Upsala J Med Sci 92: 79-83, 1987 Two Forms of az-antiplasmin: Post-traumatic Changes in the Rat H. Hogstorp and G . Carlin Institute of Forensic Medicine, Wniversity of Uppsaia, Wppsafa, Sweden ABSTRACT The plasminogen-binding (PB-AP) and non-plasminogen-binding (NPB-AP) forms ofw2-antiplasmin (AP), were assayed in rat plasma by a modified rocket immunoelectrophoretic technique before and up to 48 h after turpentine-induced trauma, using an intermediary gel containing kringles 1-3 from plasminogen. The concentration of PB-AP was signifi- cantly elevated by 2 2 % 2 4 h post-traumatically, while NPB-AP was de- creased at that point in time, leaving the total AP level unchanged. Total AP increased by 57 % during the period 2 4 - 48 h after trauma, mainly on account of increases in the NPB-AP form. I t is concluded that the plasma level of AP can remain unchanged in spite of increased fibrinolysis inhibition, owing to a relative increase in the functionally more active PB-AP. INTRODUCTION The important fibrinolysis inhibitor%-antiplasmin (AP) has been found to exist in a plasminogen-binding (PB-AP) and a non-plasminogen-binding (NPB-AP) form in man (1, 2 , 3 , 4 , 5, 1 2 , 1 4 , 15). We have previously reported on the presence of AP in the rat and described some of i t s changes after induction of trauma ( 8 , 9 , l o ) . The purpose of the present investigation was to determine the effects of turpentine trauma on the two forms of the inhibitor in the rat. MATERIALS AND METHODS R R Chemicals: French turpentine oil (Kebo AB, Sweden), CNBr-Sepharose 4 B , SepharoseR 4B (Pharmacia Fine Chemicals, Sweden) ; Agarose M 79 (LKB , Sweden). Anti-rat %AP-IgG was prepared as described previously ( 8 ) . Animals: Male Sprague-Dawley rats (300 - 380 g) from the Anticimex Farm, Stockholm, were used. They had free access to food (Ewos rat pellets) and tap water throughout the experiment. All surgical procedures were carried out under ether anaesthesia. Turpentine trauma was induced by an intramuscular injection of 0.5 ml of turpentine into each hind leg. Animals, five at each time, were killed 2 4 , 36 and 48 h after the turpentine injection. Preparation of plasma: Four millilitres of blood was drawn from the aorta into a plastic tube containing 1 ml of 3 . 8 % trisodium citrate solution. The tubes were immediately centrifuged and the plasma was aspirated with siliconised pipettes and stored at -20°C until analysed. Plasma from five untreated animals was pooled and used as a control. Assay of the different forms ofo&,-antiplasmin: An electroimmunoassay technique mainly as described by Wiman et a1 ( 1 5 ) was employed, using an intermediary gel containing kringles 1-3 from human plasminogen coupled to CNBr-activated Sepharose 4B (-5 mg kringles/ml settled gel). In this gel the PB-AP form is absorbed, while the NPB-AP form runs through and forms rockets in the subsequent anti-%-AP-IgG, containing agarose gel. The intermediary agarose gel contained 9 % "kringle-Sepharosetf. Total AP was measured on the same plate, using pure Sepharose 4B in the inter- mediary agarose gel to equate any loss of antigen due to unspecific ad- sorption. The difference i n height between the rockets was considered to represent PB-AP. Pooled normal rat plasma, 0 . 5 - 1 . 2 5 p l , diluted to 5 1.11 in 0 . 0 2 4 M Veronal buffer pH 8 . 6 , was used for standard curves. The samples contained 0 . 8 3 p1 of plasma and were diluted to 5 pl in Veronal buffer. All results are expressed in per cent of total AP found in pooled normal rat plasma. - Statistical analysis: Differences between groups were tested by Wilcoxon's rank sum test. A p value below 1 % was considered significant. RESULTS The results are presented in Fig. 1. PB-AP amounted to 6 3 . 1 % of the total AP in control animals. After 24 h PB-AP was significantly increased by 2 2 % to 7 7 + 4 %. A t the same time NPB-AP was significantly decreased to 22 + 7 % while the total AP remained unchanged. Between 24 and 48 h the total AP increased to 156 %, and this was mainly referable to an increase in the NPB form. - - 80 % 150 100 50 I I 0 24 36 40h Fig. 1 Total OC2-antiplasmin (01, plasminogen-binding ( A ) and non-plasminogen- -binding ( ) forms of* - antiplasmin 24, 36 and 48 h after induction of trauma with turpentine. d l u e s are expressed in per cent (mean 2 S D ; n = 5 ) of totalw2-antiplasmin in normal rat plasma. DISCUSSION In this study the concentration of the plasminogen-binding form of 5 - A P was increased in rat serum 24 h after turpentine trauma. There was In a previous investigation it was found that the fibrinolysis inhibition activity in serum, which involves not only plasmin inactivation but also inhibition of plasminogen activation and changes in plasmin(ogen) , fibrin interactions, was increased by 2 0 % 2 4 h after turpentine trauma even though no changes were observed in the plasmin inhibition activity in plasma or in the immunologically determined W2-antiplasmin concentration ( 9 ) . These results may be explained b y the present finding of a 2 2 % increase in PB-AP but no change i n the total AP - provided that both a concomitant decrease i n the non-plasminogen-binding form. 5 t -87857 1 81 forms of W2-AP a r e potent plasmin inhibitors and that the plasminogen- -binding form also interferes with plasminogen activation and with the , plasminogen uptake b y fibrin. Earlier investigations w i t h use of human 5 - A P have yielded results supporting this view (5, 13, 1 4 ) . Preliminary studies (11) have indicated that PB-AP is synthesized by the liver, in which case NPB-AP should be a metabolite, although the present findings do not preclude the reverse. A known metabolite of AP is i t s complex with plasmin, which in humans and in mice is rapidly removed from the circulation ( 6 , 7 ) . Preliminary attempts to measure plasmin-antiplasmin complexes by using Lysine Sepharose instead of plasminogen kringles i n the intermediary gel failed to show evidence of such complexes in the present study. ACKNOWLEDGEMENTS These investigations were supported by grants from the Swedish Medical Research Council, the Torsten and Ragnar Soderberg Foundation, the 80th Anniversary Fund of the Trygg-Hansa Insurance Company, the Medical Faculty of Uppsala University and the Swedish National Association against Heart and Lung Diseases. 1 2 3 4 5 REFERENCES Bagge, L., Jacobsson, H . & Saldeen, T . : Post-traumatic variation of the primary fibrinolysis inhibitor W2-antiplasmin. 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E . & Pizzo, S.V.: A unique pathway for the plasma elimination of d. -antiplasmin-protease complexes in mice. Thromb Haemostasis 48(2) : 208-810, 1982. 82 8 Hogstorp, H . , Jacobsson, H . & Carlin, G. : Studies on?-antiplasmin i n 9 Hogstorp, H . , Jacobsson, H. & Saldeen, T. : Effect of hepatectomy on the posttraumatic fibrinolysis inhibition and the primary fibrinolysis inhibitor in the rat. Thromb R e s 18:361-368, 1980. the r a t . Thromb Res 21:247-253, 1981. 1 0 Hogstorp, H . & Saldeen, T. : Synthesis ofDC2-antiplasmin by rat liver 11 Hogstorp, H . & Saldeen, T.: Rat hepatocytes synthesize the plasmino- gen-binding form of d2-antiplasmin. Abstr . Xth International Congress on Thrombosis and Haemostasis. San Diego July 14, 1985. Thrombosis and Haemostasis No 1, Vol 5 4 , p 1581. cells. Thromb Res 28:19-25, 1982. 1 2 Kluft, C . & Los, N . : Demonstration of two forms of u2-antiplasmin in plasma by modified crossed immunoelectrophoresis. Thromb Res 2 1 : 65- -71, 1981. 13 Moroi, M . & Aoki, N . : Inhibition of plasminogen binding to fibrin by oC2-plasmin inhibitor. Thromb Res 10:581-586,1977. plasmin. Biochem J 191:229-232, 1980. 1 4 Wiman, B . : Affinity-chromatographic purifications of human w2-anti- 15 Wiman, B., Nilsson, T . & Cedergren, B . : Studies on a form of OLZ-antiplasmin in plasma which does not interact with the lysine-binding sites in plasminogen. Thromb Res 28 : 193-200, 1982. Address for reprints: Herman Hogstorp Department of Forensic Medicine Dag Hammarskjolds vag 17 Sweden 5-752 37 UPPSALA 6-878571 83