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Altered cytoskeletal structures of thrombasthenic platelets
THROMBOSIS RESEARCH 30: 113-116, 1983 0049-3848/83/070113-04$03.00/O Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.
BRIEF
COMMUNICATION
ALTERED CYTOSKELETAL STRUCTURES OF THROMBASTHENIC PLATELETS Y. Ikeda, K. Satoh, M. Handa, Y. Yoshii, M. Imai, K. Toyama, K. Watanabex and Y. Ando* Department of Haematology and Department of Laboratory Medicine*, Keio University, Tokyo, Japan.
(Received 7.10.1982; in revised form 22.12.1982. Accepted by Editor N. Aoki)
INTRODUCTION Glanzmann's thrombasthenia is an inherited bleeding disorder associated with a failure of the platelets to aggregate and impaired or absent clot retraction (1). Many papers have been published regarding the platelet defects, which may be responsible for the lack of platelet aggregation in this disorder. Among them, deficient membrane glycoprotein IIb and IIIa and platelet a-actinin in thrombasthenic platelets are well established (2)(3). Recently, Phillips et al. have shown that the changes in the cytoskeletal structures of platelets following thrombin activation are involved in the physiological response of platelets (4). It was also shown that cytoskeletal structures from thrombin-aggregated platelets contained membrane glycoprotein IIb and III,, indicating that one or both of these glycoproteins participate in the direct interaction of platelets during aggregation (5). These observations prompted us to investigate the changes in cytoskeletal structures of thrombasthenic platelets following thrombin activation. METHODS Blood was drawn from normal volunteers and two patients with Glanzmann's thrombasthenia : The patient, Y.N., showed prolonged Ivy bleeding time ( over 15 min. ) associated with absent clot retraction. Platelet aggregation studies revealed that maximal aggregation of platelets induced by 20 PM ADP and 8 pg/ml collagen was 5 % and 4 %, respectively. The patient, S.Y., had also typical hemostatic findings of thrombasthenia. The maximal extents of platelet aggregation induced by 20 PM ADP and 8 ug/ml collagen were 0 % and 4 X, respectively. Analysis of platelet membrane glycoproteins upon SDS-polyacrylamide gel electrophoresis revealed absent or decreased amounts of GP IIb and 111,. Platelets were washed once with 10 mM tris-buffered saline containing 1 mM EDTA, pH 7.4 ( TES ), and were suspended in the same buffer at a concentration of log platelets per ml. Platelet cytoskeletal structures were prepared acceding to the method of Phillips et al. (5). To washed platelets, which were incubated Key words :
thrombasthenia, cytoskeletal proteins, thrombin. 113
THROMHASTHENIC PLATELETS
114
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with a-thrombin ( a generous gift from Dr. John W. Fenton ) for 15 min., an equal volume of the Triton extraction buffer ( 2% Triton X-100, 10 mM EGTA and 100 mM tris, pH 7.4 ) and 2 NIH units/ml of hirudin ( Sigma Chem. Co. USA ) were added. After being left at room temperature for 5 minutes, mixtures were centrifuged at 7,000 g for 5 minutes in a Fisher centrifuge, Model 59. The resulting pellets were rinsed with a one to one mixture of TES buffer and Triton extraction buffer and recentrifuged for 5 minutes to obtain platelet cytoskeletal preparation. Protein concentrations were estimated by the method of Lowry et al. (6) using bovine albumin as the standard. Platelet cytoskeletal structures thus prepared were solubilized in 2 % SDS containing 2 % 2-mercaptoethanol and were incubated at 100 'C for 10 minutes. The same volume of solubilized samples from control and thrombasthenic platelets ( usually lo-20 ug of protein ) were electrophoresed through slab gels according to the method of Laemmli using a 5-20 % exponential gradient of acrylamide in the resolving gel and 3 % acrylamide in the stacking gel (7). The protein was stained with Coomassie blue. RESULTS AND DISCUSSION Non-stimulated platelets from normal and thrombasthenic individuals yielded essentially the same amount of protein and the same polypeptide composition of Triton-precipitates. When platelets were treated with thrombin, the amount of protein in Triton-precipitates was increased in a dose-dependent manner both in control and thrombasthenic platelets. Thrombasthenic platelets, however, produced less increase in the amount of precipitates after Triton extraction in response to thrombin as shown in TABLE 1. TABLE 1 Amount of Protein in Triton-precipitates from Normal and Thrombasthenic Platelets** Normal Non-stimulated
Patient Y.N.
Patient S.Y.
161
160
148
Thrombin-activated 0.1
u/ml
261
200
200
0.5
u/ml
304
272
211
( ** ug protein / 10' platelets, mean of 3 experiments ) Composition of the polypeptides in the Triton-precipitates from non-stimulated and thrombin-activated platelets was analysed by SDS-polyacrylamide gel electrophoresis in normal subjects and two patients with thrombasthenia. ( Fig. 1 ) The Triton-insoluble materials from non-stimulated normal and thrombasthenic platelets showed essentially the same polypeptide compositions. There were three major polypeptides of molecular weight, 43,000, 200,000 and 255,000, which have been identified by Phillips et al. as actin, myosin and actin-binding protein (ABP), respectively. Analysis of cytoskeletons of thrombinactivated platelets revealed a different polypeptide composition between control and thrombasthenia. While increases in three major polypeptides ( actin, myosin and ABP ) were observed both in control and thrombasthenia, prominent increases in polypeptides of molecular weight, 52,000 (52K polypeptide), 56,000 (56K polypeptide) and 90,000 (90K polypeptide) were only demonstrated in control platelets. Thrombin-activated platelets from two patients with thrombasthenia, however, produced none or much less quantity of 52K, 56K and 90K polypeptides in Triton-
vo1.30,
No.1
THROMBASTHENIC PLATELETS
control
S.Y.
115
Y.N.
255K 200K
FIG. 1 Composition of the Triton-insoluble Materials from Normal and Thrombasthenic Platelets
90K
The Triton-insoluble materials from non-stimulated and thrombin-activated platelets were solubilized in SDS and electrophoresed on a 5-20X exponential of acrylamide. The samples are : non-stimulated platelets, lanes 1,3,5. thrombin(0.1 u/ml)-activated platelets, lanes 2,4,6.
56K 52K 43K
12
34
5
6
insoluble materials. The incorporation of 52K, 56K and 9OK polypeptides into the cytoskeletal structures after thrombin activation has been also shown by Phillips et al. and Pribluda et al. in normal platelets (4)(5)(a). However, little attention was paid on these polypeptides, although suggestion has been raised that 56K polypeptide might be the product of fibrinogen. Pribludf2st al. showed that this I-labelled fibrinogen polypeptide, which coincided with authentic band of added to the platelets, disappeared when hirudin was added together with Triton extraction buffer. We have also included hirudin in Triton extraction buffer. The reason for this discrepancy is not known at present time. Recent studies by Phillips et al. suggested the important roles of cytoskeletal proteins in platelet functions (4)(5). The facts that 52K, 56K and 90K polypeptides are soluble in Triton when platelets are non-stimulated, but are resistent to solubilization after platelets are activated by thrombin, may indicate some roles of these polypeptides in thrombin activation of platelets. The increased Triton-insolubility of actin, myosin and ABP was demonstrated both in thrombin-activated normal and thrombasthenic platelets, although to a less degree in the latter. However, thrombasthenic platelets have 52K, 56K and 90K polypeptides, which remain soluble in Triton even after thrombin activation. The altered cytoskeletal structures of thrombasthenic platelets following thrombin-activation is, therefore, of great interest. Special attention is forcused on 52K and 56K polypeptides since the detergent insoluble residues from other cell sources such as fibroblasts also contain 52-56K proteins in addition to actin and myosin (9). Since we have observed no change in the amounts of 52K, 56K and 90K between normal and thrombasthenic platelets when protein compositions of these platelets were analysed by SDS-polyacrylamide gel electrophoresis, defective association between these polypeptides and the newly formed actin filaments or molecular defects of these polypeptides from thrombasthenia is the possible explanation for our observation. The causal relationship between failure in acquiring detergentresistence of these polypeptides and aggregation defects in thrombasthenia remains
116
THROMBASTHENIC PLATELETS
Vo1.30, No.1
unknown. Further study has to be undertaken to clarify the physiological significance of these polypeptides in platelet aggregations. REFERENCE 1.
CAEN,J.P., CASTALDI,P.A., LeCLERC,J.C., INCEMAN,S., LARRIEU,M.J., PROBST,M. and BERNARD,J. Congenital bleeding disorders with long bleeding time and normal platelet count. I. Glanzmann's thrombasthenia. Amer. J. Med. 41. 4-26, 1966.
2.
HAGEN,I., NURDEN,A.T., BJERRUM,O.J., SOLUM,N.O. and CAEN,J.P. Immunological evidence for protein abnormalities in platelets from patients with Glanzmann's thrombasthenia and Bernard-Soulier syndrome. J. 722-731, ._ Clin. _ Invest. 65. 1980.
3.
GERRARD,J.M., SCHOLI&fEYER,J.F.,PHILLIPS,D.R. and WHITE,J.G. cc-actinindeficiency in thrombasthenia: possible identification of a-actinin as glycoprotein III. .Amer. J. Path. _ -94. 509-528, 1979.
4.
JENNINGS,L.K., FOX,J.E.B., EDWARDS,H.H., and PHILLIPS,D.R. Changes in the cyto. skeletal structures of human platelets following thrombin activation. J. Biol. Chem., -.. 256. 6927-6932, 1981.
5.
PHILLIPS,D.R., JENNINGS,L.K., and EDWARDS,H.H. Identification of membrane proteins mediating the interaction of human platelets. J. Cell Biol. 86. 77-86, 1980.
6.
LOWRY,O.H., ROSEBROUGH,N.J., FARR,A.L and RANDALL,R.L. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193. 265-275, 1951. ---
7.
Cleavage of structural proteins during the assembly of the head LAEMML1,U.K. Nature (London) 227. 680-685, 1970. of bacteriophage T4. -.
8.
PRIBLUDA,V. and ROTMAN,A. Dynamics of membrane-cytoskeleton interactions in Biochemistry 21. 2825-2832, 1982. activated blood platelets.
9.
Cell adhesion and acquisition of GONEN,A., WEISMAN-SHOMER,P. and FRY,M. detergent resistence by the cytoskeleton of cultured chick fibroblasts. Bioch. Biophys. Acta. 307-321, 1979. __ 552. ~...
BRIEF
COMMUNICATION
ALTERED CYTOSKELETAL STRUCTURES OF THROMBASTHENIC PLATELETS Y. Ikeda, K. Satoh, M. Handa, Y. Yoshii, M. Imai, K. Toyama, K. Watanabex and Y. Ando* Department of Haematology and Department of Laboratory Medicine*, Keio University, Tokyo, Japan.
(Received 7.10.1982; in revised form 22.12.1982. Accepted by Editor N. Aoki)
INTRODUCTION Glanzmann's thrombasthenia is an inherited bleeding disorder associated with a failure of the platelets to aggregate and impaired or absent clot retraction (1). Many papers have been published regarding the platelet defects, which may be responsible for the lack of platelet aggregation in this disorder. Among them, deficient membrane glycoprotein IIb and IIIa and platelet a-actinin in thrombasthenic platelets are well established (2)(3). Recently, Phillips et al. have shown that the changes in the cytoskeletal structures of platelets following thrombin activation are involved in the physiological response of platelets (4). It was also shown that cytoskeletal structures from thrombin-aggregated platelets contained membrane glycoprotein IIb and III,, indicating that one or both of these glycoproteins participate in the direct interaction of platelets during aggregation (5). These observations prompted us to investigate the changes in cytoskeletal structures of thrombasthenic platelets following thrombin activation. METHODS Blood was drawn from normal volunteers and two patients with Glanzmann's thrombasthenia : The patient, Y.N., showed prolonged Ivy bleeding time ( over 15 min. ) associated with absent clot retraction. Platelet aggregation studies revealed that maximal aggregation of platelets induced by 20 PM ADP and 8 pg/ml collagen was 5 % and 4 %, respectively. The patient, S.Y., had also typical hemostatic findings of thrombasthenia. The maximal extents of platelet aggregation induced by 20 PM ADP and 8 ug/ml collagen were 0 % and 4 X, respectively. Analysis of platelet membrane glycoproteins upon SDS-polyacrylamide gel electrophoresis revealed absent or decreased amounts of GP IIb and 111,. Platelets were washed once with 10 mM tris-buffered saline containing 1 mM EDTA, pH 7.4 ( TES ), and were suspended in the same buffer at a concentration of log platelets per ml. Platelet cytoskeletal structures were prepared acceding to the method of Phillips et al. (5). To washed platelets, which were incubated Key words :
thrombasthenia, cytoskeletal proteins, thrombin. 113
THROMHASTHENIC PLATELETS
114
Vo1.30, No.1
with a-thrombin ( a generous gift from Dr. John W. Fenton ) for 15 min., an equal volume of the Triton extraction buffer ( 2% Triton X-100, 10 mM EGTA and 100 mM tris, pH 7.4 ) and 2 NIH units/ml of hirudin ( Sigma Chem. Co. USA ) were added. After being left at room temperature for 5 minutes, mixtures were centrifuged at 7,000 g for 5 minutes in a Fisher centrifuge, Model 59. The resulting pellets were rinsed with a one to one mixture of TES buffer and Triton extraction buffer and recentrifuged for 5 minutes to obtain platelet cytoskeletal preparation. Protein concentrations were estimated by the method of Lowry et al. (6) using bovine albumin as the standard. Platelet cytoskeletal structures thus prepared were solubilized in 2 % SDS containing 2 % 2-mercaptoethanol and were incubated at 100 'C for 10 minutes. The same volume of solubilized samples from control and thrombasthenic platelets ( usually lo-20 ug of protein ) were electrophoresed through slab gels according to the method of Laemmli using a 5-20 % exponential gradient of acrylamide in the resolving gel and 3 % acrylamide in the stacking gel (7). The protein was stained with Coomassie blue. RESULTS AND DISCUSSION Non-stimulated platelets from normal and thrombasthenic individuals yielded essentially the same amount of protein and the same polypeptide composition of Triton-precipitates. When platelets were treated with thrombin, the amount of protein in Triton-precipitates was increased in a dose-dependent manner both in control and thrombasthenic platelets. Thrombasthenic platelets, however, produced less increase in the amount of precipitates after Triton extraction in response to thrombin as shown in TABLE 1. TABLE 1 Amount of Protein in Triton-precipitates from Normal and Thrombasthenic Platelets** Normal Non-stimulated
Patient Y.N.
Patient S.Y.
161
160
148
Thrombin-activated 0.1
u/ml
261
200
200
0.5
u/ml
304
272
211
( ** ug protein / 10' platelets, mean of 3 experiments ) Composition of the polypeptides in the Triton-precipitates from non-stimulated and thrombin-activated platelets was analysed by SDS-polyacrylamide gel electrophoresis in normal subjects and two patients with thrombasthenia. ( Fig. 1 ) The Triton-insoluble materials from non-stimulated normal and thrombasthenic platelets showed essentially the same polypeptide compositions. There were three major polypeptides of molecular weight, 43,000, 200,000 and 255,000, which have been identified by Phillips et al. as actin, myosin and actin-binding protein (ABP), respectively. Analysis of cytoskeletons of thrombinactivated platelets revealed a different polypeptide composition between control and thrombasthenia. While increases in three major polypeptides ( actin, myosin and ABP ) were observed both in control and thrombasthenia, prominent increases in polypeptides of molecular weight, 52,000 (52K polypeptide), 56,000 (56K polypeptide) and 90,000 (90K polypeptide) were only demonstrated in control platelets. Thrombin-activated platelets from two patients with thrombasthenia, however, produced none or much less quantity of 52K, 56K and 90K polypeptides in Triton-
vo1.30,
No.1
THROMBASTHENIC PLATELETS
control
S.Y.
115
Y.N.
255K 200K
FIG. 1 Composition of the Triton-insoluble Materials from Normal and Thrombasthenic Platelets
90K
The Triton-insoluble materials from non-stimulated and thrombin-activated platelets were solubilized in SDS and electrophoresed on a 5-20X exponential of acrylamide. The samples are : non-stimulated platelets, lanes 1,3,5. thrombin(0.1 u/ml)-activated platelets, lanes 2,4,6.
56K 52K 43K
12
34
5
6
insoluble materials. The incorporation of 52K, 56K and 9OK polypeptides into the cytoskeletal structures after thrombin activation has been also shown by Phillips et al. and Pribluda et al. in normal platelets (4)(5)(a). However, little attention was paid on these polypeptides, although suggestion has been raised that 56K polypeptide might be the product of fibrinogen. Pribludf2st al. showed that this I-labelled fibrinogen polypeptide, which coincided with authentic band of added to the platelets, disappeared when hirudin was added together with Triton extraction buffer. We have also included hirudin in Triton extraction buffer. The reason for this discrepancy is not known at present time. Recent studies by Phillips et al. suggested the important roles of cytoskeletal proteins in platelet functions (4)(5). The facts that 52K, 56K and 90K polypeptides are soluble in Triton when platelets are non-stimulated, but are resistent to solubilization after platelets are activated by thrombin, may indicate some roles of these polypeptides in thrombin activation of platelets. The increased Triton-insolubility of actin, myosin and ABP was demonstrated both in thrombin-activated normal and thrombasthenic platelets, although to a less degree in the latter. However, thrombasthenic platelets have 52K, 56K and 90K polypeptides, which remain soluble in Triton even after thrombin activation. The altered cytoskeletal structures of thrombasthenic platelets following thrombin-activation is, therefore, of great interest. Special attention is forcused on 52K and 56K polypeptides since the detergent insoluble residues from other cell sources such as fibroblasts also contain 52-56K proteins in addition to actin and myosin (9). Since we have observed no change in the amounts of 52K, 56K and 90K between normal and thrombasthenic platelets when protein compositions of these platelets were analysed by SDS-polyacrylamide gel electrophoresis, defective association between these polypeptides and the newly formed actin filaments or molecular defects of these polypeptides from thrombasthenia is the possible explanation for our observation. The causal relationship between failure in acquiring detergentresistence of these polypeptides and aggregation defects in thrombasthenia remains
116
THROMBASTHENIC PLATELETS
Vo1.30, No.1
unknown. Further study has to be undertaken to clarify the physiological significance of these polypeptides in platelet aggregations. REFERENCE 1.
CAEN,J.P., CASTALDI,P.A., LeCLERC,J.C., INCEMAN,S., LARRIEU,M.J., PROBST,M. and BERNARD,J. Congenital bleeding disorders with long bleeding time and normal platelet count. I. Glanzmann's thrombasthenia. Amer. J. Med. 41. 4-26, 1966.
2.
HAGEN,I., NURDEN,A.T., BJERRUM,O.J., SOLUM,N.O. and CAEN,J.P. Immunological evidence for protein abnormalities in platelets from patients with Glanzmann's thrombasthenia and Bernard-Soulier syndrome. J. 722-731, ._ Clin. _ Invest. 65. 1980.
3.
GERRARD,J.M., SCHOLI&fEYER,J.F.,PHILLIPS,D.R. and WHITE,J.G. cc-actinindeficiency in thrombasthenia: possible identification of a-actinin as glycoprotein III. .Amer. J. Path. _ -94. 509-528, 1979.
4.
JENNINGS,L.K., FOX,J.E.B., EDWARDS,H.H., and PHILLIPS,D.R. Changes in the cyto. skeletal structures of human platelets following thrombin activation. J. Biol. Chem., -.. 256. 6927-6932, 1981.
5.
PHILLIPS,D.R., JENNINGS,L.K., and EDWARDS,H.H. Identification of membrane proteins mediating the interaction of human platelets. J. Cell Biol. 86. 77-86, 1980.
6.
LOWRY,O.H., ROSEBROUGH,N.J., FARR,A.L and RANDALL,R.L. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193. 265-275, 1951. ---
7.
Cleavage of structural proteins during the assembly of the head LAEMML1,U.K. Nature (London) 227. 680-685, 1970. of bacteriophage T4. -.
8.
PRIBLUDA,V. and ROTMAN,A. Dynamics of membrane-cytoskeleton interactions in Biochemistry 21. 2825-2832, 1982. activated blood platelets.
9.
Cell adhesion and acquisition of GONEN,A., WEISMAN-SHOMER,P. and FRY,M. detergent resistence by the cytoskeleton of cultured chick fibroblasts. Bioch. Biophys. Acta. 307-321, 1979. __ 552. ~...