) of an average wet mass of 487 +/- 54 mg. To challenge local myocardial blood flow distribution, alprostadil was infused into the left circumflex artery in 3 experiments. In 2 other experiments, erythro-9-hydroxy-nonyl-adenine (EHNA) was infused to inhibit degradation of injected adenosine to inosine. RESULTS: Tracer adenosine and microspheres did not exert significant local or systemic hemodynamic effects. Both were almost completely extracted from blood within 2 min and locally retained in the tissue. Deposition densities of tracer microspheres and labeled adenosine correlated closely in each experiment, independently of the respective protocol (control, EHNA, or alprostadil), over a wide range of local myocardial blood flows (0.23-12.9 mL min(-1) g(-1)). The mean correlation coefficient (n = 293) was r = 0.93 (r(2) = 0.86; P < 0.0001), indicating that the deposition density of (3)H-adenosine could explain local blood flow as measured with the tracer microsphere technique with 86% probability. CONCLUSION: Adenosine appears to be a reliable marker of local blood flow in dog myocardium.