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CD32-dependent manner
40
109
POSTER PRESENTATIONS P-2: Interrelation
Between Coagulation
PLASMA CLOT LYSIS BY STAPHYLOKINASE IN VITRO EFFECTS ON COAGULATION AND PL.ATELETS Hauptmann .I, Stebam 1. and Research Centre for Vascular Department of Experimental Nordhauser Str 78, D-99089
Glusa E Biology and MedIcme Ang~ology and Haemostaseology Erfurt. Germany
Staphyloklnase (SAK) IS a bacterial plasmunogen activator snmlar to streptokinase (SK) m Its mode of action. Recombinant wild-type SAti and several variants are now available and have been shown fibrlnolyttally and thrombolytically effective Here, the tibrlnolytlc actlon of SAK IS compared to that of SK and t-PA m a clot lysls system usmg radioactively labelled human plasma clots The parameter clot lys~s was determIned 111comparison to the fibrinogen concentration of the plasma and to standard clottmg times (APTT, TT) The state of platelet actlvatlon in the plasma nuheu of the clots was evaluated by measuring the formation of thromboxane In separate expertmerits, platelet aggegation Induced by ADP and collagen was studled 111 whole blood and !n platelet rich plasma (PRP), moreover, flow cytometry was carried out v&h PRP after actlvatton by ADP After a 4hr uncubatlon 50 % lysls of plasma clots occurred at I3 nrnolil of SAK and at 8 mnol/l of t-PA, whereas SK did not lead to 50 l/o lysls
110 IIIJMAN
AN’I‘IHOl~lliS AN’I‘I-S7‘K~I”rOKlNAS~ AGGRliGA’l‘lON IN A I+/ C1k32-I~EFt~NI~ENT MANNIX ]J. Lehraxi, 2 L! (’ llelft 2 h Varhcron, l blbl. Samama j M. INIXJCLi
Pl~l‘El.E’l‘
Mirshahi -1
l ‘I‘. Leromnte
lIaboratoire Cardiol.
Central
d’Il~matologie-llhlel-l)ieu,
HCpital Necker,
2 Clinique
~31NSEKIl Ll 86 - Paris, l~ranc-e.
Exposure to streptokinase (SK) raises anti-SK antihodies (Abs). Their functional properties include inhibition of SK-induced fibrinolysis and induction of platelet (plt) aggregation. The mechanism of the latter seems to involve pit binding of a plasminogen--SK-anti-SK Ahs ternar? complex. Anti-SK Abs were purified hy- affinity chromatography from serum of patients having received SK for acute myocardial infarction, and were shown to be IgG. Their effects were studied with (i) human pits in titrated plasma in the presence of SK or APSAC, or (ii) washed pits. resuspended in a ‘Ij.rode huffer and after lowering the ionic strength, in the presence of APSAC (which brings both SK and (anisyolated) plasminogm).
111 BENEFICIAL EFFECTS OF HEPARIN COATED DEVICES AND APROTININ ON BLOOD CELLS AND CONTACT SYSTEM ACTIVATION IN AN IN VITRO CARDIOPULMONARY BYPASS MODEL Wendel HP, Ga llimore MJ, Heller W Dept. of Thoracic and Cardiovascular Surg., Univ. of Tuebingen, Germany We have previously described model for studying contact system activation and changes in blood cells in simulated cardiopulmonary bypass (CPB). We have also shown that aprotinin (Ap) at therapeutic concentrations reduces contact system activation in CPB surgery. In the present study we have compared heparinised (H) and non-heparinised (NH) oxygenators (Medtronic), with and without Ap (220 KIU/mI blood) in the model. Heparin was added to the blood; 1.0 IU/ml in the H-group and 3.0 IUlml in the NH-group. Eight separate CPB runs were performed with: Gpl-NH no Ap, Gp2-H no Ap, Gp3-NH plus Ap, GpCH plus Ap.
and Fibrinolysis
up to a concentranon of I pmol/l .A1 these concentrations SAK and 1PA did not lower plasma fibrmogen. whereas wth SK residual fibrinogen was _ IO % Flbrlnogenolytlcally eqmeffective concentrations of SK. t-PA and SAK were 7.2, 50. and 750 mnolil. resp AET and TT (more sensnlve) were Inversely correlated wnh residual plasma fibrinogen Up to a concentration of IO pmol/l, SAK had almost no mfluence on platelet aggre%atlon. t-PA and SK lnhlbned ADP-Induced aggregation 111whole blood and 11, PRP slgnlficantly at I pmolil Thromboxane formanon was Inhlbtted parallel to the lnhlbltlon of aarega:atlon Flow cytometry findIngs corroborated the above results v&h SAK showing no mfluence on platelets at I pmol/l In the Ilght of the above results recombinant SAK 1s an effective fibrinspecific fibrlnol_vlc agent. nearly eqmpotent to t-PA 111 lyslng SKreslstent plasma clots. wnh a low potential toward dlsturblng coagulation and platelets 111vitro
An Ah concentration response curve was obtain& with a plateau in a presence of 0.1 mg/ml of IgG. Increasing the concentration of APSAC gave an unimodal response curve, the optima1 roncentration heing 0.05 U/ml. Chelating calcium with EDTA, hlorking fibrinogen hinding with the RGDS peptide, and raising intraplatet rAblP with lloprost suppressed aggregation. Aggregation requires of the interaction of the Fr domain of the purified IgG directed against SK with the plt I+gamma receptor type II. also named CD32, sinre : (j) it is hlorked by the anti-CM2 monoclonal Ah IV-3 (ii) it does not orcur with F(ah)‘2 fragments whirh hlork the response to the intact IgG. The rliniral relevance (prothrombotic?) of these plt activating antihodies remains to he determined. Two factors might inlluence the clinical outcome: (i) the amount and t).pe of pre-existing anti-SK Abs:(ii) the known interindividual variability of the plt response to plt-hindable and plt activating IgG involving the CD.32 mrrlnr..ln . ..“._. _._.
Blood samples were taken before and after 1, 5, 10, 30, 60 and 90 minutes circulation. Platelets counts (PC), platelet factor 4 (PF4), PMN elastase-alphal-proteinase inhibitor complexes (PMNE-alPI), kallikrein-like (KK) and FXIIalike (FXIIa) activities, determined. The mean values for the various parameters for the four groups after 90 minutes CPB arc shown below (Initial values = 100%). GPl GP2 GP3 GP4 PF4 616 293 453 237 PMNEalP1 462 260 316 187 KK 209 145 197 150 FXIIa 245 174 176 161 The results show that heparinised devices together with Ap produce the least changes in blood cells and contact system activation in simulated CPB.
109
POSTER PRESENTATIONS P-2: Interrelation
Between Coagulation
PLASMA CLOT LYSIS BY STAPHYLOKINASE IN VITRO EFFECTS ON COAGULATION AND PL.ATELETS Hauptmann .I, Stebam 1. and Research Centre for Vascular Department of Experimental Nordhauser Str 78, D-99089
Glusa E Biology and MedIcme Ang~ology and Haemostaseology Erfurt. Germany
Staphyloklnase (SAK) IS a bacterial plasmunogen activator snmlar to streptokinase (SK) m Its mode of action. Recombinant wild-type SAti and several variants are now available and have been shown fibrlnolyttally and thrombolytically effective Here, the tibrlnolytlc actlon of SAK IS compared to that of SK and t-PA m a clot lysls system usmg radioactively labelled human plasma clots The parameter clot lys~s was determIned 111comparison to the fibrinogen concentration of the plasma and to standard clottmg times (APTT, TT) The state of platelet actlvatlon in the plasma nuheu of the clots was evaluated by measuring the formation of thromboxane In separate expertmerits, platelet aggegation Induced by ADP and collagen was studled 111 whole blood and !n platelet rich plasma (PRP), moreover, flow cytometry was carried out v&h PRP after actlvatton by ADP After a 4hr uncubatlon 50 % lysls of plasma clots occurred at I3 nrnolil of SAK and at 8 mnol/l of t-PA, whereas SK did not lead to 50 l/o lysls
110 IIIJMAN
AN’I‘IHOl~lliS AN’I‘I-S7‘K~I”rOKlNAS~ AGGRliGA’l‘lON IN A I+/ C1k32-I~EFt~NI~ENT MANNIX ]J. Lehraxi, 2 L! (’ llelft 2 h Varhcron, l blbl. Samama j M. INIXJCLi
Pl~l‘El.E’l‘
Mirshahi -1
l ‘I‘. Leromnte
lIaboratoire Cardiol.
Central
d’Il~matologie-llhlel-l)ieu,
HCpital Necker,
2 Clinique
~31NSEKIl Ll 86 - Paris, l~ranc-e.
Exposure to streptokinase (SK) raises anti-SK antihodies (Abs). Their functional properties include inhibition of SK-induced fibrinolysis and induction of platelet (plt) aggregation. The mechanism of the latter seems to involve pit binding of a plasminogen--SK-anti-SK Ahs ternar? complex. Anti-SK Abs were purified hy- affinity chromatography from serum of patients having received SK for acute myocardial infarction, and were shown to be IgG. Their effects were studied with (i) human pits in titrated plasma in the presence of SK or APSAC, or (ii) washed pits. resuspended in a ‘Ij.rode huffer and after lowering the ionic strength, in the presence of APSAC (which brings both SK and (anisyolated) plasminogm).
111 BENEFICIAL EFFECTS OF HEPARIN COATED DEVICES AND APROTININ ON BLOOD CELLS AND CONTACT SYSTEM ACTIVATION IN AN IN VITRO CARDIOPULMONARY BYPASS MODEL Wendel HP, Ga llimore MJ, Heller W Dept. of Thoracic and Cardiovascular Surg., Univ. of Tuebingen, Germany We have previously described model for studying contact system activation and changes in blood cells in simulated cardiopulmonary bypass (CPB). We have also shown that aprotinin (Ap) at therapeutic concentrations reduces contact system activation in CPB surgery. In the present study we have compared heparinised (H) and non-heparinised (NH) oxygenators (Medtronic), with and without Ap (220 KIU/mI blood) in the model. Heparin was added to the blood; 1.0 IU/ml in the H-group and 3.0 IUlml in the NH-group. Eight separate CPB runs were performed with: Gpl-NH no Ap, Gp2-H no Ap, Gp3-NH plus Ap, GpCH plus Ap.
and Fibrinolysis
up to a concentranon of I pmol/l .A1 these concentrations SAK and 1PA did not lower plasma fibrmogen. whereas wth SK residual fibrinogen was _ IO % Flbrlnogenolytlcally eqmeffective concentrations of SK. t-PA and SAK were 7.2, 50. and 750 mnolil. resp AET and TT (more sensnlve) were Inversely correlated wnh residual plasma fibrinogen Up to a concentration of IO pmol/l, SAK had almost no mfluence on platelet aggre%atlon. t-PA and SK lnhlbned ADP-Induced aggregation 111whole blood and 11, PRP slgnlficantly at I pmolil Thromboxane formanon was Inhlbtted parallel to the lnhlbltlon of aarega:atlon Flow cytometry findIngs corroborated the above results v&h SAK showing no mfluence on platelets at I pmol/l In the Ilght of the above results recombinant SAK 1s an effective fibrinspecific fibrlnol_vlc agent. nearly eqmpotent to t-PA 111 lyslng SKreslstent plasma clots. wnh a low potential toward dlsturblng coagulation and platelets 111vitro
An Ah concentration response curve was obtain& with a plateau in a presence of 0.1 mg/ml of IgG. Increasing the concentration of APSAC gave an unimodal response curve, the optima1 roncentration heing 0.05 U/ml. Chelating calcium with EDTA, hlorking fibrinogen hinding with the RGDS peptide, and raising intraplatet rAblP with lloprost suppressed aggregation. Aggregation requires of the interaction of the Fr domain of the purified IgG directed against SK with the plt I+gamma receptor type II. also named CD32, sinre : (j) it is hlorked by the anti-CM2 monoclonal Ah IV-3 (ii) it does not orcur with F(ah)‘2 fragments whirh hlork the response to the intact IgG. The rliniral relevance (prothrombotic?) of these plt activating antihodies remains to he determined. Two factors might inlluence the clinical outcome: (i) the amount and t).pe of pre-existing anti-SK Abs:(ii) the known interindividual variability of the plt response to plt-hindable and plt activating IgG involving the CD.32 mrrlnr..ln . ..“._. _._.
Blood samples were taken before and after 1, 5, 10, 30, 60 and 90 minutes circulation. Platelets counts (PC), platelet factor 4 (PF4), PMN elastase-alphal-proteinase inhibitor complexes (PMNE-alPI), kallikrein-like (KK) and FXIIalike (FXIIa) activities, determined. The mean values for the various parameters for the four groups after 90 minutes CPB arc shown below (Initial values = 100%). GPl GP2 GP3 GP4 PF4 616 293 453 237 PMNEalP1 462 260 316 187 KK 209 145 197 150 FXIIa 245 174 176 161 The results show that heparinised devices together with Ap produce the least changes in blood cells and contact system activation in simulated CPB.