Regulation of Rac1 and Cdc42 activation in thrombin- and collagen-stimulated CHRF-288-11 cells

Rac1 and Cdc42 are members of the Rho family of small GTPases and have been shown to promote the formation of lamellipodia and filopodia at the leading edge of motile cells and affect cell migration. In this study the authors have investigated the activation of Rac1 and Cdc42 by thrombin or collagen using the megakaryocytic cell line, CHRF-288-11. Maximal activation of Rac1 by thrombin or collagen was observed at 3 and 1 min, respectively. Similar results were obtained for the activation of Cdc42 by thrombin or collagen. The activation of Rac1 by thrombin and collagen was inhibited by the calmodulin-specific inhibitor, W7. However, W7 had no effect on the activation of Cdc42 by thrombin or collagen. The less potent calmodulin inhibitor, W5, did not have any effect on Rac1 or Cdc42 activation by thrombin or collagen. Transient over-expression of calmodulin in CHRF cells increased the basal and thrombin-mediated activation of Rac1 when compared to control but had no effect on the basal and thrombin-mediated activation of Cdc42 when compared to control. The results suggest that calmodulin regulates the activation of Rac1 in CHRF-288-11 cells in a manner similar to that in the human platelets.     

Possible involvement of cytoskeleton in collagen-stimulated activation of phospholipases in human platelets

The action of phospholipases A2 and C in the course of collagen-stimulated platelet activation and the effect of cytochalasins on the responses were studied. Stimulation of human platelets with collagen was accompanied by aggregation, Ca2+ mobilization, inositol phosphate formation, and arachidonic acid release. However, in the presence of a cyclooxygenase inhibitor or a thromboxane A2 (TXA2) receptor antagonist, collagen induced only weak arachidonic acid release and weak inositol phosphate formation. The TXA2 mimetic agonist U46619 induced all the responses except for arachidonic acid release, which was induced by synergistic action of collagen and U46619. The result that U46619 did not induce arachidonic acid release despite the activation of phospholipase C suggested that arachidonic acid was not released via phospholipase C but by phospholipase A2. These findings suggested that collagen initially induced weak activation of phospholipases A2 and C and that further activation of phospholipase C as well as Ca2+ mobilization and aggregation were induced by TXA2, whereas further activation of phospholipase A2 required the synergistic action of collagen and TXA2. Platelets pretreated with cytochalasins did not respond to collagen. Further analysis revealed that the initial activation of phospholipases A2 and C was specifically inhibited by cytochalasins, but the responses induced by U46619 or a synergistic action of collagen and U46619 were not inhibited. Therefore, we proposed that interaction of collagen receptor with actin filaments might have some roles in the collagen-induced initial activation of phospholipases.     

Mobilization of arachidonic acid in collagen-stimulated human platelets.

Stimulation of platelets with collagen results in the mobilization of arachidonic acid (AA) from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI). In this study the effect of aspirin, indomethacin, BW755C and prostaglandin H2 (PGH2) on labelled AA release in response to varied concentrations of collagen was investigated. Our results indicate that aspirin (0.56 mM) and indomethacin (5.6 microM) not only inhibited the collagen-mediated formation of cyclo-oxygenase metabolites, but also caused a significant reduction in the accumulation of free labelled AA and 12-hydroxyeicosatetraenoic acid (12-HETE) (21-64%). Aspirin and indomethacin also inhibited the release of [3H]AA from PC (37-75%) and PI (33-63%). The inhibition of AA release caused by aspirin was reversed partially by PGH2 (1 microM). In contrast, a smaller/no inhibition of collagen-stimulated labelled AA and 12-HETE accumulation (0-11%) and of collagen-stimulated AA loss from PC and PI was observed in the presence of BW755C. The results obtained in the presence of aspirin, indomethacin and BW755C at lower concentrations of collagen further demonstrate that AA release from PI (45-61% inhibition at 10 micrograms of collagen), but not from PC, was affected by the inhibition of cyclo-oxygenase. The results obtained on the effect of PGH2 further support that deacylation of phospholipids occurs independently of cyclo-oxygenase metabolites, particularly at higher concentrations of collagen. These results also demonstrate that aspirin and indomethacin, but not BW755C, cause a direct inhibition of collagen-induced [3H]AA liberation from PC as well as from PI. We also conclude that the diacylglycerol lipase pathway is a minor, but important, route for AA release from PI in collagen-stimulated human platelets. The mechanisms underlying the regulation of AA release by collagen in the absence of cyclo-oxygenase metabolites are not clear.     

Differential effects of 15-HPETE on arachidonic acid metabolism in collagen-stimulated human platelets

The 15-hydroperoxyeicosatetraenoic acid (15-HPETE) has been shown to affect platelet aggregation induced by collagen, arachidonic acid (AA), and PGH2-analogue. Furthermore, it also inhibits the platelet cyclooxygenase and lipoxygenase enzymes, and prostacyclin synthase. The present study was designed to test the effect of 15-HPETE on the mobilization of endogenous AA in collagen-stimulated human platelets. For this purpose, human platelets pretreated with BW755C (a dual inhibitor of cyclooxygenase and lipoxygenase) were stimulated with collagen in the presence of varied concentrations of 15-HPETE. We observed a significant inhibition of oxygenases at all concentrations of 15-HPETE. In contrast, our results indicate that 15-HPETE at lower concentrations (10 microM and 30 microM) significantly stimulated the collagen-induced release of AA from phospholipid sources. Although higher concentrations of 15-HPETE (50 microM and 100 microM) caused some inhibition of AA accumulation in the free fatty acid fraction (25% and 60%), the degree of inhibition was significantly lower than the inhibition observed for the oxygenases (65% and 88% for cyclooxygenase and 77% and 94% for lipoxygenase respectively). These results provide support that hydroperoxides also regulate phospholipases presumably by a different mechanism, which may be important in the detoxification of phospholipid peroxides.     

The kinetics of thrombin- and SFLLRN-induced aggregation of human platelets in flow through tubes

The kinetics of aggregation of human platelets activated by alpha-thrombin (0.17-0.35 nM) and the hexapeptide SFLLRN (2-10 microM) was studied in plasma-free washed cell suspensions undergoing Poiseuille flow at 37 degrees C using a previously described double infusion technique. Platelet-rich Tyrodes, prepared from venous blood by multiple centrifugation, and agonist were rapidly mixed in a small chamber and the suspension flowed through various lengths of 1.19 and 0.76 mm diameter polyethylene tubing at mean transit times t from 0.2 to 43 s and mean tube shear rates = 41.9, 335, and 1335 s-1. Effluent was collected in 0.5% glutaraldehyde and single cells and aggregates in the volume range 1-10(5) micron 3 counted and sized using an aperture impedance counter. The rate and extent of aggregation with thrombin increased with increasing [thrombin] and , and although characterized by a small initial lag time, exhibited a very rapid growth of aggregates to macroscopic size, > 10(5) micron 3, at low and moderate shear rates. With SFLLRN, the initial lag times were appreciably longer, but subsequently aggregates also rapidly grew to macroscopic size. We hypothesize that the initial lag time is due to the time required for sufficient secretion and surface organization of ligands such as vWF (known to be released by the platelet) to occur, in order for cross-bridging of the GPIIb-IIIa receptors on adjacent platelets to take place. It appears that thrombin, which, at the low concentrations used, primarily activates the platelet via binding to the GPIb alpha receptor, can more rapidly facilitate secretion of the ligand than SFLLRN, which activates the cell via binding to the seven transmembrane domain receptor.     

Redistribution of alpha-granules and their contents in thrombin- stimulated platelets

The redistribution of beta-thromboglobulin (beta TG), platelet Factor 4 (PF4), and fibrinogen from the alpha granules of the platelet after stimulation with thrombin was studied by morphologic and immunocytochemical techniques. The use of tannic acid stain and quick-freeze techniques revealed several thrombin-induced morphologic changes. First, the normally discoid platelet became rounder in form, with filopodia, and the granules clustered in its center. The granules then fused with one another and with elements of the surface-connected canalicular system (SCCS) to form large vacuoles in the center of the cell and near the periphery. Neither these vacuoles nor the alpha granules appeared to fuse with the plasma membrane, but the vacuoles were connected to the extracellular space by wide necks, presumably formed by enlargement of the narrow necks connecting the SCCS to the surface of the unstimulated cell. The presence of fibrinogen, beta TG, and PF4 in corresponding large intracellular vacuoles and along the platelet plasma membrane after thrombin stimulation was demonstrated by immunocytochemical techniques in saponin-permeabilized and nonpermeabilized platelets. Immunocytochemical labeling of the three proteins on frozen thin sections of thrombin-stimulated platelets confirmed these findings and showed that all three proteins reached the plasma membrane by the same pathway. We conclude that thrombin stimulation of platelets causes at least some of the fibrinogen, beta TG, and PF4 stored in their alpha granules to be redistributed to their plasma membranes by way of surface-connected vacuoles formed by fusion of the alpha granules with elements of the SCCS.     

Biphasic action of phospholipase A in collagen-stimulated rat platelets

The formation of thromboxane A2 (TXA2) in collagen-stimulated rat platelets was successfully divided into two stages, an initial and a second one, by the specific TXA2 receptor antagonist, ONO3708. In the presence of this antagonist, only the initial TXA2 production was observed, without the subsequent platelet shape change and aggregation. Collagen causes the specific cleavage of arachidonic acid from phosphatidylinositol (PI) in the initial stage, whereas in the absence of the antagonist, it caused decrease in the arachidonic acid levels in phosphatidylethanolamine (PE) and PI with concomitant formation of the respective lyso-forms. These results demonstrate that phospholipase A (PLA) preferentially acts on PI to release arachidonic acid which leads to the initial TXA2 production, which might be a trigger for the second release of arachidonic acid from PE and PI.     

Signal transduction in collagen-stimulated rat platelets is composed of three stages

A stable thromboxane A2 (TXA2) mimetic, U46619, induced a shape change of rat platelets, but did not induce phosphoinositide breakdown, aggregation or secretion. However, when U46619 was added to platelets which had been previously stimulated with collagen in the presence of indomethacin, all biological responses were induced about 1 min after the occurrence of shape change. Furthermore, two phases of phosphorylation of myosin light chain (MLC) were observed under the same conditions, one coinciding with shape change and the other with aggregation. Similar two-phase Ca2+ mobilization has been observed using aequorin (Nakano, T., Terawaki, A., & Arita, H. (1986) J. Biochem. 99, 1285-1288). From these results, collagen-induced signal transduction is considered to be composed of three stages. The first stage is the initial TXA2 generation. The second stage involves inositol trisphosphate-independent first-phase Ca2+ mobilization and the first-phase MLC phosphorylation by the action of TXA2 alone, leading to the shape change of platelets. The third stage is initiated by an abrupt phosphoinositide breakdown via the synergistic action of TXA2 and occupation of the collagen receptor, and the resulting inositol trisphosphate may induce the second-phase Ca2+ mobilization to produce the second-phase MLC phosphorylation together with aggregation and secretion.     

Mechanism of collagen activation in human platelets

The mechanism of collagen-induced human platelet activation was examined using Ca2+, Na+, and the pH-sensitive fluorescent dyes calcium green/fura red, sodium-binding benzofuran isophthalate, and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Administration of a moderate dose of collagen (10 microg/ml) to human platelets resulted in an increase in [Ca2+](i) and platelet aggregation. The majority of this increase in [Ca2+](i) resulted from the influx of calcium from the extracellular milieu via the Na+/Ca2+ exchanger (NCX) functioning in the reverse mode and was reduced in a dose-dependent manner by the NCX inhibitors 5-(4-chlorobenzyl)-2',4'-dimethylbenzamil (KD(50) = 4.7 +/- 1.1 microm) and KB-R7943 (KD(50) = 35.1 +/- 4.8 microm). Collagen-induced platelet aggregation was dependent on an increase in [Ca2+](i) and could be inhibited by chelation of intra- and extracellular calcium through the administration of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) and EGTA, respectively, or via the administration of BAPTA-AM to platelets suspended in no-Na+/HEPES buffer. Collagen induced an increase in [Ca2+](i) (23.2 +/- 7.6 mm) via the actions of thromboxane A(2) and, to a lesser extent, of the Na+/H+ exchanger. This study demonstrates that the collagen-induced increase in [Ca2+](i) is dependent on the concentration of Na+ in the extracellular milieu, indicating that the collagen-induced increase in [Ca2+](i) causes the reversal of the NCX, ultimately resulting in an increase in [Ca2+](i) and platelet aggregation.     

Inhibition of phosphatidic acid production and lysophospholipid formation in collagen-stimulated human platelets by staurosporine, a protein kinase inhibitor

To investigate a possible regulatory role of protein kinase C (PKC) on collagen-induced phospholipase activity, human platelets were prelabelled with either [3H] arachidonic acid or [14C]stearic acid and stimulated with collagen (2 micrograms/ml) in the presence or absence of the protein kinase inhibitor, staurosporine (1 microM). The collagen-induced release of [3H]arachidonic acid and formation of [14C]stearoyl-labelled lysophospholipids was inhibited by prior incubation with staurosporine, as was the formation of 3H-labelled thromboxane B2, thereby suggesting inhibition of the collagen-induced phospholipase A2 activity. The degradation of phosphatidylinositol (PI) and elevation of phosphatidic acid (PA) in platelets prelabelled with either radiotracer were also completely blocked by staurosporine pretreatment, indicating a suppression of collagen-stimulated phospholipase C activity. Suppressed phospholipase C activity may have been due to diminished thromboxane A2 formation since treatment with the dual cyclo-oxygenase/lipoxygenase inhibitor, BW755C, also resulted in an inhibition of the collagen-stimulated loss of 14C-labelled PI and rise in PA by 75-80%. Our results suggest that protein kinase, possible PKC, may be involved in the regulation of these phospholipases in collagen-stimulated human platelets.     

Role of calcium on protein phosphorylation in collagen-stimulated platelets. Effect of a new dihydropyridine derivative

We investigated the effect of extracellular calcium on protein phosphorylation stimulated by collagen in rabbit platelets. We found that collagen-induced increase in 40 kDa protein phosphorylation was maximum at 2 mM Ca2+, and was evident in buffer with zero Ca2+ but not in the presence of EGTA. We also studied the effects of a new dihydropyridine derivative, which has antithrombotic properties, on protein phosphorylation induced by collagen. This compound inhibited the phosphorylation of 40 kDa and 20 kDa protein independently of the extra-cellular Ca2+. The inhibitory effect was dose-dependent but not time-dependent and was more evident when the drug was added before or simultaneously with collagen.     

Isolation of palmitoyl-CoA hydrolases from human blood platelets.

The palmitoyl-CoA hydrolase activity, which in human blood platelets is mainly localized in the cytosol fraction [Berge, Vollset & Farstad (1980) Scand. J. Clin. Lab. Invest. 40, 271--279], was found to be extremely labile. Inclusion of glycerol or palmitoyl-CoA stabilized the activity during preparation. Gel-filtration studies revealed multiple forms of the enzyme with molecular weights corresponding to about 70 000, 40 000 and 24 000. The relative recovery of the mol.wt.-70 000 form was increased by the presence of 20% (v/v) glycerol or 10 microM-palmitoyl-CoA. The three enzyme forms are probably unrelated, since they were not interconvertible. The three different species of palmitoyl-CoA hydrolase were purified by DEAE-cellulose and hydroxyapatite chromatography, isoelectric focusing and high-pressure liquid chromatography (h.p.l.c.) to apparent homogeneity. The three enzymes had isoelectric points (pI) of 7.0, 6.1 and 4.9. The corresponding molecular weights were 27 000--33 000, 66 000--72 000 and 45 000--49 000, calculated from h.p.l.c. and Ultrogel AcA-44 chromatography. The apparently purified enzymes were unstable, as most of the activity was lost during purification. The enzyme with an apparent molecular weight of 45 000--49 000 was split into fractions with molecular weights of less than 10 000 by re-chromatography on h.p.l.c. concomitantly with a loss of activity. The stimulation of the activity by the presence of serum albumin seems to depend on the availability of palmitoyl-CoA, as has been reported for other palmitoyl-CoA hydrolases. [Berge & Farstad (1979) Eur. J. Biochem. 96, 393--401].     

Adenine nucleotide metabolism of blood platelets. VIII. Transport of adenine into human platelets

Adenine uptake into human blood platelets is a carrier-mediated process with a K_m of 159±21 nM and a V of 100±10 pmoles/min per 10⁹ platelets (in citrated platelet-rich plasma). The Q₁₀ was 2.53±0.22. A pH optimum was found at 7.5. Washing of the platelets increased the K_m to 453±33 nM and V to 397±38 pmoles/min per 10⁹ platelets. The change in shape induced in platelets by ADP was accompanied by an increase in V (2 times) and K_m (1.5 times).Guanine (K_i 50 μM), hypoxanthine (K_i 390 μM), adenine-N′-oxide (K_i 40 μM), adenosine (K_i 100 μM), RA 233 (K_i 75 μM) and papaverine (K_i 15 μM) acted as competitive inhibitors. Adenosine at low concentrations, and prostaglandin E₁ gave inhibition at only high adenine levels. A similar inhibition was obtained with 2-deoxy-d-glucose. Sulfhydryl-group inhibitors, pyrimidines and ouabain had no effect.