Growth factors--their biochemistry and role in tumour growth.
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Growth Substances↗
Biomedical subjects
H Holmsen
Publications and source records attributed to H Holmsen.
A method for production of N2 microbubbles in platelet-rich plasma in an aggregometer-like apparatus, and effect on the platelet density in vitro.
The mechanisms involved in the interactions between microgasbubbles and platelets are not clear. The platelet aggregatory response to agonist-induced, receptor-mediated stimuli has been studied extensively. As a direct in vitro approach to elucidate the interaction between gas bubbles and platelets, N2 microbubbles were used as a platelet agonist in an experimental apparatus similar to an ordinary aggregometer. N2 microbubbles of varying number and size were produced in platelet-rich plasma (PRP) and incubated at 37 degrees C. The gas-liquid interface of the gas bubbles consists of plasma proteins and lipids. In stirred PRP a considerable decrease occurred in the platelet density, which could not be attributed to formation of citrate complexes with calcium and/or the corresponding reduction in the concentration of free calcium. Gas bubbles with a diameter in the range of 40-120 micron caused the greatest reduction in the platelet density. The platelet movements in PRP and the curvature of the N2 microbubble surface seemed more important for the interaction than the total gas bubble surface available for contact.
Analysis of Variance↗
Thin-layer chromatography of polyphosphoinositides from platelet extracts: interference by an unknown phospholipid.
Different ratios of radioactive polyphosphoinositides in platelets pulse-labelled with 32p-orthophosphate have been reported by various laboratories. We studied whether these differences originate from differences in methodology. Extracts of 32p-Pi labelled human platelets were prepared at various times after gel-filtration and phosphatidylinositol (PI)-, mono (PIP)- and bisphosphate (PIP2) were separated by thin-layer chromatography using four different solvent systems. The 32p-levels in PIP and PIP2 remained constant during one hour after gel-filtration, whereas 32p-PI increased continuously and more than doubled within the first h. In two of the systems PIP co-chromatographed with a radioactive compound which separated well from PIP in the two other systems. This unknown compound was also labelled with 3H-glycerol, 3H-inositol and 3H-arachidonic acid, but it was metabolically and functionally different from the polyphosphoinositides. Both the co-chromatography of this unknown phospholipid and the increase in 32p-PI in gel-filtered platelets can explain the difference in 32p-labelling in phosphoinositides reported in the literature.
Blood Platelets↗
Synergism between thrombin and epinephrine in human platelets: different dose-response relationships for aggregation and dense granule secretion.
Synergism between epinephrine and thrombin in human gel-filtered platelets was studied. Suspensions of platelets, which did not contain added fibrinogen, were incubated at 37 degrees C to measure aggregation and dense granule secretion after stimulation. Both aggregation and secretion induced with 0.03-0.3 U/ml of thrombin were markedly potentiated by epinephrine (0.5-4 microM), which alone was without effect. Addition of epinephrine before stimulation with thrombin resulted in a marked decrease in the concentration of thrombin required for half-maximal aggregation, without affecting the maximal aggregation response. The dose-response relationship for dense granule secretion was affected in a similar way with regard to the half-maximal response; in addition, epinephrine slightly increased maximal secretion response. However, the concentration range for thrombin at which synergism occurred was distinctly lower for aggregation than for secretion. Our observations suggest that aggregation and dense granule secretion are separate steps in the sequence of stimulus-response coupling, and suggest distinct thresholds of intracellular messengers for these different responses.
Adenosine Diphosphate↗
Energy requirements for stimulus-response coupling.
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Adenosine Triphosphate↗
Lysosomotropic agents selectively potentiate thrombin-induced acid hydrolase secretion from platelets.
Thrombin induces partial secretion (up to 60%) of beta-N-acetyl-D-hexosaminidase (EC 3.2.1.52) from untreated platelets. Preincubation of platelets with 10 mM NH4Cl for up to 2 hr resulted in a time-dependent and marked stimulation of thrombin-induced secretion of both this enzyme and other acid glycosidases from platelets. The enhancement of the thrombin-induced secretion was not due to cell lysis, and NH4Cl alone did not cause leakage of lysosomal enzymes into the medium. The effect could be reversed by reincubating the platelets in NH4Cl-free medium. Stimulation of thrombin-induced secretion also was produced by a series of aliphatic primary amines from methylamine to butylamine, and by micromolar concentrations of chloroquine. The effect of weak bases on platelets appeared to be quite specific for enhancing lysosomal enzyme secretion. Thrombin-induced secretion of adenine nucleotides from dense granules and of beta-thromboglobulin from alpha granules was slightly enhanced by NH4Cl but was slightly inhibited by methylamine. The only direct effect of the weak bases on platelets was the displacement of serotonin from dense granules. Accumulation of weak bases in acidic pools in the platelets (e.g., lysosomes) might, therefore, be responsible for the enhanced secretion of lysosomal enzymes. By using controlled digitonin-induced platelet lysis, it was found that preincubation of platelets with NH4Cl lowered the digitonin concentration required for enzyme solubilization. We suggest that loading of lysosomes with weak bases dissociates already bound enzyme inside the lysosomes, resulting in a more effective discharge upon stimulation by thrombin.
Acid Phosphatase↗
Bleeding disorder associated with albumin-dependent partial deficiency in platelet thromboxane production. Effect of albumin on arachidonate metabolism in platelets.
The authors describe a patient with a longstanding bleeding disorder associated with impaired platelet aggregation and secretion despite normal granule contents. Thrombin-induced platelet thromboxane A2 production, measured using a radioimmunoassay for thromboxane B2, was markedly decreased or undetectable in platelet-rich plasma and whole blood serum. However, significant amounts of thromboxane B2 were detected on thrombin stimulation of platelets suspended in albumin-free salt medium. Malondialdehyde and 14C-hydroxyheptadecatrienoic acid production was undetectable in the patient's platelets. Liberation of free 14C-arachidonic acid from phospholipids during stimulation of prelabeled platelets was normal, indicating normal phospholipase activity. These observations indicate an albumin-dependent partial deficiency in thromboxane production resulting from a defect either in cyclooxygenase or thromboxane synthetase. Further, the authors studied the effect of albumin on arachidonic acid metabolism in normal platelets. These studies indicate that albumin enhances liberation of arachidonic acid from phospholipids but has an overall inhibitory effect on thromboxane synthesis.
Adult↗
The storage pool deficiency in platelets from humans with the Chédiak-Higashi syndrome: study of six patients.
Functional and biochemical studies of platelets from human Chédiak-Higashi syndrome (CHS) are scarce and/or incomplete. In the present report, the aggregation response to a variety of inducers of platelet aggregation, the content of the dense granule constituents ATP, ADP, serotonin and calcium, the secretion of ATP, ADP, and calcium induced by thrombin, the total content of magnesium, the incorporation of 14C-adenine in the cytoplasmic pool of adenine nucleotides, as well as the content of intracellular cyclic-AMP, have been quantitated in six patients with CHS. Furthermore, data is presented on the kinetics of uptake of radiolabelled serotonin and its storage in human CHS platelets. An abnormal aggregation behaviour was found in all patients. However, the response of CHS platelets to the different inducers studied did not show a uniform pattern. The total content and the maximal amounts of the dense granule constituents secretable by thrombin were greatly decreased in all six patients. Total magnesium content was similar to that of normal platelets. The ATP/ADP ratio was higher than in controls. Uptake of radiolabelled serotonin by CHS platelets closely followed the uptake by normal platelets; during the first 2-3 min, however, incorporation of the amine by CHS platelets came rapidly to a plateau which contrasts with the steady, linear increase in uptake found in controls. CHS platelets loaded with radiolabelled serotonin and gel-filtered, showed a spontaneous release of radioactivity not observed in normal platelets under the same conditions. The cyclic-AMP content of CHS platelets was similar to that of normals. In contrast to platelets from patients with storage pool disease, the secretable calcium from CHS platelets represents a 67% of total platelet calcium (61% in normals), suggesting that the absolute values for the non-secretable portion in CHS platelets must be very low. The results reported confirm the existence of a true storage pool deficiency of the dense granule constituents as a common defect in CHS platelets. The variety of responses among patients, to the different aggregatory stimuli studied, can not be solely ascribed to the storage pool deficiency described.
Adenine Nucleotides↗
Nucleotide metabolism of platelets.
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Adenine↗
Platelet metabolism and activation.
Stimulation and execution of platelet responses are intimately coupled to the cells' energy metabolism. The turnover of cytoplasmic ATP is higher than in most other cells; when it is lowered, platelet responses are powerfully inhibited. One-third of the total adenine nucleotides are present in the cytoplasm and a small fraction of these, 50% of the ADP, is bound to actin and not available to metabolism. These nucleotides are not synthesized de novo in platelets which readily form them from adenine, adenosine, and hypoxanthine. The remaining two-thirds are sequestered in the dense granules together with serotonin and divalent metal ions. NMR studies show that this ATP and ADP are stacked in large aggregates together with the divalent cations. Serotonin is incorporated into the aggregates by being positioned between, and in close proximity to, adjacent adenine rings. In the congenital platelet disorder, "Storage Pool Deficiency," the dense granule content is absent or markedly reduced, which may be caused by an impaired transport of ATP across the dense granule membrane. The content of the dense granules, the beta-thromboglobulin, albumin, fibrinogen, certain coagulation factors, fibronectin of the alpha-granules, and the acid hydrolases of the lysosomes are discharged by exocytosis, but the exact mechanisms are not known. This discharge, platelet secretion, is complete for the dense granule and alpha-granules contents, but is only 30% to 55% for the acid hydrolases. After pretreatment of platelets with certain amines, thrombin causes secretion of all beta-hexosaminidase, indicating that the beta-hexosaminidase usually retained during secretion is bound within the lysosomes at the acid pH but becomes dissociated by raising the pH with the amines. Agonist-induced platelet aggregation and secretion are accompanied by rapid changes in phosphoinositide metabolism which are usually monitored as the radioactivity in phospholipids of platelets preincubated with radioactive arachidonate, glycerol, inositol, or orthophosphate. The mechanisms for precursor incorporation are not well known and interpretations of published results are hampered by much uncertainty. Experimental support for the widely accepted coupling of receptor occupancy to the phosphodiesteratic hydrolysis of phosphatidylinositol or phosphatidylinositol-4,5-biphosphate is nonexistent. These two hydrolytic steps occur concomitantly and together with formation of diglyceride and phosphatidate; all four steps cannot be temporally resolved.(ABSTRACT TRUNCATED AT 400 WORDS)
Actins↗
Inhibition of ADP-induced responses in human platelets by agents elevating the cyclic AMP level: comparison of aggregation and shape change.
The inhibitory effect of cAMP-elevating agents on shape change and aggregation in human platelets was studied to improve the understanding of the sequential relationship between these two responses. Human platelet-rich plasma was preincubated for 2 min at 37 degrees C with prostaglandin E1 or adenosine, agents known to elevate the intracellular level of cAMP. Their inhibitory effects on ADP-induced shape change and aggregation were determined both separately and simultaneously. The dose-inhibition patterns for shape change and aggregation were similar for both PGE1 and adenosine. There was no distinct difference between the inhibitory action of these two inhibitors. These observations suggest that elevation of the intracellular concentration of cAMP interferes with an early step in the stimulus-response coupling that is common for aggregation and shape change.
Adenosine↗
High- and low-uptake forms of beta-hexosaminidase in human platelets. Selective retention of the high-uptake form during stimulation with thrombin.
Human platelets are rich in beta-hexosaminidase and other acid hydrolases contained in organelles (lysosomes) distinct from alpha-granules and dense granules. Incubation of platelets with bovine or human thrombin (100 U/ml for 5 min at 37 degrees C) induces the secretion of 100% of the contents of alpha- and dense granules, but only 40-60% of total beta-hexosaminidase from lysosomes. Both isozymes Hex A and Hex B are secreted in the same proportion as found intracellularly. There is no selective recapture or plasma membrane binding by platelets of secreted beta-hexosaminidase. The secreted enzyme is of the low-uptake type, i.e., it is poorly recognized by the phosphomannosyl receptor-mediated uptake mechanism of fibroblasts, while the retained enzyme is a 3-fold higher uptake form. Preincubation of platelets with NH4Cl (10 mM, 2 h), followed by thrombin stimulation, results in secretion of all beta-hexosaminidase as a low-uptake form. The data support the hypothesis that there are secretory and nonsecretory forms of lysosomes. The secretory lysosomes would contain low-uptake forms of hydrolases in addition to acid phosphatase, while the nonsecretory lysosomes would contain high-uptake hydrolases and be acid phosphatase-deficient. Conditions where the contents of both lysosomal populations were released together, i.e., amine treatment followed by thrombin induction, or extraction of unstimulated cells, would result in the exposure of high-uptake phosphomannosylated hydrolases released from one population of lysosomes to acid phosphatase released from the second population of lysosomes with their subsequent conversion to low-uptake forms.
Blood Platelets↗
Histamine uptake in pig platelets and isolated dense granules.
Histamine, a major constituent of the amine-storage organelles in pig platelets, is taken up by intact platelets in only trace amounts under conditions where 70% of 14C-serotonin is accumulated. Thrombin caused the release of 70-90% of endogenous histamine but only 5-10% of the newly absorbed 3H-amine; however, after 18 hr 30% of the 3H-amine could be specifically released by thrombin. Isolated storage organelles accumulated histamine in a reserpine-sensitive, ATP-dependent manner but at a rate 80-100-fold less than serotonin uptake. Incubation of intact platelets with 1 mM serotonin until amine uptake was saturated caused no changes in platelet histamine content. Similarly, loading of isolated storage organelles with 1 mM histamine or 1 mM serotonin did not affect the levels of the other amine. These results suggested that the storage of each amine is independent of the other. Histidine decarboxylase was not detected in platelet lysates. Since platelets have a short half-life (1-2 weeks) and pig plasma levels of histamine are higher than in other animals, it is concluded that most of the histamine in the storage organelles is probably accumulated in the platelet precursor, the megakaryocyte, either by slow uptake or by synthesis.
Animals↗
Differential effects of trifluoperazine on arachidonate liberation, secretion and myosin phosphorylation in intact platelets.
Gel-filtered platelets prelabeled with [3H]-arachidonate and [14C]-adenine or [32P]-orthophosphate were stimulated with thrombin in the presence of various concentrations of trifluoperazine (TFP). Based on the presence of [14C]- or [32P]-labeled extracellular adenine nucleotides, TFP, above 50 microM, caused platelet lysis which reached 30-40% at 100 microM. In the non-lytic range (0-50 microM) TFP caused marked inhibition of [3H]-arachidonic acid liberation and [3H]-phosphatidylcholine breakdown which was complete at 25 microM. Breakdown of [3H]-phosphatidylinositol was partially (about 50%) inhibited at 25 microM TFP and little further inhibition occurred above this concentration. These results show that thrombin-induced liberation of [3H]-arachidonic acid occurs entirely by a TFP-sensitive mechanism, and suggest that the major portion of the arachidonate is liberated from phosphatidylcholine with a possible contribution from phosphatidylinositol. Dense granule secretion and acid hydrolase secretion were progressively inhibited by TFP, while the thiazine had only a small effect on phosphorylation of myosin. These results indicate that the inhibition of the secretory processes by TFP is not caused by action of TFP on myosin light chain kinase. It is suggested that the profound effect of TFP on arachidonic acid liberation but not myosin phosphorylation is due to different subcellular localization of these calmodulin-requiring enzymes: phospholipase A2 and myosin light chain kinase. The lipophilic TFP dissolves preferentially in the membranes where it has access to phospholipase A2 but not to myosin light chain kinase.
Adenosine Diphosphate↗
Alterations in 32P-labelled intermediates during flux activation of human platelet glycolysis.
Using a newly developed isotopic tracer technique for the measurement of 32P-labelled intermediates in glycolysis and nucleotide metabolism in platelets, we studied the variations in 32P-labelled intermediates during activation of the glycolytic flux by cyanide and platelet-activating agents. The major variations occurred in [32P]Fru-1,6-P2, dihydroxy acetone phosphate, ATP and Pi. There was a quantitative covariance between the increase in lactate production and the rise in [32P]Fru-1,6-P2 induced by different platelet-activating agents. In contrast, cyanide induced weaker activation of the flux and greater accumulation of [32P]Fru-1,6-P2. Variations in 32P-labelled intermediates were apparent 5 s after flux activation, but the major changes in [32P]Fru-1,6-P2 occurred much later and fell in periods in which a constant lactate formation was maintained. The cyanide-induced changes in 32P-labelled intermediates depended on the extracellular level of glucose, showing a predominant ATP----Pi conversion in glucose-depleted medium that shifted to an ATP----Fru-1,6-P2 conversion at excess glucose. At about 50 microM glucose, flux activation occurred without major changes in [32P]Fru-1,6-P2, dihydroxy acetone phosphate and Pi, with only a small fall in [32P]ATP. The data provide evidence for a role of the aldolase reaction in flux control and demonstrate rapid changes in Fru-1,6-P2 and ATP during flux activation with an additional role for Fru-1,6-P2 as an energy buffer during post-activation periods.
Adenosine Diphosphate↗
Tight coupling of thrombin-induced acid hydrolase secretion and phosphatidate synthesis to receptor occupancy in human platelets.
Human platelets incubated with [32P]Pi and [3H]arachidonate were transferred to a Pi-free Tyrode's solution by gel filtration. The labile phosphoryl groups of ATP and ADP as well as Pi in the metabolic pool of these platelets had equal specific radioactivity which was identical to that of[32P]phosphatidate formed during treatment of the cells with thrombin for 5 min. Therefore, the 32P radioactivity of phosphatidate was a true, relative measure for its mass. The thrombin-induced formation of[32P]-phosphatidate had the same time course and dose-response relationships as the concurrent secretion of acid hydrolases. 125I-alpha-Thrombin bound maximally to the platelets within 13s and was rapidly dissociated from the cells by hirudin; readdition of excess 125I-alpha-thrombin caused rapid rebinding of radioligand. This binding-dissociation-rebinding sequence was paralleled by a concerted start-stop-restart of phosphatidate formation and acid hydrolase secretion. [3H]Phosphatidylinositol disappearance was initiated upon binding but little affected by thrombin dissociation and rebinding. ATP deprivation caused similar changes in the time courses for [32P]-phosphatidate formation and acid hydrolase secretion which were different from those of [3H]phosphatidylinositol disappearance. The metabolic stress did not alter the magnitude (15%) of the initial decrease in phosphatidylinositol-4,5-bis[32P]phosphate, but did abolish the subsequent increase of phosphatidylinositol-4,5-bis[32P]-phosphate in the thrombin-treated platelets. It is concluded that in thrombin-treated platelets (1) phosphatidate synthesis, but not phosphatidylinositol disappearance, is tightly coupled to receptor occupancy and acid hydrolase secretion in platelets, (2) successive phosphorylations to phosphatidylinositol-4,5-bisphosphate is unlikely to be the main mechanism for phosphatidylinositol disappearance, and (3) only a small fraction (15%) of phosphatidylinositol-4,5-bisphosphate is susceptible to hydrolysis.
Adenosine Diphosphate↗
Cytoplasmic free calcium concentration in porcine platelets. Regulation by an intracellular nonmitochondrial calcium pump and increase after thrombin stimulation.
Mechanisms are assumed to exist in the resting platelet which maintain the concentration of cytoplasmic free calcium below that level required to activate cellular responses. To assess such processes the porcine platelet plasma membrane was selectively lysed with digitonin and the uptake (or flux) of free calcium monitored by an extracellular calcium electrode. Lysis resulted in an immediate lowering of the extracellular free calcium, due to the action of intracellular organelle(s) acting on the extracellular space through the permeabilized plasma membrane. In resting platelets, the rate of calcium uptake was first order with respect to the extracellular prelytic calcium concentration, and hence the cytoplasmic free concentration was found to be 1 X 10(-7) M by extrapolation to a point of zero flux (i.e., the null point). This approach could not be used with thrombin-stimulated platelets, as external calcium was required for both secretion of ATP + ADP and aggregation. Nevertheless, evidence for an increase in cytoplasmic free calcium after thrombin stimulation was obtained. Metabolic inhibitors and agents known to inhibit calcium uptake by mitochondria had no effect on the calcium flux following lysis, indicating different mechanisms for calcium homeostasis in the platelet when compared with other cell types (e.g., liver). Levels of ionophore A23187, which caused platelet aggregation, gave a massive release of the nonmitochondrial pool of calcium into the cytoplasmic space. Thus, in porcine platelets an intracellular energy-requiring calcium pump, which sequesters calcium in a nonmitochondrial membranous compartment, is crucial for intracellular calcium homeostasis.
Animals↗
31P NMR studies of nucleotide storage in the dense granules of pig platelets.
Detailed 31P NMR measurements have been conducted on pig platelet dense granules and aqueous mixtures of ATP, ADP, MgCl2, and 5HT. The resonance line widths of the dense granule nucleotides were temperature independent above approximately 30 degrees C; below this temperature they exhibit a strong temperature dependence, becoming undetectably broad at approximately 5 degrees C. The temperature of transition is determined by the effective solute concentrations within the granules. Spin-spin relaxation time (T2) and line-width measurements indicate that the 31P resonances of dense granule nucleotides are not homogeneously broadened at 35 and 21 degrees C. However, the temperature-dependent changes in the intrinsic widths calculated from T2 values parallel the changes in the measured line widths. From the T1 and the T2 data, a rotational correlation time of 13 ns is calculated for the dense granule nucleotides at 35 degrees C. Removal of 5HT and HA from the dense granules induce significant but relatively small changes in the temperature dependence of the resonance line widths. Analogous effects are seen with a gel phase separated from aqueous mixtures of ATP, ADP, and MgCl2 in the presence or absence of 5HT. These results demonstrate that interactions involving the nucleotides and the divalent cations are predominant in determining the physicochemical state of the granule contents; in pig platelet dense granules the nucleotides and Mg2+ form a relatively fluid aggregate which serves as a matrix for 5HT and possibly HA binding. Incorporation of the amines into this matrix tends to increase its fluidity.
Adenosine Diphosphate↗