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G van Willigen

Publications and source records attributed to G van Willigen.

34 records · Page 2Linked to original sources
PubMed1997

Signal transduction through trimeric G proteins in megakaryoblastic cell lines.

The biogenesis of trimeric G proteins was investigated by measurement of the expression of alpha-subunits in the megakaryoblastic cell lines MEG-01, DAMI, and CHRF-288-11, representing stages of increasing maturation, and compared with platelets. Megakaryoblasts and platelets contained approximately equal amounts of Gi alpha-1/2, Gi alpha-3, Gq alpha, and G12 alpha protein. Maturation was accompanied by (1) downregulation of mRNA for Gs alpha and disappearance of iloprost-induced Ca2+ mobilization, (2) upregulation of the long form of Gs alpha protein (Gs alpha-L) and an increase in iloprost-induced cAMP formation, and (3) upregulation of G16 alpha mRNA and G16 alpha protein and appearance of thromboxane A2-induced signaling (Ca2+ mobilization and stimulation of prostaglandin I2-induced cAMP formation). Gz alpha protein was absent in the megakaryoblasts despite weak expression of Gz alpha mRNA in DAMI and relatively high levels of Gz alpha mRNA and Gz alpha protein in platelets. These findings reveal major changes in G protein-mediated signal transduction during megakaryocytopoiesis and indicate that G16 alpha couples the thromboxane receptor to phospholipase C beta.

Biological Transport↗
PubMed1997

Lateral mobility of integrin alpha IIb beta 3 (glycoprotein IIb/IIIa) in the plasma membrane of a human megakaryocyte.

The migration of integrins to sites of cell-cell and cell-matrix contact is thought to be important for adhesion strengthening. We studied the lateral diffusion of integrin alpha IIb beta 3 (glycoprotein IIb/IIIa) in the plasma membrane of a cultured human megakaryocyte by fluorescence recovery after photobleaching of FITC-labelled monovalent Fab fragments directed against the beta 3 subunit. The diffusion of beta 3 on the unstimulated megakaryocyte showed a lateral diffusion coefficient (D) of 0.37 x 10(-9) cm2/s and a mobile fraction of about 50%. Stimulation with ADP (20 microM) or alpha-thrombin (10 U/ml) at 22 degrees C induced transient decreases in both parameters reducing D to 0.21 x 10(-9) cm2/s and the mobile fraction to about 25%. The fall in D was observed within 1 min after stimulation but the fall in mobile fraction showed a lag phase of 5 min. The lag phase was absent in the presence of Calpain I inhibitor, where-as cytochalasin D completely abolished the decreased in mobile fraction. The data are compatible with the concept that cell activation induces anchorage of 50% of the mobile alpha IIb beta 3 (25% of the whole population of receptor) to the cytoplasmic actin filaments, although, as discussed, other rationals are not ruled out.

Biological Transport↗
PubMed1996

Targeting of porcine pancreatic phospholipase A2 to human platelets. Introduction of an RGD sequence and acyl-group by chemical modification.

In the present study we prepared by chemical modification a series of porcine pancreatic phospholipase A2 (PLA) derivatives, that bind to the activated glycoprotein (GP) IIb/IIIa complex and hydrolyse phospholipids in the outer leaflet of the platelet membrane. To the native enzyme, an RGD-containing peptide was coupled to introduce affinity for GPIIb/IIIa in combination with lauric acid to improve binding to the membrane. As controls, derivatives containing only one of these modifications were prepared. Acylation of the enzyme improved the affinity for densely packed phospholipids, as deduced by kinetic analyses. After stimulation of platelets, the RGD-containing PLAs bound to GPIIb/IIIa since GRGDS peptide and a monoclonal antibody against the complex interfered with binding. No binding was found with native PLA. The binding seen with lauric acid PLA was not mediated by GPIIb/IIIa. All modified PLAs induced 1-3% hydrolysis of [3H]arachidonic-acid-labelled phospholipids in resting platelets. After activation with alpha-thrombin, hydrolysis increased to 17%, corresponding to about 90% of [3H]arachidonate-labelled phospholipids in the outer leaflet of the plasma membrane. RGD-containing PLAs were more active than lauroyl PLA, and their activity was mediated via GPIIb/IIIa since GRGDS inhibited release of [3H]arachidonic acid. Acylation of the RGD-containing PLAs did not further improve the hydrolytic properties. We conclude that chemical modification of PLA leads to a targetted hydrolytic action and could be a basis for the design of enzymes that specifically destroy activated platelets.

Acylation↗
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

Identification of alpha-subunits of trimeric GTP-binding proteins in human platelets by RT-PCR.

In a search for new alpha-subunits of trimeric GTP-binding proteins in human platelets, we prepared leucocyte-free platelet concentrates and analyzed total RNA for areas homologous to known alpha-subunits. RT-PCR based on two degenerate primers revealed the expected band of 495 base pairs and an additional band of 540 base pairs reflecting the alternative splice product of Gs alpha. Following subcloning in pGEM-T vector and sequencing, we identified the alpha-subunits Gi alpha-2 and Gs alpha-S of the regulating GTP-binding proteins of adenyl cyclase as well as Gz alpha whose function is unknown, confirming earlier immunological identification. In addition, we identified Gs alpha-L (differing from Gs alpha-S by an insertion of 45 base pairs), G16 alpha, (a member of the pertussis toxin insensitive Gq-family), and two new variants of both Gs alpha-S and Gs alpha-L each containing a C-A-G triplet. With G16 we have identified another candidate for pertussis-toxin insensitive signal transduction in platelets. The C-A-G containing sequences of Gs alpha lead to an insertion of a Ser-residue, which results in the consensus sequence of a phosphorylation site for protein kinase C (Ser-X-Lys), making these variants candidates for protein kinase C-sensitive cyclic AMP formation.

Amino Acid Sequence↗
PubMed1995

Regulation of platelet glycoprotein IIb/IIIa (integrin alpha IIB beta 3) function via the thrombin receptor.

Binding sites on glycoprotein (GP) IIb/IIIa exposed by 0.5 unit/ml alpha-thrombin are insensitive to prostaglandin I2 (PGI2), in contrast with sites exposed by ADP or platelet-activating factor. Here we show that the thrombin receptor agonist peptide (TRAP) (SFLLRN; 15 microM) opens almost the same number of GPIIb/IIIa molecules as 0.5 unit/ml alpha-thrombin (64840 +/- 8920 compared with 81050 +/- 6030 molecules of fibronectin bound/platelet), but these sites rapidly close on addition of PGI2. To investigate whether alpha-thrombin and TRAP initiate different signalling pathways, we measured phospholipase C (PLC)-mediated control of GPIIb/IIIa and its sensitivity to cyclic AMP. Optimal concentrations of alpha-thrombin and TRAP activated PLC maximally, but TRAP induced only about 50% protein kinase C PKC) activation after 10 min stimulation compared with alpha-thrombin. These concentrations also suppressed PGI2-induced cyclic AMP accumulation, with alpha-thrombin inducing complete inhibition and TRAP about 10% less. Direct activation of PKC by phorbol 12-myristate 13-acetate confirmed earlier observations that PGI2-induced cyclic AMP accumulation is partly inhibited via PKC. Applying different concentration of alpha-thrombin, TRAP or a combination of alpha-thrombin and the thrombin receptor inhibitory peptide (TRIP) (Mpr-F-Cha-Cha-RKPNDK-NH2; 800 microM) (Mpr, 3-mercaptopropionic acid; Cha, cyclohexylalanine), we show that the different means of stimulating the thrombin receptor all suppressed PGI2-induced cyclic AMP accumulation via (i) activation of PKC and (ii) activation of the heterotrimeric G-protein, Gi. We conclude that complete inhibition of cyclic AMP accumulation requires activation of both PKC and Gi, as observed with 0.5 unit/ml alpha-thrombin. Although TRAP almost fully exposes GPIIb/IIIa, its activation of PKC is incomplete, enabling PGI2 to raise cyclic AMP concentration from 1.4 +/- 0.7 to 4.1 +/- 1.3 nmol/10(11) platelets (P < 0.005) which is sufficient to close exposed GPIIb/IIIa molecules.

Adenosine Diphosphate Ribose↗
PubMed1995

Targeting of porcine pancreatic phospholipase A2 to human platelets: introduction of an RGD sequence by genetic engineering.

The possibility to induce specific disruption of activated platelets by binding of porcine pancreatic phospholipase A2 (PLA2) was tested by constructing a set of PLA2-mutants containing an Arg-Gly-Asp (RGD) sequence. One mutant was made with RGD as part of a surface-exposed loop (RGDloop). Four mutants were made with RGD as part of a C-terminal extension: one with RGD directly coupled to the C-terminus (RGDc) and three mutants (CRSx) with x= 22, 42 and 82 hydrophylic non-charged amino acids between RGD and the enzyme. All mutants retained 20-80% activity of native PLA2 and showed little binding to resting platelets. The binding of the native enzyme and RGDloop was not increased following stimulation. In contrast, the mutants RGDc and CRSx showed stimulation-dependent binding to the platelet receptor GPIIb/IIIa, since GRGDS-peptide and a monoclonal antibody against the complex interfered with binding. In alpha-thrombin-stimulated platelets, CRS42 and CRS82 induced about 5% hydrolysis of [3H]-arachidonic acid-labeled phospholipids. Stimulation with a combination of alpha-thrombin and collagen (known to expose phosphatidylserine) increased hydrolysis to 11%. Despite the membrane disruption, the cells did not leak lactate dehydrogenase. We conclude that PLA2 can be targeted to activated platelets by introducing RGD in a C-terminal extension with a minimum distance (42 amino acids) between RGD and the enzyme. However, more hydrolytic activity is required to eliminate activated platelets among a suspension of resting platelets and other blood cells.

Amino Acid Sequence↗
PubMed1994

Alpha 2A-adrenergic receptors activate protein kinase C in human platelets via a pertussis toxin-sensitive G-protein.

4,4'-Diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) stimulates human platelets via alpha 2A-adrenergic receptor-mediated activation of protein kinase C (PKC) independent of the phospholipase C pathway. Here we show, that in permeabilized platelets activation of PKC by DIDS (20 microM), measured as 32P incorporation in pleckstrin, is completely inhibited by guanosine 5'-(2-O-thio)diphosphate (200 microM), an inhibitor of heterotrimeric G-proteins. Also pertussin toxin (4 micrograms/ml), which ADP-ribosylates the alpha-subunits of Gi's and Go, prevents pleckstrin phosphorylation by DIDS. N-Ethylmaleimide (50 microM), which uncouples Gi from alpha 2A-adrenoceptors, inhibits pleckstrin phosphorylation by DIDS in intact platelets. Activation of PKC by 55 nM phorbol 12-myristate 13-acetate and 500 nM platelet-activating factor are not disturbed by NEM. DIDS inhibits by 40 +/- 5% (n = 4) the pertussis toxin-catalyzed [32P]ADP-ribosylation of a 41 kDa protein fraction previously shown to contain the alpha-subunits of Gi alpha-1, Gi alpha-2 and Gi alpha-3. Thus, the alpha 2A-adrenergic receptor activates PKC via a G-protein of the Gi-family.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Different pathways for control of Na+/H+ exchange via activation of the thrombin receptor.

The aim of the present study was to clarify the control of Na+/H+ exchange in platelets activated via the thrombin receptor. When human BCECF-loaded platelets were stimulated with the thrombin-receptor-activating peptide (TRAP; amino acid sequence SFLLRN), which activates the receptor independently of proteolysis, the cytosolic pH (pHi) rose from 7.13 +/- 0.04 (n = 6) to 7.27 +/- 0.04 (n = 5), followed by a rapid decrease to resting values. Trypsin, which cleaves the receptor, induced a rapid and irreversible rise in pHi to 7.31 +/- 0.06 (n = 5). gamma-Thrombin, which cleaves the receptor but is unable to bind to the hirudin-like domain, induced a slow and irreversible rise in pHi to 7.31 +/- 0.04 (n = 14). alpha-Thrombin, which cleaves the receptor and binds to its hirudin-like domain, induced a rapid and irreversible rise in pHi to 7.31 +/- 0.04 (n = 22). Changes in pHi induced by TRAP, trypsin, gamma- and alpha-thrombin were accompanied by similar changes in cytosolic Ca2+ concentration ([Ca2+]i) and 32P-pleckstrin, a substrate of protein kinase C (PKC). The separate chelation of Ca2+i (30 microM BAPTA-AM) or inhibition of PKC (1 microM staurosporine) induced about 50% inhibition of the pHi responses triggered by TRAP, trypsin, gamma- and alpha-thrombin, but the combination induced complete inhibition. Thus the different types of activation of the thrombin receptor control Na+/H+ exchange via the same mechanism. Binding of thrombin to the hirudin-like domain accelerates exchange activation, whereas proteolysis of the receptor is essential for a sustained increase in pHi.

Alkaloids↗
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↗
PubMed1994

Suitability of low-molecular-weight heparin(oid)s and a pentasaccharide for an in vitro human thrombosis model.

There is much interest in in vitro thrombosis systems using exclusively human materials for evaluating new drugs. We have previously developed such a model using a perfusion chamber in which whole blood anticoagulated with low-molecular-weight heparin (LMWH) was circulated over the extracellular matrix of endothelial cells that had been stimulated with phorbol myristate acetate to cause tissue factor formation. Here we studied various LMWHs and a pentasaccharide to find out which was most useful in an in vitro thrombosis model. We compared unfractionated heparin, two commercial LMWHs (Fragmin and Fraxiparine), one commercial heparinoid (Orgaran), and a chemically synthesized derivative of the natural pentasaccharide (Org 31550). Blood was anticoagulated with the concentration of each glycosaminoglycan that prevented thrombin formation for at least 3 hours in the test tube (Fragmin, 20 anti-Xa U/mL; Fraxiparine, 40 anti-Xa Institute Choay U/mL; Orgaran, 15 anti-Xa U/mL; Org 31550, 200 anti-Xa U/mL; unfractionated heparin, 5 IU/mL) and recirculated over the matrix of unstimulated cells (no tissue factor in the matrix) and phorbol-stimulated endothelial cells (tissue factor in the matrix). Platelet adhesion, aggregate formation, and fibrin deposition were evaluated. In perfusions over the extracellular matrix of unstimulated cells, the highest platelet adhesion rates were observed with Orgaran. Fibrin deposition was absent with unfractionated heparin in phorbol-stimulated matrix. Increasing amounts of fibrin were observed with Orgaran, Fragmin, and Fraxiparine. Most fibrin was found with the pentasaccharide Org 31550.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Coagulation↗
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↗
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↗
PubMed1989

Different evolution rates within the lens-specific beta-crystallin gene family.

We have determined the sequence of a rat beta A3/A1-crystallin complementary DNA (cDNA) clone and the (partial) sequence of the human beta B3-crystallin gene. Calculation of the ratio of silent to nonsynonymous substitution between orthologous beta A3/A1-, beta B3-, and other beta- and gamma-crystallin sequences revealed that the region encoding the two globular domains of the beta A3/A1-crystallin sequence is the best conserved during evolution, much better than the corresponding region of the beta B1-, beta B3-, or the gamma-crystallin sequences, and even better (at least in the rodent/frog comparison) than the well-conserved alpha A-crystallin sequence. Remarkably, the rate of change of the beta A3/A1-crystallin coding sequence does not differ in the rodent and primate lineages, in contrast with previous findings concerning the evolution rates of the alpha A- or gamma-crystallin sequences in these two lineages. Comparison of the regions that encode the four motifs of the beta-crystallin between orthologous mammalian sequences showed that the extent of nonsynonymous substitution in each of these four homologous motif regions is the same. However, when the orthologous beta-crystallin genes of more distantly related species (mammals vs chicken or frog) are compared, the extent of non-synonymous substitution is higher in the regions encoding the external motifs I and III than in the regions encoding the internal motifs II and IV. This phenomenon is also observed when paralogous members of the beta/gamma-crystallin supergene family are compared.

Amino Acid Sequence↗
PubMed1984

Attachment of origins of replication to the nuclear matrix and the chromosomal scaffold.

We have investigated the attachment of DNA to the nuclear matrix and chromosomal scaffold in synchronized bovine liver cells. Label incorporated at the onset of the S phase remained preferentially associated with the matrix during the subsequent G1 phase and with a residual protein structure from dehistonized chromosomes during mitosis. On the other hand label incorporated during mid or late S phase was about equally distributed over the DNA molecule after a chase into the G1 phase. These results suggest that DNA is attached to the nuclear matrix and chromosome scaffolds by the origins of replication.

Animals↗