THROMBOSIS RESEARCH .Z Pqamon Press Ltd. 19%
Vol.
11. No. 2. PP. 183-192.
oa9-3848,-78~0801-0193 somxo
Printed in Great Britain.
STIMULATION OF PLATELET SURFACE SIALYLTRANSFERASE ACTI’JITY BY PLATELET AGGREGATING AGENTS
Kenneth K. Wu and Cecilia S. L. Ku Coagulation and Thrombosis Unit of Hematology SeCtiOn Department of Medicine, Rush Medical College and Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois
in revised form 31.5.1978. (Received 31.3.'978; Accepted by Editor K.Y. Brinkhous) ABSTRACT Effect of platelet aggregating agents and aspirin on the platelet surface sialyltransferase activity was investigated on intact twicewashed human platelet preparations. The optimum conditions for assay of the enzyme activity on intact human platelet preparations were determined as follows: a) pH 7.0-7.6, b) ternerature 24"C-37"C, c) time of incubation 60 min, d) Mn++ 8 x lo-g - 2 x lo-3M, e) fetuin concentration 0.5 mg/ml and f) CMP-14C-sialic acid concentration 12 uM. Under these optimum conditions, collagen at 100 vg/ml and 200 ug/ml induced a 3-fold increase of the surface enzyme activity. Epinephrine at 1 uM and 10 uM increased the enzyme activity to 149% and 152% respectively. There was some variation in the extent of stimulation from one platelet suspension to another. Adenosine diphosphate alone did not stimulate the enzyme activity. When fibrinogen was added, the enzyme activity was stimulated to 133% above the basal value, By contrast, aspirin at 10 ug/ml depressed the enzyme activity to 61% of the basal activity. Higher concentrations of aspirin did not depress the enzyme activity further. Similar inhibitory effect was noted when aspirin at a dosage of 650 mg was given to 2 normal subjects. The data provided suggestive evidence for a close relationship between platelet surface sialyltransferase activity and platelet aggregation.
INTRODUCTION
It has been postulated that glycosyltransferases are important not only for the synthesis of glycoproteins but also for cell to cell adhesion. A generalized role for membrane glycosyltransferases and their substrates in the intercellular adhesion was initially proposed by Roseman (1). This concept has been given further credence by identification of these enzymes on the outer membranes of a number of cells including platelets (2-4). With respect to platelets, Bosmann recently showed that sialyltransferase was present on the platelet membrane and that the enzyme activity could be inhibited by aspirin (5). He proposed a model to explain his conceptual view 183
184
PLATELET
SI_4L>-LTRXXSFER-SE
V01.13,?30.2
as to how sialyltransferase night mediate cell adhesion and disadhesion (5). In this model, sialyltransferase on one cell interacts with a glycoprotein acceptor on another cell to form a complex. Although the model appears attractive in explaining the primary event which occurs during platelet adhesion and platelet aggregation , more experimental data are needed to substantiate the validity of this hypothesis. To examine this hypothesis we have carried out experiments to determine whether the platelet surface sialyltransferase activity can be stimulated by platelet activating agents. Results show a dramatic enhancement of platelet surface sialyltransferase activity by collagen and other platelet activating agents. MATERIALS AND METHODS Preparation of suspensions of washed human platelets Blood was drawn from an antecubital vein of healthy volunteers through a gauge 19 needle into acid-citrate-dextrose anticoagulant, 1 volume of anticoagulant to 5 volumes of blood (6). Blood was centrifuged at 220 x g for 10 minutes, platelet rich plasma was removed and centrifuged at 1000 g for 15 minutes. The platelet poor plasma was discarded and the platelets were resuspended and washed twice according to a procedure described by Rossi (7). The concentration of platelets in the final suspension was adjusted to 109 cells/ml. Platelet concentrations were determined by phasecontrast microscopy and by an electronic device (Coulter ZBI, Coulter Electronic, Hialeah, Florida). The washed platelets were found to be intact by a) normal morphology under electron microscopy kindly performed by Dr. Ts'ao Northwestern University, b) normal volume distribution measured by a Channalizer 1000 (Coulter Electronics, Hialeah, Florida); the volume distribution of the washed platelets was identical to that of platelet rich plasma and c) minimal loss of lactic dehydrogenase. Assay of sialyltransferase activity The assay was based on the transfer of 14C-sialic acid from a substrate cytidine monophosphate (CMP)-14C-sialic acid (New England Nuclear, Boston, Mass.) to an exogenous acceptor, desialyzed fetuin which was prepared by a method described by Grimes (8). The assay mixture consisted of 0.1 ml platelet suspension as the source of enzyme, 0.1 ml desialyzed fetuin as acceptor, 0.01 ml CMP-14C-sialic acid, 0.02 ml MnC12 and buffer to a final volume of 1 ml. The optimum conditions with respect to pH, temperature, time of inCUbation and the optimum concentrations of substrate, acceptor and cells were determined experimentally. The reaction was terminated by adding 1% cold phosphotungstic acid (Fisher Scientific Co., Fairlawn, N.J.) to the mixture which was kept at 4'C for 30 minutes. The precipitate was then separated by centrifugation, washed with 5% trichloroacetic acid 3 times and extracted by an ether-ethanol solution. The specific radioactivity was determined with a B-liquid scintillation counter. For each assay enzyme activity due to endogenous acceptors on the platelet surface was determined and subtracted from the total enzyme activity to obtain the exogenous enzyme activity. Effect of platelet aggregating and inhibiting agents on the surface enzyme activity Acid soluble collagen from calf skin prepared by the method of Gallup and Seifter (9) was obtained from Sigma Chemical Co. (St. Louis, MO.). The preparation was kept at room temperature for 17 hours prior to the use for investigation. The collagen preparation was found to be capable of inducing platelet release and platelet aggregation in our laboratory. Epinephrine
was obtained from Parke, Davis and Co., Detroit, Mi. and adenosine diphosphate (ADP) from Sigma Chemical, St. Louis, MO.. To study the effect of these agents on platelet surface sialyltransferase activity, the platelet aggregating agent was added to washed normal human platelet suspensions which were incubated at 37°C for 10 min. In the controls, the aggregating agent was replaced with the same quantity of buffer solution. In order to avoid variation due to different batches of collagen and due to different donors, same batches of collagen and platelet suspensions from same donors
Vol.
11. No. 2. PP. 183-192.
oa9-3848,-78~0801-0193 somxo
Printed in Great Britain.
STIMULATION OF PLATELET SURFACE SIALYLTRANSFERASE ACTI’JITY BY PLATELET AGGREGATING AGENTS
Kenneth K. Wu and Cecilia S. L. Ku Coagulation and Thrombosis Unit of Hematology SeCtiOn Department of Medicine, Rush Medical College and Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois
in revised form 31.5.1978. (Received 31.3.'978; Accepted by Editor K.Y. Brinkhous) ABSTRACT Effect of platelet aggregating agents and aspirin on the platelet surface sialyltransferase activity was investigated on intact twicewashed human platelet preparations. The optimum conditions for assay of the enzyme activity on intact human platelet preparations were determined as follows: a) pH 7.0-7.6, b) ternerature 24"C-37"C, c) time of incubation 60 min, d) Mn++ 8 x lo-g - 2 x lo-3M, e) fetuin concentration 0.5 mg/ml and f) CMP-14C-sialic acid concentration 12 uM. Under these optimum conditions, collagen at 100 vg/ml and 200 ug/ml induced a 3-fold increase of the surface enzyme activity. Epinephrine at 1 uM and 10 uM increased the enzyme activity to 149% and 152% respectively. There was some variation in the extent of stimulation from one platelet suspension to another. Adenosine diphosphate alone did not stimulate the enzyme activity. When fibrinogen was added, the enzyme activity was stimulated to 133% above the basal value, By contrast, aspirin at 10 ug/ml depressed the enzyme activity to 61% of the basal activity. Higher concentrations of aspirin did not depress the enzyme activity further. Similar inhibitory effect was noted when aspirin at a dosage of 650 mg was given to 2 normal subjects. The data provided suggestive evidence for a close relationship between platelet surface sialyltransferase activity and platelet aggregation.
INTRODUCTION
It has been postulated that glycosyltransferases are important not only for the synthesis of glycoproteins but also for cell to cell adhesion. A generalized role for membrane glycosyltransferases and their substrates in the intercellular adhesion was initially proposed by Roseman (1). This concept has been given further credence by identification of these enzymes on the outer membranes of a number of cells including platelets (2-4). With respect to platelets, Bosmann recently showed that sialyltransferase was present on the platelet membrane and that the enzyme activity could be inhibited by aspirin (5). He proposed a model to explain his conceptual view 183
184
PLATELET
SI_4L>-LTRXXSFER-SE
V01.13,?30.2
as to how sialyltransferase night mediate cell adhesion and disadhesion (5). In this model, sialyltransferase on one cell interacts with a glycoprotein acceptor on another cell to form a complex. Although the model appears attractive in explaining the primary event which occurs during platelet adhesion and platelet aggregation , more experimental data are needed to substantiate the validity of this hypothesis. To examine this hypothesis we have carried out experiments to determine whether the platelet surface sialyltransferase activity can be stimulated by platelet activating agents. Results show a dramatic enhancement of platelet surface sialyltransferase activity by collagen and other platelet activating agents. MATERIALS AND METHODS Preparation of suspensions of washed human platelets Blood was drawn from an antecubital vein of healthy volunteers through a gauge 19 needle into acid-citrate-dextrose anticoagulant, 1 volume of anticoagulant to 5 volumes of blood (6). Blood was centrifuged at 220 x g for 10 minutes, platelet rich plasma was removed and centrifuged at 1000 g for 15 minutes. The platelet poor plasma was discarded and the platelets were resuspended and washed twice according to a procedure described by Rossi (7). The concentration of platelets in the final suspension was adjusted to 109 cells/ml. Platelet concentrations were determined by phasecontrast microscopy and by an electronic device (Coulter ZBI, Coulter Electronic, Hialeah, Florida). The washed platelets were found to be intact by a) normal morphology under electron microscopy kindly performed by Dr. Ts'ao Northwestern University, b) normal volume distribution measured by a Channalizer 1000 (Coulter Electronics, Hialeah, Florida); the volume distribution of the washed platelets was identical to that of platelet rich plasma and c) minimal loss of lactic dehydrogenase. Assay of sialyltransferase activity The assay was based on the transfer of 14C-sialic acid from a substrate cytidine monophosphate (CMP)-14C-sialic acid (New England Nuclear, Boston, Mass.) to an exogenous acceptor, desialyzed fetuin which was prepared by a method described by Grimes (8). The assay mixture consisted of 0.1 ml platelet suspension as the source of enzyme, 0.1 ml desialyzed fetuin as acceptor, 0.01 ml CMP-14C-sialic acid, 0.02 ml MnC12 and buffer to a final volume of 1 ml. The optimum conditions with respect to pH, temperature, time of inCUbation and the optimum concentrations of substrate, acceptor and cells were determined experimentally. The reaction was terminated by adding 1% cold phosphotungstic acid (Fisher Scientific Co., Fairlawn, N.J.) to the mixture which was kept at 4'C for 30 minutes. The precipitate was then separated by centrifugation, washed with 5% trichloroacetic acid 3 times and extracted by an ether-ethanol solution. The specific radioactivity was determined with a B-liquid scintillation counter. For each assay enzyme activity due to endogenous acceptors on the platelet surface was determined and subtracted from the total enzyme activity to obtain the exogenous enzyme activity. Effect of platelet aggregating and inhibiting agents on the surface enzyme activity Acid soluble collagen from calf skin prepared by the method of Gallup and Seifter (9) was obtained from Sigma Chemical Co. (St. Louis, MO.). The preparation was kept at room temperature for 17 hours prior to the use for investigation. The collagen preparation was found to be capable of inducing platelet release and platelet aggregation in our laboratory. Epinephrine
was obtained from Parke, Davis and Co., Detroit, Mi. and adenosine diphosphate (ADP) from Sigma Chemical, St. Louis, MO.. To study the effect of these agents on platelet surface sialyltransferase activity, the platelet aggregating agent was added to washed normal human platelet suspensions which were incubated at 37°C for 10 min. In the controls, the aggregating agent was replaced with the same quantity of buffer solution. In order to avoid variation due to different batches of collagen and due to different donors, same batches of collagen and platelet suspensions from same donors
