Nurit Rosenberg

Factor V Leiden and Antiphospholipid Antibodies Are Significant Risk Factors for Ischemic Stroke in Children

BACKGROUND AND PURPOSE The association between ischemic childhood stroke and thrombophilia has been debated. We studied the prevalence of thrombophilia risk factors in 65 unrelated children with ischemic stroke compared with 145 control subjects. METHODS Patients and control subjects were tested for antithrombin protein C and protein S deficiencies, the presence of antiphospholipid antibodies (APLA), factor V Leiden (FVL), G20210A polymorphism of factor II gene (FII G20210A), and C677T polymorphism of 5,10-methylenetetrahydrofolate reductase gene (C677T MTHFR). RESULTS Of 65 children, 7 had a stroke in the neonatal/perinatal period and therefore were analyzed separately. Thirty-one of the remaining 58 patients with pediatric stroke (53.4%) were found to have at least 1 thrombophilia marker compared with only 25.5% of control subjects. None of the patients or control subjects had protein S or antithrombin III deficiency. The prevalence of protein C deficiency was higher among pediatric stroke patients than among control subjects, but the difference was not statistically significant (OR=7, 95% CI 0.75 to 65.1). Heterozygous FII G20210A and homozygous MTHFR 677T were not associated with an increased risk for stroke (OR=1.29, 95% CI 0.2 to 8.2; and OR=1.06, 95% CI 0.4 to 2.7, respectively). In contrast, the presence of APLA was associated with a >6-fold risk of stroke (OR=6. 08, 95% CI 1.5 to 24.3), and the heterozygosity for FVL increased the risk of stroke by almost 5-fold (OR=4.82, 95% CI 1.4 to 16.5). Five patients with pediatric stroke had a combination of > or =2 thrombophilia markers, whereas none of the control subjects had a combination of the markers. Most of the patients with neonatal/perinatal stroke were found to have at least 1 thrombophilia marker. CONCLUSIONS These data suggest that the prevalence of thrombophilia markers is increased in children with stroke compared with control subjects and, specifically, that FVL and APLA contribute significantly to stroke occurrence.

Single and Combined Prothrombotic Factors in Patients With Idiopathic Venous Thromboembolism: Prevalence and Risk Assessment

The inherited thrombophilias--deficiencies of protein C, protein S, and antithrombin III--and the prothrombotic polymorphisms factor V G1691A and factor II G20210A predispose patients toward venous thromboembolism (VTE). The aim of this study was to determine the prevalence of single and combined prothrombotic factors in patients with idiopathic VTE and to estimate the associated risks. The study group consisted of 162 patients referred for work-up of thrombophilia after documented VTE. The controls were 336 consecutively admitted patients. In all subjects factor V G1691A, factor II G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T were analyzed by specific polymerase chain reactions and restriction enzymes. Activities of antithrombin III and protein C, free protein S antigen, and lupus anticoagulant were determined in a subset of 109 patients who were not receiving oral anticoagulants. The prevalences of heterozygotes and homozygotes for factor V G1691A and factor II G20210A among patients and controls were 40.1% versus 3.9% and 18.5% versus 5.4%, respectively (P=0.0001). The prevalence of homozygotes for MTHFR C677T in patients was 22.8% and in controls, 14.3% (P=0.025). Heterozygous and homozygous factor V G1691A, factor II G20210A, and homozygous MTHFR C677T were found to be independent risk factors for VTE, with odds ratios of 16.3, 3.6, and 2.1, respectively. Two or more polymorphisms were detected in 27 of 162 patients (16.7%) and in 3 of 336 controls (0.9%). Logistic regression analysis disclosed odds ratios of 58.6 (confidence interval [CI], 22.1 to 155.2) for joint occurrence of factor V and factor II polymorphisms, of 35.0 (CI, 14.5 to 84.7) for factor V and MTHFR polymorphisms, and of 7.7 (CI, 3.0 to 19.6) for factor II and MTHFR polymorphisms. Among 109 patients in whom a complete thrombophilic work-up was performed, 74% had at least 1 underlying defect. These data indicate that in most patients referred for evaluation of thrombophilia due to idiopathic VTE, 1 or more underlying genetic predispositions were discernible. The presence of >1 of the prothrombotic polymorphisms was associated with a substantial risk of VTE.

The Frequent 5,10-Methylenetetrahydrofolate Reductase C677T Polymorphism Is Associated with a Common Haplotype in Whites, Japanese, and Africans

The common 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism causes decreased activity of this enzyme and can be associated with mild-to-moderate hyperhomocysteinemia in homozygotes, particularly when there is folic acid deficiency, as well as with vascular dementia, arterial thrombosis, venous thrombosis, neural-tube defects, and fetal loss. When folic acid intake is sufficient, homozygotes for MTHFR 677T appear to be protected against colon cancer and acute lymphatic leukemia, and fetuses bearing this genotype have an augmented survival. The distribution of MTHFR 677T is worldwide, but its frequency in different populations varies extensively. In the present study, we addressed the question of whether the MTHFR 677T alteration has an ancestral origin or has occurred repeatedly. We analyzed the frequency distribution of the previously described polymorphism A1298C in exon 7 and of three intronic dimorphisms, in white Israelis (Jews and Arabs), Japanese, and Ghanaian Africans. The 677T allele was, remarkably, associated with one haplotype, G-T-A-C, in white and Japanese homozygotes. Among the Africans, analysis of maximum likelihood also disclosed an association with the G-T-A-C haplotype, although none of the 174 subjects examined was homozygous for MTHFR 677T. These results suggest that the MTHFR 677T alteration occurred on a founder haplotype that may have had a selective advantage.

Analysis of prothrombotic and vascular risk factors in patients with nonarteritic anterior ischemic optic neuropathy

OBJECTIVE To determine whether genetic or acquired thrombophilias and other risk factors are associated with nonarteritic anterior ischemic optic neuropathy (NAION). DESIGN Retrospective case-control study. PARTICIPANTS Sixty-one patients with NAION diagnosed between 1984 and 1997. Ninety consecutive patients who visited the Eye Institute made up the control group. INTERVENTION Protein C, protein S, antithrombin III, lupus anticoagulant, and three recently described prothrombotic polymorphisms (i.e., factor V G1691A, factor II G20210A, and methylenetetrahydrofolate reductase [MTHFR] C677T) were analyzed. In addition, risk factors for arteriosclerotic vascular disease were assessed. MAIN OUTCOME MEASURES Parameters of thrombophilia. RESULTS None of the thrombophilic markers (genetic and acquired) constituted a significant risk factor for NAION. Ischemic heart disease, hypercholesterolemia, and diabetes mellitus were discerned as risk factors for NAION with odds ratios of 2.9 (95% confidence interval [CI], 1.3-6.4), 2.6 (95% CI, 1.2-5.5), and 2.3 (95% CI, 1.1-4.8), respectively. Multiple logistic regression analysis indicated that ischemic heart disease and hypercholesterolemia exerted an additive risk for NAION with a combined odds ratio of 4.5 (95% CI, 1.4-14.5). However, none of these risk factors statistically predicted second eye involvement. CONCLUSION NAION was not found to be associated with thrombophilic risk factors, yet it was related to ischemic heart disease, hypercholesterolemia, and diabetes mellitus.

Inherited factor XI deficiency confers no protection against acute myocardial infarction

Summary.  Background and purpose: Factor XI (FXI) contributes to thrombin generation thereby affecting fibrin formation and to down regulation of fibrinolysis by activation of thrombin‐activatable fibrinolysis inhibitor (TAFI). The purpose of this study was to evaluate whether patients with severe FXI deficiency are protected against acute myocardial infarction (AMI). Methods: The incidence of AMI in patients with severe FXI deficiency (FXI activity less than 15 U dL−1) whose age was 35 years or more was compared to the incidence of AMI in age and gender matched persons of the general population. Atherosclerotic risk factors were assessed in FXI deficient patients and blood was tested for prothrombotic parameters such as FV Leiden, prothrombin G20210A, lupus anticoagulant, and platelet membrane polymorphisms. The common mutations causing FXI deficiency in Jews were also examined. Results: Of 96 patients with severe FXI deficiency (55 women and 41 men) 16 had a history of AMI (6 women and 10 men). The median age at the time of AMI was 64.5 for women and 58 for men. The calculated annual rate of AMI in men was similar to the expected in the general Israeli population, whereas in women it was almost 2‐fold higher, but this difference did not reach statistical significance. One or more atherosclerotic risk factors were observed in 13 of 16 patients (81.3%) with AMI compared to 44 of 79 patients (55.7%) without AMI (P < 0.001). The frequency distributions of platelet polymorphisms and of prothrombotic polymorphisms were not different between patients with severe FXI deficiency who experienced or not an AMI. None of the patients had lupus anticoagulant. The common genotypes which cause FXI deficiency in Jews were similarly distributed in patients with and without AMI. Conclusions: Severe FXI deficiency does not confer protection against AMI.

Spectrum of the Mutations in Bernard–Soulier Syndrome

Bernard–Soulier syndrome (BSS) is a rare autosomal recessive bleeding disorder characterized by defects of the GPIb‐IX‐V complex, a platelet receptor for von Willebrand factor (VWF). Most of the mutations identified in the genes encoding for the GP1BA (GPIbα), GP1BB (GPIbβ), and GP9 (GPIX) subunits prevent expression of the complex at the platelet membrane or more rarely its interaction with VWF. As a consequence, platelets are unable to adhere to the vascular subendothelium and agglutinate in response to ristocetin. In order to collect information on BSS patients, we established an International Consortium for the study of BSS, allowing us to enrol and genotype 132 families (56 previously unreported). With 79 additional families for which molecular data were gleaned from the literature, the 211 families characterized so far have mutations in the GP1BA (28%), GP1BB (28%), or GP9 (44%) genes. There is a wide spectrum of mutations with 112 different variants, including 22 novel alterations. Consistent with the rarity of the disease, 85% of the probands carry homozygous mutations with evidence of founder effects in some geographical areas. This overview provides the first global picture of the molecular basis of BSS and will lead to improve patient diagnosis and management.

Platelets mediate tumor cell adhesion to the subendothelium under flow conditions: Involvement of platelet GPIIb-IIIa and tumor cell αv integrins

The aim of our study was to explore the role of platelets and their specific integrin receptors in mediating the interaction of 4 human tumor cell lines (3 melanoma and 1 carcinoma) with the extracellular matrix (ECM) under static and arterial flow conditions. Under static conditions, all 4 cell lines adhered to the ECM. The adhesion capacity of all 4 cell lines was virtually abolished by application of flow during incubation with the ECM. Under static conditions, tumor cell adhesion was not affected by adding platelets to the cell suspension and was slightly reduced by pre‐coating the ECM with platelets prior to the addition of tumor cells. In contrast, under flow conditions, platelets significantly increased tumor cell adhesion to the ECM, the enhancing effect being more pronounced when platelets were pre‐incubated with the ECM prior to the addition of tumor cells than when incubated simultaneously with the cells. Platelet‐mediated tumor cell adhesion under flow was markedly inhibited by blockade of the platelet GPIIb‐IIIa or of the tumor cell αv integrins. Platelets of a Glanzmann thrombastenia (GT) patient were unable to support tumor cell adhesion to the ECM under flow. Our results suggest that the interaction of tumor cells with subendothelium‐bound platelets under flow conditions is mediated by platelet GPIIb‐IIIa and by tumor cell αv integrins independently of the nature of the β subunit. Int. J. Cancer, 70:201–207, 1997.

Analysis of genetic polymorphisms related to thrombosis and other risk factors in patients with retinal vein occlusion

The purpose of this study was to investigate the role of genetic polymorphisms associated with venous and arterial thrombosis in patients with retinal vein occlusion (RVO). One-hundred and two consecutive patients with RVO were examined for factor V G1691A and factor II G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T and apolipoprotein E4 by amplification of specific DNA fragments and restriction analysis. The risks exerted by these polymorphisms and by the conventional risk factors of RVO were evaluated by comparing their frequencies among patients and controls and by estimating the respective odds ratios. We found that the prevalences of the factor V G1691A, factor II G20210A, and apolipoprotein E4 polymorphisms were similar in the study and control groups. Logistic regression analysis involving the parameters for which significant differences were detected disclosed an odds ratio of 1.9 for MTHFR C677T homozygosity (95% confidence interval 0.95-3.81), an odds ratio of 2.12 for hypertension (95% confidence interval 1.16-3.73) and an odds ratio of 3.25 for a family history of stroke (95% confidence interval 1.07-9.51). Our data suggests that homozygosity for the MTHFR C677T polymorphism is a risk factor of RVO in addition to arterial hypertension and a family history of stroke.

Specific Cysteines in β3 Are Involved in Disulfide Bond Exchange-dependent and -independent Activation of αIIbβ3

Disulfide bond exchange among cysteine residues in epidermal growth factor (EGF)-like domains of β3 was suggested to be involved in activation of αIIbβ3. To investigate the role of specific β3 cysteines in αIIbβ3 expression and activation, we expressed in baby hamster kidney cells normal αIIb with normal β3 or β3 with single or double cysteine substitutions of nine disulfide bonds in EGF-3, EGF-4, and β-tail domains and assessed αIIbβ3 surface expression and activation state by flow cytometry using P2 or PAC-1 antibodies, respectively. Most mutants displayed reduced surface expression of αIIbβ3. Disruptions of disulfide bonds in EGF-3 yielded constitutively active αIIbβ3, implying that these bonds stabilize the inactive αIIbβ3 conformer. Mutants of the Cys-567–Cys-581 bond in EGF-4 were inactive even after exposure to αIIbβ3-activating antibodies, indicating that this bond is necessary for activating αIIbβ3. Disrupting Cys-560–Cys-583 in the EGF-3/EGF-4 or Cys-608–Cys-655 in β-tail domain resulted in αIIbβ3 activation only when Cys-560 or Cys-655 of each pair was mutated but not when their partners (Cys-583, Cys-608) or both cysteines were mutated, suggesting that free sulfhydryls of Cys-583 and Cys-608 participate in αIIbβ3 activation by a disulfide bond exchange-dependent mechanism. The free sulfhydryl blocker dithiobisnitrobenzoic acid inhibited 70% of anti-LIBS6 antibody-induced activation of wild-type αIIbβ3 and had a smaller effect on mutants, implicating disulfide bond exchange-dependent and -independent mechanisms in αIIbβ3 activation. These data suggest that different disulfide bonds in β3 EGF and β-tail domains play variable structural and regulatory roles in αIIbβ3.

High prevalences of vitamin B12 and folic acid deficiency in elderly subjects in Israel

Summary.  The prevalences of vitamin B12 and folic acid deficiency in the general Israeli population of elders has not been assessed. We measured plasma cobalamin and folic acid concentrations in 418 subjects from four institutions for the aged, 749 subjects attending 19 geriatric day centres and 104 healthy controls. Methylmalonic acid (MMA) and/or homocysteine concentrations were determined in subjects who had a cobalamin concentration <221 pmol/l or folic acid concentration <11 nmol/l respectively. The prevalences of vitamin B12 deficiency (cobalamin <147 pmol/l and MMA ≥0·24 μmol/l), and folic acid deficiency (folic acid <11 nmol/l and homocysteine of >15 μmol/l) in subjects from day centres were 12·6% and 16·4% respectively, and in subjects from institutions 1·2% and 2·2% respectively (P < 0·001). Multiple logistic regression analysis indicated that the relative risk of living at home versus institutions for the aged was highly significant, with odds ratios (OR) of 6·8 [95% confidence interval (CI) 2·6–18·0] for vitamin B12 deficiency and 6·6 (95% CI 2·9–13·1) for folic acid deficiency. Analysis of data for day centre patients showed that folic acid deficiency was a significant risk factor of vitamin B12 deficiency (adjusted OR 3·68, 95% CI 2·27–5·98), and vitamin B12 deficiency was a significant risk of folic acid deficiency (adjusted OR 3·69, 95% CI 2·27–6.01). These data suggest that malnutrition is a major cause of the highly prevalent deficiencies of vitamin B12 and/or folic acid in elderly Israeli subjects dwelling at home.