Biomedical subjects

P Ohlmann

Publications and source records attributed to P Ohlmann.

47 records · Page 3Linked to original sources

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

ADP receptor induced activation of guanine nucleotide binding proteins in rat platelet membranes--an effect selectively blocked by the thienopyridine clopidogrel.

The thienopyridine clopidogrel, a potent analog of ticlopidine, is a powerful inhibitor of ADP induced platelet aggregation and ADP induced inhibition of cyclic AMP accumulation in intact platelets but not of ADP induced shape change. We have recently demonstrated that ADP stimulates the binding of GTP gamma S to GTP binding proteins (G proteins) in human platelet membranes. We now studied the effects of clopidogrel, a specific inhibitor of ADP induced platelet aggregation on the stimulation of GTP gamma S binding to rat platelet membranes by ADP. Using the non hydrolyzable stable analog of ADP, 2MeSADP, we demonstrate that 2MeSADP stimulates the binding of [35S]GTP gamma S to rat platelet membranes in a concentration dependent manner, that this effect is inhibited by the specific ADP receptor antagonist Sp-ATP alpha S and that clopidogrel completely and selectively blocks the stimulation by 2MeSADP of [35S]GTP gamma S binding to platelet membranes of treated rats. We conclude that: i) rat platelet membranes possess an ADP receptor coupled to unidentified G protein(s) and ii) the thienopyridine clopidogrel impairs the interaction of the ADP receptor with its G protein by an irreversible modification the ADP receptor itself or its putative G protein.

Adenylyl Cyclases↗

PubMed1992

ADP receptor-induced activation of guanine-nucleotide-binding proteins in human platelet membranes.

ADP receptor-regulated binding of the labeled GTP analog, guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTP[gamma S]), to guanine-nucleotide-binding proteins (G proteins) was studied in human platelet membranes. The potent ADP receptor agonist, 2-methyl-thio-adenosine 5'-diphosphate (2MeSADP), a non-hydrolyzable analog of ADP, increased the binding of [35S]GTP[gamma S] without apparent lag phase. Under optimal conditions, i.e. in the presence of GDP (1-10 microM), 2MeSADP increased the binding up to about threefold, with half-maximal and maximal increase observed at 10 nM and 1 microM 2MeSADP, respectively. ADP itself increased the binding of [35S]GTP[gamma S] by maximally about twofold, with half-maximal increase occurring at 0.1 microM ADP. The agonist-induced stimulation was competitively antagonized by the ADP receptor(s) antagonist, (1S)-adenosine 5'-O-(1-thiotriphosphate) [(Sp)-ATP[alpha S]]. Other platelet receptor agonists known to act through receptors coupled to G proteins also increased binding of [35S]GTP[gamma S] in human platelet membranes, but without being inhibited by (Sp)-ATP[alpha S]. The data presented indicate that the platelet ADP receptor(s) can interact with and efficiently activate G proteins, the nature of which remains to be identified.

6-Phytase↗

PubMed1992

[Noninvasive function tests in the evaluation of vascular reactivity in pregnancy and pregnancy-induced hypertension].

At present, there is an increasing discussion regarding the change of the prostacyclin/thromboxane ratio as an aetiological factor of pregnancy-induced hypertension (PIH). By means of videophotometric capillaroscopy in the capillaries of the nailfold, it is possible to visualise the resulting changes in the microcirculation. This method employs a special illumination microscope (magnification of the optical system x 560). Red blood cell velocity is measured under test conditions and microcirculatorical reaction capacity on ischaemic stress. Studies were conducted on healthy non-pregnant women and on healthy pregnant women as well as on patients suffering from pregnancy-induced hypertension. A markedly reduced velocity of red blood cells in patients with pregnancy-induced hypertension was seen as a sign of vasoconstriction, namely, 0.53 mm/sec. versus 0.74 mm/sec. Furthermore, these patients have a higher reactivity to ischaemic stress, probably also as a result of the already existing vasoconstriction; however, the duration of the hyperaemic phases is reduced in pregnancy-induced hypertension. By this method, an insight can be gained into microcirculation in pregnancy and in pregnancy-induced hypertension. In addition, as preliminary studies have already shown, the influence exercised by different medications on microcirculation can be assessed.

Blood Flow Velocity↗

PubMed1992

Alpha IIB beta 3 integrin dissociation induced by EDTA results in formation of Birbeck granule-like structures in human blood platelets.

EDTA (5 mM at 37 degrees C and pH 7.4 for 30 min) induces morphologic changes in the platelet surface-connected canalicular system (SCCS), in particular the collapse of some portions of the canaliculi. These collapsed elements are made up of two parallel limiting membranes and a central irregular striated zone, a pentalaminar organization that clearly resembles that of Langerhans cell Birbeck granules (BG). Such BG-like structures are also seen between adjacent platelets in EDTA-treated platelet microaggregates. EDTA is known to induce an irreversible dissociation of the platelet membrane glycoprotein(GP)IIb-IIIa, the alpha IIb beta 3 platelet-specific intergrin, a calcium-dependent heterodimer that serves as an inducible receptor for fibrinogen and is essential for platelet aggregation. Hence, we looked for involvement of the GPIIb-IIIa in the formation of these BG-like modifications. We observed that these changes i) cannot be induced in type I Glanzmann's thrombasthenia, where the GPIIb-IIIa complexes are absent; ii) did not appear when human platelets were pre-incubated with MoAb anti-GPIIb-IIIa complex, which protected GPIIb-IIIa from EDTA-induced dissociation; iii) appeared only at alkaline pH and 37 degrees C, which corresponds to the range of pH and temperature where EDTA can dissociate the GPIIb-IIIa complexes; iv) are accompanied by the dissappearance on fluorescence flow cytometry analysis of the heterodimer specific epitopes; and v) do not appear in rat platelets at pH 7.4 where GPIIb-IIIa does not dissociate after EDTA treatment. Thus, the appearance of BG-like structures in the platelet SCCS is directly dependent on the EDTA-induced dissociation of the GPIIb-IIIa complexes. Furthermore, using gold-labeled MoAb concomitantly with the addition of EDTA, we observed that only GPIIb is present in the collapsed portions of the canaliculi. On Lowicryl thin sections essentially polyclonal antibodies to GPIIb labeled the central striated zone. All these observations lead us to suggest that homopolymers of GPIIb could be responsible for "zipping" of the SCCS and raise the question of the participation of a Langerhans cell integrin in the formation of Langerhans cell BG.

Blood Platelets↗

PubMed1992

Electrophoretic studies on molecular defects of von Willebrand factor and platelet glycoprotein IIb-IIIa with antibodies produced in egg yolk from laying hens.

Immunoglobulins isolated from egg yolk (IgYs) are a convenient source of polyclonal antibodies. Their purification is simple and the yields important (50 mg immunoglobulins/egg). Nevertheless their biochemical characteristics are different from those of rabbit antibodies generally used for the study of molecular defects of plasmatic and platelet proteins. Provided standardization is achieved, IgYs can be used for immunoelectrophoresis, immunoprecipitation or immunoblotting assays. We describe here the electrophoretic and immunoblotting conditions employed to explore human plasmatic and platelet von Willebrand factor (vWF) and human platelet GP IIb-IIIa using IgYs. These two proteins are involved in primary hemostasis and their absence or abnormality is responsible for hereditary bleeding disorders. The methods were applied to the characterization of patients with vWF or GP IIb-IIIa defects and compared to classical mammalian IgG immunoelectrophoretic techniques. Results were further confirmed by flow cytometric analysis.

Animals↗

PubMed1991

Normal ADP-induced aggregation and absence of dissociation of the membrane GP IIb-IIIa complex of intact rat platelets pretreated with EDTA.

ADP-induced platelet aggregation requires the presence of external calcium and fibrinogen. When human platelets are incubated for 30 min at 37 degrees C with 5 mM EDTA and then resuspended in a calcium containing medium, they lose their ability to bind fibrinogen and to aggregate in response to ADP stimulation. Under these conditions, the effect of EDTA is irreversible and accompanied by dissociation of the glycoprotein (GP) IIb-IIIa complex into its free subunits, GP IIb and GP IIIa. We studied the effect of incubation of intact rat platelets with 5 mM EDTA at 37 degrees C from 30 to 120 min. EDTA treated rat platelets showed normal aggregation in response to 5 microM ADP in the presence of added purified rat fibrinogen and bound 125I-labeled rat fibrinogen at the same rate and magnitude after stimulation with 5 microM ADP as untreated platelets. Control and EDTA treated rat platelets, labeled or not with 125I and solubilized in Triton X-100, had a similar pattern of immunoprecipitates after crossed immunoelectrophoresis (CIE) analysis. The rat GP IIb-IIIa arc was located by incorporation of an 125I-labeled polyclonal anti-human GP IIb-IIIa antibody. In contrast, in experiments using rat platelet lysates, we demonstrated that the rat GP IIb-IIIa is a Ca(2+)-dependent heterodimer as it was dissociated by EDTA. Using SDS-PAGE and two-dimensional SDS-PAGE, the rat GP IIb-IIIa complex was found to have characteristics similar to the human complex with the exception that the light chain of the rat GP IIb was undetectable after 125I surface labeling.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1991

Ultrastructural similarities between epidermal Langerhans cell Birbeck granules and the surface-connected canalicular system of EDTA-treated human blood platelets.

Birbeck granules characterize under the electron microscope epidermal Langerhans cells. These distinctive pentalaminar organelles are indeed not detectable in the possible precursors of human Langerhans cells and tend to disappear in cultured human Langerhans cells. The mechanisms that lead to the appearance of Birbeck granules in epidermal Langerhans cells and to their later disappearance still remain unknown. In the present study we show that the more or less dilated elements of the surface-connected canalicular system of human blood platelets collapse after EDTA treatment. Made up of two parallel limiting membrane and central irregular striated density, these elements show great ultrastructural similarities with the Birbeck granules of human epidermal Langerhans cells. These platelet morphologic changes i) are directly dependent on the EDTA-induced dissociation of the glycoprotein GP IIb-IIIa, the platelet-specific calcium-dependent heterodimer complex, member of the beta 3 integrin subfamily (alpha IIb beta 3) and ii) apparently result from a cross-linking of the dissociated glycoproteins. These findings lead us to propose that in the same manner cells of the Langerhans lineage, on reaching the epidermis, will find themselves in contact with an epidermal specific ligand. Interactions between this epidermal ligand and Langerhans cell receptors could then induce, all along the circuit taken by the ligand-receptor complexes, morphologic modifications, i.e., appearance of structures of Birbeck granule type.

Blood Platelets↗

PubMed1990

The thienopyridine ticlopidine selectively prevents the inhibitory effects of ADP but not of adrenaline on cAMP levels raised by stimulation of the adenylate cyclase of human platelets by PGE1.

After oral administration, ticlopidine specifically inhibits ADP-induced platelet aggregation, prolongs the bleeding time and prevents thrombosis in man. Its mechanism of action is not well known. Ticlopidine inhibits ADP-induced binding of fibrinogen to platelet glycoprotein GP IIb-IIIa but not shape change and increases deaggregation. Ticlopidine has no direct effect on the GP IIb-IIIa complex. We studied the effects of ticlopidine (500 mg/day for 8 days) in four healthy male volunteers on washed platelet aggregation induced by 5 microM ADP or thrombin (0.1 units/mL) and potentiated by 1 microM adrenaline (Adr), on basal and 1 microM PGE1-stimulated cAMP levels and on elevation of cytosolic free Ca2+ concentration ([Ca2+]i). We found that: (i) ticlopidine inhibits aggregation by ADP but not the potentiation by Adr of ADP-induced aggregation; (ii) ADP, Adr or thrombin decreases cAMP levels raised by PGE1, an effect inhibited by ticlopidine only for ADP and not for Adr or thrombin; and (iii) Ca2+ influx and Ca2+ mobilization from internal stores were not affected. These results suggested that ticlopidine or a metabolite impairs the coupling mechanism of the ADP aggregation pathway at an unknown level.

Adenosine Diphosphate↗

PubMed1990

The thienopyridine PCR 4099 selectively inhibits ADP-induced platelet aggregation and fibrinogen binding without modifying the membrane glycoprotein IIb-IIIa complex in rat and in man.

The thienopyridines, ticlopidine and PCR 4099, inhibit ex vivo aggregation in response to ADP and other agonists. It has been shown that ticlopidine induces a functional defect in the binding of fibrinogen to its platelet membrane receptor. We have studied the effects on platelet functions of PCR 4099 in rat and in man. The aim of the study was to check the possibility of a direct modification of the fibrinogen binding site on the GP IIb-IIIa complex. Washed platelet suspensions were used for aggregation and fibrinogen binding studies. Platelet lysates were submitted to SDS-polyacrylamide gel electrophoresis, crossed immunoelectrophoresis and immunoprecipitation. We found that administration of PCR 4099 inhibited selectively and irreversibly ADP-induced aggregation. Although the effect of ADP on aggregation was blocked, PCR 4099 did not modify ADP-induced shape change. Only the effects of low concentrations of thrombin on platelet aggregation were inhibited. Fibrinogen binding was dramatically inhibited in rat and in man when platelets were stimulated with ADP and low concentrations of thrombin. At high concentration of thrombin there still remained a part of fibrinogen binding inhibition although aggregation was not impaired. Electrophoretic and immunoelectrophoretic studies showed no difference before and after treatment by PCR 4099. In particular, the GP IIb-IIIa-complex was not dissociated, its electrophoretic mobility was not changed and three monoclonal anticomplex antibodies recognized it in the same manner before and after treatment. We conclude that PCR 4099 selectively inhibits the ADP aggregation pathway and that the inhibition of fibrinogen binding is probably not due to a direct modification of the GP IIb-IIIa complex.

Adenosine Diphosphate↗

PubMedDate not supplied

Effect of lipopolysaccharide treatment on neurogenic contraction and noradrenaline release in rat arteries.

In the present study, contractile responses and [3H]-noradrenaline overflow evoked by electrical field stimulation were assessed, respectively, in the small mesenteric artery and in tail artery removed from rats pre-treated with either saline or lipopolysaccharide (LPS). In small mesenteric arteries, LPS treatment did not significantly modify the contractile responses elicited by electrical stimulation, in the absence or in the presence of L-arginine. However, in arteries removed from rats treated with LPS, L-arginine addition produced relaxation of vessels pre-contracted with noradrenaline. The amplification of neurogenic contraction by the nitric oxide (NO) synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) was similar in arteries removed from saline and LPS-infused rats. In mesenteric arteries, LPS treatment suppressed the potentiation of the neurogenic responses by the alpha2-adrenoceptor antagonist, yohimbine and by the inhibitor of neuronal uptake of noradrenaline, cocaine. In rat tail artery exposed to L-arginine, LPS treatment produced an increase in [3H]-noradrenaline overflow evoked by electrical stimulation. Altogether, these data suggest that an enhanced noradrenaline release from sympathetic nerves, probably resulting from inhibition of the modulatory effect of both prejunctional alpha2-adrenoceptors and neuronal uptake mechanism, may play a role in the preservation of neurogenic response after LPS treatment despite evidence of the induction of NO synthase.

Adrenergic alpha-Antagonists↗