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Isoelectric focusing of human platelet phospholipase C: Evidence for multimolecular forms
Life Sciences, Vol. 40, pp. 161-167 Printed in the U.S.A.
Pergamon Journal
ISOELECTRIC FOCUSING OF HUMAN PLATELET PHOSPHOLIPASE C: EVIDENCE FOR MULTIMOLECULAR FORMS Richard P. Ebstein,* Estelle R. Bennett, Jochanan Stessrnan and Bernard Lerer Department of Research, Jerusalem Mental Health Center-Ezrath Nashim Hospital, P.O.B. 140, Jerusalem Israel (Received in final form October 7, 1986) Stm~nary Polyacrylamide gel isoelectric focusing was employed to characterize phospholipase C activity in the supernatant fraction after disruption of h~nan platelets. Three bands of enzyme activity were detected on focused gels: a major band of activity (B) and two additional bands (A,C) were consistently identified. The isoelectric points of the three bands were in the range of pH 7.5-8.0. Phospholipase C activity was assayed using both phosphatidylinositol and phosphatidylinositol-4-monophosphate. The prominent B hand was active against both substrates and no evidence for substrate preference towards phosphoinositides was obtained. These data suggest that isozyme forms of cystolic phospholipase C are present in hi,nan platelet supernatant and suggest the possiblity of functional and structural differentiation of ~]e various forms of the enzyme. Acc~nulating evidence has demonstrated that stimulus-response coupling for a number of extracellular signals is mediated by the enzyme-catalyzed breakdown of phosphoinositides to inositol triphosphate and diacylglycerol (i). Mammalian phosphatidylinositol (PI)-specific phospholipase C is a calciumdependent phosphodiesterase that has been implicated in the turnover of Pl and phosphoinositides (2-14). However, the exact role that phospholipase C plays in hor~one-sti~lated cleavage of phosphoinositides remains to be clarified. First of all, the great majority of enzyme activity is reported to be located in the supernatant rather than the particulate fraction after cell h(m~Dgenization (3-14). Secondly, no clear enzyme preference has been demonstrated towards polyphosphoinositides compared to PI (11-14). These findings are difficult to reconcile with the postulated role of phospholipase C in the hormone-promoted breakdown of membrane-located polyphosphoinositides and second messenger signal generation. Investigations of phosphoinositide turnover in platelets have helped establish the role of the phosphoinositide cycle in the transduction of hormonal signals (15-23): Platelets are a particularly convenient tissue for such studies since large amounts of purified cells can easily be isolated fr~n blood and a number of agents including thrcmbin, serotonin, adenosine diphosphate and vasopressin stimulate turnover of phosphoinositides. Previous investigations of phospholipase C activity in hi,nan platelets have reported only one molecular form of the enzyme located mainly in the supernatant fraction with no substrate preference towards PI, phosphatidylinositol-4-monophosphate (PIP), *Correspondence 0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
162
Isoelectric Focusing
Vol. 40, No. 2, 1987
or phosphatidylinositol-4,5-biphosphate (PIP2) (3,10,!!,13). [n the ~arrent report we used acrylamide gel isoelectric focusing to demonstrate that several molecular forms of phospholipase C are present in the sur~ernatant fraction after platelet disruption. Methods Fresh blood was collected using 15% (v:v) citrate as an anticoagulant and centrifuged for 15 min at 200xg. The p]atelet rich plasma was then pipette] off and centrifuged for 20 min at 3000xg and the packed cells were washed by resuspension 3 times with 25 mM HEPES buffer (N-2-hydroxyethylpiDerazine-N'-2ethanesulfonic acid), pH 7.0 containing 130 mM NaCl, I mM ~gTA (ethylene glycol bis-[beta-mminoethyl ether] N,N,N',N'-tetraacetic acid) and i0 ~ glucose. The platelets were disrupted in hypotonic (i0 mM) HEPES buffer using a polytron homogenizer and then centrifuged for 1 hour at 100,000xg; the supernatant and membrane fraction were stored at -100°(7_ until used. Phospholipase C was assayed in the following reaction mixture: 50 ~M Tris-maleate buffer, pH adjusted with ma[eic acid (pH with PI=5.5; pH with PIP=6.5), 50 mM N~CI, 1 mM EGTA, 2 mM CaCIg, and 0.125 ~Ci of either L-3-phosphatidyl[2--H]inositol (16 Ci/~ole) (R@diochemical Centre, Amersh~n and New England Nuclear, Boston) or phosphatidyl[2-~H]inositol-4 monophosphate (Radiochemical Centre, ~nersham) ( 1 - 5 Ci/mmole), and nonradioactive PI (0.01mM) or PIP (0.0~nM) (Sigma, St. Louis) in a total volume of 200 ul. A lower than saturating ratio of cold/radioactive PI and PIP was employed in order to detect phospholipase ~C activi~, after isoe]ectric focusing. When ~]e specific activity of the [JH]PI or [~H]PIP was lowered by the addition of non-radioactive substrate to approximately
Vol. 40, No. 2, 1987
Isoelectric Focusing
3R-Cl~l
163
B
I0¢0
3H-CPM
19
10001
A
C
pH 9-?
500} 3H-CPM
B
1000 pH 8-6
50O ..~_~J...L.*-~
~..~_L,L.3..~_L.L~_J.~_.L..C--~--~..a--L.L~..C--C--C~,-a--L~
I
I
I
.
SLICE NUMBER (EACH SLICE : 2 M~) Fig. I. Polyacrylamide gel isoelectric focusing of human platelet supernatant fraction at various pH ranges. The a;npholyte pH range employed in the top gel is 10-3.5; in the middle gel 9-7; in the bottom gel 8-6.
164
Isoelectric Focusing
Vo]. 40, No. 2, 1987
100,000xg orecioitate, data not sho~qn). Enzyme actJ~,ity was I Ln(:~a[ ~
3H-C 4000
B
3000
pH 10-3,5
2000 1000
SLICE 1411MB£11
SLICE:2 tin)
!~ig. 2. Isoelectz [c locus [ng o[ tho suf,p~ ~L a~t Fr;1(-tion from, F~{\]-tranis FoF n(~ lymphoblasto[d ceil~. Ce]
Vol. 40, No. 2, 1987
Isoelectric Focusing
165
Since increasing evidence suggests that breakdown of phosphoinositides, and not PI, is coupled to signal generation (i), it was of interest to assay platelet su£~rnatant using phosphoinositides as the substrate; the recent commercial availability of high sDecific activity tritiated PIP facilitated this investigation. When PIP was used as the substrate a similar pattern of enzyme activity was observed after isoelectric focusing (Fig. 3). The major B band was observed in the s@ne gel position when either PI or PIP was used as the substrate; the identical position of the two bands suggests that the B band cleaves both PI and phosphoinositides. In addition at least two other bands of enzyme activity, A and C, appeared to be present although they were less pr~ninent than when PI was used as the substrate. Greater heterogeneity of enzyme activity was observed after isoelectric focusing when PIP was used as the substrate.
3H-¢PN
B
1500
1000
5W
0
!
I
l
I
I
1
t
i
I
I
i
l
I
I
i.
I
i
1
i
I
l
I
i
t
I
i
I
I
i
l
;
l
I
i
l
l
i
]
SLICE BBER (F..qCHSLICE - 2 m)
Fig. 3. Isoelectric [ocusing o[ human platelet supernatant fraction with PIP as the substrate. The ampholyte pH range employed was 8-6.
Discussion What is the signi~Eicance of the multimolecular forms of human platelet phospholipase C identified by polyacrylamide gel isoelectric focusing? The substrate preference of phospholipase C towards PI and phosphoinositides is of
166
Isoelectric Focusing
Vol. 40, No. 2, 1987
particular interest. The evidence presented in the current r%oort suggests that although multimolecular forms of phospholipase C are present in the supernatant fraction after platelet homogenization, the major band of enzyme activity cleaves both PI and PIP: no evidence was obtained for substrate selectivity towards phosp~hoinositides. We note that the physical state of the phospholipase C enzyme, which is embedded in polyacrylamide gels after isoelectric focusing, makes it difficult to ascertain whether initial velocity conditions are maintained during the assay of each individual gel slice. The difficulty of determining concentration of enzyme protein in each gel slice and the diffusion rate of substrate and product as well as their accessiblity to the enzyme preclude the use of a gel-bound enzyme in the detailed evaluation of the kinetic properties of p h o s p ~ l ipase C activity. Nevertheless, we believe that the main conclusions of this report are unaffected by these considerations: several isozyme forms of PI-specific phospholipase C activity were identified and the main enzyme band cleaves both PI and PIP. On the other hand, until each of the bands of enzyme activity identified by isoelectric focusing are further characterized, it is difficult to rule out the possibility that one of the minor bands ;nay show substrate preference for phosphoinositides. Previous investigations of htmlan platelet phospholipase C failed to rec(xjnize the existence of multimolecular forms of this enzyme (2,10,11,13). Rittenhouse (Ii) has reported that partially-purified (by DEAE-cellulose chromatography) phospholipase C exhibited only one molecular form. Moreover, no substrate selectivity vis a vis PI, PIP, or PIP 2 was observed. It appears likely that multimolecular forms of the enzyn~ were discarded during the purification procedure, suggesting that accurate monitoring, preferably by gel isoelectric focusing, of phospholipase C activity is a prerequisite for further studies of this important enzyme. A number of animal studies }]ave shown the presence of multimolecular forms of phospholipase C in both brain and peripheral tissue with distinct isoelectric points (4,5,12) . No evidence for substrate specificity towards PI, PIP or PIP_ was obtained. However, the recent availability of highly purified tritiZated PIP suggests that the substrate specificity of the rat forms of phospholipase C could be profitably reinvestigated. Many different enzymes have been shown to exhibit isozyme patterns, suggesting the ubiquitous nature of this phenomenon (24). Isozymic forms, which catalyze the same chemical reaction, often differ in kinetic properties (Kin, Vmax), amino acid composition, tissue distribution, and intracel]ular location: such differences presumably reflect different functional and metabolic requirements. It is too early to speculate on the possible role of the multiple forms of phospholipase C identified in the current study in platelet physiology and transduction of hormonal signals. However, further studies of the calcium requirements, subcellular localization and substrate preferences of the several forms of human platelet phospholipase C will no doubt she~ light on the role of this enzyme in the signal-dependent turnover of phosphoinosi tides.
Acknowledgements This work was supported by grants to the Ezrath Nashim Hospital fr(>n the Herman Goldman Foundation, New York, and the United Jewish Endowment Fund of Greater Washington-Pollinger Foundation.
Vol. 40, No. 2, 1987
Isoelectric Focusing
167
References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
M.J. BERRIDGE, Bioch6m..7. 220 345-360 (1984). C.P. DOWNES and R.H. MICHELL, Biochem. J. 198 133-140 (1981). W. SIESS and E.G. LAPETINA, Biochem. Biophys. Acta 752 329-338 (1983). K. HIRASAWA, R.F. IRVINE and R.M.C. DAWSON, Biochem. Biophys. Res. Conml. 107, 533-537 (1982). K. HIRASAWA, R.F. IRVINE and R.M.C. DAWSON, Biochem. J. 205 437-442 (1982). R.F. IRVINE, A.J. LETCHER and R.M.C. DAWSON, Biochem. J. 218 177-185 (1984). K. HIRASAWA, R.F. IRVINE and R.M.C. DAWSON, Biochem. J. 206 675-678 (1982). S.L. HOFMAN and P.W. MAJERUS, J. Biol. Chem. 257 6491-6469 (1982). S.L. HOFMAN and P.W. MAJERUS, J. Biol. Chem. 257 14359-14364 (1982). S. RITTENHOUSE-SIMMONS, J. Clin. Invest. 63 580-587 (1979). S. RITTENHOUSE, Proc. Nat. Acad. Sci. U.S.A. 80 5417-5420 (1983). M.G. LOW and W.B. WEGLICKI, Biochem. J. 215 325-334 (1983). G. GRAFF, N. NAHAS, M. NIKOLOPOULOU, V. NATARAJAN and H.O. SCHMID, Archiv. Biochem. Biophys. 228 299-308 (1984). D.B. WILSON, T.E. BROSS, S.L HOFMAN and P.W. MAJERUS, J. Biol. Chem. 259 11718-11724 (1984). D. DE CHAFFOY DE COURCELLES, J.E. LEYSEN, F. DE CLERCK, H. VAN BELLE, and P.A.J. JANSSEN, ,7. Biol. Chem. 260 7603-7608 (1985). F. RENDU, P. MARCHE, J. VIRET, D. DAVELOOSE, F. LETTEREIER, S. LEVYTOLH3ANO and J.P. CAEN, Nouv. Rev. Ft. Hematol. 27 293-297 (1985). G.B. ZAVOICO, S.P. HALENDA, R.I. SHA'AFI and M.B. FBINSTEIN, Proc. Nat. Acad. U.S.A. 82 3859-3862 (1985). J.D. VICKERS, R.L. KINLOUGH-RATHBONE, and J.F. MUSTARD, Thromb. Res. 28 731-740 (1982). J.D. VICKERS, R.L. KINLOUGH-RATHBONE, and J.F. MUSTARD, Blood 60 12471250 (1982). M.J. BROEKMAN, Biochem. Biophys. Res. Comm. 120 226-231 (1984). W. SIESS, F ~ S Lett 185 151-156 (1985). B.W. AGRANOFF, P. MURTHY and E.B. SEGUIN, J. Biol. Chem. 258 2076-2078 (1983). M.M. BILLAH and E.G. LAPETINA, J. Biol. Chem. 257 2705-12708 (1982). C.L. MARKERT, in: Isozymes I. Molecular Structure, (C.L. Markert, ed.), p. 1-9, Academic Press, New York (1975). R.P. EBSTEIN, M. STEINITZ, J. MINTZER, LIPSHITZ, I. and J. STESSMAN, Experientia 41 1552-1554 (1985).
Pergamon Journal
ISOELECTRIC FOCUSING OF HUMAN PLATELET PHOSPHOLIPASE C: EVIDENCE FOR MULTIMOLECULAR FORMS Richard P. Ebstein,* Estelle R. Bennett, Jochanan Stessrnan and Bernard Lerer Department of Research, Jerusalem Mental Health Center-Ezrath Nashim Hospital, P.O.B. 140, Jerusalem Israel (Received in final form October 7, 1986) Stm~nary Polyacrylamide gel isoelectric focusing was employed to characterize phospholipase C activity in the supernatant fraction after disruption of h~nan platelets. Three bands of enzyme activity were detected on focused gels: a major band of activity (B) and two additional bands (A,C) were consistently identified. The isoelectric points of the three bands were in the range of pH 7.5-8.0. Phospholipase C activity was assayed using both phosphatidylinositol and phosphatidylinositol-4-monophosphate. The prominent B hand was active against both substrates and no evidence for substrate preference towards phosphoinositides was obtained. These data suggest that isozyme forms of cystolic phospholipase C are present in hi,nan platelet supernatant and suggest the possiblity of functional and structural differentiation of ~]e various forms of the enzyme. Acc~nulating evidence has demonstrated that stimulus-response coupling for a number of extracellular signals is mediated by the enzyme-catalyzed breakdown of phosphoinositides to inositol triphosphate and diacylglycerol (i). Mammalian phosphatidylinositol (PI)-specific phospholipase C is a calciumdependent phosphodiesterase that has been implicated in the turnover of Pl and phosphoinositides (2-14). However, the exact role that phospholipase C plays in hor~one-sti~lated cleavage of phosphoinositides remains to be clarified. First of all, the great majority of enzyme activity is reported to be located in the supernatant rather than the particulate fraction after cell h(m~Dgenization (3-14). Secondly, no clear enzyme preference has been demonstrated towards polyphosphoinositides compared to PI (11-14). These findings are difficult to reconcile with the postulated role of phospholipase C in the hormone-promoted breakdown of membrane-located polyphosphoinositides and second messenger signal generation. Investigations of phosphoinositide turnover in platelets have helped establish the role of the phosphoinositide cycle in the transduction of hormonal signals (15-23): Platelets are a particularly convenient tissue for such studies since large amounts of purified cells can easily be isolated fr~n blood and a number of agents including thrcmbin, serotonin, adenosine diphosphate and vasopressin stimulate turnover of phosphoinositides. Previous investigations of phospholipase C activity in hi,nan platelets have reported only one molecular form of the enzyme located mainly in the supernatant fraction with no substrate preference towards PI, phosphatidylinositol-4-monophosphate (PIP), *Correspondence 0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
162
Isoelectric Focusing
Vol. 40, No. 2, 1987
or phosphatidylinositol-4,5-biphosphate (PIP2) (3,10,!!,13). [n the ~arrent report we used acrylamide gel isoelectric focusing to demonstrate that several molecular forms of phospholipase C are present in the sur~ernatant fraction after platelet disruption. Methods Fresh blood was collected using 15% (v:v) citrate as an anticoagulant and centrifuged for 15 min at 200xg. The p]atelet rich plasma was then pipette] off and centrifuged for 20 min at 3000xg and the packed cells were washed by resuspension 3 times with 25 mM HEPES buffer (N-2-hydroxyethylpiDerazine-N'-2ethanesulfonic acid), pH 7.0 containing 130 mM NaCl, I mM ~gTA (ethylene glycol bis-[beta-mminoethyl ether] N,N,N',N'-tetraacetic acid) and i0 ~ glucose. The platelets were disrupted in hypotonic (i0 mM) HEPES buffer using a polytron homogenizer and then centrifuged for 1 hour at 100,000xg; the supernatant and membrane fraction were stored at -100°(7_ until used. Phospholipase C was assayed in the following reaction mixture: 50 ~M Tris-maleate buffer, pH adjusted with ma[eic acid (pH with PI=5.5; pH with PIP=6.5), 50 mM N~CI, 1 mM EGTA, 2 mM CaCIg, and 0.125 ~Ci of either L-3-phosphatidyl[2--H]inositol (16 Ci/~ole) (R@diochemical Centre, Amersh~n and New England Nuclear, Boston) or phosphatidyl[2-~H]inositol-4 monophosphate (Radiochemical Centre, ~nersham) ( 1 - 5 Ci/mmole), and nonradioactive PI (0.01mM) or PIP (0.0~nM) (Sigma, St. Louis) in a total volume of 200 ul. A lower than saturating ratio of cold/radioactive PI and PIP was employed in order to detect phospholipase ~C activi~, after isoe]ectric focusing. When ~]e specific activity of the [JH]PI or [~H]PIP was lowered by the addition of non-radioactive substrate to approximately
Vol. 40, No. 2, 1987
Isoelectric Focusing
3R-Cl~l
163
B
I0¢0
3H-CPM
19
10001
A
C
pH 9-?
500} 3H-CPM
B
1000 pH 8-6
50O ..~_~J...L.*-~
~..~_L,L.3..~_L.L~_J.~_.L..C--~--~..a--L.L~..C--C--C~,-a--L~
I
I
I
.
SLICE NUMBER (EACH SLICE : 2 M~) Fig. I. Polyacrylamide gel isoelectric focusing of human platelet supernatant fraction at various pH ranges. The a;npholyte pH range employed in the top gel is 10-3.5; in the middle gel 9-7; in the bottom gel 8-6.
164
Isoelectric Focusing
Vo]. 40, No. 2, 1987
100,000xg orecioitate, data not sho~qn). Enzyme actJ~,ity was I Ln(:~a[ ~
3H-C 4000
B
3000
pH 10-3,5
2000 1000
SLICE 1411MB£11
SLICE:2 tin)
!~ig. 2. Isoelectz [c locus [ng o[ tho suf,p~ ~L a~t Fr;1(-tion from, F~{\]-tranis FoF n(~ lymphoblasto[d ceil~. Ce]
Vol. 40, No. 2, 1987
Isoelectric Focusing
165
Since increasing evidence suggests that breakdown of phosphoinositides, and not PI, is coupled to signal generation (i), it was of interest to assay platelet su£~rnatant using phosphoinositides as the substrate; the recent commercial availability of high sDecific activity tritiated PIP facilitated this investigation. When PIP was used as the substrate a similar pattern of enzyme activity was observed after isoelectric focusing (Fig. 3). The major B band was observed in the s@ne gel position when either PI or PIP was used as the substrate; the identical position of the two bands suggests that the B band cleaves both PI and phosphoinositides. In addition at least two other bands of enzyme activity, A and C, appeared to be present although they were less pr~ninent than when PI was used as the substrate. Greater heterogeneity of enzyme activity was observed after isoelectric focusing when PIP was used as the substrate.
3H-¢PN
B
1500
1000
5W
0
!
I
l
I
I
1
t
i
I
I
i
l
I
I
i.
I
i
1
i
I
l
I
i
t
I
i
I
I
i
l
;
l
I
i
l
l
i
]
SLICE BBER (F..qCHSLICE - 2 m)
Fig. 3. Isoelectric [ocusing o[ human platelet supernatant fraction with PIP as the substrate. The ampholyte pH range employed was 8-6.
Discussion What is the signi~Eicance of the multimolecular forms of human platelet phospholipase C identified by polyacrylamide gel isoelectric focusing? The substrate preference of phospholipase C towards PI and phosphoinositides is of
166
Isoelectric Focusing
Vol. 40, No. 2, 1987
particular interest. The evidence presented in the current r%oort suggests that although multimolecular forms of phospholipase C are present in the supernatant fraction after platelet homogenization, the major band of enzyme activity cleaves both PI and PIP: no evidence was obtained for substrate selectivity towards phosp~hoinositides. We note that the physical state of the phospholipase C enzyme, which is embedded in polyacrylamide gels after isoelectric focusing, makes it difficult to ascertain whether initial velocity conditions are maintained during the assay of each individual gel slice. The difficulty of determining concentration of enzyme protein in each gel slice and the diffusion rate of substrate and product as well as their accessiblity to the enzyme preclude the use of a gel-bound enzyme in the detailed evaluation of the kinetic properties of p h o s p ~ l ipase C activity. Nevertheless, we believe that the main conclusions of this report are unaffected by these considerations: several isozyme forms of PI-specific phospholipase C activity were identified and the main enzyme band cleaves both PI and PIP. On the other hand, until each of the bands of enzyme activity identified by isoelectric focusing are further characterized, it is difficult to rule out the possibility that one of the minor bands ;nay show substrate preference for phosphoinositides. Previous investigations of htmlan platelet phospholipase C failed to rec(xjnize the existence of multimolecular forms of this enzyme (2,10,11,13). Rittenhouse (Ii) has reported that partially-purified (by DEAE-cellulose chromatography) phospholipase C exhibited only one molecular form. Moreover, no substrate selectivity vis a vis PI, PIP, or PIP 2 was observed. It appears likely that multimolecular forms of the enzyn~ were discarded during the purification procedure, suggesting that accurate monitoring, preferably by gel isoelectric focusing, of phospholipase C activity is a prerequisite for further studies of this important enzyme. A number of animal studies }]ave shown the presence of multimolecular forms of phospholipase C in both brain and peripheral tissue with distinct isoelectric points (4,5,12) . No evidence for substrate specificity towards PI, PIP or PIP_ was obtained. However, the recent availability of highly purified tritiZated PIP suggests that the substrate specificity of the rat forms of phospholipase C could be profitably reinvestigated. Many different enzymes have been shown to exhibit isozyme patterns, suggesting the ubiquitous nature of this phenomenon (24). Isozymic forms, which catalyze the same chemical reaction, often differ in kinetic properties (Kin, Vmax), amino acid composition, tissue distribution, and intracel]ular location: such differences presumably reflect different functional and metabolic requirements. It is too early to speculate on the possible role of the multiple forms of phospholipase C identified in the current study in platelet physiology and transduction of hormonal signals. However, further studies of the calcium requirements, subcellular localization and substrate preferences of the several forms of human platelet phospholipase C will no doubt she~ light on the role of this enzyme in the signal-dependent turnover of phosphoinosi tides.
Acknowledgements This work was supported by grants to the Ezrath Nashim Hospital fr(>n the Herman Goldman Foundation, New York, and the United Jewish Endowment Fund of Greater Washington-Pollinger Foundation.
Vol. 40, No. 2, 1987
Isoelectric Focusing
167
References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
M.J. BERRIDGE, Bioch6m..7. 220 345-360 (1984). C.P. DOWNES and R.H. MICHELL, Biochem. J. 198 133-140 (1981). W. SIESS and E.G. LAPETINA, Biochem. Biophys. Acta 752 329-338 (1983). K. HIRASAWA, R.F. IRVINE and R.M.C. DAWSON, Biochem. Biophys. Res. Conml. 107, 533-537 (1982). K. HIRASAWA, R.F. IRVINE and R.M.C. DAWSON, Biochem. J. 205 437-442 (1982). R.F. IRVINE, A.J. LETCHER and R.M.C. DAWSON, Biochem. J. 218 177-185 (1984). K. HIRASAWA, R.F. IRVINE and R.M.C. DAWSON, Biochem. J. 206 675-678 (1982). S.L. HOFMAN and P.W. MAJERUS, J. Biol. Chem. 257 6491-6469 (1982). S.L. HOFMAN and P.W. MAJERUS, J. Biol. Chem. 257 14359-14364 (1982). S. RITTENHOUSE-SIMMONS, J. Clin. Invest. 63 580-587 (1979). S. RITTENHOUSE, Proc. Nat. Acad. Sci. U.S.A. 80 5417-5420 (1983). M.G. LOW and W.B. WEGLICKI, Biochem. J. 215 325-334 (1983). G. GRAFF, N. NAHAS, M. NIKOLOPOULOU, V. NATARAJAN and H.O. SCHMID, Archiv. Biochem. Biophys. 228 299-308 (1984). D.B. WILSON, T.E. BROSS, S.L HOFMAN and P.W. MAJERUS, J. Biol. Chem. 259 11718-11724 (1984). D. DE CHAFFOY DE COURCELLES, J.E. LEYSEN, F. DE CLERCK, H. VAN BELLE, and P.A.J. JANSSEN, ,7. Biol. Chem. 260 7603-7608 (1985). F. RENDU, P. MARCHE, J. VIRET, D. DAVELOOSE, F. LETTEREIER, S. LEVYTOLH3ANO and J.P. CAEN, Nouv. Rev. Ft. Hematol. 27 293-297 (1985). G.B. ZAVOICO, S.P. HALENDA, R.I. SHA'AFI and M.B. FBINSTEIN, Proc. Nat. Acad. U.S.A. 82 3859-3862 (1985). J.D. VICKERS, R.L. KINLOUGH-RATHBONE, and J.F. MUSTARD, Thromb. Res. 28 731-740 (1982). J.D. VICKERS, R.L. KINLOUGH-RATHBONE, and J.F. MUSTARD, Blood 60 12471250 (1982). M.J. BROEKMAN, Biochem. Biophys. Res. Comm. 120 226-231 (1984). W. SIESS, F ~ S Lett 185 151-156 (1985). B.W. AGRANOFF, P. MURTHY and E.B. SEGUIN, J. Biol. Chem. 258 2076-2078 (1983). M.M. BILLAH and E.G. LAPETINA, J. Biol. Chem. 257 2705-12708 (1982). C.L. MARKERT, in: Isozymes I. Molecular Structure, (C.L. Markert, ed.), p. 1-9, Academic Press, New York (1975). R.P. EBSTEIN, M. STEINITZ, J. MINTZER, LIPSHITZ, I. and J. STESSMAN, Experientia 41 1552-1554 (1985).