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Biomedical subjects

G Gorter

Publications and source records attributed to G Gorter.

At least 19 recordsLinked to original sources
PubMed1996

Exposure of ligand-binding sites on platelet integrin alpha IIB/beta 3 by phosphorylation of the beta 3 subunit.

The exposure of ligand-binding sites for adhesive proteins on platelet integrin alpha IIB/beta 3 (glycoprotein IIB/IIIA) by platelet-activating (PAF) is transient, whereas sites exposed by alpha-thrombin remain accessible. The same difference is seen in the phosphorylation of the beta 3 subunit. Inhibition of protein kinases (1 microM staurosporine) and protein kinase C (10 microM bisindolylmaleimide) closes binding sites exposed by both agonists and induces dephosphorylation of beta 3. Inhibition of Tyr-kinases (20 microM Herbimycin A) has only a slight effect. Inhibition of Ser/Thr-phosphatases (1 microM okadaic acid, 30 s preincubation) changes the transient exposure and beta phosphorylation by PAF into the 'permanent' patterns induced by alpha-thrombin. Inhibition of Tyr-phosphatases (100 microM vanadate) has little effect. Preincubation with okadaic acid makes exposed binding sites and phosphorylated beta 3 insensitive to staurosporine, resulting in exposed alpha IIB/beta 3 independent of concurrent phosphorylation/dephosphorylation. The stoichiometry of beta 3 phosphorylation by alpha-thrombin is 0.80+/-0.10. Thus, one of the mechanisms that regulates exposure and closure of ligand-binding sites on the alpha IIb/beta 3 is phosphorylation/dephosphorylation of a Ser/Thr-residue in the beta 3 subunit.

Alkaloids↗
PubMed1995

Relation between membrane fluidity and signal transduction in the human megakaryoblastic cell line MEG-01.

The fluidity of the plasma membrane is thought to affect the responsiveness of blood platelets. We measured membrane fluidity in a single cell by Fluorescence Recovery after Photobleaching (FRAP) of the lipophilic probe DiIC14. Since platelets are too small for this technique, we used the human megakaryoblastic cell-line MEG-01, which shares many properties with platelets. MEG-01 cells were cultured for 44 h with simvastatin or mevalonate to change the cholesterol content, enabling analysis of signal processing at cholesterol/phospholipid ratios (C/P) between 0.20 and 0.31. The diffusion of DiIC14 correlated inversely with the C/P ratio with lateral diffusion coefficients (D) of 3.28 x 10(-9) cm2/s at a low C/P decreasing to 2.55 x 10(-9) cm2/s at a high C/P ratio. The mobile fraction was 65% and constant at the different C/P ratios. The relation between lipid diffusion and signal processing was measured following stimulation with 10 U/ml thrombin at 22 degrees C. There were only little differences in phosphatidylinositol metabolism, Ca2+ influx or mobilization and prostaglandin I2-induced formation of cyclic AMP. At 37 degrees C, cells with a high C/P ratio showed increased phosphatidylinositol metabolism, but these differences had no major effect on the Ca2+ responses. These data demonstrate that in megakaryoblasts the lateral diffusion of lipids is inversely correlated with the C/P ratio, but within the range of 0.20-0.31 the influence on signal processing is minor.

Adenylyl Cyclases↗
PubMed1994

Rapid alterations in lateral mobility of lipids in the plasma membrane of activated human megakaryocytes.

In the present study we measured membrane fluidity as the lateral mobility of the lipid probe 1,1'-ditetradecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate by fluorescence recovery after photobleaching in the plasma membrane of a single megakaryocyte, the progenitor cell of platelets. Megakaryocytes after 13 days in culture (maturation stage III) had a lateral diffusion coefficient (D) of (4.56 +/- 0.10) x 10(-9) cm2/s and a mobile fraction of 65 +/- 2% (means +/- SEM, n = 140). Megakaryocytes isolated from rib had a similar D and mobile fraction. Stimulation with alpha-thrombin (1-10 U/ml) induced a dose-dependent decrease in D to (3.40 +/- 0.22) x 10(-9) cm2/s between 1-5 min after stimulation (P < 0.001). The mobile fraction did not change. A similar decrease in D was found following stimulation with ADP (20 microM) and ionomycin (100 nM). Modulation of calpain I activity with calpain I inhibitor or tetracain had no effect. Pretreatment with cytochalasin B or colchicine decreased D to (3.64 +/- 0.29) x 10(-9) cm2/s (P < 0.003) and (3.96 +/- 0.18) x 10(-9) cm2/s (P < 0.013) respectively. After stimulation D decreased further in cytochalasin-treated cells (3.37 +/- 0.16) x 10(-9) cm2/s (P < 0.020) but remained at the same level in colchicine-treated cells. Both treatments increased the mobile fraction to 73-75% in stimulated megakaryocytes (P < 0.03). These data indicate that the diffusion velocity of lipids in megakaryocytes is low and decreases further after stimulation. These changes are independent of calpain I. Treatments that decrease the cytoskeletal mass and thereby increase the mobility of proteins in the plasma membrane increase the number of lipids that participate in this process.

Adenosine Diphosphate↗
PubMed1994

LDLs increase the exposure of fibrinogen binding sites on platelets and secretion of dense granules.

Because previous studies show that lipoproteins affect platelet aggregation, we studied the effect of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) on the binding of fibrinogen, which mediates platelet-platelet contact. Neither LDL nor HDL induced 125I-fibrinogen binding at concentrations up to 2 g protein/L. In contrast, platelets stimulated with 10 mumol/L ADP bound 63 734 +/- 2453 molecules of fibrinogen per platelet. A 5-minute preincubation with LDL (0.5 to 2 g/L protein) induced a dose-dependent increase to 91 307 +/- 2164 molecules of fibrinogen per platelet at 1.5 g/L, which is in the range found after optimal stimulation with alpha-thrombin. The increased fibrinogen binding in the presence of LDL resulted in faster aggregation with a 16% increase in single platelet disappearance and a faster optical aggregation at 5 mumol/L ADP and 1.5 g protein/L LDL. Inhibition of prostaglandin G2/H2-thromboxane A2 formation with indomethacin (30 mumol/L) did not change the stimulation by LDL. In contrast, modification of lysine residues of LDL, which is known to prevent specific binding to platelets, completely abolished the effect of LDL. Under the same conditions HDL did not change fibrinogen binding or aggregation. LDL also enhanced alpha-thrombin-induced [14C]serotonin secretion, but this property was not affected by lysine modification of LDL. These data indicate that LDL enhances platelet aggregation by stimulating the mechanisms that control exposure of fibrinogen binding sites on the glycoprotein IIB/IIIA complex via a mechanism that differs from the effect of LDL on secretion.

Adenosine Diphosphate↗
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↗
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↗
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↗
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↗
PubMed1986

Stimulus-aggregation coupling in platelets activated with PAF-acether.

Contrary to most agonists, platelet-activating factor (PAF-acether) induces a more pronounced aggregation at 22 degrees C than at 37 degrees C. A possible explanation was sought in the mechanism that couples the PAF-acether-receptor complex with exposure and occupation of fibrinogen binding sites. Comparison of studies performed at 37 degrees C with those at 22 degrees C revealed: a faster binding of [3H]PAF-acether to its receptors; more accumulation of 32P-labelled phosphatidylinositol 4-monophosphate and a slower but more abundant formation of phosphatidic acid that lasted for 5 min; a 1.4-fold increase in phosphorylation of the Mr 47,000 protein and a 2-fold increase in phosphorylation of the myosin light chain. In contrast, less secretion occurred and less [32P]phosphatidylinositol accumulated at 22 degrees C than at 37 degrees C, and also the increase in cytosolic Ca2+ content and the formation of thromboxane B2 were considerably lower. No differences were found in [32P]phosphatidylinositol 4,5-bisphosphate formation and arachidonate metabolism. Fibrinogen binding studies revealed two types of binding at both temperatures, a high-affinity and a low-affinity binding. There were 6-fold more low-affinity binding sites at 22 degrees C than at 37 degrees C, whereas high-affinity binding did not change. These data suggest that the better aggregation found at 22 degrees C is the result of exposure of an increased number of fibrinogen binding sites. The increased protein phosphorylation and phosphatidic acid accumulation and the faster binding of PAF-acether to its receptors which accompany the better aggregation responses at 22 degrees C suggest that these processes are involved in the regulation of exposure of fibrinogen binding sites.

Arachidonic Acid↗
PubMed1985

The energetics of early platelet responses. Energy consumption during shape change and aggregation with special reference to protein phosphorylation and the polyphosphoinositide cycle.

Among the different platelet responses, secretion requires the greatest amount of metabolic energy. The velocities of dense, alpha- and acid hydrolase granule secretion vary in parallel with the increase in energy consumption seen in thrombin-stimulated cells. This covariance is preceded by a phase in which energy consumption is increased without the extracellular appearance of secretion markers. By treating the platelets with thrombin and hirudin we have stimulated the platelets for short intervals and succeeded in separating shape change, single platelet disappearance and secretion to a great extent. In this report we show that the early increase in energy consumption reflects the energy requirement of aggregation but not of shape change. The cost of 100% of single platelet disappearance is 2.8 mumol of ATPeq. X (10(11) platelets)-1. Concurrent analysis of phosphorylation of Mr 20 000 and 47 000 proteins and of 32P-labelled phosphatidylinositol metabolites led to the following observations. Firstly, shape change is neither accompanied by an increase in protein phosphorylation nor by changes in the steady state levels of 32P-labelled phosphatidylinositol metabolites. Secondly, when aggregation occurs both proteins are phosphorylated, but the phosphatidylinositol metabolites do not change. Thirdly, when secretion follows, more phosphorylation of the Mr 47 000 protein occurs and initially only phosphatidic acid accumulates. At a later stage of the secretion responses, more protein phosphorylation and phosphatidic acid accumulation become evident, and are now accompanied by alterations in the steady state levels of 32P-labelled (poly)phosphoinositides. Hence, the early increase in energy consumption coincides with protein phosphorylation and, at a later stage, with alterations in (poly)phosphoinositides metabolites. This demonstrates that metabolic energy is directly involved in stimulus-response coupling in aggregating platelets.

Blood Platelets↗
PubMed1984

Platelet functions and energy metabolism in a patient with hexokinase deficiency.

We have studied the regeneration of adenosine triphosphate (ATP) in the glycolytic pathway in platelets with a 75% reduction in hexokinase (HK) activity and have investigated aggregation and Ca2+ secretion. HK-deficient platelets had a normal glycolytic flux in the resting state, but responded insufficiently to stimulation with thrombin (5 U/ml). In contrast, glycogen contents and glycogenolysis were normal. When the metabolic adenine nucleotides were labeled with 14C-adenine, the patient's platelets showed a normal adenylate energy charge and a normal level of 14C-ATP. However, the inhibitor of mitochondrial energy generation, CN-, induced a weaker fall in 14C-ATP in the patient's platelets than in the controls. Analysis of secretion markers revealed decreased amounts of granule-bound ATP and secretable Ca2+, whereas granule-bound adenosine diphosphate (ADP), beta-thromboglobulin, N-acetyl-beta-D-glucosaminidase, and beta-glucuronidase were within the normal range. Aggregation and Ca2+ secretion induced by 5 U/ml thrombin were normal and were not changed in the presence of inhibitors of mitochondrial and glycogenolytic energy generation. Aggregation was also normal at 0.1 U/ml thrombin and was independent of these inhibitors, but Ca2+ secretion was greatly impaired when mitochondrial and glycogenolytic ATP resynthesis was abolished. These findings indicate that a severe reduction in HK activity causes insufficient acceleration of the glycolytic flux during stimulation with thrombin. This leads to impaired dense granule secretion in conditions where secretion depends on concurrent ATP resynthesis and glycolysis is rate limiting.

Adenosine Triphosphate↗
PubMed1983

Close correlation between platelet responses and adenylate energy charge during transient substrate depletion.

The relation between availability of metabolic energy and thrombin-induced platelet aggregation and secretion was investigated in a system of transient substrate depletion followed by restoration of ATP resynthesis. Substrate depletion induced a fall in the concentration of metabolic ATP and in the adenylate energy charge and a concurrent decline in aggregation and secretion of dense and alpha-granule contents. Restoration of energy generation completely restored the adenylate energy charge and restored aggregation and secretion, but led to incomplete recovery of the ATP concentration. A close correlation between the adenylate energy charge and aggregation and between the adenylate energy charge and the secretion of dense and alpha-granule contents could be demonstrated. No such correlation existed between these responses and the concentration of ATP. These results show that the adenylate energy charge monitors an energetic condition which is crucial for preservation of platelet aggregation and secretion of dense and alpha-granule contents.

Adenine Nucleotides↗
PubMed1983

Properties of PAF-acether-induced platelet aggregation and secretion. Studies in gel-filtered human platelets.

Human platelets rapidly lose their responsiveness to PAF-acether after blood collection. We collected blood from fasting donors and prepared gel-filtered platelets that remained responsive to PAF-acether for about 6 hours. Log-dose response studies showed biphasic aggregation between 20 and 100 nM PAF-acether with secretion of dense-, alpha- and lysosomal granule contents during the second wave of aggregation. Between 0.2 and 10 nM PAF-acether aggregation was weak and no secretion occurred whereas 300 nM PAF-acether or more induced maximal aggregation and secretion. Secretion, however, was never more than 70, 55, and 30% of maximal secretable amount of 5HT, beta TG and beta N, respectively. Aggregation and secretion were enhanced by fibrinogen (optimal concentration 0.3-0.7 g.1(-1)), required Ca2+ or Mg2+ but were inhibited when Mg2+ or Ca2+ were present at a concentration of 2 mM or more. These data show that human platelets are almost equally sensitive to PAF-acether as rabbit platelets, and respond with incomplete secretion of dense-, alpha- and lysosomal granule contents.

Acetylglucosaminidase↗
PubMed1983

A novel technique for rapid determination of energy consumption in platelets. Demonstration of different energy consumption associated with three secretory responses.

A novel method has been developed for rapid and quantitative determination of the rate of energy consumption in platelets. In platelets suspended in a cyanide-containing medium. ATP resynthesis is abruptly blocked by addition of 2-deoxyglucose and D-glucono-1,5-lactone. We demonstrate that the subsequent changes in the levels of cytoplasmic ATP and ADP reflect the velocity of energy consumption in the platelets immediately before addition of the inhibitors. Despite the arrest in ATP resynthesis the platelets remain responsive to stimulation by thrombin (5 units x ml-1) which triggers the secretion of the contents of dense, alpha- and acid hydrolase granules. Unstimulated platelets were found to consume about 3.5 and 0.5 mumol of ATP equivalents x min-1 x (10(11) cells)-1 at 37 degrees C and 15 degrees C, respectively; the thrombin-treated platelets consumed respectively 16 and 2 mumol of ATP equivalents x min-1 x (10(11) cells)-1 at these temperatures. When the velocity of energy consumption was varied by (a) changing the temperature and (b) preincubation with glyco(geno)lytic inhibitors, it was found to be linearly related to the initial rate of secretion from the three types of granules. The precise nature of this relationship differed between the three types of secretion responses and indicated an increasing requirement for metabolic energy for secretion from the three types of granules in the order: dense granule less than alpha-granule less than acid hydrolase granule. The results obtained with changes in temperature were superimposable on those obtained with the glyco(geno)lytic inhibitors for dense granule secretion and alpha-granule secretion, suggesting an apparent coupling between energy consumption and the rate of these secretion responses. The rate of secretion of acid hydrolase was always higher when energy consumption was varied by temperature changes than when glyco(geno)lytic inhibitors were used, probably as a result of metabolic changes prior to induction of secretion. On the basis of these experiments, we calculated an incremental energy consumption during complete secretion of dense, alpha- and acid hydrolase granule contents of 2.5, 4.2 and 6.7 mumol of ATP equivalents x (10(11) platelets)-1, respectively.

Adenosine Triphosphate↗
PubMed1983

ATP-ADP compartmentation in storage pool deficient platelets: correlation between granule-bound ADP and the bleeding time.

Platelets contain two major compartments of ATP and ADP, the cytosol and the dense granules. We separated the two compartments by controlled digitonin-induced cell lysis and measured both fractions directly in the platelets of 16 patients with storage pool deficiency. The total contents of ATP and ADP in the platelets of these patients was significantly lower than in normal controls. This was primarily caused by decreased amounts of ATP and ADP in the granule compartment, but also cytosolic ADP was low in these patients. In contrast, cytosolic ATP was not significantly decreased. The lower the amount of granule-bound ATP and ADP, the longer was the bleeding time in these patients. The best correlation was found with granule ADP. These data may indicate that ADP stored in platelet dense granules plays a role in the arrest of bleeding.

Adenosine Diphosphate↗
PubMed1982

Kinetic analysis of alpha-granule secretion by platelets. A methodological report.

A method is described for the kinetic measurement of alpha-granule secretion by platelets. The method uses formaldehyde as a secretion-blocking reagent. This treatment alters the antigenecity of beta-thromboglobulin but not of Platelet Factor 4, both measured with commercially available reagents. Evidence is shown that this formaldehyde effect does not alter the secretion kinetics when the data are expressed as a percentage of a similarly treated reference sample. The method shows that following stimulation with thrombin or A 23187 alpha-granule secretion is much slower than dense granule secretion.

Blood Platelets↗