Scand J Haematol(1976) 16, 75-80

Reptilase Clot Retraction Induced by Electrical Stimulation DOMENICO

BOTTECCHIA, M.D., GIANPAOLO FANTIN, B.SC., PAOLO GRUPPO, M.D., GINONASSUATO & PIETRO ZATTI,M.D.

lstituto di Fisiologia Umana dell'llniversitci di Padova (Chief, P . Zatti), Padova, Italy

Retraction of platelet rich plasma clotted by reptilase is induced by electrical stimulation. Optimal retraction is obtained by stimuli, applied for more than 4 min, with the following characteristics: intensity = 150 volts, duration = 50 msec each, frequency = lO/sec. Electrically induced reptilase clot retraction is shoswn t o be inhibited by EDTA, EGTA, methyl-xanthines, PGEi, acetylsalicylic acid, indomethacin, but not by apyrase or by phosphoenolpyruvate-pyruvate kinase and MgCIz. The results indicate that electrical stimulation induces retraction of PRP clotted by reptilase by triggering off an increased availability of Cap in the intracellular space. K e y words: platelets - clot retraction - reptilase

Accepted for publication November 29, 1975 Correspondence to: Dr. B. Bottecchia, Istituto di Fisiologia Umana, Universita degli Studi, Via Marzolo, 3, 35100 Padova, Italy

Platelet rich plasma (PRP) clotted by reptilase docs not retract unless platelet activation occurs (de Gaetano et a1 1973). Platelet contractile system activation can be obtained by adding aggregating agents (de Gaetano et al 1973), or by exposing clots to low temperature (Kubisz 1974). Recent work from our laboratory has demonstrated that reptilase clot retraction can also be induced by electrical or mechanical stimulation; the phenomenon certainly depends on platelet activation, as it is not present in platelet poor plasma (PPP) (Fantin et a1 1975). The purpose of the present experiments

was to investigate the mechanism(s) involved in the electrically induced reptilase clot retraction (EICR). This paper reports the results obtained: a) by changing the characteristics of the electrical stimuli; b) by changing the concentration of extracellular Ca2+,MgZ+,and K+; c) by adding some inhibitors of platelet functiomns. MATERIALS AND METHODS Human blood was collected from the antecubital vein of healthy donors. PRP and PPP were prepared as described previously (Bottecchia & Doni 1973).

76

D. BOTTECCHIA, G. FANTIN, P. GRUPPO, G. NASSUATO & P. ZATTI

Clot retraction was evaluated as follows: in glass tubes 0.3 ml PRP and 0.1 ml reptilase were added in succession; when platelet function inhibitors were studied, 0.3 ml PRP was preincubated for 3 rnin with either 0.1 ml buffer or test substance before the addition of 0.1 ml reptilase. The mixture was rapidly mixed with a nonsiliconized glass rod which was left inside the tube; at the end of the experiment the rod to which the clot was attached, was gently removed and the serum expressed was carefully measured by means of a Hamilton Microliter syringe. The degree of clot retraction was calculated by relating this volume of serum otm the total amount of the initial mixture. The mean of two simultaneous determinations was always used. The inhibition of clot retraction was calculated as follows: the retraction obtained in the presence of the inhibitor was confronted with the retraction obtained in the absence of any inhibitor, following the same electrical stimulation; the decrease of the response induced by the inhibitor was calculated in percent. Electrical stimulation of reptilase clots was performed according to our method recently described (Fantin et al 1975), by means of two platinum electrodes connected with a stimulator (S 8 Stimulator, Grass Instruments, Quincy, Mass., USA). Electrical stimulation always was started 10 rnin after reptilase addition, and was prolonged, in a first group of experiments, for different periods of time. The samples were then transferred at 37O C and clot retraction was measured 60 rnin after the end of the electrical stimulation. Figure 1. Retraction of reptilase clots induced by electrical stimulation applied for different periods of time. Test system: 0.3 ml PRP (or PPP) + 0.1 ml reptilase solution. Number of platelets in PRP: 3aO,OOO/pl. Electrical stimulation started 10 rnin after reptilase addition; after 60 rnin at 37O C the clot retraction was evaluated by measuring the serum expressed. Electrical stimuli: intensity = 150 volts; duration = 50 msec; frequency = lO/sec.

In a preliminary group of experiments we found that maximal clot retraction is induced by an electrical stimulation longer than 4 min (see below). Therefore, in all the following experiments electrical stimulation was applied for 6 min. Chemicals used included the following: Reptilase reagent (Pentapharm, Basel, Switzerland), ethylenediaminetetracetic acid (EDTA) dinatrium salt (Merck, Darmstadt, Germany), ethyleneglicol 2(2-aminocthyl)tetraceticacid (EGTA) (Carlo Erba, Milano, Italy), caffeine (Carlo Erba, Milano, Italy), theophylline (Boehringer, Mannheim, Germany), aminophylline (Carlo Erba, Milano, Italy), acetylsalicylic acid (ASA) (Carlo Erba, Milano, Italy), adrenaline (Merck, Darmstadt, Germany), indomethacin (Merck Sharp and Dohme Italia, Pavia, Italy), apyrase (Sigma Chemical Co., St. Louis, Mo., USA; 0.89 U/mg ADPase activity), phospho(eno1)pyruvate and pyruvate kinase (Sigma Chemical Co., St. Louis, Mo., USA). Prostaglandin-El (PGEr), 10315 - VDV - 60 B (Upjohn Company, Kalamazoo, Mich., USA) was dissolved as described by Kinlough-Rathbone et al (1970) and stored at -2OO C until use. Other chemicals were of the best quality a t disposal. RESULTS

a) EfJect of difJerent electrical stimuli

The effectiveness of electrical stimuli (intensity = 150 volts, frequency = 10/sec,

I 2

TIME

OF

I

I

4

6

STIMULATION

(rn

I

I

8

10

minuter)

RETRACTION OF REPTILASE CLOTS

duration = 50 msec) depended on the duration of their application. The retraction reached its maximal value on a stimulation longer than 4 min (Figure 1). The high voltage of the stimuli was required by the high electrical resistance (about 3,000 ohms) of our experimental system (PRP, electrodes, connections with the stimulator); therefore the current actually passing through the clot had relatively low intensity. Figure 2 shows the relationship between clot retraction and the different voltage of the electrical stimuli. It appears that clot retraction was strictly dependent on the voltage to about 100 volts, while further increase of the intensity did not produce any increase of the response. Figures 3 and 4 show the results of two typical experiments with different frequency or length of the stimuli. Optimal retraction followed stimuli with frequency 10/sec and

77

90-

z

80-

0 70-

0'

60-

a

0: 5 0 -

IIJJ 40-

e

0

10 I

I

I

5

10

15

FREQUENCY

(

pulses / s e c I

Figure 3. Clot retraction induced by electrical stimuli of different frequency. Electrical stimulation started 10 min after reptilase addition. The intensity (150 volts) and the duration (50 msec) of the stimuli were constant. Test system: 0.3 ml PRP + 0.1 ml reptilase solution. Number of platelets = 300,000/,~~1.

' 090 °1

2 30i c

20

0

'"1

1

10

I

I

25

50

75

100

125

, 10

20

I

I

I

I

I

I

30

40

50

60

70

80

150

I NT ENS1T V Iv o i t \ I

Figure 2. Clot retraction induced by electrical stimuli of different intensity. Electrical stimulation started 10 min after reptilase addition. The frequency (10/sec) and the duration (50 msec) of the stimuli were constant. Test system: 0.3 ml PRP + 0.1 ml reptilase solution. Number of platelets = 300,000/,~1.

DURATION ( r n s e c )

Figure 4. Clot retraction induced by electrical stimuli of different duration. Electrical stimulation started 10 min after reptilase addition. The intensity (150 volts) and the frequency (lO/sec) of the stimuli were constant. Test system: 0.3 ml PRP + 0.1 ml reptilase solution. Number of platelets = 3aO,OOO/pl.

78

D. BOITECCHIA, G. FANTIN, P. GRUPPO, G. NASSUATO & P. ZATTI TABLE I1 Final concentrations of cafleine, theophylline, aminophylline, P G E I , acetylsalicylic acid (ASA) and indomethacin giving 50 % inhibition of electrically induced reptilase-clot retraction

Compound

Final concentration giving 80 % inhibition

No. of experiments

EDTA

5x1e3M

7

EGTA

2 x 10-2 M

7

1.5 x lCi M

7

KCl

duration 50 msec. Consequently in all the following experiments the stimulation of reptilase clots (for 6 min) was performed according to the optimal conditions (frequency 10/sec; duration 50 msec; intensity 150 volts). b) Influence of calcium, magnesium and potassium

Compound

Final conceatration giving 50 % inhibition

No. osf experi-

ments

Caffeine

2 x 10-4 M

7

Theophylline

2 x 1 ~ 3 ~

6

Aminophylline

4x 1 P M

7

0.2-0.02 , u g h 1

5

PGEi ASA Indomethacin

2 x 1 w ~ 0.001 mg/ml

19

6

c) Effect of some inhibitors of the platelet functions Methyl-xanthines. Table I1 indicates the inhibitory effect of three methyl-xanthines on EICR. Caffeine was the most active compound of this group, followed by aminophylline and theophylline.

Calcium and magnesium were necessary for EICR induced, in a reptilase system, by electrical stimuli; this fact appeared from experiments performed with EDTA. Following preincubation for 3 min of PRP Prostaglandin-El. PGEl was very active in with EDTA, before adding reptilase, EICR inhibiting EICR: at a final concentration was inhibited. We tested EGTA as well as ranging between 0.2 and 0.02 pg/ml, PGEi EDTA. The inhibitory effect of EGTA induced 50 % inhibition (Table 11). (chelating only Ca2+and not Mg2+)was constantly less marked than that of EDTA. Acetylsalicylic acid. ASA inhibited EICR: The effect of these two drugs was dose 50 % inhibition was found at a final condependent. centration of 2 x M (Table 11). Table I shows the minimal final concentrations of EDTA and EGTA which Indomethacin. This drug also inhibited gave 80 % inhibition of EICR. EICR: a final concentration of 0.001 mg/ Potassium chloride was also able to in- ml gave 50 % inhibition (Table 11). hibit EICR. At a final concentration 2 x 10-1 M KC1 completely inhibited clot retraction; Apyrase. This enzyme is known to remove at concentrations lower than 4 x M its ADP. At final concentrations up to 0.2 mg/ effect was not evident; 80 % inhibition was ml (0.89 U/mg ADPase activity) it did not generally given by a final concentration of significantly inhibit EICR. 1.5 x 10-1 M (Table I).

RETRACTION OF REPTILASE CLOTS

Phosphoenolpyruvate (PEP) pyruvate kinase (PK) and MgB. (Final concentration: PEP 0.3 mM; PK 20 U/ml; MgClz 2 mM): this system is known to remove ADP; it was completely ineffective on EICR.

79

to maintain the resting potential and for the normal development of the action potential (Davies 1968ah). The inhibitory effect on platelet of high extracellular concentrations of K could indicate that for several platelet functions membrane depolarization is required, as it DISCUSSION occurs in muscular cells. Present experiments confirm that electrical Thus, further evidence is available in fastimulation of reptilase clots can induce clot vour of the assumption that contractile acretraction. tivity in platelets is controlled by a mechIn a previous paper (Fantin et a1 1975) anism which is in every respect comparable we assumed that the electrical stimulation to that one in muscle. of the platelets within the clot induced Acetylsalicylic acid and indomethacine, modifications of the permeability of the which are ineffective on reptilase clot replatelet membrane(s), with consequent re- traction induced by aggregating agents (de lease of intracellular calcium ions, which Gaetano et a1 1973), can inhibit the EICR. in turn trigger off the contractile activity of This fact could indicate that release reacplatelets. tion is necessary for the latter type of rePresent experiments demonstrate that traction. However, the lack of inhibition availability of Ca2+ ions is necessary for by apyrase and by phosphoenolpyruvatethe development of the retraction of rep- pyruvate kinase and Mg2+, known to detilase-clots following electrical stimulation, grade ADP, indicates that this is not the as chelating agents inhibit EICR. case. On the other hand it is known that there It is known that ASA and indomethacin is an interplay of adenosine-3’-5’-cyclic stabilize the platelet membrane and inhibit monophosphate (CAMP) level and Ca2’ ions the lysis of platelet provoked by repeated level in the regulation of platelet functions freezing and thawing (Vermylen et a1 1973). (Liischer & Bettex-Galland 1972). MethylWe venture to suggest that the stabilizing xanthines and PGEI, which are known to effect of ASA and indomethacin is the reaincrease the platelet content of CAMP (Salz- son for the refractariness of the platelet man & Weisemberger 1972) were very ac- membrane to electrical stimulation. tive in inhibiting EICR probably by reducing the intracellular availability of Ca2+ ACKNOWLEDGEMENTS ions. High concentration of K ions in the ex- The technical assistance of Mr. Maurizio Travan, tracellular fluid prevented complete EICR. Mr. Renzo Ferronato and Miss Marisa Pagin was appreciated. It is known that many platelet functions require high cellular potassium content and REFERENCES that the potassium gradient is of primary importance for platelet activation (Gross Battecchia D & Doni M G (1973) Artero-venous & Schneider 1971). On the other hand K differences in blood platelet clumping by ADP. gradient is necessary in the muscular cell Experientia 29, 211-12.

D. BOTT'ECCHIA, G. FANTIN, P. GRUPPO, G. NASSUATO & P. ZATTI

80

Davies P W (1968a) Membrane theory and resting potential. In V B Mountcastle (ed) Medical Physiology, 12th ed, pp 1082-93. The C V Mosby Co., St. Louis. Davies P W (1968b) The action potential. In V B Mountcastle fed) Medical Physiology, 12th ed, pp 1094-1120. The C V Mosby Co., St. Louis. de Gaetano G, Bottecchia D & Vermylen J (1973) Retraction of reptilase clots in the presence of agents inducing or inhibiting the platelet adhesion-aggregation reaction. Thrombos Res 2, 71-84. Fantin G, Bottecchia D, Nassuato G, Gruppo P & Zatti P (1975) Evidence that electrical and mechanical stimulation can induce reptilaseclot retraction. Thrombos Res (submitted for publication). Gross R & Schneider W (1971) Energy metabolism. I n S A Johnson (ed) The Circulating

Platelet, pp 123-88. Academic Press, New York. Kinlough-Rathbone R L, Packham M A & Mustard J F (1970) The effect of prostaglandin-Ei on platelet function in vitro and in vivo. Br J Haematol 19, 559-71. Kubisz P (1974) Cold induced retraction of reptilase clots. Scand J Haematol 13, 175-78. Liischer E F & Bettex-Galland M (1972) Thrombosthenin, the contractile protein of blood platelets. New facts and problems. Path Biol 20, Suppl, 89-101. Salzman E W & Weisemberger H (1972) Role of cyclic AMP in platelet function. Advanc Cycl Nucleotide Res 1, 231-47. Vermylen J, de Gaetano G & Verstraete M (1973) Platelets and thrombosis. In L Poller (ed) Recent Advances in Thrombosis, pp 113-50. Churchill, London.

BOOKS

Proceedings o f the first national symposium on sickle cell disease - John I. Hercules, Alan N. Schechter, William A. Eaton & Rudolph E. Jackson (eds). National Institutes of Health, Bethesda, M.D. - 1974. 413 pp. (Available fro'm the Sickle cell Disease Branch, NIH, Bethesda, M.D. 20014).

Francis S. Morrison (ed) (1975) Self-assessment of current knowledge in hematology. Part 2: Literature review. 1,022 multiple choise questions and referenced answers. Medical Examination Publishing Co. Inc. New York. 185 pp. Price: US $ 12.00. Jorgen Silwer (1973) von Willebrand's disease in Sweden. Acta pzdiatr suppl 238; pp 159. Almqvist & Wiksell Periodical Co. Stockholm, Sweden. 0. N. Lutin & I. R. Peake (eds) (1975) Haemophilia. Proc IX Congr World Fed Haemophilia, Istanbul, Aug, 1974. Full length of 43 papers dealing with different aspects of haemophilia and von Willebrand's disease (detections of carriers of haemophilia A and B, Synovectomy, Incidence, Factor VIII concentrates, Factor VIII inhibitors, Home treatment of von Willebrand's disease and haemophilia). Excerpta Medica, Amsterdam, American Elsevier Publishing Co. Inc., New York. 293 pp. Price: US $ 37.50, Dfl. 90.00. Paperback.