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Biomedical subjects

J M Gerrard

Publications and source records attributed to J M Gerrard.

At least 19 recordsLinked to original sources
PubMed1992

Biochemical and ultrastructural studies suggest that the effects of thapsigargin on human platelets are mediated by changes in intracellular calcium but not by intracellular histamine.

The involvement of intracellular histamine in thapsigargin (Tg)-induced platelet aggregation was studied. Platelet aggregation induced by 0.25 and 0.5 microM Tg was not accompanied by a rise in intracellular histamine but a significant (p < 0.01) increase in the level of intracellular histamine was observed at 1 microM Tg. Preincubation of platelets with inhibitors of histamine metabolizing enzymes had little effect on intracellular histamine levels in platelets stimulated by 0.5 microM Tg. In addition, the inhibitors of histidine decarboxylase (HDC), alpha-methyl histidine (alpha-MH) and alpha-fluoromethyl histidine (alpha-FMH) failed to inhibit Tg-induced aggregation. The intracellular histamine receptor antagonist, N,N-diethyl-2-[4-(phenylmethyl)phenoxy] ethanamine. HCl (DPPE), inhibited Tg-induced aggregation but with IC50 values dependent on the concentration of agonist used. The inhibitory effects of DPPE on Tg-induced aggregation were not reversed by the addition of histamine to saponin-permeabilized platelets suggesting non-histamine mediated effects of DPPE on Tg-induced aggregation. Tg stimulated an increase in the cytosolic free calcium concentration which was unaffected by DPPE indicating that the effects of DPPE are also not due to the inhibition of mobilization of cytosolic calcium. The ultrastructural studies suggest that the major Tg-induced changes (pseudopod formation and granule centralization) are consistent with a primary role for Tg to mobilize calcium; DPPE had very little effect on these ultrastructural changes. The results indicate that the effects of Tg on human platelets are mediated by an increase in cytosolic calcium but not by intracellular histamine.

Blood Platelets↗
PubMed1992

Rapid ultrastructural changes in the dense tubular system following platelet activation.

The dense tubular system (DTS) functions to regulate platelet activation by sequestering or releasing calcium, similar to the sarcotubules of skeletal muscle. In resting platelets, the DTS exists as thin elongated membranes. Within 10 seconds of the addition of thrombin, platelets show a major ultrastructural change in their DTS: from the thin elongated form to a rounded vesicular form. These morphologic changes were demonstrated with two different stains and two different fixation methods. Platelets exposed to the calcium ionophore A23187 showed the same ultrastructural changes in the DTS. In contrast, the DTS remains in a thin elongated form when platelets are stimulated by the protein kinase C activators phorbol 12-myristate 13-acetate (PMA) and oleoylacetylglycerol (OAG). These morphologic changes may be related to the discharge of calcium from the DTS because this is stimulated by thrombin and A23187, but not by PMA. Preincubation of the platelets with the intracellular calcium chelator 5,5'-dimethyl-bis-(0-aminophenoxy)-ethane-N,N,N',N tetra acetic acid (BAPTA) largely prevented both the thrombin-induced rise in intracellular calcium and the changes in DTS morphology, suggesting that the changes in DTS morphology are secondary to the increase in cytosolic calcium. The results provide a morphologic correlate to existing biochemical evidence showing that the DTS is involved early during paltelet activation.

Blood Platelets↗
PubMed1992

Platelet dense granule membranes contain both granulophysin and P-selectin (GMP-140).

We recently reported the characterization of a platelet granule membrane protein of molecular weight (mol wt) 40,000 called granulophysin (Gerrard et al: Blood 77:101, 1991), identified by a monoclonal antibody (MoAb D545) raised to purified dense granule membranes. Using immunoelectron-microscopic techniques on frozen thin sections, this protein was localized in resting and thrombin-stimulated platelets. In resting platelets, labeled with antigranulophysin antibodies and immunogold probes, label was localized to the membranes of one or two clear granules per platelet thin section. D545 also labeled dense granules in permeabilized whole platelets and isolated dense granule preparations examined by whole-mount techniques. Expression of granulophysin on the platelet surface paralleled dense granule secretion as measured by 14C-serotonin release under conditions in which lysosomal granule release, as measured by beta-glucuronidase secretion, was less than 5%. After thrombin stimulation, both the surface-connected canalicular system and the plasma membrane were labeled, demonstrating redistribution of granulophysin associated with degranulation. Double labeling experiments with D545 and antibodies to the alpha-granule membrane protein, P-selectin, demonstrated labeling of both P-selectin and granulophysin on dense granule membranes. Distribution of both proteins on the plasma membrane after platelet stimulation was similar. The results demonstrate that granulophysin is localized to the dense granules of platelets and is redistributed to the plasma membrane after platelet activation.

Antibodies, Monoclonal↗
PubMed1992

Sharing of antigenic epitopes between synaptophysin and granulophysin.

The immunological crossreactivity between the two granule-specific membrane glycoproteins, synaptophysin and granulophysin, was studied using a series of site-specific monoclonal and polyclonal antibodies. The epitope relatedness of six monoclonal antibodies against granulophysin was examined by competitive ELISA. The antibodies are shown to recognize distinct, but overlapping epitopes within a compact region that is constructed by the three-dimensional configuration of the molecule. All these antibody clones also recognize rat neuronal synaptophysin. Two monoclonal antibodies against synaptophysin, of which one is the well-characterized SY38 antibody, directed against the carboxy terminal of the molecule, are also shown to react with granulophysin. Characterized polyclonal antibodies against different peptide antigens of synaptophysin failed to recognize granulophysin. Synaptophysin and granulophysin are distinctly recognized in brain cell (white matter) and the pituitary both qualitatively and quantitatively. Based on these and other observations, it is suggested that the repeat motif in the cytoplasmic tail of synaptophysin represents an immunodominant construct that is the target for the observed crossreactive antibodies and that a similar tertiary construct has been preserved in granulophysin and in other transmembrane proteins.

Animals↗
PubMed1992

Essential fatty acid status of neonates born to Inuit mothers: comparison with Caucasian neonates and effect of diet.

Fatty acid compositions were determined of phospholipids isolated from venous cord plasma and from the walls of umbilical arteries and veins, collected from healthy, a terme, Inuit and Caucasian (Dutch) neonates. The Inuit fatty acid profiles were characterized by a lower essential fatty acid (EFA) status, with higher levels of monounsaturated fatty acids, of Mead acid [20:3(n-9)] and its direct elongation product, and with lower amounts of the longer chain (greater than or equal to 20 carbon atoms), highly unsaturated (greater than or equal to 4 double bonds) fatty acids of both the (n-3) and (n-6) families. Levels of linoleic- and dihomo-gamma-linolenic acids were higher in Inuit as compared to Caucasian neonates, which suggests a low activity of the delta-5-desaturase in the Inuit. Within the Inuit group, a higher intake of marine food was associated with a better neonatal (n-3) status. Although the differences between Inuit and Caucasian neonates may be of genetic rather than of dietary origin, the results imply that dietary long-chain (n-3) or (n-6) fatty acids may be particularly important during pregnancy in Inuit mothers. Further studies are indicated with respect to the EFA content of the habitual Inuit diet and levels of delta-5-desaturase activity in the Inuit.

Diet↗
PubMed1992

Aerobic fitness level (VO2max) moderates the increased cardiovascular function and basal thromboxane formation of young healthy Type A males.

The authors evaluated the interaction between physical fitness and Type A behavior on vascular production of prostacyclin and platelet thromboxane in response to a standard vessel injury. Ninety-seven male university students were classified as Type A or B on the basis of the Structured Interview of Rosenman. Reactivity, as measured by changes in systolic blood pressure (SBP), and heart rate (HR), measured before and after a stressful Stroop task 1 week later, showed that Type A individuals were more reactive to the stressor than Type Bs, providing independent confirmation of the behavioral interview assessment procedure. Fitness level was determined by a graded treadmill test and was not different for Type As and Type Bs. Basal thromboxane production, measured as the primary metabolite, thromboxane B2, in blood oozing from the bleeding-time site, was highest among unfit Type A subjects. Fit Type As and Type Bs showed significantly lower thromboxane production. No significant differences in prostacyclin production were seen. This study marks the first time that behavior pattern has been linked to an adverse aspect of a thrombosis-related parameter likely to be involved in the genesis of cardiovascular disease. The present results also indicate that physical fitness may, in some fashion, ameliorate the "toxic" effects of Type A behavior.

Adolescent↗
PubMed1992

Platelet storage pool deficiency, leukemia, and myelodysplastic syndromes.

Abnormalities in platelet dense granules, small intracellular organelles containing ATP, ADP, calcium, serotonin, and pyrophosphate, have frequently been reported in patients with leukemia and myeloproliferative disorders, particularly acute and chronic myelogenous leukemia. Recent studies of a family which includes several members with an autosomal dominant dense granule deficiency condition show an association between the presence of this form of dense granule deficiency and the development of acute myelogenous leukemia. Studies in two additional patients, one with the Monosomy 7 syndrome and the second with a myelodysplastic syndrome, revealed a defect in platelet dense granules. This defect appears to be due to an abnormality in the formation of these granules rather than the presence of empty vesicular structures or decreased contents due to activation associated secretion. The results suggest that the defect in platelet dense granules associated with leukemia or myelodysplastic syndromes may result from a chromosome alteration in the megakaryocyte cell line leading to decreased formation of dense granules. Studies in the family with an inherited bleeding disorder suggest that a gene coding for a protein important for the formation of dense granules is located adjacent to a gene which, when abnormal, may predispose to the development of leukemia.

Adult↗
PubMed1992

A role for prostacyclin in bruising symptomatology.

The relationship between bleeding and bruising and the production of prostacyclin and thromboxane was assessed in children who were to have a tonsillectomy and/or an adenoidectomy. Eicosanoids in the blood oozing from the bleeding time incision were measured and correlated with the reported frequency of bruising and epistaxis. A striking association (P = .0003) between prostacyclin production and the frequency of bruising was found; children reporting bleeding at least biweekly had the highest prostacyclin synthesis. Successively lower levels of the prostacyclin metabolite, 6-keto-prostaglandin F1 alpha, were found in children reporting less frequent bruising. Prostacyclin production in bleeding time blood was also correlated inversely with systolic blood pressure and hemoglobin level, although neither of these variables could explain the association between prostacyclin production and bruising. There was no correlation between thromboxane formation, systolic blood pressure, hemoglobin level, age, or bleeding time and the frequency of bruising. The ratio of thromboxane B2 to 6-keto-prostaglandin F1 alpha was correlated inversely with the length of the bleeding time (P = .016). It is concluded that vascular prostacyclin production may have a role in bruising symptomatology. It is suggested that prostacyclin formed at the injured vessel surface collects within the first few seconds after injury inside the tissue space at the site of the bruise and, by influencing the formation of the platelet/fibrin plug and/or the leakage of blood from the vessels, plays a significant role in modifying the development of bruising.

6-Ketoprostaglandin F1 alpha↗
PubMed1991

The inhibition of platelet aggregation of metastatic H-ras-transformed 10T1/2 fibroblasts with castanospermine, an N-linked glycoprotein processing inhibitor.

A series of T24-H-ras-transformed 10T1/2 fibroblasts with varying metastatic potential was tested for the ability to aggregate platelets. Results indicate that although platelet activation was always detected in the highly metastatic cells, some non-metastatic cells also have the ability to cause platelet aggregation, suggesting that this is a necessary but not sufficient characteristic of the metastatic phenotype. Apyrase, an ADP scavenger, effectively inhibited platelet aggregation by metastatic cells, however, there was no significant increase in ADP secretion or relation to the ability of the tumor cells to activate platelets. Hirudin, a thrombin inhibitor, did not affect aggregation, suggesting that the pathway of activation is thrombin-independent. The glycoprotein processing inhibitor, castanospermine, which reduces glycosidase I activity and metastatic capability, inhibited the ability of metastatic cells to cause platelet aggregation. However, another inhibitor of oligosaccharide processing, swainsonine, which inhibits mannosidase II activity and does not reduce metastasis, had no effect on platelet aggregation. These results show that the integrity of N-linked oligosaccharide structure of glycoproteins is an important feature of the ability of ras-transformed fibroblasts to activate platelets.

Adenosine Diphosphate↗
PubMed1991

Leukophysin: a 28-kDa granule membrane protein of leukocytes.

A membrane glycoprotein of human platelet dense granules, called granulophysin, with serologic homology to synaptophysin has recently been identified. To determine if this protein was present in granulated leukocytes, we examined several cell types for the presence of the protein by indirect immunofluorescence. Antigranulophysin mAb staining was detected in a granular pattern in the cytoplasm of permeabilized IL-2-stimulated CD3+ peripheral lymphocytes, neutrophils, U937 monocytes, and mast cells. Immunohistochemistry of human lymph nodes showed cytoplasmic staining of macrophages, neutrophils, and some dendritic cells. Induction of granule exocytosis in granulated CD3+ lymphocytes after stimulation with PMA and calcium ionophore A23187 resulted in a redistribution of the reactive epitope from the cytoplasm to the plasma membrane. Subcellular fractions contained two peaks of reactivity; the first peak coincided with N-benzyloxycarbonyl-L-lysine thiobenzyl ester-esterase activity in dense granules whereas the second peak was present in lighter fractions. The affinity purified protein from both peaks was identical in Western blot analysis and had a molecular mass of 28 kDa under reducing conditions. The protein could only be solubilized in detergent suggesting that it was an integral membrane protein. We have named this protein leukophysin to differentiate it from the 40-kDa granulophysin of platelets. Monocytes contained a protein with identical m.w. to leukophysin, whereas a protein of a slightly higher m.w. was detected in neutrophils. We propose that leukophysin is a common granule membrane protein of leukocytes.

Antibodies, Monoclonal↗
PubMed1991

Blood pressure during pregnancy in Canadian Inuit: community differences related to diet.

OBJECTIVE: To assess a possible relation between the incidence of hypertension during pregnancy and the consumption of fatty acids found in fish and sea mammals. DESIGN: Retrospective survey of pregnancy-induced hypertension; prospective diet survey. SETTING: Inuit women from seven communities in the Keewatin region of the Northwest Territories. PATIENTS: All women from Arviat (formerly Eskimo Point), Baker Lake, Chesterfield Inlet, Coral Harbour, Repulse Bay, Sanikiluaq and Whale Cove who gave birth between Sept. 1, 1984, and Aug. 31, 1987. MAIN OUTCOME MEASURES: All blood pressure measurements recorded during the pregnancy, incidence of pregnancy-induced hypertension in the seven communities, harvest of country food (food obtained from the land or sea rather than bought in a store) for six of the communities, self-reported consumption of fish, sea mammals and terrestrial mammals by a subgroup of the subjects and levels of phospholipid fatty acids in cord serum samples from a subgroup of the infants. MAIN RESULTS: Significantly lower mean diastolic blood pressure values during the last 6 hours of pregnancy were noted for the women from the three communities with a higher consumption of fish and sea mammals (78.2 [95% confidence limits (CL) 76.6 and 79.9] mm Hg) than for those from the four communities with a lower consumption of such food (81.5 [95% CL 80.1 and 82.9] mm Hg) (p less than 0.005). The relation between community diet type and blood pressure was independent of other factors. Correspondingly, the women from communities with a lower consumption of marine food were 2.6 times more likely to be hypertensive during the pregnancy than those from communities with a higher consumption of marine food (p less than 0.007). Parity (p less than 0.05) and prepregnancy weight (p less than 0.005) were also significantly associated with pregnancy-induced hypertension; however, the relation between hypertension and community diet type remained significant in logistic regression analysis (odds ratio 2.56, p = 0.03). The differences between the community groups were substantiated by the results of the diet survey, the levels of eicosapentaenoic acid (EPA) in the cord serum phospholipids and the harvest data. CONCLUSIONS: Increased consumption of fish may be beneficial for women at risk for hypertension during pregnancy. A prospective randomized trial of fish or EPA supplementation during pregnancy is warranted.

Animals↗
PubMed1991

Bleeding/bruising symptomatology in children with and without bleeding disorders.

A questionnaire, designed to assess bleeding/bruising tendencies, was administered to 251 otherwise healthy children undergoing a tonsillectomy and/or adenoidectomy. 23 children with excessive bleeding during or after the operation, with a long bleeding time or who reported taking aspirin recently were excluded, to give a population of 228 non-bleeders. For comparative purposes, 31 patients with bleeding disorders (von Willebrand's disease and/or platelet function defects) were studied. A considerable proportion of "non-bleeding" children reported easy bruising (24%), had bruises at least once a week (36%) and suffered from nosebleeds (39%). The respective frequencies (67%, 68% and 69%) for children with bleeding disorders were significantly higher. Occurrence of bruises usually on more than one part of the body, frequent large bruises or hematomas were rare in "non-bleeders" (4.9%, 3.5% and 2.7% respectively), but more common in "bleeders" (38.5%, 29.6% and 21.7% respectively).

Adenoidectomy↗
PubMed1991

Synthesis of intracellular histamine in platelets is associated with activation of protein kinase C, but not with mobilization of Ca2+.

In previous reports, we have provided evidence indicating that newly formed histamine is an intracellular messenger in human platelets. The involvement of protein kinase C (PKC) and intracellular calcium (Ca2+i) in the synthesis of histamine was investigated. Human platelets were stimulated by phorbol 12-myristate 13-acetate (PMA), collagen and the Ca2+ ionophore A23187, with or without the PKC inhibitor staurosporine. Aggregation, histamine synthesis and phosphorylation of pleckstrin (47 kDa; P47) and myosin light chain (20 kDa; P20) proteins were monitored. Staurosporine inhibited PMA- and collagen-induced aggregation, histamine synthesis and phosphorylation of 47 kDa and 20 kDa proteins in a dose-dependent manner. For PMA, median inhibitory concentrations (IC50 values) for staurosporine inhibition of aggregation, histamine synthesis and phosphorylation were similar, suggesting that histamine synthesis induced by this agonist may be a consequence of PKC activation. Conversely, collagen-stimulated histamine synthesis was inhibited by staurosporine at concentrations significantly higher than those required to inhibit aggregation (P less than 0.005) or pleckstrin phosphorylation (P less than 0.01), indicating the possible involvement of non-PKC mechanism(s) in the synthesis of histamine induced by this agonist. A23187 failed to induce the synthesis of intracellular histamine in platelets, whereas staurosporine blocked A23187-induced aggregation and phosphorylation of the 20 kDa protein at significantly higher concentrations than those needed to inhibit PKC. When platelets were stimulated with a combination of A23187 and PMA, the increase in platelet histamine was less than that with PMA alone. The results provide evidence that the synthesis of intracellular histamine in platelets occurs as a consequence of PKC activation and may be down-regulated under conditions where there is a substantial rise in [Ca2+]i.

Adenosine Diphosphate↗
PubMed1991

Identification of a platelet dense granule membrane protein that is deficient in a patient with the Hermansky-Pudlak syndrome.

Monoclonal antibodies were raised after injecting mice with isolated human dense granules. Several of these monoclonals were found to recognize a 40-Kd dense granule membrane protein. Western blot and immunofluorescent analysis confirmed the dense-granule specificity. After thrombin activation, the protein was found in patches on the external platelet membrane. By Western blot and slot blot analysis, the protein was found to be markedly deficient in a patient with the Hermansky-Pudlak syndrome. Studies of neutrophils and endothelial cells show the presence of immunologically related granule-membrane protein(s). Western blots using four anti-synaptophysin antibodies and three antibodies to the platelet 40-Kd protein suggest that the protein may share some homology with, but is not identical to, the synaptosomal membrane protein synaptophysin.

Albinism, Oculocutaneous↗
PubMed1991

The interactive effects of Type A behavior and hostility on bleeding time thromboxane and prostacyclin formation.

Sixty-six male university students were classified as Type A or B on the basis of the Structured Interview of Rosenman and as hostile or non-hostile on the basis of the Cook-Medley scale. Vascular production of prostacyclin and platelet thromboxane in response to a standard vessel injury was evaluated. Basal thromboxane production, measured as the primary metabolite, thromboxane B2, in blood oozing from the bleeding-time site, was highest among hostile Type A subjects with significantly lower thromboxane production in hostile Type Bs and all non-hostile groups combined. Following an exercise treadmill test hostile subjects produced more thromboxane than non-hostile ones, and hostile Type As had significantly shorter bleeding times than hostile Type Bs. No significant differences on any measure were observed following a stressful color naming task. The observed interaction of hostility and Type A behavior on bleeding time thromboxane formation links behavior to an adverse aspect of a thrombosis-related parameter thought to be involved in the genesis of cardiovascular disease.

6-Ketoprostaglandin F1 alpha↗
PubMed1991

Enhanced electron transfer by GTP: cross-membrane electron signaling by G-proteins?

Activation of several receptor types is followed by their binding to a G-protein. Prior to transmission of the agonist signal, the G-protein which had affinity for guanosine 5-diphosphate (GDP) binds guanosine 5-triphosphate (GTP) instead. Because evidence exists that several agonist groups activate their receptors by reduction, we evaluated whether the nucleotide associated with G-proteins could enhance electron flow. Using a model system of ferrous iron and ferric cytochrome c, it was determined that substitution of GTP for GDP led to an enhanced reduction of ferric cytochrome c. These results support the concept that cellular activation by certain receptors may involve reductive activation with the participation of GTP and G-proteins. We speculate that GTP, when bound to G-protein, can facilitate electron transfer perhaps from the receptor or the G-protein to the catalytic subunit of the adenylate cyclase enzyme.

Adenylyl Cyclases↗
PubMed1991

Inherited platelet-storage pool deficiency associated with a high incidence of acute myeloid leukaemia.

A family with an inherited bleeding disorder extending over four generations, and multiple cases of myeloblastic and myelomonoblastic leukaemia was studied. Ten members of the family had, by history, a haemorrhagic diathesis. There were three documented cases of myeloblastic leukaemia, two documented cases of myelomonoblastic leukaemia and two more cases of leukaemia by history. In four of the cases the bleeding diathesis clearly antedated the leukaemia, in two by many years. The bleeding disorder is characterized by a long bleeding time, abnormal platelet aggregation, low platelet ADP and decreased numbers of platelet dense bodies consistent with a dense granule storage pool deficiency. The number of dense granules was decreased by immunofluorescence employing quinacrine or using an antibody to the dense granule membrane protein, granulophysin, confirming an absolute decrease in dense granule numbers rather than the presence of empty granule sacs. This congenital storage pool deficiency is associated with a high incidence of acute myeloid leukaemia in this family.

Acute Disease↗