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J Amiral

Publications and source records attributed to J Amiral.

At least 55 records · Page 3Linked to original sources
PubMed1998

One-step assay for the determination of free protein S antigen in plasma using real-time biospecific interaction analysis.

Real-time biospecific interaction analysis based on optical detection by surface plasmon resonance was used to develop an accurate one-step method for the direct measurement of free protein S in human plasma. This assay was validated, compared with classical immunological methods and shown to be suitable for the routine clinical diagnosis of protein S deficiency. The method relies on the specific capture of free protein S directly from plasma by a monoclonal antibody (mAb), 34G2, immobilized on a sensor chip surface. A calibration curve was established with serial dilutions of standard plasma (working range 5-50%) and a linear relationship was found to exist between the relative response in resonance units (RU) and the concentration of free protein S expressed as percentage plasma dilution (r = 0.99). The specificity of the assay was confirmed using purified human protein S and polyethylene glycol treated plasma. In addition, it could be demonstrated that no dissociation of C4b-BP-protein S complexes occurred under the chosen experimental conditions. The technique was reproducible with inter-assay, intra-assay and inter-sensor chip variation coefficients of 1.5-5.4%, 2-3.1% and 4.4-4.9%, respectively, as evaluated in two different plasma samples. Since all tests are automatic and long series of analyses can be performed with the same sensor chip, the method was applied to the determination of free protein S antigen in plasma from 20 normal blood donors and 38 thrombophilic patients. Results displayed excellent correlation with those of free protein S enzyme-linked immunosorbent assay (r = 0.99) and rocket immunoelectrophoresis of polyethylene glycol-treated plasma (r = 0.93).

Adolescent↗
PubMed1998

Assessment of activated protein C resistance using a new and rapid venom-based test: STA Staclot APC-R.

Activated protein C (APC) resistance is related to a single point mutation in the factor V gene (FV:Q506) and appears to be the most common inherited risk factor for venous thromboembolism. A reliable screening test is therefore useful. We aimed to evaluate a new APC resistance test, on the basis of the procoagulant activity present in one snake venom of a crotalidae family: STA Staclot APC-R. We studied 36 consecutive patients with an acute deep venous thrombosis (DVT) confirmed by compression ultrasonography and carrying the FV:Q506 allele, assessed by DNA analysis, 103 of their family members and 35 consecutive patients with a proven DVT but who did not carry the FV:Q506 allele. Blood samples were collected within 24 h of admission for the DVT cases and on the day of medical registration for the family members. Tests were performed blind. The STA Staclot APC-R test, using a cut-off value of 0.80, had an overall sensitivity of 100% (95% CI, 95-100) and a specificity of 98.8% (95% CI, 92.0-99.6). An acute thrombosis process did not influence the performance of the test. We conclude that this test is easy and rapid to perform in every day practice and fulfills the criteria for a screening test.

Adult↗
PubMed1998

Prevalence of antiphospholipid-related antibodies in unselected patients with history of venous thrombosis.

Antiphospholipid antibodies (aPL) are heterogeneous and are now accepted to be mainly phospholipid-protein-dependent antibodies. Although these antibodies are classically associated with thrombosis, their clinical relevance remains to be established. The subgroups of antibodies characterized by their proteic targets were reported to be more appropriate thrombotic markers. We analysed the prevalence of a large panel of antiphospholipid-related antibodies (aPLR), comprising antibodies directed to phospholipid-protein complexes and to different protein cofactors (beta2GPI, prothrombin, annexin V and protein S), in 122 consecutive unselected patients who had experienced at least one venous thrombotic event. The presence of lupus anticoagulants was assessed with an integrated assay using hexagonal phase phospholipids. Two types of aPL (APA and anti-beta2GPI-PL) were measured using a mixture of phospholipids containing cardiolipin and goat serum or human beta2GPI, respectively, as a source of protein cofactor. Our results show a similar prevalence, close to 15%, of lupus anticoagulants, APA and anti-beta2GPI-PL. In contrast, antibodies to beta2GPI were detected in only 8% of the patients, and very few patients had antibodies directed to other proteins. Of the 35 patients having at least one positive aPLR, 17 were classified as severe, because they had recurrent or early onset of thrombosis (< 35 years). The distribution of aPLR between severe and mild cases was not significantly different except for lupus anticoagulants. Our results clearly indicate that lupus anticoagulant is the only aPLR test to be strongly associated with the severity of thrombosis.

Annexin A5↗
PubMed1998

Soluble adhesion molecules, endothelial markers and atherosclerosis risk factors in abdominal aortic aneurysm: a comparison with claudicants and healthy controls.

Development of an abdominal aortic aneurysm (AAA) may be a product of generalised atherosclerosis. If that is indeed the case, we would expect similarities in various risk factors and other markers in common with occlusive peripheral arterial disease (peripheral arterial disease), and less congruity with healthy controls. To test this hypothesis, we recorded the major risk factors for atherosclerosis, two markers of endothelial dysfunction, and soluble adhesion molecules in 21 patients with an uncomplicated AAA free of symptomatic peripheral arterial disease, 42 patients with peripheral arterial disease, and 42 healthy controls who were matched, as a group, for age and sex. After adjusting for smoking, there were no significant differences in blood pressure, fibrinogen, soluble intercellular adhesion molecule-1, soluble vascular cell adhesion molecule-1 or lipoproteins between the groups. However, markers of endothelial integrity von Willebrand factor and soluble thrombomodulin were both higher (P < 0.05) only in peripheral arterial disease patients. Relative to the controls, platelet marker soluble P-selectin was increased in AAA (P < 0.01) and in the peripheral arterial disease patients (P < 0.05). Levels were higher in AAA patients than in peripheral arterial disease patients (P < 0.05). Our laboratory data suggest that the pathophysiology AAA and peripheral arterial disease are not identical.

Aged↗
PubMed1998

A supersulfated low-molecular-weight heparin (IK-SSH) increases plasma levels of free and total tissue factor pathway inhibitor after intravenous and subcutaneous administration in humans.

Unfractionated as well as low-molecular-weight heparins (LMWH) are known to cause an increase in blood levels of tissue factor pathway inhibitor (TFPI). To study the effect of a newly developed supersulfated LMWH (IK-SSH, Iketon Farmaceutici) on TFPI concentrations in human plasma, the compound was injected into volunteers at doses of 0.14, 0.33 and 0.66 mg/kg intravenously or 0.33, 0.66 and 1.0 mg/kg subcutaneously. At certain known times blood was drawn and plasma levels of both total and free TFPI were measured using enzyme-linked immunosorbent assay methodology. Baseline plasma concentrations of TFPI were 72.2+/-3.1 ng/ml for total and 10.8+/-0.8 ng/ml for free TFPI. Intravenous or subcutaneous injection of IK-SSH led to a strong and long-lasting rise in TFPI levels which were increased more than 5-fold for total TFPI and more than 30-fold for free TFPI. Maximum TFPI levels were reached 5-10 min after intravenous and 60 min after subcutaneous administration. IK-SSH caused prolongation of ex-vivo clotting times in the APTT and Heptest assay, whereas thrombin time was not affected. Anticoagulant actions of IK-SSH showed a significant correlation to plasma concentrations of TFPI and they are thought to be based at least partially on the release of TFPI from vascular sites.

Adult↗
PubMed1998

Evaluation of a new, rapid, and quantitative D-Dimer test in patients with suspected pulmonary embolism.

Previous studies have suggested the utility of D-Dimer ELISA assays in eliminating a diagnosis of pulmonary embolism (PE). Our objectives were to evaluate the performance of a new, rapid, quantitative, and automated Liatest D-Dimer Assay in patients with suspected PE. Three hundred eighty-six consecutive patients referred to our institution between March 1992 and December 1996 for clinically suspected PE, with recent clinical signs not exceeding 1 wk, were included in this study. Diagnosis of PE was based on clinical evaluation, radionuclide lung imaging, lower limb examination, and, when required, pulmonary angiography. D-Dimer performances, for both Liatest D-Dimer and standard D-Dimer ELISA (Asserachrom DDi), assays, were assessed at the end of the study. Among the 386 patients tested, 146 (37.8%) were classified as PE-positive. Liatest D-Dimer assay had a 100% sensitivity (95% confidence interval, 97 to 100%) and a negative predictive value of 100% (95% confidence interval, 94 to 100%). A normal result, below the cutoff of 500 ng/ml, occurred in 83 of the 386 (21%) patients. There was a strong agreement between Liatest D-Dimer and Asserachrom DDi analyses. These findings suggest that this rapid, quantitative, and automated D-Dimer assay provides a useful diagnostic tool for the clinician with regard to exclusion of PE.

Adolescent↗
PubMed1998

Hyperhomocyst(e)inemia and endothelial dysfunction in IDDM.

OBJECTIVE: Considering that elevated blood levels of homocyst(e)ine represent a known independent risk factor for macrovascular disease, we assessed the link between hyperhomocyst(e)inemia and diabetic microvascular complications. RESEARCH DESIGN AND METHODS: Homocyst(e)ine and thrombomodulin plasma levels, a marker of endothelial cell damage, were measured before and 3 h after oral methionine loading in 75 patients with stable, well-controlled IDDM and 40 healthy control subjects matched for sex and age. Exclusion criteria were hyperlipidemia, hypertension, smoking, or positive family history for cardiovascular disease. RESULTS: IDDM patients had higher pre- and postload homocyst(e)ine plasma levels than did healthy control subjects (12.0 vs. 7.7 mumol/l and 27.6 vs. 16.0 mumol/l; P < 0.001). Of 75 IDDM patients, 26 had homocyst(e)ine plasma levels above the normal range (defined as mean +2 SD of values obtained in the control group). The IDDM patients with hyperhomocyst(e)inemia had higher thrombomodulin plasma levels (62.2 vs. 38.2 ng/ml; P < 0.001), higher albumin excretion rates (485 vs. 115 mg/l; P < 0.005), and a higher prevalence of late diabetic complications (nephropathy, 76 vs. 33%; retinopathy, 69 vs. 51%; neuropathy, 57 vs. 41%; macroangiopathy, 57 vs. 33%) compared with IDDM patients with normal plasma homocyst(e)ine. In vitro experiments with human umbilical vein cells show an increased release of thrombomodulin into the culture supernatant only when endothelial cells were pretreated with advanced glycation end product (AGE)-albumin before L-homocystine was added. A synergistic action of homocyst(e)ine and AGEs might contribute to vascular complications of patients with diabetes. CONCLUSIONS: Hyperhomocyst(e)inemia is common in nephropathic diabetic patients and may contribute to the enhanced morbidity and mortality from cardiovascular diseases characteristically observed in IDDM patients with diabetic nephropathy.

Adult↗
PubMed1998

Thrombomodulin levels during normal pregnancy, at delivery and in the postpartum: comparison with tissue-type plasminogen activator and plasminogen activator inhibitor-1.

Some studies suggest that soluble thrombomodulin (TM) could be used as a marker of preeclampsia or eclampsia. However little is known about the sequential changes of TM during the course of normal pregnancy. Levels of TM were determined in 100 women with uneventful pregnancies. Samples (n = 394) were divided into five study intervals, three during pregnancy, one at delivery and one three days postpartum. As compared with TM levels (median 34.3 ng/ml, range 17.6-61) of a control group of 60 healthy non-pregnant women, TM levels were shown to increase throughout pregnancy, median (and range) values being respectively 38.5 (17.6-72.7) from 11 to 20 weeks, 45.2 (22.6-75.2) from 21 to 30 weeks and 54.3 (25.1-114.5) ng/ml from 31st week to delivery. One hour after delivery TM levels were still elevated and dropped three days postpartum to 40.5 (20.9-79.4) ng/ml. The increase of TM levels was correlated with those of tissue-type plasminogen activator and plasminogen activator inhibitor-1 antigens. The large overlap in TM levels between the study periods seems to preclude a clinical use of TM based on reference values from a control group. Our data suggest that it would be more appropriate to take into account TM baseline values in a given woman to examine her TM increase during pregnancy.

Adult↗
PubMed1998

Predictive value of plasma thrombomodulin in preeclampsia and gestational hypertension.

In a prospective longitudinal study, 130 primigravidae at risk for preeclampsia were examined and plasma sampling performed in 45 of them. Plasma thrombomodulin (pTM) was sequentially measured at weeks 12, 24 and 32 of gestation and after delivery in 20 primigravidae who developed either mild preeclampsia (n = 8) or gestational hypertension (n = 12) between weeks 32 and 39 of gestation and in 25 (age-matched) primigravidae who had uneventful pregnancies. pTM elevations were not observed until week 32 in uneventful pregnancies, but were present by week 24 (p = 0.002) in patients who later developed hypertensive complications. A net individual pTM increase > or = 4.2 ng/ml between weeks 12 and 24 (more than 8 times that of normotensive primigravidae) and/or pTM level > or = 47.5 ng/ml at week 32 predicted the development of hypertensive complications with 80% accuracy. Serial pTM determinations can be useful to select pregnancies who may benefit from early pharmacological intervention.

Adult↗
PubMed1998

Localisation of protein Z in vascular lesions of patients with atherosclerosis.

BACKGROUND: The deposition of protein Z was investigated in atherosclerotic vascular lesions of patients with diabetes mellitus or atherosclerotic vascular disease without diabetes in comparison to controls. PATIENTS AND METHODS: Protein Z antigen was evidenced by immunohistochemistry in arteries of 5 healthy control patients, 11 diabetic patients with arterio-occlusive disease and 7 patients suffering from arterio-occlusive disease without diabetes. For immunohistochemistry, a commercially available antibody was taken as first antibody, and immunopositivity was evaluated independently by two investigators (J.G.; I.K.) as negative (0), positive (+) and strongly positive (++). The results were assessed by the Whitney-Mann-Wilcoxon test. RESULTS: Macrovascular endothelial cells were stained positive for protein Z in all arteries studied. Arteries of controls did not show significant immunopositivity in cells other than macrovascular endothelial cells, while the microvascular endothelial cells of control arteries were largely negative. The proliferating subendothelial space in atherosclerotic vascular lesions showed significant immunopositivity for protein Z. In contrast to control arteries, the microvascular endothelial cells of the proliferating areas stained positive. The staining pattern of the subendothelial space was similar in atherosclerotic vessels independent of the risk factor for atherosclerosis. Plaques were immunopositive for protein Z, too. CONCLUSIONS: Protein Z is present in atherosclerotic vascular lesions of diabetic and non-diabetic patients, but not in the subendothelial space and microvascular endothelial cells of healthy controls. Since protein Z-positivity was detected in microvascular endothelium as well as in extra-vascular deposits around plaques, it may play a role in the development of these lesions.

Arterial Occlusive Diseases↗
PubMed1998

Effect of the antioxidants selenium and beta-carotene on HIV-related endothelium dysfunction.

Patients infected with HIV are at increased risk of atherosclerosis, and have evidence of endothelium dysfunction. The hypothesis was tested that HIV-related endothelium dysfunction is related to loss of antioxidants. This was done by the supplementation of the antioxidants selenium and beta-carotene. We supplemented the diet of 10 HIV-seropositive subjects with 100 microg selenium daily, 11 subjects with 30 mg beta-carotene twice daily while 15 subjects were not supplemented. Plasma was obtained at outset and after a year, and tested by ELISA for endothelial cell, platelet and inflammatory markers. The non-supplemented patients experienced increases in von Willebrand factor and soluble thrombomodulin (both p <0.01). There were no changes in any of the indices in the patients taking selenium or beta-carotene. Increased von Willebrand factor and soluble thrombomodulin in the non-supplemented patients imply increased damage to the endothelium over the year of the study. Therefore we interpret the lack of increase in the patients taking antioxidants as evidence of the protection of the endothelium by these agents.

Antioxidants↗
PubMed1997

Prevalence of heparin-associated antibodies without thrombosis in patients undergoing cardiopulmonary bypass surgery.

BACKGROUND: Patients with cardiovascular disease almost invariably receive heparin before cardiopulmonary bypass surgery, which places them at risk of developing heparin-associated antibodies with a risk of thromboembolic complications. This study was designed to determine the prevalence of heparin-induced antibodies in patients before and after cardiopulmonary bypass surgery. METHODS AND RESULTS: Plasma from 111 patients was tested before surgery and 5 days after surgery for heparin-dependent platelet-reactive antibodies with a 14C-serotonin-release assay (SRA) and for antibodies to heparin/platelet factor 4 complexes with an ELISA. Heparin exposure after surgery was minimized. Heparin-dependent antibodies were detected before surgery in 5% of patients with SRA and 19% of patients with ELISA. By the fifth postoperative day, there was a marked increase in patients positive on the SRA or ELISA (13% and 51%, respectively; P < .01 for each). Patients who had received heparin therapy earlier in their hospitalization were more likely to have a positive ELISA before surgery (35%; P = .017) and a positive ELISA (68%; P = .054) or SRA (30%; P = .002) after surgery. However, there was no difference in the prevalence of thrombocytopenia or thromboembolic events between the antibody-positive and-negative groups. CONCLUSIONS: Approximately one fifth of patients undergoing cardiopulmonary bypass surgery have heparin-induced platelet antibodies detectable before the procedure as a result of prior heparin exposure, and many more develop antibodies after surgery. The absence of an association between these antibodies and thromboembolic complications in this study may be, in part, attributable to careful avoidance of heparin after surgery. The high prevalence of heparin-induced antibodies in this setting suggests that these patients may be at risk of developing thrombotic complications with additional heparin exposure.

Antibodies↗
PubMed1997

Identification of cell adhesive active sites in the N-terminal domain of thrombospondin-1.

Using a series of fusion proteins that span almost all of the thrombospondin-1 (TSP-1) molecule, we observed in this study that Chinese hamster ovary (CHO) K1 cells strongly attached to the N-terminus but not to the other domains of TSP-1 (e.g. the C-terminus, and type 1, type 2 and type 3 repeats). In addition, attachment to the N-terminus of CHO S745 cells defective in cell-surface glycosaminoglycans (GAGs) was decreased by 47% compared with that observed with CHO K1 cells, indicating the presence of GAG-dependent cell adhesive sites. With the aim of identifying these cell adhesive sites, a series of synthetic peptides, overlapping heparin-binding sequences ARKGSGRR (residues 22-29), MKKTRG (residues 79-84) and TRDLASIARLRIAKGVNDNF (residues 170-189), were synthesized and tested for their ability to support CHO cell attachment. Using both centrifugation and cell-attachment assays, MKKTRG-containing peptides promoted CHO K1 cell adhesion, while ARKGSGRR-containing peptides and peptide TRDLASIARLRIAKGVNDNF did not. CHO S745 cell attachment to MKKTRG-containing peptides was partially decreased. A 36% decrease in CHO K1 cell attachment to the N-terminus was also observed when the heparin-binding consensus sequence KKTR was mutated to QNTR. In addition, peptide MKKTRG partially inhibited (25% inhibition) CHO K1 cell attachment to the N-terminus. However, peptide MKKTRG was not sufficient to fully promote cell attachment to the N-terminus of TSP-1. Peptides VDAVRTEKGFLLLASLRQ and TLLALERKDHS also supported CHO K1 cell attachment in a GAG-dependent and -independent manner respectively. Moreover, CHO K1 cell attachment to MKKTRG was found to be markedly enhanced when flanked with the sequences VDAVRTEKGFLLLASLRQ and TLLALERKDHS. Peptide VDAVRTEKGFLLLASLRQMKKTRG nearly abolished (98% inhibition) CHO K1 cell attachment to the N-terminus, while peptides MKKTRG, MKKTRGTLLALERKDHS and VDAVRTEKGFLLLASLRQ had only a moderate inhibitory effect (25, 27 and 53% inhibition respectively). These data indicate that the sequence VDAVRTEKGFLLLASLRQMKKTRGTLLALERKDHS (residues 60-94) constitutes a GAG-dependent cell adhesive site in the N-terminus of TSP-1. Moreover, a GAG-independent site, encompassing residues 189-200 (FQGVLQNVRFVF), has been identified. These two adhesive sites supported the attachment of a wide variety of cells (human breast carcinoma, melanoma and osteosarcoma cells), and a high degree of sequence homology was found between TSP-1 and TSP-2 between residues 60 and 94 (48% identity) and 189-200 (67% identity), further suggesting the functional importance of these two cell adhesive sites in the N-terminus of TSP-1.

Amino Acid Sequence↗
PubMed1997

A 13-24 C-terminal peptide related to PF4 accelerates hematopoietic recovery of progenitor cells in vivo in mice treated with 5-fluorouracil.

We have recently reported that platelet factor 4 (PF4), a megakaryocyte-platelet protein, is a potent inhibitor of human and murine megakaryocytopoiesis. In addition, PF4 accelerated the recovery of the marrow precursor cells in 5-fluorouracil (5-FU) treated mice. We show in this study that a slight modification of the C-terminal peptide related to PF4 (C13-24DE), which was previously reported as the carboxy terminal region of PF4 implicated in PF4 inhibitory activity, is also able to significantly increase murine high proliferating-potential-colony forming cells (HPP-CFC), colony-forming-unit megakaryocyte (CFU-MK) and colony-forming unit granulocyte-macrophage (CFU-GM) progenitor number, eight days after 5-FU administration, when it was given intraperitoneally twice a day (200 micrograms/kg/inj) prior to 5-FU administration (150 mg/kg). Furthermore, the C13-24DE pretreatment enhanced both the number and the diameter of single megakaryocyte (MK) by day 8. These data indicate that the C13-24DE peptide related to PF4 accelerated the in vivo recovery of stem cells, progenitors (CFU-GM, CFU-MK) and single MK after 5-FU treatment and may have a hemoprotective effect against chemotherapeutic agents.

Amino Acid Sequence↗
PubMed1997

Annexin V, a new marker of platelet storage lesion: correlation with dMPV.

Released annexin V, an intracellular platelets glycoprotein, was used to determine the cellular injury which occurred during storage of platelet concentrates. Twenty-eight units of leuco-reduced apheresis platelet concentrates, obtained without leucocyte filtration, were analysed. Released annexin V showed a significant correlation with EDTA-induced shape changes of platelet (r = 0.62, P < 0.01) while poor correlation was found between released annexin V and pH or MPV. The combination of released annexin V with dMPV provides excellent markers of the platelet storage lesion for quality monitoring, based on morphological/functional integrities and cellular injury, which are of direct relevance to clinical efficacy of platelet concentrates.

Annexin A5↗
PubMed1997

Immunomodulation, autoimmunity and haemostatic dysfunctions.

Many links exist between the two major host defence mechanisms, haemostasis and immunity. Blood cells and endothelium are involved in these systems and their dysfunctions. The dual function of these cells is discussed. Once activated, cell-cell interactions enhance the biological response and contribute to development of pathology. Furthermore, autoimmunity presents a special clinical relevance, and could be involved in many haemostatic disorders. A hypothesis is presented on how some auto-antibodies are generated and can trigger pathology, by targeting the immune response onto blood or endothelial cells, or to body's organs.

Adjuvants, Immunologic↗