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J W Akkerman

Publications and source records attributed to J W Akkerman.

At least 73 records · Page 4Linked to original sources
PubMed1990

Na+/H+ exchange modulates Ca2+ mobilization in human platelets stimulated by ADP and the thromboxane mimetic U 46619.

According to recent observations ADP stimulates platelets via activation of Na+/H+ exchange which increases cytosolic pH (pHi). This event initiates formation of thromboxane A2 (via phospholipase A2) and, thereafter, inositol 1,4,5-trisphosphate (via phospholipase C) which is known to mobilize Ca2+ from intracellular storage sites. We investigated changes in pHi and cytosolic free Ca2+, [Ca2+]i, activating platelets with ADP and the thromboxane mimetic U 46619. We found that ADP (5 microM) increased pHi from 7.15 +/- 0.08 to 7.35 +/- 0.04 (n = 8) in 2'-7'-bis-(carboxyethyl)-5,6-carboxyfluorescein-loaded platelets, whereas thromboxane A2 formation was inhibited by indomethacin. ADP also induced a dose-dependent Ca2+ mobilization in fura2-loaded platelets which again was not affected by indomethacin. [Ca2+]i increased by 54 +/- 10 nM (n = 8) at 1 microM and by 170 +/- 40 nM (n = 7) at 10 microM ADP above the resting value of 76 +/- 12 nM (n = 47). Inhibition of Na+/H+ exchange by ethylisopropylamiloride (EIPA) reduced ADP-induced Ca2+ mobilization by more than 65% in indomethacin-treated platelets. This inhibition could be completely overcome by artificially raising pHi using either NH4Cl or the Na+/H+ ionophore monensin. We found that U 46619 increased pHi by 0.18 +/- 0.05 at 0.1 microM and by 0.29 +/- 0.07 (n = 7) at 1.0 microM above the resting value via an EIPA-sensitive mechanism. In conflict with the proposed role of the Na+/H+ exchange we found that U 46619 raised [Ca2+]i via a mechanism that for more than 50% depended on intact Na+/H+ exchange. Again, artificially elevating pHi restored U 46619-induced Ca2+ mobilization despite the presence of EIPA. Thus, our data show that Na+/H+ exchange is a common step in platelet activation by prostaglandin endoperoxides/thromboxane A2 and ADP and enhances Ca2+ mobilization independently of phospholipase A2 activity.

Benzofurans↗
PubMed1990

Interference of deoxyadenosine with transmembrane signaling events in human T lymphocytes.

Deoxyadenosine (dAdo) has been recognized as the toxic metabolite in the immunodeficiency disease associated with adenosine deaminase (ADA) deficiency. Under ADA deficient conditions, dAdo accumulates intracellularly as deoxyadenosine triphosphate (dATP) which by interference with ribonucleotide reductase, prevents DNA synthesis. Recently, we and others have demonstrated that in cells rendered ADA deficient by treatment with deoxycoformycin, dAdo affects T-cell activation events which precede DNA synthesis, such as interleukin 2 receptor (IL-2R) expression and IL-2 production. Here we have analyzed interference of dAdo with the early events of T-cell activation. It is shown that dAdo affects the mitogen induced phosphatidyl inositol turnover. Furthermore dAdo interferes with increase of intracellular calcium. Deoxycytidine, although capable of preventing intracellular accumulation of dATP, cannot reverse the functional consequences of dAdo treatment. The ability of a cell to increase its cytoplasmic free Ca2+, as induced by ionomycin, is not affected by dAdo. The exact target for this novel effect of dAdo is at the present unknown.

Adenosine Deaminase↗
PubMed1990

Filtration. A method to prepare white cell-poor platelet concentrates with optimal preservation of platelet viability.

HLA alloimmunization is a major problem for thrombocytopenic patients receiving long-term platelet support. It is caused by white cells (WBCs) that are present as contaminants in platelet concentrates (PCs). Recent data have shown that filtration is an effective means to reduce WBC contamination, but it has little effect on the recovery of platelets. The present report evaluates two filters, a cellulose acetate (CA) filter requiring the inactivation of platelets with prostacyclin and a cotton wool (CW) filter requiring no platelet inactivation. The results show that, using fresh pooled PCs from six random donors, both filters reduce WBC contamination below 10(7) per PC, the likely threshold below which alloimmunization does not develop. With platelets stored for 2 to 3 days the efficacy of the CW filter decreases. Neither filter inflicts important damage to the platelets, as there is no considerable platelet activation or cell disruption. Moreover, PCs prepared by both filters show normal survival and effectively reduce the bleeding times. Thus, filtration of PCs results in platelets with optimal responsiveness both in vitro and in vivo.

Animals↗
PubMed1990

Evaluation of a new UVB source for irradiation of platelet concentrates.

The application of ultraviolet B (UVB) radiation has been proposed as a new technology to decrease immunogenicity of leucocytes in platelet transfusions. UV radiation also induces platelet aggregation, which occurs most effectively at wavelengths between 240 and 280 nm and falls off sharply above 300 nm. In order to minimize the effects of UV energy on the platelets we evaluated in this study the expected benefit of a new narrow-band UVB source, emitting a narrow peak around 312 nm. Exposure of platelet or lymphocyte suspensions to this source induced in the platelets both aggregation and functional defects at a dose of 12 J/cm2 and in the lymphocytes inhibition of in vitro function at a dose of 2 J/cm2. A conventional UVB source, emitting a broad spectrum between 280 and 340 nm, was more deleterious for the cells and induced similar defects in the platelets at a dose of 6 J/cm2 and inhibition of lymphocyte function at a dose of 1 J/cm2. These data indicate no benefit for the new UVB source, since the ratio of the doses to induce platelet defects and inhibition of lymphocyte function is identical for each of the two sources. Absorption of UV energy by plasma and the plastic material of platelet containers is another criterium for selection of UV sources. In view of the better transmission characteristics of long wavelength UV energy we propose that there is a preference for the new narrow-band UVB source.

Blood Platelets↗
PubMed1990

Irradiation of platelets with UV-B light exposes fibrinogen binding sites via an intracellular mechanism.

Previous studies have shown that ultraviolet irradiation (UVI) causes platelet aggregation. In the present study we exposed platelet suspensions to a relatively high dose of UV-B (8 J/cm2) under conditions comparable to those of UVI of platelet concentrates in order to obtain more insight into the UV-induced aggregation response and to evaluate the significance of this phenomenon for the clinical use of UV-irradiated platelet concentrates. This study provides evidence that UV-B induced aggregation is mediated by a Ca2(+)-dependent process of fibrinogen binding to an intact glycoprotein IIb-IIIa complex on platelet membranes. Although UV-induced platelet aggregation is independent of thromboxane A2 formation and ADP secretion, it requires metabolic energy, cytosolic Ca2+ and a low cyclic-AMP level. Thus, UV-B irradiation causes platelet aggregation by exposing fibrinogen binding sites via an intracellular mechanism. Since the amount of bound fibrinogen following UVI is relatively low (about 2,300 molecules platelet) and the binding remains reversible, its effect on platelet behaviour after transfusion may be minor.

Binding Sites↗
PubMed1989

Na+/H+ exchange and Ca2+ influx.

Cell stimulation raised the cytosolic free Ca2+ concentration, [Ca(2+]i and induces activation of Na+/H+ exchange which raises the cytosolic pH, pHi. Recent studies have addressed the question whether Na+/H+ exchange plays a role in Ca(2+) influx and, specifically, whether a rise in pHi alone suffices to open Ca(2+) channels in the plasma membrane. Artificial cytosolic alkalinization can induce Ca(2+) uptake across the plasma membrane of endothelial cells, lymphocytes and smooth muscle cells. Furthermore, inhibition of Na+/H+ exchange reduces agonist-induced Ca(2+) influx in endothelial cells and platelets which supports the concept that pHi may regulate the opening of Ca(2+) channels in the plasma membrane. Although these findings argue in favour of a role of Na+/H+ exchange in Ca(2+) influx, the onset of pHi and Ca(2+) rises, measured with fluorescent indicators, suggests that the increase in [Ca(2+)]i distinctly precedes the increase in pHi. This challenges the concept that alkalinization per se is a sufficient signal for the opening of Ca(2+) channels in the plasma membrane.

Animals↗
PubMed1989

Cytosolic alkalinization alone is not sufficient for Ca2+ mobilization, phosphatidic acid formation, and protein phosphorylation in human platelets.

One of the earliest events following stimulation of human platelets with thrombin is a rise in the cytosolic pH, pHi, mediated by Na+/H+ exchange, and an increase in the cytosolic free Ca2+ concentration, [Ca2+]i. In the present study we investigated whether an increase in pHi alone, induced by the Na+/H+ ionophore monensin, is sufficient for platelet activation. Although monensin (20 microM) raised pHi from 7.10 +/- 0.05 (n = 21) to 7.72 +/- 0.17 (n = 13), neither Ca2+ influx nor mobilization were detectable upon this treatment in fura2-loaded platelets. In contrast, thrombin (0.05 U/ml) raised pHi to 7.31 +/- 0.10 (n = 10) and increased [Ca2+]i by more than 250 nM both in the presence and absence of extracellular Ca2+. Thrombin also caused the formation of phosphatidic acid and phosphorylation of the 20 kDa and 47 kDa proteins in platelets labeled with 32P. Monensin, however, induced none of these responses. It is concluded that an increase in pHi alone is not a sufficient trigger for platelet activation but enhances intracellular signal transduction in platelets stimulated by natural agonists.

Blood Platelets↗
PubMed1989

Activation of Na+/H+ exchange and Ca2+ mobilization start simultaneously in thrombin-stimulated platelets. Evidence that platelet shape change disturbs early rises of BCECF fluorescence which causes an underestimation of actual cytosolic alkalinization.

Although an increase in cytosolic pH (pHi) caused by Na+/H+ exchange enhances Ca2+ mobilization in platelets stimulated by low concentrations of thrombin [Siffert & Akkerman (1987) Nature (London) 325, 456-458], studies using fluorescent indicators for pHi (BCECF) and [Ca2+]i (fura2) suggest that Ca2+ is mobilized while the cytosolic pH decreases. Several lines of evidence indicate that the initial fall in BCECF fluorescence is not due to cytosolic acidification but is caused by a platelet shape change. (1) Pulse stimulation of platelets by successive addition of hirudin (4 unit/ml) and thrombin (0.2 unit/ml) induced a shape change of 43 +/- 8% and a fall in BCECF fluorescence, which both remained unchanged when Na+/H+ exchange was inhibited by ethylisopropylamiloride (EIPA, 100 microM). (2) Increasing the thrombin concentration to 0.4 unit/ml doubled the shape change and the fall in BCECF fluorescence, but again EIPA had no effect on these responses. (3) Treating platelets with 2 microM-ADP induced shape change and a decline in BCECF fluorescence that was unaffected by EIPA. (4) A second addition of thrombin to platelets that had already undergone shape change induced an immediate increase in BCECF fluorescence without a prior decrease. (5) Activation of protein kinase C by 1,2-dioctanoyl-sn-glycerol (DiC8) neither induced shape change nor a decline in BCECF fluorescence; in contrast BCECF fluorescence rapidly increased indicating an immediate cytosolic alkalinization. Concurrent analysis of [Ca2+]i under conditions in which shape change did not interfere with BCECF fluorescence showed that cytosolic alkalinization and Ca2+ mobilization started almost simultaneously. These observations suggest that cytosolic alkalinization is not preceded by a fall in pHi and can support Ca2+ mobilization induced by weak agonists.

Blood Platelets↗
PubMed1989

Impaired energy metabolism in platelets from patients with Wiskott-Aldrich syndrome.

The platelet function defect seen in patients with Wiskott-Aldrich syndrome (WAS) has been ascribed to abnormal mitochondrial energy generation. The present study reveals a reduced energy content and low adenylate energy charge in platelets from two WAS-patients. Energy consumption in the resting platelets is slightly beyond the normal range, especially when ATP-resynthesis is primarily glycolytic. When platelets are stimulated with thrombin, the increase in energy consumption is 40-60% lower than in controls, both when energy is produced in glycolysis as when the mitochondria supply most of the energy. Analysis of the electron transport chain reveals no abnormalities. In contrast, the balance between glycolytic and mitochondrial ATP resynthesis is disturbed with a lowered contribution of oxidative ATP production. No such abnormalities are found in two WAS-carriers with the exception of a slight impairment in energy consumption during stimulation with thrombin. Thus, the platelet malfunction in WAS may be caused by a defect in the regulation of energy generation.

Adenosine Diphosphate↗
PubMed1988

Protein kinase C enhances Ca2+ mobilization in human platelets by activating Na+/H+ exchange.

Control of cytoplasmic pH (pHi) by a Na+/H+ antiport appears a general property of most eukaryotic cells. In human platelets activation of the Na+/H+ exchanger enhances Ca2+ mobilization and aggregation induced by low concentrations of thrombin (Siffert, W., and Akkerman, J. W. N. (1987) Nature 325, 456-458). Several observations indicate that the exchanger is regulated by protein kinase C. (i) Inhibitors of protein kinase C (trifluoperazine, sphingosine) inhibit the increase in pHi seen during thrombin stimulation as well as Ca2+ mobilization; artificially increasing pHi by monensin or NH4Cl then restores Ca2+ mobilization. (ii) Direct activation of protein kinase C by 1-oleoyl-2-acetylglycerol initiates an increase in pHi that depends on the presence of extracellular Na+ and is sensitive to inhibition by ethylisopropylamiloride. The pHi sensitivity of thrombin-induced Ca2+ mobilization is particularly evident in the range between pH 6.8 and 7.4 and at low thrombin concentrations, whereas thrombin concentrations of more than 0.2 unit/ml bypass the pH sensitivity. In the absence of thrombin an increase in pHi, either induced artificially (by addition of the ionophores nigericin or monensin) or via activation of protein kinase C (by addition of 1-oleoyl-2-acetylglycerol), does not induce Ca2+ mobilization. We conclude that activation of protein kinase C is essential for Ca2+ mobilization in platelets stimulated by low concentrations of thrombin and that protein kinase C exerts this effect via activation of the Na+/H+ exchanger.

Blood Platelets↗
PubMed1987

Inhibition of Na+/H+ exchange reduces Ca2+ mobilization without affecting the initial cleavage of phosphatidylinositol 4,5-bisphosphate in thrombin-stimulated platelets.

Stimulation of human platelets increases cytoplasmic pH (pHi) via activation of Na+/H+ exchange. We have determined the effect of inhibiting Na+/H+ exchange on (i) thrombin-induced Ca2+ mobilization and (ii) turnover of 32P-labelled phospholipids. Blocking Na+/H+ exchange by removal of extracellular Na+ or by ethylisopropylamiloride (EIPA) inhibited Ca2+ mobilization induced by 0.2 U/ml thrombin, whereas increasing pHi by NH4Cl enhanced the thrombin-induced increase in cytosolic free Ca2+. The effect of EIPA was bypassed after increasing pHi by moneasin. The thrombin-induced cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) was unaffected by treatments that blocked Na+/H+ exchange or increased pHi. It is concluded that activation of Na+/H+ exchange is a prerequisite for Ca2+ mobilization in human platelets but not for the stimulus-induced hydrolysis of PIP2.

Amiloride↗
PubMed1987

Cryopreservation disturbs stimulus-response coupling in a platelet subpopulation.

Cryopreservation of platelet suspensions leads to a severe reduction in the functional responses of these cells. This defect primarily resides in a subpopulation which consists of about half the total platelet mass and shows an increased susceptibility to the cryopreservation procedure. Following freeze-thawing this subpopulation failed to aggregate upon stimulation with collagen despite a normal appearance of membrane glycoproteins. Also the contents of alpha granules was similar to the whole suspension and since changes in alpha and dense granule contents often occur in parallel, the poor aggregability could not be attributed to a defect in secretion granules. In contrast, the generation of phosphatidyl-inositol metabolites, the increase in cytosolic Ca2+ content and the phosphorylation of proteins of mol wt 47,000 and 20,000 seen when normal platelets are activated, were completely absent in the subpopulation. These data reveal a severe defect in stimulus-response coupling mechanisms in about half of the platelets after freeze-thawing. The other half of the suspension endures this treatment relatively unharmed and may be used for transfusion.

Blood Platelets↗
PubMed1987

The influence of cyclandelate on Ca++ translocation in human platelets.

A major event in signal transduction in platelets is the mobilisation of Ca++ ions by 2 mechanisms: influx of Ca++ from the extracellular medium and release from intracellular storage sites. The calcium modulating drug cyclandelate is known to inhibit platelet aggregation induced by various agonists. Thus, by studying the effects of cyclandelate on cytosolic Ca++ concentrations in gel-filtered human platelets, an explanation was sought of the mechanism which couples receptor stimulation with the processes that execute aggregation and secretion. Cyclandelate in concentrations between 10 and 50 mumol/L induced a dose-dependent increase in the basal level of cytosolic Ca++ of unstimulated platelets. In platelets stimulated with thrombin (5 U/ml) or platelet activating factor (1 mumol/L), cyclandelate strongly inhibited the increase of cystolic Ca++ in the presence of extracellular Ca++, but in the absence of extracellular Ca++ only a weak inhibition was observed. This inhibition was dose dependent and optimal at about 50 mumol/L, the concentration at which Ca++ mobilisation was suppressed to about 10% of control values, and at which cyclandelate inhibits platelet aggregation. In contrast, the metabolites cyclandelate alcohol and cyclandelate acid were without effect at the same concentrations (50 mumol/L). Concentrations of cyclandelate greater than 50 mumol/L were found to further increase the concentration of Ca++ in the unstimulated platelet, an effect which interfered with the stimulus-induced Ca++ translocation. Thus, at concentrations which inhibit platelet aggregation, cyclandelate prevents the mobilisation of Ca++ ions induced by such agonists as thrombin and platelet activating factor. Further study is required to determine the mechanism by which cyclandelate inhibits these platelet responses.

Biological Transport↗
PubMed1987

Clinical experience with transfusion of leukocyte-poor platelet concentrates prepared by filtration with prostacyclin.

Repeated transfusions with platelets from randomly selected donors lead to HLA alloimmunization in about 50% of patients due to lymphocyte contamination of platelet concentrates. Attempts to remove the leukocytes from the platelet concentrates by additional centrifugation steps led to substantial loss of platelets. We report a new procedure for removal of almost all leukocytes with excellent platelet recoveries. Single donor concentrates are treated with 50 ng/mL prostacyclin to inactivate the platelets transiently. The concentrates are then passed through a cellulose-acetate filter to remove the leukocytes. In 30 concentrates this treatment reduced the contamination by leukocytes to less than 0.1 million per concentrate with a platelet recovery of 89% +/- 1% (mean +/- SEM). Thirty filtered platelet concentrates transfused to ten thrombocytopenic patients within one hour after filtration were well tolerated and led to corrected count increments of (22.0 +/- 1.1) X 10(6)/mL blood after one hour and normal survival thereafter. In four of five patients these concentrates reduced the bleeding time. We conclude that transient inactivation of platelets by prostacyclin enables optimal removal of leukocytes and may help to reduce alloimmunization during frequent transfusions with platelet concentrates.

Adenosine Diphosphate↗
PubMed1987

Patients with a prolonged bleeding time and normal aggregation tests may have storage pool deficiency: studies on one hundred six patients.

One hundred six patients with storage pool deficiency (SPD) were studied with respect to platelet count, bleeding time, total platelet ATP and ADP, platelet serotonin, and in vitro aggregation. The diagnosis of SPD was made on basis of a prolonged bleeding time, a decreased total platelet ADP, and a diminished level of serotonin. Fifty-one patients from 34 unrelated families had congenital SPD, and 55 patients had acquired SPD. Congenital SPD was a common disorder in patients with a lifelong bleeding tendency and a prolonged bleeding time. The frequency in this group of patients was 18%, about one-half the frequency of von Willebrand's disease (vWd). Twenty-three percent of all patients had normal aggregation responses to ADP, epinephrine, and collagen; 33% had aggregation tracings typical for a secretion defect; and 44% had miscellaneous aggregation abnormalities. These findings indicate that SPD is common, heterogeneous, and not necessarily associated with in vitro aggregation abnormalities.

Bleeding Time↗
PubMed1986

Platelet-activating factor (PAF-acether) induces high- and low-affinity binding of fibrinogen to human platelets via independent mechanisms.

When human platelets are incubated with 500 nM-PAF-acether (platelet-activating factor. 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) under equilibrium conditions (60 min, 22 degrees C, non-stirred suspensions), two classes of fibrinogen binding sites are exposed: one class with a high affinity [Kd (7.2 +/- 2.1) X 10(-8) M, 2367 +/- 485 sites/platelet, n = 9] and one class with a low affinity [Kd (5.9 +/- 2.4) X 10(-7) M, 26972 +/- 8267 sites/platelet]. Preincubation with inhibitors of cyclo-oxygenase (acetylsalicylic acid, indomethacin) or thromboxane synthetase (UK 38.485) completely abolishes high-affinity binding, leaving low-affinity binding unchanged. In contrast, ADP scavengers (phosphocreatine/creatine kinase or phosphoenol pyruvate/pyruvate kinase) completely prevent low-affinity binding, leaving high-affinity binding unaltered. Initial binding studies (2-10 min incubation) confirm these findings with a major part of the binding being sensitive to ADP scavengers, a minor part sensitive to indomethacin and complete blockade with both inhibitors. Increasing the temperature to 37 degrees C decreases the number of low affinity-binding sites 6-fold without changing high-affinity binding. Aggregation, measured as the rate of single platelet disappearance, then depends on high-affinity binding at 10 nM-fibrinogen or less, whereas at 100 nM-fibrinogen or more low-affinity binding becomes predominant. These findings point at considerable platelet activation during binding experiments. However, arachidonate metabolism [( 3H]arachidonate mobilization and thromboxane synthesis) and secretion [( 14C]serotonin and beta-thromboglobulin) are about 10% or less of the amounts found under optimal conditions (5 units of thrombin/ml 37 degrees C, stirring). We conclude that PAF-acether induces little platelet activation under binding conditions. The amounts of thromboxane A2 and secreted ADP, however, are sufficient for initiating high- and low-affinity fibrinogen binding via mutually independent mechanisms.

Adenosine Diphosphate↗