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Biomedical subjects

H Holmsen

Publications and source records attributed to H Holmsen.

At least 91 records · Page 5Linked to original sources
PubMed1987

Requirement for thrombin receptor occupancy during platelet secretion under aggregating and non-aggregating conditions.

The requirement for receptor occupancy in thrombin-induced secretion in human platelets has been studied. When increasing concentrations of thrombin were added to gel-filtered platelets containing a constant, high concentration of hirudin, dense granule secretion was initiated at lower thrombin concentrations than those required for alpha-granule secretion and aggregation; acid hydrolase secretion required higher concentrations. A 62-fold excess of hirudin produced abrupt stop of dense granule secretion and alpha-granule secretion when added to non-aggregating (no stirring) platelets shortly after thrombin; it had no affect after these secretory process had reached about 30% of their maximal values. Acid hydrolase secretion was, however, abruptly stopped by hirudin at any stage. When the platelets were allowed to aggregate, the three secretory processes increased their rates and were abruptly stopped by hirudin at any stage. Aggregation (optical) occurred slower than dense granule and alpha-granule secretion, and was reversed by hirudin when added before it had reached 30% of its maximum. It is concluded that alpha-granule secretion, like dense granule secretion, only requires a short receptor occupancy to be completed, in contrast to the requirement for sustained occupancy for acid hydrolase secretion. alpha-Granule secretion might, however, require longer occupancy than dense granule secretion. Aggregation is believed to potentiate secretion through close cell contact and the secretion processes were inhibited by hirudin through hirudin's effect on aggregation.

Adenosine Diphosphate↗
PubMed1987

Studies on the preferential incorporation of [3H]glycerol over [32P]phosphate into major phospholipids of human platelets.

It is well known that platelets readily incorporate radioactive glycerol, but not radioactive phosphate into phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in vitro, thus not in accordance with de novo synthesis according to the Kennedy pathway. In attempts to understand the reason for the discrepancy, gel-filtered platelets were incubated simultaneously with [32P]Pi and [3H]glycerol, and the specific and relative radioactivities of products and intermediates were determined. Both precursors were incorporated into phosphatidylinositol (PI) with a 32P/3H ratio similar to that in glycerol 3-phosphate (in accordance with the Kennedy pathway). However, PC and PE obtained a much lower ratio. The specific 32P radioactivity in phosphorylcholine was similar to that of the gamma-phosphoryl of ATP and 650-times higher than that of PC. The specific 32P radioactivity of phosphorylethanolamine was 20-times less than that of phosphorylcholine. Both mass and 32P labelling of CDP-choline were below the detection limits. It is concluded that the incorporation of [32P]Pi into PC via phosphorylcholine is insignificant while the preferential incorporation of [3H]glycerol could be explained by exchange of diacyl[3H]glycerol in the reversible choline phosphotransferase (CDP-choline: 1,2-diacylglycerol cholinephosphotransferase) reaction. The same mechanism would explain the preferential incorporation of 3H over 32P into PE, although dilution of 32P at the phosphorylethanolamine stage would account for part of the feeble 32P incorporation. Although other mechanisms are also possible, our results clearly show that the appearance of [3H]glycerol in PC and PE is not a reliable method of monitoring de novo synthesis of these phospholipids.

Blood Platelets↗
PubMed1987

Turnover of the phosphomonoester groups of polyphosphoinositol lipids in unstimulated human platelets.

The metabolic activity of the polyphosphoinositol lipids in unstimulated human platelets was studied by short-term labelling with [32P]Pi, by replacement of [32P]Pi from pre-labelled platelets with unlabelled phosphate, and by depriving the cells of metabolic ATP. Under short-term labelling conditions, the 4- and 5-phosphate groups of phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] had the same specific 32P radioactivity as the gamma-phosphate of metabolic ATP. The specific 32P radioactivity of the 1-phosphates of phosphatidylinositol, PtdIns4P and PtdIns(4,5)P2 was similar, but only 4-13% compared to that of the ATP-gamma-phosphate. When [32P]Pi pre-labelled platelets were incubated with up to 25 mM of unlabelled phosphate, the displacement of the 32P label from PtdIns4P, PtdIns(4,5)P2 and metabolic ATP followed similar kinetics. Inhibition of ATP regeneration in platelets pre-labelled with [32P]Pi resulted in a rapid fall in metabolic ATP with a much slower fall in [32P]PtdIns(4,5)P2, whereas [32P]PtdIns4P increased initially. However, ATP turnover was not abolished, as indicated by the marked (25% of the control) incorporation of extracellular [32P]Pi into PtdIns4P and PtdIns(4,5)P2 in metabolically inhibited platelets. This low phosphate turnover may explain the relative resistance of PtdIns4P and PtdIns(4,5)P2 to metabolic inhibition. We conclude that PtdIns4P and PtdIns(4,5)P2 are present as a single metabolic pool in human platelets. Turnover of the 4- and 5-phosphates of PtdIns4P and PtdIns(4,5)P2 in unstimulated platelets is as rapid as that of the gamma-phosphate of metabolic ATP, and accounts for about 7% of basal ATP consumption.

Adenosine Diphosphate↗
PubMed1987

Phosphoinositide metabolism in resting and thrombin-stimulated human platelets. Evidence against metabolic heterogeneity.

The specific radioactivity of the phosphodiester and phosphomonoester moieties of the phosphoinositides was determined in resting and thrombin-stimulated platelets that were prelabelled with [32P] Pi. In the unstimulated cells, the specific radioactivity of the monoester phosphates of PIP and PIP2 was similar. Both prolonged incubation at 37 degrees C and upon stimulation with 0.5 U/ml of thrombin, the specific radioactivity of the monoester phosphates decreased 10-15% in both polyphosphoinositides. In the unstimulated cells, the specific radioactivity of the diester phosphates was similar in PI, PIP and PIP2 but amounted only to 3% of the activity of the monoester groups. Prolonged incubation of the unstimulated cells as well as stimulation with thrombin induced a similar 5-6 fold increase in specific radioactivity of the diester phosphate of PI, PIP and PIP2. The results indicate that the phosphoinositides in both resting and thrombin-stimulated platelets exist in a metabolically homogeneous pool.

Adenosine Triphosphate↗
PubMed1987

Stimulation of phosphate uptake in human platelets by thrombin and collagen. Changes in specific 32P labeling of metabolic ATP and polyphosphoinositides.

The uptake of [32P]phosphate by human, gel-filtered blood platelets and its incorporation into cytoplasmic ATP and polyphosphoinositides was studied. In unstimulated platelets, uptake was Na+o-dependent and saturable at approximately 20 nmol/min/10(11) cells with a half-maximal rate at 0.5 mM extracellular phosphate. Upon stimulation with thrombin or collagen, net influx of [32P]Pi was accelerated 5- to 10-fold. With thrombin, [32P]Pi efflux was also increased. After the first 2 min, efflux exceeded influx, resulting in the net release of [32P]Pi from the platelets. Since the stimulus-induced burst in [32P]Pi uptake paralleled the secretory responses, it might be an integral part of stimulus-response coupling in platelets. The stimulus-induced burst in net [32P]Pi uptake led to an enhanced labeling of metabolic ATP, which was already detectable at 5 s after stimulation with thrombin. Concomitantly, the incorporation of [32P]Pi into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was accelerated. The thrombin-induced increase in specific 32P radioactivity of cytoplasmic ATP fully accounted for the simultaneous increase in specific 32P radioactivity of these phosphoinositides. In studying the extent of 32P labeling of phosphorylated compounds in response to a cellular stimulus, it is therefore essential to measure the effect of the stimulus on the specific radioactivity of cytoplasmic ATP.

Adenosine Triphosphate↗
PubMed1987

Neomycin inhibits agonist-stimulated polyphosphoinositide metabolism and responses in human platelets.

The effect of neomycin on agonist-induced changes in polyphosphoinositide metabolism was studied in human platelets. Neomycin induced no changes in the 32P-labeled phospholipids of unstimulated cells. Between 1 and 5 mM of neomycin the initial thrombin-induced decrease in [32P]phosphatidylinositol-4,5-bis-phosphate and production of [32P]phosphatidic acid were gradually inhibited. In contrast, the production of [32P]phosphatidylinositol-4-phosphate was increased. The thrombin-induced decrease in mass of phosphatidylinositol was completely inhibited at 5 mM of neomycin. Collagen- and PAF acether-induced changes in platelet phosphoinositide metabolism as well as aggregation and dense granule secretion induced by all three agonists were inhibited by neomycin. The results indicate that neomycin inhibit platelet responses by selective interference with the interconversions and hydrolysis of polyphosphoinositides upon thrombin stimulation.

Blood Platelets↗
PubMed1987

Evidence that neomycin inhibits binding of herpes simplex virus type 1 to the cellular receptor.

The effect of neomycin, a phosphoinositide-binding aminoglycoside, on herpes simplex virus type 1 (HSV-1) infection of BHK cells was studied. We showed earlier that it specifically inhibits HSV-1 production but not HSV-2 production (Langeland et al., Biochem Biophys. Res. Commun. 141:198-203, 1986). We now show that neomycin had no effect on cellular protein synthesis, as judged by the appearance of 35S-labeled polypeptides separated by polyacrylamide gel electrophoresis. Virus-induced polypeptides, however, were strongly inhibited at neomycin concentrations above 2 mM. Comparison among different aminoglycosides showed a variation in inhibition of HSV-1 production that paralleled the cationic charge of the aminoglycosides. HSV-1 receptor binding at 4 degrees C was completely inhibited by neomycin. At 37 degrees C both receptor binding and internalization, as measured by an indirect assay, appeared to be inhibited by more than 90%. The effect of neomycin on the infection was almost immediate upon the addition of the drug and preceded virus internalization. Possible mechanisms of the neomycin effect are discussed.

Animals↗
PubMed1987

Familial bleeding disorder associated with deficiencies in platelet signal processing and glycoproteins.

Aggregation responses to low concentrations of ADP, epinephrine, collagen and cationophore A23187 in platelets from two family members with marked bleeding tendencies were virtually absent, whereas shape change with ADP was normal. The contribution to factor X activation by collagen-treated platelets was markedly decreased. Glycoproteins IIb and III were also significantly reduced. The patients' platelets had normal stores of secretable constituents, but secretion of adenine nucleotides and acid hydrolases in response to low concentrations of thrombin and A23187 was drastically reduced compared to normal platelets; secretion of platelet factor 4 was normal. Agonist concentrations that normally produce maximal responses induced only partial aggregation and secretion in the patients' platelets. After prelabelling with [3H]arachidonate thrombin caused less changes in the [3H]phosphatidylinositol, [3H]phosphatidylcholine and free [3H]arachidonate in platelets from the patients than in platelets from normals. We conclude that the patients' platelets have an impairment in part of the signal processing mechanism that is common for all agonists and responses. This platelet abnormality, which has a superficial resemblance to thrombasthenia, represents a hitherto undescribed qualitative platelet disorder.

Adenosine Diphosphate↗
PubMed1987

Transmission and scanning electron microscopy of N2 microbubble-activated human platelets in vitro.

When N2 microbubbles are stirred in platelet-rich plasma, they cause a fall in the number of free platelets. Changes in the platelets ultrastructure during this interaction between gas bubbles and platelets have been studied by transmission and scanning electron microscopy. Platelets, and aggregates of platelets, adhere to the surface of the N2 microbubbles. This adhesion induces ultrastructural changes (shape change, pseudopod formation, granule centralization, fusion, and disappearance) that are similar to "classical" agonists like ADP, collagen, and thrombin. These ultrastructural studies further strengthen our previous contention that N2 microbubbles activate platelets in a way similar to these physiologic agonists, and show that the previously reported fall in the number of free platelets is due to platelet aggregation. Platelet aggregates are also present in the interstices between gas bubbles. Fixed N2 microbubbles have been demonstrated, and these can be broken like cracked eggs by means of the electron beam in the electron microscope. Possible mechanisms for activation of the platelets by the gas bubbles are: through ADP released from some few platelets; by diffusion of gas after bubble-platelet interaction; and through certain fractions of the plasma proteins and lipids in the bubble surface that may act as binding sites for the platelets and promote adhesion and spreading of the platelets over the surface.

Blood Platelets↗
PubMed1986

Phosphate turnover of phosphatidylinositol in resting and thrombin-stimulated platelets.

Human platelets were pulse-labelled with [32P]Pi and extracts were analyzed for masses and radioactivities of ATP and phosphoinositides. Immediately after pulse-labelling, the specific 32P radioactivity of phosphatidylinositol (PI) was only 3.4% of that of the gamma-phosphoryl of ATP. Upon incubation of the platelets at 37 degrees C, the specific 32P radioactivity of ATP (beta- and gamma-phosphoryls) remained constant. However, specific 32P radioactivity in PI increased continuously to 17% of specific [gamma-32P]ATP at 90 min of incubation. Stimulation with 0.5 U/ml of thrombin induced a 35% decrease in mass of PI which was unaffected by the time after the pulse-labelling. In contrast, the thrombin-induced changes in [32P]PI differed markedly at the various times after the [32P]Pi-pulse. Immediately after pulse-labelling, [32P]PI initially decreased but increased thereafter to 260% of control values after 180 s. With increasing specific 32P-radioactivity in PI before stimulation, the thrombin-induced increase in [32P]PI gradually disappeared. After 90 min of incubation, thrombin induced a continuous decrease in [32P]PI that almost parallelled mass. The data are explained by an initial breakdown of PI to diacylglycerol through the PI cycle or the polyphosphoinositide cycle, followed by resynthesis of PI through phosphatidic acid. In contrast to pre-existing PI, the resynthesized PI is in full isotopic equilibrium with ATP. This allowed us to estimate that 14% of the PI that is consumed between 30 and 180 s of stimulation, is recycles. From our data we calculate that the rate of PI resynthesis increased from 2.4 to 20 nmol/min per 10(11) cells upon thrombin stimulation of platelets.

Blood Platelets↗
PubMed1986

Evidence that neomycin inhibits HSV 1 infection of BHK cells.

The effect of neomycin on the Herpes Simplex Virus (HSV) type 1 and 2 infection of baby hamster kidney cells was studied. Neomycin concentrations of 3 mM or more caused a more than 90% inhibition of HSV 1 proliferation, while it had no effect on HSV 2 proliferation, measured as plaque-forming units. Furthermore, neomycin must be present at the time of infection in order to exert full effect, addition 1 hour postinfection was comparable to untreated cells. This indicates that neomycin may specifically interfere with very early stages of HSV 1 infection.

Animals↗
PubMed1986

Differential effects of chlorpromazine on secretion, protein phosphorylation and phosphoinositide metabolism in stimulated platelets.

Increasing concentrations of chlorpromazine (30-500 microM) caused a progressive lysis of gel-filtered platelets, as monitored by the extracellular appearance of cytoplasmic ([14C]adenine-labelled) adenine nucleotides. The chlorpromazine-induced lysis was markedly enhanced by thrombin and phorbol ester, and complete cytolysis was found at chlorpromazine concentrations of 100 microM and above in the presence of thrombin. At non-lytic concentrations, chlorpromazine caused a dramatic increase in the thrombin- or phorbol ester-mediated incorporation of 32P into phosphatidylinositol 4-phosphate and, to a lesser extent, into phosphatidylinositol 4,5-bisphosphate in platelets pulse-labelled with [32P]Pi. Chlorpromazine alone also caused an incorporation of 32P into the phosphoinositides. Non-lytic concentrations of chlorpromazine had no effect on the phosphorylation of the 47 kDa protein (regarded as the substrate for protein kinase C), but markedly inhibited the accompanying secretion of ATP + ADP and beta-hexosaminidase when platelets were incubated with 0.17 microM-phorbol ester or 0.1-0.2 unit of thrombin/ml. At lower concentrations of thrombin, chlorpromazine did not inhibit, but slightly enhanced, secretion. A protein of 82 kDa was phosphorylated during the interaction of platelets with thrombin and phorbol ester, and this phosphorylation was enhanced by chlorpromazine (non-lytic). These results suggest that the previously reported inhibition of protein kinase C by chlorpromazine is probably non-specific and due to cytolysis. However, since non-lytic concentrations of chlorpromazine inhibit secretion, but not protein kinase C, in platelets, activation of protein kinase C is not involved in the stimulation-secretion coupling, or chlorpromazine acts at a step after kinase activation. Possible mechanisms of this inhibition by chlorpromazine are discussed in the light of its effect on phosphoinositide metabolism and protein phosphorylation.

Blood Platelets↗
PubMed1986

Polyphosphoinositide metabolism in baby-hamster kidney cells infected with herpes simplex virus type 1.

The incorporation of [32P]Pi and [3H]inositol into the inositol lipids of baby-hamster kidney cells was studied in herpes-simplex-virus-type-1(HSV-1)-infected and mock-infected cells. The infection was conducted during incorporation of, as well as after prelabelling with, the precursors. These methods were used in order to study both synthesis de novo of, and steady-state changes in, the phosphoinositides. Both with infection during labelling, and after prelabelling, we found increased [32P]- and [3H]-phosphatidylinositol 4,5-bisphosphate (PIP2) and decreased [32P]- and [3H]-phosphatidylinositol 4-monophosphate in infected as compared with mock-infected cells, whereas no effect was observed on phosphatidylinositol. This altered inositol-lipid metabolism was (at least in the case of PIP2) not present until 3-6 h after infection and remained stable, or increased slightly, throughout the infection period. Polyphosphoinositide metabolism constitutes an important step in signal processing in many forms of cellular stimulation, and the results obtained suggest that HSV-1 infection may induce such events in our cell system.

Animals↗
PubMed1986

Nucleotide exchange between cytosolic ATP and F-actin-bound ADP may be a major energy-utilizing process in unstimulated platelets.

About 40% of the cytosolic ADP of human platelets is tightly bound to protein and the complex is precipitated from the cells by 43% ethanol. We show here that this ADP is bound to F-actin by three criteria (a) copurification with F-actin, (b) specific extraction with water and (c) by specific interaction with DNase I. Platelets contain 0.3 mumol/10(11) cells of this F-actin--ADP complex compared to the total actin content of 0.8 mumol/10(11) cells, which is consistent with the view that actin is maintained in different pools (F-actin--ADP, profilactin, G-actin). In intact platelets the F-actin-bound ADP turns over rapidly and we have determined a turnover rate at 37 degrees C of 0.1 +/- 0.025 s-1 by using a double-labelling procedure. This rapid turnover indicates that F-actin in intact platelets is in a very dynamic state, which may be necessary for rapid responses to stimuli. If it is assumed that the source of the ADP bound to F-actin is cytosolic ATP, the turnover of F-actin ADP measured represents an ATP-consuming process that would account for up to 50% of total ATP consumption in resting platelets.

Actins↗
PubMed1986

Effects of platelet antagonists on the reduction in platelet density caused by microbubbles in vitro.

Platelet-rich plasma (PRP) was stirred and incubated at 37 degrees C with N2 microbubbles in vitro in the presence and absence of platelet antagonists. The N2 microbubbles acted as a platelet agonist, like "classical" agonists such as ADP, collagen, and thrombin, causing an agonist-induced aggregation requiring extracellular Ca2+. This aggregation is abolished by 2-ethylamineglycolether (N',N',N',N') tetraacetate (chelator of extracellular Ca2+), and 2-deoxy-D-glucose plus antimycin A (inhibitors of glycolysis and electron transport, and thereby of ATP production). Furthermore, this aggregation could be depressed pharmacologically by several antagonists of the aggregation induced by "classical" platelet agonists. The greatest inhibition of microbubble-induced aggregation was obtained by substances that increase the intracellular levels of cyclic AMP. Medications that are in common clinical use, such as theophylline, seem promising in this respect. The prostaglandin-thromboxane pathway did not seem to be involved in the N2 microbubble-induced platelet aggregation in vitro, since acetylsalicylic acid and indomethacin were without effect, nor did the 3',5'-cyclic guanosine monophosphate pathway seem to be linked to this aggregation mechanism.

3',5'-Cyclic-AMP Phosphodiesterases↗