On a new function of lipoprotein lipase in blood

E.A.l~1alakhova, G.G.Dazasian: T.P.Levchti< and V.A.Yakovlev Biochemical Laboratory, All-Union Research Vitamine Institute, Moscow and Laboratory of Physiology and Biochemistry of Blood Coagulation, f:oscowState University: Yoscow, U.S.S.R. (Received

2.11.1977.

Accepted

by- Editg:lr B. Kudr-jashov)

A3STRACT Qeriments on complexes of lipoprotein lipase with heparin and artificial chylomicrons revealed the ability of these complexes to lyse non-crosslinked fibrin clots, which is not typical of lipoprotein lipase 'per se'; its own 1ypolW.c activity is retained. It is suggested that due to their new property the complex es may exert in the organism a protective effect against intravascular blood coagulation.

INTRODUCTION Lipoprotein lipase (LPL; triacylglycero-proteinacylhydrolase, l% 3.1.1.34) plays an important role in the metabolism of lipids in blood (1). It catalyzes the release of free fatty acids from an emulsion of triacylglycerols in the presence of a specific protein cofactor present in some blood plasma lipoproteins (2, 3). The natural substrates for this enzyme are the triacyiglycerols of chylomicrons and very low density lipoproteins (VLDL). One of the most typical characteristics of Ll?Lis its rapid release into the blood from the endothelical membrane of the blood capillaries as a response to intravenous injection of heparin or heparin-like substances. It is assumed that the solubilized IPL circulates in the blood in a heparin-bound state (4, 5).

209

210

LIPOPROTEIN

LiPXSE

IS BLOOD

l-nf.12.Sn.2

LPL has also been referred to as a humoral agent of the physiological anticoagulant system (6-8). Recently B.A.Kudrjashov et al. have reported the existence of a new type of fibrinolysis, which was called non-enzymatic (9, IO). Nonenzymatic fibrinolysis is carried out by complexes of heparin with some components of the plasma, particularly with some proteins of the blood coagulant system. These proteins, being combined with heparin, lose their ability for thrombogenesis. Contrary to enzymatic fibrinolysis which is accomplished by plasmin, non-enzymatic fibrinolysis proceeds without disruption of the peptide bonds and is observed only in fibrin clots, which are non-crosslinked. It has been recently demonstrated that the intravenous injection of I& to rats fed on an atherogenic diet favours the functioning of the anticoagulant system (II). In particular, the animals showed a tendency to normalization of the fibrinolytic activity of blood. It has been supposed that the normalizing effect of LPL on the state of the anticoagulant system may be due to an appearance of the fibrinolytic activity of IPL as a result of its complexing with heparin. Therefore we tried to isolate the LPL-heparin complex from the post-heparin plasma of rats and to reveal its fibrinolgtic activity. JdETHODS Blood (5-8 ml) was taken from rat jugular vein into a 0.1 volume of 3.8% sodium citrate IO min after an intravenous injection of 100 intern. units of heparin. Post-heparin plasma was obtained by centrifugation at 2500 g for 30 min. Purification of I&% from the post-heparin plasma was carried out according to Fielding (51, up to the calcium phosphate gel step. IPL activity was assayed by continuous titration of free fatty acids with 0.005 N NaOH, pH 8.5 on a "Radioaeter'1 pH-state (12). The reaction mixture contained: 1% chylomicrons (with respect to triacylglycerols content), 8.10e3M CaC12 and 5.10'2M Xl. LPL activity is expressed in units of ,umolesof free fatty acids released per 1 br at 37°C. Artificial LPL substrate, here termed as 'chylomicrons',was

obtained 2:; activation of Intralipid (Vitrum, Sweden) by incubation of IO ml of 202 Intralipid and 90 ml human blood plasma for 30 min at 37°C. The resulting substrate was separated from other plasma components by centrifugatioa at 100 GGG g for I hr and subsequent suspending in G.%

XaCl.

son-crosslinked fibrin gel was obtained in flat-bottomed Petri dishes as described in (13), by mixing 0.1 ml (2 'kozlaw units) of thrombin in 0.9% NaCl with 5 ml of fibrinogen solution iPith inhibitors. To prepare the latter, 203 ng of fibrinogen and IGO mg of EDT,; were dissolved in a mixture of 50 ml of 0.05 ?,!tris-HCl btifer, pR 7.35 and 50 ml of 3.8;; sodium

citrate; then 40 ml of 0.05 M tris-Xl buffer containing 864 mg of F-aminocaproic acid ( g-ACA) and 120 mg of monoiodoacetic acid were added to the mixture. The mixture was heated for 15 min at 37°C and poured into the dishes.Total (enzmatic

and non-enzymatic) fibrinolgsis was assayed from

the areas of lysis zones on the plates of non-crosslinked fibrin gel and expressed in m2

(14). Aliquots of 50~1

of

the sample under analysis were pipetted on the gel and the dishes were incubated for 2 hrs at 37°C. The areas of lysis zones were calculated after staining the gel with water-soluble ink. ITon-enzymatic fibrinolysis was measured similarly, but in order to inhibit enzymatic fibrinolysis an equal volu-

me of 6%

E-ACA was added to the sample.

RESULTS In the course of LPL purification both ly-polytic and fibrinolytic activities were controlled. The results are given in Table 1. As seen from Table I, the fraction containing the I&% -1ntralipid complex exerts the strongest 1Ytic effect

on the

non-crosslinked fibrin gel. This effect is also observed in E-ACA, which is indicative of a non-enzymthe presence of atic nature of the fibrinolysis. Solubilized LPL possesses a two times as low non-enzymatic fibrinolytic activity as compared to that of the adsorbed one, whereas acetone-extracted LPI, is found incapable to lyse the gel. Recently Fielding (5) has reported that 35 S-heparin injected intravenously to rats is then found in LPL adsorbed on the intralipid and also

21

?

LIPOPROTEIB

LIPXSE

IS BLOOD

vo1.12,xo.2

TABLE I Lysis of Non-crosslinked Fibrin Gel by LPL at Different Stages of Purification Stages of Purification

Volume, IJ?L activity Fibrinolysis (lgsis ml units/ml units/mg ~~n~Li~c&_ 2 per unit

Post-heparin plasma 120 Adsorption on 5 Intralipid Solubilization 4 Acetone treatment 2

total 44.4

0.76

316

-

288

200

480

450

95,+11 IlO_+ 533

none

non-enzymatic 65~7 125_+10 55*

none

in the LFL following solubilization. On the contrary, a highly purified LPL preparation practically does not contain heparin. The above-mentioned data suggest that heparin is necessary for the formation of LPL complex, which possesses the fibrinolytic activity. Since we have established that the fibrinolysis accomplished by solubilized LPL is considerably lower than that induced by chylomicron-bound LPL, it seems of interest to elucidate whether the chylomicrons, parallel with heparin, also participate in the formation of the IPL complex to produce fibrin lysis. To study this problem we have tried to reconstruct this complex from acetonated IPL, heparin and chylomicrons. The reconstruction of the complex was performed at room temperature and pH 7.6 .

TlLEIz

II

Reconstruction of the IX, Complex, Possessing the Ability to Lyse Non-Crosslinked Fibrin Gel

IJ?Lactivity (units in the sample)

Heparin (pg in the sample)

Chvlomicrons Eon-enzymatic (migtriacyl- fibrinolysis glycerols in (lysis sones) the sample m-n2 Eml2/unit IPL activity

5

0

0

3

0,42 6 6 6 495

0,42 0,42 0,42 0,42 0,42

0

0

0

0

0

64

296

59

91

70

403

As seen from Table II, the complexes ~lLPL-heparin~q and "ISLchylomicrons", as well as heparin, IPL and chylomicrons taken separately, have no lytic effect on the fibrin gel. The complex "chylomicrons-heparin"reveals very low fibrinoly-ticactivity. However, after addition of acetonated LPL to the "chylomicrons-heparin"complex its ability to lyse the fibrin gel is sharply increased. Beparin and chylomicrons do not affect the active site of LPL, as is evidenced from the fact that LPL, being a constituent of the complex, retains its lipolytic activity. In such a complex LPL may hydrolyse triacylglycerols of the chylomicrons, which are components of the complex itself. However, such "self-digestion" is po_ssibleonly in the presence of a free fatty acid acceptor, e.g. albumin or calcium ions. The complex is relatively thermostable and is inactivated only at temperature exceding 70" with the loss of lipoprotein lipase and fibrinolytic nonenzymatic activities.

213

LIPOPROTEIS

LIPXSE

IX BLOOD

\-01.12,X0.2

It aprears that heparin and chylomicrons to some extent protect LPL against the denaturating effect of high temperatures. It has been shown that the level of fiblinolytic activity of the complex depends on the proportion of its constituents. As seen from Figure I, the fibrinolgtic activity of the complex increases sharply with an increase in chylomicron concentration and remains unchanged within a wide range of chylomicron concentrations after the maximal value of lysis has been reached. Further increase in chylomicron concentration causes a decrease of the fibrinolysis. At chylo-

200

d "N 100

FIG.1 Dependence of non-crosslinked fibrin plates lysis by 'LPI-heparin-chylomicrons'complex on chylomicron concentration. The sample contained: IS u_l. of acetone-extracted LPL (3.6 units), 15 pl heparin (0.75 pg) and 2O)il chglomicrons (different concentrations). micron concentration equal to 28 mg/ml (with respect to triacylglycerols) the inhibition of the fibrinolysis reaches 35%. Next it seemed essential to find out, which of the chylomicron components are involved in the formation of a complex, active with respect to fibrinolysis. For this purpose a total protein fraction was isolated from chylomicrons by

i. ~1

, -_

.14,l-n.2

solubilization aTit& 0.5 x lo-‘?! decxvcholate, ccnsotilm " taining 0.25 x 1O'3?~ potassium oleate. The detergent contaminations were removed

from tine sample by a prolonged di-

alysis, lipid admixtures - by treatment acetone. The

FIG.2 Lysis of non-crosslinked fibrin plates by complexes of acetone-extracted IPL. Left - experimental samples (a - e>; right- controls (a' - e'), in which the enzyme was substituted by an equal volume of ammonium buffer pH 7.6; sample volume - 50 @L. a - LPL-heparin-chylomicrons (4.5 units: 1 )~g: 0.42 mg with respect to triacylglycerols) b - LPL-heparin-protein cofactor (4.5 units:O.l ,ug:9.7,ug:> c - LX-heparin-protein cofactor (4.5 units:1 ,ug:?.7 .ug> (6 units:O.l pg:O.42 mg d- IPLheparin-chylomicrons with respect to triacylglycerols); e - LX-heparin-protein cofactor (6 units:O.l ug:2.9 ,ug> protein cofactor (6 units: f - LPEheparin-acetone-extracted O.l,ug:2.7 ,ug>. resulting preparation had a high LF'L cofactor activity and was therefore termed as "protein-LPL cofactor". As seen from Figure 2 f, the complex reconstructed from LPL, heparin and acetone-extracted protein cofactor did not possess lytic properties. The reconstructed complex was capable to lgse non-crosslinked fibrin when protein cofactor was obtained Ttithout treatment of acetone (cf. Pigs. 2 b and 2 e>.

216

LIPOPROTEIS

LIPXSE

I?; BLOOD

v01.12,s0.2

However, its fibrinolytic activity was lower than that of the "native" system, i.e. the IPEheparin-chylomicrons complex (Figs. 2 a and 2 d), the degree of reactivation being approximately 7% (Figs. 2 a and 2 b). Presumably, the presence of lipids in the complex is necessary for the manifestation of the fibrinolytic activity. A comparison of Figs, 2 a and 2 d shows that the fibrinolytic activity is increased with an increase in heparin concentration and is completely inhibited by excess amounts of heparin (Figs. 2 b and 2 c). It is probable that the dependence of fibrinolytic activity of the LPL complex on heparin and protein cofactor concentrations has the same character as does the dependence of the activity on the chylomicron concentration. DISCUSSION Summing up the results of the experiments, it may be assumed that LPL complexes with heparin and crudely disperse lipoproteins acquire a new property, namely the ability to lyse non-crosslinked fibrin clots. A question arises as to the physiological role of the ternary LPLheparin-chylomicrons complex. Normally LPL appears in the blood as a result of food lipemia, which may provoke thrombinogenesis due to the thromboplastic activity of chylomicrons. Our present data enables us to suppose that IPL, when forming conplexes with chylomicrons in the presence of heparin, deprives them of their tnromboplastic properties, since the complex formed possesses the ability to lyse non-crosslinked fibrin gel. LPL, being a constituent of the complex, retains its lipolytic activity and hydrolyzes the triacylglycerol core of chylomicrons, thus facilitating the removing of them from the blood stream. The present work is the first demonstration of the fact that the LPL complex with heparin and chylomicrons, apart from previously known heparin complexes with catecholamines and specific proteins of the blood,participate in the lysie of non-crosslinked fibrin, thus protecting the organism against intravascular thrombogenesis. Since LPL is not the only enzyme appearing in the blood as a response to intravenous injection of heparin, it is probable that other

heparin-dependent

lipolytic enzymes may also be involved in

the formation of complexes possessing the fibrinolytic activity.

REFERENCES 1. ROBINSON, D.So, CRYER, A., and DAVIES, P. The role of clearing-factor lipase (lipoprotein lipase) in the trana7;;; of plasma triglycerides. Proc. Nutr. SOC., 34, 211, .

2. FIELDING, C.J., LLM, C.T., and SCANU, A.&l. A protein component of serum High density lipoprotein with cofactor activity against purufied lipoprotein lipase. Biochem. Biophys. Res. Conr;nuns.,39, 889, 1970. 3.

HAVEL, R.J., FIELDIlVG,C.J., OLIVECRONA, T., SHORE, V.G., FIELDING, P.E., and EGELRUD, T. Cofactor activity of protein components of human very low density lipoproteins in the hydrolysis of triglycerides by lipoprotein lipase from different sources. Biochemistry, 12, 1828, 1973.

4. PORTE, D., and WILLIAMS, R.H. Post-hepy3in

tivity following intravenous heparin-S Biol. Med., 118, 639, 1965. 5.

lipolytic ac. Proc. of Exptl.

FIELDING, P.E., SHORE, V.G., and FIELDING, CjJ. Lipoprotein lipase: properties of the enzyme isolated from postheparin plasma. Biochemistry, 13, 4318, 1974.

6. KUDRJASHOV, B.A. The role of the physiological anticoagulant system. Voprosy meditsinskoj khimii, 6, 3, 1960. 7. KUDRJASHOV, B.A., and

BAZASIAN, G.G. Lipoprotein lipase as a humoral agent of the physiological anticoagulant system. J. Atheroscler. Res., No. 3, 199, 1963.

8. KUDRJASROV, B.A., BAZASIAN, G.G., and BONFITTO, L.L. Lipoprotein lipase of blood and its properties as a component of the physiological anticoagulant system. Voprosy meditsinskoj khimii, 9, 533, 1963. 9. KUDRJASHOV, B.A., LYAI'INA, L.A., MOLCHABOVA, L.V., and RUSTAMOVA, B.A. On the natare of lytic effect of fibrinogen-heparin and tyroxine-heparin complexes on fibrin. Voprosy meditsinskoj khimii, 76, 161, 1970. IO. KUDRJASHOV, B.A. Humorale Factoren des anticoagulatorischen Systems. Folia Haematol., (Leipzig), 98, 391, 1972.

218

Il.

LIPOPROTEIS

LIPASE

IS BLOOD

Yo1.12,?;0.2

BAZASIAU, G.G., iXVCXE,.T.?., :.WXZ@VA, X.A., SME?37OVA, V .F_.,SYTINA, X.P., and YAKOVLE3, V,A. Isolation of lipoprotein lipase preparation from rat post-heparin plasma and examination of its biological activity. Voprosy meditsinskoj 'khimii,20, 14, 1974.

12. YtiOVL.EV,V.A., LEVC_XJK,T-P., and ?&IA.KXOVA,E.A. %timation of lipoprotein lipase activity using the method of potentiometric titration at constant pH. Voprosy meditsinskoj khimii, 22, 417, 1976. 13. KUDRJASUOV, B.A., LYAPI?lA,L.A., and BASKOVA, I.P. A modified procedure for the estimation of non-enzymatic fibrinolytic activity of plasma and some of its fractions. Vestnik MGU (biology, pedology series), No.5, 35, 19740 '4. KUDRJASFIOV,B.A., and LYAPINA, L.A. A method for estimation of the activity of physiological solvents of unstabilized fibrin. Laboratornoe delo, No. 6, 326, 1971.