Biomedical subjects

C Ravanat

Publications and source records attributed to C Ravanat.

27 records · Page 2Linked to original sources

PubMed1996

Xenograft rejection: molecular mechanisms and therapeutic prospects.

The use of animal organs for transplantation in humans is seen as a potential solution to the short supply of human donor organs available for clinical transplantation. However, while several attempts at clinical xenografting have been made over the last ninety years, xenotransplantation has not matched the success of allotransplantation, because of a vigorous rejection response. Xenograft rejection is mediated by mechanisms that differ from those involved in alloreactivity and which are inadequately controlled by conventional immunosuppressive agents. Xenotransplantation therefore requires the development of specific strategies to overcome rejection, through modification of the host immunity or production of genetically engineered pig organs. This article reviews the cellular and molecular events underlying xenograft rejection and the potential strategies of prevention and gives a brief history of the main attempts at clinical xenotransplantation since the beginning of the century.

Animals↗

PubMed1996

Species specific immunoassays to measure blood platelet and coagulation activation in the rat.

The purpose of this study was to develop specific and sensitive immunoassays to detect early indices of hypercoagulability in the rat. Rat platelet factor 4 (rPF4) and rat fibrinopeptide A (rFPA) assays, tools for the detection of activation of platelets and coagulation respectively, were designed using antibodies raised against purified rPF4 and against synthetic rFPA. The relevance of these new assays and of the commercially available ELISA kit for thrombin-antithrombin III (TAT) complexes was demonstrated in a rat model of a prethrombotic state induced by intravenous infusion of varying doses of thrombo-plastin (90 to 2400 microliters/kg/h). In this model, the immunoassays allowed simultaneous detection of low levels of rFPA and rPF4 which were correlated with fibrinogen and platelet consumption and TAT generation and further proved to be of higher sensitivity than the classical methods of platelet count or measurement of fibrinogen levels. Plasma concentrations of rFPA, rPF4 and TAT were dependent on infusion time and thromboplastin dose, while hirudin (1 mg/kg) prevented their appearance. Thus the new specific immunoassays for rPF4 and rFPA and the commercial human TAT assay represent useful tools for pathophysiological studies or the screening of antithrombotic drugs in rats.

Animals↗

PubMed1995

Cross-reactivity of human molecular markers for detection of prethrombotic states in various animal species.

The aim of the present study was to investigate the reactivity of immunoreagents developed for clinical applications in humans in different animal species (hen, mouse, rat, rabbit, guinea-pig, dog, pig, sheep, baboon). Prothrombin fragment 1 + 2, thrombin-antithrombin III complex and fibrinopeptide A were tested for coagulation, platelet factor 4 and beta-thromboglobulin for platelet activation, glycoprotein IIb-IIIa, glycoprotein Ib and P-selectin for platelet membrane glycoproteins, D-dimers for fibrinolysis, thrombomodulin for activation of endothelial cells and thrombospondin and von Willebrand factor for adhesive proteins. Prothrombin fragment 1 + 2, platelet factor 4, beta-thromboglobulin and D-dimers were revealed only in baboons. Fibrinopeptide A was well detected in baboons but weakly in mice, dogs, pigs and sheep. Whereas glycoprotein IIb-IIIa was revealed on guinea-pig, dog and sheep platelets and glycoprotein Ib on rabbit and dog platelets, P-selectin and thrombomodulin were never detected. Thrombospondin was revealed in hens, mice, rats, guinea-pigs, pigs, sheep and baboons and von Willebrand factor in mice, rats, guinea-pigs, dogs, pigs, sheep and baboons. Interestingly, thrombin-antithrombin III complex (TAT) was detected in all species tested except the hen. A time- and dose-dependent increase in TAT was observed when rats, dogs or pigs were infused with thromboplastin (4.5-450 microliters/kg/h), while administration of hirudin (1 mg/kg) abolished this TAT generation. Thus, the TAT immunoassay could provide a tool for the screening of antithrombotic drugs in a number of animal species. However, the possibility of using a wider panel of human immunoreagents would appear to be restricted to baboons which display good species cross-reactivity.

Animals↗

PubMed1994

Rat platelets contain glycosylated and non-glycosylated forms of platelet factor 4. Identification and characterization by mass spectrometry.

Platelet factor 4 is a heparin-binding protein released from the alpha granules of activated platelets. This study describes the purification and identification of two forms of rat platelet factor 4, the previously characterized non-glycosylated form of 7 kDa and an additional glycosylated form of molecular mass 9 kDa. The two proteins both neutralized the antithrombin-III-dependent inhibitory activity of heparin. Although their amino acid composition was found to be the same, in the N-terminal sequence of the 9-kDa protein, the second threonine residue could not be detected and a difference of 976Da was determined by mass spectrometry. After digestion with O-glycanase and sialidase, the two proteins showed the same molecular mass. Overall consideration of these data led to identification of the higher-molecular-mass protein as a glycosylated form of rat platelet factor 4 with O-glycosylation at the second N-terminal amino acid, while the structure of the oligosaccharide core was established by mass spectrometry and sugar differentiation with lectins. The two forms of platelet factor 4 are both present in platelets and secreted after platelet activation.

Amino Acid Sequence↗

PubMed1993

The Arg-4 mutant factor IX Strasbourg 2 shows a delayed activation by factor XIa.

We have characterized at the DNA and protein levels a mutant factor IX, factor IX Strasbourg 2, which is responsible for a severe form (< 0.01 U/ml) of haemophilia B. Factor IX Strasbourg 2 has a higher molecular weight than normal factor IX. A mutation G-->A at position 6365 of the gene was demonstrated by DNA sequencing and confirmed by restriction mapping which showed absence of a Hae III site. This leads to the substitution of glutamine for arginine at position -4 of the propeptide. Factor IX Strasbourg 2 was purified from plasma by DEAE Sepharose chromatography and immunoaffinity and relative to normal factor IX, binding of calcium to the mutant protein was clearly reduced in calcium lactate agarose gel. Quantification of gamma-carboxyglutamic acid residues gave about 50% carboxylation as compared to normal factor IX. Microsequencing of the NH2-terminal part of factor IX Strasbourg 2 confirmed the attachment of the propeptide and the mutation Arg-->Gln. Activation of factor IX Strasbourg 2 by purified factor XIa was found to be retarded as compared to normal factor IX, but after activation the mutant factor IXa was able to activate factor X. In conclusion, factor IX Strasbourg 2 circulates with the attached propeptide and shows reduced gamma-carboxylation and delayed activation by factor XIa but a normal capacity to activate factor X after total cleavage by factor XIa.

1-Carboxyglutamic Acid↗

PubMed1992

A neutron solution scattering study of the structure of annexin-V and its binding to lipid vesicles.

Low-angle neutron solution scattering has been used to study the structure of annexin-V and its interaction with small single-bilayer vesicles consisting of phosphatidylserine and phosphatidylcholine at a 33:66 (mol:mol) ratio. There was no evidence for a change in the state of aggregation of annexin-V, which remained as a monomer in the presence of 3 mM-free calcium. The only difference between presence and absence of free calcium was the increase of the radius of gyration, from 19(+/- 0.4) A to 22(+/- 0.4) A in 2H2O buffer and from 19.7(+/- 1.2) A to 22.2(+/- 1.2) A in H2O buffer. The relative molecular weight, outer radius and average surface area per lipid of vesicles alone were respectively 2.5(+/- 0.5) x 10(6), 127 A and 90(+/- 19) A2. These parameters were not modified in the presence of free calcium, which testified to the absence of vesicle coalescence. The calcium-dependent binding of annexin-V was essentially interfacial and therefore did not alter significantly the structural characteristics of the vesicles. At saturation, 80(+/- 10) annexin-V molecules were bound per vesicle, the available area per molecule being 2500(+/- 300) A2 thus covering approximately 28 lipid head groups. The protein shell was approximately 35 A thick. The apparent dissociation constant was probably less than 1 nM. These data contribute to a more accurate definition of annexin-V as a possible probe of those cytodynamic events involving exposure of sequestered membrane aminophospholipids.

Annexin A5↗

PubMed1992

Use of annexin-V to demonstrate the role of phosphatidylserine exposure in the maintenance of haemostatic balance by endothelial cells.

Annexin-V (PAP-I, lipocortin-V) acts as a potent anticoagulant in vitro by binding to negatively charged phospholipids with higher affinity than vitamin K-dependent proteins, with a Kd in the 10(-10) M range. The purpose of the present study was to use annexin-V as a probe to assess the catalytic potential of phospholipids in pro- and anti-coagulant reactions in purified systems and at the surface of endothelial cells in culture after stimulation. Procoagulant tissue factor and anticoagulant thrombomodulin activities were compared by using specific two-stage amidolytic assays performed with purified proteins. Procoagulant activity was estimated by the generation of Factor Xa by the Factor VII(a)-tissue factor complex. Anticoagulant activity was estimated by the generation of activated protein C by either the thrombin-thrombomodulin complex or Factor Xa. Annexin-V induced a decrease of 70% of thrombomodulin activity when thrombomodulin (5.4-214 nM) was reconstituted into phosphatidylcholine/phosphatidylserine (1:1, mol/mol) vesicles at 37.5 or 75 microM-phospholipid concentration, the apparent Ki being 0.5 microM at 75 microM-lipid. The saturating concentration of annexin-V was dependent on phospholipid concentration, but was independent of the phospholipid/thrombomodulin ratio. By contrast, when thrombomodulin was not reconstituted in vesicles, annexin-V had no effect. At 2 microM, annexin-V totally inhibited the generation of activated protein C by Factor Xa in the presence of 75 microM-lipid, the saturating inhibitory concentration being dependent on phospholipid concentration. At 0.1 microM, annexin-V totally inhibited tissue-factor activity present in crude brain thromboplastin. In the absence of stimulation, human endothelial cells in culture expressed significant thrombomodulin activity and no detectable tissue-factor activity. Basal thrombomodulin activity was only slightly inhibited (less than 15%) by 0.5 microM-annexin-V. Phorbol myristate acetate (PMA) induced the expression of tissue-factor activity and decreased thrombomodulin activity at the endothelial-cell surface. Annexin-V, at a concentration of 16 microM, caused an 80% decrease of tissue-factor activity induced by PMA at 10 ng/ml, whereas it inhibited thrombomodulin activity by only 15% on the same stimulated cells. Our results confirm that annexin-V inhibits, in vitro, procoagulant tissue-factor activity and anticoagulant activities (activation of protein C by the thrombin-thrombomodulin complex and by Factor Xa), through phospholipid-dependent mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Annexin A5↗

PubMed1991

Sub-domain structure of lipid-bound annexin-V resolved by electron image analysis.

Two-dimensional crystals of annexin-V bound to lipid layers containing dioleoylphosphatidylserine have been obtained in the presence of Ca2+. The crystals diffract to 20 A resolution and have the symmetry of the plane group p3 (unit cell dimensions: a = b = 94 A, gamma = 120 degrees). Electron image analysis revealed that the crystals are composed of trimers of annexin-V forming triskelion-like motifs. Each annexin-V molecule has a characteristic elongated shape, about 65 A by 20 A, when observed perpendicularly to the crystal plane. It is composed of two staggered domains of similar size, about 40 A by 20 A. Both domains are made of two sub-domains. The present data suggest that the four resolved sub-domains represent the folding units corresponding to the four 70 amino acid repeating segments characteristic of all annexins.

Annexin A5↗

PubMed1991

The catalytic role of anionic phospholipids in the activation of protein C by factor Xa and expression of its anticoagulant function in human plasma.

Phospholipids bearing a proportion of anionic species such as phosphatidylserine are necessary to promote the anticoagulant potential of the protein C pathway. Factor Xa (200 or 350 pM) was found to activate protein C in a thrombomodulin-independent reaction requiring only phospholipids in Al(OH)3,-adsorbed plasma resupplemented with physiological concentrations of protein C (70 nM) and protein S (130 nM). All experiments were performed in the presence of an excess of hirudin. The activity of activated protein C was assessed by the survival of factor Va. The optimal phospholipid concentration range was 5 to 25 microM with a proportion of phosphatidylserine of 50% (mol/mol) resulting in a half-life of factor Va of 7.5 min in the absence of protein S and 4.2 min in its presence. Dns-EGR-Xa, an inactive derivative of factor Xa, behaved as an apparent protector of factor Va. When replacing factor Xa, thrombin at 10 nM was not an efficient protein C activator in the absence of purified human placenta thrombomodulin. In the presence of 100 pM activated protein C, factor Va half-life was 2 min in the absence of protein S and 1.1 min in its presence in the above optimal phospholipid concentration range. The presence of protein S allowed reduction of phospholipid requirements. Annexin-V (placental anticoagulant protein-I), a potent phospholipid antagonist, fully protected factor Va from degradation by phospholipid-dependent mechanisms. Factor Va was partially protected in the plasma of a patient having experienced thrombosis associated with lupus-like anticoagulant and anti-phospholipid auto-antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Anions↗