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Biomedical subjects

J Caen

Publications and source records attributed to J Caen.

At least 55 records · Page 3Linked to original sources
PubMed1989

Prelining of polytetrafluoroethylene grafts with cultured human endothelial cells isolated from varicose veins.

Prelining graft material with autologous functioning endothelial cells might be one of the ultimate requirements to obtain a biocompatible surface. Accordingly, endothelial cells from stripped varicose veins were enzymatically harvested and grown on a fibronectin matrix. Proliferation was investigated in defined medium supplemented with various concentrations of endothelial cell growth supplement (ECGS) (25, up to 150 micrograms/ml) and heparin (10(-8), up to 10(-5)mol/L): optimal growth required both 150 micrograms/ml of ECGS and 10(-5)mol/L heparin. Under these conditions, cell culture achieved cell densities at a confluence of 1.2 +/- 1.1 10(5) cells/cm2 with a doubling time of 1 day. During subcultivation cultured cells consistently exhibited characteristic cobblestone morphology and immunofluorescent staining for factor VIII-related antigen, whereas prostacyclin production determined by enzyme-linked immunosorbent assay for 6-keto-prostaglandin F1 alpha reached 21.1 +/- 1.2 ng/10(6) cells after 15-minute stimulation with 1 U/ml of thrombin. Heparin-containing culture medium-endothelial cell interactions were particularly studied, and with iodine 125-heparin, binding was demonstrated with an apparent dissociation constant (Kd) of 0.36 +/- 0.04 mumol/L. A cold storage technique at -80 degrees C was sought, and freezed cells were used to coat in vitro polytetrafluoroethylene grafts. Protein-treated material allowed cell attachment and growth to a confluent monolayer as assayed by light and scanning electron microscopy. These data validate the feasibility of prelining grafts in vitro with autologous functioning endothelial cells. This approach may be useful in improving the performance of small-caliber vascular grafts according to prostacyclin production and surface-bound heparin of these cells.

Adult↗
PubMed1988

New carbohydrate site in mutant antithrombin (7 Ile----Asn) with decreased heparin affinity.

A mutant antithrombin was isolated from the plasma of a patient with pulmonary embolism. The new protein, which accounted for 55% of the antithrombin, had decreased heparin affinity and contained two components when analysed on the basis of either charge or molecular mass. Sialidase and endo-beta-N-acetylglucosaminidase F treatment suggested that this heterogeneity was due to a partial glycosylation occurring at a new carbohydrate attachment sequence. Peptide mapping by reverse-phase HPLC showed that the abnormality involved the N-terminal tryptic peptide. Sequence analysis demonstrated that the underlying mutation was 7 Ile----Asn which introduces a new Asn-Cys-Thr glycosylation sequence. This new oligosaccharide attachment site occupies the base of the proposed heparin-binding site, and the finding explains the consequent decrease in heparin affinity.

Antithrombin III↗
PubMed1988

Effect of lupus anticoagulant on antithrombogenic properties of endothelial cells--inhibition of thrombomodulin-dependent protein C activation.

We have investigated the effect of purified immunoglobulin G (IgG) on endothelial cell functions in 16 patients with lupus anticoagulant, 9 of whom had systemic lupus erythematosus (SLE). Spontaneous or thrombin-stimulated secretion of prostacyclin (PGI2) by cultured human endothelial cells from umbilical cord vein (HUVEC) was not inhibited by the patient's IgG. Nor was spontaneous release of tissue plasminogen activator (t-PA) or of its inhibitor (PAI) modified in the presence of patient's IgG. The rate of activation of purified protein C (PC) by HUVEC in the presence of thrombin was significantly lowered by patient's IgG or Fab' fragment (inhibition of 43%). Neutralization of this effect was obtained by incubation of a greater quantity of phospholipids (phosphatidylcholine, phosphatidylserine) with the patient's IgG. Activation of PC was also performed using purified rabbit thrombomodulin (TM) and a similar inhibition of the patient IgG was observed (inhibition of 48%) but the activation of Gla-domainless PC was not modified.

Animals↗
PubMed1988

Changes in plasma fibrin degradation products as a marker of thrombus evolution in patients with deep vein thrombosis.

The level of fibrin degradation products (fdp) as a marker of fibrin clot dissolution was studied prospectively in 51 patients with phlebographically identified deep vein thrombosis (DVT). For this purpose a highly sensitive fdp assay using an anti D neo monoclonal antibody (McAb) was used. At the onset of the hospitalization, in 47 (92%) of the 51 patients tested, the plasma fdp level performed was high, but does not reflect the size of the thrombus, demonstrating that spontaneous thrombus lysis varies from one patient to another. During 10 days of standard heparin or low molecular weight heparin treatment, two different patterns of fdp evolution could be identified in these patients, independent of the type of heparin used. The first was characterized by a gradual decrease in fdp level and a corresponding reduction in the thrombus size. The second pattern showed a persistence of high levels of fdp after 10 days of therapy although the phlebographic score reveals a poor or partial response indicating that fibrinolysis or the balance of thrombus formation/fibrinolysis did not insure total thrombus dissolution. The 4 patients whose initial plasma fdp levels were only slightly increased during the 10 days, seem to have poor thrombolysis, as was shown by the unmodified phlebographic score after 10 days of treatment. Consequently, we conclude that the investigation of plasma fdp levels with a highly sensitive assay should contribute to the evaluation of thrombus evolution in DVT.

Enzyme-Linked Immunosorbent Assay↗
PubMed1988

Binding of heparin to human microvascular endothelial cells and the effect on proliferation.

We have studied the binding of 125I-heparin to human omental microvascular endothelial cells (HOME cells) and investigated its effect on cell proliferation. At 20 degrees C, the binding reached a steady state from 4 hours onwards. Saturation of 125I-heparin binding occurred at 200 nM. Scatchard analysis indicated one class of binding sites (KD = 0.023 microM, 2 X 10(-6) sites/cell). Using fractionated heparin and other sulfated polysaccharides it was demonstrated that the binding was dependent on the charge and the molecular weight of the compounds. The binding was followed by the partial internalization of the bound ligand (23.8%). Heparin and Stipocus Japonicus mucopolysaccharide (SJAMP) inhibited the proliferation of exponentially growing HOME cells and human umbilical vein endothelial cells (HUVE cells). However, serum-deprived HOME cells were not inhibited by heparin.

Binding, Competitive↗
PubMed1988

Proposed heparin binding site in antithrombin based on arginine 47. A new variant Rouen-II, 47 Arg to Ser.

Antithrombin Rouen-II, a new inherited variant of antithrombin-III, was found in two members of a family with no definite history of thrombosis. The subjects had normal antigenic concentrations of antithrombin and normal progressive inhibitory activity. However, the variant had defective heparin and heparan sulfate cofactor activities, and was not activated by a synthetic pentasaccharide representing the minimum heparin sequence. The abnormal antithrombin was isolated using heparin-Sepharose chromatography, and on electrophoresis at pH 8.6 migrated more anodally than normal. Two-dimensional peptide mapping of tryptic and Staphylococcus aureus V8 protease digests was performed and the abnormal peptide was located by tryptophan staining. Amino acid sequence studies demonstrated a substitution of arginine at residue 47 by a serine. Evidence strongly suggests that arginine 47 is a prime heparin binding site in antithrombin and that it forms part of a proposed positively charged linear site (to which heparin binds) that stretches across the surface of the molecule from the A to the D helix.

Adult↗
PubMed1988

Rapid evolution in the immunochemical findings of a gamma heavy chain disease.

We noted the following major immunochemical changes in the final course of a gamma heavy-chain disease associated with a myelomonocytic leukemia: an important oligoclonal increase of IgG1/kappa, during sepsis development, whereas the lambda molecules decreased and IgG2 and IgG3 completely disappeared in a one-month interval. Initial concentrations of IgG4 were stable during that period, and gamma 1 heavy chains were slightly increased. This is a very unusual observation of the successive immunochemical events terminating a gamma heavy-chain disease.

Aged↗
PubMed1988

[New treatments for Raynaud's syndrome].

Raynaud's phenomenon, either idiopathic or secondary is often severe. It is due to a vasospasm in response to cold, and an increase in sympathetic tone, hyperviscosity and sometimes hyperactivation of platelets and erythrocytes have been reported. New drugs have been developed such as ketanserin, which is a specific inhibitor of serotonin S2 receptors, and has led to a positive response in 38%-80% of patients. The prostaglandins PGE1 and PGI2 were given i.v. in refractory patients and led to a beneficial effect in 50%-70% of cases. These data seem to justify the development of prostaglandin analogues.

Alprostadil↗
PubMed1987

Increased adhesion to fibronectin and Mo-1 expression by diabetic monocytes.

As excessive monocyte adhesion to blood vessel wall could produce endothelial cell injury, we undertook comparative studies on the monocyte adhesiveness in insulin-dependent diabetics with vascular complications (n = 26) and healthy, normal subjects (n = 36). In the diabetic group, the extent of monocyte adhesion on fibronectin- and autologous plasma-coated surfaces was significantly increased compared with that in the control group (p less than 0.01 on both types of surfaces). Monocytes adhesion to the plasma-coated surface, but not to the fibronectin-coated surface, could be inhibited by monoclonal anti-Mo-1 antibody in a dose-dependent manner. For diabetic monocyte adhesion, a higher amount of anti-Mo-1 antibody was required to produce a similar extent of inhibition as observed with control monocytes. This indication of increased Mo-1 expression on diabetic monocytes was further confirmed by analyzing the fluorescence intensity of monocytes labeled with the antibody anti-Mo-1. The results of the present study suggest that diabetic monocytes have increased adhesiveness as the result of increased expression of fibronectin and Mo-1 receptors.

Antibodies, Monoclonal↗
PubMed1987

Selective binding of heparins to human endothelial cells. Implications for pharmacokinetics.

Cultured endothelial cells isolated from human umbilical vein bind heparin and heparin fragments. The binding capacity of endothelial cells for 35S-heparin was for 38% composed of high affinity binding sites (Kd = 11 X 10(-8) M) and for 62% of sites with much lower affinity (Kd = 14 X 10(-7) M). The affinity of unlabeled compounds for heparin binding sites was determined by competition with binding of 125I-heparin. I50 found for unlabeled heparin was 16 X 10(-8) M, which is in agreement with the Kd for binding of heparin to high affinity sites. PK 10169, a low molecular weight fragment of heparin, competed only at relatively high concentrations (I50 = 10(-5) M). Competition experiments with subfractions of PK 10169 showed that I50 was inversely correlated with molecular weight. Gelfiltration of 35S-heparin and 35S-PK 10169 before and after binding to endothelial cells demonstrated a selective binding of high molecular weight molecules from polydisperse heparin and PK 10169 preparations. Bound heparin and PK 10169 molecules were detached from the cell-surface by proteolytic treatment and tested for antifactor-Xa and antifactor-IIa activity. Released heparin is slightly more active in antifactor-Xa and antifactor-IIa activity than its parent preparation. Released PK 10169 was 4 fold more active in antifactor-Xa and 8 fold more active in antifactor-IIa assays than heparin.

Binding Sites↗
PubMed1987

Hermansky-Pudlak platelets: further studies on release reaction and protein phosphorylations.

Platelets from a patient with the Hermansky-Pudlak syndrome were studied. These platelets had decreased amounts of serotonin and adenine nucleotides, and a decreased number of mepacrine-labeled dense bodies. beta-Thromboglobulin and acid hydrolases contained in alpha-granules and lysosomes respectively were present in normal amount. Platelets in platelet-rich plasma did not respond to collagen, but arachidonic acid and ionophore A 23187 induced normal aggregation and normal thromboxane (TX) synthesis. Alpha-granule release was found impaired and remained subnormal even with high doses of inducers. In response to thrombin aggregation, release and TX synthesis of isolated metrizamide gradient platelets were found at lower than normal levels. Phosphorylation of P20 and P43 proteins was normal. Only a combination of ADP plus thrombin could restore a normal aggregation, with normal alpha-granule and lysosome release and normal TX synthesis. These results indicated that in the absence of dense bodies: the release of other granules is impaired; the TX synthesis is delayed except when induced by arachidonic acid and A 23187 ionophore; the absence of dense bodies could be compensated for by the addition of ADP which restores the impaired release reaction and TX formation; and P20 and P43 polypeptides were phosphorylated as rapidly as those in normal platelets.

Adenine Nucleotides↗
PubMed1987

Signal transduction in normal and pathological thrombin-stimulated human platelets.

Human blood platelets stimulated by thrombin undergo very rapid morphological changes, the most characteristic of which are pseudopod formation and granule centralization. These early changes in shape are accompanied by a transient decrease (30%) in phosphatidyl inositol 4,5-bisphosphate (PIP2) which occurs in the first 10 s after thrombin addition. Transient decreases in phosphatidyl inositol 4-phosphate (PIP) and phosphatidyl inositol (PI) occur later (20-30 s). These events lead to the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) and hence phosphatidate (PA). Two polypeptides are phosphorylated during the same time span: the myosin light chain (P20) and a 43 kDa protein (P43). Concomitant with these molecular changes, platelet 'release reaction' occurs, i.e., liberation of the different granule constituents into the external medium: the earliest concerns dense bodies which liberate adenine nucleotides, calcium and serotonin; alpha-granules then liberate adhesive and specific proteins and are followed by lysosomes which liberate hydrolases. Pathological platelets from patients with inherited disorders, presenting well-characterized and specific defects of either the platelet membrane (GT) or storage granules (GPS and HPS), have also been studied. The results obtained lead to the following conclusions: (1) the transducing system is normal in platelets unable to aggregate; (2) phosphorylation of P20 and P43 proteins can be complete with impaired release; and (3) when platelets lack alpha-granules the transducing system as well as the release of other granule populations are impaired. These results evidence the relationship between the absence of intraplatelet components and metabolic events.

Blood Platelet Disorders↗
PubMed1987

Heparin binding defect in a new antithrombin III variant: Rouen, 47 Arg to His.

Antithrombin III (AT-III) Rouen is a hereditary abnormal antithrombin with normal progressive inhibitory activity and reduced heparin cofactor activity. It was isolated from the plasma of a woman who suffered a sudden idiopathic sensorineural hearing loss and balance impairment. There was no familial history of thrombosis. By heparin-Sepharose chromatography, AT-III Rouen was separated from the normal antithrombin on elution with increasing concentrations of NaCl. AT-III Rouen eluted earlier than is normal at both pH 7.4 and pH 6.0. At the lower pH, the antithrombins bound more avidly to the column, with the abnormal AT-III eluting closer to the normal than at the higher pH. Two-dimensional peptide mapping of tryptic and Staphylococcus aureus V8 protease digests of carboxymethylated antithrombins was performed on thin-layer silica plates. The abnormal peptide was located by tryptophan staining, and amino acid analysis and sequence studies demonstrated a substitution of an arginine at residue 47 for a histidine. Results from this study suggest that replacement of arginine 47 by a partially positively charged histidine has less effect on the heparin binding affinity than dose replacing it with a neutral cysteine side chain as in AT-III Toyama, in which no heparin binding was observed. In addition, heparin binding per se is not a sufficient condition to activate AT-III.

Adult↗
PubMed1986

Increased procoagulant response of monocytes from patients with familial Mediterranean fever.

Familial Mediterranean Fever (FMF) is an inherited disease of unknown etiology characterized by recurrent inflammatory episodes. Circulating fibrin was found in patients with FMF in absence of clinical manifestation of thrombosis and was statistically less frequently observed in patients treated with colchicine. These results suggest a cellular dysfunction. Therefore, we examined the procoagulant activity (PCA) of isolated mononuclear leukocytes and purified monocytes from FMF patients (n = 20). No PCA was detectable on freshly-isolated monocytes. After several hours of culture. FMF monocytes contained more PCA than control cells and the difference was more marked after endotoxin stimulation. Data obtained with coagulation factor-deficient plasma and anti-human apoprotein III antiserum indicated that the enhanced PCA in FMF monocytes is thromboplastin-like. Lysozyme and interleukin 1 production by monocytes were similar in patients and controls. The increased monocyte PCA appears to be due to an intrinsic and selective higher responsiveness of monocytes.

Adult↗
PubMed1986

[Course of the manifestations associated with the antiprothrombinase circulating anticoagulant].

Fifteen patients (9 females and 6 males) with the "lupus type" circulating anticoagulant have been studied. The underlying disease was an auto-immune disorder in 11 cases and a malignant hemopathy in 4 cases. The manifestations frequently associated with the lupus inhibitor, such as thrombosis, thrombocytopenia and false-positive VDRL test were analysed. Hemorrhagic syndrome occurred only when thrombocytopenia or acquired abnormality of Willebrand's factor was present. Thrombotic events (8 cases) were frequent. Deep venous thrombosis was complicated with pulmonary embolism in 4 patients. Platelet abnormality, decreased fibrinolytic response or acquired Willebrand's syndrome were found in all patients with a thrombotic event. These different manifestations followed diverging courses in some patients with persistent thrombocytopenia although the anticoagulant had disappeared in 3 cases, negativation of the false-positive VDRL test while the anticoagulant remained unchanged in 1 case, occurrence of a thromboembolic episode although the anticoagulant had disappeared in 1 case.

Adult↗
PubMed1986

PDGF modifies phosphoinositide metabolism and inhibits aggregation and release in human platelets.

While platelet derived growth factor (PDGF) did not induce any platelet aggregation nor secretion, it modified the polyphosphoinositide metabolism of human platelets prelabeled with 32P-orthophosphate. We found a decrease of 32P associated with phosphatidylinositol 4,5 bisphosphate after 3 min, with parallel increase of 32P-phosphatidylinositol 4 phosphate and 32P-phosphatidylinositol using 100 ng/ml of PDGF. This modification was PDGF concentration dependent. PDGF inhibited thrombin and collagen induced platelet aggregation and 14C-serotonin release in a dose dependent manner, but was without effect when arachidonic acid was used. These results suggest that PDGF (i) stimulated the hydrolysis of polyphosphoinositides (ii) and could exert a negative feedback control on platelet activation induced by thrombin or collagen.

Arachidonic Acid↗