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Biomedical subjects

R M Hardisty

Publications and source records attributed to R M Hardisty.

At least 19 recordsLinked to original sources
PubMed1991

Effects of a monoclonal antibody to glycoprotein IIb/IIIa (P256) and of enzymically derived fragments of P256 on human platelets.

The anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10(-9)-3 x 10(-8) M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10(-7) M. P256-induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In-111-labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications.

Adult↗
PubMed1990

The action of ticlopidine on human platelets. Studies on aggregation, secretion, calcium mobilization and membrane glycoproteins.

The effects on platelet function of a 5-day course of Ticlopidine (Tcl) have been studied in two groups of volunteers receiving different dosage schedules. Tcl had a relatively greater inhibitory effect on aggregation induced by ADP than by other agonists, and a greater effect, in contrast to that of an ADP receptor antagonist, on the second phase than on the initial rate of aggregation. Tcl inhibited ATP secretion in response to ADP and 0.05 u/ml thrombin, but not to higher concentrations of thrombin or to calcium ionophores. No inhibitory effect was observed on Ca2+ influx or intracellular mobilization, on the binding of monoclonal antibodies to the glycoprotein IIb-IIIa complex or on the state of association of the complex. We suggest that Tcl neither inhibits the binding of ADP to its receptor nor acts directly on the fibrinogen binding site, but that it may inhibit a step in signal transduction between these two events.

Adenosine Triphosphate↗
PubMed1990

Measurement of fibrinogen binding to platelets in whole blood by flow cytometry: a micromethod for the detection of platelet activation.

Platelet fibrinogen binding in whole blood has been measured in vitro by flow cytometry using a commercially available, fluorescein isothiocyanate (FITC)-conjugated polyclonal antifibrinogen antibody. Fibrinogen-antifibrinogen immune complexes were formed in experimental conditions approaching antigen-antibody equivalence, but optimal reaction conditions in which their formation was prevented or minimized could be achieved. Immune complex formation was associated with fibrinogen binding to unstimulated platelets but did not significantly affect ADP-induced fibrinogen binding. Half-maximal fibrinogen binding occurred at about 0.4 microM ADP, and ADP-induced fibrinogen binding continued progressively during 20 min incubation with 10 microM ADP. Fibrinogen binding correlated closely with platelet glycoprotein IIb-IIIa expression in members of a family with Glanzmann's thrombasthenia, and, in double labelling experiments, with the binding of PAC1, a monoclonal antibody that binds to GP IIb-IIIa only after the exposure of fibrinogen receptors. These studies show that platelet fibrinogen binding can be reliably measured in whole blood by means of a polyclonal antifibrinogen antibody which does not discriminate between plasma and platelet-bound fibrinogen, despite the presence of an approximately 100-fold excess of the former.

Adenosine Diphosphate↗
PubMed1988

Potentiation by adrenaline of Ca2+ influx and mobilization in stimulated human platelets: dissociation from thromboxane generation and aggregation.

In a medium containing 1 mM extracellular Ca2+ (Ca2+o), the prior addition of 0.5 microM adrenaline to quin 2-loaded human platelets increased both the rate and amplitude of the rise in cytosolic free Ca2+ (Ca2+i) in response to sub-threshold concentrations of thrombin and PAF and these effects were not prevented by blocking either fibrinogen binding and aggregation or cyclo-oxygenase. In the presence of 2 mM EGTA [( Ca2+o] less than 100 nM), the rate, but not the extent of rise of [Ca2+i] was enhanced by adrenaline, and this was also unaffected by blockade of cyclo-oxygenase. Addition of adrenaline 1 min after the other agonist in the presence of 1 mM Ca2+o resulted in aggregation without further elevation of [Ca2+i]. Adrenaline thus enhances both influx and intracellular mobilization of Ca2+ by a mechanism independent of both fibrinogen binding and thromboxane production, but these effects do not fully explain its potentiation of aggregation by other agonists.

Blood Platelets↗
PubMed1988

Intravascular platelet activation in the hemolytic uremic syndrome.

We studied intravascular platelet activation in patients with typical (epidemic) and atypical (sporadic) HUS and found defective aggregation, decreased platelet and increased plasma serotonin in both groups. The findings were present not only on admission during the thrombocytopenic stage of the disease, but persisted for several weeks after recovery of the platelet count. Reduced endothelial PGI2 production was significantly more common in plasma from atypical than typical cases. Plasma from both typical and atypical HUS patients induced aggregation of normal platelets, but this phenomenon was unrelated to both the presence of thrombocytopenia or the stage of the disease. Serum platelet aggregating activity was higher in the atypical HUS patients, and was not associated with immune complexes detectable by polyethylene glycol precipitation. The data indicate that intravascular platelet activation is a feature of both forms of HUS, and may be initiated by different mechanisms in the two subgroups. While abnormal PGI2 synthesis appears to be important in the atypical cases, neither defective PGI2 production nor platelet aggregation by plasma factors adequately accounts for platelet activation in the majority of typical cases.

Adolescent↗
PubMed1987

Long survival in childhood lymphoblastic leukaemia.

Long term follow-up of 378 children with acute lymphoblastic leukaemia (ALL) treated at a single centre showed that at six years from diagnosis 202 (53%) were alive, of whom 140 (37%) remained in first remission. Only three children had a first relapse after six years. Children who survived six years despite a single extramedullary relapse in the testis or CNS were likely to remain in second remission but patients with previous marrow or with multiple relapses continued at risk for up to ten years from diagnosis. Presenting factors influencing event-free survival were: leucocyte count, age and sex. After allowing for these factors morphological (FAB) subtype and liver enlargement retained their prognostic significance. Immunological type of ALL was not of independent prognostic significance, except for the small number of patients with B-ALL. Most factors lost their significance after 2-4 years. It is concluded that patients alive 6 years from diagnosis without relapse or even with a single extramedullary relapse of ALL, have a high chance of prolonged survival and cure.

Child↗
PubMed1987

Decreased sensitivity to heparin in vitro in steroid-responsive nephrotic syndrome.

The in vitro heparin sensitivity of 18 nephrotic children was compared with that of 10 normal children and 13 children with other renal diseases. The influence of age on the heparin sensitivity of 52 normal subjects (aged 12 to 85 years) was also studied. The heparin sensitivity was calculated from the dose-response curve obtained when increasing amounts of heparin were added to plasma and the kaolin partial thromboplastin time (KPTT) was measured. There was a significantly-reduced heparin sensitivity in nephrotic children compared to the control children and a progressive decline in heparin sensitivity with age. In the nephrotic syndrome heparin-sensitivity correlated with albumin and triglyceride concentrations but not with antithrombin III, platelet factor 4, cholesterol, fibrinogen, heparin cofactor II or histidine-rich glycoprotein. Addition of exogenous albumin did not restore the heparin sensitivity of nephrotic plasma. Four patients with Type II hyperlipidemia had a normal sensitivity to heparin. The decreased sensitivity to heparin thus does not appear to be a consequence of the nephrotic state, and may be a reflection of an underlying disturbance of charged macromolecules in steroid-responsive nephrotic syndrome.

Adolescent↗
PubMed1987

Grey platelet syndrome: studies on platelet alpha-granules, lysosomes and defective response to thrombin.

The platelets of a young man with the grey platelet syndrome were severely depleted of all seven alpha-granule proteins assayed as well as partially deficient in alpha-mannosidase and alpha-fucosidase; four other lysosomal enzymes were present in normal concentrations. Total platelet 5-hydroxytryptamine (5HT) and adenine nucleotides were normal, and 14C-5HT uptake reached normal levels only slightly more slowly than a control. Aggregation and dense body secretion occurred normally in response to ADP, adrenaline, collagen, PAF-acether, sodium arachidonate, A23187, Ionomycin, TPA and U44069, but were very delayed in response to thrombin. The increase in cytosolic free calcium in response to thrombin was very slow and much reduced in amplitude, whether in the presence or absence of extracellular Ca2+. These defects in response to thrombin were not corrected by the separate addition of purified alpha-granule proteins or by a whole releasate from normal platelets. It is suggested that these platelets, in addition to their alpha-granule deficiency, may have a specific defect of thrombin receptor-mediated activation of phospholipase C.

Blood Platelet Disorders↗
PubMed1987

Oral methotrexate is as effective as intramuscular in maintenance therapy of acute lymphoblastic leukaemia.

It has been postulated that variations in methotrexate absorption may influence the outcome of treatment in lymphoblastic leukaemia. One hundred and forty four children with acute lymphoblastic leukaemia not of the T cell type were randomised to receive continuing treatment with daily 6-mercaptopurine, vincristine, and prednisolone six weekly and methotrexate once weekly, either as a single oral dose or an intramuscular injection. Analysis of results with a minimum follow up of three and a half years has shown that the route of administration of methotrexate has had no influence on relapse at any site, but more children receiving intramuscular methotrexate died in remission. These results indicate that oral methotrexate is as effective as intramuscular methotrexate in continuing treatment of lymphoblastic leukaemia.

Administration, Oral↗
PubMed1986

Differential effects of 12-O-tetradecanoylphorbol 13-acetate on platelet responses to various agonists in the presence and absence of extracellular Ca2+.

In the presence of 1 mM extracellular Ca2+ (Ca2+o), incubation of washed, quin 2 loaded platelets with a low concentration (1 nM) of 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited platelet shape change, aggregation, ATP secretion and cytosolic calcium (Ca2+i) fluxes in response to thrombin, platelet activating factor (PAF), adenosine diphosphate (ADP), vasopressin (VP) and the thromboxane mimetic U46619, but potentiated platelet activation induced by the calcium ionophores, A23187 and ionomycin, the Ca2+ flux remaining unaltered. In the presence of less than 100 nM Ca2+o, TPA again inhibited shape change but potentiated ATP secretion induced by all agonists, even those in which the cytosolic calcium flux was inhibited. Addition of TPA 30 seconds after a low dose of agonist, sufficient to elevate [Ca2+i] to about 250 nM without causing aggregation, induced substantial aggregation and dense granule secretion without affecting Ca2+ flux. These results confirm that very slight elevation of [Ca2+i] above resting levels greatly enhances the effect of low concentrations of TPA, and suggest that protein kinase C activation may exert a feedback inhibition of receptor-mediated shape change, aggregation, ATP secretion and Ca2+ mobilization.

Adenosine Triphosphate↗
PubMed1986

Acute myeloid leukaemia in childhood: clinical features and prognosis.

Clinical and laboratory features at presentation were correlated with morphological (FAB) subclass of AML in a group of 112 children diagnosed between 1972 and 1982. Patients with a monocytic component of AML (M4, M5) had higher initial leucocyte counts, a higher incidence of extramedullary infiltration and of CNS involvement. In M4 AML CNS relapse occurred in patients with a high initial leucocyte count whereas in M5 AML CNS involvement tended to occur at presentation in children with low initial counts. Two-thirds of patients treated achieved remission and most failures were due to inadequate chemotherapy, although haemorrhage, leucostasis or metabolic complications caused early death in patients with M4 and M5 AML. With a minimum follow up of 3 years only 12% of patients are alive; these figures have not improved in consecutive series despite increasing intensity of induction and more recent availability of bone marrow transplantation. No features predictive of long-term survival were identified, but patients with myeloid differentiation (M1, M2, M3) did better than those with a monocytic or erythroid component (M4, M5, M6). The proportion of patients with AML curable by chemotherapy seems unlikely to increase without marked intensification of post-remission chemotherapy. More aggressive CNS prophylaxis may be of benefit in cases with a monocytic component.

Brain Neoplasms↗
PubMed1985

First trimester prenatal diagnosis and detection of carriers of haemophilia A using the linked DNA probe DX13.

Although the use of a gene specific deoxyribonucleic acid (DNA) probe is the method of choice for detecting carriers of genes for rare genetic disorders, there will always be families in which such probes cannot be used because key subjects are not informative for restriction fragment length polymorphisms in or around the gene. In these cases closely linked DNA markers have to be used. An X chromosome specific DNA probe, DX13, which is closely linked to the haemophilia A locus on the X chromosome, was used for early prenatal diagnosis in two cases and to detect carriers in a series of nine possible heterozygote women. The first reported crossover between DX13 and the factor VIII:C locus was observed in this study. There are complexities inherent in using any linked DNA probe for assignment of genes, but such techniques are clinically important.

Female↗
PubMed1985

Cyclic AMP inhibits platelet activation independently of its effect on cytosolic free calcium.

Stimulation of platelets with the ionophore A23187, thrombin, ADP or PAF-acether resulted in a rapid increase of cytosolic free Ca2+, as measured with Quin-2, and in aggregation, 5HT secretion and - in the case of the first two agonists - thromboxane generation. PGI2 and dibutyryl cyclic AMP inhibited all these responses, except in the case of A23187, in response to which the increase in Ca2+ was unaffected, although the other responses were inhibited. The inhibition of aggregation and secretion in response to the combination of thrombin and A23187 was indistinguishable from that in response to thrombin alone. It thus appears that cAMP inhibits these responses independently of its effect in lowering cytosolic free Ca2+.

Adenosine Diphosphate↗
PubMed1985

Glycoprotein IIb-IIIa complex and Ca2+ influx into stimulated platelets.

Changes in intracellular Ca2+ concentrations [( Ca2+]i) in platelets stimulated with aggregating agents were measured with the fluorescent indicator dye quin 2. Ca2+ influx, but not intracellular mobilization, in response to adenosine diphosphate (ADP), platelet aggregating factor (PAF-acether), and sodium arachidonate was significantly inhibited by monoclonal antibodies against the glycoprotein (GP) IIb-IIIa complex; inhibition of thrombin-stimulated influx was inhibited to a lesser extent and reached statistical significance only at thrombin concentrations of 0.1 U/mL and below. Anti-GP Ib and HLA-ABC monoclonal antibodies had no effect on Ca2+ influx in response to any agonist. Thrombasthenic platelets gave normal [Ca2+]i responses to ADP and thrombin, which were not inhibited by an anti-GP IIb-IIIa antibody. It is suggested that Ca2+ influx in response to weak agonists occurs predominantly via a channel closely adjacent to the GP IIb-IIIa complex, but that higher concentrations of thrombin and A23187 also stimulate influx via another pathway.

Adenosine Diphosphate↗
PubMed1984

Von Willebrand factor has more than one binding site for platelets.

By the use of a series of monoclonal antibodies against platelet membrane glycoproteins (GP) and the von Willebrand factor (vWF), it is shown that thrombin-induced binding to GP IIb/IIIa involves a different site on vWF from ristocetin-induced binding to GP Ib.

Antibodies, Monoclonal↗
PubMed1984

A monoclonal antibody binding to human medulloblastoma cells and to the platelet glycoprotein IIB-IIIA complex.

A monoclonal antibody, designated M148, produced by the hybridoma technique from spleen cells of mice immunized with human medulloblastoma, was found by indirect immunofluorescence to bind to normal human platelets (both PlA1 positive and PlA1 negative) and megakaryocytes, as well as to some medulloblastoma and neuroblastoma cells and cell lines and certain other solid tumours. No binding was observed to other marrow constituents, nor to any other normal tissue examined. The antibody bound to platelets from a patient with the Bernard-Soulier syndrome but not to thrombasthenic platelets. It immunoprecipitated glycoproteins IIb and IIIa from 125I-labelled normal platelet membranes, and completely inhibited ADP-induced fibrinogen binding and aggregation of platelets. Aggregation was also inhibited in response to adrenaline, collagen, thrombin, sodium arachidonate and the ionophore A23187; clot retraction was partially inhibited. The antibody was without effect on thromboxane formation or 5-hydroxytryptamine (5HT) secretion in response to thrombin, but inhibited 5HT secretion in response to arachidonate. It did not inhibit factor VIII binding or agglutination in response to ristocetin, but completely inhibited factor VIII binding in response to thrombin. These findings suggest that the epitopes are close to the fibrinogen and factor VIII binding sites on glycoproteins IIb/IIIa, and that the lack of these glycoproteins is sufficient explanation for the pattern of dysfunction observed in thrombasthenic platelets, without invoking any other membrane abnormality.

Animals↗