THROMBOSIS RESEARCH Printed in the United
Vol.
States
3, PP. Pergamon
43-50, Press,
1976 Inc.
SPIN-LABELLED HUMAN PLATELETS
Frederick Sachs and Richard D. Feinman
Laboratory of Biophysics, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20014, and Department of Biochemistry, State University of New York Downstate Medical Center, Brooklyn, New York 11203.
in revised form 12.8.1975. (Received 27.5.1975; Accepted by Editor S. Niewiarowski. Received by Executive Editorial Office 24.11.1975)
ABSTRACT Human platelets were labelled with a variety of lipophilic and thiol reagent spin probes. Aggregation was not affected by the lipid probes but was blocked by the covalent protein-directed probes. ESR spectra before and after thrombin treatment indicated that lipid structure of the platelet membrane was not affected to any measurable extent by the thrombin interaction, although there was a change in the partition coefficient of the probe between solution and the membrane. From the fact that four different probes for different parts of the membrane lipid showed the same result, we conclude that thrombin interaction and resultant changes in the platelet membrane do not involve any substantial change in lipid organization.
INTRODUCTION Platelets undergo profound biochemical and morphological changes when stimulated by thrombin or other agents (review (1) ).
Those responses which
are generally believed to involve the membrane or surface of the platelet include:
binding of thrombin to the platelet (2,3), morphologic changes
including pseudopod formation (4), aggregation, as well as changes in surface change (5).
In addition, changes in lipid synthesis on thrombin stimulation
have been reported (12).
It is, thus, of interest to ask whether 47
s4
SPIN-LABELLED
PUTELETS
Vol.8,No.l
or not physical alterations in membrane structure accompany platelet responses.
We have attempted to measure the effects of thrombin stimulus on
human platelets using spin probes.
Our results indicate that lipid spin
labels are taken up by platelets but that motion of these lipids within the membrane is not affected by thrombin treatment under conditions where aggregation occurs.
METHODS Washed human platelets were prepared and suspended in buffers as described by Detwiler and Feinman (2).
Human thrombin was the generous gift of
Dr. John Fenton II and has been prepared essentially by the method of Fasco and Fenton (6). Spin labels were obtained from Synvar (Palo Alto, Calif.).
The lipid
spin labels (Figure 1) were a cholesterol derivative; Synvar No. 612: 17-~-hydroxy-4'-dimethylspiro-(5-a-androstane-3,2'-o~zoladin)-3'-yloxyl, three stearic acid analogs, Synvar No. 618:
and
2-(3-carboxypropyl)-4,4-dimethyl-
2-tridecyl-3-oxazolidinyloxyl; Synvar No. 616:
2-(14-carboxytetradecyl)-2-
ethyl-4,4-dimethyl-3-oxazolidinyloxyl; Synvar No. 614:
2-(lO-carboxydecyl)-
2-hexyl-4,4-dimethyl-3-oxazolidinyloxyl. The following covalent thiol and amine reagents were used: 100:
Synvar No.
4-maleimido-2,2,6,6_tetramethyl piperidinooxyl; Synvar No. 101:
iodoacetamido)-2,2,6,6_tetramethyl piperidinooxyl; Synvar No. 610:
4-(2-
2-
(acetoxymercurio)-4,4,5,5-tetramethyl-2-imidazolin-l-yloxyl-3-oxide, Synvar No. 103:
4-isothiocyanato-2,2,6,6_tetramethyl piperidinooxyl.
The lipid labels were introduced by gently swirling a platelet suspension (typically 10' platelets/ml) in a tube containing dried label. Labelled platelets were kept on ice until used.
(Storage of labelled plate-
lets at room temperature led to loss of both ESR signal and sensitivity to thrombin-induced aggregation.) Covalent labels (l&f) were incubated with platelets in buffer for times of up to 1 hour and then washed to remove free label. Several thrombin concentrations from 0.1 u/ml to 3 u/ml were added either by addition of 20 X of stock thrombin to
0.5 ml of platelet suspen-
sion, or by mixing the cell suspension with a thrombin solution in a rapidmix flow cell in the method.
ESR spectrometer.
Results were identical by either
L-01.8,50.1
SPIK-LMELLED
The instrument was a Varian E-3.
PLATELETS
All measurements were made at room
temperature (25 ? 2°C) unless otherwise noted. We used a simple, semi-quantitative method to determine whether or not the probe significantly altered platelet sensitivity to thrombin.
Small
aliquots of thrombin stock solution were placed in a 3 ml vial and 100x of platelet suspension (log/ml) was added. at room temperature.
The vial was swirled for one minute
Threshold was defined as the lowest thrombin concen-
tration at which visible aggregates formed within the test period.
We did
not use an aggregometer since dilution of the platelets to levels normally used in the aggregometer would result in redistribution of the probe and not permit comparison with ESR data. Lactic dehydrogenase (LDH) assays were performed on a DuPont automated analyzer. The net probe concentration in the sample was measured by comparing the area under the absorption curve (double integration of the ESR spectra) with that of a known concentration of probe. Platelet concentrations were measured using an automatic cell counter.
CH:i(CHd:pC~CHd
,,, COOH
614 O";"' CH:,CH,>C+CHd,,
COOH
616 O’;““’ CHn(CH-)
,:yCqUZHd::
COOH
618 O4” OH
612
FIG. 1 Lipid Spin Labels
46
SPIN-LABELLED
PLATELETS
Vol.8,No.l
RESULTS AND DISCUSSION
Human platelets were labelled with the lipidic probes shown in Figure 1. Labelling did not alter the sensitivity to thrombin based on the procedure described in METHODS:
labelled platelets were aggregated at the same thres-4 hold as native platelets (5 x 10 u/ml). They were intact as judged by LDH leakage.
Thus, platelets were essentially unaltered by labels dissolved in
the membrane. The ESR spectra of platelets spin labelled with stearic acid derivative 618 before and after addition of thrombin are shown in Figure 2. aggregation occurred a few seconds after addition of thrombin).
(Visible The spectra
contain two components, a freely tumbling component in an aqueous environment, contributing the sharp lines marked "a" in Figure 2, and a membrane bound fraction contributing the broader lines.
CONTROL
.,
I
I,:,.,
* I
,.,.
I,.
Vol.
States
3, PP. Pergamon
43-50, Press,
1976 Inc.
SPIN-LABELLED HUMAN PLATELETS
Frederick Sachs and Richard D. Feinman
Laboratory of Biophysics, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20014, and Department of Biochemistry, State University of New York Downstate Medical Center, Brooklyn, New York 11203.
in revised form 12.8.1975. (Received 27.5.1975; Accepted by Editor S. Niewiarowski. Received by Executive Editorial Office 24.11.1975)
ABSTRACT Human platelets were labelled with a variety of lipophilic and thiol reagent spin probes. Aggregation was not affected by the lipid probes but was blocked by the covalent protein-directed probes. ESR spectra before and after thrombin treatment indicated that lipid structure of the platelet membrane was not affected to any measurable extent by the thrombin interaction, although there was a change in the partition coefficient of the probe between solution and the membrane. From the fact that four different probes for different parts of the membrane lipid showed the same result, we conclude that thrombin interaction and resultant changes in the platelet membrane do not involve any substantial change in lipid organization.
INTRODUCTION Platelets undergo profound biochemical and morphological changes when stimulated by thrombin or other agents (review (1) ).
Those responses which
are generally believed to involve the membrane or surface of the platelet include:
binding of thrombin to the platelet (2,3), morphologic changes
including pseudopod formation (4), aggregation, as well as changes in surface change (5).
In addition, changes in lipid synthesis on thrombin stimulation
have been reported (12).
It is, thus, of interest to ask whether 47
s4
SPIN-LABELLED
PUTELETS
Vol.8,No.l
or not physical alterations in membrane structure accompany platelet responses.
We have attempted to measure the effects of thrombin stimulus on
human platelets using spin probes.
Our results indicate that lipid spin
labels are taken up by platelets but that motion of these lipids within the membrane is not affected by thrombin treatment under conditions where aggregation occurs.
METHODS Washed human platelets were prepared and suspended in buffers as described by Detwiler and Feinman (2).
Human thrombin was the generous gift of
Dr. John Fenton II and has been prepared essentially by the method of Fasco and Fenton (6). Spin labels were obtained from Synvar (Palo Alto, Calif.).
The lipid
spin labels (Figure 1) were a cholesterol derivative; Synvar No. 612: 17-~-hydroxy-4'-dimethylspiro-(5-a-androstane-3,2'-o~zoladin)-3'-yloxyl, three stearic acid analogs, Synvar No. 618:
and
2-(3-carboxypropyl)-4,4-dimethyl-
2-tridecyl-3-oxazolidinyloxyl; Synvar No. 616:
2-(14-carboxytetradecyl)-2-
ethyl-4,4-dimethyl-3-oxazolidinyloxyl; Synvar No. 614:
2-(lO-carboxydecyl)-
2-hexyl-4,4-dimethyl-3-oxazolidinyloxyl. The following covalent thiol and amine reagents were used: 100:
Synvar No.
4-maleimido-2,2,6,6_tetramethyl piperidinooxyl; Synvar No. 101:
iodoacetamido)-2,2,6,6_tetramethyl piperidinooxyl; Synvar No. 610:
4-(2-
2-
(acetoxymercurio)-4,4,5,5-tetramethyl-2-imidazolin-l-yloxyl-3-oxide, Synvar No. 103:
4-isothiocyanato-2,2,6,6_tetramethyl piperidinooxyl.
The lipid labels were introduced by gently swirling a platelet suspension (typically 10' platelets/ml) in a tube containing dried label. Labelled platelets were kept on ice until used.
(Storage of labelled plate-
lets at room temperature led to loss of both ESR signal and sensitivity to thrombin-induced aggregation.) Covalent labels (l&f) were incubated with platelets in buffer for times of up to 1 hour and then washed to remove free label. Several thrombin concentrations from 0.1 u/ml to 3 u/ml were added either by addition of 20 X of stock thrombin to
0.5 ml of platelet suspen-
sion, or by mixing the cell suspension with a thrombin solution in a rapidmix flow cell in the method.
ESR spectrometer.
Results were identical by either
L-01.8,50.1
SPIK-LMELLED
The instrument was a Varian E-3.
PLATELETS
All measurements were made at room
temperature (25 ? 2°C) unless otherwise noted. We used a simple, semi-quantitative method to determine whether or not the probe significantly altered platelet sensitivity to thrombin.
Small
aliquots of thrombin stock solution were placed in a 3 ml vial and 100x of platelet suspension (log/ml) was added. at room temperature.
The vial was swirled for one minute
Threshold was defined as the lowest thrombin concen-
tration at which visible aggregates formed within the test period.
We did
not use an aggregometer since dilution of the platelets to levels normally used in the aggregometer would result in redistribution of the probe and not permit comparison with ESR data. Lactic dehydrogenase (LDH) assays were performed on a DuPont automated analyzer. The net probe concentration in the sample was measured by comparing the area under the absorption curve (double integration of the ESR spectra) with that of a known concentration of probe. Platelet concentrations were measured using an automatic cell counter.
CH:i(CHd:pC~CHd
,,, COOH
614 O";"' CH:,CH,>C+CHd,,
COOH
616 O’;““’ CHn(CH-)
,:yCqUZHd::
COOH
618 O4” OH
612
FIG. 1 Lipid Spin Labels
46
SPIN-LABELLED
PLATELETS
Vol.8,No.l
RESULTS AND DISCUSSION
Human platelets were labelled with the lipidic probes shown in Figure 1. Labelling did not alter the sensitivity to thrombin based on the procedure described in METHODS:
labelled platelets were aggregated at the same thres-4 hold as native platelets (5 x 10 u/ml). They were intact as judged by LDH leakage.
Thus, platelets were essentially unaltered by labels dissolved in
the membrane. The ESR spectra of platelets spin labelled with stearic acid derivative 618 before and after addition of thrombin are shown in Figure 2. aggregation occurred a few seconds after addition of thrombin).
(Visible The spectra
contain two components, a freely tumbling component in an aqueous environment, contributing the sharp lines marked "a" in Figure 2, and a membrane bound fraction contributing the broader lines.
CONTROL
.,
I
I,:,.,
* I
,.,.
I,.
