BBALIP 54031

Activation of phospholipases in platelets by polyclonal antibodies against a surface membrane protein

(Received 4 June 1992)

Key words: Platelet: Polyclonal antibody: Phosphoiipase; Thromboxane A2 paper we demonstrated using immuno~hemi~a~ techniques that pro~~y~~t~de of van Willeb~a~d factor was present on the surface of resting platelets. In the present paper we show that pdycfonat ~~ti~~~j~~ against propo~y~e~t~de ofvon Wi~Iebrand factor induce activation of ~hosphol~~a~~~s~ in ptatelets and lead to pIatelet aggregation. The antibody-stimulation of platelets induced the synthesis of thromboxan~ A2 ITXA,). Furthermore, the aggregation was inhibited by aspirin and an antagonist of TXA,. Aspirin inhibited not only the aggregation but also the activation of arachidonic acid liberation from phospholipids, but the effect of aspirin on arachidonic acid liberation was cancelled by the combined effect of the antibodies and a TXA, mimetic agonist, which itself did not activate arachidonic acid Iibcration. The antibody-induced activation of arachidonic acid liberation and the aggregation were blocked by cytochalasin B. All these results obtained with antibodies were quite similar to the results obtained with collagen. In a previous


play a central

role in ~a~rnostas~s_ Re-

sponding to various kinds of agonists, they are activated and aggregated. The chemical nature of agonists

differ very widely. They include smal1 molecules such as ADP, epinephrine, serotonin, platelet-activating factor, thromboxane (TX) A,, pharbol ester and calcium ionophore, and macromolecules such as collagen and thrombin. Precise mechanisms through which platelets are activated by those agonists are very complicated and under intensive jnves~igatio~s. Since the dramatic activation reactions proceed very rapidly, platefets offer a good model to study intrac~Iln~ar signal transduction.

Correspondence to: Y. Saito, Department of Biological Sciences, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo 152, Japan. Abbreviations: TX, thromboxane; pp-vWF, propolypeptide of von Wikbrand factor: GHETE, 12%hydroxyeicosatetraenoic acid; PRP, platelet-rich ptasma; Hepes, N-2-Hydroxyethylpiperazine-N’-2~thane~s~~fon~c acid; ACD, acid-citrate dextrose; TLC. thin-layer chro~ato~raph~~ HHT, hydroxfheptadecatrienoif acid: PLA,, phospbolipase A,: PLC, ~~ospholipase C.

In addition to those above rne~~io~cd agcanists, there are quite a few membrane protc~~-directed mo~oclo~al arttibodies which activate platelets and induce aggregation. Antigens of some of those antibodies are well characterized [l-53, but some. are not [f&83. On the other hand, studies dealing with platelet aggregation induced by polyclonal antibodies are much less frequently reported. We have found that propolypeptide of von Willebrand factor (pp-vWF) binds to collagen and inhibits collagen-induced platelet aggregation {9,103. We have also determined the ~llagen-binding domain in ppvWF using monoclona~ antibodies [ll] and fragments obtained by a proteolyt~c enzyme 1121. We have reported in a recent paper that a ~o~~eg~igible amount of pp-vWF is present on the surface membrane of platelets [13], although the majority is found in a-granulcs and is released upon activation of platelets by various agonists. There we also showed that polyclonal antibodies against pp-vWF decreased the turbidity of platelet suspension. The present study was initiated to clarify whether this turbidity change is the result of mere agglutination of platelets or aggregation accompanied by various bi~~emical reactions especially phosphofipase activation and TXA, formation, In the

28 present paper we have presented evidence to support the notion that polyclonal antibodies against pp-vWF can induce biochemical reactions, including activation of phospholipase(s) leading to platelet activation and aggregation, Similarity between the antibody- and collagen-induced activations was also revealed. Experimental procedures

Bovine serum albumin (fatty acid free) and cytochalasin B were obtained from Sigma Chemical (St. Louis, MO). A TXA, mimetic agonist U46619 was from Cayman Chemical (Ann Arbor, MI). [1-14Cl Arachidonic acid (2 GBq/mmol) was purchased from New England Nuclear (Boston, MA). Fibrillar type I collagen from horse tendon was obtained from Hormon-Chimie (Munich, Germany). TXB, lZ5I-radioimmunoassay kit was from Amersham (Buckinghamshire, UK). A monoclonal antibody (IV.31 against Fc receptor Fc,RII was purchased from Medarex (West Lebanon, NH). A TXA, antagonist ONO-3708 was kindly provided by Ono Pharmaceutical (Osaka, Japan). 12S-Hydroxyeicosatetraenoic acid (1ZHETE) was synthesized as described before 1141. All other reagents were of analytical grade. Platelet preparation

Venous blood was obtained from healthy volunteers, who had not taken any drugs for the previous 14 days, into one-tenth vol. of 3,8% trisodium citrate. Plateletrich plasma (PRP) was obtained by centrifugation of blood at 150 x g for 15 min. Platelets were precipitated by centrifugation at 900 X g for 5 min in the presence of 10% acid-citrate dextrose (ACD; 60 mM citric acid, 90 mM trisodium citrate and 100 mM glucose). Preparation of Flab ‘Jz fragments

Polyclonal antibodies against pp-vWF were prepared as described in a previous paper [131. FCab’), fragments were prepared by digesting antibodies with immobilized pepsin (Pierce, Rockford, IL). Undigested IgG and,pFc’ fragments were removed by using Protein A affinity column (Pierce, Rockford, IL). The purity of F(ab’), fragments was checked by sodium dodecyl sulfate-polyac~lamide gel electrophoresis. Measurement of platelet aggregation

Platelets were suspended in Hepes-Tyrode buffer containing 137 mM NaCl, 2.7 mM KCl, 3.8 mM NaHCO,, 4 mM Hepes (N-2-HydroxyethylpiperazineN’-2-ethanesulfonic acid) and 5.5 mM glucose or TrisACD (2 . 1O8 cells/ml), and bovine serum albumin (1 mg/ml) and CaCl, (2 mM) were then added. Piatelet suspension was prewarmed at 37°C for a few minutes with constant stirring at 1000 rpm. Platelet aggregation

was initiated by the addition of agonists and was monitored turbidimetrically by an aggregometer Hematracer Model 601 (Niko Bioscience, Tokyo, Japan). Measurement of thromboxane formation An aliquot (10 ~1) was taken from stimulated platelet

suspension and mixed with 190 ,ul of phosphatebuffered saline containing 50 PM indomethacin, 10 mM EDTA and 0.005% Triton X-100 (pH 6.81, piaced in an ice bath. Samples were diluted to adequate concentration with the assay buffer. TXB, radioimmunoassay was performed as suggested by the manufacturer. Analysis of arachidonic acid metabolites

PRP was added to 2 vol. of Tris-ACD buffer and was incubated with 700 Bq/ml [‘4C]arachidonic acid for 1 h at 30°C. Unincorporated arachidonic acid was removed by centrifugation. Platelets were stimulated as indicated in the legends for figures. The reactions were stopped by the addition of 8 vol. of chloroform/ methanol (1 : 2, v/v> and lipids were extracted essentially by the method of Bligh and Dyer [15]. Arachidonic acid metabolites were separated by thin-Iayer chromatography (TLC) with the solvent system of the organic solvent phase of isooctane/ ethyl acetate/ acetic acid/water (50: 90 : 20: 100, v/v) and analyzed by Bioimaging Analyzer BAS 2000 system (Fuji Film, Tokyo, Japan). Results Lectins are known to agglutinate platelets as well as red blood cells even if cells are fixed by aldehydes. This is because lectins and the membrane both have multivalent binding sites. The situation is very similar to antigen-antibody complex formation by polyclonal antibodies. However, paraformaldehyde-fixed platelets were not agglutinated by the polyclonal antibodies against pp-vWF 1131. This indicated to us that the polyclonal antibodies might induce aggregation of platelets accompanied by activation processes, but not mere agglutination. This notion was further supported by the next experiment. F(ab’j2 fragments obtained from the antibodies did not induce the turbidity change of washed platelet suspension even at 600 pg/ml, although antibodies themselves induced very clear turbidity change at 200 pg/ml. However, preincubation of platelets with the fragments at 300 pg/ml completely inhibited the antibody (200 pg/ml)-induced aggregation of platelets (data not shown). These fragments are divalent and should be able to make antigen-antibody complex, just as well as the intact antibodies do. The involvement of the Fe portion of the antibodies in the activation process was strongly suggested. In fact, a monoclonal antibody against

29 platelet Fc receptor (Fc,,RII) inhibited the antibodyinduced platelet aggregation in a dose-dependent manner (data not shown). It is intriguing to know how Fc receptors are involved in the activation process. Is an Fc portion necessary to make a bridge between surface antigen and Fc receptor on the platelet surface, or else

is the occupancy of the Fc receptor by the antigenbound antibody the key reaction to initiate the whole series of biochemical reactions? Several experiments conducted with monoclonal antibodies against platelet membrane proteins proved the importance of an Fc portion of immunoglobulin to aggregate platelets [16,17]. Fig. 1 reveals that the antibody stimulation induced the synthesis of TXA,. Although the extent was very nominal, TXA, synthesis was already initiated even before the onset of aggregation. The synthesis was continued further, along with the aggregation. In order to find out whether TXA, synthesis is obligatory for the aggregation of platelets or TXA, is a non-essential by-product of the aggregation, the following experiments using various kinds of inhibitors were conducted (Fig. 2). A cyclooxygenase inhibitor aspirin at 1 mM clearly inhibited the aggregation. A TXA, antagonist ONO-3708 also strongly inhibited at 6 PM. 12-HETE, which was discovered to be a potent inhibitor of arachidonic acid liberation from phospholipids [ 181, also inhibited the aggregation at 20 PM. As expected a Ca*+/metal chelator EDTA (1 mM) and an activator of adenylate cyclase prostaglandin E, (2 PM) also unequivocally inhibited the aggregation (data not shown). All of these data strongly suggest that the antibody-induced turbidity change of platelets represents aggregation of platelets and TXA, synthesis, is in fact a prerequisite for the aggregation.

,2 min,


Fig. 2. The effect of inhibitors on the antibody-induced aggregation. The antibodies (the final concentration 150 pg/ml) were added as soon as possible after the addition of 12-HETE or ONO-3708, or a 5-min later aspirin addition. (A) The antibodies alone (the control); (B) with 12-HETE (20 PM); (C) with ONO-3708 (6 FM); (D) with aspirin (1 mM).


TXB,-+ PL+ ABC 0






Fig. 1. Thromboxane synthesis in platelets induced by the antibodies. Aliquots were taken at the times indicated during the aggregation. The upper continuous tracing lower line with dots indicates sized. The final concentration

indicates platelet aggregation the amounts of thromboxane of the antibodies added pg/ml.

and the synthewas 132


Fig. 3. Synergistic effect of TXA, mimetic agonist and the antibodies on arachidonic acid liberation. [‘4C]Arachidonic acid-labeled platelets were pretreated with (lanes C and D) or without (lanes A and B) 1 mM aspirin for 5 min at room temperature prior to the activation by 200 pg/ml antibody (lanes B, C and D) or 100 nM U46619 (lanes A and D). 10 min later, lipids were extracted and analyzed by TLC as described in Experimental Procedures. AA, arachidonic acid; IZHETE, 12S-hydroxyeicosatetraenoic acid; HHT, hydroxyheptadecatrienoic acid; TXB,, thromboxane B,; PL, phospholipids.



f-TX& +PL ab Fig. 4. The effect of cytochalasin B on the antibody-induced arachidonic acid liberation and platelet aggregation. [“C]Arachidonic acid labeled-platelets were preincubated with (b) or without (a) 40 PM cytochalasin B for 5 min at room temperature prior to the activation by ‘200 ,ug/mt antibody. A, platelet aggregation. B, TLC profiles of arachidonic acid metabolit~s. 8 min after the addition of the antibodies, lipids were extracted from platelets and analyzed by TLC as described in experimental Procedures. Abbreviations are indicated in the legend for Fig. 3.

We then investigated the mechanism through which antibodies stimulated TXA, synthesis. As is clearly shown in Fig. 3, arachidonic acid was liberated from phospholipid and was converted into TXA, and hydroxyheptadecatrienoic acid (HHT) (cyclooxygenase products) and I2-HETE (a 1Zlipoxygenase product) upon stimulation with the antibodies (lane B). The addition of aspirin, however, almost completely abolished the production of these compounds without extensive accumulation of free arachidonic acid, although a small accumulation of unidentified less polar metabolites was detected (lane C). This result strongly suggests that the preceding synthesis of TXA, is required for the massive increase in arachidonic acid liberation induced by anti-pp-vWF antibody. In fact, the addition of a TXA, mimetic agonist U46619 restored the ability for the liberation of arachidonic acid and the synthesis of IZHETE (lane D>, although the mimetic agonist alone did not induce the liberation at all (lane A). In addition to TXA2, intact cytoskeletal machinery was also necessary for the antibody-induced liberation of arachidonic acid, because cytochalasin B, which inhibits the formation of normal microfilament organization, very strongly inhibited the liberation of arachidonic acid at 40 PM (Fig. 4B). Antibody-induced aggregation was also very much inhibited by cytochalasin B (Fig. 4A). It is, however, not clear whether this inhibition of aggregation is a result of inhibition of phospholipases, or a reflection of disruption of cytoskeletal assembly, or is due to both.

Discussion Platelet activation by the antibodies is accompanied with the synthesis of TXA,. Both cyclooxygenase inhibitor (aspirin) and a TXA, antagonist (ONO-3708) almost completely abolished the antibody-induced aggregation. Furthermore IZ-HETE also inhibited the aggregation, probably by inhibiting the activation of phospholipase A, @LA,) which is a preceding step of TXA, formation. Apparently, the synthesis of TXA, was a prerequisite for the induction of the antibody-induced aggregation. The arachidonic acid liberation that we detected should be mediated by PLA,, because the majority of arachidonic acid is thought to be provided by the action of PLA, along with platelet activation [191. Contribution of the combined effects of phospholipase C (PLC) and diacylglycerol lipase was also suggested in certain conditions [20]. In the present study, however, arachidonic acid liberation seems to be mainly mediated by PLA, because the labeling technique we employed with [‘*C]arachidonic acid was reported to result in preferential incorporation into phosphatidylcholine molecules 1211, which are good substrates for PLA, but poor substrates for PLC. importance of TXA, is further investigated at the level of PLA, activation. The antibodies activated PLA, and liberated arachidonic acid from phospholipids and arachidonic acid was then converted to TXA,, KHT and 12-HETE. In the presence of aspirin, activation of PLA, was completely blocked. However, when the

31 TXA, mimetic agonist U46619 was simultaneously added, liberation of arachidonic acid was quite normal, although U46619 alone did not have any ability to activate PLA 2 at all. The mechanism by which PLA 2 is activated by the antibodies is not understood, but it is quite sure that TXA, would play a central role. These data suggest that small amounts of TXA, have to be initially synthesized without the help of TXA, and the activation process should then be accelerated to produce more ara~hidoni~ acid and finally TXA,. A question remains as to the involvement of multiple kinds of PLAz in the antibody-induced platelet aggregation. It may be conceivable that one of them is aciivated by the antibodies without the help of TXA, and that the other is only activated in the presence of TXA,. The mechanism has to be clarified by further investigation. Early change of intracellular Ca’+ concentration and phosphorylation of 20- and 47 kDa proteins (data not shown) would suggest the activation of PLC in the early phase of activation by the antibodies. The involvement of PLC and dia~lglycerol lipase for TXA, production also remains to be elucidated. A monoclonal antibody against CD9 antigen was reported to induce two-step mobilization of arachidonic acid 133.It was proposed that the first step was mediated by PLA, and the second step was mediated either by PLA, or PLC, or by both and was accelerated by TXA, and other unidentified factors. A similar kind of proposal was made about collagen-induced platelet aggregation

Ku Ail of the data presented so far were completely in line with the experiments simultaneously conducted with collagen (data not shown). It was reported by Nakano et al. [23] that a cyclooxygenase inhibitor or a TXA, receptor antagonist inhibited arachidonic acid release from phospholipids induced by collagen stimulation, as we have shown in the present study using antibodies and collagen (data not shown). They also reported that the inhibitory effect of a cyclooxygenase inhibitor was canceled by the synergistic effect between collagen and U46619 and that cytochalasin B strongly inhibited the stimulation of collagen. They have suggested that cytoskeleton plays an important role in collagen-stimulated activation of pbospholipases. This was also shown in the present study by the experiment using cytochalasin B with the antibodies and collagen (data not shown), which completely blocked the liberation of arachidonic acid and aggregation. We do not understand why anti-pp-vWF antibodies and collagen seem to share, at least superficially, signal transduction pathways. This remains to be further investigated. We believe this system will give us a good model to understand in the future how phospholipases are activated by external stimuli.

Acknowledgements This work was supported in part by Grants-in-Aid for General Scientific Research and for Co-operative Research from the Ministry of Education, Science and Culture of Japan and by grants from The Ryoichi Naito Foundation for Medical Research and Ito Memorial Foundation. Special appreciation should be extended to nurses in Health Service Center in Tokyo Institute of Technolo~ for collecting blood from volunteers. References 1 Slupsky, J.R., Seehafer, J.G., Tang, S.-C., Masellis-Smith, A. and Shaw, A.R.E. (1989) J. Biol. Chem. 264, 12289-12293. 2 Carroll, R.C., Worthington, R.E. and Boucheix, C. (1990) Biochem. J. 266, 527-535. 3 Ozaki, Y., Matsumoto, Y., Yatomi, Y., Higashihara, M. and Kume, S. (1991) Eur. J. Biochem. 199, 347-354. 4 Boucheix, C., Benoit, P., Frachet, P.. Billard, M., Worthington, R.E., Gagnon, J., and Uzan, 6. (1991) J. Biol. Chem. 266, 117-122. 5 Gulino, I)., Ryckewaert, J.-J., Andrieux, A., Rabiet, M.-J. and Marguerie, G. (1990) J. Biol. Chem. 265, 9575-9581. 6 Scott, J.L., Dunn, S.M., Jin, B., Hillam, A. J., Walton, S., Berndt, M.C., Murray, A.W., Krissanssen, G.W. and Burns, G.F. (1989) J. Biol. Chem. 264, 13475-13482. 7 Shah, V.G., Zamora, P.O., Mills, S.L., Mann, P.L. and Comp, P.C. (1990) Thromb. Res. %X,493-504. 8 Kornecki, E., Walkowiak, B., Naik, U.P. and Ehrlich Y.H. (1990) J. Biol. Chem. 265, 10042-10048. 9 Takagi, J., Sekiya, F., Kasahara, K., Inada, Y. and Saito, Y. (1989) J. Biol. Chem. 264, 6017-6020. 10 Takagi, J., Kasahara, K., Sekiya, F., Inada, Y. and Saito, Y. (1989) J. Biol. Chem. 264, 1~25-1043~. 11 Fujisawa, T., Takagi, J., Sekiya, F., Miake, F., Goto, A. and Saito, Y. (1991) Eur. J. Biochem. 196.673-677. 12 Takagi, J., Fujisawa, T., Sekiya, F. and Saito, Y. (1991) J. Biol. Chem. 266, 5575-5579. 13 Hashimoto, K., Usui, T., Sasaki, K., Fujisawa, T., Sekiya, F., Takagi, J., Tsukada, T. and Saito, Y. (1991) Biochem. Biophys. Res. Commun. 176, 1571-1576. 14 Shimazaki, T., Kobayashi, Y. and Sato, F, (1988) Chem. Lett. 1785-1788. 15 Bligh, E.A. and Dyer, W.J. (19.59) Can. J. Biochem. Physiol. 37, 911-917. 16 Worthington, R.E., Carroll, R.C. and Boucheix, C. (1990) Br. J. Haematol. 74, 216-222. 17 RubiRstein, E., Kouns, W.C., Jennings, L.K., Boucheix, C. and Carroll, R.C. (1991) Br. J. Haematol. 77, 80-86. 18 Sekiya, F., Takagi, J., Sasaki, K., Kawajiri, K.. Kobayashi, Y., Sato, F. and Saito, Y. (19901 Biothim. Biophys. Acta 1044, 165-168. 19 Irvine, R.F. (1982) Biochem. J. 204, 3-16. 20 Bell, R.L., Kennerly, D.A., Stanford, N. and Majerus, P.W. (1979) Proc. Natl. Acad. Sci. USA 76, 3238-3241. 21 Bills, T.K., Smith, J.B. and Silver, M.J. (1976) Biochim. Biophys. Acta 424, 303-314. 22 Hanasaki, K., Nakano, T. and Arita, H. (1987) Thromb. Res. 46, 425-436. 23 Nakano, T., Hanasaki, K. and Arita, H. (1989) J. Biol. Chem. 264, 5400-5406.

Activation of phospholipases in platelets by polyclonal antibodies against a surface membrane protein.

In a previous paper we demonstrated using immunochemical techniques that propolypeptide of von Willebrand factor was present on the surface of resting...
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