305
Biochem. J. (1990) 265, 305-307 (Printed in Great Britain)
BUCCHLEBilUAL LETTERS L- i
Rapid blood platelet activation: continuousand quenched-flow versus stopped-flow approaches Efficient haemostasis is estimated to require platelet activation times on the order of 0.1 s [1]. The quenchedflow approach of stimulating platelets under physiological rheological conditions similar to those in the arterial circulation [2] has been used to study morphological changes [3], secretion [4], protein phosphorylation [5,6], and aggregation [2] approaching this time scale. Both aggregation, as detected by single-particle counting [7], and phosphorylation of the 20 kDa and 47 kDa proteins are significant within 0.3 s and reach a plateau by 3-5 s [5]. The roles of intracellular messengers, such as Ca2l and inositol trisphosphate, in the activation of platelets by various agonists are not yet clearly understood during this initial phase from 0 to 5 s. Two fundamentally different methods have been used to study the rapid dynamics of cytosolic free calcium concentration [Ca2l], in platelets loaded with the Ca2l-sensitive dyes indo-1 or fura-2. We have developed a continuous-flow fluorimetric approach [8] to measure [Ca2+]i under physiological blood-flow conditions, with shear rates between 1258 and 236 s-5 [9,10] and observed a biphasic [Ca2+]i increase induced by ADP or thrombin that was significant within 0.5 s. Sage & Rink, on the other hand, have used a more traditional stopped-flow fluorimetric method [11,12]. Although this approach has the advantage of permitting measurements at shorter reaction times, there may be problems associated with its use in the study of blood platelets. Unlike our quenched- and continuousflow approaches, the stopped-flow method involves high shear rates for ramming the platelet and agonist solutions into the mixing and observation chamber. Fluorescent changes in this chamber are then monitored in the absence of shear. High shear stresses have been previously shown to cause platelet lysis, activation [13,14], and rises in [Ca2+]i [15]. Despite these risks, Sage & Rink did not provide control data for platelets studied in the absence of agonist or report resting [Ca2+]i levels [11,12]. Similar deficiencies are also present in a more recent publication [16]. We are therefore interested to know whether the stopped-flow technique itself may cause some significant artifactual [Ca2"], increases. Although their recent Mn2+entry studies [16] are potentially very useful, interpretation of the results would be helped by availability of control experiments. In addition, their method of pre-
*
Present address: American Red Cross, Biomedical Research and
Rockville, MD 20855, U.S.A.
Vol. 265
paring washed platelets [12] uses a medium lacking calcium; Ca2" is only added just prior to each experiment. It would be important to know whether ADP-responsive platelets isolated in the presence of physiological levels of Ca2" [6] would behave similarly in the stopped-flow experiments. We have reported that the rapid initial increase in [Ca2"], to about 2 gm, caused by ADP or thrombin [8], is derived only from internal sites. This is followed by calcium entry beginning near 2 s. Control runs indicate that the physiological shear stresses of pumping the platelets through the apparatus only slightly increased the [Ca2+]i to 0.12 ,aM, from a static non-flowing level of IuM [8]. Extrapolation backwards of the initial rates 0.10 of [Ca2"], increases for ADP and thrombin gave 'onset' times of 0.06 and 0.19 s, respectively [8]. We therefore believe that the similar initial (0.5-2 s) Ca2" response of platelets to either ADP or thrombin represents a mobilization of Ca2" from internal site(s), via a common pathway. Additional studies are required to understand better the mechanism of this mobilization. The quenchedflow approach should permit determination of the kinetics and extent of early inositol lipid turnover; the stopped-flow method is not applicable to these biochemical measurements. Glen D. JONES* and Adrian R. L. GEAR Department of Biochemistry, University of Virginia, Charlottesville, VA 22908, U.S.A. 1. Born, G. V. & Richardson, P. D. (1980) J. Membr. Biol. 57, 87-90 2. Gear, A. R. L. (1982) J. Lab. Clin. Med. 100, 866-886 3. Gear, A. R. L. (1984) Br. J. Haematol. 56, 387-398 4. Gear, A. R. L. & Burke, D. (1982) Blood 60, 1231-1234 5. Carty, D. J., Spielberg, F. & Gear, A. R. L. (1986) Blood 67, 1738-1743 6. Carty, D. J., Freas, D. L. & Gear, A. R. L. (1987) Blood 70, 511-515 7. Gear, A. R. L. (1986) in Platelet Responses and Metabolism (Holmsen, H., ed.), vol. 1, pp. 97-114, CRC Press, Boca Raton 8. Jones, G. D. & Gear, A. R. L. (1988) Blood 71, 1539-1543 9. Carty, D. J., Jones, G. D., Freas, D. L. & Gear, A. R. L. (1988) J. Lab. Clin. Med. 112, 603-611 10. Jones, G. D., Carty, D. J., Freas, D. L., Spears, J. T. & Gear, A. R. L. (1989) Biochem. J. 262, 611-616 11. Sage, S. O. & Rink, T. J. (1986) Biochem. Biophys. Res. Commun. 136, 1124-1129 12. Sage, S. 0. & Rink, T. J. (1987) J. Biol. Chem. 262, 16364-16369
Development, Jerome H. Holland Laboratories,
15601 Crabbs Branch
Way,
BJ Letters
306 13. Brown, C. H., III, Leverett, L. B., Lewis, C. W., Alfrey, C. P., Jr. & Hellums, J. D. (1975) J. Lab. Clin. Med. 86, 462-471 14. Ramstack, J. M., Zuckerman, L. & Mockros, L. F. (1979) J. Biomech. 12, 113 15. Giorgio, T. D. & Hellums, J. D. (1986) Thromb. Res. 41, 353-359 16. Sage, S. O., Merritt, J. E., Hallam, T. J. & Rink, T. J. (1989) Biochem. J. 258, 923-926 Received 15 September 1989
Reply to 'Rapid blood platelet activation: continuous- and quenched-flow versus stopped-flow approaches' Jones & Gear [1] raise the potential difficulty of platelet activation by high shear stresses in continuous-flow or stopped-flow experiments. They suggest that detection of Ca2l influx by stopped-flow fluorimetry at early times of activation, before the release of Ca2l from intracellular stores [2-5], could be due to such non-specific, rather than agonist-evoked, effects. Their suggestion is not supported by inspection of our published data.
We have reported that in the presence of external Ca2+, ADP evokes a rise in [Ca2+]i without measurable delay (less than 20 ms) [2,3]. ADP-evoked Mn2" entry shows similar kinetics [4]. These events are attributable to divalent cation influx. In the absence of external Ca2+, the ADP-evoked rise in [Ca2+]i is delayed in onset by about 200 ms [2-4]. If the early influx of Ca2" or Mn2+ were due to shear activation, then this should also be present in analogous stopped-flow experiments using other agonists such as thrombin. This is not the case; there is no detectable rise in [Ca2]i or Mn2" entry during the first 300 ms of activation in experiments employing any agonist other than ADP [2-4]. Naturally, in developing our whole-cell stopped-flow fluorimetric technique, we paid careful attention to the potential problem of non-specific activation. Fig. I explicitly demonstrates our control experiments; comparing the results obtained when stimulating fura-2loaded platelets with ADP in the presence of external Ca2` or Mn2+, with agonist-free controls under the same conditions. Fluorescence records obtained with excitation wavelengths of 340 nm and 360 nm are shown. At 340 nm, fura-2 fluorescence is increased by a rise in [Ca2"] and quenched by the binding of Mn2+. At 360 nm, the fluorescence is insensitive to changes in [Ca2+]i but still quenched by Mn2"; thus this signal gives a selective indication of Mn2+ entry [4]. Figs. 1 (b) and 1 (d) show the
1 mM-Ca2+
1 mM-Mn2+
(c)
(a)
(L)i--n340nm
k
340 nm
,
,
,
ADP
ADP
360 nm
F
1~
A ADP
ADP
(d)
(b) F
340 nm
F
360 nm
0
360 nm
_
1
3 2 Time (s)
4
5
nm ~~~~~~~340
F
F
fvv ill
0
1
3 2 Time (s)
4
5
Fig. 1. Stopped-flow fluorescence records of fura-2-loaded human platelets Platelets, prepared as previously described [3], were suspended in medium containing: 145 mM-NaCl, 5 mM-KCl, 1 mM-MgCl2, 10 mM-Hepes, pH 7.4 at 37 'C. Panels (a) and (b) show traces obtained with 1 mM-Ca2+ in the mixing chamber and (c) and (d) with 1 mM-Mn2+. (a) and (c) show the result of applying 40,uM-ADP in the mixing chamber at the point the recording was triggered (time zero). In (b) and (d), the conditions were identical except that ADP was absent. Each of the eight traces were separately obtained by signal averaging records of 10 sequential runs. All traces were obtained using the same preparation of platelets. Excitation was at 340 nm or 360 nm as indicated, emission was at 500 nm in all cases. The fluorescence axis is in arbitrary units and the scales are identical for each trace. The initial (resting) fluorescence is indicated by the horizontal mark.
1990
Biochem. J. (1990) 265, 305-307 (Printed in Great Britain)
BUCCHLEBilUAL LETTERS L- i
Rapid blood platelet activation: continuousand quenched-flow versus stopped-flow approaches Efficient haemostasis is estimated to require platelet activation times on the order of 0.1 s [1]. The quenchedflow approach of stimulating platelets under physiological rheological conditions similar to those in the arterial circulation [2] has been used to study morphological changes [3], secretion [4], protein phosphorylation [5,6], and aggregation [2] approaching this time scale. Both aggregation, as detected by single-particle counting [7], and phosphorylation of the 20 kDa and 47 kDa proteins are significant within 0.3 s and reach a plateau by 3-5 s [5]. The roles of intracellular messengers, such as Ca2l and inositol trisphosphate, in the activation of platelets by various agonists are not yet clearly understood during this initial phase from 0 to 5 s. Two fundamentally different methods have been used to study the rapid dynamics of cytosolic free calcium concentration [Ca2l], in platelets loaded with the Ca2l-sensitive dyes indo-1 or fura-2. We have developed a continuous-flow fluorimetric approach [8] to measure [Ca2+]i under physiological blood-flow conditions, with shear rates between 1258 and 236 s-5 [9,10] and observed a biphasic [Ca2+]i increase induced by ADP or thrombin that was significant within 0.5 s. Sage & Rink, on the other hand, have used a more traditional stopped-flow fluorimetric method [11,12]. Although this approach has the advantage of permitting measurements at shorter reaction times, there may be problems associated with its use in the study of blood platelets. Unlike our quenched- and continuousflow approaches, the stopped-flow method involves high shear rates for ramming the platelet and agonist solutions into the mixing and observation chamber. Fluorescent changes in this chamber are then monitored in the absence of shear. High shear stresses have been previously shown to cause platelet lysis, activation [13,14], and rises in [Ca2+]i [15]. Despite these risks, Sage & Rink did not provide control data for platelets studied in the absence of agonist or report resting [Ca2+]i levels [11,12]. Similar deficiencies are also present in a more recent publication [16]. We are therefore interested to know whether the stopped-flow technique itself may cause some significant artifactual [Ca2"], increases. Although their recent Mn2+entry studies [16] are potentially very useful, interpretation of the results would be helped by availability of control experiments. In addition, their method of pre-
*
Present address: American Red Cross, Biomedical Research and
Rockville, MD 20855, U.S.A.
Vol. 265
paring washed platelets [12] uses a medium lacking calcium; Ca2" is only added just prior to each experiment. It would be important to know whether ADP-responsive platelets isolated in the presence of physiological levels of Ca2" [6] would behave similarly in the stopped-flow experiments. We have reported that the rapid initial increase in [Ca2"], to about 2 gm, caused by ADP or thrombin [8], is derived only from internal sites. This is followed by calcium entry beginning near 2 s. Control runs indicate that the physiological shear stresses of pumping the platelets through the apparatus only slightly increased the [Ca2+]i to 0.12 ,aM, from a static non-flowing level of IuM [8]. Extrapolation backwards of the initial rates 0.10 of [Ca2"], increases for ADP and thrombin gave 'onset' times of 0.06 and 0.19 s, respectively [8]. We therefore believe that the similar initial (0.5-2 s) Ca2" response of platelets to either ADP or thrombin represents a mobilization of Ca2" from internal site(s), via a common pathway. Additional studies are required to understand better the mechanism of this mobilization. The quenchedflow approach should permit determination of the kinetics and extent of early inositol lipid turnover; the stopped-flow method is not applicable to these biochemical measurements. Glen D. JONES* and Adrian R. L. GEAR Department of Biochemistry, University of Virginia, Charlottesville, VA 22908, U.S.A. 1. Born, G. V. & Richardson, P. D. (1980) J. Membr. Biol. 57, 87-90 2. Gear, A. R. L. (1982) J. Lab. Clin. Med. 100, 866-886 3. Gear, A. R. L. (1984) Br. J. Haematol. 56, 387-398 4. Gear, A. R. L. & Burke, D. (1982) Blood 60, 1231-1234 5. Carty, D. J., Spielberg, F. & Gear, A. R. L. (1986) Blood 67, 1738-1743 6. Carty, D. J., Freas, D. L. & Gear, A. R. L. (1987) Blood 70, 511-515 7. Gear, A. R. L. (1986) in Platelet Responses and Metabolism (Holmsen, H., ed.), vol. 1, pp. 97-114, CRC Press, Boca Raton 8. Jones, G. D. & Gear, A. R. L. (1988) Blood 71, 1539-1543 9. Carty, D. J., Jones, G. D., Freas, D. L. & Gear, A. R. L. (1988) J. Lab. Clin. Med. 112, 603-611 10. Jones, G. D., Carty, D. J., Freas, D. L., Spears, J. T. & Gear, A. R. L. (1989) Biochem. J. 262, 611-616 11. Sage, S. O. & Rink, T. J. (1986) Biochem. Biophys. Res. Commun. 136, 1124-1129 12. Sage, S. 0. & Rink, T. J. (1987) J. Biol. Chem. 262, 16364-16369
Development, Jerome H. Holland Laboratories,
15601 Crabbs Branch
Way,
BJ Letters
306 13. Brown, C. H., III, Leverett, L. B., Lewis, C. W., Alfrey, C. P., Jr. & Hellums, J. D. (1975) J. Lab. Clin. Med. 86, 462-471 14. Ramstack, J. M., Zuckerman, L. & Mockros, L. F. (1979) J. Biomech. 12, 113 15. Giorgio, T. D. & Hellums, J. D. (1986) Thromb. Res. 41, 353-359 16. Sage, S. O., Merritt, J. E., Hallam, T. J. & Rink, T. J. (1989) Biochem. J. 258, 923-926 Received 15 September 1989
Reply to 'Rapid blood platelet activation: continuous- and quenched-flow versus stopped-flow approaches' Jones & Gear [1] raise the potential difficulty of platelet activation by high shear stresses in continuous-flow or stopped-flow experiments. They suggest that detection of Ca2l influx by stopped-flow fluorimetry at early times of activation, before the release of Ca2l from intracellular stores [2-5], could be due to such non-specific, rather than agonist-evoked, effects. Their suggestion is not supported by inspection of our published data.
We have reported that in the presence of external Ca2+, ADP evokes a rise in [Ca2+]i without measurable delay (less than 20 ms) [2,3]. ADP-evoked Mn2" entry shows similar kinetics [4]. These events are attributable to divalent cation influx. In the absence of external Ca2+, the ADP-evoked rise in [Ca2+]i is delayed in onset by about 200 ms [2-4]. If the early influx of Ca2" or Mn2+ were due to shear activation, then this should also be present in analogous stopped-flow experiments using other agonists such as thrombin. This is not the case; there is no detectable rise in [Ca2]i or Mn2" entry during the first 300 ms of activation in experiments employing any agonist other than ADP [2-4]. Naturally, in developing our whole-cell stopped-flow fluorimetric technique, we paid careful attention to the potential problem of non-specific activation. Fig. I explicitly demonstrates our control experiments; comparing the results obtained when stimulating fura-2loaded platelets with ADP in the presence of external Ca2` or Mn2+, with agonist-free controls under the same conditions. Fluorescence records obtained with excitation wavelengths of 340 nm and 360 nm are shown. At 340 nm, fura-2 fluorescence is increased by a rise in [Ca2"] and quenched by the binding of Mn2+. At 360 nm, the fluorescence is insensitive to changes in [Ca2+]i but still quenched by Mn2"; thus this signal gives a selective indication of Mn2+ entry [4]. Figs. 1 (b) and 1 (d) show the
1 mM-Ca2+
1 mM-Mn2+
(c)
(a)
(L)i--n340nm
k
340 nm
,
,
,
ADP
ADP
360 nm
F
1~
A ADP
ADP
(d)
(b) F
340 nm
F
360 nm
0
360 nm
_
1
3 2 Time (s)
4
5
nm ~~~~~~~340
F
F
fvv ill
0
1
3 2 Time (s)
4
5
Fig. 1. Stopped-flow fluorescence records of fura-2-loaded human platelets Platelets, prepared as previously described [3], were suspended in medium containing: 145 mM-NaCl, 5 mM-KCl, 1 mM-MgCl2, 10 mM-Hepes, pH 7.4 at 37 'C. Panels (a) and (b) show traces obtained with 1 mM-Ca2+ in the mixing chamber and (c) and (d) with 1 mM-Mn2+. (a) and (c) show the result of applying 40,uM-ADP in the mixing chamber at the point the recording was triggered (time zero). In (b) and (d), the conditions were identical except that ADP was absent. Each of the eight traces were separately obtained by signal averaging records of 10 sequential runs. All traces were obtained using the same preparation of platelets. Excitation was at 340 nm or 360 nm as indicated, emission was at 500 nm in all cases. The fluorescence axis is in arbitrary units and the scales are identical for each trace. The initial (resting) fluorescence is indicated by the horizontal mark.
1990
