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Tyrosine phosphorylation events during different stages of collagen-platelet activation
Biochimica et Biophysica Acta 1405 (1998) 128^138
Tyrosine phosphorylation events during di¡erent stages of collagen-platelet activation Urszula Kralisz *, Czeslaw S. Cierniewski Department of Biophysics, Institute of Physiology and Biochemistry, Medical University of Lodz, Lindleya 3, 90-131 Lodz, Poland Received 13 May 1998; revised 30 July 1998; accepted 10 August 1998
Abstract Three groups of phosphoproteins have been distinguished, basing on the velocity and extent of phosphorylation in platelets stimulated with collagen. pp60cÿsrc constituted the first group; the increase in its phosphorylation was the highest and most rapid (maximal in 30 s after the addition of collagen). pp80/85 and non-identified protein of 65 kDa formed the second group; the increase in their phosphorylation was twice smaller than that of pp60cÿsrc , and reached its maximum 60 s after the addition of collagen. pp120, pp72syk , and two non-identified phosphoproteins of 90 and 75 kDa constituted the third group; the increase in their phosphorylation was 4^10-fold lower than that of pp60cÿsrc and reached its maximum after 180 s. We conclude that the phosphorylation of pp60cÿsrc is important for the change of shape of platelets, the phosphorylation of pp80/85 and pp65 for the initiation of the formation of aggregates and the phosphorylation of the third group of phosphoproteins for the formation of massive aggregates. This conclusion was supported by using a monoclonal anti-GPIb antibody, which did not inhibit the shape change of platelets and did not inhibit pp60cÿsrc phosphorylation. This antibody inhibited aggregate formation as well as tyrosine phosphorylation of proteins belonging to the second and the third group of phosphoproteins. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Platelet; Collagen; Activation; Aggregation; Tyrosine phosphorylation
1. Introduction Fibrillar collagen, one of the most thrombogenic agents of the vessel wall [1], interacts with platelets by several mechanisms which can be divided into direct (primary) and indirect (secondary). Primary
Abbreviations: PGE1 , prostaglandin E1 ; SDS, sodium dodecyl sulfate; PMSF, phenylmethylsulfonyl£uoride ; NEM, N-ethylmaleimide ; PAGE, polyacrylamide gel electrophoresis ; TxA2 , thromboxane A2 ; HEPES, 4-(2-hydroxyethyl)1-piperazine-ethanesulfonic acid; PBS, phosphate-bu¡ered saline ; BME, 2-mercaptoethanol; PAF, platelet -activating factor; ASA, acetylsalicylic acid * Corresponding author. Fax: +48 (42) 678-9433.
mechanisms are mediated by components present on the surface of unactivated platelets, while secondary mechanisms involve receptors expressed only upon activation of platelets. Although several platelet proteins have been proposed to be primary collagen receptors, i.e. collagen-glucosyl transferase [2], ¢bronectin [3], 65-kDa protein [4], GPIIb^IIIa [5,6], GPIa^IIa [7^12], factor XIII [13], glycoprotein VI [14^16] and glycoprotein IV [17^19], only a few of them have gained general acceptance. The strongest evidence supporting such a role for GPIa^IIa and GPVI has come from clinical studies. Patients de¢cient in GPIa^IIa and GPVI were described to have impaired or lack collagen-induced aggregation and adhesion [7,8,14^16]. There is some evidence that
0167-4889 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 8 9 ( 9 8 ) 0 0 1 0 0 - 1
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glycoprotein IV (CD36) functions at the earliest stages of collagen adhesion as a platelet collagen receptor although CD36 de¢cient platelets adhere normally to collagen types I, III and IV. CD36 de¢cient platelets show also normal aggregation induced by collagen. CD36 seems to be essential for collagen type V-induced platelet interaction although the physiological importance of such interaction is questionable [19^23]. There is general agreement on the involvement of GPIIb^IIIa in mediating secondary adhesion through macromolecules released from activated platelets or present in plasma, such as ¢bronectin, von Willebrand's factor (vWf) and thrombospondin (TSP) [24]. The role of GPIb in mediating shear-dependent platelet adhesion is also well characterized through studies with Bernard^Soulier platelets. The primary role of GPIb is to bind to vWF on exposed subendothelium. The GpIb is expressed on unactivated platelets, but is active at high shear rates [25,26]. Platelets show high levels of non-receptor protein tyrosine kinase (PTK) activity, largely due to the high concentration of pp60cÿsrc and pp72syk as well as other PTKs, such as pp60fyn , pp54/58lyn , pp62hck , pp62yes and pp125FAK [27^30]. The activation of platelets with many agonists, such as thrombin, collagen and platelet -activating factor (PAF), leads to many events (including shape change, secretion, binding of soluble ¢brinogen to its receptor and aggregation) that are thought to be regulated through a number of intracellular signalling pathways, including PTK activation [31^34]. Collagen-induced platelet activation di¡ers in many ways from the activation induced by soluble activators, such as thrombin. Firstly, cyclic AMP does not inhibit collagen-induced platelet signal transduction, whereas it inhibits the signal transduction induced by ADP, thrombin and a thromboxane mimetic U-46619 [35,36]. Secondly, collagen-induced platelet stimulation involves the tyrosine phosphorylation-dependent activation of phospholipase CQ2, while that induced by several other agonists does not [37^39]. Thirdly, collagen is the only physiological platelet agonist that has been identi¢ed to induce phosphorylation of the FcRQ chain enabling binding of pp72syk and the assembly of a signaling complex independently from phospholipase C (PLC) activity [39^41]. The cytoplasmic tail of the FcRQ chain contains an immunoreceptor tyro-
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sine-based activation motif (ITAM). The association of pp72syk with FcRQ chain appears to be mediated through the tandem SH2 domains in pp72syk and the ITAM motif of this receptor [38,40,41]. For the purpose of this study, we have concentrated on the role of protein phosphorylation during di¡erent stages of platelet activation induced by collagen. The data demonstrated an increase in tyrosine phosphorylation of many proteins upon collagen treatment. Three groups of phosphoproteins were distinguished based on the velocity and extent of phosphorylation. The pp60cÿsrc belongs to the ¢rst group since the increase in its phosphorylation was the highest and most rapid. The phosphorylation of pp60cÿsrc was found to be important for the early activation events concerning the change of shape of platelets. The phosphorylation of the second and third group of proteins was important for the later stages of collagen-induced activation connected with the aggregates formation. These ¢ndings were supported by the use of anti-Ib monoclonal antibody PO14, which neither inhibited the shape change nor the pp60cÿsrc phosphorylation. PO14 inhibited aggregation as well as tyrosine phosphorylation of the proteins belonging especially to the third group. 2. Materials and methods Fibrillar equine tendon collagen type I was from Chrono-Log. Anti-phosphotyrosine monoclonal IgG 4G10 and monoclonal anti-src, pp80/85, pp120 as well as polyclonal anti-syk antibodies were from Upstate Biotechnology. A monoclonal antibody PO14 (clone name 105.12B12H3) against GPIb was a generous gift from Dr. C. de Romeuf (Hemostasis Research Laboratories, Lille, France). Rainbow protein molecular weight markers were from Amersham. All other chemicals were purchased from Sigma. 2.1. Preparation of human platelets Washed platelets were prepared from human blood anticoagulated with ACD (citrite acid^citrate^dextrose) obtained from volunteer donors as described previously, with some minor modi¢cation [17,42]. Brie£y, platelet-rich plasma was obtained by centrifuging the whole blood at 2000Ug for 20 min
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at 22³C. For the preparation of the washed platelets, the pH of the platelet-rich plasma was adjusted to 6.5 with citric acid (0.2 M) and made 1 Wg/ml with respect to prostaglandin E1 . Then, the platelets were sedimented by centrifugation at 1000Ug for 10 min and the pellet was resuspended in the platelet wash bu¡er (5.5 mM dextrose, 128 mM NaCl, 4.26 mM Na2 HPO4 , 7.46 mM NaH2 PO4 , 4.77 mM trisodium citrate, and 2.35 mM citric acid, pH 6.5) supplemented with 0.35% bovine serum albumin. The platelet suspension was then centrifuged at 2000Ug for 10 min to sediment the platelets. The platelets were washed twice with the platelet wash bu¡er and ¢nally were suspended in the modi¢ed HEPES^Tyrode's bu¡er (136 mM NaCl, 5.5 mM dextrose, 2 mM MgCl2 , 0.47 mM NaH2 P04 , 16.6 mM NaHC03 , 2.7 mM KCl, 10 mM HEPES, pH 7.35). The platelet were counted by the photometric method as described by Walkowiak et al. [43]. 2.2. Collagen-induced platelet aggregation Washed platelets suspended in modi¢ed HEPES^ Tyrode' bu¡er at 4U108 /ml were used for aggregation study. The platelets were incubated with monoclonal anti-Ib antibody for 30 min at 22³C. Platelet aggregation was monitored using the Labor Apact aggregometer. The aggregation was terminated 10, 30, 60, 120, and 180 s after the addition of collagen (1 Wg/ml) by solubilizing platelets in the sample buffer (2% SDS, 0.062 M Tris-HCl, 0.01% Bromophenol blue, 10% glycerol, pH 6.8) containing 2 mM PMSF, 2 mM NEM and 1 mM sodium orthovanadate. The samples were immediately boiled for 10 min and stored at 320³C, until used for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Stock solution of 100 mM sodium orthovanadate was prepared according to Goodno [44]. Brie£y, Na3 V04 was dissolved in water, pH was adjusted to 10.0 with 6 N HCl. Polymeric species of vanadate (orange-yellow) were destroyed by boiling the solution until colorless, followed by rechecking the pH of the cooled solution. 2.3. SDS-PAGE SDS-PAGE was performed by the method of Laemmli [45] using a 7% running gel. The samples
were reduced by heating at 100³C for 5 min with 5% BME. Rainbow molecular weight markers, containing myosin (200 kDa), phosphorylase (97 kDa), bovine serum albumin (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (21.5 kDa) and lysozyme (14.3 kDa) were run on each gel. Proteins from 2U107 platelets per lane (30 Wg) were separated by SDS-PAGE and transferred onto nitrocellulose ¢lters. The parallel gels were stained with Coommassie blue to ensure that equivalent amounts of proteins were added to each lane. 2.4. Immunoblotting The Bio-Rad trans-blot cell was used to transfer proteins from polyacrylamide gels onto nitrocellulose sheets. The transfer was done at 50 mA overnight. The detection of phosphotyrosine-containing proteins was carried out according to the UBI protocol, using a Western-blot kit for anti-phosphotyrosine monoclonal antibody. Brie£y, the blots were incubated in PBS containing 3% skim milk for 1 h at room temperature. After blocking with milk, the blots were incubated with anti-phosphotyrosine monoclonal antibody at the concentration of 1 Wg/ ml overnight at 4³C, washed and incubated with alkaline phosphatase-conjugated secondary antibody at the concentration of 0.5 Wg/ml for 2 h at room temperature. Protein bands were visualized by alkaline phosphatase detection system (UBI). Before the detection of tyrosine phosphorylated proteins, nitrocelluloses were stained with Panceau S to ensure that equivalent amounts of proteins were added to each lane. Speci¢city of the 4G10 anti-phosphotyrosine antibody was con¢rmed by the method of Nakamura and Yamamura [31]. Tyrosine-phosphorylated and Coommassie-stained protein bands were quantitated using the GelImage system, Pharmacia LKB. To quantify the densitometric scans, the background was subtracted and the area for each protein peak was determined. The identi¢cation of phosphotyrosine-containing proteins was performed using monoclonal anti-src, anti-pp120, and anti-pp80/85 antibodies and antisyk rabbit serum. Protein transfer and blots processing was done as in the case of phosphotyrosine antibodies with the exception that all monoclonal anti-
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Fig. 1. Protein tyrosine phosphorylation during collagen-induced activation and aggregation. Washed platelets suspended in a modi¢ed HEPES^Tyrode's bu¡er were aggregated as described in Section 2. (A) Proteins were analyzed by SDS-PAGE on 8% polyacrylamide gels and by subsequent Western blotting with an anti-phosphotyrosine antibody, followed by alkaline phosphatase visualization. Lanes 1, 2, 3, 4, 5, platelets 180, 120, 60, 30 and 10 s after the addition of collagen (1 Wg/ml); lane 6, resting platelets. Molecular weight markers are shown on the right. Each lane contains proteins from 2U107 platelets. (B) Platelets aggregated by collagen after 180 s were solubilized and proteins were separated by SDS-PAGE on 8% polyacrylamide gels and then transferred to nitrocellulose. The platelet proteins were stained with antibodies speci¢c to phosphotyrosine (lane 1), pp60cÿsrc (lane 2), pp72syk (lane 3), pp80/85 (lane 4) and pp120 (lane 5). The molecular masses of marker proteins are shown on the right. (C) Time-course aggregation was monitored in the Labor-Apact aggregometer as described in Section 2. Arrows indicate the addition of collagen (1 Wg/ml) (representative experiment). The experiments shown were reproduced on at least three occasions.
bodies were used at the concentration of 2 Wg/ml and the anti-syk rabbit serum was used at dilution 1:1000. 3. Results Washed platelets were activated with collagen (1 Wg/ml) and the extent of aggregation was monitored in an aggregometer. The aggregation of human platelets was terminated before the shape change took place (10 s after the addition of collagen), after
the shape change (30 s after the addition of collagen), at the onset of aggregate formation (60 s after the addition of collagen), at the beginning of the completion of aggregation (120 s after the addition of collagen), and at the completion of aggregation (180 s after the addition of collagen) by solubilizing platelets in the sample bu¡er. Solubilized platelets were transferred to nitrocellulose and phosphotyrosine containing proteins were detected using 4G10 monoclonal anti-phosphotyrosine antibody. As shown in Fig. 1A, in unstimulated platelets, four tyrosine phosphorylated proteins were detected:
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a major band of 60 kDa and minor bands at 120, 80 and 65 kDa. Activation with collagen increased the phosphorylation of these bands and caused a number of proteins to become phosphorylated at tyrosine. After 30 s, there were 13 proteins with molecular weight of 120, 115, 112, 90, 82, 80, 70, 67, 65, 60, 58, 56 and 48 kDa phosphorylated. The extent of phosphorylation of these proteins increased after 60 and 120 s. At 180 s, after the addition of collagen, two additional phosphoproteins of 78 and 75 kDa appeared. To identify some of these proteins, platelet aggregates obtained 180 s after the addition of collagen were separated by SDS-PAGE, followed by transferring to nitrocellulose and blotting with the anti-phosphotyrosine antibodies. Protein bands were identi¢ed by: (a) apparent molecular weight; and (b) immunoblotting with mono- and polyclonal antibodies speci¢c to major phosphoproteins. As shown in Fig. 1B, the major band, showing molecular weight of 60 kDa, corresponded to pp60cÿsrc . The other bands, showing molecular weights of 120 and 70 kDa, corresponded to pp120 (the pp60cÿsrc substrate) and pp72syk , respectively. A triad at about 82, 80 and 78 kDa corresponded to the forms of pp80/85, the substrate of pp60cÿsrc . The other phosphoproteins were not identi¢ed; however, the proteins of molecular masses of 65, 58 and 56 kDa may represent the src family kinases pp62yes and pp54/58lyn , respectively. The identi¢cation of some of these proteins, like pp72syk and pp60cÿsrc , by immunoprecipitation followed by immunoblotting with anti-phosphotyrosine antibody gave identical results (not shown). In order to compare quantitatively the appearance of phosphoproteins in collagen-induced activation and aggregation of platelets nitrocelluloses containing phosphorylated proteins were densitometrically scanned and quantitated using the GelImage system. Fig. 2 shows that three groups of phosphoproteins can be distinguished, basing on the velocity and extent of phosphorylation. pp60cÿsrc constituted the ¢rst group; the increase in its phosphorylation was the highest and most rapid up to 30 s after the addition of collagen. The phosphorylation of this tyrosine kinase remained basically at the same level 180 s after the addition of collagen A kinase assay of pp60cÿsrc immunoprecipitates showed similar increase in pp60cÿsrc phosphorylation during a time
Fig. 2. Time course of platelet protein phosphorylation during collagen-induced platelet activation and aggregation. Platelet activation and aggregation was terminated 10, 30, 60, 120 and 180 s after the addition of collagen as described in Section 2. Platelet proteins were analyzed by SDS-PAGE on 8% polyacrylamide gels and by subsequent Western blotting with an anti-phosphotyrosine antibody, followed by alkaline phosphatase visualisation. Phosphoprotein bands were quantitated using GelImage system. To quantify densitometric scans, the background was subtracted and the area for each peak was determined. The results are the mean from three experiments.
course of collagen-induced platelet activation (results not shown). The second group consisted of pp80/85 and nonidenti¢ed protein of 65 kDa. The increase in their phosphorylation was 2-fold lower than that of pp60cÿsrc (i.e. 180 s after the addition of collagen), and reached its maximum 60 s after the addition of collagen. pp120, pp72syk , and two non-identi¢ed phosphoproteins of 90 and 75 kDa were in the third group. The increase in their phosphorylation was 4^10-fold lower than that of pp60cÿsrc (i.e. 180 s after the addition of collagen) and reached its maximum at 180 s. To help evaluate the role of tyrosine phosphorylation in platelet activation and aggregation we have tested the in£uence of monoclonal anti-Ib (PO14) antibody. Platelet aggregation in the presence of increasing concentration of PO14 was measured 180 s after the addition of collagen. The result in Fig. 3 shows that PO14 inhibited platelet aggregation in a concentration-dependent manner. PO14 had no e¡ect on platelet shape change, as shown in Fig. 3A. In order to compare quantitatively the e¡ect of PO14 on the phosphorylation of platelet proteins,
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nitrocelluloses containing the proteins obtained from the aggregated platelets, following immunoblotting with the anti-phosphotyrosine antibodies, were densitometrically scanned and quantitated using the GelImage system. Since not all the phosphoproteins
Fig. 4. The e¡ect of anti-Ib antibody on tyrosine phosphorylation of pp60cÿsrc , pp65, pp72syk and pp80/85. Platelets were aggregated in the presence and absence of the anti-Ib antibody. Platelets were incubated with the anti-Ib antibody for 30 min at room temperature. Phosphotyrosine bands were visualised as described in Section 2. Phosphoprotein bands were quantitated using the GelImage system. Relative peak area (y-axis) is the value obtained by dividing the peak area of the band from control aggregation (in the absence of antibody) by the peak area in the presence of antibody. The results are the mean þ S.D. from four experiments.
Fig. 3. The in£uence of anti-Ib antibody on platelet aggregation. Platelets were incubated with the anti-Ib antibody for 30 min at room temperature before aggregation. The aggregation of platelets was measured 3 min after the addition of collagen (1 Wg/ml), as described in Section 2. (A) Platelet aggregation tracings from control platelets (not treated with PO14) (line 1) and incubated with PO14 at concentrations 1, 5, 10 and 30 Wg/ ml (lines 2, 3, 4 and 5, respectively) are from one representative of six independent experiments with similar results. Collagen was added at the arrow. (B) Results are expressed as % of control aggregation (without antibody). The aggregation of control platelets (not incubated with antibody was 70^90%. The results are the mean þ S.D. from six experiments.
were detectable by this system, Fig. 4 shows only the inhibitory e¡ect of PO14 on the phosphorylation of 4 proteins. As it is seen in Fig. 4, P014 a¡ected mostly the phosphorylation of pp72syk which was decreased 3^6-fold when the antibody was used at the concentration of 5^30 Wg/ml. The inhibition of pp80/85 and pp65 was approximately twice smaller than that seen in the case of pp72syk . The phosphorylation of pp60cÿsrc was not a¡ected even at the concentration of 30 Wg/ml of anti-Ib, the highest concentration tested which inhibited platelet aggregation by 90%. The inhibition of phosphorylation of pp120 was complete even at the concentration of 5 Wg/ml of anti-Ib, the lowest concentration tested in these sets of experiments (not shown). 4. Discussion Activation and aggregation of platelets by collagen is associated with tyrosine phosphorylation of many platelet proteins. Based on the rate and extent of phosphorylation they may be divided into three groups: (a) pp60cÿsrc formed the ¢rst group, since
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the increase in the phosphorylation of this kinase was the most rapid one. A two-fold increase was already visible after 10 s (before the shape change) and an almost 10-fold increase after 30 s (after the shape change). The events taking place in platelets after 10 and 30 s are de¢ned as early activation events induced by collagen. Since the activation of pp60cÿsrc was almost complete during 30 s it can be concluded that the phosphorylation of this kinase is very important for the early activation manifested by the shape change. This conclusion was supported by using monoclonal anti-Ib antibodies PO14, which neither inhibited the shape change of platelets nor the pp60cÿsrc phosphorylation. The rate and extent of pp60cÿsrc phosphorylation induced by collagen is comparable to that described with other agonists [46]. Since the collagen-induced phosphorylation of pp60cÿsrc was the most rapid one it is very likely, as indicated earlier with thrombin [47], that the activation of this kinase is a prerequisite for phosphorylation of other kinases. (b) The second group consisted of pp80/85 and non-identi¢ed protein of 65 kDa. The increase in their phosphorylation reached maximum after 60 s. The onset of aggregate formation was observed at the same time. Thus, these two phosphoproteins seem to be responsible for this event. pp80/ 85 was primarily identi¢ed as a cytoskeleton-associated substrate for v-src in transformed cells. It did not show any identity with the 85 kDa subunit of phosphatidylinositol-3 kinase. The sequence of the SH3 domain of pp80/85 showed the greatest identity with other cytoskeleton-associated protein, such as fodrin and myosin 1 [48]. pp80/85 was shown to be present in platelets and was found previously to associate with pp60cÿsrc in thrombin-activated platelets. Similarly to collagen, thrombin induced the phosphorylation of this protein as soon as after 1 min [46]. In thrombin-activated platelets, pp60cÿsrc was postulated to participate in the cytoskeletal changes by phosphorylation of pp80/85 [46]. Probably the same mechanism is responsible for shape change in collagen-activated platelets. (c) pp72syk , pp120, and two phosphoproteins of 75 and 90 kDa belonged to the third group of phosphoproteins. The increase in their phosphorylation started basically 120 s after the addition of collagen. At this time, a massive aggregate formation occurred, and the phosphoproteins
from the third group can be responsible for this phenomenon. pp72syk was found previously not to be phosphorylated on tyrosine in resting platelets [34]. Many agonists, such as wheat germ agglutinin, thrombin, PAF, collagen and thromboxane A2 mimetic U 44069, were described previously to induce the activation of pp72syk in platelets [49^53]. Thrombin rapidly induced the phosphorylation of pp72syk [50]. The increase in pp72syk activity was observed as early as 5 s upon the thrombin treatment, it reached its maximum in 10 s and decreased to the basal level within 60 s in a calcium-dependent manner [50]. Our data are consistent with this observation and indicate the lack of phosphorylation of pp72syk in resting platelets. They also show that the kinetics of the pp72syk phosphorylation in the collagen-treated platelets differs from that described for the thrombin-treated platelets. A very small increase in the phosphorylation of pp72syk (together with three other proteins of 120, 90 and 75 kDa) was observed up to 120 s after the collagen addition. A rapid increase in its phosphorylation was seen after 120 s, which was coincident with massive aggregates formation. This may suggest that thrombin elicits some intracellular signals which are not shared with collagen. Our data also show some di¡erences from that were described previously for the collagen-treated platelets [39,53]. First, proteins of 54, 65, 75, 97 and 125 kDa were phosphorylated in the collagentreated platelets as described by Asazuma et al. [53]. The 75 kDa band consisted of two proteins, cortactin and pp72syk . Thus, a total number of six phosphoprotein bands was found. We found 15 proteins to be phosphorylated upon the comparable time of collagen treatment. Secondly, the activation of pp60cÿsrc showed only minimal changes upon the collagen treatment in the previous work, while we found that pp60cÿsrc phosphorylation increased several times and that was much higher and faster than other proteins. Thirdly, Asazuma et al. [53] described that the phosphorylation of pp72syk was very rapid; it reached its peak after 30^60 s and then gradually decreased. We show that the phosphorylation level of pp72syk was very low up to 120 s after the addition of collagen and a rapid increase in its phosphorylation occurred thereafter. Several factors can account for
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the above discrepancies. Collagen at the concentration 50 Wg/ml from Hormon-Chemie was required for the stimulation of platelets pretreated with 1 mM acetylsalicylic acid (ASA), as described by Asazuma et al. [53]. We used the equine-tendon collagen and the concentration of 1 Wg/ml of this collagen was enough to induce the activation and aggregation of platelets. The platelets used in our experiments were not subjected to ASA treatment, thus secondary e¡ects of thromboxane A2 generation may contribute to our results. The present data do not con¢rm observation that there is a signi¢cant increase in tyrosine phosphorylation of pp72syk within 1 min after collagen treatment of platelets [39]. The possible explanation of this discrepancy is that the experimental conditions used in both studies were di¡erent, i.e. treatment of platelets with soluble collagen from bovine skin at concentration 200 Wg/ml under non-stirring conditions may account for the di¡erences. The general importance of pp72syk activation for the collagen-induced aggregation of platelets was shown previously [39] by using a pp72syk -selective kinase inhibitor, piceatannol, which inhibited both kinase activity of pp72syk and platelet aggregation. Piceatannol also completely inhibited platelet shape change implying a role for pp72syk in the initial collagen-induced events. Additionally, since activation of pp72syk occurred even in the absence of platelet aggregation and K2 L1 integrin engagement a role for pp72syk upstream of platelet aggregation was postulated [39]. Our ¢ndings on the kinetics of pp72syk phosphorylation are in accordance with the report of Fuji et al. [51]. pp120, a phosphoprotein which like pp72syk belongs to the third group, was previously identi¢ed in chicken embryo ¢broblasts. Tyrosine phosphorylation of this protein correlated with src transformation in ¢broblasts. Monoclonal antibody against this protein used in this study are speci¢c for pp120. This antibody does not react with pp125FAK [54]. The identity and function of pp120 as well as other proteins of the molecular masses of 90, 75, 67, 65, 58, 56 and 48 have yet to be determined. We found that the phosphorylation of pp72syk as well as pp120 was inhibited by PO14 antibody which inhibited the platelet aggregation and did not inhibit the shape change of platelets. It clearly shows that these proteins play
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a role in later signal transduction events associated with the aggregation of platelets. Previous studies, by using of anti-K2 L1 monoclonal antibody P1E6, showed that interaction of collagen with K2 L1 is directly connected with the phosphorylation of PLCQ2 and pp72syk [39]. However, the existence of K2 L1 -independent pathway of PLCQ2 and pp72syk phosphorylation was indicated by the use of two anti-K2 L1 mAbs, 6F1 and mAb13, which did not inhibit tyrosine phosphorylation of pp72syk and PLCQ2 induced by collagen-related triple helical peptide [55]. The use of K2 L1 integrin blocking monoclonal antibodies 6F1 and P1E6 demonstrated that tyrosine phosphorylation of FcRQ chain in response to collagen is not dependent on the integrin K2 L1 [39^ 41]. The identity of the collagen receptor which leads to tyrosine phosphorylation of FcRQ chain is not known. However, recent studies indicate that GPVI may be the signalling collagen receptor whose activation result in the tyrosine phosphorylation of the FcRQ chain [56]. In response to collagen, the activity of pp60cÿsrc from GPVI de¢cient platelets was comparable to normal platelets. Such activation of pp60cÿsrc was found to be K2 L1 -dependent because it was signi¢cantly inhibited in the presence of anti-K2 L1 mAb Gi9. By contrast, GPVI de¢cient platelets did not exhibit detectable tyrosine phosphorylation of pp72syk , pp125FAK and PLC-Q2 in response to collagen. Therefore, the requirement of GPVI for the collagen-stimulated tyrosine phosphorylation of these proteins was indicated [57]. To evaluate the contribution of GPIV to collagen induced protein tyrosine phosphorylation GPIV de¢cient platelets were used [58]. Since GPIV de¢cient platelets exhibited normal protein tyrosine phosphorylation in response to collagen, it seems that the role of this protein in collagen-induced signalling events is minor when compared with those of K2 L1 and GPVI. Ruan et al. initially reported that an anti-GPIbK antibody SZ 2 inhibited platelet aggregation induced by collagen and PAF [59]. However, the e¡ect of this antibody on protein tyrosine phosphorylation during collageninduced aggregation was not studied. It is noteworthy that anti-Ib antibody PO14 blocks both platelet aggregation and protein tyrosine phosphorylation. PO14 antibody was described previously as strong inhibitors of ristocetin/vWf induced platelet aggluti-
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nation and thrombin-induced platelet aggregation [60]. In addition, we demonstrate that PO14 inhibited the aggregation of platelets as well as tyrosine phosphorylation of proteins belonging to the second and third groups. These results suggest that GPIb plays an important role in the collagen-mediated platelet signalling occurring at the later stages of aggregation, i.e. at the stage of aggregate formation.
[10]
[11]
[12]
Acknowledgements This work was supported by Project 6 P207 055 06 from the State Committee for Scienti¢c Research. C.S.C. is HHMI international investigator. We thank Dr C. de Romeuf (Hemostasis Research Laboratories, Lille, France) for providing anti-GPIb antibody.
References
[13]
[14]
[15]
[1] H.R. Baumgartner, Platelet interaction with collagen ¢brils in £owing blood. I. Reaction of human platelets with K chymotrypsin-digested subendothelium, Thromb. Haemost. 37 (1977) 1^16. [2] G.A. Jamieson, C.L. Urban, A.J. Barber, Enzymatic basis for platelet-collagen adhesion as the primary step in haemostasis, Nature 234 (1971) 5^7. [3] H.B. Bensusan, T.L. Koh, K.G. Henry, B.A. Marray, L.A. Culp, Evidence that ¢bronectin is the collagen receptor on platelet membranes, Proc. Natl. Acad. Sci. USA 75 (1978) 5864^5868. [4] T.M. Chiang, A.H. Kang, Isolation and puri¢cation of collagen K1 (I) receptor from human platelet membrane, J. Biol. Chem. 257 (1982) 7581^7586. [5] S. Tsenuhisa, T. Tsuji, H. Tohyama, T. Osawa, Interaction of human platelet membrane glycoproteins with collagen and lectins, Biochim. Biophys. Acta 797 (1984) 10^19. [6] N.J. Kotite, J.V. Staros, L.W. Cunningham, Interaction of speci¢c platelet membrane proteins with collagen: evidence from chemical cross-linking, Biochemistry 23 (1984) 3099^ 3104. [7] H.K. Nieuwenhuis, J.W.N. Akkerman, W.P.M. Houdijk, J.J. Sixma, Human blood platelets showing no response to collagen fail to express surface glycoprotein Ia, Nature 318 (1985) 470^472. [8] S.A. Santoro, Identi¢cation of a 160,000 dalton platelet membrane protein that mediates the initial divalent cationdependent adhesion of platelets to collagen, Cell 46 (1988) 913^920. [9] B. Kehrel, L. Balleisen, R. Kokott, R. Mesters, W. Stenzinger, K.J. Clemetson, J. van de Loo, De¢ciency of intact
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
thrombospodin and membrane glycoprotein Ia in platelets with defective collagen-induced aggregation and spontaneous loss of disorder, Blood 71 (1988) 1074^1078. S.A. Santoro, S.M. Rajpara, W.D. Staatz, V.L. Woods, Isolation and characterization of a platelet surface collagen binding complex related to VLA-2, Biochem. Biophys. Res. Commun. 153 (1988) 217^223. W.D. Staatz, S.M. Rajpara, E.A. Wayner, W.G. Carter, S.A. Santoro, The membrane glycoprotein Ia^IIa (VLA2 ) complex mediates the Mg -dependent adhesion of platelets to collagen, J. Cell Biol. 108 (1989) 1917^1924. S.A. Santoro, J.J. Walsh, W.D. Staatz, K.J. Baranski, Distinct determinants on collagen support K2 L1 integrin-mediated platelet adhesion and platelet activation, Cell Regul. 2 (1991) 905^913. Y. Saito, T. Imada, J. Takagi, T. Kikuchi, Y. Inada, Platelet factor XIII. The collagen receptor?, J. Biol. Chem. 261 (1986) 1355^1358. T. Sugiyama, M. Okuma, F. Ushikubi, S. Sensaki, K. Kanaji, H. Uchino, A novel platelet aggregating factor found in a patient with defective collagen-induced platelet aggregation and autoimmune thrombocytopenia, Blood 69 (1987) 1712^ 1720. M. Moroi, S.M. Jung, M. Okuma, K. Shinmyozu, A patient with platelets de¢cient in glycoprotein VI that lack both collagen-induced aggregation and adhesion, J. Clin. Invest. 84 (1989) 1440^1445. M. Moroi, S.M. Jung, K. Shinmyozu, Y. Tomiyama, A. Ordinas, M. Diaz-Ricart, Analysis of platelet adhesion to a collagen-coated surface under £ow conditions: the involvement of glycoprotein VI in the platelet adhesion, Blood 88 (1996) 2081^2092. N.N. Tandon, U. Kralisz, G.A. Jamieson, Identi¢cation of GPIV(CD36) as a primary receptor for platelet-collagen adhesion, J. Biol. Chem. 264 (1989) 7576^7583. N.N. Tandon, Ch.F. Ockenhouse, N.J. Greco, G.A. Jamieson, Adhesive functions of platelets lacking glycoprotein IV (CD36), Blood 78 (1991) 2809^2813. M. Diaz-Ricart, N.N. Tandon, M. Carretero, A. Ordinas, E. Bastida, G.A. Jamieson, Platelets lacking functional CD36 (glycoprotein IV) show reduced adhesion to collagen in £owing whole blood, Blood 82 (1993) 491^496. L. McKeown, M. Vail, S. Williams, W. Kramer, K. Hansmann, H. Gralnick, Platelet adhesion to collagen in individuals lacking glycoprotein IV, Blood 83 (1994) 2866^2871. E.U.M. Saelman, B. Kehrel, K.M. Hese, P.G. de Groot, J.J. Sixma, K.H. Nieuwenhuis, Platelet adhesion to collagen and endothelial cell matrix under £ow conditions is not dependent on glycoprotein IV, Blood 83 (1994) 3240^3244. B. Kehrel, A. Kronenberg, J. Rauterberg, D. Niesing-Bresch, U. Niehues, J. Kardoeus, B. Schwippert, D. Tschope, J. van de Loo, K.J. Clemetson, Platelet de¢cient in glycoprotein IIIb aggregate normally to collagens type I and III but not to collagen type V, Blood 82 (1993) 3364^3370. N. Yamamoto, N. Akamatsu, H. Yamazaki, K. Tanoue, Normal aggregations of glycoprotein IV (CD36)-de¢cient
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[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32] [33]
[34]
[35]
[36]
[37]
[38]
[39]
platelets from seven healthy Japanese donors, Br. J. Haematol. 81 (1992) 86^92. B.S. Coller, J.H. Beer, L.E. Scudder, M.H. Steinberg, Collagen^platelet interactions: evidence for a direct interaction of collagen with platelet GPIa/IIa and indirect interaction with platelet GPIIb/IIIa mediated by adhesive proteins, Blood 74 (1989) 182^192. H. Weiss, V.T. Turitto, H.R. Baumgartner, Platelet adhesion and thrombus formation on subendothelium in platelets de¢cient in glycoproteins IIb^IIIa Ib, and storage granules, Blood 67 (1986) 322^330. K.S. Sakariassen, P.F.E.M. Nievelstein, B.S. Coller, J.J. Sixma, The role of platelet membrane glycoproteins Ib and IIb^ IIIa in platelet adherence to human artery subendothelium, Br. J. Haematol. 63 (1986) 681^691. A. Golden, S.P. Nemeth, J.S. Brugge, Blood platelets express high levels of the pp60cÿsrc -speci¢c tyrosine kinase activity, Proc. Natl. Acad. Sci. USA 83 (1986) 852^856. I.D. Horak, M.L. Corcoran, P.A. Thompson, L.M. Wahl, J.B. Bolen, Expression of p60fyn in human platelets, Oncogene 5 (1990) 597^602. S. Ohta, T. Taniguchi, M. Asahi, M. Kato, G. Nakagawara, H. Yamamura, Protein tyrosine kinase p72syk is activated by wheat germ agglutinin in platelets, Biochem. Biophys. Res. Commun. 185 (1992) 1128^1132. L. Lipfert, B. Haimovich, M.D. Schaller, B.S. Cobb, J.T. Parsons, J.S. Brugge, Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets, J. Cell Biol. 119 (1992) 905^912. S. Nakamura, H. Yamamura, Thrombin and collagen induce rapid phosphorylation of a cellular proteins on tyrosine in human platelets, J. Biol. Chem. 264 (1989) 7089^7091. M.H. Kroll, A.I. Schafer, Biochemical mechanisms of platelet activation, Blood 74 (1989) 1181^1195. A. Dhar, S.D. Shukla, Involvement of pp60cÿsrc in plateletactivating factor-stimulated platelets, J. Biol. Chem. 266 (1991) 18797^18801. E.A. Clark, S.J. Shattil, J.S. Brugge, Regulation of protein tyrosine kinases in platelets, Trends Biochem. Sci. 19 (1994) 464^469. J.B. Smith, C. Dangelmaier, M.a. Selak, B. Ashby, J. Daniel, Cyclic AMP does not inhibit collagen-induced platelet signal transduction, Biochem. J. 283 (1992) 889^892. J.B. Smith, C. Dangelmaier, J.D. Daniel, Elevation of cAMP in human platelets inhibits thrombin- but not collagen-induced tyrosine phosphorylation, Biochem. Biophys. Res. Commun. 191 (1993) 695^700. J.J. Daniel, C. Dangelmaier, J.B. Smith, Evidence for a role for tyrosine phosphorylation of phospholipase CQ2 in collagen induced platelet cytosolic calcium mobilization, Biochem. J. 302 (1994) 617^622. R.A. Blake, G.L. Schieven, S.P. Watson, Collagen stimulates tyrosine phosphorylation of phospholipase C-Q2 but phospholipase C-Q1 in human platelets, FEBS Lett. 353 (1994) 212^216. P.J. Keely, L.V. Parise, The K2 L1 integrin is a necessary co-
[40]
[41]
[42]
[43]
[44] [45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
137
receptor for collagen-induced activation of syk and subsequent phosphorylation of phospholipase CQ2 in platelets, J. Biol. Chem. 271 (1996) 26668^26676. F. Yanaga, A. Poole, J. Asselin, R. Blake, G.L. Schieven, E.A. Clark, C.-L. Law, S.P. Watson, Syk interacts with tyrosine-phosphorylated proteins in human platelets activated by collagen and cross-linking of the FcQ-IIa receptor, Biochem. J. 311 (1995) 471^478. J. Gibbins, J. Asselin, R. Farndale, M. Barnes, C.-L. Law, S.P. Watson, Tyrosine phosphorylation of the Fc receptor Q-chain in collagen-stimulated platelets, J. Biol. Chem. 271 (1996) 18095^18099. C. Watala, T. Pietrucha, K. Gwozdzinski, U. Kralisz, C.z.S. Cierniewski, Microenvironment changes in human blood platelet membranes associated with binding of tissue-type plasminogen activator, Eur. J. Biochem. 215 (1993) 867^ 871. B. Walkowiak, E. Michalak, W. Koziolkiewicz, C.S. Cierniewski, Rapid photometric method for estimation of platelet count in blood plasma or platelet suspension, Thromb. Res. 56 (1989) 763^766. C.C. Goodno, Myosin active-site trapping with vanadate ion, Methods Enzymol. 85 (1982) 116^123. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4 , Nature 227 (1970) 680^685. S. Wong, A.B. Reynolds, J. Papko¡, Platelet activation leads to increase c-src kinase activity and association of c-src with an 85-kDa tyrosine phosphoprotein, Oncogene 7 (1992) 2407^2415. R. Polanowska-Grabowska, M. Geanacopoulos, A.R.L. Gear, Platelet adhesion to collagen via the K2 L1 integrin under arterial £ow conditions causes rapid tyrosine phosphorylation of pp125FAK , Biochem. J. 296 (1993) 543^547. H. Wu, A.B. Reynolds, S.B. Kanner, R.R. Vines, J.T. Parsons, Identi¢cation and characterization of a novel cytoskeleton-associated pp60cÿsrc substrate, Mol. Cell. Biol. 11 (1991) 5113^5124. H. Maeda, T. Taniguchi, T. Inazu, Ch. Yang, G. Nakagawara, H. Yamamura, Protein-tyrosine kinase p72syk is activated by thromboxane A2 mimetic u44069 in platelets, Biochem. Biophys. Res. Commun. 197 (1993) 62^67. T. Taniguchi, H. Kitagawa, S. Yasue, S. Yanagi, K. Sakai, M. Asahi, S. Ohta, F. Takeuchi, S. Nakamura, H.P. Yamamura, Protein-tyrosine kinase p72syk is activated by thrombin and is negatively regulated through Ca2 mobilization in platelets, J. Biochem. Chem. 268 (1993) 2277^2279. C. Fuji, S. Yanagi, K. Sada, K. Nagai, K. Taniguchi, H. Yamamura, Involvement of protein-tyrosine kinase p72syk in collagen-induced signal transduction in platelets, Eur. J. Biochem. 226 (1994) 243^248. K. Rezaul, S. Yanagi, K. Sada, T. Taniguchi, H. Yamamura, Protein-tyrosine kinase p72syk is activated by platelet activating factor in platelets, Thromb. Haemost. 72 (1994) 637^641. N. Asazuma, Y. Yatomi, Y. Ozaki, R. Qi, K. Kuroda, K.
BBAMCR 14372 13-10-98
138
[54]
[55]
[56]
[57]
U. Kralisz, C.S. Cierniewski / Biochimica et Biophysica Acta 1405 (1998) 128^138 Satoh, S. Kume, Protein-tyrosine phosphorylation and p72syk activation in human platelets stimulated with collagen is dependent upon glycoprotein Ia/IIa and actin polymerization, Thromb. Haemost. 75 (1996) 648^654. S.B. Kanner, A.B. Reynolds, R.R. Vines, J.T. Parsons, Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases, Proc. Natl. Acad. Sci. USA 87 (1990) 3328^3332. J. Asselin, J.M. Gibbins, M. Achison, Y. Han Lee, L.F. Morton, R.W. Farndale, M.J. Barnes, S.P. Watson, A collagen-like peptide simulates tyrosine phosphorylation of syk and phospholipase CQ2 in platelets independent of the integrin K2 L1 , Blood 89 (1997) 1235^1242. J.M. Gibbins, M. Okuma, R. Farndale, M. Barnes, S.P. Watson, Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor Q-chain, FEBS Lett. 413 (1997) 255^259. T. Ichinohe, H. Takayama, Y. Ezumi, M. Arai, N. Yama-
moto, H. Takahashi, M. Okuma, Collagen-stimulated activation of syk but not c-src is severely compromised in human platelets lacking membrane glycoprotein VI, J. Biol. Chem. 272 (1997) 63^68. [58] J.A. Daniel, C. Dangelmaier, R. Strouse, J.B. Smith, Collagen induces normal signal transduction in platelets de¢cient in CD36 (platelet glycoprotein IV), Thromb. Haemost. 71 (1994) 353^356. [59] C.G. Ruan, X.P. Du, X.D. Xi, P.A. Castaldi, M.C. Berndt, A murine antiglycoprotein Ib complex monoclonal antibody, SZ 2, inhibits platelet aggregation induced by both ristocetin and collagen, Blood 69 (1987) 570^577. [60] K.J Clemetson, B. Hugli, V. von Tscharner, E¡ects of antibodies to the platelet GPIb-V-IX (CD42b, CD42c, CD42a) complex on ristocetin/vWF- and thrombin-induced platelet activation, in: S. Schlossman et al. (Eds.), Leucocyte Typing V, Vol. 2, Oxford University Press, Oxford, 1995, pp. 1328^ 1330.
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Tyrosine phosphorylation events during di¡erent stages of collagen-platelet activation Urszula Kralisz *, Czeslaw S. Cierniewski Department of Biophysics, Institute of Physiology and Biochemistry, Medical University of Lodz, Lindleya 3, 90-131 Lodz, Poland Received 13 May 1998; revised 30 July 1998; accepted 10 August 1998
Abstract Three groups of phosphoproteins have been distinguished, basing on the velocity and extent of phosphorylation in platelets stimulated with collagen. pp60cÿsrc constituted the first group; the increase in its phosphorylation was the highest and most rapid (maximal in 30 s after the addition of collagen). pp80/85 and non-identified protein of 65 kDa formed the second group; the increase in their phosphorylation was twice smaller than that of pp60cÿsrc , and reached its maximum 60 s after the addition of collagen. pp120, pp72syk , and two non-identified phosphoproteins of 90 and 75 kDa constituted the third group; the increase in their phosphorylation was 4^10-fold lower than that of pp60cÿsrc and reached its maximum after 180 s. We conclude that the phosphorylation of pp60cÿsrc is important for the change of shape of platelets, the phosphorylation of pp80/85 and pp65 for the initiation of the formation of aggregates and the phosphorylation of the third group of phosphoproteins for the formation of massive aggregates. This conclusion was supported by using a monoclonal anti-GPIb antibody, which did not inhibit the shape change of platelets and did not inhibit pp60cÿsrc phosphorylation. This antibody inhibited aggregate formation as well as tyrosine phosphorylation of proteins belonging to the second and the third group of phosphoproteins. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Platelet; Collagen; Activation; Aggregation; Tyrosine phosphorylation
1. Introduction Fibrillar collagen, one of the most thrombogenic agents of the vessel wall [1], interacts with platelets by several mechanisms which can be divided into direct (primary) and indirect (secondary). Primary
Abbreviations: PGE1 , prostaglandin E1 ; SDS, sodium dodecyl sulfate; PMSF, phenylmethylsulfonyl£uoride ; NEM, N-ethylmaleimide ; PAGE, polyacrylamide gel electrophoresis ; TxA2 , thromboxane A2 ; HEPES, 4-(2-hydroxyethyl)1-piperazine-ethanesulfonic acid; PBS, phosphate-bu¡ered saline ; BME, 2-mercaptoethanol; PAF, platelet -activating factor; ASA, acetylsalicylic acid * Corresponding author. Fax: +48 (42) 678-9433.
mechanisms are mediated by components present on the surface of unactivated platelets, while secondary mechanisms involve receptors expressed only upon activation of platelets. Although several platelet proteins have been proposed to be primary collagen receptors, i.e. collagen-glucosyl transferase [2], ¢bronectin [3], 65-kDa protein [4], GPIIb^IIIa [5,6], GPIa^IIa [7^12], factor XIII [13], glycoprotein VI [14^16] and glycoprotein IV [17^19], only a few of them have gained general acceptance. The strongest evidence supporting such a role for GPIa^IIa and GPVI has come from clinical studies. Patients de¢cient in GPIa^IIa and GPVI were described to have impaired or lack collagen-induced aggregation and adhesion [7,8,14^16]. There is some evidence that
0167-4889 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 8 9 ( 9 8 ) 0 0 1 0 0 - 1
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glycoprotein IV (CD36) functions at the earliest stages of collagen adhesion as a platelet collagen receptor although CD36 de¢cient platelets adhere normally to collagen types I, III and IV. CD36 de¢cient platelets show also normal aggregation induced by collagen. CD36 seems to be essential for collagen type V-induced platelet interaction although the physiological importance of such interaction is questionable [19^23]. There is general agreement on the involvement of GPIIb^IIIa in mediating secondary adhesion through macromolecules released from activated platelets or present in plasma, such as ¢bronectin, von Willebrand's factor (vWf) and thrombospondin (TSP) [24]. The role of GPIb in mediating shear-dependent platelet adhesion is also well characterized through studies with Bernard^Soulier platelets. The primary role of GPIb is to bind to vWF on exposed subendothelium. The GpIb is expressed on unactivated platelets, but is active at high shear rates [25,26]. Platelets show high levels of non-receptor protein tyrosine kinase (PTK) activity, largely due to the high concentration of pp60cÿsrc and pp72syk as well as other PTKs, such as pp60fyn , pp54/58lyn , pp62hck , pp62yes and pp125FAK [27^30]. The activation of platelets with many agonists, such as thrombin, collagen and platelet -activating factor (PAF), leads to many events (including shape change, secretion, binding of soluble ¢brinogen to its receptor and aggregation) that are thought to be regulated through a number of intracellular signalling pathways, including PTK activation [31^34]. Collagen-induced platelet activation di¡ers in many ways from the activation induced by soluble activators, such as thrombin. Firstly, cyclic AMP does not inhibit collagen-induced platelet signal transduction, whereas it inhibits the signal transduction induced by ADP, thrombin and a thromboxane mimetic U-46619 [35,36]. Secondly, collagen-induced platelet stimulation involves the tyrosine phosphorylation-dependent activation of phospholipase CQ2, while that induced by several other agonists does not [37^39]. Thirdly, collagen is the only physiological platelet agonist that has been identi¢ed to induce phosphorylation of the FcRQ chain enabling binding of pp72syk and the assembly of a signaling complex independently from phospholipase C (PLC) activity [39^41]. The cytoplasmic tail of the FcRQ chain contains an immunoreceptor tyro-
129
sine-based activation motif (ITAM). The association of pp72syk with FcRQ chain appears to be mediated through the tandem SH2 domains in pp72syk and the ITAM motif of this receptor [38,40,41]. For the purpose of this study, we have concentrated on the role of protein phosphorylation during di¡erent stages of platelet activation induced by collagen. The data demonstrated an increase in tyrosine phosphorylation of many proteins upon collagen treatment. Three groups of phosphoproteins were distinguished based on the velocity and extent of phosphorylation. The pp60cÿsrc belongs to the ¢rst group since the increase in its phosphorylation was the highest and most rapid. The phosphorylation of pp60cÿsrc was found to be important for the early activation events concerning the change of shape of platelets. The phosphorylation of the second and third group of proteins was important for the later stages of collagen-induced activation connected with the aggregates formation. These ¢ndings were supported by the use of anti-Ib monoclonal antibody PO14, which neither inhibited the shape change nor the pp60cÿsrc phosphorylation. PO14 inhibited aggregation as well as tyrosine phosphorylation of the proteins belonging especially to the third group. 2. Materials and methods Fibrillar equine tendon collagen type I was from Chrono-Log. Anti-phosphotyrosine monoclonal IgG 4G10 and monoclonal anti-src, pp80/85, pp120 as well as polyclonal anti-syk antibodies were from Upstate Biotechnology. A monoclonal antibody PO14 (clone name 105.12B12H3) against GPIb was a generous gift from Dr. C. de Romeuf (Hemostasis Research Laboratories, Lille, France). Rainbow protein molecular weight markers were from Amersham. All other chemicals were purchased from Sigma. 2.1. Preparation of human platelets Washed platelets were prepared from human blood anticoagulated with ACD (citrite acid^citrate^dextrose) obtained from volunteer donors as described previously, with some minor modi¢cation [17,42]. Brie£y, platelet-rich plasma was obtained by centrifuging the whole blood at 2000Ug for 20 min
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at 22³C. For the preparation of the washed platelets, the pH of the platelet-rich plasma was adjusted to 6.5 with citric acid (0.2 M) and made 1 Wg/ml with respect to prostaglandin E1 . Then, the platelets were sedimented by centrifugation at 1000Ug for 10 min and the pellet was resuspended in the platelet wash bu¡er (5.5 mM dextrose, 128 mM NaCl, 4.26 mM Na2 HPO4 , 7.46 mM NaH2 PO4 , 4.77 mM trisodium citrate, and 2.35 mM citric acid, pH 6.5) supplemented with 0.35% bovine serum albumin. The platelet suspension was then centrifuged at 2000Ug for 10 min to sediment the platelets. The platelets were washed twice with the platelet wash bu¡er and ¢nally were suspended in the modi¢ed HEPES^Tyrode's bu¡er (136 mM NaCl, 5.5 mM dextrose, 2 mM MgCl2 , 0.47 mM NaH2 P04 , 16.6 mM NaHC03 , 2.7 mM KCl, 10 mM HEPES, pH 7.35). The platelet were counted by the photometric method as described by Walkowiak et al. [43]. 2.2. Collagen-induced platelet aggregation Washed platelets suspended in modi¢ed HEPES^ Tyrode' bu¡er at 4U108 /ml were used for aggregation study. The platelets were incubated with monoclonal anti-Ib antibody for 30 min at 22³C. Platelet aggregation was monitored using the Labor Apact aggregometer. The aggregation was terminated 10, 30, 60, 120, and 180 s after the addition of collagen (1 Wg/ml) by solubilizing platelets in the sample buffer (2% SDS, 0.062 M Tris-HCl, 0.01% Bromophenol blue, 10% glycerol, pH 6.8) containing 2 mM PMSF, 2 mM NEM and 1 mM sodium orthovanadate. The samples were immediately boiled for 10 min and stored at 320³C, until used for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Stock solution of 100 mM sodium orthovanadate was prepared according to Goodno [44]. Brie£y, Na3 V04 was dissolved in water, pH was adjusted to 10.0 with 6 N HCl. Polymeric species of vanadate (orange-yellow) were destroyed by boiling the solution until colorless, followed by rechecking the pH of the cooled solution. 2.3. SDS-PAGE SDS-PAGE was performed by the method of Laemmli [45] using a 7% running gel. The samples
were reduced by heating at 100³C for 5 min with 5% BME. Rainbow molecular weight markers, containing myosin (200 kDa), phosphorylase (97 kDa), bovine serum albumin (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (21.5 kDa) and lysozyme (14.3 kDa) were run on each gel. Proteins from 2U107 platelets per lane (30 Wg) were separated by SDS-PAGE and transferred onto nitrocellulose ¢lters. The parallel gels were stained with Coommassie blue to ensure that equivalent amounts of proteins were added to each lane. 2.4. Immunoblotting The Bio-Rad trans-blot cell was used to transfer proteins from polyacrylamide gels onto nitrocellulose sheets. The transfer was done at 50 mA overnight. The detection of phosphotyrosine-containing proteins was carried out according to the UBI protocol, using a Western-blot kit for anti-phosphotyrosine monoclonal antibody. Brie£y, the blots were incubated in PBS containing 3% skim milk for 1 h at room temperature. After blocking with milk, the blots were incubated with anti-phosphotyrosine monoclonal antibody at the concentration of 1 Wg/ ml overnight at 4³C, washed and incubated with alkaline phosphatase-conjugated secondary antibody at the concentration of 0.5 Wg/ml for 2 h at room temperature. Protein bands were visualized by alkaline phosphatase detection system (UBI). Before the detection of tyrosine phosphorylated proteins, nitrocelluloses were stained with Panceau S to ensure that equivalent amounts of proteins were added to each lane. Speci¢city of the 4G10 anti-phosphotyrosine antibody was con¢rmed by the method of Nakamura and Yamamura [31]. Tyrosine-phosphorylated and Coommassie-stained protein bands were quantitated using the GelImage system, Pharmacia LKB. To quantify the densitometric scans, the background was subtracted and the area for each protein peak was determined. The identi¢cation of phosphotyrosine-containing proteins was performed using monoclonal anti-src, anti-pp120, and anti-pp80/85 antibodies and antisyk rabbit serum. Protein transfer and blots processing was done as in the case of phosphotyrosine antibodies with the exception that all monoclonal anti-
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Fig. 1. Protein tyrosine phosphorylation during collagen-induced activation and aggregation. Washed platelets suspended in a modi¢ed HEPES^Tyrode's bu¡er were aggregated as described in Section 2. (A) Proteins were analyzed by SDS-PAGE on 8% polyacrylamide gels and by subsequent Western blotting with an anti-phosphotyrosine antibody, followed by alkaline phosphatase visualization. Lanes 1, 2, 3, 4, 5, platelets 180, 120, 60, 30 and 10 s after the addition of collagen (1 Wg/ml); lane 6, resting platelets. Molecular weight markers are shown on the right. Each lane contains proteins from 2U107 platelets. (B) Platelets aggregated by collagen after 180 s were solubilized and proteins were separated by SDS-PAGE on 8% polyacrylamide gels and then transferred to nitrocellulose. The platelet proteins were stained with antibodies speci¢c to phosphotyrosine (lane 1), pp60cÿsrc (lane 2), pp72syk (lane 3), pp80/85 (lane 4) and pp120 (lane 5). The molecular masses of marker proteins are shown on the right. (C) Time-course aggregation was monitored in the Labor-Apact aggregometer as described in Section 2. Arrows indicate the addition of collagen (1 Wg/ml) (representative experiment). The experiments shown were reproduced on at least three occasions.
bodies were used at the concentration of 2 Wg/ml and the anti-syk rabbit serum was used at dilution 1:1000. 3. Results Washed platelets were activated with collagen (1 Wg/ml) and the extent of aggregation was monitored in an aggregometer. The aggregation of human platelets was terminated before the shape change took place (10 s after the addition of collagen), after
the shape change (30 s after the addition of collagen), at the onset of aggregate formation (60 s after the addition of collagen), at the beginning of the completion of aggregation (120 s after the addition of collagen), and at the completion of aggregation (180 s after the addition of collagen) by solubilizing platelets in the sample bu¡er. Solubilized platelets were transferred to nitrocellulose and phosphotyrosine containing proteins were detected using 4G10 monoclonal anti-phosphotyrosine antibody. As shown in Fig. 1A, in unstimulated platelets, four tyrosine phosphorylated proteins were detected:
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a major band of 60 kDa and minor bands at 120, 80 and 65 kDa. Activation with collagen increased the phosphorylation of these bands and caused a number of proteins to become phosphorylated at tyrosine. After 30 s, there were 13 proteins with molecular weight of 120, 115, 112, 90, 82, 80, 70, 67, 65, 60, 58, 56 and 48 kDa phosphorylated. The extent of phosphorylation of these proteins increased after 60 and 120 s. At 180 s, after the addition of collagen, two additional phosphoproteins of 78 and 75 kDa appeared. To identify some of these proteins, platelet aggregates obtained 180 s after the addition of collagen were separated by SDS-PAGE, followed by transferring to nitrocellulose and blotting with the anti-phosphotyrosine antibodies. Protein bands were identi¢ed by: (a) apparent molecular weight; and (b) immunoblotting with mono- and polyclonal antibodies speci¢c to major phosphoproteins. As shown in Fig. 1B, the major band, showing molecular weight of 60 kDa, corresponded to pp60cÿsrc . The other bands, showing molecular weights of 120 and 70 kDa, corresponded to pp120 (the pp60cÿsrc substrate) and pp72syk , respectively. A triad at about 82, 80 and 78 kDa corresponded to the forms of pp80/85, the substrate of pp60cÿsrc . The other phosphoproteins were not identi¢ed; however, the proteins of molecular masses of 65, 58 and 56 kDa may represent the src family kinases pp62yes and pp54/58lyn , respectively. The identi¢cation of some of these proteins, like pp72syk and pp60cÿsrc , by immunoprecipitation followed by immunoblotting with anti-phosphotyrosine antibody gave identical results (not shown). In order to compare quantitatively the appearance of phosphoproteins in collagen-induced activation and aggregation of platelets nitrocelluloses containing phosphorylated proteins were densitometrically scanned and quantitated using the GelImage system. Fig. 2 shows that three groups of phosphoproteins can be distinguished, basing on the velocity and extent of phosphorylation. pp60cÿsrc constituted the ¢rst group; the increase in its phosphorylation was the highest and most rapid up to 30 s after the addition of collagen. The phosphorylation of this tyrosine kinase remained basically at the same level 180 s after the addition of collagen A kinase assay of pp60cÿsrc immunoprecipitates showed similar increase in pp60cÿsrc phosphorylation during a time
Fig. 2. Time course of platelet protein phosphorylation during collagen-induced platelet activation and aggregation. Platelet activation and aggregation was terminated 10, 30, 60, 120 and 180 s after the addition of collagen as described in Section 2. Platelet proteins were analyzed by SDS-PAGE on 8% polyacrylamide gels and by subsequent Western blotting with an anti-phosphotyrosine antibody, followed by alkaline phosphatase visualisation. Phosphoprotein bands were quantitated using GelImage system. To quantify densitometric scans, the background was subtracted and the area for each peak was determined. The results are the mean from three experiments.
course of collagen-induced platelet activation (results not shown). The second group consisted of pp80/85 and nonidenti¢ed protein of 65 kDa. The increase in their phosphorylation was 2-fold lower than that of pp60cÿsrc (i.e. 180 s after the addition of collagen), and reached its maximum 60 s after the addition of collagen. pp120, pp72syk , and two non-identi¢ed phosphoproteins of 90 and 75 kDa were in the third group. The increase in their phosphorylation was 4^10-fold lower than that of pp60cÿsrc (i.e. 180 s after the addition of collagen) and reached its maximum at 180 s. To help evaluate the role of tyrosine phosphorylation in platelet activation and aggregation we have tested the in£uence of monoclonal anti-Ib (PO14) antibody. Platelet aggregation in the presence of increasing concentration of PO14 was measured 180 s after the addition of collagen. The result in Fig. 3 shows that PO14 inhibited platelet aggregation in a concentration-dependent manner. PO14 had no e¡ect on platelet shape change, as shown in Fig. 3A. In order to compare quantitatively the e¡ect of PO14 on the phosphorylation of platelet proteins,
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nitrocelluloses containing the proteins obtained from the aggregated platelets, following immunoblotting with the anti-phosphotyrosine antibodies, were densitometrically scanned and quantitated using the GelImage system. Since not all the phosphoproteins
Fig. 4. The e¡ect of anti-Ib antibody on tyrosine phosphorylation of pp60cÿsrc , pp65, pp72syk and pp80/85. Platelets were aggregated in the presence and absence of the anti-Ib antibody. Platelets were incubated with the anti-Ib antibody for 30 min at room temperature. Phosphotyrosine bands were visualised as described in Section 2. Phosphoprotein bands were quantitated using the GelImage system. Relative peak area (y-axis) is the value obtained by dividing the peak area of the band from control aggregation (in the absence of antibody) by the peak area in the presence of antibody. The results are the mean þ S.D. from four experiments.
Fig. 3. The in£uence of anti-Ib antibody on platelet aggregation. Platelets were incubated with the anti-Ib antibody for 30 min at room temperature before aggregation. The aggregation of platelets was measured 3 min after the addition of collagen (1 Wg/ml), as described in Section 2. (A) Platelet aggregation tracings from control platelets (not treated with PO14) (line 1) and incubated with PO14 at concentrations 1, 5, 10 and 30 Wg/ ml (lines 2, 3, 4 and 5, respectively) are from one representative of six independent experiments with similar results. Collagen was added at the arrow. (B) Results are expressed as % of control aggregation (without antibody). The aggregation of control platelets (not incubated with antibody was 70^90%. The results are the mean þ S.D. from six experiments.
were detectable by this system, Fig. 4 shows only the inhibitory e¡ect of PO14 on the phosphorylation of 4 proteins. As it is seen in Fig. 4, P014 a¡ected mostly the phosphorylation of pp72syk which was decreased 3^6-fold when the antibody was used at the concentration of 5^30 Wg/ml. The inhibition of pp80/85 and pp65 was approximately twice smaller than that seen in the case of pp72syk . The phosphorylation of pp60cÿsrc was not a¡ected even at the concentration of 30 Wg/ml of anti-Ib, the highest concentration tested which inhibited platelet aggregation by 90%. The inhibition of phosphorylation of pp120 was complete even at the concentration of 5 Wg/ml of anti-Ib, the lowest concentration tested in these sets of experiments (not shown). 4. Discussion Activation and aggregation of platelets by collagen is associated with tyrosine phosphorylation of many platelet proteins. Based on the rate and extent of phosphorylation they may be divided into three groups: (a) pp60cÿsrc formed the ¢rst group, since
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the increase in the phosphorylation of this kinase was the most rapid one. A two-fold increase was already visible after 10 s (before the shape change) and an almost 10-fold increase after 30 s (after the shape change). The events taking place in platelets after 10 and 30 s are de¢ned as early activation events induced by collagen. Since the activation of pp60cÿsrc was almost complete during 30 s it can be concluded that the phosphorylation of this kinase is very important for the early activation manifested by the shape change. This conclusion was supported by using monoclonal anti-Ib antibodies PO14, which neither inhibited the shape change of platelets nor the pp60cÿsrc phosphorylation. The rate and extent of pp60cÿsrc phosphorylation induced by collagen is comparable to that described with other agonists [46]. Since the collagen-induced phosphorylation of pp60cÿsrc was the most rapid one it is very likely, as indicated earlier with thrombin [47], that the activation of this kinase is a prerequisite for phosphorylation of other kinases. (b) The second group consisted of pp80/85 and non-identi¢ed protein of 65 kDa. The increase in their phosphorylation reached maximum after 60 s. The onset of aggregate formation was observed at the same time. Thus, these two phosphoproteins seem to be responsible for this event. pp80/ 85 was primarily identi¢ed as a cytoskeleton-associated substrate for v-src in transformed cells. It did not show any identity with the 85 kDa subunit of phosphatidylinositol-3 kinase. The sequence of the SH3 domain of pp80/85 showed the greatest identity with other cytoskeleton-associated protein, such as fodrin and myosin 1 [48]. pp80/85 was shown to be present in platelets and was found previously to associate with pp60cÿsrc in thrombin-activated platelets. Similarly to collagen, thrombin induced the phosphorylation of this protein as soon as after 1 min [46]. In thrombin-activated platelets, pp60cÿsrc was postulated to participate in the cytoskeletal changes by phosphorylation of pp80/85 [46]. Probably the same mechanism is responsible for shape change in collagen-activated platelets. (c) pp72syk , pp120, and two phosphoproteins of 75 and 90 kDa belonged to the third group of phosphoproteins. The increase in their phosphorylation started basically 120 s after the addition of collagen. At this time, a massive aggregate formation occurred, and the phosphoproteins
from the third group can be responsible for this phenomenon. pp72syk was found previously not to be phosphorylated on tyrosine in resting platelets [34]. Many agonists, such as wheat germ agglutinin, thrombin, PAF, collagen and thromboxane A2 mimetic U 44069, were described previously to induce the activation of pp72syk in platelets [49^53]. Thrombin rapidly induced the phosphorylation of pp72syk [50]. The increase in pp72syk activity was observed as early as 5 s upon the thrombin treatment, it reached its maximum in 10 s and decreased to the basal level within 60 s in a calcium-dependent manner [50]. Our data are consistent with this observation and indicate the lack of phosphorylation of pp72syk in resting platelets. They also show that the kinetics of the pp72syk phosphorylation in the collagen-treated platelets differs from that described for the thrombin-treated platelets. A very small increase in the phosphorylation of pp72syk (together with three other proteins of 120, 90 and 75 kDa) was observed up to 120 s after the collagen addition. A rapid increase in its phosphorylation was seen after 120 s, which was coincident with massive aggregates formation. This may suggest that thrombin elicits some intracellular signals which are not shared with collagen. Our data also show some di¡erences from that were described previously for the collagen-treated platelets [39,53]. First, proteins of 54, 65, 75, 97 and 125 kDa were phosphorylated in the collagentreated platelets as described by Asazuma et al. [53]. The 75 kDa band consisted of two proteins, cortactin and pp72syk . Thus, a total number of six phosphoprotein bands was found. We found 15 proteins to be phosphorylated upon the comparable time of collagen treatment. Secondly, the activation of pp60cÿsrc showed only minimal changes upon the collagen treatment in the previous work, while we found that pp60cÿsrc phosphorylation increased several times and that was much higher and faster than other proteins. Thirdly, Asazuma et al. [53] described that the phosphorylation of pp72syk was very rapid; it reached its peak after 30^60 s and then gradually decreased. We show that the phosphorylation level of pp72syk was very low up to 120 s after the addition of collagen and a rapid increase in its phosphorylation occurred thereafter. Several factors can account for
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the above discrepancies. Collagen at the concentration 50 Wg/ml from Hormon-Chemie was required for the stimulation of platelets pretreated with 1 mM acetylsalicylic acid (ASA), as described by Asazuma et al. [53]. We used the equine-tendon collagen and the concentration of 1 Wg/ml of this collagen was enough to induce the activation and aggregation of platelets. The platelets used in our experiments were not subjected to ASA treatment, thus secondary e¡ects of thromboxane A2 generation may contribute to our results. The present data do not con¢rm observation that there is a signi¢cant increase in tyrosine phosphorylation of pp72syk within 1 min after collagen treatment of platelets [39]. The possible explanation of this discrepancy is that the experimental conditions used in both studies were di¡erent, i.e. treatment of platelets with soluble collagen from bovine skin at concentration 200 Wg/ml under non-stirring conditions may account for the di¡erences. The general importance of pp72syk activation for the collagen-induced aggregation of platelets was shown previously [39] by using a pp72syk -selective kinase inhibitor, piceatannol, which inhibited both kinase activity of pp72syk and platelet aggregation. Piceatannol also completely inhibited platelet shape change implying a role for pp72syk in the initial collagen-induced events. Additionally, since activation of pp72syk occurred even in the absence of platelet aggregation and K2 L1 integrin engagement a role for pp72syk upstream of platelet aggregation was postulated [39]. Our ¢ndings on the kinetics of pp72syk phosphorylation are in accordance with the report of Fuji et al. [51]. pp120, a phosphoprotein which like pp72syk belongs to the third group, was previously identi¢ed in chicken embryo ¢broblasts. Tyrosine phosphorylation of this protein correlated with src transformation in ¢broblasts. Monoclonal antibody against this protein used in this study are speci¢c for pp120. This antibody does not react with pp125FAK [54]. The identity and function of pp120 as well as other proteins of the molecular masses of 90, 75, 67, 65, 58, 56 and 48 have yet to be determined. We found that the phosphorylation of pp72syk as well as pp120 was inhibited by PO14 antibody which inhibited the platelet aggregation and did not inhibit the shape change of platelets. It clearly shows that these proteins play
135
a role in later signal transduction events associated with the aggregation of platelets. Previous studies, by using of anti-K2 L1 monoclonal antibody P1E6, showed that interaction of collagen with K2 L1 is directly connected with the phosphorylation of PLCQ2 and pp72syk [39]. However, the existence of K2 L1 -independent pathway of PLCQ2 and pp72syk phosphorylation was indicated by the use of two anti-K2 L1 mAbs, 6F1 and mAb13, which did not inhibit tyrosine phosphorylation of pp72syk and PLCQ2 induced by collagen-related triple helical peptide [55]. The use of K2 L1 integrin blocking monoclonal antibodies 6F1 and P1E6 demonstrated that tyrosine phosphorylation of FcRQ chain in response to collagen is not dependent on the integrin K2 L1 [39^ 41]. The identity of the collagen receptor which leads to tyrosine phosphorylation of FcRQ chain is not known. However, recent studies indicate that GPVI may be the signalling collagen receptor whose activation result in the tyrosine phosphorylation of the FcRQ chain [56]. In response to collagen, the activity of pp60cÿsrc from GPVI de¢cient platelets was comparable to normal platelets. Such activation of pp60cÿsrc was found to be K2 L1 -dependent because it was signi¢cantly inhibited in the presence of anti-K2 L1 mAb Gi9. By contrast, GPVI de¢cient platelets did not exhibit detectable tyrosine phosphorylation of pp72syk , pp125FAK and PLC-Q2 in response to collagen. Therefore, the requirement of GPVI for the collagen-stimulated tyrosine phosphorylation of these proteins was indicated [57]. To evaluate the contribution of GPIV to collagen induced protein tyrosine phosphorylation GPIV de¢cient platelets were used [58]. Since GPIV de¢cient platelets exhibited normal protein tyrosine phosphorylation in response to collagen, it seems that the role of this protein in collagen-induced signalling events is minor when compared with those of K2 L1 and GPVI. Ruan et al. initially reported that an anti-GPIbK antibody SZ 2 inhibited platelet aggregation induced by collagen and PAF [59]. However, the e¡ect of this antibody on protein tyrosine phosphorylation during collageninduced aggregation was not studied. It is noteworthy that anti-Ib antibody PO14 blocks both platelet aggregation and protein tyrosine phosphorylation. PO14 antibody was described previously as strong inhibitors of ristocetin/vWf induced platelet aggluti-
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nation and thrombin-induced platelet aggregation [60]. In addition, we demonstrate that PO14 inhibited the aggregation of platelets as well as tyrosine phosphorylation of proteins belonging to the second and third groups. These results suggest that GPIb plays an important role in the collagen-mediated platelet signalling occurring at the later stages of aggregation, i.e. at the stage of aggregate formation.
[10]
[11]
[12]
Acknowledgements This work was supported by Project 6 P207 055 06 from the State Committee for Scienti¢c Research. C.S.C. is HHMI international investigator. We thank Dr C. de Romeuf (Hemostasis Research Laboratories, Lille, France) for providing anti-GPIb antibody.
References
[13]
[14]
[15]
[1] H.R. Baumgartner, Platelet interaction with collagen ¢brils in £owing blood. I. Reaction of human platelets with K chymotrypsin-digested subendothelium, Thromb. Haemost. 37 (1977) 1^16. [2] G.A. Jamieson, C.L. Urban, A.J. Barber, Enzymatic basis for platelet-collagen adhesion as the primary step in haemostasis, Nature 234 (1971) 5^7. [3] H.B. Bensusan, T.L. Koh, K.G. Henry, B.A. Marray, L.A. Culp, Evidence that ¢bronectin is the collagen receptor on platelet membranes, Proc. Natl. Acad. Sci. USA 75 (1978) 5864^5868. [4] T.M. Chiang, A.H. Kang, Isolation and puri¢cation of collagen K1 (I) receptor from human platelet membrane, J. Biol. Chem. 257 (1982) 7581^7586. [5] S. Tsenuhisa, T. Tsuji, H. Tohyama, T. Osawa, Interaction of human platelet membrane glycoproteins with collagen and lectins, Biochim. Biophys. Acta 797 (1984) 10^19. [6] N.J. Kotite, J.V. Staros, L.W. Cunningham, Interaction of speci¢c platelet membrane proteins with collagen: evidence from chemical cross-linking, Biochemistry 23 (1984) 3099^ 3104. [7] H.K. Nieuwenhuis, J.W.N. Akkerman, W.P.M. Houdijk, J.J. Sixma, Human blood platelets showing no response to collagen fail to express surface glycoprotein Ia, Nature 318 (1985) 470^472. [8] S.A. Santoro, Identi¢cation of a 160,000 dalton platelet membrane protein that mediates the initial divalent cationdependent adhesion of platelets to collagen, Cell 46 (1988) 913^920. [9] B. Kehrel, L. Balleisen, R. Kokott, R. Mesters, W. Stenzinger, K.J. Clemetson, J. van de Loo, De¢ciency of intact
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
thrombospodin and membrane glycoprotein Ia in platelets with defective collagen-induced aggregation and spontaneous loss of disorder, Blood 71 (1988) 1074^1078. S.A. Santoro, S.M. Rajpara, W.D. Staatz, V.L. Woods, Isolation and characterization of a platelet surface collagen binding complex related to VLA-2, Biochem. Biophys. Res. Commun. 153 (1988) 217^223. W.D. Staatz, S.M. Rajpara, E.A. Wayner, W.G. Carter, S.A. Santoro, The membrane glycoprotein Ia^IIa (VLA2 ) complex mediates the Mg -dependent adhesion of platelets to collagen, J. Cell Biol. 108 (1989) 1917^1924. S.A. Santoro, J.J. Walsh, W.D. Staatz, K.J. Baranski, Distinct determinants on collagen support K2 L1 integrin-mediated platelet adhesion and platelet activation, Cell Regul. 2 (1991) 905^913. Y. Saito, T. Imada, J. Takagi, T. Kikuchi, Y. Inada, Platelet factor XIII. The collagen receptor?, J. Biol. Chem. 261 (1986) 1355^1358. T. Sugiyama, M. Okuma, F. Ushikubi, S. Sensaki, K. Kanaji, H. Uchino, A novel platelet aggregating factor found in a patient with defective collagen-induced platelet aggregation and autoimmune thrombocytopenia, Blood 69 (1987) 1712^ 1720. M. Moroi, S.M. Jung, M. Okuma, K. Shinmyozu, A patient with platelets de¢cient in glycoprotein VI that lack both collagen-induced aggregation and adhesion, J. Clin. Invest. 84 (1989) 1440^1445. M. Moroi, S.M. Jung, K. Shinmyozu, Y. Tomiyama, A. Ordinas, M. Diaz-Ricart, Analysis of platelet adhesion to a collagen-coated surface under £ow conditions: the involvement of glycoprotein VI in the platelet adhesion, Blood 88 (1996) 2081^2092. N.N. Tandon, U. Kralisz, G.A. Jamieson, Identi¢cation of GPIV(CD36) as a primary receptor for platelet-collagen adhesion, J. Biol. Chem. 264 (1989) 7576^7583. N.N. Tandon, Ch.F. Ockenhouse, N.J. Greco, G.A. Jamieson, Adhesive functions of platelets lacking glycoprotein IV (CD36), Blood 78 (1991) 2809^2813. M. Diaz-Ricart, N.N. Tandon, M. Carretero, A. Ordinas, E. Bastida, G.A. Jamieson, Platelets lacking functional CD36 (glycoprotein IV) show reduced adhesion to collagen in £owing whole blood, Blood 82 (1993) 491^496. L. McKeown, M. Vail, S. Williams, W. Kramer, K. Hansmann, H. Gralnick, Platelet adhesion to collagen in individuals lacking glycoprotein IV, Blood 83 (1994) 2866^2871. E.U.M. Saelman, B. Kehrel, K.M. Hese, P.G. de Groot, J.J. Sixma, K.H. Nieuwenhuis, Platelet adhesion to collagen and endothelial cell matrix under £ow conditions is not dependent on glycoprotein IV, Blood 83 (1994) 3240^3244. B. Kehrel, A. Kronenberg, J. Rauterberg, D. Niesing-Bresch, U. Niehues, J. Kardoeus, B. Schwippert, D. Tschope, J. van de Loo, K.J. Clemetson, Platelet de¢cient in glycoprotein IIIb aggregate normally to collagens type I and III but not to collagen type V, Blood 82 (1993) 3364^3370. N. Yamamoto, N. Akamatsu, H. Yamazaki, K. Tanoue, Normal aggregations of glycoprotein IV (CD36)-de¢cient
BBAMCR 14372 13-10-98
U. Kralisz, C.S. Cierniewski / Biochimica et Biophysica Acta 1405 (1998) 128^138
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32] [33]
[34]
[35]
[36]
[37]
[38]
[39]
platelets from seven healthy Japanese donors, Br. J. Haematol. 81 (1992) 86^92. B.S. Coller, J.H. Beer, L.E. Scudder, M.H. Steinberg, Collagen^platelet interactions: evidence for a direct interaction of collagen with platelet GPIa/IIa and indirect interaction with platelet GPIIb/IIIa mediated by adhesive proteins, Blood 74 (1989) 182^192. H. Weiss, V.T. Turitto, H.R. Baumgartner, Platelet adhesion and thrombus formation on subendothelium in platelets de¢cient in glycoproteins IIb^IIIa Ib, and storage granules, Blood 67 (1986) 322^330. K.S. Sakariassen, P.F.E.M. Nievelstein, B.S. Coller, J.J. Sixma, The role of platelet membrane glycoproteins Ib and IIb^ IIIa in platelet adherence to human artery subendothelium, Br. J. Haematol. 63 (1986) 681^691. A. Golden, S.P. Nemeth, J.S. Brugge, Blood platelets express high levels of the pp60cÿsrc -speci¢c tyrosine kinase activity, Proc. Natl. Acad. Sci. USA 83 (1986) 852^856. I.D. Horak, M.L. Corcoran, P.A. Thompson, L.M. Wahl, J.B. Bolen, Expression of p60fyn in human platelets, Oncogene 5 (1990) 597^602. S. Ohta, T. Taniguchi, M. Asahi, M. Kato, G. Nakagawara, H. Yamamura, Protein tyrosine kinase p72syk is activated by wheat germ agglutinin in platelets, Biochem. Biophys. Res. Commun. 185 (1992) 1128^1132. L. Lipfert, B. Haimovich, M.D. Schaller, B.S. Cobb, J.T. Parsons, J.S. Brugge, Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets, J. Cell Biol. 119 (1992) 905^912. S. Nakamura, H. Yamamura, Thrombin and collagen induce rapid phosphorylation of a cellular proteins on tyrosine in human platelets, J. Biol. Chem. 264 (1989) 7089^7091. M.H. Kroll, A.I. Schafer, Biochemical mechanisms of platelet activation, Blood 74 (1989) 1181^1195. A. Dhar, S.D. Shukla, Involvement of pp60cÿsrc in plateletactivating factor-stimulated platelets, J. Biol. Chem. 266 (1991) 18797^18801. E.A. Clark, S.J. Shattil, J.S. Brugge, Regulation of protein tyrosine kinases in platelets, Trends Biochem. Sci. 19 (1994) 464^469. J.B. Smith, C. Dangelmaier, M.a. Selak, B. Ashby, J. Daniel, Cyclic AMP does not inhibit collagen-induced platelet signal transduction, Biochem. J. 283 (1992) 889^892. J.B. Smith, C. Dangelmaier, J.D. Daniel, Elevation of cAMP in human platelets inhibits thrombin- but not collagen-induced tyrosine phosphorylation, Biochem. Biophys. Res. Commun. 191 (1993) 695^700. J.J. Daniel, C. Dangelmaier, J.B. Smith, Evidence for a role for tyrosine phosphorylation of phospholipase CQ2 in collagen induced platelet cytosolic calcium mobilization, Biochem. J. 302 (1994) 617^622. R.A. Blake, G.L. Schieven, S.P. Watson, Collagen stimulates tyrosine phosphorylation of phospholipase C-Q2 but phospholipase C-Q1 in human platelets, FEBS Lett. 353 (1994) 212^216. P.J. Keely, L.V. Parise, The K2 L1 integrin is a necessary co-
[40]
[41]
[42]
[43]
[44] [45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
137
receptor for collagen-induced activation of syk and subsequent phosphorylation of phospholipase CQ2 in platelets, J. Biol. Chem. 271 (1996) 26668^26676. F. Yanaga, A. Poole, J. Asselin, R. Blake, G.L. Schieven, E.A. Clark, C.-L. Law, S.P. Watson, Syk interacts with tyrosine-phosphorylated proteins in human platelets activated by collagen and cross-linking of the FcQ-IIa receptor, Biochem. J. 311 (1995) 471^478. J. Gibbins, J. Asselin, R. Farndale, M. Barnes, C.-L. Law, S.P. Watson, Tyrosine phosphorylation of the Fc receptor Q-chain in collagen-stimulated platelets, J. Biol. Chem. 271 (1996) 18095^18099. C. Watala, T. Pietrucha, K. Gwozdzinski, U. Kralisz, C.z.S. Cierniewski, Microenvironment changes in human blood platelet membranes associated with binding of tissue-type plasminogen activator, Eur. J. Biochem. 215 (1993) 867^ 871. B. Walkowiak, E. Michalak, W. Koziolkiewicz, C.S. Cierniewski, Rapid photometric method for estimation of platelet count in blood plasma or platelet suspension, Thromb. Res. 56 (1989) 763^766. C.C. Goodno, Myosin active-site trapping with vanadate ion, Methods Enzymol. 85 (1982) 116^123. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4 , Nature 227 (1970) 680^685. S. Wong, A.B. Reynolds, J. Papko¡, Platelet activation leads to increase c-src kinase activity and association of c-src with an 85-kDa tyrosine phosphoprotein, Oncogene 7 (1992) 2407^2415. R. Polanowska-Grabowska, M. Geanacopoulos, A.R.L. Gear, Platelet adhesion to collagen via the K2 L1 integrin under arterial £ow conditions causes rapid tyrosine phosphorylation of pp125FAK , Biochem. J. 296 (1993) 543^547. H. Wu, A.B. Reynolds, S.B. Kanner, R.R. Vines, J.T. Parsons, Identi¢cation and characterization of a novel cytoskeleton-associated pp60cÿsrc substrate, Mol. Cell. Biol. 11 (1991) 5113^5124. H. Maeda, T. Taniguchi, T. Inazu, Ch. Yang, G. Nakagawara, H. Yamamura, Protein-tyrosine kinase p72syk is activated by thromboxane A2 mimetic u44069 in platelets, Biochem. Biophys. Res. Commun. 197 (1993) 62^67. T. Taniguchi, H. Kitagawa, S. Yasue, S. Yanagi, K. Sakai, M. Asahi, S. Ohta, F. Takeuchi, S. Nakamura, H.P. Yamamura, Protein-tyrosine kinase p72syk is activated by thrombin and is negatively regulated through Ca2 mobilization in platelets, J. Biochem. Chem. 268 (1993) 2277^2279. C. Fuji, S. Yanagi, K. Sada, K. Nagai, K. Taniguchi, H. Yamamura, Involvement of protein-tyrosine kinase p72syk in collagen-induced signal transduction in platelets, Eur. J. Biochem. 226 (1994) 243^248. K. Rezaul, S. Yanagi, K. Sada, T. Taniguchi, H. Yamamura, Protein-tyrosine kinase p72syk is activated by platelet activating factor in platelets, Thromb. Haemost. 72 (1994) 637^641. N. Asazuma, Y. Yatomi, Y. Ozaki, R. Qi, K. Kuroda, K.
BBAMCR 14372 13-10-98
138
[54]
[55]
[56]
[57]
U. Kralisz, C.S. Cierniewski / Biochimica et Biophysica Acta 1405 (1998) 128^138 Satoh, S. Kume, Protein-tyrosine phosphorylation and p72syk activation in human platelets stimulated with collagen is dependent upon glycoprotein Ia/IIa and actin polymerization, Thromb. Haemost. 75 (1996) 648^654. S.B. Kanner, A.B. Reynolds, R.R. Vines, J.T. Parsons, Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases, Proc. Natl. Acad. Sci. USA 87 (1990) 3328^3332. J. Asselin, J.M. Gibbins, M. Achison, Y. Han Lee, L.F. Morton, R.W. Farndale, M.J. Barnes, S.P. Watson, A collagen-like peptide simulates tyrosine phosphorylation of syk and phospholipase CQ2 in platelets independent of the integrin K2 L1 , Blood 89 (1997) 1235^1242. J.M. Gibbins, M. Okuma, R. Farndale, M. Barnes, S.P. Watson, Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor Q-chain, FEBS Lett. 413 (1997) 255^259. T. Ichinohe, H. Takayama, Y. Ezumi, M. Arai, N. Yama-
moto, H. Takahashi, M. Okuma, Collagen-stimulated activation of syk but not c-src is severely compromised in human platelets lacking membrane glycoprotein VI, J. Biol. Chem. 272 (1997) 63^68. [58] J.A. Daniel, C. Dangelmaier, R. Strouse, J.B. Smith, Collagen induces normal signal transduction in platelets de¢cient in CD36 (platelet glycoprotein IV), Thromb. Haemost. 71 (1994) 353^356. [59] C.G. Ruan, X.P. Du, X.D. Xi, P.A. Castaldi, M.C. Berndt, A murine antiglycoprotein Ib complex monoclonal antibody, SZ 2, inhibits platelet aggregation induced by both ristocetin and collagen, Blood 69 (1987) 570^577. [60] K.J Clemetson, B. Hugli, V. von Tscharner, E¡ects of antibodies to the platelet GPIb-V-IX (CD42b, CD42c, CD42a) complex on ristocetin/vWF- and thrombin-induced platelet activation, in: S. Schlossman et al. (Eds.), Leucocyte Typing V, Vol. 2, Oxford University Press, Oxford, 1995, pp. 1328^ 1330.
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