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Platelet α2-antiplasmin is located in the platelet α-granules
THROHBOSTSRESC.mCH 31; 387-390,1983 0049-3348/83$3.00 + .OO Printed in the USA. Copyright (c) I.953?ergamon Press Ltd. -Allrights reserved.
BRIEF
PLATELET
COMMUNICATION
4-ANTIPLASMIN
G.0,
Gogstad,
IS LOCATED IN THE PLATELET H. Stonnorken
*GRANULES
and N-0. Solum
Research Institute for Internal Medicine, Section on Hemostasis and Thrombosis, University of Oslo, Oslo 1, Norway.
(Received7.2.83;Accepted in revised form 25.4.83 by Editor U. Abildgaard)
INTRODUCTION The protein a -antiplasmin is the primary inhibitor of the fibrinolytic ensyme plasmin in blood (l-3). The main part of this inhibitory protein is located in the plasma, but a certain fraction has also been reported to be associated with the platelets (4,5). a -antiplasmin is released from platelets when these are trig 4ered by various stimuli (6). Recently, Plow and Collen (7) reported that a2 -antiplasmin was secreted from thrombin-stimulated platelets in a manner which was consistent with a subcellular location in the a-granules. In the present study we confirm this location by subcellular fractionation and also identify a -antiplasmin in the pattern obtained after crossed immunoe f ectrophoresis of Triton X-100-solubilized platelets against antibodies to whole platelets. MATERIALS AND METHODS Chemicals: Agarose was from Litex, Glostrup, Denmark and Triton X-100 was from Sigma Chemical Company, St. Louis, USA. Antibodies: Antibodies to whole human platelets were obtained as descri'bed by Hagen et al. (8). Antiserum monospecific to
Key
words:
c2-antiplasmin,
a-granules, 387
platelets.
human a -antiplasmin was a generous gift from >j. Eizarson, Kabi, S$ockholm. Platelets and a-qranules were obtained as described previously (9). Crossed immunoelectrophoresis was performed as described by Hagen et al. (8). RESULTS AND DISCUSSION Platelet a-granules were isolated by a previously described procedure (9). A satisfactory purity of preparations was revealed from the distribution of subcellular markers (91, the quali, tative distinction from the platelet soluble cytoplasm as judged from crossed immunoelectrophoresis (lo), and the finding that all of the non-membraneous proteins consistently found in these preparations could be released from platelets that had been triggered by thrombin or polystyrene latex particles (11). a-granules were solubilized in Triton X-100 and applied to crossed immunoelectrophoresis against antibodies to whole platelets (Fig. 1A). Some of the immunoprecipitates which have been identified in previous work (8,12,13) are named in the figure. Unidentified immunoprecipitates are numbered as suggested previously (8,lO). When solubilized platelets were subjected to cros sed immunoelectrophoresis against an antiserum monospecific to a -antiplasmin, a single immunoprecipitate was formed (data n 8t shown) thus showing that this antiserum contained precipiWhen tating antibodies against a single platelet component. this antiserum was included in the intermediate
FIG. 1 Crossed immunoelectrophoresis of Triton X-100 solubilized a-granules against antibodies to whole platelets. antibody-free intermediate gel A: antibodies to a -antiplasmin in intermediate gel. B: TSP: thrombospondin, *PF-4 : platelet factor 4, GP IIB-IIIA: glycoprotein IIb-IIIa-complex, Fgn: Fibrinogen
vo1.31,
No.2
PLATELET az-ANTIPLASMIN
gel in crossed immunoelectrophoresis the position of immunoprecipitate No. 7 was lowered (Fig. 1A compared to Fig. 1B). This shows that precipitate No. 7 represents a2-antiplasmin which means that this antigen is present in the a-granule preparation. We have shown previously that antigen No. 7 is also present in the material which is released from platelets after stimulation by thrombin or latex particles (11). Performing crossed immunoelectrophoresis of the released material, using the same experimental setup as shown in Fig. 1, the presence of %-antiplasmin in the releasable pool was demonstrated directly (data not shown). Plow and Collen (7) have shown that -antiplasmin is relc-aSea from platelets in a way which woul% be consistent with a subcellular location in the crgranules. The present study thus confirms this location by subcellular fractionation. In previous studies, applying crossed affinity immunoelectrophoresis with various lectins (10,111, we have also shown that the antigen contained in precipitate No. 7 was glycosylated. Thus, these data are consistent with the fact that 02-antiplasmin is a glycoprotein (2,141. In conclusion, platelet -antiplasmin is located in the a-granules and corresponds % o the immunoprecipitate termed No. 7 in the previously presented patterns obtained after crossed immu. noelectrophoresis of platelet material against anti-platelet antibodies.
ACKNOWLEDGEMENTS This work was financially supported by The Norwegian Council on Cardiovascular Diseases and Anders Jahres Fond til Vitenskapens Fremme.
REFERENCES 1.
COLLEN, D. Identification and some properties of a new fastreacting plasmin inhibitor in human plasma. Eur. J. Biochem. 69, 209-216, 1976
2.
MOROI, M. and AOKI, N. Isolation and characterization of -plasmin inhibitor from human plasma. J. Biol. Chem. 5956-5965, 1976
3.
SLLERZ, S. and CLEMMENSEN, I. The primary inhibitor of plasmin in human plasma. Biochem. J. 159, 545-553, 1972.
4.
JOIST, J.H. Platelets and fibrinolysis. 38_, 955-962, 1978.
5.
WAKABAYASHI, K., FUJIKAWA, K. and ABE, T. Some properties of an antiplasmin substance from rabbit platelets. Thromb. Diathes. Haemorrh. 24, 76-85, 1970.
Thromb. Haemostas.
390
PLATELETa2-ILUTIPLASHIN
Vo1.31,.No.3
6.
MORRE, S., PEPPER, D.S. and CASH, C.D. The isolation and characterization of a platelet-specific f3-globulin (Bthrom. boglobulin) and the detection of antiurokinase and antiplas. min released from thrombin-aggregated washed human platelets. Biochim. Biophys. Acta 379, 360-369, 1975.
7.
PLOW, E.F. and COLLEN, D. The presence and release of -antiplasmin from human platelets. -Blood 58, 1069-1074, "481. 1
8.
HAGEN, I., BJERRUM, O.J. and SOLUM, N.O. Characterization of human platelet proteins soiubilized with Triton X-100 and examined by crossed immunoelectrophoresis. Eur. J. Biothem. 99, 9-22, 1979.
9.
GOGSTAD, G.O. A method for the isolation of *granules from human platelets. Thromb. Res. 20, 669-681, 1980.
10.
GOGSTAD, G.O., HAGEN, I., KORSMO, R. and SOLUM, N.O. Characterization of isolated human platelet &granules. Evidence for a separate a-granule pool of the glycoproteins IIb and IIIa. Biochim. Biophys. Acta 670, 150-162, 1981.
11.
GOGSTAD, G.O., HAGEN, I., KORSMO, R. and SOLUM, N.O. Evidence for release of soluble but not of membrane-integrated proteins from human platelet a-granules. Biochim. Biophys. Acta 702, 81-89, 1982. --
12.
HAGEN, I., NURDEN, A., BJERRUM, O.J., SOLUM, N.O. and CAEN, J.P. Immunochemical evidence for protein abnormalities in platelets from patients with Glanzmann's thrombasthenia and Bernard-Soulier syndrome. J. Clin. Invest. 65, 722-731, 1980.
13.
KUNICKI, T.J., PIDAB, D., ROSA, J.-P. and NURDEN, A.T. The formation of Ca -dependent complexes of platelet membrane glycoproteins IIb and IIIa in solution as determined by crossed immunoelectrophoresis. -v Blood 58, 268-278, 1981.
14.
WIMAN, B. and COLLEN, D. Purification of human antiplasmin, the fast-acting Eur. J. Biochem. 78, 19-26, 1977.
and characterization inhibitor in plasma.
BRIEF
PLATELET
COMMUNICATION
4-ANTIPLASMIN
G.0,
Gogstad,
IS LOCATED IN THE PLATELET H. Stonnorken
*GRANULES
and N-0. Solum
Research Institute for Internal Medicine, Section on Hemostasis and Thrombosis, University of Oslo, Oslo 1, Norway.
(Received7.2.83;Accepted in revised form 25.4.83 by Editor U. Abildgaard)
INTRODUCTION The protein a -antiplasmin is the primary inhibitor of the fibrinolytic ensyme plasmin in blood (l-3). The main part of this inhibitory protein is located in the plasma, but a certain fraction has also been reported to be associated with the platelets (4,5). a -antiplasmin is released from platelets when these are trig 4ered by various stimuli (6). Recently, Plow and Collen (7) reported that a2 -antiplasmin was secreted from thrombin-stimulated platelets in a manner which was consistent with a subcellular location in the a-granules. In the present study we confirm this location by subcellular fractionation and also identify a -antiplasmin in the pattern obtained after crossed immunoe f ectrophoresis of Triton X-100-solubilized platelets against antibodies to whole platelets. MATERIALS AND METHODS Chemicals: Agarose was from Litex, Glostrup, Denmark and Triton X-100 was from Sigma Chemical Company, St. Louis, USA. Antibodies: Antibodies to whole human platelets were obtained as descri'bed by Hagen et al. (8). Antiserum monospecific to
Key
words:
c2-antiplasmin,
a-granules, 387
platelets.
human a -antiplasmin was a generous gift from >j. Eizarson, Kabi, S$ockholm. Platelets and a-qranules were obtained as described previously (9). Crossed immunoelectrophoresis was performed as described by Hagen et al. (8). RESULTS AND DISCUSSION Platelet a-granules were isolated by a previously described procedure (9). A satisfactory purity of preparations was revealed from the distribution of subcellular markers (91, the quali, tative distinction from the platelet soluble cytoplasm as judged from crossed immunoelectrophoresis (lo), and the finding that all of the non-membraneous proteins consistently found in these preparations could be released from platelets that had been triggered by thrombin or polystyrene latex particles (11). a-granules were solubilized in Triton X-100 and applied to crossed immunoelectrophoresis against antibodies to whole platelets (Fig. 1A). Some of the immunoprecipitates which have been identified in previous work (8,12,13) are named in the figure. Unidentified immunoprecipitates are numbered as suggested previously (8,lO). When solubilized platelets were subjected to cros sed immunoelectrophoresis against an antiserum monospecific to a -antiplasmin, a single immunoprecipitate was formed (data n 8t shown) thus showing that this antiserum contained precipiWhen tating antibodies against a single platelet component. this antiserum was included in the intermediate
FIG. 1 Crossed immunoelectrophoresis of Triton X-100 solubilized a-granules against antibodies to whole platelets. antibody-free intermediate gel A: antibodies to a -antiplasmin in intermediate gel. B: TSP: thrombospondin, *PF-4 : platelet factor 4, GP IIB-IIIA: glycoprotein IIb-IIIa-complex, Fgn: Fibrinogen
vo1.31,
No.2
PLATELET az-ANTIPLASMIN
gel in crossed immunoelectrophoresis the position of immunoprecipitate No. 7 was lowered (Fig. 1A compared to Fig. 1B). This shows that precipitate No. 7 represents a2-antiplasmin which means that this antigen is present in the a-granule preparation. We have shown previously that antigen No. 7 is also present in the material which is released from platelets after stimulation by thrombin or latex particles (11). Performing crossed immunoelectrophoresis of the released material, using the same experimental setup as shown in Fig. 1, the presence of %-antiplasmin in the releasable pool was demonstrated directly (data not shown). Plow and Collen (7) have shown that -antiplasmin is relc-aSea from platelets in a way which woul% be consistent with a subcellular location in the crgranules. The present study thus confirms this location by subcellular fractionation. In previous studies, applying crossed affinity immunoelectrophoresis with various lectins (10,111, we have also shown that the antigen contained in precipitate No. 7 was glycosylated. Thus, these data are consistent with the fact that 02-antiplasmin is a glycoprotein (2,141. In conclusion, platelet -antiplasmin is located in the a-granules and corresponds % o the immunoprecipitate termed No. 7 in the previously presented patterns obtained after crossed immu. noelectrophoresis of platelet material against anti-platelet antibodies.
ACKNOWLEDGEMENTS This work was financially supported by The Norwegian Council on Cardiovascular Diseases and Anders Jahres Fond til Vitenskapens Fremme.
REFERENCES 1.
COLLEN, D. Identification and some properties of a new fastreacting plasmin inhibitor in human plasma. Eur. J. Biochem. 69, 209-216, 1976
2.
MOROI, M. and AOKI, N. Isolation and characterization of -plasmin inhibitor from human plasma. J. Biol. Chem. 5956-5965, 1976
3.
SLLERZ, S. and CLEMMENSEN, I. The primary inhibitor of plasmin in human plasma. Biochem. J. 159, 545-553, 1972.
4.
JOIST, J.H. Platelets and fibrinolysis. 38_, 955-962, 1978.
5.
WAKABAYASHI, K., FUJIKAWA, K. and ABE, T. Some properties of an antiplasmin substance from rabbit platelets. Thromb. Diathes. Haemorrh. 24, 76-85, 1970.
Thromb. Haemostas.
390
PLATELETa2-ILUTIPLASHIN
Vo1.31,.No.3
6.
MORRE, S., PEPPER, D.S. and CASH, C.D. The isolation and characterization of a platelet-specific f3-globulin (Bthrom. boglobulin) and the detection of antiurokinase and antiplas. min released from thrombin-aggregated washed human platelets. Biochim. Biophys. Acta 379, 360-369, 1975.
7.
PLOW, E.F. and COLLEN, D. The presence and release of -antiplasmin from human platelets. -Blood 58, 1069-1074, "481. 1
8.
HAGEN, I., BJERRUM, O.J. and SOLUM, N.O. Characterization of human platelet proteins soiubilized with Triton X-100 and examined by crossed immunoelectrophoresis. Eur. J. Biothem. 99, 9-22, 1979.
9.
GOGSTAD, G.O. A method for the isolation of *granules from human platelets. Thromb. Res. 20, 669-681, 1980.
10.
GOGSTAD, G.O., HAGEN, I., KORSMO, R. and SOLUM, N.O. Characterization of isolated human platelet &granules. Evidence for a separate a-granule pool of the glycoproteins IIb and IIIa. Biochim. Biophys. Acta 670, 150-162, 1981.
11.
GOGSTAD, G.O., HAGEN, I., KORSMO, R. and SOLUM, N.O. Evidence for release of soluble but not of membrane-integrated proteins from human platelet a-granules. Biochim. Biophys. Acta 702, 81-89, 1982. --
12.
HAGEN, I., NURDEN, A., BJERRUM, O.J., SOLUM, N.O. and CAEN, J.P. Immunochemical evidence for protein abnormalities in platelets from patients with Glanzmann's thrombasthenia and Bernard-Soulier syndrome. J. Clin. Invest. 65, 722-731, 1980.
13.
KUNICKI, T.J., PIDAB, D., ROSA, J.-P. and NURDEN, A.T. The formation of Ca -dependent complexes of platelet membrane glycoproteins IIb and IIIa in solution as determined by crossed immunoelectrophoresis. -v Blood 58, 268-278, 1981.
14.
WIMAN, B. and COLLEN, D. Purification of human antiplasmin, the fast-acting Eur. J. Biochem. 78, 19-26, 1977.
and characterization inhibitor in plasma.