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Lipoprotein (a) regulates plasmin generation and inihibition
Chemistry and Physics of Lipids 67/68 (1994) 363-368
ELSEVIER SCIENCE IRELAND
Chemistry and Physics of LIPID$
Lipoprotein (a) regulates plasmin generation and inihibition Jay Edelberg, S.V. Pizzo* Duke University Medical Center, Box 3712, Durham, NC 27710, USA
(Accepted 12 November 1992)
Abstract The relationship between lipoprotein (a) (Lp(a)) and atherosclerosis has been appreciated for a number of years. Only in recent years, however, has the structural relationship of Lp(a) to plasminogen resulted in studies of the effect of this lipoprotein on fibrinolysis. Lp(a) inhibits activation of plasminogen by tissue-type (t-PA) and urinary-type (u-PA) plasminogen activators. These inhibitory reactions are surface-dependent. When Lp(a) binds to fibrin, fibrinogen, heparin or cells it blocks activation of plasminogen by t-PA. u-PA-mediated activation of plasminogen is blocked on surfaces including heparin and chondroitin sulfate. Lp(a) also favors inhibition of plasmin by c~2-antiplasmin (c~2-AP). The ability of Lp(a) to compete with plasmin for fibrin binding displaces plasmin into solution where c~2-AP rapidly inhibits this proteinase. These effects are all antifibrinolytic. Lp(a) also exhibits one profibrinolytic effect, since it blocks inhibition of t-PA by plasminogen activator type 1 in the presence of fibrinogen or heparin. Thus, Lp(a) modulates most of the reactions involved in plasmin generation and inhibition. Its overall effect will depend primarily on the concentrations of Lp(a), PAl-1 and t-PA in vivo. Key words: Lipoprotein (a) and fibrinolysis; c~2-Antiplasmin; Plasminogen activation; Tissue-type plasminogen
activator
1. Lipoprotein (a) and Atherosclerosis Lipoprotein (a) (Lp(a)) is a low-density plasma lipoprotein first identified by Berg (1963). Elevated levels of Lp(a) (greater than 30 mg/dl) are associated with a risk of atherosclerosis two to five times that of control subjects (Albers et al., 1977; Frick et al., 1978; Rhoads et al., 1986; Dalhen et al., 1986). The elevated Lp(a) levels are linked not only to coronary artery disease but also to stenosis
* Corresponding author.
of carotid and cerebral arteries (Murai et al., 1986; Zenker et al., 1986). Structural studies of Lp(a) demonstrate that, like low-density lipoproteins, it contains a lipid core and an apoprotein B (apo B) sub-unit. However, it also contains an apoprotein (a) (apo(a)) sub-unit disulfide-linked to apo B (for detailed review see Scanu, 1988). The apo B sub-unit is an Mr - 510 000 polypeptide (Law et al., 1986; Knott et al., 1986), and the apo(a) sub-unit is also a large, but heterogenous, apoprotein with isoforms larger than, smaller than and equal in size to the apo B sub-unit (Gaubatz et al., 1983; Arm-
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364
strong et al., 1985; Fless et al., 1986; Eaton et al., 1987; Karadi et al., 1988). Protein and cDNA sequences of the apo(a) sub-unit reveal extensive homology between the sub-unit and the fibrinolytic zymogen plasminogen (McLean et al., 1987; Eaton et al., 1987). The apo(a) sub-unit contains the lysine binding domains, termed kringles, found in plasminogen and other coagulation and fibrinolytic serine proteinases; for example, tissue-type plasminogen activator (t-PA), urinary-type plasminogen activator (u-PA) and prothrombin (Patthy, 1985; Furie and Furie, 1988). While plasminogen has single copies of kringles 1 to 5, the apo(a) sub-unit has up to 37 copies of kringle 4 and a single copy of kringle 5, with 75-85% and 95% homology, respectively, to their plasminogen counterparts. The apo(a) sub-unit also contains a region 94% homologous to the plasminogen proteinase domain. This proteinase domain has an intact catalytic triad essential for serine proteinase activity (Walsh, 1979), but the sub-unit lacks a critical plasminogen cleavage site, Arg560-Va156b necessary for zymogen activation (Robbins et al., 1967). The apo(a) sub-unit has a Ser in the place of an Arg found in plasminogen, and it cannot be converted to a proteinase by typical plasminogen activators (Eaton et al., 1987).
2. The relationship of lipoprotein (a) and fibrinolysis The association between the increased incidence of atherosclerosis and elevated levels of Lp(a) may be due to suppression of normal fibrinolytic activity. Several clinical studies have demonstrated an inverse correlation between fibrinolytic activity and the risk of coronary artery disease (Walker et ai., 1977; Hamsten et al., 1987). Recent studies also demonstrate that Lp(a) accumulates in the lesions of the affected vessels (Rath et ai., 1989; Smith and Cochran, 1990; Beisiegel et al., 1990), which suggests that Lp(a) may have a direct role in the pathogenous of these lesions. The homology between apo(a) and plasminogen suggests that this accumulation of Lp(a) may result in a competition with plasminogen for cellular and matrix binding sites and that the association between the increased incidence of atherosclerosis and the elevated
Z Edelberg, S.V. Pizzo / Chem. Phys. Lipids 67/68 (1994) 363-368
levels of Lp(a) may be due to suppression of normal fibrinolytic activity at such sites. Fibrinolysis may be regulated by controlling the generation of plasmin, the activity of plasmin and the inhibition of plasmin. The regulation of plasmin generation, or plasminogen activation, is dependent on many factors, including the local concentrations of plasminogen, plasminogen activators and plasminogen activator inhibitors, as well as the kinetic rates of these activators and their inhibitors. The homology between the apo(a) sub-unit of Lp(a) and plasminogen led several investigators to examine the potential roles of Lp(a) in the fibrinolytic system. Recent studies demonstrate that Lp(a) may down-regulate in vivo fibrinolysis by competing with plasminogen for various vascular pro-fibrinolytic surfaces, including cellular binding sites, fibrinogen fragments and heparin. Cellular binding sites, by concentrating plasminogen and plasminogen activators on the vascular surface, are critical in fibrinolytic initiation (for review see Miles and Plow, 1988). Plasminogen binds these sites through its kringle domains. Recent studies demonstrate that Lp(a) competes with plasminogen via its kringle 4 domains for cellular binding sites on monocytes, macrophages and endothelial cells (Gonzalez-Gronow et al., 1989; Miles et al., 19.89; Hajjar et al., 1989; Zionchech et al., 1991). Lp(a) displacement of plasminogen from these cellular binding sites may prevent plasminogen from interacting with t-PA and u-PA, thereby locally depressing fibrinolysis. Lp(a) inhibits t-PA-mediated plasminogen activation in the presence of fibrinogen fragments and fibrin (Edelberg et al., 1990; Loscalzo et al., 1990; Rouy et al., 1991). Kinetic analysis demonstrates that Lp(a) acts as a competitive inhibitor of this activation in the presence of fibrinogen fragments (Edelberg et al., 1990). Lp(a) does not inhibit t-PA amidolytic activity, indicating that the lipoprotein does not occupy the active site of t-PA. Lp(a) does not displace t-PA from fibrinogen fragments, since these interactions would have been reflected in an uncompetitive or non-competitive inhibition constant. Importantly, Lp(a) has no effect on the rate of the reaction in the absence of fibrinogen
J. Edelberg, S.V. Pizzo / Chem. Phys. Lipids 67/68 (1994) 363-368
fragments. Together these data suggest, then, that Lp(a) competes for a plasminogen binding site on fibrinogen fragments adjacent to the bound t-PA, thereby inhibiting the rate of plasmin generation. Lp(a) also inhibits heparin-enhanced plasminogen activation. Heparin, an anticoagulant glycosaminoglycan located on both the endothelial surface and the basement membrane (for brief review see Rosenberg, 1986), stimulates the rate of both t-PA and u-PA-mediated plasminogen activation (Edelberg and Pizzo, 1990b; Edelberg et al., 1991b). Heparin enhances the rate of plasmin generation by increasing the catalytic activity of both t-PA and u-PA. Lp(a) inhibits these heparinenhanced reactions by competing with plasminogen for access to the heparin-bound plasminogen activator (Edelberg and Pizzo, 1990b; Edelberg et al., 1991b). Lp(a) does not inhibit the amidolytic activity of the plasminogen activators, nor does it effect plasmin generation in the absence of heparin similar to its effect on fibrinogen fragmentstimulated activation. These data suggest that Lp(a) binds to heparin at a site adjacent to the activator and thereby prevents plasminogen from interacting with these enzymes. o~2-Antiplasmin is the primary inhibitor of plasmin formed in the plasma (for review see Lijnen and Collen, 1986). Inhibition is rapid and involves an interaction with both the catalytic domain of plasmin and the amino terminal regulatory kringle domains of the proteinase. Modulators that block the interaction between the kringles and a2-antiplasmin will decrease the rate of inhibition (Wiman and Collen, 1978; Wiman et al., 1978, 1979; Christensen and Clemmensen, 1978; Anonick and Gonias, 1991). Kinetic analysis shows that CNBr-derived fibrinogen fragments competitively prevent inhibition (Edelberg and Pizzo, 1992). The decreases in the rate of inhibition may result from the prevention of a kringleinhibitor interaction. The initial interaction between o~2-antiplasmin and the kringle domains of plasmin may involve one of the first three lysine binding sites of the enzyme. Previous studies demonstrated that c~2antiplasmin binds the kringles of plasminogen, and that domains 1-3 are preferentially involved
365
(Christensen and Clemmensen, 1978; Wiman et al., 1979; Sugiyama et al., 1988). More recent studies by Sugiyama et al. (1988) demonstrate that tryptic fragments of the carboxyl terminal of ct2antiplasmin have a higher affinity for the plasminogen kringle 1-3 domains than for the kringle 4 domain. In addition, e-aminocaproic acid decreases the rate of the inhibition approximately fivefold, e-Aminocaproic acid binds to plasmin with a dissociation constant of 120/~M, a binding constant consistent with binding to plasminogen kringle 1 (Markus et al., 1978). These data suggest that the reaction between plasmin and o~2-antiplasmin is mediated by an initial interaction between the kringle 1 domain of the enzyme and the kringle binding site of the inhibitor. Lp(a) contains only kringles 4 and 5 and does not, on its own, effect the rate of the inhibition of plasmin by et2-antiplasmin. However, in the presence of CNBr-derived fibrinogen fragments, Lp(a) increases the rate of the inhibition. The kinetics demonstrate that Lp(a) acts as a competitive suppressor of fibrinogen fragment-modulated inhibition. Lp(a) competes with plasmin for binding sites on the CNBr-derived fibrinogen fragments, resulting in plasmin dissociation from the fragments followed by rapid inhibition by ct2antiplasmin. These results suggest that in the vasculature, Lp(a) may promote the premature inhibition of fibrinolysis by promoting plasmin inhibition. Unlike these anti-fibrinolytic effects, Lp(a) can also have pro-fibrinolytic activity. Lp(a) protects t-PA from irreversible enzymatic inhibition by plasminogen activator inhibitor type I (PAl-l). In the vasculature, the major irreversible inhibitor of t-PA is PAl-1 (for brief review see Sprengers and Kluft, 1987). Lp(a) decreases the rate of t-PA inhibition by PAI-1 in a manner analogous to Lp(a) inhibition of t-PA-mediated plasminogen activation (Edelberg et al., 1990, 1991a; Edelberg and Pizzo, 1991). Moreover, similar to the Lp(a) effect on plasminogen activation, the decrease in PAI-1 inhibition is dependent on the presence of either fibrinogen or heparin. Kinetic analysis of the Lp(a) depression in t-PA irreversible inhibition indicates that Lp(a) competes with PAI-1 for
366
J. Edelberg, S.V. Pizzo / Chem. Phys. Lipids 67/68 (1994) 363-368
access to the template-bound plasminogen activator. Lp(a) decreases the rate o f the inhibition by sterically preventing P A I - I from interacting with fibrinogen-bound and heparin-bound t-PA similar to Lp(a) effects on plasminogen activation.
3. Lipoprotein(a) physiology and pathophysiology These studies demonstrate that in vitro Lp(a) competes with plasminogen for various vascular binding sites, inhibits plasmin generation by plasminogen activators and promotes plasmin inhibition but protects plasminogen activators from inhibition. This suggests that Lp(a) may both depress and prolong in vivo fibrinolysis. Lp(a), at physiological levels, may regulate the production of plasmin by protecting plasminogen activators from inactivation by PAI-1. In addition, it competes with plasminogen for access to surfacebound plasminogen activators as well as competing with plasmin for fibrin. Elevated levels of Lp(a) may depress fibrinolytic activity to a degree that is not compensated for by the prolonged availability o f the plasminogen activators. At these pathophysiological levels, Lp(a) should prevent a large fraction o f the available plasminogen from accessing the surface-bound activators, and promote the inhibition o f plasmin by u2-antiplasmin.
Endothelial Cells
I
Fibrin
P eP P ~P@P
Endothelial Ceils tPA (~
PAl-1
<:
Pg 0
Fibrin Pm ~
~2AP
~ Lp(a) p
Fig. 1. A scheme of the effects of lipoprotein (a) on fibrinolysis. The interaction of tissue-type plasminogen activator, plasminogen, plasminogen activator inhibitor 1 and cte-antiplasmin on both the endothelial cell and fibrin clot surfaces governs the rate of fibrinolysis. The addition of lipoprotein (a) to the system can disrupt these surface interactions, thereby regulating the fibrinolytic activity.
The net effect is to greatly depress fibrinolysis, even though the plasminogen activators are not inhibited by PAI-1 as rapidly as in the absence o f Lp(a). In addition, high circulating levels o f PAl- 1 would tend to overcome the protective effect o f Lp(a) on the inhibition reaction between PAI-1 and t-PA. Under these conditions, the overall effect o f elevated Lp(a) levels would be a suppression o f fibrinolysis. It is particularly noteworthy that both elevated Lp(a) and PAI-1 are associated with atherosclerosis and myocardial infarction. A model of this Lp(a) regulation of the fibrinolytic system is shown in Fig. 1. These data, combined with the kinetic results described above, may indicate that the pathogenous o f elevated levels of Lp(a) may be due to a chronic depression in endogenous fibrinolysis. Such a depression may in turn lead to thrombus stability and promote atherosclerosis.
4. References Albers, J.J., Adolphson, J.L., and Hazzard, W.I., 1977, Radioimmunoassay of human plasma Lp(a) lipoprotein. J. Lipid Res. 18, 331-338. Anonick, P.K., and Gonias, S.L., 1991, Soluble fibrin preparations inhibit the reaction of plasmin with u2-macroglobulin. Biochem. J. 275, 53-59. Armstrong, V.W., Walli, A.K., and Slidel, D., 1985, Isolation, characterization and uptake in human fibroblasts of an apo(a)-free lipoprotein obtained on reduction of lipoprotein (a). J. Lipid Res. 26, 1314-1323. Beisiegel, U., Niendorf, A., Wolf, K., Reblin T., and Rath, M., 1990, Lipoprotein (a) in the arterial wall. Eur. Heart J. 11, Suppl. E, 174-183. Berg, K., 1963, A new serum type system in man: the Lp system. Acta Pathol. Microbiol. Scand., Suppl. 59, 166-167. Christensen, U., and Clemmensen, I., 1978, Purification and reaction mechanisms of the primary inhibitor of plasmin from human plasma. Biochem. J. 175, 635-641. Dahlen, G.H., Guyton, J.R., Attar, M., et al., 1986, Association of levels of lipoproroteins with coronary artery disease documented by angiography. Circulation 74, 758-765. Eaton, D.L., Fless, G.M., Hohr, W.J., et al., 1987, Partial amino acid sequence of apolipoprotein (a) shows that it is homologous to plasminogen. Proc. Natl. Acad. Sci. USA 84, 3224-3228. Edelberg, J.M., and Pizzo, S.V., 1990, Kinetic analysis of the effects of heparin and lipoproteins on tissue plasminogen activator-mediated plasminogen activation. Biochemistry 29, 5906-591 I.
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Edelberg, J.M., and Pizzo, S.V., 1991, Novel regulatory mechanisms in fibrinolysis. Fibrinolysis 5, 135-143. Edelberg, J.M., and Pizzo, S.V., 1992, Plasmin inhibition by ~2 M-antiplasmin: a kinetic model of inhibition in the vasculature. Biochem. J. 286, 79-84. Edelberg, J.M., Gonzalez-Gronow, M., and Pizzo, S.V., 1990, Lipoprotein(a) inhibition of plasminogen activation by tissue-type plasminogen activator. Thromb. Res. 57, 155-162. Edelberg, J.M., Reilly, C.F., and Pizzo, S.V., 1991a, The effects of vascular components on the inhibition of tissue-type plasminogen activator by plasminogen activator inhibitor1. J. Biol. Chem. 266, 7488-7493. Edelberg, J.M., Weissler, M., and Pizzo, S.V., 1991b, Kinetic analysis of the effects of glycosaminoglycans and lipoproteins on urokinase-mediated plasminogen activator. Biochem. J. 276, 785-791. Fless, G.M., ZumMallen, M.E., and Scanu, A.M., 1986, Physicochemical properties of apolipoprotein(a) and lipoprotein(a) derived from the dissociation of human plasma lipoprotein(a). J. Biol. Chem. 261, 8712-8718. Frick, M.M., Dalhen, G.H., Berg, K., et al., 1978, Serum lipids in angiographically assessed coronary atherosclerosis. Chest 73, 62-65. Furie, B., and Furie, B.C., 1988, The molecular basis of blood coagulation. Cell 53, 505-518. Gaubatz, J.W., Heidmean, C., Gotto, A.M., et al., Human plasma lipoprotein(s): structural properties. J. Biol. Chem. 258, 4582-4589. Gonzalez-Gronow, M., Edelberg, J.M., and Pizzo, S.V., 1989, Further characterization of the cellular plasminogen binding site: evidence that plasminogen 2 and lipoprotein(a) compete for the same site. Biochemistry 28, 2375-2377. Hajjar, H.A., Gavish, D., Breslow, J.L., and Nachman, R.L., 1989, Lipoprotein(a) modulation of endothelial cell surface fibrinolysis and its potential role in atherosclerosis. Nature 339, 303-305. Hamsten, A., Walldius, G., Szamosi, A., et al., 1987, Plasminogen activator inhibitor in plasma: risk factor for recurrent myocardial infarction. Lancet ii, 3-9. Karadi, I., Hostner, G.M., Gries, A., et al., Lipoprotein(a) and plasminogen are immunochemically related. Biochim. Biophys. Acta 960, 91-97. Knott, T.J., Pease, R.J., Powell, L.M., et al., Complete protein sequence and identification of structural domains of human apolipoprotein B. Nature 323, 734-738. Law, S.W., Grant, S.M., Higuchi, H., et al., 1986, Human liver apolipoprotein B-100 cDNA: complete nucleic acid and derived amino acid sequence. Proc. Natl. Acad. Sci. USA 83, 8142-8146. Lijnen, H.R., and Collen, D., 1986, ~2-Antiplasmin, in: A.J. Barret and G. Salvesen (Eds.), Proteinase Inhibitors, Elsevier, Amsterdam, pp. 457-476. Loscalzo, J., 1990, Lipoprotein(a): a unique risk factor for atherothrombotic disease. Arteriosclerosis 10, 672-679. Loscalzo, J., Weinfeld, M., Fless, G.M., and Scanu, A.M.,
~
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1990, Lipoprotein(a), fibrin binding and plasminogen activation. Atherosclerosis 10, 240-245. Markus, G., DePasquale, J.L., and Wissler, F.C., 1978, Quantitative determination of the binding of 6-aminocaproic acid to native plasminogen. J. Biol. Chem. 253, 727-732. McLean, J.W., Tomlinson, J.E., Kuang, K., et al., 1987, cDNA sequence of human apolipoprotein(a) is homologous to plasminogen. Nature (Lond.) 330, 132-137. Miles, L.A., and Plow, E.F., 1987, Receptor mediated binding of the fibrinolytic components, plasminogen and urokinase, to peripheral blood cells. Thromb. Haemostasis 58, 936-942. Miles, L.A., Fless, G.M., Levine, E.G., et al., 1989, A potential basis for the thrombotic risks associated with lipoprotein(a). Nature 339, 301-303. Muria, A., Miyahara, T., Fujimoto, N., et al., 1986, Lp(a) lipoprotein as a risk factor for coronary heart disease and cerebral infarction. Atherosclerosis 59, 199-204. Patthy, L., 1985, Evolution of the proteases of blood coagulation and fibrinolysis by assembly from modules. Cell 41, 647-663. Rath, M., Niendorf, A., Reblin, T., et al., 1989, Detection and quantification of lipoprotein(a) in the arterial wall of 107 coronary bypass patients. Arteriosclerosis 9, 579-592. Rhoads, G.G., Dahlen, G., Berg, K., et al., 1986, Lp(a), lipoprotein as a risk factor for myocardial infarction. J. Am. Med. Assoc. 256, 2540-2544. Robbins, K.C., Summaria, L., Hsieh, B., and Shah, R., 1967, The primary structure of human plasminogen. J. Biol. Chem. 247, 6757-6762. Rosenberg, R.D., Bauer, H.A., and Marcum, J.A., 1986, The heparin-antithrombin system, in: E. Murano (Ed.), Reviews in Hematology, PJD, New York, pp 351-416. Rouy, D., Grailhe, P., Nigon, F., et al., 1991, Lipoprotein(a) impairs generation of plasmin by fibrin-bound tissue-type plasminogen activator: in vitro studies in a plasma milieu. Atheroscler. Thromb. 11, 629-638. Scanu, A.M., and Fless, G.M., 1990, Lipoprotein (a) heterogeneity and biological relevance. J. Clin. Invest. 85, 1709-1715. Smith, E.B., and Cochran, S., 1990, Factors influencing the accumulation in fibrous plaques of lipid derived from lowdensity lipoproteins. Atherosclerosis 84, 173-181. Sprengers, E.D., and Kluft, C., 1987, Plasminogen activator inhibitors. Blood 69, 381-387. Sugiyama, N., Sasaki, T., lwamoto, M., and Abiki, Y., 1988, Binding site of ~2-plasmin inhibitor to plasminogen. Biochem. Biophys. Acta 952, 1-7. Walker, I.D., Davidson, J.F., Hutton, I., Lawrie, T.D., 1977, Disordered 'fibrinolytic potential' in coronary heart disease. Thromb. Res. 10, 509-510. Walsh, C., 1979, Enzymatic Reaction Mechanisms, Freeman, San Francisco, pp. 55-97. Wiman, B., and Collen, D., 1978, On the kinetics of the reaction between human antiplasmin and a low-molecularweight form of plasmin. Eur. J. Biochem. 84, 573-578.
368 Wiman, B., Boman, L., and Collen, D., 1978, On the kinetics of the reactions between human antiplasmin and a low molecular weight form of plasmin. Eur. J. Biochem. 87, 143-146. Wiman, B., Lijnen, H.R., and Collen, D., 1979, On the kinetics of the reaction between human antiplasmin and plasmin. Biochem. Biopbys. Acta 579, 142-154.
J. Edelberg, S.V. Pizzo / Chem. Phys. Lipids 67/68 (1994) 363-368 Zenker, G., Koltringer, P., Bone, G., et al., 1986, Lipoprotein(a) as a strong indicator for cerebrovascular disease. Stroke 17, 942-947. Zioncheck, T.F., Powell, L.M., Rice, G.C., et al., 1991, Interaction of recombinant apolipoprotein(a) and lipoprotein(a) with macrophages. J. Clin. Invest. 87, 767-771.
ELSEVIER SCIENCE IRELAND
Chemistry and Physics of LIPID$
Lipoprotein (a) regulates plasmin generation and inihibition Jay Edelberg, S.V. Pizzo* Duke University Medical Center, Box 3712, Durham, NC 27710, USA
(Accepted 12 November 1992)
Abstract The relationship between lipoprotein (a) (Lp(a)) and atherosclerosis has been appreciated for a number of years. Only in recent years, however, has the structural relationship of Lp(a) to plasminogen resulted in studies of the effect of this lipoprotein on fibrinolysis. Lp(a) inhibits activation of plasminogen by tissue-type (t-PA) and urinary-type (u-PA) plasminogen activators. These inhibitory reactions are surface-dependent. When Lp(a) binds to fibrin, fibrinogen, heparin or cells it blocks activation of plasminogen by t-PA. u-PA-mediated activation of plasminogen is blocked on surfaces including heparin and chondroitin sulfate. Lp(a) also favors inhibition of plasmin by c~2-antiplasmin (c~2-AP). The ability of Lp(a) to compete with plasmin for fibrin binding displaces plasmin into solution where c~2-AP rapidly inhibits this proteinase. These effects are all antifibrinolytic. Lp(a) also exhibits one profibrinolytic effect, since it blocks inhibition of t-PA by plasminogen activator type 1 in the presence of fibrinogen or heparin. Thus, Lp(a) modulates most of the reactions involved in plasmin generation and inhibition. Its overall effect will depend primarily on the concentrations of Lp(a), PAl-1 and t-PA in vivo. Key words: Lipoprotein (a) and fibrinolysis; c~2-Antiplasmin; Plasminogen activation; Tissue-type plasminogen
activator
1. Lipoprotein (a) and Atherosclerosis Lipoprotein (a) (Lp(a)) is a low-density plasma lipoprotein first identified by Berg (1963). Elevated levels of Lp(a) (greater than 30 mg/dl) are associated with a risk of atherosclerosis two to five times that of control subjects (Albers et al., 1977; Frick et al., 1978; Rhoads et al., 1986; Dalhen et al., 1986). The elevated Lp(a) levels are linked not only to coronary artery disease but also to stenosis
* Corresponding author.
of carotid and cerebral arteries (Murai et al., 1986; Zenker et al., 1986). Structural studies of Lp(a) demonstrate that, like low-density lipoproteins, it contains a lipid core and an apoprotein B (apo B) sub-unit. However, it also contains an apoprotein (a) (apo(a)) sub-unit disulfide-linked to apo B (for detailed review see Scanu, 1988). The apo B sub-unit is an Mr - 510 000 polypeptide (Law et al., 1986; Knott et al., 1986), and the apo(a) sub-unit is also a large, but heterogenous, apoprotein with isoforms larger than, smaller than and equal in size to the apo B sub-unit (Gaubatz et al., 1983; Arm-
0009-3084/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0009-3084(93)0222 I-C
364
strong et al., 1985; Fless et al., 1986; Eaton et al., 1987; Karadi et al., 1988). Protein and cDNA sequences of the apo(a) sub-unit reveal extensive homology between the sub-unit and the fibrinolytic zymogen plasminogen (McLean et al., 1987; Eaton et al., 1987). The apo(a) sub-unit contains the lysine binding domains, termed kringles, found in plasminogen and other coagulation and fibrinolytic serine proteinases; for example, tissue-type plasminogen activator (t-PA), urinary-type plasminogen activator (u-PA) and prothrombin (Patthy, 1985; Furie and Furie, 1988). While plasminogen has single copies of kringles 1 to 5, the apo(a) sub-unit has up to 37 copies of kringle 4 and a single copy of kringle 5, with 75-85% and 95% homology, respectively, to their plasminogen counterparts. The apo(a) sub-unit also contains a region 94% homologous to the plasminogen proteinase domain. This proteinase domain has an intact catalytic triad essential for serine proteinase activity (Walsh, 1979), but the sub-unit lacks a critical plasminogen cleavage site, Arg560-Va156b necessary for zymogen activation (Robbins et al., 1967). The apo(a) sub-unit has a Ser in the place of an Arg found in plasminogen, and it cannot be converted to a proteinase by typical plasminogen activators (Eaton et al., 1987).
2. The relationship of lipoprotein (a) and fibrinolysis The association between the increased incidence of atherosclerosis and elevated levels of Lp(a) may be due to suppression of normal fibrinolytic activity. Several clinical studies have demonstrated an inverse correlation between fibrinolytic activity and the risk of coronary artery disease (Walker et ai., 1977; Hamsten et al., 1987). Recent studies also demonstrate that Lp(a) accumulates in the lesions of the affected vessels (Rath et ai., 1989; Smith and Cochran, 1990; Beisiegel et al., 1990), which suggests that Lp(a) may have a direct role in the pathogenous of these lesions. The homology between apo(a) and plasminogen suggests that this accumulation of Lp(a) may result in a competition with plasminogen for cellular and matrix binding sites and that the association between the increased incidence of atherosclerosis and the elevated
Z Edelberg, S.V. Pizzo / Chem. Phys. Lipids 67/68 (1994) 363-368
levels of Lp(a) may be due to suppression of normal fibrinolytic activity at such sites. Fibrinolysis may be regulated by controlling the generation of plasmin, the activity of plasmin and the inhibition of plasmin. The regulation of plasmin generation, or plasminogen activation, is dependent on many factors, including the local concentrations of plasminogen, plasminogen activators and plasminogen activator inhibitors, as well as the kinetic rates of these activators and their inhibitors. The homology between the apo(a) sub-unit of Lp(a) and plasminogen led several investigators to examine the potential roles of Lp(a) in the fibrinolytic system. Recent studies demonstrate that Lp(a) may down-regulate in vivo fibrinolysis by competing with plasminogen for various vascular pro-fibrinolytic surfaces, including cellular binding sites, fibrinogen fragments and heparin. Cellular binding sites, by concentrating plasminogen and plasminogen activators on the vascular surface, are critical in fibrinolytic initiation (for review see Miles and Plow, 1988). Plasminogen binds these sites through its kringle domains. Recent studies demonstrate that Lp(a) competes with plasminogen via its kringle 4 domains for cellular binding sites on monocytes, macrophages and endothelial cells (Gonzalez-Gronow et al., 1989; Miles et al., 19.89; Hajjar et al., 1989; Zionchech et al., 1991). Lp(a) displacement of plasminogen from these cellular binding sites may prevent plasminogen from interacting with t-PA and u-PA, thereby locally depressing fibrinolysis. Lp(a) inhibits t-PA-mediated plasminogen activation in the presence of fibrinogen fragments and fibrin (Edelberg et al., 1990; Loscalzo et al., 1990; Rouy et al., 1991). Kinetic analysis demonstrates that Lp(a) acts as a competitive inhibitor of this activation in the presence of fibrinogen fragments (Edelberg et al., 1990). Lp(a) does not inhibit t-PA amidolytic activity, indicating that the lipoprotein does not occupy the active site of t-PA. Lp(a) does not displace t-PA from fibrinogen fragments, since these interactions would have been reflected in an uncompetitive or non-competitive inhibition constant. Importantly, Lp(a) has no effect on the rate of the reaction in the absence of fibrinogen
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fragments. Together these data suggest, then, that Lp(a) competes for a plasminogen binding site on fibrinogen fragments adjacent to the bound t-PA, thereby inhibiting the rate of plasmin generation. Lp(a) also inhibits heparin-enhanced plasminogen activation. Heparin, an anticoagulant glycosaminoglycan located on both the endothelial surface and the basement membrane (for brief review see Rosenberg, 1986), stimulates the rate of both t-PA and u-PA-mediated plasminogen activation (Edelberg and Pizzo, 1990b; Edelberg et al., 1991b). Heparin enhances the rate of plasmin generation by increasing the catalytic activity of both t-PA and u-PA. Lp(a) inhibits these heparinenhanced reactions by competing with plasminogen for access to the heparin-bound plasminogen activator (Edelberg and Pizzo, 1990b; Edelberg et al., 1991b). Lp(a) does not inhibit the amidolytic activity of the plasminogen activators, nor does it effect plasmin generation in the absence of heparin similar to its effect on fibrinogen fragmentstimulated activation. These data suggest that Lp(a) binds to heparin at a site adjacent to the activator and thereby prevents plasminogen from interacting with these enzymes. o~2-Antiplasmin is the primary inhibitor of plasmin formed in the plasma (for review see Lijnen and Collen, 1986). Inhibition is rapid and involves an interaction with both the catalytic domain of plasmin and the amino terminal regulatory kringle domains of the proteinase. Modulators that block the interaction between the kringles and a2-antiplasmin will decrease the rate of inhibition (Wiman and Collen, 1978; Wiman et al., 1978, 1979; Christensen and Clemmensen, 1978; Anonick and Gonias, 1991). Kinetic analysis shows that CNBr-derived fibrinogen fragments competitively prevent inhibition (Edelberg and Pizzo, 1992). The decreases in the rate of inhibition may result from the prevention of a kringleinhibitor interaction. The initial interaction between o~2-antiplasmin and the kringle domains of plasmin may involve one of the first three lysine binding sites of the enzyme. Previous studies demonstrated that c~2antiplasmin binds the kringles of plasminogen, and that domains 1-3 are preferentially involved
365
(Christensen and Clemmensen, 1978; Wiman et al., 1979; Sugiyama et al., 1988). More recent studies by Sugiyama et al. (1988) demonstrate that tryptic fragments of the carboxyl terminal of ct2antiplasmin have a higher affinity for the plasminogen kringle 1-3 domains than for the kringle 4 domain. In addition, e-aminocaproic acid decreases the rate of the inhibition approximately fivefold, e-Aminocaproic acid binds to plasmin with a dissociation constant of 120/~M, a binding constant consistent with binding to plasminogen kringle 1 (Markus et al., 1978). These data suggest that the reaction between plasmin and o~2-antiplasmin is mediated by an initial interaction between the kringle 1 domain of the enzyme and the kringle binding site of the inhibitor. Lp(a) contains only kringles 4 and 5 and does not, on its own, effect the rate of the inhibition of plasmin by et2-antiplasmin. However, in the presence of CNBr-derived fibrinogen fragments, Lp(a) increases the rate of the inhibition. The kinetics demonstrate that Lp(a) acts as a competitive suppressor of fibrinogen fragment-modulated inhibition. Lp(a) competes with plasmin for binding sites on the CNBr-derived fibrinogen fragments, resulting in plasmin dissociation from the fragments followed by rapid inhibition by ct2antiplasmin. These results suggest that in the vasculature, Lp(a) may promote the premature inhibition of fibrinolysis by promoting plasmin inhibition. Unlike these anti-fibrinolytic effects, Lp(a) can also have pro-fibrinolytic activity. Lp(a) protects t-PA from irreversible enzymatic inhibition by plasminogen activator inhibitor type I (PAl-l). In the vasculature, the major irreversible inhibitor of t-PA is PAl-1 (for brief review see Sprengers and Kluft, 1987). Lp(a) decreases the rate of t-PA inhibition by PAI-1 in a manner analogous to Lp(a) inhibition of t-PA-mediated plasminogen activation (Edelberg et al., 1990, 1991a; Edelberg and Pizzo, 1991). Moreover, similar to the Lp(a) effect on plasminogen activation, the decrease in PAI-1 inhibition is dependent on the presence of either fibrinogen or heparin. Kinetic analysis of the Lp(a) depression in t-PA irreversible inhibition indicates that Lp(a) competes with PAI-1 for
366
J. Edelberg, S.V. Pizzo / Chem. Phys. Lipids 67/68 (1994) 363-368
access to the template-bound plasminogen activator. Lp(a) decreases the rate o f the inhibition by sterically preventing P A I - I from interacting with fibrinogen-bound and heparin-bound t-PA similar to Lp(a) effects on plasminogen activation.
3. Lipoprotein(a) physiology and pathophysiology These studies demonstrate that in vitro Lp(a) competes with plasminogen for various vascular binding sites, inhibits plasmin generation by plasminogen activators and promotes plasmin inhibition but protects plasminogen activators from inhibition. This suggests that Lp(a) may both depress and prolong in vivo fibrinolysis. Lp(a), at physiological levels, may regulate the production of plasmin by protecting plasminogen activators from inactivation by PAI-1. In addition, it competes with plasminogen for access to surfacebound plasminogen activators as well as competing with plasmin for fibrin. Elevated levels of Lp(a) may depress fibrinolytic activity to a degree that is not compensated for by the prolonged availability o f the plasminogen activators. At these pathophysiological levels, Lp(a) should prevent a large fraction o f the available plasminogen from accessing the surface-bound activators, and promote the inhibition o f plasmin by u2-antiplasmin.
Endothelial Cells
I
Fibrin
P eP P ~P@P
Endothelial Ceils tPA (~
PAl-1
<:
Pg 0
Fibrin Pm ~
~2AP
~ Lp(a) p
Fig. 1. A scheme of the effects of lipoprotein (a) on fibrinolysis. The interaction of tissue-type plasminogen activator, plasminogen, plasminogen activator inhibitor 1 and cte-antiplasmin on both the endothelial cell and fibrin clot surfaces governs the rate of fibrinolysis. The addition of lipoprotein (a) to the system can disrupt these surface interactions, thereby regulating the fibrinolytic activity.
The net effect is to greatly depress fibrinolysis, even though the plasminogen activators are not inhibited by PAI-1 as rapidly as in the absence o f Lp(a). In addition, high circulating levels o f PAl- 1 would tend to overcome the protective effect o f Lp(a) on the inhibition reaction between PAI-1 and t-PA. Under these conditions, the overall effect o f elevated Lp(a) levels would be a suppression o f fibrinolysis. It is particularly noteworthy that both elevated Lp(a) and PAI-1 are associated with atherosclerosis and myocardial infarction. A model of this Lp(a) regulation of the fibrinolytic system is shown in Fig. 1. These data, combined with the kinetic results described above, may indicate that the pathogenous o f elevated levels of Lp(a) may be due to a chronic depression in endogenous fibrinolysis. Such a depression may in turn lead to thrombus stability and promote atherosclerosis.
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