The formation of an anti-restenotic/anti-thrombotic surface by immobilization of nitric oxide synthase on a metallic carrier.

Coronary stenosis due to atherosclerosis, the primary cause of coronary artery disease, is generally treated by balloon dilatation and stent implantation, which can result in damage to the endothelial lining of blood vessels. This leads to the restenosis of the lumen as a consequence of migration and proliferation of smooth muscle cells (SMCs). Nitric oxide (NO), which is produced and secreted by vascular endothelial cells (ECs), is a central anti-inflammatory and anti-atherogenic player in the vasculature. The goal of the present study was to develop an enzymatically active surface capable of converting the prodrug l-arginine, to the active drug, NO, thus providing a targeted drug delivery interface. NO synthase (NOS) was chemically immobilized on the surface of a stainless steel carrier with preservation of its activity. The ability of this functionalized NO-producing surface to prevent or delay processes involved in restenosis and thrombus formation was tested. This surface was found to significantly promote EC adhesion and proliferation while inhibiting that of SMCs. Furthermore, platelet adherence to this surface was markedly inhibited. Beyond the application considered here, this approach can be implemented for the local conversion of any systemically administered prodrug to the active drug, using catalysts attached to the surface of the implant.

Cooperating attackers in neural cryptography.

A successful attack strategy in neural cryptography is presented. The neural cryptosystem, based on synchronization of neural networks by mutual learning, has been recently shown to be secure under different attack strategies. The success of the advanced attacker presented here, called the "majority-flipping attacker," does not decay with the parameters of the model. This attacker's outstanding success is due to its using a group of attackers which cooperate throughout the synchronization process, unlike any other attack strategy known. An analytical description of this attack is also presented, and fits the results of simulations.

Hyperhomocysteinemia is common in patients with antiphospholipid syndrome and may contribute to expression of major thrombotic events.

Homocysteine plasma levels were measured by high-performance liquid chromatography in 52 patients with primary antiphospholipid syndrome (APS). Elevated homocysteine concentrations (>or= 15 micromol/l) were found in 16/52 (30.8%) APS patients. Elevated homocysteine levels were found to be associated with an increased risk for a major thromboembolic event (50 versus 16.7%, P = 0.014). Hyperhomocysteinemia is a common finding in APS patients, and may contribute to severity of thrombotic tendency observed in this syndrome.

Prophylactic therapy with enoxaparin during L-asparaginase treatment in children with acute lymphoblastic leukemia.

Forty-one consecutive children with acute lymphoblastic leukemia (ALL) received prophylaxis therapy with the low molecular weight heparin (LMWH) enoxaparin during L-asparaginase treatment. Enoxaparin was given every 24 h subcutaneously at a median dose of 0.84 mg/kg per day (range, 0.45-1.33 mg/kg per day) starting at the first dose of L-asparaginase until 1 week after the last dose. Molecular analysis for thrombophilic polymorphisms documented prothrombin G20210A mutation in 3/27 (11%), homozygosity for MTHFR C677T mutation in 5/27 (18.5%, and heterozygosity for factor V Leiden mutation in 5/27 (18.5%) children. There were no thrombotic events during 76 courses of L-asparaginase in 41 patients who had received enoxaparin. One patient suffered brain infarct 7 days after enoxaparin was stopped. There were no bleeding episodes. In a historical control group of 50 ALL children who had not received prophylactic enoxaparin during L-asparaginase treatment, two had thromboembolisms (one deep vein thrombosis and one pulmonary embolism). Enoxaparin is safe and seems to be effective in prevention of thromboembolism in ALL patients during L-asparaginase therapy. This study provides pilot data for a future randomized trial of the use of LMWH during ALL therapy for the prevention of asparaginase-associated thrombotic events.

Acquired activated protein C resistance is common in cancer patients and is associated with venous thromboembolism.

PURPOSE: Cancer patients have an increased risk for venous thromboembolism. Because activated protein C resistance is a common risk factor for venous thromboembolism, we prospectively evaluated the activated protein C sensitivity ratio and factor V Leiden mutation in cancer patients with and without venous thromboembolism. SUBJECTS AND METHODS: We studied 55 consecutive cancer patients with deep vein thrombosis, 58 cancer patients with no history of venous thromboembolism, 54 patients with venous thromboembolism without malignancy, and 56 healthy controls. The presence of factor V Leiden mutation was determined by polymerase chain reaction and allele specific restriction digestion. The activated protein C sensitivity ratio was expressed as the ratio of activated partial thromboplastin times measured in the presence and absence of activated protein C; a ratio <2.0 in patients who did not have factor V Leiden was considered to indicate acquired activated protein C resistance. RESULTS: The prevalence of factor V Leiden mutation in cancer patients with thromboembolism (1 of 55, 2%) did not differ significantly from those in cancer patients without thromboembolism (4 of 58, 7%) or normal controls (2 of 56, 4%), but was significantly lower than that of patients with thromboembolism without cancer (18 of 54, 33%, P <0.001). The prevalence of acquired activated protein C resistance was significantly greater in cancer patients with thromboembolism (29 of 54, 54%, P = 0.001) compared with the other groups: 9 of 54 (17%) in cancer patients without thromboembolism, 7 of 36 (19%) in patients with thromboembolism without cancer, and none of the normal controls. CONCLUSION: Although factor V Leiden is not a major risk factor for thrombosis in cancer patients, acquired activated protein C resistance is common and may contribute to the thrombotic tendency in these patients.

Molecular analysis of the genotype-phenotype relationship in factor VII deficiency.

Factor VII (FVII) deficiency is a rare haemorrhagic condition, normally inherited as an autosomal recessive trait, in which clinical presentation is highly variable and correlates poorly with laboratory phenotype. The FVII (F7) gene was sequenced in 48 unrelated individuals with FVII deficiency, yielding a total of 23 novel lesions including 15 missense mutations, 2 micro-deletions, 5 splice junction mutations and a single base-pair substitution in the 5' untranslated region. Family studies were performed in order to distinguish the contributions of individual mutant F7 alleles to the clinical and laboratory phenotypes. Specific missense mutations were evaluated by molecular modelling in the context of the FVIIa-tissue factor crystal structure. Single base-pair substitutions in splice sites and the 5' untranslated region were studied by in vitro splicing assay and luciferase reporter gene assay, respectively. All probands were also typed for four previously reported F7 polymorphisms. In the majority of cases of FVII deficiency studied here, consideration of both mutational and polymorphism data permitted the derivation of plausible explanations for the FVII activity and antigen levels measured in the laboratory. Inter-familial variation in FVII activity and the antigen levels of heterozygous relatives of probands was found to be significantly higher than intra-familial variation, consistent with the view that the nature of the F7 gene lesion(s) segregating in a given family is a prime determinant of laboratory phenotype. Although no relationship could be discerned between laboratory phenotype and polymorphism genotype, the frequencies of the A2 and M2 polymorphic alleles were significantly higher in the FVII-deficient individuals tested than in controls. This suggests that the presence of these alleles may have served to increase the likelihood of pathological F7 gene lesions coming to clinical attention.

The effect of thrombophylaxis on pregnancy outcome in patients with recurrent pregnancy loss associated with factor V Leiden mutation.

OBJECTIVE: To observe the effect of thrombophylaxis on pregnancy in women with a history of unexplained recurrent pregnancy loss also carrying the factor V Leiden mutation. METHODS: Between 1 January and 31 December 1996, activated protein C (APC) resistance and factor V Leiden mutation were prospectively measured in 56 nonpregnant women, with a history of two or more unexplained recurrent pregnancy losses. During the same study period, seven women carrying the factor V Leiden mutation conceived, and were subsequently followed throughout their pregnancy. Subcutaneous low molecular weight heparin (LMWH, enoxaparin, 40 mg/day) and oral low dose aspirin (100 mg/day) were administered throughout the pregnancies, starting at early first trimester. Ultrasound and Doppler umbilical and fetal middle cerebral arterial flow studies were performed in the second and third trimesters, and the course and outcome of the pregnancies were documented. RESULTS: Activated protein C resistance and factor V Leiden were found in 20 (36%) and 12 (21%) women of the study, respectively. Five of the seven pregnancies occuring progressed uneventfully to term with normal fetal growth, normal Doppler flow studies and uneventful neonatal outcome. Two of the seven women had early missed abortions. CONCLUSIONS: Thrombophylaxis, beginning in early pregnancy, in women with unexplained recurrent pregnancy loss associated with factor V Leiden mutation, seems to be safe and allow normal fetal development and good neonatal outcome. To prove the efficacy of thrombophylaxis by LMWH and low dose aspirin in this setting prospective controlled studies seem to be justified.

Activated protein C resistance and factor V Leiden mutation can be associated with first-as well as second-trimester recurrent pregnancy loss.

PROBLEM: To examine whether the occurrence of activated protein C resistance (APCR) and factor V Leiden mutation differs in women with first- compared to women with second-trimester unexplained recurrent pregnancy loss. METHOD OF STUDY: Seventy eight consecutive women with two or more unexplained post-embryonic recurrent pregnancy losses and 139 fertile women with at least one successful pregnancy and no abortions were prospectively investigated for APCR and the factor V Leiden mutation. No women were pregnant at the time of investigation. APCR was defined as APC sensitivity ratio (APC SR) of < or = 2.0. All patients with an APC SR < or = 2.4 were investigated for the factor V Leiden mutation. Women in this study were divided into three groups. Group A included only women with a history of recurrent first-trimester embryonic loss (37 women) and Group B included women with second-trimester abortions with or without additional first-trimester abortions (41 women). Group C included the controls (139 women). RESULTS: APCR and factor V Leiden mutations were significantly more prevalent in all recurrent pregnancy loss patients in this study as compared to controls. 38%(30/78) and 19%(15/78) in contrast to 8% (11/139) and 6% (8/139), respectively. All three groups in the study were comparable regarding age, parity, and number of living children, whereas Groups A and B were also comparable regarding gravidity. Mean APC SRs were significantly higher in Group C as compared to Groups A and B. The incidence of APCR was significantly higher in Groups A and B, as compared to controls, 27 and 49% in contrast to 8%, respectively. Moreover, the incidence of the factor V Leiden mutation was significantly higher in Groups A and B as compared to Group C, 16 and 22% as distinct from 6%, respectively. The incidence of APCR was higher in Group B as compared to Group A, 49% in contrast to 27%, with borderline significance: however, the factor V Leiden mutation did not significantly differ between the two groups. CONCLUSIONS: APCR and factor V Leiden are associated with unexplained recurrent pregnancy loss. The occurrence of APCR and factor V Leiden seems to be linked to post-embryonic first-trimester as well as second-trimester recurrent pregnancy loss. The significance of acquired, non-heritable APCR in recurrent fetal loss patients, especially in the second-trimester aborters, is still to be determined.

Multiple retinal arteriolar occlusions associated with coexisting primary antiphospholipid syndrome and factor V Leiden mutation.

PURPOSE: To investigate a case of a young woman with both primary antiphospholipid syndrome and factor V Leiden mutation who developed multiple retinal arteriolar occlusions. METHOD: Case report of a 25-year-old woman with history and laboratory tests confirming the diagnosis of both primary antiphospholipid syndrome and factor V Leiden mutation who presented with blurred vision in both eyes. RESULTS: Multiple retinal arteriolar occlusions were observed in both of her eyes. The patient was treated first with heparin and then with warfarin. CONCLUSIONS: Primary antiphospholipid syndrome and factor V Leiden mutation, as well as other forms of thrombophilia, should be considered in the differential diagnosis of unexplained retinal vascular occlusions. The coexistence of several thrombophilic disorders may carry a particularly high risk for thrombotic manifestations.

Molecular characterization of four novel mutations causing factor VII deficiency.

INTRODUCTION: Hereditary deficiency of factor VII (FVII) is a rare coagulation defect. We previously studied the molecular basis of the FVII deficiency in Israeli patients and found that the majority of them bore the Ala244Val mutation. In the present study we further analysed FVII deficient patients. PATIENTS AND METHODS: Three patients with severe FVII deficiency (FVII activity < or =1%) and one with partial deficiency (25%) were studied. In all four patients, the FVII gene was amplified and sequenced. RESULTS: Four novel mutations have been identified: IVS 2+1G-->C Phe 24 deletion, Leu300Pro and Arg277His. Homozygosity for the IVS2+1G-->C mutation was lethal, whereas homozygosity for the Phe 24 deletion was accompanied by a severe bleeding tendency. FVII modeling showed that Phe 24 is located in the Gla domain. Both Arg 277 and Leu 300 are within the catalytic domain, although Arg 277 is also involved in tissue factor binding. CONCLUSION: We have analysed four mutations, two of which (IVS2+1G-->C, Phe 24 deletion) were associated with severe bleeding tendency in the homozygous state, facilitating prenatal diagnosis. Hypothetically, using FVII modeling, Arg 277 replacement by histidine may weaken the tissue factor, while deletion of Phe 24 and Leu300Pro mutation might be associated with abnormal folding of the Gla and catalytic domains, respectively.

High prevalence of activated protein C resistance due to factor V leiden mutation in cases of intrauterine fetal death.

OBJECTIVE: To test a possible association between activated protein C resistance and intrauterine fetal death. METHODS: The activated protein C anticoagulant activity and factor V R506Q mutation were assessed in 14 nonpregnant women with a history of intrauterine fetal death and 14 healthy controls. RESULTS: Four women in the study group were heterozygotes for the factor V mutation and none of the controls. The mean activated protein C activity of the study group was statistically significantly lower than that of the controls (P = 0.013). CONCLUSION: Resistance to activated protein C activity may be of etiologic importance in some cases of intrauterine fetal death.

Thrombophilic polymorphisms are common in women with fetal loss without apparent cause.

An association between fetal loss and thrombophilia has recently been described but has not been yet fully elucidated. We have evaluated prospectively the prevalence of the three common thrombophilic polymorphisms (TP) factor V G1691A (Leiden), thermolabile-methylenetetrahydrofolate reductase (TL-MTHFR) C677T and factor II G20210A mutations, in 76 women with fetal loss (> or =3 in first, > or =2 in second, > or =1 in third trimester) without apparent cause and 106 controls without fetal loss. Thirty seven out of 76 (49%) of the women in the fetal loss group had at least one TP compared to only 23/106 (22%) in the control group (p = 0.0001 ). Factor V-Leiden was more common in the fetal loss group 24/76 (32%) compared to the control group 11/106 (10%) (OR = 4.0, 95% CI: 1.8-8.8, p <0.001). Five of the 76 patients (7%) were homozygous for factor V-Leiden compared to none of the controls (p = 0.012). A trend, albeit no statistically significant difference was found between women with fetal loss and control groups regarding factor II G20210A (8% vs. 4% respectively, OR = 2.2, 95% CI: 0.6-8.0, p = 0.23) and MTHFR C677T (18% vs. 10% respectively, OR = 1.95, 95% CI: 0.83-4.6, p = 0.12). Combined TP were documented in 6/76 (8%) patients compared to 1/106 (1%) in controls (OR = 9.0, 95% CI: 1.1-76, p = 0.02). Second or third trimester fetal loss were more common cause of pregnancy termination in 37 patients with TP compared to 39 patients without TP (57/158 (36%) vs. 23/135 (17%) respectively, (p = 0.0004). Thrombophilic polymorphisms are common in women with fetal loss without apparent cause and are associated with late pregnancy wastage. Combinations of TP increase the risk for fetal loss.

Budd-Chiari syndrome associated with factor V leiden mutation: a report of 6 patients.

Budd-Chiari syndrome is characterized by hepatic venous outflow obstruction. Although myeloproliferative disorders are usually responsible for this severe thrombotic disorder, deficiency or dysfunction of the natural anticoagulants can be involved. Resistance to activated protein C caused by factor V Leiden mutation has been recently identified as a major cause of thrombophilia. We report 6 patients with Budd-Chiari syndrome associated with factor V Leiden mutation combined with another acquired thrombophilic state (myeloproliferative disorder and lupus anticoagulant in 3 cases) and without another thrombophilic disorder in the other 3 cases. We conclude that factor V Leiden mutation should be evaluated in any case of hepatic vein occlusion because the prevalence of this mutation in the general population is high.

A missense mutation in gamma-glutamyl carboxylase gene causes combined deficiency of all vitamin K-dependent blood coagulation factors.

To identify potential mutations in the gamma-glutamyl carboxylase gene, the sequence of all exons and intron/exon borders was determined in 4 patients from a consanguineous kindred with combined deficiency of all vitamin K-dependent procoagulants and anticoagulants and results were compared with normal genomic sequence. All 4 patients were homozygous for a point mutation in exon 9 that resulted in the conversion of an arginine codon (CTG) to leucine codon (CGG) at residue 394. Screening of this mutation based on introduction of Alu I site in amplified fragment from normal allele but not from the mutated allele showed that 13 asymptomatic members of the kindred were heterozygous for the mutation. The mutation was not found in 340 unrelated normal chromosomes. The segregation pattern of the mutation which is the first reported in the gamma-glutamyl carboxylase gene fits perfectly with phenotype of the disorder and confirms the suggested autosomal recessive pattern of inheritance of combined deficiency of all vitamin K-dependent procoagulants and anticoagulants in this kindred. The mutated carboxylase protein expressed in Drosophila cells was stable but demonstrated threefold reduced activity compared with WT carboxylase, confirming that the L394R mutation results in a defective carboxylase.

Long-lasting protective immunity to experimental autoimmune encephalomyelitis following vaccination with naked DNA encoding C-C chemokines.

DNA vaccination represents a novel means of expressing Ag in vivo for the generation of both humoral and cellular immune responses. The current study uses this technology to elicit protective immunity against experimental autoimmune encephalomyelitis (EAE), a T cell-mediated autoimmune disease of the central nervous system that serves as an experimental model for multiple sclerosis. RT-PCR verified by Southern blotting and sequencing of PCR products of four different C-C chemokines, macrophage-inflammatory protein-1alpha (MIP-1alpha), monocyte-chemotactic protein-1 (MCP-1), MIP-1beta, and RANTES, were performed on brain samples from EAE rats to evaluate mRNA transcription at different stages of disease. Each PCR product was then used as a construct for naked DNA vaccination. The subsequent in vivo immune response to MIP-1alpha or MCP-1 DNA vaccines prevented EAE, even if disease was induced 2 mo after administration of naked DNA vaccines. In contrast, administration of the MIP-1beta naked DNA significantly aggravated the disease. Generation of in vivo immune response to RANTES naked DNA had no notable effect on EAE. MIP-1alpha, MCP-1, and MIP-1beta mRNA transcription in EAE brains peaked at the onset of disease and declined during its remission, whereas RANTES transcription increased in EAE brains only following recovery. Immunization of CFA without the encephalitogenic epitope did not elicit the anti-C-C chemokine regulatory response in DNA-vaccinated rats. Thus, modulation of EAE with C-C chemokine DNA vaccines is dependent on targeting chemokines that are highly transcribed at the site of inflammation at the onset of disease.

Reactivity patterns of antiphospholipid antibodies and endothelial cells: effect of antiendothelial antibodies on cell migration.

Antiphospholipid syndrome (APS) is characterized by the presence of a heterogeneous class of antibodies directed against phospholipids and associated with high occurrence of thrombotic complications. Antiendothelial cell antibodies (AECAs) have been identified in various autoimmune disorders including APS, but their reactivity patterns remain unclear. We used eluted endothelial membrane-bound antibodies (EC eluates) to investigate possible cross-reactivity of AECAs and their pathogenic effects on endothelial cell integrity. The heterogeneous and nonspecific nature of AECAs was confirmed by our finding that they cross-react with fibroblasts and platelets and bind to cardiolipin. In addition, platelet-bound antibodies from sera of patients with APS reacted with endothelial cells. A dose-dependent binding of human monoclonal anticardiolipin antibody was demonstrated, but this antibody did not compete with AECAs in EC eluates, indicating that only small portion of AECAs are directed against cardiolipin. Although sera from APS patients prolonged coagulation tests, EC eluates did not affect coagulation, suggesting that AECAs may belong to antiphospholipid antibodies subsets that does not interfere with coagulation. Vascular damage is a common feature of autoimmune disorders associated with AECAs. Possible effects of AECAs on vascular perturbance were investigated by cytotoxicity, attachment, and migration assays. Although AECAs were not shown to be cytotoxic or to affect cell attachment, sera from APS patients caused reduced cellular migration (by 30%), and EC eluates caused even more significant inhibition (by 50%). These findings suggest possible interference of AECAs in vascular repair mechanisms and provide an explanation for the thrombotic complications frequently seen in APS patients.

Cone and plate(let) analyzer: monitoring glycoprotein IIb/IIIa antagonists and von Willebrand disease replacement therapy by testing platelet deposition under flow conditions.

Exposure of citrated whole blood samples to polystyrene plates under flow conditions with the cone and plate(let) analyzer (CPA) results in surface immobilization of plasma von Willebrand factor (vWF) followed by platelet deposition. Staining the plates allows measurement of the percentage of surface covered (SC) by the adhered particles and their average size (AS). Both SC and AS parameters depend on platelet count and hematocrit level and reach maximal values after 2 minutes; only AS is shear rate dependent. Under optimal assay conditions (2 minutes at 1800 sec(-1)) normal blood samples yielded an SC of 14.9% +/- 2.5% and an AS of 39.4 +/- 5.2 microm2. Severe von Willebrand disease (eight patients) yielded low SC (5.2% +/- 2.1%), which was restored to normal by testing with vWF precoated surfaces. Glanzmann's thrombasthenia (six patients) samples demonstrated no adhesion of platelet at all. Blocking of the glycoprotein (GP) IIb/IIIa receptor by the chimeric antibody abciximab, and of the GPIb by a recombinant vWF fragment, have yielded a dose-response inhibition demonstrating the crucial role of these receptors in platelet deposition on polystyrene plates in this system. Evaluation of a patient with severe von Willebrand disease receiving replacement vWF factor VIII therapy revealed a comparable response as tested by both the CPA and the Ricof methods. In vitro testing of the GPIIb/IIIa blocking by a nonpeptidic analogue tirofiban and abciximab revealed a good correlation between the CPA test and the routine aggregometry. Eleven patients treated by abciximab after coronary angioplasty were studied by the CPA during the first 24 hours, demonstrating a marked decrease in SC and AS, with some diversity in the responses. We conclude that the CPA test is suitable for evaluation of primary hemostasis and for monitoring of anti-platelet drugs.