THROXBOSI3 RESEXCH Printed in the UniT;ed states
BRIEF
Vol.
8, pp. 523-528, 1976 Pergamon Press, Inc.
COMMUNICATION
EVIDENCE THAT ELECTRICAL AND MECHANICAL STIMULATION CAN INDUCE REPTILASE-CLOT RETRACTION
G.P. Fantin, D. Bottecchia, G. Nassuato, P. Gruppo and P. Zatti Istituto di Fisiologia Umana - Universita di Padova 35100 - Padova - Italy (Received 26.10.1975; in revised form 23.2.1976. Accepted by Editor H.H. Hartert)
Due to the lack of platelet activation, human titrated platelet-rich plasma (PRP) clotted by reptilase, does not retract (1). In the presence of aggregating agents a strong reptilaseclot retraction occurs (1); recently Kubisz has shown that low temperature can also induce reptilase-clot retraction (2). The existence of analogies between clot retraction
and mu-
scle contraction is supported by many arguments (3,4,5)
even
if the two systems are based on different types of membrane activation: the muscular contraction is generally triggered off by electrical changes in the membrane,while clot retraction is generally due to chemical induction; still unexplored
are the
electrical changes of the platelet membrane. Present experiments demonstrate that it is possible to indu ce reptilase-clot retraction by means of electrical or mechani cal stimulation. Electrical stimulation of the clot was performed by means of two platinum electrodes (diameter 0.5 mm) connected to a stl mulator (S 8 Stimulator,Grass Instruments,Quincy,Mass. U.S.A.). One of the electrodes was inserted through the bottom of a glass 523
REFTILASE CLOT-RETRACTIOK
j24
SO-
60-
I 2
I
I 6
4
TIME
OF
STIMULATION
I
I 8 (in
10
minutes)
FIG. 1 Retraction of reptilase-clots induced by electrical stimulation applied for different times. Test system: 0.3 ml PRP (or PPP) + 0.1 ml reptilase solution. Number of platelets in PRP=300,000/ul. Electrical stimulation started 10 min after reptilase addition; after 60 min at 37OC the clot retraction was evaluated by measu ring the serum expressed (1). Electrical stimuli: 150 volts, 50 msec, lO/sec.
0I
I
10
I
I
20
TIME
d0
(in
40
I
50
6’0
minutes)
FIG. 2 Dynographic recording of the mechanical strength produced by PRP clotted by reptilase. The recording began 47 min after reptilase addition to PRP. Number of platelets =340,000/@. The bath contained Tyrode's modified solution (5) without Ca2+ and Mg2+.
REPTIW3E
vo1.8,50.4
523
CLOT-RETRACTION
tube (internal diameter 8nun, length 78mm) jutting 2mm out inside the tube: the second electrode (length 2mm) was immersed
at
the top of PRP before adding reptilase. (Penta-
Human PRP, platelet-poor plasma (PPP) and reptilase
pharm, Basel) were prepared as described previously (1). IO min after reptilase addition electrical stimuli (frequency lO/sec, duration 50 msec, intensity 150 volts)
were applied
to the clot for different times (I-IO min). After the end of the electrical stimulation the tube was immediately transferred at 37Oc and clot retraction was evaluated 60 min later, by
measu-
ring serum expressed (1). A marked clot retraction was always observed in the clots sti mulated for more than 4 min (fig. 1). PPP clots do not after similar stimulation, thus confirming that the
retract
electrical
stimulation activates platelets. As the retraction,following tha electrical stimulation, is not immediate but it retains its usu al characteristics of a slowly evolving process, we may conclude that the stimulation is only a trigger for the retraction. In a previous work (5) we have shown the possibility to measure the strength of clot retraction by dynographic method . By using
a
similar method de Gaetano et al. (6) found that
clotted by reptilase does not develop any mechanical
PRP
strength,
but all our attempts to confirme this result were unsuccessful, as after a latent period (about 30 min) all the reptilase-clots were always retracting. A typical dynographic tracing
obtained
with PRP clotted by reptilase is reported in figure 2. Consequently we supposed that even a slight mechanical stimu lation of the clot, as that unavoidable by handling (while trans ferring the clot from the tube to the pharmacological bath)and/or as that due to the stretch of the clot in the pharmacological bath (due to its own weight), may be a stimulus sufficient for inducing clot retraction. This hypothesis was supported by the fact that a sample of the same PRP clotted in a test tube and
526
REPTIWSE
CLOT-RETRACTION
observed in avoidance of any mechanical stimulation, does not show any retraction after 2 hours (18 experiments, with 3 diffe rent batches of reptilase, showed the same result). To confirme further this hypothesis, we did another series of experiments, in which reptilase-PRP-clots were stimulated mecha nically, using an experimental system like that described in Pi gure 3. The result was constantly a marked clot retraction (figure 4). In contrast, PPP-clots, which underwent the same mecha nical stimulation, do not retract.
c
4
-7
p”“cy
silk
29r
plastic ring
B
FIG. 3 Mechanical stimulation of the clot. A: control sample; B: a pl$ stic ring was inserted in PRP before adding reptilase. 30 min after clotting a weight of 2 grams was attached to the hook,and removed 10 min later. In A and B 0.3 ml of reptilase solution were added to 3 ml of PRP, and the tubes were incubated at 37Y. Present results seem interesting because until the present tL me it has not been demonstrated that the platelet contractile sy stem can be activated by electrical or mechanical stimulation, i.e. two typical triggers of the contraction in the muscular cells. Human platelets contain both a contractile protein regulato-
REFERENCES
1.
de GAETANO G., BOTTECCHIA D. and VERMYLEN J. Retraction of Reptilase Clots in the Presence or Absence of Agents Inducing or Inhibiting the Platelet Adhesion-Aggregation ReacThrombos. Res.: 2, 71, 1973. tion.
2.
KUBISZ P. Cold Induced Retraction of Reptilase Clots. Stand. J. Haemat.: 13, 175, 1974.
3.
L:SCHER E-F., BETTEX-GALLAND M. Thrombosthenin, the Contrac tile Protein of Blood Platelets. New Facts and Problems. Pathologie-Biologie: 20, supplement, 89, 1972.
4.
L;;SCHERE.F., PROBST E. and BETTEX-GALLAND M. Thrombosthenin: Structure and Function. Annals New York Acad. of Sci.: 202, 122, 1972.
5.
BOTTECCHIA D. and FANTIN G. Platelet and Clot Retraction. Effect of Divalent Cations and Several Drugs. Thromb. Diath. Haemorrh.: 30, 576, 1973.
6.
de GAETANO G., FRANC0 R., DONATI M.B., BONACCORSI A. and GARATTINI S. Mechanical Recording of Reptilase-clot Retraction: Effect of Adenosine-5'-Diphosphate and Prostaglandin E1 Thrombos. Res.: 4, 189, 1973.
7.
COHEN I. and COHEN C.: Tropomyosin-like protein from Human Platelets. J. Mol. Biol.: 68, 383, 1972.
8.
THORENS S., SCHAUB M.C. and L&HER E.F. A Calcium-Sensiti zing System from Human Platelets and its Activity on Muscle and Platelet Actomyosin. Experientia: 29, 349, 1973.
9.
GRETTE K. Relaxing Factor in Extracts of Blood Platelets and its Function in the Cells. Nature: 198, 488, 1963.
10.
STATLAND B.E., HEGAN B.M. and WHITE J.G. Uptake of Calcium by Platelet Relaxing Factor. Nature: 233, 527, 1969.
11.
ZIERLER K.L.: Mechanism of Muscle Contraction and its Energs tics. in "Medical Physiology" edited by Mountcastle V.B. 12th edition, Mosby Company 1968.
A
B
Fig. 4 A: control; B: clot stimulated mechanically as described in figs_ re 3. The picture was taken 44 min after the end of the mechanical stimulation. Number of platelets in PRP: 300,000 pl. ry system (7,3,4,8) and a membrane associated relaxing factor resembling the muscle sarcotubular system which, upon stimulation, discharge their contents into the cytoplasm (g-10). It is likely that the electrical stimulation of the platelets within the clot, induces some modifications of the permeability of the platelet membrane(s) with following availability of intracellular calcium ions which in turn control the contractile activity of platelets (3)* It is well known that, when a smooth muscle is stretched, a membrane depolarization occurs, with subsequent release of intra cellular calcium ions and muscular contraction (11). Since an evident clot retraction is induced by stretching reptilase-clots (which do not retract unless platelet activation occurs) the pre viously formulated hypothesis about analogies between clot and smooth muscle (5) is further supported by present experiments.
ACKNOWLEDGEMENTS: Reptilase was a gift of Penthapharm (Basel). The technical assistance of M??.Maurizio Travan and of Mr. Renzo Ferronato was appreciated.
BRIEF
Vol.
8, pp. 523-528, 1976 Pergamon Press, Inc.
COMMUNICATION
EVIDENCE THAT ELECTRICAL AND MECHANICAL STIMULATION CAN INDUCE REPTILASE-CLOT RETRACTION
G.P. Fantin, D. Bottecchia, G. Nassuato, P. Gruppo and P. Zatti Istituto di Fisiologia Umana - Universita di Padova 35100 - Padova - Italy (Received 26.10.1975; in revised form 23.2.1976. Accepted by Editor H.H. Hartert)
Due to the lack of platelet activation, human titrated platelet-rich plasma (PRP) clotted by reptilase, does not retract (1). In the presence of aggregating agents a strong reptilaseclot retraction occurs (1); recently Kubisz has shown that low temperature can also induce reptilase-clot retraction (2). The existence of analogies between clot retraction
and mu-
scle contraction is supported by many arguments (3,4,5)
even
if the two systems are based on different types of membrane activation: the muscular contraction is generally triggered off by electrical changes in the membrane,while clot retraction is generally due to chemical induction; still unexplored
are the
electrical changes of the platelet membrane. Present experiments demonstrate that it is possible to indu ce reptilase-clot retraction by means of electrical or mechani cal stimulation. Electrical stimulation of the clot was performed by means of two platinum electrodes (diameter 0.5 mm) connected to a stl mulator (S 8 Stimulator,Grass Instruments,Quincy,Mass. U.S.A.). One of the electrodes was inserted through the bottom of a glass 523
REFTILASE CLOT-RETRACTIOK
j24
SO-
60-
I 2
I
I 6
4
TIME
OF
STIMULATION
I
I 8 (in
10
minutes)
FIG. 1 Retraction of reptilase-clots induced by electrical stimulation applied for different times. Test system: 0.3 ml PRP (or PPP) + 0.1 ml reptilase solution. Number of platelets in PRP=300,000/ul. Electrical stimulation started 10 min after reptilase addition; after 60 min at 37OC the clot retraction was evaluated by measu ring the serum expressed (1). Electrical stimuli: 150 volts, 50 msec, lO/sec.
0I
I
10
I
I
20
TIME
d0
(in
40
I
50
6’0
minutes)
FIG. 2 Dynographic recording of the mechanical strength produced by PRP clotted by reptilase. The recording began 47 min after reptilase addition to PRP. Number of platelets =340,000/@. The bath contained Tyrode's modified solution (5) without Ca2+ and Mg2+.
REPTIW3E
vo1.8,50.4
523
CLOT-RETRACTION
tube (internal diameter 8nun, length 78mm) jutting 2mm out inside the tube: the second electrode (length 2mm) was immersed
at
the top of PRP before adding reptilase. (Penta-
Human PRP, platelet-poor plasma (PPP) and reptilase
pharm, Basel) were prepared as described previously (1). IO min after reptilase addition electrical stimuli (frequency lO/sec, duration 50 msec, intensity 150 volts)
were applied
to the clot for different times (I-IO min). After the end of the electrical stimulation the tube was immediately transferred at 37Oc and clot retraction was evaluated 60 min later, by
measu-
ring serum expressed (1). A marked clot retraction was always observed in the clots sti mulated for more than 4 min (fig. 1). PPP clots do not after similar stimulation, thus confirming that the
retract
electrical
stimulation activates platelets. As the retraction,following tha electrical stimulation, is not immediate but it retains its usu al characteristics of a slowly evolving process, we may conclude that the stimulation is only a trigger for the retraction. In a previous work (5) we have shown the possibility to measure the strength of clot retraction by dynographic method . By using
a
similar method de Gaetano et al. (6) found that
clotted by reptilase does not develop any mechanical
PRP
strength,
but all our attempts to confirme this result were unsuccessful, as after a latent period (about 30 min) all the reptilase-clots were always retracting. A typical dynographic tracing
obtained
with PRP clotted by reptilase is reported in figure 2. Consequently we supposed that even a slight mechanical stimu lation of the clot, as that unavoidable by handling (while trans ferring the clot from the tube to the pharmacological bath)and/or as that due to the stretch of the clot in the pharmacological bath (due to its own weight), may be a stimulus sufficient for inducing clot retraction. This hypothesis was supported by the fact that a sample of the same PRP clotted in a test tube and
526
REPTIWSE
CLOT-RETRACTION
observed in avoidance of any mechanical stimulation, does not show any retraction after 2 hours (18 experiments, with 3 diffe rent batches of reptilase, showed the same result). To confirme further this hypothesis, we did another series of experiments, in which reptilase-PRP-clots were stimulated mecha nically, using an experimental system like that described in Pi gure 3. The result was constantly a marked clot retraction (figure 4). In contrast, PPP-clots, which underwent the same mecha nical stimulation, do not retract.
c
4
-7
p”“cy
silk
29r
plastic ring
B
FIG. 3 Mechanical stimulation of the clot. A: control sample; B: a pl$ stic ring was inserted in PRP before adding reptilase. 30 min after clotting a weight of 2 grams was attached to the hook,and removed 10 min later. In A and B 0.3 ml of reptilase solution were added to 3 ml of PRP, and the tubes were incubated at 37Y. Present results seem interesting because until the present tL me it has not been demonstrated that the platelet contractile sy stem can be activated by electrical or mechanical stimulation, i.e. two typical triggers of the contraction in the muscular cells. Human platelets contain both a contractile protein regulato-
REFERENCES
1.
de GAETANO G., BOTTECCHIA D. and VERMYLEN J. Retraction of Reptilase Clots in the Presence or Absence of Agents Inducing or Inhibiting the Platelet Adhesion-Aggregation ReacThrombos. Res.: 2, 71, 1973. tion.
2.
KUBISZ P. Cold Induced Retraction of Reptilase Clots. Stand. J. Haemat.: 13, 175, 1974.
3.
L:SCHER E-F., BETTEX-GALLAND M. Thrombosthenin, the Contrac tile Protein of Blood Platelets. New Facts and Problems. Pathologie-Biologie: 20, supplement, 89, 1972.
4.
L;;SCHERE.F., PROBST E. and BETTEX-GALLAND M. Thrombosthenin: Structure and Function. Annals New York Acad. of Sci.: 202, 122, 1972.
5.
BOTTECCHIA D. and FANTIN G. Platelet and Clot Retraction. Effect of Divalent Cations and Several Drugs. Thromb. Diath. Haemorrh.: 30, 576, 1973.
6.
de GAETANO G., FRANC0 R., DONATI M.B., BONACCORSI A. and GARATTINI S. Mechanical Recording of Reptilase-clot Retraction: Effect of Adenosine-5'-Diphosphate and Prostaglandin E1 Thrombos. Res.: 4, 189, 1973.
7.
COHEN I. and COHEN C.: Tropomyosin-like protein from Human Platelets. J. Mol. Biol.: 68, 383, 1972.
8.
THORENS S., SCHAUB M.C. and L&HER E.F. A Calcium-Sensiti zing System from Human Platelets and its Activity on Muscle and Platelet Actomyosin. Experientia: 29, 349, 1973.
9.
GRETTE K. Relaxing Factor in Extracts of Blood Platelets and its Function in the Cells. Nature: 198, 488, 1963.
10.
STATLAND B.E., HEGAN B.M. and WHITE J.G. Uptake of Calcium by Platelet Relaxing Factor. Nature: 233, 527, 1969.
11.
ZIERLER K.L.: Mechanism of Muscle Contraction and its Energs tics. in "Medical Physiology" edited by Mountcastle V.B. 12th edition, Mosby Company 1968.
A
B
Fig. 4 A: control; B: clot stimulated mechanically as described in figs_ re 3. The picture was taken 44 min after the end of the mechanical stimulation. Number of platelets in PRP: 300,000 pl. ry system (7,3,4,8) and a membrane associated relaxing factor resembling the muscle sarcotubular system which, upon stimulation, discharge their contents into the cytoplasm (g-10). It is likely that the electrical stimulation of the platelets within the clot, induces some modifications of the permeability of the platelet membrane(s) with following availability of intracellular calcium ions which in turn control the contractile activity of platelets (3)* It is well known that, when a smooth muscle is stretched, a membrane depolarization occurs, with subsequent release of intra cellular calcium ions and muscular contraction (11). Since an evident clot retraction is induced by stretching reptilase-clots (which do not retract unless platelet activation occurs) the pre viously formulated hypothesis about analogies between clot and smooth muscle (5) is further supported by present experiments.
ACKNOWLEDGEMENTS: Reptilase was a gift of Penthapharm (Basel). The technical assistance of M??.Maurizio Travan and of Mr. Renzo Ferronato was appreciated.
