Phosphorylation reactions and the affinity state of platelet integrin alphaIIbbeta3.
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Journal Article↗
Biomedical subjects
J W Akkerman
Publications and source records attributed to J W Akkerman.
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↗
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↗
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↗
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↗
Platelet activation via trimeric GTP-binding proteins.
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Calcium↗
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↗
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↗
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↗
Pilot trial of the effects of low-dose acetylsalicylic acid on platelet thromboxane B2 production.
OBJECTIVE: It has been suggested that certain foods of plant origin contain milligram-quantities of acetylsalicylate which could exert an anti-thrombotic effect. Acetylsalicylic acid prevents cardiovascular diseases through inhibition of platelet endoperoxide thromboxane production and platelet aggregation. We investigated whether a daily intake of 3 mg acetylsalicylic acid causes a measurable decrease of platelet cyclo-oxygenase activity assessed by in vitro thromboxane B2 production. DESIGN: We carried out a randomised, double-blind, placebo-controlled cross-over study. SUBJECTS: Ten healthy volunteers (5 men, 5 women) aged 22 +/- 3 years (mean +/- s.d) participated in the study; there were no drop-outs. INTERVENTIONS: Participants took 3 mg/d of acetylsalicylic acid or a placebo for 2 weeks each. At the end of each treatment period venous blood was drawn, and platelet-rich plasma was stimulated with arachidonic acid. RESULTS: Treatment with acetylsalicylic acid caused a 39 +/- 8% decrease in maximal thromboxane B2 production (P = 0.000), which was independent of treatment order. CONCLUSIONS: Quantitative data on acetylsalicylate in foods and the possible antithrombotic action of a diet rich in acetylsalicylate deserve closer investigation.
Adult↗
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↗
Cytosolic calcium ions regulate lipid mobility in the plasma membrane of the human megakaryoblastic cell line MEG-01.
The fluidity of the plasma membrane is thought to play a role in the activation of blood platelets. We investigated the lateral diffusion of the lipophilic probe 1,1'-ditetradecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiIC14) and derivatives in the plasma membrane of the megakaryoblast MEG-01 by fluorescence recovery after photobleaching. The lateral diffusion coefficient (D) of DiIC14 in an unstimulated cell was (3.53 +/- 0.06) x 10(-9) cm2/s with a mobile fraction of 75%. Similar data were found with DiIC12 and DiIC18, but lipophilic probes specific for the outer leaflet showed a slower diffusion with a D value of (2.99 +/- 0.31) x 10(-9) cm2/s and a mobile fraction of 58%. Stimulation with platelet-activating agents decreased the diffusion of DiIC14 within 2 min, but left the mobile fraction unchanged. Signal processing was required for the decrease in D as D-Phenylalanyl-L-prolyl-L-arginyl-chloromethane-treated thrombin, which binds normally to the thrombin receptor but fails to activate the cell, had no effect. The decrease in D was accompanied by an increase in cytosolic Ca2+ content, [Ca2+]i, and studies using different concentrations of thrombin, the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethylester and the Ca2+ ionophore ionomycin revealed that lipid mobilty in the plasma membrane is regulated by Ca2+. In contrast, treatments thought to interfere with the mobility of membrane proteins had little effect. We conclude that the rigidification of the plasma membrane during cell activation is caused by an increase in [Ca2+]i and is therefore a late event and might only contribute to signal transduction at steps downstream of the mobilization/influx of Ca2+.
Adenosine Diphosphate↗
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↗
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↗
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↗
Development of the Baylor/NASA axial flow ventricular assist device: in vitro performance and systematic hemolysis test results.
Our newly developed axial flow pump consists of a flow tube, an internal rotating impeller, and a fixed flow stator (we call the stator) behind the impeller. This pump produces a flow of 3 to 8 L/min against 50 to 150 mm Hg pressure difference, respectively, in the range of 10,000 to 16,000 rpm. An axial flow pump that will be used as a ventricular assist device (VAD) has to have low hemolytic and good antithrombogenic characteristics. This paper will show how to decrease the hemolytic properties of this axial flow pump systematically using a test matrix. The test variables evaluated were impeller blade tip geometry, impeller flow tube clearance (radial clearance), impeller stator clearance (axial clearance), impeller blade number, stator blade number, and impeller length. All in vitro hemolysis tests were performed at 5.0 L/min against 100 mm Hg pressure difference using a total of 83 bags of fresh bovine blood. The results were as follows: the impeller blade tip geometry did not significantly effect hemolysis, a 0.005-inch and a 0.009-inch radial clearance were significantly (p < 0.01 or 0.001) less hemolytic than the other clearances, a 0.075-inch axial clearance was significantly (p < 0.05) more hemolytic than the other clearances, two- and six-bladed impellers were significantly (p < 0.01 and 0.02, respectively) less hemolytic than a four-bladed impeller, a five-bladed stator was significantly (p < 0.05 or 0.01) less hemolytic than the other stators, and the impeller length did not make a significant difference. Currently, the best index of hemolysis is 0.031 +/- 0.018 g/100 L, and using parameters from these results, implantable devices are being fabricated.
Animals↗
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↗
4,4'-Di-isothiocyanatostilbene-2,2'-disulphonic acid ('DIDS') activates protein kinase C and Na+/H+ exchange in human platelets via alpha 2A-adrenergic receptors.
Most agonists stimulate platelets by inducing Ca2+ mobilization, Ca2+ influx and protein kinase C (PKC) activation leading to Na+/H+ exchange, exposure of fibrinogen-binding sites and aggregation. In contrast, previous studies showed that adrenaline induces exposure of fibrinogen-binding sites and aggregation without appreciable changes in cystolic Ca2+ content or PKC activity. In the present study we investigated platelet responses mediated via alpha 2A-adrenergic receptors, using 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS), which is known to bind to this type of receptor. Addition of DIDS (2-20 microM) induced (i) a rise in cytosolic pH of 0.23 +/- 0.05 pH unit (n = 5) as detected by BCECF fluorescence, due to activation of the Na+/H+ exchanger, (ii) a 3.5-4-fold increase in the phosphorylation of the 47 kDa protein, a major substrate of PKC, (iii) exposure of 81,072 +/- 7293 (n = 3) binding sites for 125I-fibrinogen per platelet, and (iv) irreversible aggregation. These responses occurred without changes in cytosolic [Ca2+], secretion of dense-granule contents and enhanced phosphoinositide metabolism, and were not affected by inhibition of thromboxane A2 generation (30 microM indomethacin). The alpha 2A-adrenergic-receptor antagonists oxymetazoline (500 microM) and yohimbine (1 mM) completely abolished DIDS-induced responses. Inhibition of PKC (1 microM staurosporine) prevented phosphorylation of the 47 kDa protein, the increase in Na+/H+ exchange and exposure of fibrinogen-binding sites. Thus our present data suggest that activation of PKC is an early event in DIDS-induced platelet activation via the alpha 2A-adrenergic receptor, which precedes any of the other known signal-transducing sequences.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗