THROMBOSIS RESEARCH Printed in the United States
BRIEF
Vol.
a, PP* 70 -7111 1976
Pergamon i: ress, Inc.
COMMUNICATION
FIBRIN CLOT RETRACTION BY A RAT RHABDOMYOSARCOMA CELL LINE + M.B. Donati, E. Dolfini,
L. Morasca and G. de Gaetano
Laboratory for Haemostasis and Thrombosis Research and Laboratory of Cancer Chemotherapy in vitro. Istituto di Ricerche Farmacologiche ‘Mario Negri’ Via Eritrea, 62 - 20157 MILANO,Italy.
(Received 31.12.1975; in revised form 5.1.1976. Accepted by Editor S. Niewiarowski. Received by Executive Editorial Office 18.3.1976) The capacity to induce fibrin clot retraction had been considered to be a specific property of platelets (l-3), until Niewiarowski et al. (4,s) showed that undifferentiated mouse fibroblasts and human skin fibroblasts were The purpose of this study was to capable of causing fibrin clot retraction. investigate the possibility that fibrin clot retraction is a property of several types of cells. Therefore the following cell lines were studied : KB (human cervix carcinoma) ; HeLa (human cervix carcinoma) ; Chang Liver (humannormal epithelium) ; Chang Conjunctiva (humannormal epithelium) ;NCTC clone 929 (L) fibroblasts from C3H/ANmice) and BA 1112 (rhabdomyosarcoma developed on WAG/Rji inbred rats) (6-8) . Cells were cultured in plastic flasks in Eagle’s MEM,Henk’s balanced salt solution plus 10% calf serum, harvested from monolayer by 5 minute exposure to 0.25% trypsin solution,washed 3 times and resuspended in Tyrode albumin solution containing Ca++ and Mg++ (2x10-3Ml . The cell suspension was adjusted to 2x106 cells/ml . Fibrin clot retraction was studied in a system composed of 0.8 ml cell suspension, Cl2 ml platelet-poor plasma from human, rat or mouse (according to the origin of the cell line used) and 0.1 ml of a 2 NIH U/ml thrombin solution (Topostasine, Roche, Milano, Italy) . In some experiments 0.1 ml of Botrops atrox thrombinlike enzyme (Reptilase-r; Pentapharm, Ba.sle,Switzerland and Onnonoterapia Richter,Milano, Italy) was used as the clotting enzyme. The reagent used (lot No. 7130) did not cause spontaneous clot retraction in a platelet-rich plasma system. Clots were held in a 37°C waterbath and retraction was measured after 3 hour incubation as previously described (9) . + This investigation
was supported by Grant NIH-FHRB-1ROlCA 12764-01. This study was presented at the Vth Congress of the International Society on Thrombosis and Haemostasis, Paris, July 1975, Abstracts, p. 548 .
FIBRIN CLOT RETRACTION
708
Vo1.8,No.5
TABLE I Retractionof thrombin-induced fibrinclotsby cultured cell lines.Means2 S.E. of at least5 experiments. Cell Line
I
2”” HeLa L 929 ChangConjunctiva Chang Liver BA 1112 BA 1112 (disrupted) BA 1112 (sedimented)
% Clot Retraction 10.4 + 1.6 11.4T 1.2 10.6+ 1.5 15.4T 1.6 7.6 -i: 1.1 7.6 7 1.0 60.5-+3.9 11.07 2.5 11.27 1.8
TABLE II Effectof severalcompoundson BA 1112 cell-induced fibrin clot retraction. The clottingenzymein all experiments was thrombin.Means2 S.E. of at least5 experiments. Compounds Isotonicsaline Na -EDTA P&l m744 m744 m744 vK744 Acetylsalicylic acid
FinalConcentration
0.3% 3.3 Clg/ml 5.10-3M l.lO-3M 1.10'4M 1.10'5M 2.10-3M
% Clot Retraction 60.5 + 3.9 11.47 1.0 22.2‘r2.5 11.8+ 1.5 21.0-i1.9 30.2'71.6 63.4T 2.1 61.05 2.5
Thrombelastographic studieswere performedby mixingat 37'C 0.2 ml of cell 0.1 ml platelet-poor plasmaand 0.05 ml thrombin(2 NIH U/ml) in suspension, the cuvetteof a Clotscanner(Elvi-Logos, Milano,Italy)(11 ) 9 Among the cells studied,anly the BA 1112 linewas foundcapableof inducingretraction(table1) . The retractionbeganwithin 30 minutesand was completedwithinthreehours,the degreeof finalretractionbeing dependentupon the cell ntier (Fig.1). BA 1112 cellswere devoidof both procoagulant and fibrinolytic activity(7) . Integrityand homogeneous distribution of the cellswere essentialfor fibrin clot retractionto occur , since this phenomenonwas absentaftereither disruptionof the cells (by three timesfreezingand thawing)(10)or pelletingthem at the bottomof the test tube by centrifugation . The presenceof Ca++ or otherbivalentcationswas also required, as shown by inhibitionof fibrinclot retractionin the presenceof Na2-EDTA(Table II) . Thrombelastography confirmedthat, amongthe cell linesstudied,only BA 1112 cells,likeplatelets(11)were able to retractfibrinclots formed by thrombinas indicatedby the maximalamplitudeof thrombelastogram (Fig.2). Prostaglandin El (PCEl)and VK 744, a pyrimido-pyrimidine derivative
FIBRIN CLOT RETRACTION
Vo1.8,N0.5
709
TABLE III Failure of BA 111.2 cells to retract reptilase-induced fibrin. Means 2 S.E. of at least 5 experiments . Final Concentration
Cells Preincubated (3min) with
I
% Clot Retraction I
Isotonic saline ADP Collagen Adrenaline
l. lO-4M 0.1 mg/ml 0.1 mg/ml
11.6 11.2 13.2 10.4
+ 5 + z
3.0 2.3 2.8 1.6
(2/Z-aminoethyl)amino- 7-4-morpholinothieno (3,2-d)-pyrimidine-dihydrochloride-) inhibited the BA ill2 cell-induced fibrin clot retraction , whereas when (Table II) . No retraction-occurred acetylsalicylic acid was ineffective reptilase instead of thrombin was used as the clotting agent. However, in
FIG. 1
i 1...,......
lo5
10
20
30
46 50 60 70 % clot retraction
60
90
1M)
Retraction of thrombin-induced fibrin clots in the presence of different concentrations of BA 1112 cells .
contrast to platelets (9), preincubation of BA 1112 cells with ADP,collagen or adrenaline did not result in subsequent retraction of reptilase-induced clots (Table III) . This suggests that these cells are insensitive to the above mentioned substances (at least at the concentrations used) and may possess some membrane sites specifically susceptible to the action of thrcnnbin. A similar peculiarity has been recently reported by Niewiarowski et al. (12) for human skin fibroblasts . Thrcanbin has been shown to be a potent mitogenic agent on chick embryo fibroblasts (13); however , it does not cause any detectable alteration of surface protein profile of these cells (14) . A deeper insight in the mechanisms underlying cultured cell-thrombin interactionis therefore warranted . The intracellular level of cyclic AMPinfluences the ability of platelets to retract fibrin clots (15). Possibly, cyclic AMPlevels are also important in BA 1112 cell-fibrin retraction, as suggested by the inhibitory effect of PGEl (an activator of adenyl cyclase) and of a pyrimido-pyrimidine compound (an inhibitor of phosphodiesterase) . The failure of acetylsalicylic acid to inhibit both platelet-and BA 1112 cell-dependent fibrin clot retraction is of interest, although it does not allow to speculate on the role of intra-
Vol.8,No.j
FIBRIN CLOT RETRACTION
710
KB
BA1112 I I 10min.
FIG. 2 Thrombelastographic pattern of thrombiri-formed clots in the presence of either BA 1112 or KB cells . cellular
prostaglandin
biosynthesis
in these phenomena .
In conclusion, the present paper, together with the observations of Niewiarowski et al. (4), demonstrates that fibrin clot retraction is not a specific function of platelets . ACKNOWLEDGEMENTS
The skilful technical assistance of Miss Annalisa Cavenaghi was highly appreciated . .Mrs. Amy Crook, Mrs. Graziella Scalvini and Miss Gigliola Brambilla valaubly helped us in preparing the manuscript . Topostasine was a gift of Roche, Milano, Italy. .Reptilase-r was kindly provided by Pentapharm Ltd., Basle, Switzerland and by Ormonoterapia Richter, Milano, Italy . PGEl was a gift of Professor D.A. Van Dorp, Unilever Research Laboratories, Vlaardingen, The Netherlands. VK 744 was kindly supplied by Karl Thomae, Biberach an der Riss, West Germany. Acetylsalicilye acid was kindly provided by Bayer Italia , Milan0 , Italy . REFERENCES
1.
Budtz-Olsen,
O.E. “Clot Retraction”.
Oxford : Blackwell.
1951 .
2.
Bettex-Galland, M. and Liischer, E.F. Thrombostenin -- a contractile protein from thrombocytes. Its extraction from humanblood platelets and some of its properties . Biochim. Biophys. Acta 49, 536, 1961 .
Vo1.8,No.5
FIBRIN CLOT RETRACTION
711
3.
Nachman, R.L., Marcus, A.J. and Safier, L.B. Platelet subcellular localization and function . J. Clin. Invest. 46 , 1380, 1967 .
4.
Niewiarowski, S., Regoeczi,E. and Mustard,J.F. to polymerizing fibrin and retraction of fibrin Proc. Sot. Exp. Biol. Med. 140, 199, 1972 .
5.
Niewiarowski, S., and Goldstein, S. Interaction of cultured human fibroblasts with fibrin : modification by drugs and aging in vitro . J. Lab. Clin. Med. 82 , 605, 1973 .
6.
Hermens, A.F., and Barendsen, G.W. Cellular proliferation an experimental rhabdomyosarcoma in the rat . Europ. J. Cancer 2 , 361, 1967 .
7.
Morasca, L., Donati, M.B., Dolfini, E., Roncaglioni, M.C. and de Gaetano, G. Procoagulant and fibrinolytic activity of humancancer cell lines . Abstracts of the 11th International Cancer Congress, Florence, Italy, October , 1974,vol. 4, p. 635 .
8.
Donati, M.B.! Dolfini, E., Roncaglioni, M.C., de Gaetano, G., and Morasca, L. Humanepithelial cell lines : A source of both procoagulant and fibrinolytic activity. Circulation 50 , suppl. 3, 295, 1974
9.
de Gaetano, G., Bottecchia, D., and Vermylen, J. Retraction of reptilase clots in the presence of agents inducing or inhibiting the platelet adhesion-aggregation reaction . Thromb. Res. 2 , 71, 1973 .
thrombostenin :
Adhesion of fibroblasts induced by fibroblasts.
patterns
in
10.
de Gaetano, G., Donati,M.B. and Vermylen, J. A simple method to study the “stabilising” effect of chemicals and drugs on the platelet membrane. Thromb. Res. 1, 631, 1972 .
11.
de Gaetano, G., Bottecchia, D. and Vermylen, J. Effect of platelets on clot structuration. A Thrombelastographic study . Thromb. Res. 2 , 425, 1973 .
12.
Niewiarowski, S. and Goldstein, S. Fibrin clot retraction by human skin fibroblasts . Effects of ADP and thrombin. Proc. Sot. Exp. Biol. Med. , 1976,in press .
13.
Chen, L.B. and Buchanan, J.M. Mitogenic activity of blood components. 1. Thrombin and prothrombin . Proc. Nat. Acad. Sci., U.S.A. 72 , 131, 1975 .
14.
Teng, N.N.H. and Chen, L.B. The role of surface proteins proliferation as studied with thrombin and other proteases Acad. Sci., U.S.A. 72 , 413, 1975 .
15.
Miirer, E.H. Compoundsknown to affect the cyclic adenosine monophosphate level in blood platelets : effect on thrombin-induced clot retraction and platelet release . Biochim. Biophys. Acta 237 , 310, 1971 .
in cell . Proc. Nat.
BRIEF
Vol.
a, PP* 70 -7111 1976
Pergamon i: ress, Inc.
COMMUNICATION
FIBRIN CLOT RETRACTION BY A RAT RHABDOMYOSARCOMA CELL LINE + M.B. Donati, E. Dolfini,
L. Morasca and G. de Gaetano
Laboratory for Haemostasis and Thrombosis Research and Laboratory of Cancer Chemotherapy in vitro. Istituto di Ricerche Farmacologiche ‘Mario Negri’ Via Eritrea, 62 - 20157 MILANO,Italy.
(Received 31.12.1975; in revised form 5.1.1976. Accepted by Editor S. Niewiarowski. Received by Executive Editorial Office 18.3.1976) The capacity to induce fibrin clot retraction had been considered to be a specific property of platelets (l-3), until Niewiarowski et al. (4,s) showed that undifferentiated mouse fibroblasts and human skin fibroblasts were The purpose of this study was to capable of causing fibrin clot retraction. investigate the possibility that fibrin clot retraction is a property of several types of cells. Therefore the following cell lines were studied : KB (human cervix carcinoma) ; HeLa (human cervix carcinoma) ; Chang Liver (humannormal epithelium) ; Chang Conjunctiva (humannormal epithelium) ;NCTC clone 929 (L) fibroblasts from C3H/ANmice) and BA 1112 (rhabdomyosarcoma developed on WAG/Rji inbred rats) (6-8) . Cells were cultured in plastic flasks in Eagle’s MEM,Henk’s balanced salt solution plus 10% calf serum, harvested from monolayer by 5 minute exposure to 0.25% trypsin solution,washed 3 times and resuspended in Tyrode albumin solution containing Ca++ and Mg++ (2x10-3Ml . The cell suspension was adjusted to 2x106 cells/ml . Fibrin clot retraction was studied in a system composed of 0.8 ml cell suspension, Cl2 ml platelet-poor plasma from human, rat or mouse (according to the origin of the cell line used) and 0.1 ml of a 2 NIH U/ml thrombin solution (Topostasine, Roche, Milano, Italy) . In some experiments 0.1 ml of Botrops atrox thrombinlike enzyme (Reptilase-r; Pentapharm, Ba.sle,Switzerland and Onnonoterapia Richter,Milano, Italy) was used as the clotting enzyme. The reagent used (lot No. 7130) did not cause spontaneous clot retraction in a platelet-rich plasma system. Clots were held in a 37°C waterbath and retraction was measured after 3 hour incubation as previously described (9) . + This investigation
was supported by Grant NIH-FHRB-1ROlCA 12764-01. This study was presented at the Vth Congress of the International Society on Thrombosis and Haemostasis, Paris, July 1975, Abstracts, p. 548 .
FIBRIN CLOT RETRACTION
708
Vo1.8,No.5
TABLE I Retractionof thrombin-induced fibrinclotsby cultured cell lines.Means2 S.E. of at least5 experiments. Cell Line
I
2”” HeLa L 929 ChangConjunctiva Chang Liver BA 1112 BA 1112 (disrupted) BA 1112 (sedimented)
% Clot Retraction 10.4 + 1.6 11.4T 1.2 10.6+ 1.5 15.4T 1.6 7.6 -i: 1.1 7.6 7 1.0 60.5-+3.9 11.07 2.5 11.27 1.8
TABLE II Effectof severalcompoundson BA 1112 cell-induced fibrin clot retraction. The clottingenzymein all experiments was thrombin.Means2 S.E. of at least5 experiments. Compounds Isotonicsaline Na -EDTA P&l m744 m744 m744 vK744 Acetylsalicylic acid
FinalConcentration
0.3% 3.3 Clg/ml 5.10-3M l.lO-3M 1.10'4M 1.10'5M 2.10-3M
% Clot Retraction 60.5 + 3.9 11.47 1.0 22.2‘r2.5 11.8+ 1.5 21.0-i1.9 30.2'71.6 63.4T 2.1 61.05 2.5
Thrombelastographic studieswere performedby mixingat 37'C 0.2 ml of cell 0.1 ml platelet-poor plasmaand 0.05 ml thrombin(2 NIH U/ml) in suspension, the cuvetteof a Clotscanner(Elvi-Logos, Milano,Italy)(11 ) 9 Among the cells studied,anly the BA 1112 linewas foundcapableof inducingretraction(table1) . The retractionbeganwithin 30 minutesand was completedwithinthreehours,the degreeof finalretractionbeing dependentupon the cell ntier (Fig.1). BA 1112 cellswere devoidof both procoagulant and fibrinolytic activity(7) . Integrityand homogeneous distribution of the cellswere essentialfor fibrin clot retractionto occur , since this phenomenonwas absentaftereither disruptionof the cells (by three timesfreezingand thawing)(10)or pelletingthem at the bottomof the test tube by centrifugation . The presenceof Ca++ or otherbivalentcationswas also required, as shown by inhibitionof fibrinclot retractionin the presenceof Na2-EDTA(Table II) . Thrombelastography confirmedthat, amongthe cell linesstudied,only BA 1112 cells,likeplatelets(11)were able to retractfibrinclots formed by thrombinas indicatedby the maximalamplitudeof thrombelastogram (Fig.2). Prostaglandin El (PCEl)and VK 744, a pyrimido-pyrimidine derivative
FIBRIN CLOT RETRACTION
Vo1.8,N0.5
709
TABLE III Failure of BA 111.2 cells to retract reptilase-induced fibrin. Means 2 S.E. of at least 5 experiments . Final Concentration
Cells Preincubated (3min) with
I
% Clot Retraction I
Isotonic saline ADP Collagen Adrenaline
l. lO-4M 0.1 mg/ml 0.1 mg/ml
11.6 11.2 13.2 10.4
+ 5 + z
3.0 2.3 2.8 1.6
(2/Z-aminoethyl)amino- 7-4-morpholinothieno (3,2-d)-pyrimidine-dihydrochloride-) inhibited the BA ill2 cell-induced fibrin clot retraction , whereas when (Table II) . No retraction-occurred acetylsalicylic acid was ineffective reptilase instead of thrombin was used as the clotting agent. However, in
FIG. 1
i 1...,......
lo5
10
20
30
46 50 60 70 % clot retraction
60
90
1M)
Retraction of thrombin-induced fibrin clots in the presence of different concentrations of BA 1112 cells .
contrast to platelets (9), preincubation of BA 1112 cells with ADP,collagen or adrenaline did not result in subsequent retraction of reptilase-induced clots (Table III) . This suggests that these cells are insensitive to the above mentioned substances (at least at the concentrations used) and may possess some membrane sites specifically susceptible to the action of thrcnnbin. A similar peculiarity has been recently reported by Niewiarowski et al. (12) for human skin fibroblasts . Thrcanbin has been shown to be a potent mitogenic agent on chick embryo fibroblasts (13); however , it does not cause any detectable alteration of surface protein profile of these cells (14) . A deeper insight in the mechanisms underlying cultured cell-thrombin interactionis therefore warranted . The intracellular level of cyclic AMPinfluences the ability of platelets to retract fibrin clots (15). Possibly, cyclic AMPlevels are also important in BA 1112 cell-fibrin retraction, as suggested by the inhibitory effect of PGEl (an activator of adenyl cyclase) and of a pyrimido-pyrimidine compound (an inhibitor of phosphodiesterase) . The failure of acetylsalicylic acid to inhibit both platelet-and BA 1112 cell-dependent fibrin clot retraction is of interest, although it does not allow to speculate on the role of intra-
Vol.8,No.j
FIBRIN CLOT RETRACTION
710
KB
BA1112 I I 10min.
FIG. 2 Thrombelastographic pattern of thrombiri-formed clots in the presence of either BA 1112 or KB cells . cellular
prostaglandin
biosynthesis
in these phenomena .
In conclusion, the present paper, together with the observations of Niewiarowski et al. (4), demonstrates that fibrin clot retraction is not a specific function of platelets . ACKNOWLEDGEMENTS
The skilful technical assistance of Miss Annalisa Cavenaghi was highly appreciated . .Mrs. Amy Crook, Mrs. Graziella Scalvini and Miss Gigliola Brambilla valaubly helped us in preparing the manuscript . Topostasine was a gift of Roche, Milano, Italy. .Reptilase-r was kindly provided by Pentapharm Ltd., Basle, Switzerland and by Ormonoterapia Richter, Milano, Italy . PGEl was a gift of Professor D.A. Van Dorp, Unilever Research Laboratories, Vlaardingen, The Netherlands. VK 744 was kindly supplied by Karl Thomae, Biberach an der Riss, West Germany. Acetylsalicilye acid was kindly provided by Bayer Italia , Milan0 , Italy . REFERENCES
1.
Budtz-Olsen,
O.E. “Clot Retraction”.
Oxford : Blackwell.
1951 .
2.
Bettex-Galland, M. and Liischer, E.F. Thrombostenin -- a contractile protein from thrombocytes. Its extraction from humanblood platelets and some of its properties . Biochim. Biophys. Acta 49, 536, 1961 .
Vo1.8,No.5
FIBRIN CLOT RETRACTION
711
3.
Nachman, R.L., Marcus, A.J. and Safier, L.B. Platelet subcellular localization and function . J. Clin. Invest. 46 , 1380, 1967 .
4.
Niewiarowski, S., Regoeczi,E. and Mustard,J.F. to polymerizing fibrin and retraction of fibrin Proc. Sot. Exp. Biol. Med. 140, 199, 1972 .
5.
Niewiarowski, S., and Goldstein, S. Interaction of cultured human fibroblasts with fibrin : modification by drugs and aging in vitro . J. Lab. Clin. Med. 82 , 605, 1973 .
6.
Hermens, A.F., and Barendsen, G.W. Cellular proliferation an experimental rhabdomyosarcoma in the rat . Europ. J. Cancer 2 , 361, 1967 .
7.
Morasca, L., Donati, M.B., Dolfini, E., Roncaglioni, M.C. and de Gaetano, G. Procoagulant and fibrinolytic activity of humancancer cell lines . Abstracts of the 11th International Cancer Congress, Florence, Italy, October , 1974,vol. 4, p. 635 .
8.
Donati, M.B.! Dolfini, E., Roncaglioni, M.C., de Gaetano, G., and Morasca, L. Humanepithelial cell lines : A source of both procoagulant and fibrinolytic activity. Circulation 50 , suppl. 3, 295, 1974
9.
de Gaetano, G., Bottecchia, D., and Vermylen, J. Retraction of reptilase clots in the presence of agents inducing or inhibiting the platelet adhesion-aggregation reaction . Thromb. Res. 2 , 71, 1973 .
thrombostenin :
Adhesion of fibroblasts induced by fibroblasts.
patterns
in
10.
de Gaetano, G., Donati,M.B. and Vermylen, J. A simple method to study the “stabilising” effect of chemicals and drugs on the platelet membrane. Thromb. Res. 1, 631, 1972 .
11.
de Gaetano, G., Bottecchia, D. and Vermylen, J. Effect of platelets on clot structuration. A Thrombelastographic study . Thromb. Res. 2 , 425, 1973 .
12.
Niewiarowski, S. and Goldstein, S. Fibrin clot retraction by human skin fibroblasts . Effects of ADP and thrombin. Proc. Sot. Exp. Biol. Med. , 1976,in press .
13.
Chen, L.B. and Buchanan, J.M. Mitogenic activity of blood components. 1. Thrombin and prothrombin . Proc. Nat. Acad. Sci., U.S.A. 72 , 131, 1975 .
14.
Teng, N.N.H. and Chen, L.B. The role of surface proteins proliferation as studied with thrombin and other proteases Acad. Sci., U.S.A. 72 , 413, 1975 .
15.
Miirer, E.H. Compoundsknown to affect the cyclic adenosine monophosphate level in blood platelets : effect on thrombin-induced clot retraction and platelet release . Biochim. Biophys. Acta 237 , 310, 1971 .
in cell . Proc. Nat.
