The interaction of lipoprotein lipase (LPL) with heparan sulfate and with size-fractionated fragments of heparin was characterized by several approaches (stabilization, sedimentation, surface plasmon resonance, circular dichroism, fluorescence). The results show that heparin decasaccharides form a 1:1 complex with dimeric LPL and that decasaccharides are the shortest heparin fragments which can completely satisfy the heparin binding regions in dimeric LPL. Equimolar concentrations of octasaccharides also stabilized dimeric LPL, while shorter fragments (hexa- and tetrasaccharides) were less efficient. Binding of heparin did not induce major rearrangements in the conformation of LPL, supporting the view that the heparin binding region is preformed in the native structure. Interaction of LPL with heparan sulfate, as studied by surface plasmon resonance, was found to be a fast exchange process characterized by a high value for the association rate constant, 1.7 x 10(8) M-1 s-1, a relatively high dissociation rate constant, 0.05 s-1, and as a result a very low equilibrium dissociation constant equal to 0.3 nM at 0.15 M NaCl. The contribution of electrostatics was estimated to be 44% for the binding of LPL to heparan sulfate, 49% for the binding of LPL to unfractionated heparin, and 60% for the binding of LPL to affinity-purified heparin decasaccharides at 0.15 M NaCl. The number of ionic interactions between LPL and high-affinity decasaccharides was estimated to be 10. We propose an essential role of electrostatic steering in the association. Monomeric LPL had 6000-fold lower affinity for heparin than dimeric LPL had, expressed as a ratio of equilibrium dissociation constants. A model for binding of LPL to heparan sulfate-covered surfaces is proposed. Due to the fast rebinding, LPL is concentrated to the close proximity of the heparan sulfate surface. As the dissociation is also fast, the enzyme exchanges rapidly between specific binding sites on the immobilized heparan sulfate, without leaving the surface. This model may also apply to LPL at the endothelium of blood vessels.

In cultured human and rat cells, the lipolysis-stimulated receptor (LSR), when activated by free fatty acids (FFA), mediates the binding of apoprotein B- and apoprotein E-containing lipoproteins and their subsequent internalization and degradation. To better understand the physiological role of LSR, we developed a biochemical assay that optimizes both the activation and binding steps and, thus, allows the estimation of the number of LSR binding sites expressed in the livers of living animals. With this technique, a strong inverse correlation was found in rats between the apparent number of LSR binding sites in liver and the postprandial plasma triglyceride concentration (r = -0.828, p < 0.001, n = 12). No correlation existed between the number of LSR and plasma triglycerides measured in the same animals after 24 h of fasting. The same membrane binding assay was used to elucidate the mechanism by which FFA induce lipoprotein binding to LSR. The LSR activation step was mediated by direct interaction of FFA with LSR candidate proteins of apparent molecular masses of 115 and 90 kDa and occurred independently of the membrane lipid environment. The FFA-induced conformational shift that revealed the lipoprotein binding site remained fully reversible upon removal of the FFA. However, occupancy of the site by the apoprotein ligand stabilized the active form of LSR. Comparison of the effect of different FFA alone or in combination indicated that the same binding site is revealed by different FFA and that the length and saturation of the FFA monomeric carbon chain are critical in determining the potency of the FFA activating effect. We propose that the LSR pathway represents a limiting step for the cellular uptake of intestinally derived triglyceride-rich lipoproteins and speculate that FFA liberated by lipolysis initiate this process by altering the conformation of LSR to reveal the lipoprotein binding site.

Some or most of the turnover of lipoprotein lipase (LPL) occurs by dissociation from vascular endothelial sites in extrahepatic tissues and further degradation in the liver. Heparin greatly enhances this dissociation and delays but does not abolish uptake in the liver, raising the possibility that heparin could lead to accelerated catabolism of functional LPL. To investigate this, we determined time curves for heparin (anti-factor Xa activity) and for LPL and hepatic lipase after injection in rats of two doses of conventional unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH). The high dose (250 U/kg) of both heparins resulted in similar initial levels of LPL activity in plasma, but at 30 minutes the activity with LMWH had declined by more than 80%, whereas with UFH it remained essentially unchanged during this time. In contrast, time curves for heparin activity in blood were similar for the two heparins. The low dose (50 U/kg) led to lower initial levels of LPL activity with LMWH in spite of slower elimination of heparin activity from the blood. These results agree with previous studies that indicate that LMWH has a similar ability as UFH to release LPL, but a lesser ability to delay its removal by the liver. Only slight differences were noted in the time curves for hepatic lipase with the two heparins. To assess the possible depletion of the Upases, we administered a second large dose of conventional heparin. One hour after the first injection, the second injection resulted in lower plasma LPL activities in all four groups. This depletion of releasable LPL was more pronounced with high-dose LMWH (49% of that in saline-treated controls versus about 60% in the other groups). The LPL activity released by the second injection remained significantly depressed with low-dose LMWH and the high dose of either heparin at 4 hours, but had returned to normal after 24 hours. By contrast, no depletion of hepatic lipase activity could be shown at any time. The results showed that release of LPL into the circulating blood is followed by a period during which time the stores of functional LPL are depleted. This occurs with both UFH and LMWH; the difference between the two heparins lies more in the kinetics of the LPL removal process than in its ultimate result {Arterioscler Thromb. 1993;13:1391-1396 KEY WORDS • low-molecular-weight heparin heparin clearance • in vivo • rats• hepatic lipase • endothelium • lipase clearance H eparin has been used clinically for about 50 years, and its efficacy in the prophylaxis and treatment of thrombosis is well documented. In addition to its anticoagulant activity, heparin greatly increases the activity of two Upases in plasma: lipoprotein lipase (LPL) of extrahepatic origin, which hydrolyzes mainly triglycerides in chylomicra and very-lowdensity lipoproteins, and hepatic lipase (HL), which acts preferentially on remnants from chylomicra and very-low-density lipoproteins and on high-density lipoproteins. 1 The "lipolytic effect" of heparin comes from its ability to displace t...

Lipoprotein lipase (LPL) and hepatic lipase (HL) are two enzymes which participate in metabolism of plasma lipoproteins. The enzymes are located at vascular surfaces and are released from their binding sites on injection of heparin. In this paper we give a short overview of the structure of the lipases and their role in lipoprotein metabolism. Earlier studies had shown that low molecular weight (LMW) heparin preparations result in lower LPL activities in blood than do corresponding amounts of conventional heparin. Studies with organ perfusion in rats show that the two types of heparin have similar ability to release the lipases from their binding sites in extrahepatic tissues, but that LMW heparin is less effective than conventional heparin in preventing rapid uptake and degradation of LPL by the liver. After injection of heparin the metabolism of triglyceride-rich lipoproteins is initially accelerated, presumably as a result of the high levels of circulating LPL. Then follows a phase when lipoprotein metabolism is slower than normal, perhaps because endothelial LPL has been depleted by accelerated transport to and degradation in the liver.