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Biomedical subjects

J W Akkerman

Publications and source records attributed to J W Akkerman.

At least 55 records · Page 3Linked to original sources
PubMed1993

Effect of epinephrine on the regulation of Na+/H+ exchange in human platelets.

In the present study, we investigated whether stimulation of platelets by epinephrine affects the Na+/H+ exchanger, an antiport that regulates the cytosolic pH (pHi). Epinephrine alone failed to modulate Na+/H+ exchange, as reflected by a constant fluorescence of the pHi indicator BCECF. In contrast, epinephrine accelerated Na+/H+ exchange upon stimulation with a threshold concentration of platelet activating factor (PAF). The extra Na+/H+ exchange was not caused by a better binding of PAF to platelets and occurred also in the presence of indomethacin, excluding a role for cyclooxygenase products. Epinephrine failed to mobilize Ca2+i (measured by fura-2 fluorescence) and did not activate protein kinase C ([32P]pleckstrin) or phospholipase C ([32P]phosphatidic acid). In combination with PAF, epinephrine left the PAF-induced mobilization of Ca2+i and accumulation of [32P]phosphatidic acid unchanged, but induced a 1.3-fold increase in the phosphorylation of pleckstrin. These data indicate that epinephrine enhances Na+/H+ exchange via a direct effect of alpha 2A-adrenergic receptors on protein kinase C.

Blood Platelets↗
PubMed1993

Regulation of the receptor for platelet-activating factor on human platelets.

Human platelets possess about 300 receptors for platelet-activating factor (PAF) per cell with a Kd of about 0.2 nM. In the present study we investigated whether these receptors are subject to intracellular control mechanisms. Preincubation with the protein kinase C inhibitor staurosporine had no effect, and also agents that increase cyclic AMP failed to change the binding of [3H]PAF. The Ca(2+)-calmodulin inhibitors W-7 and sphingosine decreased PAF binding by 50-80%. Inhibition of energy metabolism induced a fall in adenylate energy charge [AEC = ([ATP] + 1/2[ADP])([ATP + ADP + AMP])] and an almost parallel decrease in specific [3H]PAF binding without changing the Kd. Restoration of the AEC restored the [3H]PAF binding. Abrupt arrest of energy metabolism during binding of [3H]PAF left the binding unchanged until the metabolic ATP level had decreased by about 90%. These data indicate that PAF receptors on human platelets are under close intracellular control, possibly via a Ca(2+)-calmodulin-dependent phosphorylation/dephosphorylation process.

Adenosine Diphosphate↗
PubMed1993

Human platelets secrete chemotactic activity for eosinophils.

Thrombin-stimulated platelets liberate factors that induce chemotaxis of eosinophils and raise their cytosolic Ca2+ content ([Ca2+]i). The sources of this activity are the dense- and alpha-granules because inhibition of prostaglandin endoperoxide/thromboxane A2 formation and the platelet-activating factor receptor-antagonist WEB 2086 have no effect. Platelets from patients with Storage-Pool Deficiency show about 60% of the normal chemotactic activity with little effect on [Ca2+]i, whereas completely degranulated platelets fail to affect eosinophils. In concentrations secreted by the platelets, adenosine diphosphate (ADP), and platelet factor 4 have no effect, whereas adenosine triphosphate (ATP) induces a strong chemotactic response and increases [Ca2+]i. However, apart from ATP other modulating factors must be involved as platelet releasates induce more chemotaxis than ATP alone. Thus, platelets secrete factors that activate eosinophils and may contribute to inflammatory and allergic processes.

Adenosine Diphosphate↗
PubMed1993

Ultraviolet-B irradiation of platelets induces a dose-dependent increase in the expression of platelet activation markers with storage.

Ultraviolet B (UVB) irradiation of platelet concentrates (PCs) has been proposed as a novel technology to prevent HLA sensitization. We have recently reported that 2 J/cm2 of UVB radiation abolishes alloreactive lymphocyte responses in vitro. In order to increase the efficacy of UV irradiation for the prevention of HLA sensitization, we exposed PCs to 4 or 8 J/cm2 of UVB and evaluated the effect of UV radiation on platelet integrity during storage. We report here that UV exposed platelets show a progressive increase in the expression of activation markers P-selectin (GMP-140; CD62) and LIMP-CD63 (GP-53; CD63) on the platelet membrane over time in a dose-dependent manner compared to age-matched controls. Platelet metabolism was also enhanced as evidenced by significant changes in lactate and pH during post-irradiation storage. Based on these findings we transfused PCs within 4 h after UV irradiation. PCs exposed to 4 J/cm2 showed normal post-transfusion recoveries and haemostatic functions, while poor platelet recoveries were found after administration of PCs exposed to 8 J/cm2. We hypothesize that the rapid expression of P-selectin on platelets exposed to the higher dose of UVB leads to an increased binding of these platelets to leucocytes in the circulation resulting in poor platelet recoveries.

Antigens, CD↗
PubMed1993

UVB radiation exposes fibrinogen binding sites on platelets by activating protein kinase C via reactive oxygen species.

Previous studies have shown that ultraviolet B (UVB) radiation causes platelet aggregation by exposing fibrinogen binding sites via activation of an intracellular mechanism. In the present study we have further investigated the routes of platelet activation following UVB exposure. Evidence is provided that UVB radiation does not activate the platelets via the classical Phospholipase A2 and Phospholipase C routes. Despite this observation, UVB-induced fibrinogen binding was found to be correlated with a 40% increase in phosphorylated 47 kD protein. Both findings could be completely inhibited in the presence of staurosporine, a potent inhibitor of protein kinase C (PK-C). In efforts to explain the mechanism of PK-C activation by UV radiation we found that both UV-induced PK-C activation and platelet aggregation were significantly reduced in the presence of specific scavengers for reactive oxygen species including superoxide dismutase and catalase. We conclude that exposure of platelets to UVB radiation can activate PK-C via oxygen radicals, resulting in exposure of fibrinogen binding sites and subsequent platelet aggregation.

Binding Sites↗
PubMed1993

Effects of low-dose EPA-E on glycemic control, lipid profile, lipoprotein(a), platelet aggregation, viscosity, and platelet and vessel wall interaction in NIDDM.

OBJECTIVE: To assess the effects of low-dose eicosapentaenoic acid-ethyl-ester on diabetes regulation, lipid metabolism, blood rheology, and platelet reactivity. RESEARCH DESIGN AND METHODS: In a double-blind, randomized, placebo-controlled study, 24 NIDDM subjects received 1800 mg of EPA-E, 900 mg of EPA-E, or a placebo (1656 mg olive oil) daily for 8 wk. RESULTS: The EPA:arachidonic acid plasma ratio increased over an 8-wk period, then declined after a 4-wk wash-out period in the fish-oil groups in a dose-dependent way. Platelet-activating factor-induced platelet aggregation decreased from 75 +/- 7% at wk 0 to 35 +/- 21% at wk 8 in the 900-mg group (P = 0.016) and from 72 +/- 11 to 40 +/- 30% in the 1800-mg group (P = 0.039), but did not change in the placebo group. No effects on ADP- or collagen-induced aggregation could be attributed to EPA-E. In the 1800-mg group low-density-lipoprotein cholesterol increased significantly, without concomitant rise in apolipoprotein B. Triglycerides, glycemic control, lipoprotein (a), blood and plasma viscosity, erythrocyte deformability, and platelet adhesion to and aggregate formation on extracellular endothelial cell matrix were not significantly influenced. CONCLUSIONS: Purified EPA-E in doses of 900 and 1800 mg reduces Platelet-activating factor-induced platelet aggregation without negatively affecting glycemic control. Low-density-lipoprotein cholesterol was elevated in the 1800-mg group.

Adenosine Diphosphate↗
PubMed1993

Occupancy of platelet receptors for platelet-activating factor in asthmatic patients during an allergen-induced bronchoconstrictive reaction.

The administration of platelet-activating factor (PAF) to human subjects triggers asthma-like responses. We investigated whether a bronchoconstrictive reaction was accompanied by the release of PAF in the circulation of allergic asthmatics. The appearance of PAF was assessed by measuring the number of freely accessible PAF-receptors on platelets in vitro, assuming that the contact between platelets and PAF in vivo would reduce the receptor binding of [3H]PAF in vitro. 16 asthmatics were challenged twice, first with buffer and the next day with allergen. A comparison between receptor binding after provocation with the data of the same patients after allergen challenge revealed a significant difference in PAF binding (P = 0.034), with an average decrease of 14% immediately after allergen challenge followed by a return to control values after about 4 h and a transient increase of 9% at 7 h after provocation. The decrease in accessible PAF receptors was accompanied by a slight decrease in platelet count in peripheral blood between 30 min and 4 h after allergen challenge. The platelet counts recovered to the original values afterwards. These data support the concept that in patients with allergic asthma PAF is secreted in the circulation. The contact between PAF and the platelets may trigger the transient sequestration of platelets, possibly in the lung. Thus, PAF and platelets may contribute to the pathogenesis of allergic asthma.

Adolescent↗
PubMed1992

Arachidonate transfer between platelets and lipoproteins.

Although platelets have specific bindingsites for LDL and HDL, it is doubtful whether lipoproteins modulate platelet functions via receptor-mediated processes. We investigated platelet-lipoprotein interaction during prolonged incubation with concentrations of LDL and HDL that saturate the bindingsites within a few minutes. When [3H]arachidonate-labeled human platelets were incubated for 4 h with lipoproteins, part of the 3H-radioactivity transferred to LDL and to a lesser extent to HDL. The transfer was temperature-sensitive, unaffected by modification of lysine in LDL or indomethacin treatment of the platelets, and almost irreversible. [3H]arachidonate transfer to lipoproteins could be mimicked by incubating platelets with a high concentration of fatty acid free albumin. This showed, that the loss of 3H-radioactivity reflected a decrease in endogenous arachidonate, leading to impaired aggregation, secretion and thromboxane B2 formation in platelets after stimulation with thrombin but not with arachidonate. Thus, the decrease in platelet functions seen after long incubation with HDL is caused by depletion of platelet arachidonate. Despite an even stronger arachidonate depletion by LDL, this lipoprotein initiated arachidonate metabolism and secretion independent of specific binding sites for LDL on the platelet. Surprisingly, the major part of the secretion was preserved when the formation of prostaglandin endoperoxides/thromboxane A2 was inhibited with indomethacin. These findings argue against a role for LDL and HDL receptors in the modulation of platelet functions and are more in favor of lipid exchange processes between platelets and lipoproteins.

Adenosine Triphosphate↗
PubMed1992

Enhancement of platelet functions by low density lipoproteins.

Platelet suspensions, that secreted about 50% of their dense granule contents upon stimulation with alpha-thrombin, showed a dose-dependent increase in secretion after 30 min preincubation with 0.5-3.0 g low density lipoprotein (LDL) protein/1. A 1-5 min preincubation had no effect. The enhancement by LDL only occurred at about 20% secretion or more, indicating that a minimal degree of activation was required for LDL to become effective. Lysine-modified LDL was equally effective as native LDL. The effect of LDL on secretion was accompanied by enhanced thromboxane B2 formation caused by stimulation of the liberation of arachidonate from phosphatidylcholine and/or phosphatidylinositol. However, when thromboxane formation was inhibited or the prostaglandin H2-thromboxane A2-receptor was blocked, LDL remained a potent stimulator of the secretion response. Thus, LDL enhances platelet secretion by a thromboxane A2-dependent and a thromboxane A2-independent mechanism via an effect that is independent of specific binding sites on the platelet.

Arachidonic Acids↗
PubMed1992

Regulation of glycoprotein IIB/IIIA exposure on platelets stimulated with alpha-thrombin.

Previous studies have shown that binding sites for fibrinogen on platelets stimulated with platelet-activating factor (PAF), adenosine diphosphate or epinephrine rapidly close in the absence of fibrinogen. In the present study we investigated whether alpha-thrombin induced similar changes in the glycoprotein (GP) IIB/IIIA-complex. Whereas 80% of binding sites exposed by PAF closed within 30 minutes (22 degrees C), alpha-thrombin (0.1 U/mL) triggered long-lasting exposure of binding sites for [125I]-fibrinogen and [125I]-fibronectin. Even removal of alpha-thrombin with an excess of hirudin failed to close the binding sites. Similar to PAF, alpha-thrombin-exposed sites rapidly closed after addition of the protein kinase C inhibitor staurosporine (1 mumol/L) or dibutyryl cyclic adenosine monophosphate (250 mumol/L). In contrast, prostacyclin (PGI2, 10 ng/mL), which induced rapid closure of binding sites in platelets stimulated with PAF, failed to close the sites in alpha-thrombin-treated platelets. Removal of alpha-thrombin from the platelets restored the PGI2-sensitivity. These data indicate that a short interaction between alpha-thrombin and platelets triggers long-lasting exposure of GPIIB/IIIA. Furthermore, as long as alpha-thrombin remains bound to the platelets, agonists that activate the PGI2-receptor are unable to close GPIIB/IIIA.

Alkaloids↗
PubMed1992

Platelet Na(+)-H+ exchanger activity in normotensive and hypertensive subjects: effect of enalapril therapy upon antiport activity.

OBJECTIVE: Primary hypertension has been reported to be associated with an enhancement of Na(+)-H+ exchange. However, details of the kinetic properties of the Na(+)-H+ exchanger in hypertensives and its dependence upon age and gender in normotensives are unknown. PARTICIPANTS: We determined the activity of the platelet Na(+)-H+ exchanger in 20 normotensives and 26 untreated primary hypertensives. INTERVENTIONS: In eight hypertensive individuals antihypertensive treatment was interrupted for 1 week. Treatment for 6 weeks with a daily single dose of 10 mg enalapril decreased mean arterial pressure to 105.7 +/- 11.6 mmHg. METHODS: Platelets were loaded with the intracellular pH (pHi) indicator 2'-7'-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF) and acidified by propionic acid. Initial velocities of pH recovery were determined and used for calculation of maximum velocity (Vmax), baseline pHi and the pHi value for half maximal activation (pH0.5) of the Na(+)-H+ exchanger in each individual. RESULTS: In normotensives, Vmax averaged 0.05 +/- 0.01 dpHi/min independently of age, gender and actual diastolic blood pressure. In hypertensives, two different subgroups were defined bearing either low or high Na(+)-H+ exchange activity. Values of pHi and pH0.5 were identical in all subgroups irrespective of Vmax. The twofold enhancement of Na(+)-H+ exchange in the second group was preserved in thrombin-stimulated platelets. Vmax values remained unaffected by enalapril treatment. CONCLUSIONS: Enhanced Na(+)-H+ exchange activity in hypertensives is primarily characterized by an increase in Vmax. This enhancement is refractory to antihypertensive treatment and therefore appears to be a relatively fixed parameter.

Age Factors↗
PubMed1992

Effect of platelet agonists on the binding of platelet-activating factor to human platelets.

The binding of PAF in vitro to its receptors on platelets gradually becomes irreversible. This property has been applied to the measurement of PAF in circulating blood, the concept being that in vivo contact with endogenous PAF diminishes subsequent in vitro binding of 3H-labeled PAF. Here we show that, when platelets are stimulated with thrombin, the subsequent binding of [3H]PAF is greatly reduced, probably due to proteolytic damage of the PAF receptors. In addition, [3H]PAF binding is decreased, when the cell's energy status is low, which is seen during thrombin-induced secretion. Thus, the detection of PAF in circulating blood based on the binding to PAF receptors on platelets is severely hampered when platelets make contact with thrombin.

Adenosine Diphosphate↗
PubMed1991

Abnormal platelet functions in a patient with abetalipoproteinemia.

Studies with isolated lipoproteins and washed platelets suggest that lipoproteins may affect platelet functions. We investigated platelet-rich plasma (PRP) from a patient with abetalipoproteinemia (ABL), whose plasma lacks apo-B containing lipoproteins (VLDL, LDL and chylomicrons). ABL-PRP aggregated poorly with different agonists and failed to respond to arachidonate. Thromboxane B2 (TxB2) formation was severely impaired. After gel-filtration most of the aggregation defects persisted in agreement with reduced metabolism of endogenous arachidonate. However, arachidonate-induced aggregation and TxB2 production partially normalized. Normal platelets suspended in ABL-plasma showed similar defects in aggregation and TxB2 production but arachidonate-induced aggregation was much lower than expected on the basis of TxB2. We conclude that the abnormal platelet functions in ABL-PRP are caused by (i) an intrinsic platelet abnormality due to reduced arachidonate mobilization and (ii) a property in ABL plasma that inhibits aggregation partially by trapping the arachidonate and partially by an unidentified mechanism. The latter properties may be the result of the abnormal lipid composition of ABL-plasma.

Abetalipoproteinemia↗
PubMed1991

Protein kinase C and cyclic AMP regulate reversible exposure of binding sites for fibrinogen on the glycoprotein IIB-IIIA complex of human platelets.

Platelet aggregation is mediated via binding of fibrinogen to sites on the membrane glycoprotein IIB-IIIA complex which become exposed when the cells are stimulated. We report here evidence of a dynamic and reversible exposure of binding sites for fibrinogen. In the absence of fibrinogen, exposed sites (B*) gradually lose their capacity to bind fibrinogen and close (Bo). On stimulation with platelet-activating factor (PAF, 500 nM) at 22 degrees C, closing of B* is enhanced by agents that raise cyclic AMP levels (10 ng of prostaglandin I2/ml; 5 mM-theophylline), inhibit protein kinase C (PKC; 25 microM-sphingosine; 1 microM-staurosporine), or disrupt the energy supply (30 mM-2-deoxy-D-glucose + 1 mM-CN-), or by raising the temperature to 37 degrees C. Conversely, activation of PKC 1 microM-1,2-dioctanoyl-sn-glycerol; 55 nM-phorbol 12-myristate 13-acetate) and an increase in intracellular [Ca2+] (100 nM-ionomycin + extracellular Ca2+) oppose the disappearance of B*. Phosphorylation of the 47 kDa protein illustrates the tight coupling between PKC and B* under all conditions tested, except when the cyclic AMP level is raised, and B* is converted to Bo without affecting PKC activity. Although the increase in PKC activity is much smaller with ADP or even absent upon stimulation with adrenaline, the control of B* is equally sensitive to modulation of cyclic AMP and PKC activity. We conclude that PAF, ADP and adrenaline regulate exposure of fibrinogen binding sites through a common mechanism consisting of two independent pathways, one dominated by PKC and the other by an as yet unidentified cyclic AMP-sensitive step.

Adenosine Diphosphate↗
PubMed1991

Use of leukocyte-depleted platelet concentrates for the prevention of refractoriness and primary HLA alloimmunization: a prospective, randomized trial.

Compared with conventional transfusion regimes a strong reduction in HLA alloimmunization and refractoriness to platelet transfusions is obtained when both red blood cell concentrates (RBCs) and platelet concentrates (PCs) are depleted of leukocytes by filtration. Because most of the leukocyte contamination is introduced by transfusion of RBCs, filtration of RBCs appears rational, but uncertainty exists regarding the degree of leukocyte-depletion of PCs needed for the prevention of HLA alloimmunization and refractoriness. We conducted a prospective trial and randomized patients with acute leukemia to receive leukocyte-depleted PCs prepared either by centrifugation (mean leukocyte count 35 x 10(6)/PC of 6 U) or by filtration (mean leukocyte count less than 5 x 10(6)/PC of 6 U). Both groups received RBCs that were filtered after prior removal of the buffy coat. Clinical refractoriness occurred in 46% (12 of 26) of the evaluable patients that were transfused with centrifuged PCs and only in 11% (3 of 27) in the filtered group (P less than .005). De novo anti-HLA antibodies were detected in 42% (11 of 26) patients in the centrifuged group and only in 7% (2 of 27) of the patients receiving filtered PCs (P less than .004). In 8 of 11 alloimmunized patients in the centrifuged group antibodies were detected in the first 4 weeks of transfusion therapy while none of the patients in the filtered group became immunized against HLA antigens during that period. We conclude that for the prevention of HLA alloimmunization and refractoriness to platelet transfusions from random donors, both RBCs and PCs have to be leukocyte-depleted by filtration.

Acute Disease↗
PubMed1990

Sodium fluoride prevents receptor- and protein kinase C-mediated activation of the human platelet Na+/H+ exchanger without inhibiting its basic pHi-regulating activity.

When human platelets are stimulated with thrombin or activators of protein kinase C, cytosolic pH (pHi) increases due to activation of Na+/H+ exchange. In order to further elucidate the molecular mechanisms that regulate the exchanger, we used sodium fluoride, which is a known activator of guanine nucleotide-binding proteins (G proteins) in platelets. Although NaF induced the mobilization of Ca2+ from intracellular storage sites in fura2-loaded platelets, it failed to raise pHi as determined from the fluorescence of 2,7-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein-loaded platelets. Furthermore, when thrombin (0.1 unit/ml) or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) had raised pHi from 7.13 +/- 0.05 to 7.35 +/- 0.07 (n = 30), addition of NaF (2.5-10 mM) rapidly restored pHi to values found before stimulation. Conversely, preincubation of platelets with low concentrations of NaF (2.5 mM) completely prevented alkalinization in response to thrombin or TPA. Unlike ethylisopropylamiloride, which completely blocked Na+/H+ exchange, NaF did not prevent the recovery of pHi from an artificial acid load. Hence, the inhibitory action of NaF is restricted to receptor-mediated activation of the antiport. In order to investigate whether the NaF effect was attributable to a G protein, platelets were preincubated with N-ethylmaleimide (50 microM), which is known to inhibit the adenylyl cyclase-inhibitory G protein. N-Ethylmaleimide treatment not only prevented inhibition of adenylyl cyclase by epinephrine but also completely reversed the inhibitory effect of NaF on the Na+/H+ exchanger. Our data suggest the existence of a novel G protein which is activated by fluoride and functions as a negative regulator of the Na+/H+ exchanger in platelets.

Adenosine Diphosphate↗
PubMed1990

Stimulation of human platelets by collagen occurs by a Na+/H+ exchanger independent mechanism.

In stimulated human platelets dense-granule secretion in response to the 'weak agonists' ADP, adrenaline, platelet activating factor and low concentrations of thrombin as well as Ca2+ mobilisation in response to thrombin are enhanced by a Na+/H+ exchanger. In the present study the role of this antiport in collagen stimulated human platelets was examined. While stimulation of platelets loaded with the fluorescent intracellular pH-sensitive dye, bis-carboxyethyl-5-(6)-carboxyfluorescein (BCECF) with thrombin resulted in the activation of the Na+/H+ exchanger, activation of this antiport did not occur in collagen-stimulated platelets. The lack of antiport activity in response to collagen using BCECF-loaded platelets correlated with the lack of any functional role of the antiport in collagen stimulated platelets. In the presence of a Na+/H+ exchange inhibitor, ethylisopropylamiloride, neither collagen-induced platelet aggregation or dense-granule secretion was affected. Furthermore, while the removal of extracellular Na+ (Na+ext), a condition that also prevents activation of the antiport, inhibited dense-granule secretion in response to a low concentration of thrombin, collagen-induced secretion was potentiated. This potentiatory effect could not be attributed to changes in either the membrane potential or in collagen-induced phospholipase C or protein kinase C activity. The present results indicate that in contrast to the 'weak agonists' (1) collagen-induced platelet activation does not require activation of the Na+/H+ exchanger and (2) Na+ext per se is an inhibitor of collagen-induced secretion.

Blood Platelets↗