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Two dimensional immunoelectrophoresis of antithrombin III during disseminated intravascular coagulation in acute leukemia
rHRoMBOSIS RESEARCH Printed in Great
BRIEF
Vol. Britain
PP. 191-196, 1977 Pergamon Press, Ltd.
12,
COMMUNICATION
TWO DIMENSIONAL IMMUNOElECTROPHORESISOF ANTITHROMBIN III DURING DISSEMINATED INTRAVASCUUR COAGULATION IN ACUTE LEDEEMIA Francesco Rodeghiero and Tiziano Barbui Division of Haematology "San BortolollHospital Vicenza,Italy
(Received
19.8.1977; Accepted by
in revised Editor P.M.
form 7.11.1977. Mannucci)
INTRODUCTION Antithrombin III (AT-1II)inactivatesblood coagulation factors such as thrombin,factorXa,factors IXa and XIa,in addition to kallikrein and plasmin,throu& the formation of stoichiometric complexes (4,8,9,15).Ihedetection of these complexes would be indicative of physiologicalor pathologicalactivation of blood coagulation factors.Saset al.(lO,ll)foundthat three separate precipitationpeaks of Isnmmo-AT-III(IAT-III) could be obtained when heparin was added to the agarose gel in the first dimension.If the binding sites on antithroaibin III are wholly or partly occupied by activated clotting factors,theinmnmoprecipitation pattern changes and shows an increase of the fractions IAT-IIIgand IAT-III3 concomitantlywith a decrease of the main molecular fraction called IAT-1111. We report here the modifications of IAT-III electrophoretic pattern during disseminated intravascularcoagulation(DIC)observed in patients with acute leukemia. 191
192
AT-III
DURING
DIC IN LEUKEMIA
Vol.12,No.l
MATERIAL AND METHODS Acute leukemia was diagnosed on the basis of the bone marrow and peripheral examinations including also cytochemical atains. . Blood was collected into 0.1~01 3.8% sodium citrate anticoagulant containing 0.1 epsilon-amino-caproicacid,centrifuat 1,500 g for 20 minutes and deep frozen(-30°C).l'he one ged stage prothrombin time(PT the thrombin time(TT) performed with standard techniques using Behringwerkereagents.Fibrinogen levels were measured according to a heat precipitation method (14)and immunologicallyusing Partigen-fibrinogenBehringwerke.Ihe fibrinogen-fibrindegradation products(FDP)in serum were tested using latex particles agglutinationmethod (Thrombo-Wellcotest,Wellcome).The Ethanol Gelation Test(EGT) and the ProtamLneGelation Test(PGT)were performed according to Godal and Abildgaard(3)and to Niewiarowski and Gurewich(7) respectively;onlyformation of fibrin-like structure of gel (but not precipitate)wasconsidered positive.FactorXIII subunits-A and-S were measured as previously described(l,2).Plasminogen and antithrombin III were determined immunologicallyusing the Laurel1 technique(5,6):theagarose concentrationwas 1.2% in Tris-sodium-barbitalbuffer pH 8.8,ionic strenght 0.02, the antiserum concentrationwas 0.77.and 0.8% for plasminogen and antithrombin III respectively.Crossedelectrophoresisof AT-III was performed according to Sas et a1.(10):13 ml of 1.2% agarose solution in Tris-sodium-barbitalbuffer pH 8.8,ionic strenght O.OS,containing16.6 U/ml heparintliquemin)were poured onto a glass plate 100x100 mm in size;after solidification 5 ul of plasma sample were applied in a well of 2.5 mm diameter. After the first dimension run at 10 V/cm until the marker dye (albumin stained with bromophenol blue)added to samples migrated 5.5 cm (approximately2 hours),the gel was cut # cm internal to the sample well and parallel to the direction of electrophoresis.lhelarge segment of agarose was removed and replaced by 10 ml of agarose containing instead of heparin, 1% antithrombin III antiserum(Behringwerke).Ihe plate was then rotated at 90Oand electrophoresiscontinued at 2.5 V/cm wernight.After electrophoresisthe plates remained in saline for 12 hours and distilled water 1 hour,then dried and stained with coomassie brillant blue(Serva). RESULTS AND DISCUSSION Coagulation findings consistent with DIC are reported in Table 1. Patients 1,2,3 presented
severe defibrinogenation;theiussu-
THROMBOSIS RESEARCH Printed in Great
BRIEF
vol. Britain
12, pp. 191-196, 1977 Pergamon Press, Ltd.
COMMUNICATION
TWO DIMENSIONAL IMMUNOELECTROPHORESISOF ANTITEROMBIN III DURING DISSEMINATED INTRAVASCULARCOAGULATION IN ACUTE LEUREMIA Francesco Rodeghiero and Tiziano Barbui Division of Haematology "San BortololtHospital Vicensa,Italy
(Received
19.8.1977; Accepted by
in revised Editor P.M.
form 7.11.1977. Mannucci)
INTRODUCTION Antithrombin III (AT-1II)inactivatesblood coagulation factors such as thrombin,factorXa,factors IXa and XIa,in addition to kallikrein and plasmin,throughthe formation of stoichiometric complexes (4,8,9,15).Ihedetection of these complexeswould be indicative of physiologicalor pathologicalactivation of blood coagulation factors.Saset al.(lO,ll)foundthat three separate precipitationpeaks of Iamum-AT-III (IAT-III) could be obtained nhen heparin was added to the agarose gel in the first dimension.Ifthe binding sites on antithrombin III are holly or partly occupied by activated clotting factors,the lmwnoprecipltation pattern changes and shows an increase of the fractious IAT-IIQand IAT-III3 concomitantlywith a decrease of the main molecular fraction called IAT-III1. We report here the modificationsof IAT-IXI electrophoretic pattern during disseminated intravascularcoagulation(DIC)observed in patients with acute leukemia. 191
192
AT-III
DURING
DIC IN LEUKEMIA
Vol.12,No.l
MATERIAL AND METHODS Acute leukemia was diagnosed on the basis of the bone marrow and peripheral examinations including also cytochemical stains. Blood was collected Into 0.1~01 3.8% sodium citrate anticoagulant containing 0.1 epsilon-amino-caproicacid,centrifuat 1,500 g for 20 minutes and deep frozen(-30°C).Iheone ged stage prothrombin time(PT the thrombin time(TT) performed with standard techniques using Behringwerkereagents.Fibrinogen levels were measured according to a heat precipitation method (14)and inmamologicallyusing Partigen-fibrinogenBehringwerke.The fibrinogen-fibrindegradation products(FDP)in serum were tested using latex particles agglutinationmethod (Ihrombo-Wellcotest,Wellcome).Ihe Ethanol Gelation Test(EGT) and the ProtandneGelation Test(PGT)wereperformed according to Godal and Abildgaard(3)and to Niewiarowski and Gurewich(7) respectively;onlyformation of fibrin-like structure of gel (but not precipitate)wasconsidered positive.FactorXIII subunits-A and-S were measured as previously described(l,2) .Plasminogen and antithrombin III were determined immunologicallyusing the Laurel1 technique(5,6):theagarose concentrationwas 1.22 in Tris-sodium-barbitalbuffer pH 8.8,ionic strenght 0.02, the antiserum concentrationwas 0.7% and 0.82 for plasmlnogen and antithrombin III respectively.Crossedelectrophoresisof AT-III was performed according to Sas et a1.(10):13ml of 1.2% agarose solution in Tris-sodium-barbitalbuffer pH 8.8,ionic strengFt O.OP,containing16.6 U/ml heparin(Liquemin)were poured onto a glass plate 100x100 mn in sixe;after solidification 5 ul of plasma sample were applied in a well of 2.5 nundiameter. After the first dimension run at 10 V/cm until the marker dye (albumin stained with bromophenol blue)added to samples mfgrated 5.5 cm (approximately2 hours),the gel was cut # cm internal to the sample well and parallel to the direction of electrophoresis.Ihelarge segment of agarose was removed and replaced by 10 ml of agarose containing instead of heparln, 1% antithrombin III antiserum(Behringwerke).The plate was then rotated at 90Oand electrophoreslscontinued at 2.5 V/cm overnight.After electrophoresisthe plates remained in saline for 12 hours and distilled water 1 hour,then dried and stained with coomassie brillant blue(Serva). RESULTS AND DISCUSSION Coagulation findings consistent with DIC are reported in Table 1. Patients 1,2,3 presented
severe defibrlnogenatioqthe inssu-
Vol.12,No.l
AT-III
DURING
DIC
195
IN LEUKEMIA
showed a clear increase of AT-1112,not observed in the other cases.This finding corresponds to the results obtained by Sas et al.(l2)uho found that the AT-III2 arc results from the corn-plex
thrombin-antithrombin,experimentally produced "in vitro'@. ThetIn vivo*lthrombin generation,asshown in the case with
severe DIC,reproducesthis pattem,which occurs only in
some
of the typical DIC cases.This observation confirms recent results obtained by Sas et a1.(13). REFERENCES ~.BARBUI T.,CARTEI C.,CHISESI T.,DINI E.:Electroimmunoassayof plasma subunits-Sand A in a case of congenital fibrin stabilizing factor deficiency.'Ihromb.Diath.Haemorrh.32,124,1974. 2.BARBUI T.,RODEGHIEROF.,DINI E.,MARIANI G.,PAPA M.L.,DE BIASI R.,CORDERO MURILLG R.,MONTERO UMANA C.:Subunits-Aand-s inheritance in four families with congenital factor-XIIIdeficiency.Brit.J.Haemat.1977,in press. 3.GODAL H.C.,ABILDGAARDU.:Gelation of soluble fibrin in plasma by ethanol.Scand.J.Haemat.3,342,1966. 4.LAHIRI B.,ROSENBERG R.D.,TALAMO R.L.,BAGDASARIANA.,COIMAN R. W.:AntithrombinIII:an inhibitor of human plasma kallikrein. Fed.Proc.33,642,1974. S.LAURELL C.B.:Quantitativeestimation of proteins by electrophoresis in agarose gel containing antibodies.Anal.Bioch. 15,45,1966. 6.LAURELL C.B.:Electroimmunoassay.Scand.J.Clin.Lab.Invest. 29(suppl.l24)21,1972. 7.NIEWIAROWSKI S.,GUREWIQlV.:Laboratory identificationof intravascularcoagulation.J.Lab.Clin.Med.77,665,1971. 8.ROSENBERG R.D.,DAMIS P.S.:The purification and mechanism of action of human antithrombin-heparincofactor.J.Biol.Chem. 248,6490,1973. 9.ROSWBERG R.D.:The effect of heparin on factor Ma and plasmin.l'hromb.Diath.Haemorrh.33,51,1974. lO.SAS G.,PEPPER D.S.,CASH J.D.:Investigationon antithrombin III in normal plasma and serum.Brit.J.Haemat.30,265,1975. ll.SAS G.,PEPPER D.S.,CASH J.D.:Plasma and serum antithrombin III:Differentiationby crossed imnnmoelectrophoresis. Thromb.Diath.Haemorrh.6,87,1975. 12.SAS G.,PEPPER D.S.:Detectionof thrombin-antithrombinIII cow plex by crossed inmnmoelectrophoresis.Ihromb.Res.9,95,1976. 13.SAS G.,KC%ES A.,PETO I.:Detectionof antithrombin-III(AT-III)
196
AT-III DURING DIC IN LEUKEMIA
Vol.l2,No.1
complexes in %ypercoagulableffand hyperfibrinolyticstates. Itmxnb,Haemost.(Abstract)38,164,1977. 14.SCHULTZF.H.:Eine einfache volumetrishe fibrinbestimung. Art.Lab.l,107,1955. &YIN E.T.:Effect of heparin on the neutralizationof factor xa and throntbinby the plasma alpha-2-globulininhibitor. 'Ihromb.Diath.Haemorrh.33,43,1974.
BRIEF
Vol. Britain
PP. 191-196, 1977 Pergamon Press, Ltd.
12,
COMMUNICATION
TWO DIMENSIONAL IMMUNOElECTROPHORESISOF ANTITHROMBIN III DURING DISSEMINATED INTRAVASCUUR COAGULATION IN ACUTE LEDEEMIA Francesco Rodeghiero and Tiziano Barbui Division of Haematology "San BortolollHospital Vicenza,Italy
(Received
19.8.1977; Accepted by
in revised Editor P.M.
form 7.11.1977. Mannucci)
INTRODUCTION Antithrombin III (AT-1II)inactivatesblood coagulation factors such as thrombin,factorXa,factors IXa and XIa,in addition to kallikrein and plasmin,throu& the formation of stoichiometric complexes (4,8,9,15).Ihedetection of these complexes would be indicative of physiologicalor pathologicalactivation of blood coagulation factors.Saset al.(lO,ll)foundthat three separate precipitationpeaks of Isnmmo-AT-III(IAT-III) could be obtained when heparin was added to the agarose gel in the first dimension.If the binding sites on antithroaibin III are wholly or partly occupied by activated clotting factors,theinmnmoprecipitation pattern changes and shows an increase of the fractions IAT-IIIgand IAT-III3 concomitantlywith a decrease of the main molecular fraction called IAT-1111. We report here the modifications of IAT-III electrophoretic pattern during disseminated intravascularcoagulation(DIC)observed in patients with acute leukemia. 191
192
AT-III
DURING
DIC IN LEUKEMIA
Vol.12,No.l
MATERIAL AND METHODS Acute leukemia was diagnosed on the basis of the bone marrow and peripheral examinations including also cytochemical atains. . Blood was collected into 0.1~01 3.8% sodium citrate anticoagulant containing 0.1 epsilon-amino-caproicacid,centrifuat 1,500 g for 20 minutes and deep frozen(-30°C).l'he one ged stage prothrombin time(PT the thrombin time(TT) performed with standard techniques using Behringwerkereagents.Fibrinogen levels were measured according to a heat precipitation method (14)and immunologicallyusing Partigen-fibrinogenBehringwerke.Ihe fibrinogen-fibrindegradation products(FDP)in serum were tested using latex particles agglutinationmethod (Thrombo-Wellcotest,Wellcome).The Ethanol Gelation Test(EGT) and the ProtamLneGelation Test(PGT)were performed according to Godal and Abildgaard(3)and to Niewiarowski and Gurewich(7) respectively;onlyformation of fibrin-like structure of gel (but not precipitate)wasconsidered positive.FactorXIII subunits-A and-S were measured as previously described(l,2).Plasminogen and antithrombin III were determined immunologicallyusing the Laurel1 technique(5,6):theagarose concentrationwas 1.2% in Tris-sodium-barbitalbuffer pH 8.8,ionic strenght 0.02, the antiserum concentrationwas 0.77.and 0.8% for plasminogen and antithrombin III respectively.Crossedelectrophoresisof AT-III was performed according to Sas et a1.(10):13 ml of 1.2% agarose solution in Tris-sodium-barbitalbuffer pH 8.8,ionic strenght O.OS,containing16.6 U/ml heparintliquemin)were poured onto a glass plate 100x100 mm in size;after solidification 5 ul of plasma sample were applied in a well of 2.5 mm diameter. After the first dimension run at 10 V/cm until the marker dye (albumin stained with bromophenol blue)added to samples migrated 5.5 cm (approximately2 hours),the gel was cut # cm internal to the sample well and parallel to the direction of electrophoresis.lhelarge segment of agarose was removed and replaced by 10 ml of agarose containing instead of heparin, 1% antithrombin III antiserum(Behringwerke).Ihe plate was then rotated at 90Oand electrophoresiscontinued at 2.5 V/cm wernight.After electrophoresisthe plates remained in saline for 12 hours and distilled water 1 hour,then dried and stained with coomassie brillant blue(Serva). RESULTS AND DISCUSSION Coagulation findings consistent with DIC are reported in Table 1. Patients 1,2,3 presented
severe defibrinogenation;theiussu-
THROMBOSIS RESEARCH Printed in Great
BRIEF
vol. Britain
12, pp. 191-196, 1977 Pergamon Press, Ltd.
COMMUNICATION
TWO DIMENSIONAL IMMUNOELECTROPHORESISOF ANTITEROMBIN III DURING DISSEMINATED INTRAVASCULARCOAGULATION IN ACUTE LEUREMIA Francesco Rodeghiero and Tiziano Barbui Division of Haematology "San BortololtHospital Vicensa,Italy
(Received
19.8.1977; Accepted by
in revised Editor P.M.
form 7.11.1977. Mannucci)
INTRODUCTION Antithrombin III (AT-1II)inactivatesblood coagulation factors such as thrombin,factorXa,factors IXa and XIa,in addition to kallikrein and plasmin,throughthe formation of stoichiometric complexes (4,8,9,15).Ihedetection of these complexeswould be indicative of physiologicalor pathologicalactivation of blood coagulation factors.Saset al.(lO,ll)foundthat three separate precipitationpeaks of Iamum-AT-III (IAT-III) could be obtained nhen heparin was added to the agarose gel in the first dimension.Ifthe binding sites on antithrombin III are holly or partly occupied by activated clotting factors,the lmwnoprecipltation pattern changes and shows an increase of the fractious IAT-IIQand IAT-III3 concomitantlywith a decrease of the main molecular fraction called IAT-III1. We report here the modificationsof IAT-IXI electrophoretic pattern during disseminated intravascularcoagulation(DIC)observed in patients with acute leukemia. 191
192
AT-III
DURING
DIC IN LEUKEMIA
Vol.12,No.l
MATERIAL AND METHODS Acute leukemia was diagnosed on the basis of the bone marrow and peripheral examinations including also cytochemical stains. Blood was collected Into 0.1~01 3.8% sodium citrate anticoagulant containing 0.1 epsilon-amino-caproicacid,centrifuat 1,500 g for 20 minutes and deep frozen(-30°C).Iheone ged stage prothrombin time(PT the thrombin time(TT) performed with standard techniques using Behringwerkereagents.Fibrinogen levels were measured according to a heat precipitation method (14)and inmamologicallyusing Partigen-fibrinogenBehringwerke.The fibrinogen-fibrindegradation products(FDP)in serum were tested using latex particles agglutinationmethod (Ihrombo-Wellcotest,Wellcome).Ihe Ethanol Gelation Test(EGT) and the ProtandneGelation Test(PGT)wereperformed according to Godal and Abildgaard(3)and to Niewiarowski and Gurewich(7) respectively;onlyformation of fibrin-like structure of gel (but not precipitate)wasconsidered positive.FactorXIII subunits-A and-S were measured as previously described(l,2) .Plasminogen and antithrombin III were determined immunologicallyusing the Laurel1 technique(5,6):theagarose concentrationwas 1.22 in Tris-sodium-barbitalbuffer pH 8.8,ionic strenght 0.02, the antiserum concentrationwas 0.7% and 0.82 for plasmlnogen and antithrombin III respectively.Crossedelectrophoresisof AT-III was performed according to Sas et a1.(10):13ml of 1.2% agarose solution in Tris-sodium-barbitalbuffer pH 8.8,ionic strengFt O.OP,containing16.6 U/ml heparin(Liquemin)were poured onto a glass plate 100x100 mn in sixe;after solidification 5 ul of plasma sample were applied in a well of 2.5 nundiameter. After the first dimension run at 10 V/cm until the marker dye (albumin stained with bromophenol blue)added to samples mfgrated 5.5 cm (approximately2 hours),the gel was cut # cm internal to the sample well and parallel to the direction of electrophoresis.Ihelarge segment of agarose was removed and replaced by 10 ml of agarose containing instead of heparln, 1% antithrombin III antiserum(Behringwerke).The plate was then rotated at 90Oand electrophoreslscontinued at 2.5 V/cm overnight.After electrophoresisthe plates remained in saline for 12 hours and distilled water 1 hour,then dried and stained with coomassie brillant blue(Serva). RESULTS AND DISCUSSION Coagulation findings consistent with DIC are reported in Table 1. Patients 1,2,3 presented
severe defibrlnogenatioqthe inssu-
Vol.12,No.l
AT-III
DURING
DIC
195
IN LEUKEMIA
showed a clear increase of AT-1112,not observed in the other cases.This finding corresponds to the results obtained by Sas et al.(l2)uho found that the AT-III2 arc results from the corn-plex
thrombin-antithrombin,experimentally produced "in vitro'@. ThetIn vivo*lthrombin generation,asshown in the case with
severe DIC,reproducesthis pattem,which occurs only in
some
of the typical DIC cases.This observation confirms recent results obtained by Sas et a1.(13). REFERENCES ~.BARBUI T.,CARTEI C.,CHISESI T.,DINI E.:Electroimmunoassayof plasma subunits-Sand A in a case of congenital fibrin stabilizing factor deficiency.'Ihromb.Diath.Haemorrh.32,124,1974. 2.BARBUI T.,RODEGHIEROF.,DINI E.,MARIANI G.,PAPA M.L.,DE BIASI R.,CORDERO MURILLG R.,MONTERO UMANA C.:Subunits-Aand-s inheritance in four families with congenital factor-XIIIdeficiency.Brit.J.Haemat.1977,in press. 3.GODAL H.C.,ABILDGAARDU.:Gelation of soluble fibrin in plasma by ethanol.Scand.J.Haemat.3,342,1966. 4.LAHIRI B.,ROSENBERG R.D.,TALAMO R.L.,BAGDASARIANA.,COIMAN R. W.:AntithrombinIII:an inhibitor of human plasma kallikrein. Fed.Proc.33,642,1974. S.LAURELL C.B.:Quantitativeestimation of proteins by electrophoresis in agarose gel containing antibodies.Anal.Bioch. 15,45,1966. 6.LAURELL C.B.:Electroimmunoassay.Scand.J.Clin.Lab.Invest. 29(suppl.l24)21,1972. 7.NIEWIAROWSKI S.,GUREWIQlV.:Laboratory identificationof intravascularcoagulation.J.Lab.Clin.Med.77,665,1971. 8.ROSENBERG R.D.,DAMIS P.S.:The purification and mechanism of action of human antithrombin-heparincofactor.J.Biol.Chem. 248,6490,1973. 9.ROSWBERG R.D.:The effect of heparin on factor Ma and plasmin.l'hromb.Diath.Haemorrh.33,51,1974. lO.SAS G.,PEPPER D.S.,CASH J.D.:Investigationon antithrombin III in normal plasma and serum.Brit.J.Haemat.30,265,1975. ll.SAS G.,PEPPER D.S.,CASH J.D.:Plasma and serum antithrombin III:Differentiationby crossed imnnmoelectrophoresis. Thromb.Diath.Haemorrh.6,87,1975. 12.SAS G.,PEPPER D.S.:Detectionof thrombin-antithrombinIII cow plex by crossed inmnmoelectrophoresis.Ihromb.Res.9,95,1976. 13.SAS G.,KC%ES A.,PETO I.:Detectionof antithrombin-III(AT-III)
196
AT-III DURING DIC IN LEUKEMIA
Vol.l2,No.1
complexes in %ypercoagulableffand hyperfibrinolyticstates. Itmxnb,Haemost.(Abstract)38,164,1977. 14.SCHULTZF.H.:Eine einfache volumetrishe fibrinbestimung. Art.Lab.l,107,1955. &YIN E.T.:Effect of heparin on the neutralizationof factor xa and throntbinby the plasma alpha-2-globulininhibitor. 'Ihromb.Diath.Haemorrh.33,43,1974.