THROMBOSIS RESEARCH 63; 395398,1991 0049-3848/91 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.
BRIEF
COMMUNICATIOIj
ANTI-GELSOLINANTIBODY FOR DF3ECTION OF PLASMA AND PLATELET GELSOLIN Motofumi HIYOSHI,Takahisa YAMANE, Takeshi INOUE, Yoshio FURUKAWA. and Noriyuki TATSUMI Department of Laboratory Medicine,Osaka City UniversityMedical School, l-5-7,Asahimachi,Abeno, Osaka, 545, Japan. (Received 4.4.1991; accepted in revised form 30.5.1991 by Editor A. Takada)
INTRODUCTION Gelsolinis a protein that regulates actin polymerization(1)and that is found in macrophages (2).platelets(3)and plasma (4). Details about how gelsolinbehaves in the process of thrombus formationin -- vivo are not known and the roles of gelsolincan be studied by the use of appropriate antibodies. Preparation of anti-gelsolinantibodieswith the use of plateletsis not efficientbecause it is difficultto obtain enough platelets. Since there is a high amount of gelsolinin plasma, we prepared the antibody by the use of human plasma gelsolin. METHODS Fresh human plasma (1.2L), obtained from healthy donors and prevented from coagulationby treatment with citrate phosphate-dextrose solution,was fractionatedwith 30-50X saturationof (NH4)2S04. The sample was dialyzed against 10 mM Tris-HCl(pH 8.0;buffer A) and loaded on a DE53 (Whatman)column equilibratedin buffer A. After the column was washed with 0.05 M NaCl in buffer A, it was eluted with a gradient of 0.05-0.25 M NaCl in buffer A. The fractionsthat depolymerized Factin were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),and the fractionsthat contained gelsolinwere dialyzed against 50 mM Tris-HCl(pH 7.0;buffer B). Column chromatography with Blue Sepharose CL-6B (Pharmacia)equilibratedin buffer B was carried out to the dialysate. The column was eluted with a gradient of O-l.5M KC1 in buffer B. The pooled active fractionswere dialyzed against 0.025M imidazole-HCl(pH 7.4,buffer C) and loaded on a PBE94 column (Pharmacia)equilibratedin buffer C. The column was eluted with a gradient of pH 7.4-4.0with 0.0094M/pH unit Polybuffer 74 (Pharmacia). to give purifiedplasma gelsolin. All procedures were carried out at 4'C and redistilledwater was used for all experiments.
Key words: Gelsolin,platelets,monoclonal antibody,plasma protein. 395
ANTIBODY TO HUMAN GELSOLIN
Vol. 63, No. 3
BALB/c mice 4 weeks old were immunized intraperitoneallywith the purifiedprotein (0.01mg/mouse) and Freund's complete adjuvant. Ten days later the mice were immunized intraperitoneallywith plasma gelsolin(0.005mg/mouse) and Freund'sincomplete adjuvant. The immunization with use of Freud'sincomplete adjuvant was repeated for a total of four times every 10 days. Three days later each mouse was immunized intravenouslywith 0.01 mg of plasma gelsolin. Serum was obtained from blood samples from the mice and its antibody titer was evaluated by immunodiffusion(5)and electroblotting(6). The immunoglobulin(Ig) class was identifiedby enzyme-linkedimmunosorbent assay (ELISA),and the IgG fractionwas purifiedas previously described (7). Human platelet gelsolinwas purifiedas previously described (6). RESULTS AND DISCUSSION Human plasma constituentsare differentfrom cytosolicproteins obtained from cells such as platelets. Several procedures, therefore, were modified to purify gelsolinfrom plasma. After the first DE53 chromatography,the pooled active fractions had major bands and several minor bands on SDS-PAGE. With the two steps of further purification, a single band with the molecular weight of 93,000was obtained (Fig.lA, lane 3). The purifiedprotein was at the positionof 93,000dalton,which was the molecular weight of human plasma gelsolin(2). The purifiedprotein also showed viscosity decrease activityof gelsolinto F-actin described previously (2). We, therefore,concluded the purifiedprotein was gelsolin. The final yield of gelsolinwas 5 mg from 1.2 L of fresh plasma. The purifiedprotein was kept at -20 'C and later used in antibody experiments. Gamma globulin fused strongly with both purifiedplasma gelsolin and purifiedplatelet gelsolinon the immunodiffusionplates. On the electroblottednitrocellulosesheet, the serum reacted with both gelsolins(Fig.1B). Thus, the polyclonal antiserum reacted with both plasma gelsolinand platelet gelsolin. The polyclonal antibody recognized epitopes common to plasma and platelet gelsolins. The immunoglobulin class of purifiedpolyclonal antibody was IgG1. Chaponnier et al. (9)reported the preparation of a monoclonal antibody to gelsolinfrom human plasma. The antibody could bind to a protein from human platelets. In their paper, detailsabout how the protein was checked to find if it was gelsolinare not given. The antigelsolinantibody we obtained reacted with platelet gelsolin. The role of cellular gelsolinin cell activationhas been studied. When cells are activated (for example,when plateletsare activatedby thrombin).gelsolincontributesto actin polymerizationby regulation of Ca++ and phosphatidylinositolsin the cells (10,ll).However, this idea is inconsistentwith the known in vitro biochemicalproperties of gelsolin,actin,phosphatidylinositog,c and profilin(12). To predict on the basis of these properties,a cell stimulusthat elevated Ca++ and transientlydepressed phosphatidylinositol4,5-bisphosphate would accelaratethe severing actin filaments,increase the actin monomer pool by capping its barbed ends, increase the amount of profilactin formed, and promote actin depolymerizationoverall. However, what is
Vol. 63, No. 3
ANTIBODY TO HUMAN GELSOLIN
23845
AI
66,200-
42.699-
397
!WMB
--
31,000 7 FRONT-&
Fig.
1.
Specificity
_
of the polyclonal
anti-gelsolin
antibody.
Plasma gelsolinor platelet gelsolinfrom human materialswas electrophoresed on a 9% SDS-polyacrylamidegel and stained with Coomassie blue (panel A). After SDS-PAGE, the proteins were electroblotted on a nitrocellulosesheet and stained with polyclonal antigelsolinantibody (panel B). Lane 1, molecular weight standards;lanes 3 and 4, plasma gelsolin;lanes 2 and 5, platelet gelsolin. Actin is also seen on SDS-PAGE (lane 2) because gelsolin-actin complex is obtained by the method previously described (8).
observed is that stimulatedcells respond by assemblingactin,that is, by polymerizingactin. Therefore,many questions about the role of gelsolinin cell activationare unanswered. In studies done to answer such questions,the antibody reported here might be useful. A simple method is currently prepared to measure the amount of gelsolinboth in cytosolic fraction of human plateletsand in human plasma with use of the anti-gelsolinantibodiesconjugated to latex particles. We are now making monoclonal antibody to human plasma gelsolin,too. When the monoclonal antibody is obtained,a sandwich method by ELISA with use of the monoclonal antibody and polyclonal one could be used to measure the amount of gelsolin. REFERENCES I. JANMEY, P. A., CHAPONNIER, C., LIND, S. E.. ZANER, K. S..STOSSEL, T. P.,and YIN, H. L. Interactionsof gelsolinand gelsolin-actin complexes with actin: effects of calcium on actin nucleation, filamentsevering,and end blocking. Biochemistry 24, 3714-3723,
ANTIBODY TO HUMAN GELSOLIN
Vol. 63, No. 3
1985.
2. YIN, H. L..and STOSSEL, T. P. Control of cytoplasmicactin gel-sol transformationby gelsolin,a calcium-dependentregulatory protein. Nature (London) 281, 583-586,1979. 3. LIND. S. E.,YIN, H. L.,and STOSSEL, 'I'. P. Human plateletscontain gelsolin: a regulator of actin filamentlength. 2" J Clin Invest @, 1384-1387,1982. 4. HARRIS, D. A., and SCHWARTZ, J. H. Characterizationof brevin,a serum protein that shortens actin filaments. Proc. Natl. Acad. Sci. USA 2, 6798-6802,1981. 5. FAHEY. J. L.,and MCKELVEY, E. M. Quantitativedeterminationof serum immunoglobulinsin antibody-agarplates. J- Immunol. 94, 8490, 1965. 6. TOWBIN. H., STAEHELIN,T.,and GORDON, J. Electrophoretictransfer of proteins from polyacrylamidegels to nitrocellulosesheets: procedure and some applications.--Proc. Natl.Acad. -Sci. USA 76, 4350-4354,1979. 7. ~KERSTRZM. B.,BRODIN, T..REIS, K.,and BJ~~RCK,L. Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies. J- Immunol. 135, 2589-2592,1985. 8. HIYOSHI,M., IM, T.,SASAKI,A., HASHIMOTO, K., and TATSUMI,N. Involvement of calcium ion in the interactionbetween the actingelsolincomplex and phosphatidylinositol 4-monophosphate or 4,5bisphosphate. Biochem.Int. 2, 1009-1015,1989. 9. CHAPONNIER, C.. JANMEY, P. A., and YIN, H. L. The actin filamentJ. Cell Biol 103, 1473-1481, severing domain of plasma gelsolin. _-A 1988. 10. KWIATKOWSKI,D. J.,and YIN, H. L. Molecular biology of gelsolin,a calcium-regulatedactin filamentsevering protein. Biorheology 24, 643-647,1987. 11. JANMEY, P. A., IIDA,K.,YIN, H. L.,and STOSSEL. T. P. Polyphosphoinositide micellesand polyphosphoinositide-containing vesiclesdissociateendogenous gelsolin-actin complexes and promote actin assembly from the fast-growingend of actin filamentsblocked 1987. by gelsolin. J- &.& Chem. 262, 12228-12236. 12. HARRIS, H. Microfilamentdynamics. Nature (London) 330, 310-311, 1987.
BRIEF
COMMUNICATIOIj
ANTI-GELSOLINANTIBODY FOR DF3ECTION OF PLASMA AND PLATELET GELSOLIN Motofumi HIYOSHI,Takahisa YAMANE, Takeshi INOUE, Yoshio FURUKAWA. and Noriyuki TATSUMI Department of Laboratory Medicine,Osaka City UniversityMedical School, l-5-7,Asahimachi,Abeno, Osaka, 545, Japan. (Received 4.4.1991; accepted in revised form 30.5.1991 by Editor A. Takada)
INTRODUCTION Gelsolinis a protein that regulates actin polymerization(1)and that is found in macrophages (2).platelets(3)and plasma (4). Details about how gelsolinbehaves in the process of thrombus formationin -- vivo are not known and the roles of gelsolincan be studied by the use of appropriate antibodies. Preparation of anti-gelsolinantibodieswith the use of plateletsis not efficientbecause it is difficultto obtain enough platelets. Since there is a high amount of gelsolinin plasma, we prepared the antibody by the use of human plasma gelsolin. METHODS Fresh human plasma (1.2L), obtained from healthy donors and prevented from coagulationby treatment with citrate phosphate-dextrose solution,was fractionatedwith 30-50X saturationof (NH4)2S04. The sample was dialyzed against 10 mM Tris-HCl(pH 8.0;buffer A) and loaded on a DE53 (Whatman)column equilibratedin buffer A. After the column was washed with 0.05 M NaCl in buffer A, it was eluted with a gradient of 0.05-0.25 M NaCl in buffer A. The fractionsthat depolymerized Factin were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),and the fractionsthat contained gelsolinwere dialyzed against 50 mM Tris-HCl(pH 7.0;buffer B). Column chromatography with Blue Sepharose CL-6B (Pharmacia)equilibratedin buffer B was carried out to the dialysate. The column was eluted with a gradient of O-l.5M KC1 in buffer B. The pooled active fractionswere dialyzed against 0.025M imidazole-HCl(pH 7.4,buffer C) and loaded on a PBE94 column (Pharmacia)equilibratedin buffer C. The column was eluted with a gradient of pH 7.4-4.0with 0.0094M/pH unit Polybuffer 74 (Pharmacia). to give purifiedplasma gelsolin. All procedures were carried out at 4'C and redistilledwater was used for all experiments.
Key words: Gelsolin,platelets,monoclonal antibody,plasma protein. 395
ANTIBODY TO HUMAN GELSOLIN
Vol. 63, No. 3
BALB/c mice 4 weeks old were immunized intraperitoneallywith the purifiedprotein (0.01mg/mouse) and Freund's complete adjuvant. Ten days later the mice were immunized intraperitoneallywith plasma gelsolin(0.005mg/mouse) and Freund'sincomplete adjuvant. The immunization with use of Freud'sincomplete adjuvant was repeated for a total of four times every 10 days. Three days later each mouse was immunized intravenouslywith 0.01 mg of plasma gelsolin. Serum was obtained from blood samples from the mice and its antibody titer was evaluated by immunodiffusion(5)and electroblotting(6). The immunoglobulin(Ig) class was identifiedby enzyme-linkedimmunosorbent assay (ELISA),and the IgG fractionwas purifiedas previously described (7). Human platelet gelsolinwas purifiedas previously described (6). RESULTS AND DISCUSSION Human plasma constituentsare differentfrom cytosolicproteins obtained from cells such as platelets. Several procedures, therefore, were modified to purify gelsolinfrom plasma. After the first DE53 chromatography,the pooled active fractions had major bands and several minor bands on SDS-PAGE. With the two steps of further purification, a single band with the molecular weight of 93,000was obtained (Fig.lA, lane 3). The purifiedprotein was at the positionof 93,000dalton,which was the molecular weight of human plasma gelsolin(2). The purifiedprotein also showed viscosity decrease activityof gelsolinto F-actin described previously (2). We, therefore,concluded the purifiedprotein was gelsolin. The final yield of gelsolinwas 5 mg from 1.2 L of fresh plasma. The purifiedprotein was kept at -20 'C and later used in antibody experiments. Gamma globulin fused strongly with both purifiedplasma gelsolin and purifiedplatelet gelsolinon the immunodiffusionplates. On the electroblottednitrocellulosesheet, the serum reacted with both gelsolins(Fig.1B). Thus, the polyclonal antiserum reacted with both plasma gelsolinand platelet gelsolin. The polyclonal antibody recognized epitopes common to plasma and platelet gelsolins. The immunoglobulin class of purifiedpolyclonal antibody was IgG1. Chaponnier et al. (9)reported the preparation of a monoclonal antibody to gelsolinfrom human plasma. The antibody could bind to a protein from human platelets. In their paper, detailsabout how the protein was checked to find if it was gelsolinare not given. The antigelsolinantibody we obtained reacted with platelet gelsolin. The role of cellular gelsolinin cell activationhas been studied. When cells are activated (for example,when plateletsare activatedby thrombin).gelsolincontributesto actin polymerizationby regulation of Ca++ and phosphatidylinositolsin the cells (10,ll).However, this idea is inconsistentwith the known in vitro biochemicalproperties of gelsolin,actin,phosphatidylinositog,c and profilin(12). To predict on the basis of these properties,a cell stimulusthat elevated Ca++ and transientlydepressed phosphatidylinositol4,5-bisphosphate would accelaratethe severing actin filaments,increase the actin monomer pool by capping its barbed ends, increase the amount of profilactin formed, and promote actin depolymerizationoverall. However, what is
Vol. 63, No. 3
ANTIBODY TO HUMAN GELSOLIN
23845
AI
66,200-
42.699-
397
!WMB
--
31,000 7 FRONT-&
Fig.
1.
Specificity
_
of the polyclonal
anti-gelsolin
antibody.
Plasma gelsolinor platelet gelsolinfrom human materialswas electrophoresed on a 9% SDS-polyacrylamidegel and stained with Coomassie blue (panel A). After SDS-PAGE, the proteins were electroblotted on a nitrocellulosesheet and stained with polyclonal antigelsolinantibody (panel B). Lane 1, molecular weight standards;lanes 3 and 4, plasma gelsolin;lanes 2 and 5, platelet gelsolin. Actin is also seen on SDS-PAGE (lane 2) because gelsolin-actin complex is obtained by the method previously described (8).
observed is that stimulatedcells respond by assemblingactin,that is, by polymerizingactin. Therefore,many questions about the role of gelsolinin cell activationare unanswered. In studies done to answer such questions,the antibody reported here might be useful. A simple method is currently prepared to measure the amount of gelsolinboth in cytosolic fraction of human plateletsand in human plasma with use of the anti-gelsolinantibodiesconjugated to latex particles. We are now making monoclonal antibody to human plasma gelsolin,too. When the monoclonal antibody is obtained,a sandwich method by ELISA with use of the monoclonal antibody and polyclonal one could be used to measure the amount of gelsolin. REFERENCES I. JANMEY, P. A., CHAPONNIER, C., LIND, S. E.. ZANER, K. S..STOSSEL, T. P.,and YIN, H. L. Interactionsof gelsolinand gelsolin-actin complexes with actin: effects of calcium on actin nucleation, filamentsevering,and end blocking. Biochemistry 24, 3714-3723,
ANTIBODY TO HUMAN GELSOLIN
Vol. 63, No. 3
1985.
2. YIN, H. L..and STOSSEL, T. P. Control of cytoplasmicactin gel-sol transformationby gelsolin,a calcium-dependentregulatory protein. Nature (London) 281, 583-586,1979. 3. LIND. S. E.,YIN, H. L.,and STOSSEL, 'I'. P. Human plateletscontain gelsolin: a regulator of actin filamentlength. 2" J Clin Invest @, 1384-1387,1982. 4. HARRIS, D. A., and SCHWARTZ, J. H. Characterizationof brevin,a serum protein that shortens actin filaments. Proc. Natl. Acad. Sci. USA 2, 6798-6802,1981. 5. FAHEY. J. L.,and MCKELVEY, E. M. Quantitativedeterminationof serum immunoglobulinsin antibody-agarplates. J- Immunol. 94, 8490, 1965. 6. TOWBIN. H., STAEHELIN,T.,and GORDON, J. Electrophoretictransfer of proteins from polyacrylamidegels to nitrocellulosesheets: procedure and some applications.--Proc. Natl.Acad. -Sci. USA 76, 4350-4354,1979. 7. ~KERSTRZM. B.,BRODIN, T..REIS, K.,and BJ~~RCK,L. Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies. J- Immunol. 135, 2589-2592,1985. 8. HIYOSHI,M., IM, T.,SASAKI,A., HASHIMOTO, K., and TATSUMI,N. Involvement of calcium ion in the interactionbetween the actingelsolincomplex and phosphatidylinositol 4-monophosphate or 4,5bisphosphate. Biochem.Int. 2, 1009-1015,1989. 9. CHAPONNIER, C.. JANMEY, P. A., and YIN, H. L. The actin filamentJ. Cell Biol 103, 1473-1481, severing domain of plasma gelsolin. _-A 1988. 10. KWIATKOWSKI,D. J.,and YIN, H. L. Molecular biology of gelsolin,a calcium-regulatedactin filamentsevering protein. Biorheology 24, 643-647,1987. 11. JANMEY, P. A., IIDA,K.,YIN, H. L.,and STOSSEL. T. P. Polyphosphoinositide micellesand polyphosphoinositide-containing vesiclesdissociateendogenous gelsolin-actin complexes and promote actin assembly from the fast-growingend of actin filamentsblocked 1987. by gelsolin. J- &.& Chem. 262, 12228-12236. 12. HARRIS, H. Microfilamentdynamics. Nature (London) 330, 310-311, 1987.
