Vol.
170,
August
3, 1990
No.
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 1121-1127
16, 1990
Effect
Walter
W. Chien,
Divisions Received
of Platelet
Release Products on Cytosolic Cardiac Myocytesl
Rajendra Leung+,
Mohabir, David Newman, and William T. Clusin*
of Cardiology and +-Hematology, Stanford of Medicine, Stanford, CA 94305 July
2,
Calcium
in
Lawrence
University
L. K.
School
1990
The effect of platelet release products on cytosolic Summary: calcium ([Ca++]i) was examined by monitoring the fluorescence of chick embryonic heart cells loaded with the fluorescent calcium indicator indo-l AM. Cell free filtrate of platelet release products was obtained from rabbit platelets activated with thrombin or collagen. This filtrate caused a rapid increase in both systolic and diastolic [Ca++]i in a dose-dependent manner. The effect was not blocked by pretreating the platelets with aspirin or a thromboxane synthetase inhibitor. It was not mimicked by a thromboxane analog, or by several substances known to be released from platelets including ADP, serotonin, or platelet activating factor. Apyrase or ATP-gamma S had no effect on the activity. The responsible product was heat-sensitive, trypsin-sensitive, and partitioned into the aqueous phase of a chloroform suspension. It has a low molecular weight (< 3kD) and is sensitive to 2-mercaptoethanol. Protease inhibitor appears to prolong the activity. These results suggest that trypsin-sensitive peptide released from activated platelets can increase [Ca++]i in cardiac cells. 0 1990Academrc mess, 1°C. Platelet activation and thrombus formation is the most common cause of cardiac &hernia. Thrombus formation is also a common Platelets have been shown to cause of sudden cardiac death (1). exacerbate the arrhythmogenic and electrophysiological effects of it has been shown that ischemia and reperfusion (2). Recently, [Ca++]i increases in the heart during early ischemia (3) which may be lW.W.C. is the recipient of a fellowship from the American Heart Association, California Affiliate. R.M. is the recipient of a Canadian Heart Foundation Fellowship. D.N. is the recipient of an award from the Medical Research Council, Canada. W.T.C. and L.L.K.L. are Established Investigators of the American Heart Association. *To
whom
correspondence
should
be addressed. 0006-291x/W 1121
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
170,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
ischemia-induced arrhythmias (4). We important in causing therefore examined the effect of platelet release products on [Ca++]i in chick embryonic heart cells. We further characterized the active substance by comparing its effect with compounds known to be released by platelets and by attempting to block the action of the substance with specific agents. Methods Chick embryonic heart cell aggregates were prepared from 912 day embryos as described previously (5). Spontaneously beating aggregates were formed within 3 days and all experiments were performed in aggregates cultured for 3-5 days. Cell aggregates were loaded with 20 uM indo-l AM for 45 minutes followed by removal The of extracellular indo-l AM by washing with physiological saline. cells were then transferred to a temperature regulated (370C ) Fluorescence chamber on the stage of an inverted microscope. excitation was provided by a 1OOW ultraviolet source, filtered at 360 nm. Fluorescence emissions were filtered at 400 nm or 550 nm before reaching photomultiplier tubes. The photomultiplier output at the two wavelengths was entered into an electronic ratio circuit to obtain the [Ca++]i-specific F4OO/F550 ratio. Platelets were prepared from fresh blood obtained from the central ear-lobe artery of rabbits. They were washed and resuspended at a concentration of 107 to 1081ml in Tyrodes-Hepes buffered saline solution containing no calcium. Platelets were then activated either with thrombin (10 unit/ml), collagen (0.02 mg/ml), or fibrinogen (1 mg/ml)/ADP (20 FM) in the presence of 1.8 mM calcium before being passed through a 0.45 urn Millipore filter to obtain a cell-free filtrate. CS- 13080, a thromboxane synthetase inhibitor was kindly provided by Dr. Edmond Ku of Ciba-Geigy Pharmaceutical (Princeton, NJ). U46619, a thrombroxane A2 analog was a gift from Upjohn Pharmaceutical (Kalamazoo, MI). PPACK, DPhenylalanyl-prolyl-arginyl chloromethyl ketone and indo-l AM were purchased from Calbiochem (La Jolla, CA). All other chemicals were purchased from Sigma (St. Louis, MO). For the estimation of the size of the active substance, the platelet filtrate was centrifuged in a centricon (Amicon, Beverly, MA) with a 3,000 molecular weight cutoff at 7,500 rpm at 4oC for 2 hours. A higher concentration of platelets, 2 x107 to 2 x108, was used in these experiments. After centrifugation, the retente and filtrate fractions would include predominantly compounds with molecular weight >3 kD and 10 kD, and neither compound has been shown to increase [Ca++]i in cardiac myocytes. In summary, we have shown that a novel low molecular weight, trypsin and 2-mercaptoethanol sensitive peptide secreted by platelets can increase [Ca++]i in cardiac myocytes. Acknowledgments. their expert technical
We thank Rita assistance.
Petriceks
and Yin-Gail
Yee for
References 1. 2. 3. 4. 5. 6. 7.
8. 9.
10. 11.
12. 13. 14.
Davies, M. and Thomas, A. (1984) N Engl J Med 310:1137-40. Flores, N. A., Seghatchian, M. J., and Sheridan, D. J. (1989) Circulation Vol 80, No 4:supplement 11-149. Lee, H. C., Mohabir, R., Smith, N., and Clusin, W. T. (1987) Proc. Natl. Acad. Sci. U.S.A. 85:7793-7797. Lee, H. C., Mohabir, R., Smith, N., Franz, M., and Clusin, W.T. (1988) Circulation 78:1047-1059. Clusin, W. T. (1981) J. Physiol. (London) 320:149-174. Chien, W., Mohabir, R., and Clusin, W. T. (1990) J. Clin. Invest. 85: 1436-1443. Danzinger, R.S., Raffaeli, S., Moreno-Sanchez, R., Sakai, M., Capogrossi, M.C., Spurgeon, H.A., Hansford, R.G., and Lakatta, E.G. (1988) Cell Calcium 9:193-199. Lucchesi, B.R., Mickelson, J.K., Homeister, J.W., and Jackson, C.V. (1987) Federation Proc. 46: 63-72. Montrucchio, G., Alloatti, G., Tetta, C., De Luca, R., Saunders, R.N., Emanuelli, G., and Camussi, G. (1989) Am. J. Physiol. 256 (Heart Circ. Physiol. 25): H1236-H1246. Baumgartner, H.R., and Hosang, M.(1988)Experientia44:109-12 Holmsen, H. (1987) In Hemostasis and Thrombosis (Colman, R.W., Hirsh, J., Marder, V.J., and Salzman, E.W., Eds.), pp. 606617. J.B. Lippincott Co, Philadelphia, PA. Russell, R. and Vogel, A. (1978) Cell. 14:203-210. Assoian, R.K., Komoriya, A., Meyers, C.A., Miller, D.M., and Sporn, M.B. (1983) J. Biol. Chem. 258:7155-7160. Van Nostrand, W.E., Schmaier, A.H., Farrow, J., and Cunningham D.D. (1990) Science. 248:745-748. 1127
170,
August
3, 1990
No.
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 1121-1127
16, 1990
Effect
Walter
W. Chien,
Divisions Received
of Platelet
Release Products on Cytosolic Cardiac Myocytesl
Rajendra Leung+,
Mohabir, David Newman, and William T. Clusin*
of Cardiology and +-Hematology, Stanford of Medicine, Stanford, CA 94305 July
2,
Calcium
in
Lawrence
University
L. K.
School
1990
The effect of platelet release products on cytosolic Summary: calcium ([Ca++]i) was examined by monitoring the fluorescence of chick embryonic heart cells loaded with the fluorescent calcium indicator indo-l AM. Cell free filtrate of platelet release products was obtained from rabbit platelets activated with thrombin or collagen. This filtrate caused a rapid increase in both systolic and diastolic [Ca++]i in a dose-dependent manner. The effect was not blocked by pretreating the platelets with aspirin or a thromboxane synthetase inhibitor. It was not mimicked by a thromboxane analog, or by several substances known to be released from platelets including ADP, serotonin, or platelet activating factor. Apyrase or ATP-gamma S had no effect on the activity. The responsible product was heat-sensitive, trypsin-sensitive, and partitioned into the aqueous phase of a chloroform suspension. It has a low molecular weight (< 3kD) and is sensitive to 2-mercaptoethanol. Protease inhibitor appears to prolong the activity. These results suggest that trypsin-sensitive peptide released from activated platelets can increase [Ca++]i in cardiac cells. 0 1990Academrc mess, 1°C. Platelet activation and thrombus formation is the most common cause of cardiac &hernia. Thrombus formation is also a common Platelets have been shown to cause of sudden cardiac death (1). exacerbate the arrhythmogenic and electrophysiological effects of it has been shown that ischemia and reperfusion (2). Recently, [Ca++]i increases in the heart during early ischemia (3) which may be lW.W.C. is the recipient of a fellowship from the American Heart Association, California Affiliate. R.M. is the recipient of a Canadian Heart Foundation Fellowship. D.N. is the recipient of an award from the Medical Research Council, Canada. W.T.C. and L.L.K.L. are Established Investigators of the American Heart Association. *To
whom
correspondence
should
be addressed. 0006-291x/W 1121
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
170,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
ischemia-induced arrhythmias (4). We important in causing therefore examined the effect of platelet release products on [Ca++]i in chick embryonic heart cells. We further characterized the active substance by comparing its effect with compounds known to be released by platelets and by attempting to block the action of the substance with specific agents. Methods Chick embryonic heart cell aggregates were prepared from 912 day embryos as described previously (5). Spontaneously beating aggregates were formed within 3 days and all experiments were performed in aggregates cultured for 3-5 days. Cell aggregates were loaded with 20 uM indo-l AM for 45 minutes followed by removal The of extracellular indo-l AM by washing with physiological saline. cells were then transferred to a temperature regulated (370C ) Fluorescence chamber on the stage of an inverted microscope. excitation was provided by a 1OOW ultraviolet source, filtered at 360 nm. Fluorescence emissions were filtered at 400 nm or 550 nm before reaching photomultiplier tubes. The photomultiplier output at the two wavelengths was entered into an electronic ratio circuit to obtain the [Ca++]i-specific F4OO/F550 ratio. Platelets were prepared from fresh blood obtained from the central ear-lobe artery of rabbits. They were washed and resuspended at a concentration of 107 to 1081ml in Tyrodes-Hepes buffered saline solution containing no calcium. Platelets were then activated either with thrombin (10 unit/ml), collagen (0.02 mg/ml), or fibrinogen (1 mg/ml)/ADP (20 FM) in the presence of 1.8 mM calcium before being passed through a 0.45 urn Millipore filter to obtain a cell-free filtrate. CS- 13080, a thromboxane synthetase inhibitor was kindly provided by Dr. Edmond Ku of Ciba-Geigy Pharmaceutical (Princeton, NJ). U46619, a thrombroxane A2 analog was a gift from Upjohn Pharmaceutical (Kalamazoo, MI). PPACK, DPhenylalanyl-prolyl-arginyl chloromethyl ketone and indo-l AM were purchased from Calbiochem (La Jolla, CA). All other chemicals were purchased from Sigma (St. Louis, MO). For the estimation of the size of the active substance, the platelet filtrate was centrifuged in a centricon (Amicon, Beverly, MA) with a 3,000 molecular weight cutoff at 7,500 rpm at 4oC for 2 hours. A higher concentration of platelets, 2 x107 to 2 x108, was used in these experiments. After centrifugation, the retente and filtrate fractions would include predominantly compounds with molecular weight >3 kD and 10 kD, and neither compound has been shown to increase [Ca++]i in cardiac myocytes. In summary, we have shown that a novel low molecular weight, trypsin and 2-mercaptoethanol sensitive peptide secreted by platelets can increase [Ca++]i in cardiac myocytes. Acknowledgments. their expert technical
We thank Rita assistance.
Petriceks
and Yin-Gail
Yee for
References 1. 2. 3. 4. 5. 6. 7.
8. 9.
10. 11.
12. 13. 14.
Davies, M. and Thomas, A. (1984) N Engl J Med 310:1137-40. Flores, N. A., Seghatchian, M. J., and Sheridan, D. J. (1989) Circulation Vol 80, No 4:supplement 11-149. Lee, H. C., Mohabir, R., Smith, N., and Clusin, W. T. (1987) Proc. Natl. Acad. Sci. U.S.A. 85:7793-7797. Lee, H. C., Mohabir, R., Smith, N., Franz, M., and Clusin, W.T. (1988) Circulation 78:1047-1059. Clusin, W. T. (1981) J. Physiol. (London) 320:149-174. Chien, W., Mohabir, R., and Clusin, W. T. (1990) J. Clin. Invest. 85: 1436-1443. Danzinger, R.S., Raffaeli, S., Moreno-Sanchez, R., Sakai, M., Capogrossi, M.C., Spurgeon, H.A., Hansford, R.G., and Lakatta, E.G. (1988) Cell Calcium 9:193-199. Lucchesi, B.R., Mickelson, J.K., Homeister, J.W., and Jackson, C.V. (1987) Federation Proc. 46: 63-72. Montrucchio, G., Alloatti, G., Tetta, C., De Luca, R., Saunders, R.N., Emanuelli, G., and Camussi, G. (1989) Am. J. Physiol. 256 (Heart Circ. Physiol. 25): H1236-H1246. Baumgartner, H.R., and Hosang, M.(1988)Experientia44:109-12 Holmsen, H. (1987) In Hemostasis and Thrombosis (Colman, R.W., Hirsh, J., Marder, V.J., and Salzman, E.W., Eds.), pp. 606617. J.B. Lippincott Co, Philadelphia, PA. Russell, R. and Vogel, A. (1978) Cell. 14:203-210. Assoian, R.K., Komoriya, A., Meyers, C.A., Miller, D.M., and Sporn, M.B. (1983) J. Biol. Chem. 258:7155-7160. Van Nostrand, W.E., Schmaier, A.H., Farrow, J., and Cunningham D.D. (1990) Science. 248:745-748. 1127
