[10]
HUMANPLATELETP235: TALIN
99
7.5 containing 5 mM EGTA. The elution can be followed using SDS-gel electrophoresis. The use of these particular antibodies to purify actin-bound gelsolin is described in more detail in Hwo and Bryan. 24 Chaponnier et al. 25 have described the use of murine monoclonal antibodies to study changes in the concentration of gelsolin-actin complexes in macrophages. Acknowledgments The author wishes to thank ShuyingHwo, RobynLee, Wen-GenLin, David Hou, and Perry Sedlar for their excellent technical assistance during the years this work was done. Special thanks also to Brenda Cipriano, who provided help and careful preparation of the manuscript. This work was supported by NIH Grants HL26973 and GM26091 to J.B.
[10] P u r i f i c a t i o n a n d P r o p e r t i e s o f Human Platelet P235: Talin
By
NANCY
C.
COLLIER
and
KUAN
WANG
Introduction When a resting platelet is activated, a dramatic reorganization of the cytoskeleton occurs. Although few microfilaments are observed in the resting platelet, activation results in a burst of microfilament formation.l These changes in microfilament morphology are temporally and spatially controlled by a large number of actin-modulating proteins that have been identified in the platelet. These proteins serve to sequester monomeric actin, restrict length of actin filaments, modulate rate and polarity of actin polymerization, sever polymeric actin, or cross-link actin filaments. 2 One of these proteins, P235, is a major cytoplasmic component (3-5% of total protein)? P235 is implicated in the contractile activities ofplatelet, because the specific in s i t u degradation of P235 and filamin accompanies the loss of the ability of the platelet to extend pseudopodia. 4 Furthermore, both proteins are degraded by endogenous calcium-dependent proteases during
I j. C. Lewis, Cell Muscle Motil. 5, 341 (1984). 2 j. E. B. Fox, in "Biochemistry of Platelets" (D. R. Philips and M. A. Shuman, eds.), p. 115, Academic Press, Orlando, Florida, 1986. 3 N. C. Collier and K. Wang, J. Biol. Chem. 257, 6937 (1982). 4 V. T. Nachmias, J. S. Sullender, and J. R. Fallon, Blood 53, 63 (1979).
METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
100
ISOLATIONOF PLATELETCOMPONENTS
[10]
thrombin-induced platelet aggregation. 5 In solution, purified P235 affects actin polymerization. W h e n present with G-actin during salt-induced polymerization, P235 reduces the rate of actin polymerization in a calciumand calmodulin-sensitive manner. 6-9 The resultant actin filaments are shorter but the total a m o u n t of actin filaments remains unaffected. 6-9 Thus, P235 m a y play an important role in the actin-mediated changes in the platelet and its activity m a y be regulated by its phophorylation state, proteolytic processing, a c c e s s o r y proteins, and calcium level. P235 has been s h o w n to bear similarity to talin, a vinculin- and integrinbinding p h o s p h o p r o t e i n found in other cell types at the adhesion plaques where actin filaments terminate as the plasma membrane.l°-13 Its role in t r a n s m e m b r a n e linkage of extracellular matrix to actin cytoskeleton in platelet is suggested by its redistribution to s u b m e m b r a n o u s location in activated platelets. 13
Purification of H u m a n Platelet P235 Platelet Concentrates
Our starting material is h u m a n platelet concentrates. The use of concentrates has the advantage o v e r whole blood since it eliminates several centrifugal purification steps. A small amount of contaminating erythrocytes and l e u k o c y t e s is easily r e m o v e d by differential centrifugation. Concentrates, which quickly b e c o m e outdated (within 3 days) for transfusion purposes, are usually available at minimal or no cost from blood banks and hospitals. Since we h a v e found that P235 and filamin remain intact in freshly outdated c o n c e n t r a t e s (within 3 to 7 days of venipuncture), we routinely use these for purification of contractile proteins. Fresh platelet concentrates, needed to confirm the results of some experiments, are available at cost. 5 j. E. B. Fox, D. E. Goll, C. C. Reynolds, and D. R. Phillips, J. Biol. Chem. 260, 1060 (1985), and references listed therein. 6 N. C. Collier and K. Wang, FEBS Lett. 143, 205 (1982). 7 N. C. Collier and K. Wang, Biophys. J. 41, 86a (1983). 8 N. C. Collier and K. Wang, J. Cell Biol. 97, 289a (1983). 9 N. C. Collier, Ph.D. dissertation, University of Texas at Austin (1982). to T. O'Halloran, M. C. Beckerle, and K. Burridge, Nature (London) 317, 449 (1985). 11L. Molony, D. McCaslin, J. Abernethy, B. Paschal, and K. Burridge, J. Biol. Chem. 262, 7790 (1987). t2 A. Horwitz, K. Duggan, C. Buck, M. C. Beckerle, and K. Burridge, Nature (London) 320, 531 (1986). t3 M. C. Beckerle, D. E. Miller, M. E. Bertagonolli, and S. J. Locke, J. Cell Biol. 109, 3333 ( 1989).
[10]
HUMANPLATELETP235: TALIN
101
Proteolytic Degradation P235 is highly susceptible to proteolytic degradation, in particular to the endogenous Ca 2+-dependent proteases. 3-5 Several measures are therefore taken to minimize proteolysis. First, platelets are washed at room temperature in the presence of EDTA in plasticware to avoid activation that leads to P235 degradation. 14 Second, EDTA is included in all steps during and after the extraction of platelet proteins to prevent proteolysis induced by Ca2+-activated proteases. We have found that when the EDTA concentration in the extraction buffer was reduced below 0.3 mM, extensive degradation of P235 occurred. The inclusion of leupeptin (at 20 /zg/ml), an inhibitor of calcium-dependent protease, did not further improve P235 stability. Covalent inhibitors such as iodoacetamide or N-ethylmaleimide were not used to avoid potential alkylation of P235. Even with these precautions, purified P235 is slowly degraded into large fragments of 220 and 200 kDa on storage at 4° for several days.
Assay The purity and yield of P235 are assayed by sodium dodecyl sulfate (SDS)-gel electrophoresis. Polyclonal antiserum directed against P235 is used, when needed, to monitor P235 degradation by immunoblot techniques. The discontinuous SDS-gel method of Laemmli 15 is modified as follows: Proteins are analyzed on a 3 to 12% linear gradient polyacrylamide separating gel without the stacking gel. This modification results in a sharp protein banding pattern while resolving both high and low molecular weight proteins. Protein in the presence of Triton X-100 is determined by the method of Bensadoun and Weinstein.16 Protein in other buffers is determined by the Hartree method, j7 Crystalline bovine serum albumin (BSA, type V; Sigma, St. Louis, MO) is used as the standard. The concentration of BSA was determined by absorbance t~-~ ~L'280 = 6.6).
Buffers Platelet Washing Buffers Buffer 1:0.12 M NaC1, 0.129 M sodium citrate, 0.03 M glucose, pH 7.4 Buffer 2:0.154 M NaCI, 0.005 M EDTA, 0.01 M Tris-HC1, pH 7.4 14Other investigators have used aspirin or prostacyclin in the wash medium to prevent platelet activation. 15U. K. Laemmli,Nature (London) 227, 680 (1970). 16A. Bensadounand D. Weinstein,Anal. Biochem. 70, 241 (1976). 17E. F. Hartree, Anal. Biochem. 48, 422 (1972).
102
ISOLATION OF PLATELET COMPONENTS
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HUMANPLATELETP235: TALIN
99
7.5 containing 5 mM EGTA. The elution can be followed using SDS-gel electrophoresis. The use of these particular antibodies to purify actin-bound gelsolin is described in more detail in Hwo and Bryan. 24 Chaponnier et al. 25 have described the use of murine monoclonal antibodies to study changes in the concentration of gelsolin-actin complexes in macrophages. Acknowledgments The author wishes to thank ShuyingHwo, RobynLee, Wen-GenLin, David Hou, and Perry Sedlar for their excellent technical assistance during the years this work was done. Special thanks also to Brenda Cipriano, who provided help and careful preparation of the manuscript. This work was supported by NIH Grants HL26973 and GM26091 to J.B.
[10] P u r i f i c a t i o n a n d P r o p e r t i e s o f Human Platelet P235: Talin
By
NANCY
C.
COLLIER
and
KUAN
WANG
Introduction When a resting platelet is activated, a dramatic reorganization of the cytoskeleton occurs. Although few microfilaments are observed in the resting platelet, activation results in a burst of microfilament formation.l These changes in microfilament morphology are temporally and spatially controlled by a large number of actin-modulating proteins that have been identified in the platelet. These proteins serve to sequester monomeric actin, restrict length of actin filaments, modulate rate and polarity of actin polymerization, sever polymeric actin, or cross-link actin filaments. 2 One of these proteins, P235, is a major cytoplasmic component (3-5% of total protein)? P235 is implicated in the contractile activities ofplatelet, because the specific in s i t u degradation of P235 and filamin accompanies the loss of the ability of the platelet to extend pseudopodia. 4 Furthermore, both proteins are degraded by endogenous calcium-dependent proteases during
I j. C. Lewis, Cell Muscle Motil. 5, 341 (1984). 2 j. E. B. Fox, in "Biochemistry of Platelets" (D. R. Philips and M. A. Shuman, eds.), p. 115, Academic Press, Orlando, Florida, 1986. 3 N. C. Collier and K. Wang, J. Biol. Chem. 257, 6937 (1982). 4 V. T. Nachmias, J. S. Sullender, and J. R. Fallon, Blood 53, 63 (1979).
METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
100
ISOLATIONOF PLATELETCOMPONENTS
[10]
thrombin-induced platelet aggregation. 5 In solution, purified P235 affects actin polymerization. W h e n present with G-actin during salt-induced polymerization, P235 reduces the rate of actin polymerization in a calciumand calmodulin-sensitive manner. 6-9 The resultant actin filaments are shorter but the total a m o u n t of actin filaments remains unaffected. 6-9 Thus, P235 m a y play an important role in the actin-mediated changes in the platelet and its activity m a y be regulated by its phophorylation state, proteolytic processing, a c c e s s o r y proteins, and calcium level. P235 has been s h o w n to bear similarity to talin, a vinculin- and integrinbinding p h o s p h o p r o t e i n found in other cell types at the adhesion plaques where actin filaments terminate as the plasma membrane.l°-13 Its role in t r a n s m e m b r a n e linkage of extracellular matrix to actin cytoskeleton in platelet is suggested by its redistribution to s u b m e m b r a n o u s location in activated platelets. 13
Purification of H u m a n Platelet P235 Platelet Concentrates
Our starting material is h u m a n platelet concentrates. The use of concentrates has the advantage o v e r whole blood since it eliminates several centrifugal purification steps. A small amount of contaminating erythrocytes and l e u k o c y t e s is easily r e m o v e d by differential centrifugation. Concentrates, which quickly b e c o m e outdated (within 3 days) for transfusion purposes, are usually available at minimal or no cost from blood banks and hospitals. Since we h a v e found that P235 and filamin remain intact in freshly outdated c o n c e n t r a t e s (within 3 to 7 days of venipuncture), we routinely use these for purification of contractile proteins. Fresh platelet concentrates, needed to confirm the results of some experiments, are available at cost. 5 j. E. B. Fox, D. E. Goll, C. C. Reynolds, and D. R. Phillips, J. Biol. Chem. 260, 1060 (1985), and references listed therein. 6 N. C. Collier and K. Wang, FEBS Lett. 143, 205 (1982). 7 N. C. Collier and K. Wang, Biophys. J. 41, 86a (1983). 8 N. C. Collier and K. Wang, J. Cell Biol. 97, 289a (1983). 9 N. C. Collier, Ph.D. dissertation, University of Texas at Austin (1982). to T. O'Halloran, M. C. Beckerle, and K. Burridge, Nature (London) 317, 449 (1985). 11L. Molony, D. McCaslin, J. Abernethy, B. Paschal, and K. Burridge, J. Biol. Chem. 262, 7790 (1987). t2 A. Horwitz, K. Duggan, C. Buck, M. C. Beckerle, and K. Burridge, Nature (London) 320, 531 (1986). t3 M. C. Beckerle, D. E. Miller, M. E. Bertagonolli, and S. J. Locke, J. Cell Biol. 109, 3333 ( 1989).
[10]
HUMANPLATELETP235: TALIN
101
Proteolytic Degradation P235 is highly susceptible to proteolytic degradation, in particular to the endogenous Ca 2+-dependent proteases. 3-5 Several measures are therefore taken to minimize proteolysis. First, platelets are washed at room temperature in the presence of EDTA in plasticware to avoid activation that leads to P235 degradation. 14 Second, EDTA is included in all steps during and after the extraction of platelet proteins to prevent proteolysis induced by Ca2+-activated proteases. We have found that when the EDTA concentration in the extraction buffer was reduced below 0.3 mM, extensive degradation of P235 occurred. The inclusion of leupeptin (at 20 /zg/ml), an inhibitor of calcium-dependent protease, did not further improve P235 stability. Covalent inhibitors such as iodoacetamide or N-ethylmaleimide were not used to avoid potential alkylation of P235. Even with these precautions, purified P235 is slowly degraded into large fragments of 220 and 200 kDa on storage at 4° for several days.
Assay The purity and yield of P235 are assayed by sodium dodecyl sulfate (SDS)-gel electrophoresis. Polyclonal antiserum directed against P235 is used, when needed, to monitor P235 degradation by immunoblot techniques. The discontinuous SDS-gel method of Laemmli 15 is modified as follows: Proteins are analyzed on a 3 to 12% linear gradient polyacrylamide separating gel without the stacking gel. This modification results in a sharp protein banding pattern while resolving both high and low molecular weight proteins. Protein in the presence of Triton X-100 is determined by the method of Bensadoun and Weinstein.16 Protein in other buffers is determined by the Hartree method, j7 Crystalline bovine serum albumin (BSA, type V; Sigma, St. Louis, MO) is used as the standard. The concentration of BSA was determined by absorbance t~-~ ~L'280 = 6.6).
Buffers Platelet Washing Buffers Buffer 1:0.12 M NaC1, 0.129 M sodium citrate, 0.03 M glucose, pH 7.4 Buffer 2:0.154 M NaCI, 0.005 M EDTA, 0.01 M Tris-HC1, pH 7.4 14Other investigators have used aspirin or prostacyclin in the wash medium to prevent platelet activation. 15U. K. Laemmli,Nature (London) 227, 680 (1970). 16A. Bensadounand D. Weinstein,Anal. Biochem. 70, 241 (1976). 17E. F. Hartree, Anal. Biochem. 48, 422 (1972).
102
ISOLATION OF PLATELET COMPONENTS
I
I
I
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