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Actinoidin: A new inhibitor of ristocetin- and ristomycin-induced platelet agglutination
IIifio>4BOSI.S RESEXRCH 17; 603-610 @pergamon Press Ltd. 1980. Printed
in the rJnited Starves ootig-38~8/80/050603-08$0~.00/0
A NEW INHIBITOR OF RISTOCETIN- AND RISTOACTINOIDIN: :4YCIN-INDUCED PLATELET AGGLUTINATION Z. Boda, N.O. Solum, F. Sztaricskai and K. Rak IInd Department of Medicine, University Medical School, Debrecen, Hungary Research Institute for Internal Medicine, University of Oslo, Rikshospitalet, Oslo 1, Norway The Research Group of Antibiotics, Hungarian Academy of Sciences, Lajos Kossuth University, Debrecen, Hungary (Received
16.10.1979.
Accepted
by
Editor
H.C.
Godal)
ABSTRACT Actinoidin, like vancomycin, inhibits ristocetin-and ristomycin-induced VIIIR:WF-dependent agglutination of untreated or formaldehyde-fixed human platelets but does not interfere with bovine factor VIII-induced agglutination. The socalled "direct effect" of ristocetin and ristomycin on fixed platelets, which is represented by an immediate increase in light absorbancy in the absence of cofactor, was not blocked by actinoidin at concentrations which totally inhibited agglutination in the presence of VIIIR:WF. Actinoidin does not inhibit thrombin-induced platelet aggregation. This represents a further argument against a common platelet membrane receptor site for thrombin and ristocetin or a ristocetin-VIIIR:WF complex on the platelet membrane. INTRODUCTION The mode of action of ristocetin in the VIIIR:WF-dependent platelet agglutination is not established. The opinion dominates that most likely ristocetin binds to the platelet membrane and increases affinity of that to VIIIR:WF which at the end of events may act as a bridging protein between platelets (1,2,3). Other findings are consistent with the hypothesis that modification of VIIIR:WF by ristocetin precedes and initiates platelet agglutination. In fact, ristocetin has been observed to aggregate or polymerize VIII:WF in vitro at a concentration which agglutinates platelets (4). Binding of ristocetin to platelets was recently demonstrated by an isotope technique; the process was independent
Key Words:
Actinoidin,
platelet
agglutination
inhibitor.
EFFECTS
OF ACTIXGIDIX
vol.l;,~o.j
platelets of the presence of VIIIR:WF, and chymotrypsin-treated though unable to agglutinate, did not bind less ristocetin than the untreated platelets (5). It has also been demonstrated that VIIIR:WF binds to the platelet membrane in the presence of ristocetin (6) and that the binding sites are specific for VIIIR:WF(7). It is not yet decided whether attachment of ristocetin to platelets or to VIIIR:WF is the more important in respect to platelet agglutination. In a previous work (8) we have demonstrated that a striking similarity exists between ristocetin and ristomycin as inducers of platelet agglutination both in qualitative and quantitative terms which means that ristomycin can substitute ristocetin in practical and experimental laboratory work. In fact, ristomycin is advantageous in theoretical studies as its structure is better known and derivatives and fragments of ristomycin are available. We reported in the same paper that still another member of the vancomycin group, i.e. actinoidin, is a potent inhibitor of ristocetin-and ristomycin-induced platelet agglutination. The purpose of this paper is to present some more details on the effects of this antibiotic. MATERIALS
AND METHODS
Platelet rich plasma (PRP) was obtained from centrifugation of The blood was anticoaaulated whole blood at 320 x g for 15 min. by either 11 mM sodium citrate (citratg PRP) or 5.4 mM E&A (EDTA. Platelet count was 300-400 x 10 11. PRP). Platelet poor plasma (PPP) was prepared from whole blood by cenPlatelet count was below trifugabion at 2200 x g for 20 min. 10 x 10 /l. Formaldehyde-fixed telet concentrates
platelets (FFP) were prepared from fresh plaas described by Evans and Austen (9).
Reference plasma was obtained by pooling equal volumes of titrated PPP from eight healthy individuals (4 men and 4 women). Tris-buffered saline (TS) consists HCl pH 7.4, 280 mOsM.
of 148 mM NaCl and 20 mtM Tris-
Ristocetin sulphate was purchased from Lundbeck Copenhagen, Denmark, and dissolved in TS.
and Co. A/S,
Ristomycin A mono-sulphate and actinoidin A + B were obtained from Institute of New Antibiotics, Academy of Medical Sciences, Moscow, USSR,and dissolved in TS. Actinoidin was isolated from Proactimyces actinoides in 1957 (10). It consists of two components: actinoidin A and B _(ll) . Both components have three primary amino, four phenolic hydroxyl, one carboxyl and more carbohydrate hydroxyl groups. The total structure of actinoidin is not yet known. Both components possess phenylalanin and actinoic acid, but there are also known differences (12,13,14). Two new aminohexoses, acosamine and actosamine were found besides the neutral carbohydrates in both components (15,16).
Acrgregating materials were epinepnrine (Adrenalin, Richter Cc., Budapest, Hungary) in the concentration of 1%: ,$g/ml, ADP, Recezl, Budapest, Hungary) in i.5 and 0.3 pg/ml concentrations,collagen (Reagent Ho-q, Hunich, West Germany) in 4.2 and 3.3 pg/ml concentrations, thrombin !Topostasin, La Roche, Basel, Switzerland) in the concentrations of 3.0 and 1.0 U/ml and bovrne factor VIII related protein purified as previously described by Solum and Peterka (17) and used either as "intermediate" or "highly" purified material. Plasma protein precipitation was studied by addition of 100 ~1 of antibiotic in various dilutions to 900 ~1 of titrated plasma in the cuvette of a Beckman spectrophotometer model 35. After mixing and incubation at room temperature for 3 min, the absorbancy was read at 600 nm and used as a parameter of precipitation. Platelet counting or electronically Ltd., England) .
was performed by phase contrast microscopy (18) using a Thrombocounter-C (Coulter Electronics
Platelet agglutination was investigated at 37'C in a Payton Dual Channel Aggregometer or Chrono-Log Aggregometer (Model 330). In the standard system 400 ~1 citrate- or EDTA-PRP were preheated at 37OC for 3 min. Then 50 ~1 of ristomycin or ristocetin were added. The 8ystem containing FFP consisted of 400 J.L~of FFP preheated at 37 C for 3 min, 50 ~1 of actinoidin or TS (control), 50 ~1 of ristomycin or ristocetin and 50 ~1 of undiluted reference plasma. The Payton aggregometer was calibrated in such a way that the difference in light absorption between PRP and PRP diluted l/2 with autologous PPP corresponded to a recorder deflection of 50 chart division (50 mV) The Chrono-Log-Aggregometer was calibrated so that the difference in light absorption between PRP and PPP corresponded to a recorder deflection of 80 chart division (80 mv).
RESULTS Incubation of citrate- or EDTA-PRP at 37OC for 3 min with actinoidin in concentrations varying from 0.6 to 2.5 mg/ml resulted in a complete inhibition of ristocetin- or ristomycininduced platelet agglutination at a concentration of 1.5 mg/ml of these A partial inhibition was observed at actinoidin concenagents. trations between 0.15 and 0.6 mg/ml (Fig. 1). Actinoidin did notinhibitplatelet agglutination induced by bovine factor VIII related protein or aggregation induced by This is demonstrated in thrombin, epinephrine, ADP or collagen. Fig. 2 for bovine factor VIII related protein and thrombin. Actinoidin completely inhibited ristocetin- or ristomycininduced platelet agglutination of formaldehyde-treated platelets at concentrations which did not affect the "direct effect" seen as an increase in absorbancy in the absence of cofactor (Fig.3). All antibiotics
of the vancomycin
group induced
a protein
606
EFFECTS
Yol.l,,So.~
OF ACTIXOIDI?;
At the same concenprecipitation when added to titrated plasma. tration the degree of protein precipitation increased in sequence (Table 1). from actinoidin, ristocetin, ristomycin and vancomycin
b 40 -
20 Jo4050607080-
-*
“\
b
c
FIG. 1 platelet agglutination by actiInhibition of ristomycin-induced Final concentranoidin in (a): EDTA-PRP and (b) : citrate-PRP. tions of actinoidin were (A): 2.5 mg/ml and (B): 0.4 mg/ml. Control (C) was with TS only. Concentration of ristomycin 1.5 mg/ml
0 40 20 30 40 50
60 70 80
a
% ,),,,, C
b
A
4
8
B
3042042
nME/fliN./
FIG. 2 Effect of actinoidin on (a): bovine factor VIII-induced or (b = Final c): thrombin-induced platelet agglutination in citr-PRP. Control with concentration of actinoidin was (A): 2.5 mg/ml. (B) : Final concentrations of thrombin were (b): 0.3 U/ml and TS only. (cl: 0.1 U/ml.
EFFECT3
i 0;
C)F XCTII\;OIDIX
I
FIG.
TmlMN/
3
Effect of actinoidin on the "direct effect" of ristomycin on formaldehyde-treated platelets and on ristomycin-induced agglutination of formaldehyde-treated platelets in the presence of The following reactants were added at the points stated. VIIIR:WF. platelets, (B): 50 ~1 of acti(A): 400 ~1 formaldehyde-treated noidin (0.6 mg/ml), (Cl: 50 Fl TS as control, (D): 50 ~1 ristomycin (1.0 mg/ml) and (E): 50 ~1 undiluted reference plasma as source of VIIIR:WF.
TABLE
1
PROTEIN PRECIPITATION RESULTING FROM THE ADDITION OF ANTIBIOTICS OF THE VANCOMYCIN GROUP TO A CITRATED PLASMA (POOLED REFERENCE (See MatePLASMA) MEASURED AS INCREASE IN ABSORBANCY AT 600 nm. rials and Methods). CONCENTRATION
ANTIBIOTIC
OF ANTIBIOTIC
1.5 mg/ml
3.0 mg/ml
3.5 mg/ml
ACTINOIDIN
0
0
0
RISTOCETIN
0
0.587
1.548
RISTOMYCIN
0
1.184
1.952
2.123
2.125
VANCOMYCIN
1.565
EFFECTS
OF XCTIXOIDIS
DISCUSSIOX Actinoidin is another specific inhibitor of ristocetin- and ristomycin-induced platelet agglutination. its action is similar to that of vancomycin, but it has a great advantage in experimental studies in that its tendency to produce plasma protein precipitation is much less than that of vancomycin. As actinoidin totally biocks the ristocetin- and ristonycininduced agglutination of formaldehyde-treated platelets in concentrations which do not inhibit the cofactor-independent increase in absorbancy, this may mean that some binding of ristocetin or ristomycin to the platelet membrane may occur which is unrelated to the agglutination phenomenon. Agglutination of human platelets induced by the bovine factor VIII related protein was not affected by actinoidin. Many observations point to a common receptor site on the platelet membrane for both the bovine and the human factor VIII related proIf so, tein when the latter is added together with ristocetin. the inhibitory effect of actinoidin is not directed towards this binding site. Okumura and Jamieson (19) assumed that glycocalicin functioned as a common receptor both in ristocetin- and thrombininduced platelet aggregation. Weinger and Moake (20) could not confirm this in the sense that vancomycin was found to inhibit ristocetinSimilarly, our but not thrombin-induced platelet aggregation. observation that actinoidin also inhibits ristocetin- and ristomycin-induced platelet agglutination but not thrombin-induced aggregation, does not favour the concept of a common receptor. It is not possible from the present data to decide whether the antibiotics act on the platelet membrane or on the protein cofactor when exerting their agglutinating effect, or whether the inhibitory and the agglutination promoting antibiotics act at the same step in the sequence of events leading to the platelet Nevertheless, our data suggest that ristocetin or agglutination. ristomycin may in some way modify VIIIR:WF which then acts as bovine factor VIII related protein regarding platelet agglutination. These problems, first of all the question of the physiological counterpart of these activating agents, are considered to be of great importance and require further studies. ACKNOWLEDGEMENT The authors are indebted New Antibiotics, Moscow, actinoidin.
to Professor G.F. Gauze (Institute of USSR) for the samples of ristomycin and
The work was in part supported Cardiovascular Diseases.
by The Norwegian
Council
on
vo:.
iy,l‘;o.j
EFFECTS
OF XCTISOIDIS
REFERENCES 1.
XOAKE, J-L., CIlclO,P.L., PETERSON, D.M., ROPER, P. and NATELSON, E.A. Inhibition of ristocetin-induced platelet agglutination by vancomycin. Blood 50, 397-406, 1977. --
2.
COLLER, B.S. and GRALNICK, H.R. Studies on the mechanisms of ristocetin-induced platelet agglutination. J. Clin. Invest. 60, 302-312, 1977. -
3.
COLLER, B.S. The effects of ristocetin and von Willebrand factor on platelet electrophoretic mobility. J. Clin. Invest. 78, 1168-1175, 1978. -
4.
FLOYD, M., BURNS, K:. and GREEN, D. On the interaction between ristocetin and the ristocetin cofactor (RCOF). Thromb. Res. 10, 841-850, 1977.
5.
JENKINS, C.S.P., CLEMETSON, K.J. and LUSCHER, E.F. Studies on the mechanism of ristocetin-induced platelet agglutination: binding of ristocetin to platelets. J.Lab.Clin.Med. 93 -' 220-231, 1979.
6.
Platelet GREEN, D. and MULLER, H.P. brand factor. Thrombos. haemostas.
7.
Demonstration and KAO, K-J., PIZZO, S.V. and MCKEE, P-A. characterization of specific binding sites for factor VIII/ von Willebrand factor on human platelets. J.Clin.Invest. 63, 656-665, 1979.
a.
study of BODA, Z., SOLUM, N.O., SZTARICSKAI, F. and RAK, K. platelet agglutination induced by the antibiotics of the vancomycin group: Ristocetin, ristomyciil,actinoidin and vancomycin. Thrombos. haemostas. (in press).
9.
EVANS, R.J. and AUSTEN, D.E.G. Assay of ristocetin cofactor using fixed platelets and a platelet counting technique. Brit. J. Haematol. 37, 289-294, 1977.
binding of the von Wille39_, 689-694, 1978.
10. SORIN, V-A., JUDINCEV, S.D., KUHRAT, J-A., GOLDBERG, L.E., PEUZNER, N.S., BRAZHNIKOVA, M-G., LOMAKINA, N.N. and A new antibiotic, the actinoidine. OPAPUSEVA, E.F. Antibiotiki 2, 44-49, 1957. 11. LOMAKINA, N-N., YURINA, M-S., LAVROVA, M.F. and BRAZHNIKOVA, Actinoidin and its separation into biologically active M.G. variants. Antibiotiki 5, 609-619, 1961. 12. BOGNAR, R., MAKLETT, S., SZTARICSKAI, F., LOMAKINA, N.N. and Synthesis of the amino-acids of the hydroYURINA, M.S. lysate of actinoidins. Antibiotiki 2, 875-880, 1964.
fj 1 c>
EFFECTS
OF ACTIXOIDIS
i-ol.l,,No,j
13.
LOMAKINA, N.N., Yurina, M.S. and B.RAZHNiKOVA, X-G. Isolation and analysis of actinoidin hydrolysate amino-acid components. Antibiotiki, 2, 880-885, 1964.
14.
LOMAXINA, N-N., ZENKOVA, V.A. and YURINA, M.S. Absolute configurations of amino-acids of actinoidins. Khim. Prir. Soedin. 2, 43-46, 1969.
IS.
SZTARICSKAI, F., LOMAKINA, N.N., SPIRIDINOVA, I-A., YURINA, MS. and PUSKAS, M. Quantitative determination of carbohydrates in actinoidines A and B. Antibiotiki -12, 126-129, ,nc-f
16.
LOMAKINA, N.N., SPIRIDINOVA, I.A., SHEINKER, J.N. and VLASOVA, T.F. Structures of aminosugars isolated from actinoidine. Khim. Prir. Soedin. 1, 101-107, 1973.
17.
SOLUM, factor Serono (Ed.)
18.
FISCHER, W. and GERMER, W.D. Uber eine einfache Methode der direkten phasenoptischen Thrombocytenzghlung. Rantgen und Lab.-Praxis 10, 49-52, 1957.
19.
OKUMURA, T. and JAMIESON, G.A. Platelet single receptor for platelet aggregation or ristocetin. Thromb. Res. 8, 701-706,
20.
Vancomycin effect on thrombinWEINGER, R.S. and MOAKE, J.L. evidence against a common induced platelet aggregation: platelet membrane receptor for thrombin and ristocetin. 911-915, 1978. Thromb. Res. 12,
N.O. and PETERKA, M. Interactions of platelets with VIII related protein. In: Platelets and Thrombosis, Symposium, Milan 1976. D.C.B. Mills and F.I. Pareti New York and LondonAcademic Press. 1977, p.21.
glycocalicin: a induced by thrombin 1976.
in the rJnited Starves ootig-38~8/80/050603-08$0~.00/0
A NEW INHIBITOR OF RISTOCETIN- AND RISTOACTINOIDIN: :4YCIN-INDUCED PLATELET AGGLUTINATION Z. Boda, N.O. Solum, F. Sztaricskai and K. Rak IInd Department of Medicine, University Medical School, Debrecen, Hungary Research Institute for Internal Medicine, University of Oslo, Rikshospitalet, Oslo 1, Norway The Research Group of Antibiotics, Hungarian Academy of Sciences, Lajos Kossuth University, Debrecen, Hungary (Received
16.10.1979.
Accepted
by
Editor
H.C.
Godal)
ABSTRACT Actinoidin, like vancomycin, inhibits ristocetin-and ristomycin-induced VIIIR:WF-dependent agglutination of untreated or formaldehyde-fixed human platelets but does not interfere with bovine factor VIII-induced agglutination. The socalled "direct effect" of ristocetin and ristomycin on fixed platelets, which is represented by an immediate increase in light absorbancy in the absence of cofactor, was not blocked by actinoidin at concentrations which totally inhibited agglutination in the presence of VIIIR:WF. Actinoidin does not inhibit thrombin-induced platelet aggregation. This represents a further argument against a common platelet membrane receptor site for thrombin and ristocetin or a ristocetin-VIIIR:WF complex on the platelet membrane. INTRODUCTION The mode of action of ristocetin in the VIIIR:WF-dependent platelet agglutination is not established. The opinion dominates that most likely ristocetin binds to the platelet membrane and increases affinity of that to VIIIR:WF which at the end of events may act as a bridging protein between platelets (1,2,3). Other findings are consistent with the hypothesis that modification of VIIIR:WF by ristocetin precedes and initiates platelet agglutination. In fact, ristocetin has been observed to aggregate or polymerize VIII:WF in vitro at a concentration which agglutinates platelets (4). Binding of ristocetin to platelets was recently demonstrated by an isotope technique; the process was independent
Key Words:
Actinoidin,
platelet
agglutination
inhibitor.
EFFECTS
OF ACTIXGIDIX
vol.l;,~o.j
platelets of the presence of VIIIR:WF, and chymotrypsin-treated though unable to agglutinate, did not bind less ristocetin than the untreated platelets (5). It has also been demonstrated that VIIIR:WF binds to the platelet membrane in the presence of ristocetin (6) and that the binding sites are specific for VIIIR:WF(7). It is not yet decided whether attachment of ristocetin to platelets or to VIIIR:WF is the more important in respect to platelet agglutination. In a previous work (8) we have demonstrated that a striking similarity exists between ristocetin and ristomycin as inducers of platelet agglutination both in qualitative and quantitative terms which means that ristomycin can substitute ristocetin in practical and experimental laboratory work. In fact, ristomycin is advantageous in theoretical studies as its structure is better known and derivatives and fragments of ristomycin are available. We reported in the same paper that still another member of the vancomycin group, i.e. actinoidin, is a potent inhibitor of ristocetin-and ristomycin-induced platelet agglutination. The purpose of this paper is to present some more details on the effects of this antibiotic. MATERIALS
AND METHODS
Platelet rich plasma (PRP) was obtained from centrifugation of The blood was anticoaaulated whole blood at 320 x g for 15 min. by either 11 mM sodium citrate (citratg PRP) or 5.4 mM E&A (EDTA. Platelet count was 300-400 x 10 11. PRP). Platelet poor plasma (PPP) was prepared from whole blood by cenPlatelet count was below trifugabion at 2200 x g for 20 min. 10 x 10 /l. Formaldehyde-fixed telet concentrates
platelets (FFP) were prepared from fresh plaas described by Evans and Austen (9).
Reference plasma was obtained by pooling equal volumes of titrated PPP from eight healthy individuals (4 men and 4 women). Tris-buffered saline (TS) consists HCl pH 7.4, 280 mOsM.
of 148 mM NaCl and 20 mtM Tris-
Ristocetin sulphate was purchased from Lundbeck Copenhagen, Denmark, and dissolved in TS.
and Co. A/S,
Ristomycin A mono-sulphate and actinoidin A + B were obtained from Institute of New Antibiotics, Academy of Medical Sciences, Moscow, USSR,and dissolved in TS. Actinoidin was isolated from Proactimyces actinoides in 1957 (10). It consists of two components: actinoidin A and B _(ll) . Both components have three primary amino, four phenolic hydroxyl, one carboxyl and more carbohydrate hydroxyl groups. The total structure of actinoidin is not yet known. Both components possess phenylalanin and actinoic acid, but there are also known differences (12,13,14). Two new aminohexoses, acosamine and actosamine were found besides the neutral carbohydrates in both components (15,16).
Acrgregating materials were epinepnrine (Adrenalin, Richter Cc., Budapest, Hungary) in the concentration of 1%: ,$g/ml, ADP, Recezl, Budapest, Hungary) in i.5 and 0.3 pg/ml concentrations,collagen (Reagent Ho-q, Hunich, West Germany) in 4.2 and 3.3 pg/ml concentrations, thrombin !Topostasin, La Roche, Basel, Switzerland) in the concentrations of 3.0 and 1.0 U/ml and bovrne factor VIII related protein purified as previously described by Solum and Peterka (17) and used either as "intermediate" or "highly" purified material. Plasma protein precipitation was studied by addition of 100 ~1 of antibiotic in various dilutions to 900 ~1 of titrated plasma in the cuvette of a Beckman spectrophotometer model 35. After mixing and incubation at room temperature for 3 min, the absorbancy was read at 600 nm and used as a parameter of precipitation. Platelet counting or electronically Ltd., England) .
was performed by phase contrast microscopy (18) using a Thrombocounter-C (Coulter Electronics
Platelet agglutination was investigated at 37'C in a Payton Dual Channel Aggregometer or Chrono-Log Aggregometer (Model 330). In the standard system 400 ~1 citrate- or EDTA-PRP were preheated at 37OC for 3 min. Then 50 ~1 of ristomycin or ristocetin were added. The 8ystem containing FFP consisted of 400 J.L~of FFP preheated at 37 C for 3 min, 50 ~1 of actinoidin or TS (control), 50 ~1 of ristomycin or ristocetin and 50 ~1 of undiluted reference plasma. The Payton aggregometer was calibrated in such a way that the difference in light absorption between PRP and PRP diluted l/2 with autologous PPP corresponded to a recorder deflection of 50 chart division (50 mV) The Chrono-Log-Aggregometer was calibrated so that the difference in light absorption between PRP and PPP corresponded to a recorder deflection of 80 chart division (80 mv).
RESULTS Incubation of citrate- or EDTA-PRP at 37OC for 3 min with actinoidin in concentrations varying from 0.6 to 2.5 mg/ml resulted in a complete inhibition of ristocetin- or ristomycininduced platelet agglutination at a concentration of 1.5 mg/ml of these A partial inhibition was observed at actinoidin concenagents. trations between 0.15 and 0.6 mg/ml (Fig. 1). Actinoidin did notinhibitplatelet agglutination induced by bovine factor VIII related protein or aggregation induced by This is demonstrated in thrombin, epinephrine, ADP or collagen. Fig. 2 for bovine factor VIII related protein and thrombin. Actinoidin completely inhibited ristocetin- or ristomycininduced platelet agglutination of formaldehyde-treated platelets at concentrations which did not affect the "direct effect" seen as an increase in absorbancy in the absence of cofactor (Fig.3). All antibiotics
of the vancomycin
group induced
a protein
606
EFFECTS
Yol.l,,So.~
OF ACTIXOIDI?;
At the same concenprecipitation when added to titrated plasma. tration the degree of protein precipitation increased in sequence (Table 1). from actinoidin, ristocetin, ristomycin and vancomycin
b 40 -
20 Jo4050607080-
-*
“\
b
c
FIG. 1 platelet agglutination by actiInhibition of ristomycin-induced Final concentranoidin in (a): EDTA-PRP and (b) : citrate-PRP. tions of actinoidin were (A): 2.5 mg/ml and (B): 0.4 mg/ml. Control (C) was with TS only. Concentration of ristomycin 1.5 mg/ml
0 40 20 30 40 50
60 70 80
a
% ,),,,, C
b
A
4
8
B
3042042
nME/fliN./
FIG. 2 Effect of actinoidin on (a): bovine factor VIII-induced or (b = Final c): thrombin-induced platelet agglutination in citr-PRP. Control with concentration of actinoidin was (A): 2.5 mg/ml. (B) : Final concentrations of thrombin were (b): 0.3 U/ml and TS only. (cl: 0.1 U/ml.
EFFECT3
i 0;
C)F XCTII\;OIDIX
I
FIG.
TmlMN/
3
Effect of actinoidin on the "direct effect" of ristomycin on formaldehyde-treated platelets and on ristomycin-induced agglutination of formaldehyde-treated platelets in the presence of The following reactants were added at the points stated. VIIIR:WF. platelets, (B): 50 ~1 of acti(A): 400 ~1 formaldehyde-treated noidin (0.6 mg/ml), (Cl: 50 Fl TS as control, (D): 50 ~1 ristomycin (1.0 mg/ml) and (E): 50 ~1 undiluted reference plasma as source of VIIIR:WF.
TABLE
1
PROTEIN PRECIPITATION RESULTING FROM THE ADDITION OF ANTIBIOTICS OF THE VANCOMYCIN GROUP TO A CITRATED PLASMA (POOLED REFERENCE (See MatePLASMA) MEASURED AS INCREASE IN ABSORBANCY AT 600 nm. rials and Methods). CONCENTRATION
ANTIBIOTIC
OF ANTIBIOTIC
1.5 mg/ml
3.0 mg/ml
3.5 mg/ml
ACTINOIDIN
0
0
0
RISTOCETIN
0
0.587
1.548
RISTOMYCIN
0
1.184
1.952
2.123
2.125
VANCOMYCIN
1.565
EFFECTS
OF XCTIXOIDIS
DISCUSSIOX Actinoidin is another specific inhibitor of ristocetin- and ristomycin-induced platelet agglutination. its action is similar to that of vancomycin, but it has a great advantage in experimental studies in that its tendency to produce plasma protein precipitation is much less than that of vancomycin. As actinoidin totally biocks the ristocetin- and ristonycininduced agglutination of formaldehyde-treated platelets in concentrations which do not inhibit the cofactor-independent increase in absorbancy, this may mean that some binding of ristocetin or ristomycin to the platelet membrane may occur which is unrelated to the agglutination phenomenon. Agglutination of human platelets induced by the bovine factor VIII related protein was not affected by actinoidin. Many observations point to a common receptor site on the platelet membrane for both the bovine and the human factor VIII related proIf so, tein when the latter is added together with ristocetin. the inhibitory effect of actinoidin is not directed towards this binding site. Okumura and Jamieson (19) assumed that glycocalicin functioned as a common receptor both in ristocetin- and thrombininduced platelet aggregation. Weinger and Moake (20) could not confirm this in the sense that vancomycin was found to inhibit ristocetinSimilarly, our but not thrombin-induced platelet aggregation. observation that actinoidin also inhibits ristocetin- and ristomycin-induced platelet agglutination but not thrombin-induced aggregation, does not favour the concept of a common receptor. It is not possible from the present data to decide whether the antibiotics act on the platelet membrane or on the protein cofactor when exerting their agglutinating effect, or whether the inhibitory and the agglutination promoting antibiotics act at the same step in the sequence of events leading to the platelet Nevertheless, our data suggest that ristocetin or agglutination. ristomycin may in some way modify VIIIR:WF which then acts as bovine factor VIII related protein regarding platelet agglutination. These problems, first of all the question of the physiological counterpart of these activating agents, are considered to be of great importance and require further studies. ACKNOWLEDGEMENT The authors are indebted New Antibiotics, Moscow, actinoidin.
to Professor G.F. Gauze (Institute of USSR) for the samples of ristomycin and
The work was in part supported Cardiovascular Diseases.
by The Norwegian
Council
on
vo:.
iy,l‘;o.j
EFFECTS
OF XCTISOIDIS
REFERENCES 1.
XOAKE, J-L., CIlclO,P.L., PETERSON, D.M., ROPER, P. and NATELSON, E.A. Inhibition of ristocetin-induced platelet agglutination by vancomycin. Blood 50, 397-406, 1977. --
2.
COLLER, B.S. and GRALNICK, H.R. Studies on the mechanisms of ristocetin-induced platelet agglutination. J. Clin. Invest. 60, 302-312, 1977. -
3.
COLLER, B.S. The effects of ristocetin and von Willebrand factor on platelet electrophoretic mobility. J. Clin. Invest. 78, 1168-1175, 1978. -
4.
FLOYD, M., BURNS, K:. and GREEN, D. On the interaction between ristocetin and the ristocetin cofactor (RCOF). Thromb. Res. 10, 841-850, 1977.
5.
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