S H 0 R T C 0 M M U N I CAT10 N

Scand J H a e m a t o l ( l 9 7 6 ) 16, 263-265

Mechanism of Calcium-Induced Reptilase Clot Retraction PETERKUBISZ, M.D., JANA SURANOVA & STIGCRONBERG, M.D. Division of Hematology, University Hospital, Oran, Algeria, Hemostasis Laboratory, Departmental Hospital, Cadca, Czechoslovakia and Department of Infectious Diseases and Coagulation Laboratory. Allmanna sjukhuset, Malmo, Sweden

Calcium ions induce retraction of reptilase clots. This could also be induced by thrombin. Heparin inhibited the reptilase clot retraction induced either by thrombin or calcium, but did not influence the clot retraction induced by ADP. This indicated that the clot retraction induced by Ca2' was mediated by an activation of the coagulation system with the formation of thrombin. Key words: reptilase - clot retraction - Ca2'

Accepted for publication January 4, 1976 Correspondence to: Dr. S. Cronberg, Coagulation Laboratory, Malmo General Hospital, S-21401 Malmo, Sweden

When citrated platelet-rich plasma (c-PRP) is clotted by thrombin the clot retracts. This retraction does not occur if EDTA is added to the PRP. This indicates that a small amount of calcium is necessary for clot retraction by thrombin. When citrated PRP is clotted by reptilase a clot is formed which will not contract unless the platelets are activated by ADP, adrenaline, collagen, thrombin (Niewiarowski et a1 1975) or some other inducer which will cause a change in shape of the platelets. Retraction of reptilase clots can also be performed when ADP is added after the formation of a clot (de Gaetano et a1 1974). It has been shown that calcium potentiates reptilase clot retraction (Kubisz &

Suranovi 1975). Further investigations showed that Ca2+'sthemselves could act as an inducer. The aim of the present study was to elucidate the effect of calcium on the reptilase clot retraction. MATERIAL AND METHODS Platelet and plasma preparations. Human citrated platelet-rich plasma (c-PRP) and platelet-poor plasma were used. Blood was collected in citrate solution (0.11 M). The ratio of blood to anticoagulant was 9 : l . Platelet-rich plasma was prepared by centrifuging the blood at 280 g at 22O C for 10 min and immediately used. Reagents. ADP from Caibiochem, Lucerne, Switzerland, snake venom from Bothrops atrox either as reptilase (lot 791) from Pentapharm, Basle, Swit-

PETER KUBISZ, JANA SURANOVA & STIG CRONBERG

264

TABLE I Reptilase clot retraction induced by A D P , calcium or thrombin Influence of heparin Clot retraction in per cent was measured 15, 30 and 45 min after the addition of the inducer. It was induced by ADP (final concentration 0.1 mmol), calcium ions (final concentration 10 mmol) or thrombin 0.75 U per ml PRP. The inhibitory effect of heparin was tested at a concentration of 10 U/ml PRP. Mean and SD of 11 experiments.

Addition of inducer

Clot retraction induced by Calcium Thrombin

mance I ADP Iretraction of clot without with

I I I (min)

hefpah

I

heparin

(%I

2 min before reptilase

15 30 45

23f13 44f15 55f17

27f17 47+15 56f14

5 2 f 11 80+ 5 85k 4

o+o Of0 o+o

73f 7 84f 6 88+ 4

Of0 Of0 Of0

10 sec after reptilase

15 30 45

44f15 63k 9 66+13

41f14 64f10 67f10

5 3 f 13 85f 4 85f 2

Of0 Of0

69f10 85k 3 89k 4

Of0

15 30 45

44f20 63f14 66f20

42f13 64f12 67f17

55+ 5 80f 4 82f 3

Of0 Of0

46f 10 55f11 65f 6

Of0 Of0

15 30 45

3 9 1 12 59217 66f16

41 11 63+13 66fll

47 f 12 68f 5 75+ 4

Of0 Of0

38f 8 57f 3 60f 4

Of0

15 30 45

32 f 18 52+'17 58f15

35 k 14 58f10 67f13

50f 4 72f 3 80+ 4

Of0 Of0

4 2 2 14 62f 7 67f 9

Of0

10 sec after clotting wih reptilase

10 min after clotting with reptilase

30 rnin after clotting with reptilase

zerland or as lot 36 from Stago, Asnitres, France, bovine thrombin (Topostasin&) from Roche and heparin from Spofa, Prague, Czechoslovakia. All agents were dissolved in Tris-buffered saline. Measurement of reptilase clot retraction This was measured by a slight modification of the method of Kubisz & Suranov; (1974). This experimental system consisted of: 0.7 ml PRP 0.1 ml buffer or 7 U heparin in 0.1 ml buffer 0.1 ml inducer (1 mmol ADP, 0.1 mol calcium chloride or 0.75 U thrombin) 0.1 ml reptilase corresponding to 1 U thrombin. The inducer was added at various intervals from 2 min before until 30 min after the reptilase. The assay was performed in a water bath at 37O C. The volume of fluid separated from the clot after 15, 30 and 45 rnin was measured using a 1 ml tuberculin syringe. The degree of retraction was expressed in per cent to the initial volume of the sample. I

+ +

+

*

o+o o+o

Of0

o+o

Of0 Of0

o+o

Of0 Of0

Of0 Of0

RESULTS

ADP, calcium and thrombiq were all capable of inducing retraction of reptilase clots (Table I). This also occurred when they were added after the clot had formed. In reptilase clots heparin inhibited the clot retraction induced by thrombin or calcium, but not that induced by ADP (Table I). DISCUSSION

Judging from the results not only ADP but also calcium and thrombin can diffuse into a reptilase clot and cause retraction. Both the calcium-induced and the thrombininduced reptilase clot retraction was in-

Ca-INDUCED REPTILASE CLOT RETRACTION

hibited by heparin, which did not inhibit the clot retraction induced by ADP. All evidence therefore suggested that the clot retraction induced by calcium is mediated by the formation of thrombin. It is surprising, but in agreement with earlier observations, that the coagulation process is not activated in the course of clotting with snake venom (Esnouf & Tunnah 1967, O’Brien 1968). On the other hand, the present findings show that the coagulation process can be activated inside the reptilase clots by the addition of calcium ions. REFERENCES de Gaetano G, Franco R, Donati M B, Bonaccorsi A & Garattini S (1974) Mechanical re-

265

cording of reptilase-clot retraction. Effect of adenosine-5’-diphosphateand prostaglandin E L Thrombos Res 4, 189-92. Esnouf M P & Tunnah G W (1967) The isolation and properties of the thrombin-like activity from Agkistrodon rhodostoma venom. Br J Haematol 13, 581-90. Kubisz P & Suranovl J (1974) Further variant of the thrombocytopathy with abnormalities in platelet release reaction. Thrombos Res 4, 11930. Kubisz P & Suranovl J (1975)Reptilase clot retraction test. Its cofactors and relation to other platelet functions. Pathol Biol 23, 269-75. Niewiarowski S, Stewart G J, Nath N, Sha A T & Lieberman G E (1975) ADP, thrombin and Bothrops atrox enzyme in platelet dependent fibrin reaction. A m J Physiol 229, 737-45. O’Brien J R (1968)Arvin and platelets. Lancet 1, 889.

Mechanism of calcium-induced reptilase clot retraction.

S H 0 R T C 0 M M U N I CAT10 N Scand J H a e m a t o l ( l 9 7 6 ) 16, 263-265 Mechanism of Calcium-Induced Reptilase Clot Retraction PETERKUBISZ,...
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