- Email: [email protected]
Investigations on the effect of thrombocytin on platelets
THRU@OSIS RESEARCH 33; 225-227, 1984 0049-3848/84 $3.00 + .OO Printed in the USA. Copyright (cl 1984 Pergamon Press Ltd. All rights reserved.
BRIEF
COMMUNICATION
INVESTIGATIONS ON THE EFFECT OF THROMBOCYTIN ON PLATELETS P. Kubisz, A. Arabi, F. Seghier and S. Cronberg Division of Hematology, University Hospital, Oran, Algeria, Division of Hematology, General Hospital, Cadca, Czechoslovakia, Department of Infectious Diseases, General Hospital, Malmo, Sweden. (Received 8.7.1983; Accepted in revised form 19.8.1983 by Editor H.-P. KlScking) (Received in final form by Executive Editorial Office 8.11.1983) INTRODUCTION Thrombocytin is a serine protease from Bothrops atrox venom. It is similar to thrombin in many of its activities. It aggregates platelets and causes release of their granular content, but its fibrinogen clotting activity is less than 0.001 times that of thrombin (I, 2). The mechanism by which thrombocytin causes aggregation and release is incompletely known and was therefore studied in more detail in this work. MATERIAL AND METHODS Blood samples were obtained from healthy volunteers by allowing 9 parts of venous blood to flow directly into test tubes containing 1 part of 0.11 M trisodium citrate. Platelet-rich plasma (PRP) was obtained by centrifuging the blood at 280 g for 20 min at 16 C. The platelet count was adjusted to 300 000 per ~1 by addition of platelet-poor plasma (PPP) from the same vol#nteer. PPP was prepared by centrifuging PRP at 12 000 g for 20 min at 12 C. Heparin (Spofa, Prague, CSSR), EDTA (Calbiochem), PGE (J.E.Pike, Upjohn, Kalamazoo Mich. USA), cytochalasin B (J.E.Pike LIdjohn, Kalamazoo, Mich. USA), amitriptyline (Spofa, Prague, CSSR), adenosine diphosphate (ADP, Sigma), pyruvate kinase (PK) and phosphoenolpyruvate (PEP) (Sigma), apyrase (Sigma) and serotonin-2-14C, (CFA 170, Batch 91, Radiochemical Centre Amersham, UK) were used. Thrombocytin was obtained from dr K. Stocker, Pentapharm, Basle, Switzerland. Thrombocytin and inhibitors of aggregation were diluted to the desired concentrations by addition of Michaeli's buffer at pH 7.35. Platelet aggregation was measured by platelet aggregometer (Evans Electroselenium) connected to a pen recorder to permit automatic registration of transmitted light. Sensitivity was adjusted so that PPP was set close to 100 % transmission and PRP to 0 %. Reagents were added to the cuvette of the aggregometer as follows: 0.9 ml PRP, 0.1 ml buffer of inhibitor, 0.1 ml buffer of thrombocytin. Platelet aggregation was performed KEY
WORDS:
Platelets,
thrombocytin
225
PLATELETS AND THROMBOCYTIN
226
Vo1.33, No.2
at .37'Cwith automatic stirring. Platelet aggregation was evaluated as maximal slope, deepest deflection within 4 minutes and presence of second wave. The release of platelet factor 4 (PF4) was determined as earlier described in detail (3). The release of serotonin was measured after prglabelling citrate PRP with 0.5 $1 (14C) 5HT (58 mCi/mmol) for 20 min at 37 C. Labelled PRP was incubated 3 min at 37'C with buffer or appropriate inhibitor. It was then stirred in an aggregometer tube with the inducer (TCN) or buffer for 3 min. The reaction was stopped by adding ice-cold 0.1 M EDTA I:5 (v/v) and immediately centrifuging for 60 s at 3 500 g. The radioactivity of 0.1 ml of the supernatant was measured by liquid scintillation counting. The results are expressed as per cent release of the total (14C) 5HT uptake in the platelets. RESULTS Thrombocytin induced aggregation of platelets in titrated plateletrich plasma (Table I). TABLE I Platelet Aggregation and Release of PF4 and Serotonin Induced by Thrombocytin in Platelet-rich Plasma Thrombocytin mg/l : : 1;
Platelet aggregation Deepest deflection within 4 minutes % 0 :; 5"; 63
Release of PF4 % 4 6 1; 49 57
Release of serotonin % : 8 ;: 57
Concentrations up to 4 mg/l induced only the first wave of aggregation and did not release PF4 or serotonin. For concentrations of 5 mg/l or more the aggregation became irreversible. At the same time PF4 and serotonin were released. Aggregation by thrombocytin was completely blocked by EDTA and PGE, (Table II). It was also inhibited by heparin, amitriptyline and cytochalasin B. Inhibition of platelet aggregation also resulted in inhibition of the release of PF4 and serotonin. Addition of low concentrations of ADP acted synergistically with thrombocytin in causing aggregation and release. Apyrase and PK/PEP degrade ADP and inhibited both aggregation and release induced by thrombocytin.
PLATELETS AND THRDMBOCYTIN
Vo1.33, No.2
227
TABLE II Effect of Various Inhibitors on Platelet Aggregation and Release Induced by Thrombocytin in Platelet-rich Plasma Inhibitor in final concentration
buffer buffer heparin 5 IU/ml
Inducer in final concentration
Platelet aggregation Deepest deflection %
buffer thrombocytin 5 mg/l I, 5 mg/l
EDTA PGE 1 10mg/l PM am1.1riptyline 500 PM cytochalasin B 50 PM
II 0 ' '
5 mg/l 5 mg/l 5 mg/l
Release of PF4
Release of serotonin
%
0 58 4 0" 6 20
DISCUSSION The investigations showed that thrombocytin activated the platelets and caused aggregation. If this was sufficiently intense a secondary release of the granular contents occurred. The inhibitory action of heparin was probably due to the biological relationship of thrombocytin with thrombin. The inhibitory effect of EDTA, PGE amitriptyline and cytochalasin B on platelet aggregation induced by thrdmbocytin was similar to that when platelet assresation is induced bv ADP or adrenaline. 'The investiga:ions thus suggest that the release reaction induced in platelets by thrombocytin was a secondary phenomenon to the aggregation of platelets. REFERENCES 1.
KIRBY, E.P., NIEWIAROWSKI, S., STOCKER, K., KETTNER, C., SHAW, E. and BRUDZYNSKI, T.M. Thrombocytin, a serine protease from Bothrops atrox venom. I. Purification and characterization of the enzyme. Biochemistry2,3564-3569, 1979.
2.
NIEWIAROWSKI, S., KIRBY, E.P., BRUDZYNSKI, T.M. and STOCKER, K. Thrombocytin, a serine protease from Bothrox atrox venom. 2. Interaction with platelets and plasma-clotting factors. Biochemistq .I& 3570-3576, 1979.
3.
KLENER, P., KUBISZ, P. and SURANOVA, J. Influence of cytotoxic drugs on platelet functions and coagulation in vitro. IV. Melphalan. l'hromb. Haemostasis x,53-61, 1977.
BRIEF
COMMUNICATION
INVESTIGATIONS ON THE EFFECT OF THROMBOCYTIN ON PLATELETS P. Kubisz, A. Arabi, F. Seghier and S. Cronberg Division of Hematology, University Hospital, Oran, Algeria, Division of Hematology, General Hospital, Cadca, Czechoslovakia, Department of Infectious Diseases, General Hospital, Malmo, Sweden. (Received 8.7.1983; Accepted in revised form 19.8.1983 by Editor H.-P. KlScking) (Received in final form by Executive Editorial Office 8.11.1983) INTRODUCTION Thrombocytin is a serine protease from Bothrops atrox venom. It is similar to thrombin in many of its activities. It aggregates platelets and causes release of their granular content, but its fibrinogen clotting activity is less than 0.001 times that of thrombin (I, 2). The mechanism by which thrombocytin causes aggregation and release is incompletely known and was therefore studied in more detail in this work. MATERIAL AND METHODS Blood samples were obtained from healthy volunteers by allowing 9 parts of venous blood to flow directly into test tubes containing 1 part of 0.11 M trisodium citrate. Platelet-rich plasma (PRP) was obtained by centrifuging the blood at 280 g for 20 min at 16 C. The platelet count was adjusted to 300 000 per ~1 by addition of platelet-poor plasma (PPP) from the same vol#nteer. PPP was prepared by centrifuging PRP at 12 000 g for 20 min at 12 C. Heparin (Spofa, Prague, CSSR), EDTA (Calbiochem), PGE (J.E.Pike, Upjohn, Kalamazoo Mich. USA), cytochalasin B (J.E.Pike LIdjohn, Kalamazoo, Mich. USA), amitriptyline (Spofa, Prague, CSSR), adenosine diphosphate (ADP, Sigma), pyruvate kinase (PK) and phosphoenolpyruvate (PEP) (Sigma), apyrase (Sigma) and serotonin-2-14C, (CFA 170, Batch 91, Radiochemical Centre Amersham, UK) were used. Thrombocytin was obtained from dr K. Stocker, Pentapharm, Basle, Switzerland. Thrombocytin and inhibitors of aggregation were diluted to the desired concentrations by addition of Michaeli's buffer at pH 7.35. Platelet aggregation was measured by platelet aggregometer (Evans Electroselenium) connected to a pen recorder to permit automatic registration of transmitted light. Sensitivity was adjusted so that PPP was set close to 100 % transmission and PRP to 0 %. Reagents were added to the cuvette of the aggregometer as follows: 0.9 ml PRP, 0.1 ml buffer of inhibitor, 0.1 ml buffer of thrombocytin. Platelet aggregation was performed KEY
WORDS:
Platelets,
thrombocytin
225
PLATELETS AND THROMBOCYTIN
226
Vo1.33, No.2
at .37'Cwith automatic stirring. Platelet aggregation was evaluated as maximal slope, deepest deflection within 4 minutes and presence of second wave. The release of platelet factor 4 (PF4) was determined as earlier described in detail (3). The release of serotonin was measured after prglabelling citrate PRP with 0.5 $1 (14C) 5HT (58 mCi/mmol) for 20 min at 37 C. Labelled PRP was incubated 3 min at 37'C with buffer or appropriate inhibitor. It was then stirred in an aggregometer tube with the inducer (TCN) or buffer for 3 min. The reaction was stopped by adding ice-cold 0.1 M EDTA I:5 (v/v) and immediately centrifuging for 60 s at 3 500 g. The radioactivity of 0.1 ml of the supernatant was measured by liquid scintillation counting. The results are expressed as per cent release of the total (14C) 5HT uptake in the platelets. RESULTS Thrombocytin induced aggregation of platelets in titrated plateletrich plasma (Table I). TABLE I Platelet Aggregation and Release of PF4 and Serotonin Induced by Thrombocytin in Platelet-rich Plasma Thrombocytin mg/l : : 1;
Platelet aggregation Deepest deflection within 4 minutes % 0 :; 5"; 63
Release of PF4 % 4 6 1; 49 57
Release of serotonin % : 8 ;: 57
Concentrations up to 4 mg/l induced only the first wave of aggregation and did not release PF4 or serotonin. For concentrations of 5 mg/l or more the aggregation became irreversible. At the same time PF4 and serotonin were released. Aggregation by thrombocytin was completely blocked by EDTA and PGE, (Table II). It was also inhibited by heparin, amitriptyline and cytochalasin B. Inhibition of platelet aggregation also resulted in inhibition of the release of PF4 and serotonin. Addition of low concentrations of ADP acted synergistically with thrombocytin in causing aggregation and release. Apyrase and PK/PEP degrade ADP and inhibited both aggregation and release induced by thrombocytin.
PLATELETS AND THRDMBOCYTIN
Vo1.33, No.2
227
TABLE II Effect of Various Inhibitors on Platelet Aggregation and Release Induced by Thrombocytin in Platelet-rich Plasma Inhibitor in final concentration
buffer buffer heparin 5 IU/ml
Inducer in final concentration
Platelet aggregation Deepest deflection %
buffer thrombocytin 5 mg/l I, 5 mg/l
EDTA PGE 1 10mg/l PM am1.1riptyline 500 PM cytochalasin B 50 PM
II 0 ' '
5 mg/l 5 mg/l 5 mg/l
Release of PF4
Release of serotonin
%
0 58 4 0" 6 20
DISCUSSION The investigations showed that thrombocytin activated the platelets and caused aggregation. If this was sufficiently intense a secondary release of the granular contents occurred. The inhibitory action of heparin was probably due to the biological relationship of thrombocytin with thrombin. The inhibitory effect of EDTA, PGE amitriptyline and cytochalasin B on platelet aggregation induced by thrdmbocytin was similar to that when platelet assresation is induced bv ADP or adrenaline. 'The investiga:ions thus suggest that the release reaction induced in platelets by thrombocytin was a secondary phenomenon to the aggregation of platelets. REFERENCES 1.
KIRBY, E.P., NIEWIAROWSKI, S., STOCKER, K., KETTNER, C., SHAW, E. and BRUDZYNSKI, T.M. Thrombocytin, a serine protease from Bothrops atrox venom. I. Purification and characterization of the enzyme. Biochemistry2,3564-3569, 1979.
2.
NIEWIAROWSKI, S., KIRBY, E.P., BRUDZYNSKI, T.M. and STOCKER, K. Thrombocytin, a serine protease from Bothrox atrox venom. 2. Interaction with platelets and plasma-clotting factors. Biochemistq .I& 3570-3576, 1979.
3.
KLENER, P., KUBISZ, P. and SURANOVA, J. Influence of cytotoxic drugs on platelet functions and coagulation in vitro. IV. Melphalan. l'hromb. Haemostasis x,53-61, 1977.