Affinity and Kinetics of Different Heparins Binding to P- and L-Selectin

ABSTRACT

Selectins are adhesion receptors that participate in inflammation and tumor cell metastasis. The anti-inflammatory and antimetastatic activities of heparins have been related partly to their ability to interact with P- and L-selectin. The recent findings that various heparins differ in antimetastatic activity were explained by differences in their P- and L-selectin binding ability. To obtain data to illustrate the binding characteristics, we detected for the first time the binding kinetics and affinity of the two low molecular weight heparins (LMWHs) enoxaparin and nadroparin, and of the unfractionated heparin Liquemin N to P- and L-selectin using a quartz crystal microbalance biosensor. Enoxaparin and nadroparin behave nearly identical in their binding affinity to both P-selectin (K_D 4.60 × 10^{- 6} M versus 7.61 × 10^{- 6} M) and L-selectin (K_D 2.01 × 10^{- 6} M versus 2.84 × 10^{- 6} M). Liquemin N displayed slightly higher affinities to both selectins (K_D 6.07 × 10^{- 7} M versus 1.07 × 10^{- 7} M). The differences are caused by a higher association rate compared with that of the LMWHs. These data support recent findings of antimetastatic activities, but illustrate that the intrinsic selectin binding does not entirely reflect the antimetastatic activities in vivo.

Binding kinetics - quartz crystal microbalance (QCM) - biosensor - heparin - selectins

REFERENCES

• 1 Kakkar A K, Levine M N, Kadziola Z et al.. Low molecular weight heparin, therapy with dalteparin, and survival in advanced cancer: the fragmin advanced malignancy outcome study (FAMOUS).  J Clin Oncol. 2004;  22(10) 1944-1948
• 2 Altinbas M, Coskun H S, Er O et al.. A randomized clinical trial of combination chemotherapy with and without low-molecular-weight heparin in small cell lung cancer.  J Thromb Haemost. 2004;  2(8) 1266-1271
• 3 Klerk C P, Smorenburg S M, Otten H M et al.. The effect of low molecular weight heparin on survival in patients with advanced malignancy.  J Clin Oncol. 2005;  23(10) 2130-2135
• 4 Lee A Y, Levine M N, Baker R I et al.. Low-molecular-weight heparin versus a coumarin for the prevention of recurrent venous thromboembolism in patients with cancer.  N Engl J Med. 2003;  349(2) 146-153
• 5 Lee A Y, Rickles F R, Julian J A et al.. Randomized comparison of low molecular weight heparin and coumarin derivatives on the survival of patients with cancer and venous thromboembolism.  J Clin Oncol. 2005;  23(10) 2123-2129
• 6 Zacharski L R, Ornstein D L. Heparin and cancer.  Thromb Haemost. 1998;  80(1) 10-23
• 7 Smorenburg S M, Van Noorden C J. The complex effects of heparins on cancer progression and metastasis in experimental studies.  Pharmacol Rev. 2001;  53(1) 93-105
• 8 Skinner M P, Fournier D J, Andrews R K et al.. Characterization of human platelet GMP-140 as a heparin-binding protein.  Biochem Biophys Res Commun. 1989;  164(3) 1373-1379
• 9 Skinner M P, Lucas C M, Burns G F, Chesterman C N, Berndt M C. GMP-140 binding to neutrophils is inhibited by sulfated glycans.  J Biol Chem. 1991;  266(9) 5371-5374
• 10 Nelson R M, Cecconi O, Roberts W G et al.. Heparin oligosaccharides bind L- and P-selectin and inhibit acute inflammation.  Blood. 1993;  82(11) 3253-3258
• 11 Koenig A, Norgard-Sumnicht K, Linhardt R, Varki A. Differential interactions of heparin and heparan sulfate glycosaminoglycans with the selectins. Implications for the use of unfractionated and low molecular weight heparins as therapeutic agents.  J Clin Invest. 1998;  101(4) 877-889
• 12 Ley K. The role of selectins in inflammation and disease.  Trends Mol Med. 2003;  9(6) 263-268
• 13 Vestweber D, Blanks J E. Mechanisms that regulate the function of the selectins and their ligands.  Physiol Rev. 1999;  79(1) 181-213
• 14 Borsig L, Wong R, Feramisco J et al.. Heparin and cancer revisited: mechanistic connections involving platelets, P-selectin, carcinoma mucins, and tumor metastasis.  Proc Natl Acad Sci USA. 2001;  98(6) 3352-3357
• 15 Kim Y J, Borsig L, Varki N M, Varki A. P-selectin deficiency attenuates tumor growth and metastasis.  Proc Natl Acad Sci USA. 1998;  95(16) 9325-9330
• 16 Ludwig R J, Boehme B, Podda M et al.. Endothelial P-selectin as a target of heparin action in experimental melanoma lung metastasis.  Cancer Res. 2004;  64(8) 2743-2750
• 17 Borsig L, Wong R, Hynes R O, Varki N M, Varki A. Synergistic effects of L- and P-selectin in facilitating tumor metastasis can involve non-mucin ligands and implicate leukocytes as enhancers of metastasis.  Proc Natl Acad Sci USA. 2002;  99(4) 2193-2198
• 18 Varki N M, Varki A. Heparin inhibition of selectin-mediated interactions during the hematogenous phase of carcinoma metastasis: rationale for clinical studies in humans.  Semin Thromb Hemost. 2002;  28(1) 53-66
• 19 Laubli H, Stevenson J L, Varki A, Varki N M, Borsig L. L-selectin facilitation of metastasis involves temporal induction of Fut7-dependent ligands at sites of tumor cell arrest.  Cancer Res. 2006;  66(3) 1536-1542
• 20 Stevenson J L, Choi S H, Varki A. Differential metastasis inhibition by clinically relevant levels of heparins-correlation with selectin inhibition, not antithrombotic activity.  Clin Cancer Res. 2005;  11(19 pt 1) 7003-7011
• 21 Ludwig R J, Alban S, Bistrian R et al.. The ability of different forms of heparins to suppress P-selectin function in vitro correlates to their inhibitory capacity on bloodborne metastasis in vivo.  Thromb Haemost. 2006;  95(3) 535-540
• 22 Sauerbrey G. Verwendung von Schwingquarzen zur Wägung dünner Schichten und zur Mikrowägung.  Zeitschrift der Physil. 1959;  155(2) 206-222
• 23 Fritzsche J, Alban S, Ludwig R J et al.. The influence of various structural parameters of semisynthetic sulfated polysaccharides on the P-selectin inhibitory capacity.  Biochem Pharmacol. 2006;  72(4) 474-485
• 24 Xie X, Rivier A S, Zakrzewicz A et al.. Inhibition of selectin-mediated cell adhesion and prevention of acute inflammation by nonanticoagulant sulfated saccharides. Studies with carboxyl-reduced and sulfated heparin and with trestatin a sulfate.  J Biol Chem. 2000;  275(44) 34818-34825
• 25 Wang L, Brown J R, Varki A, Esko J D. Heparin's anti-inflammatory effects require glucosamine 6-O-sulfation and are mediated by blockade of L- and P-selectins.  J Clin Invest. 2002;  110(1) 127-136
• 26 Borsig L. Selectins facilitate carcinoma metastasis and heparin can prevent them.  News Physiol Sci. 2004;  19 16-21
• 27 Mousa S A. Heparin and low molecular weight heparin in thrombosis, cancer, and inflammatory diseases.  Methods Mol Med. 2004;  93 35-48
• 28 Nicholson M W, Barclay A N, Singer M S, Rosen S D, van der Merwe P A. Affinity and kinetic analysis of L-selectin (CD62L) binding to glycosylation-dependent cell-adhesion molecule-1.  J Biol Chem. 1998;  273(2) 763-770
• 29 Mehta P, Cummings R D, McEver R P. Affinity and kinetic analysis of P-selectin binding to P-selectin glycoprotein ligand-1.  J Biol Chem. 1998;  273(49) 32506-32513
• 30 Wang J G, Geng J G. Affinity and kinetics of P-selectin binding to heparin.  Thromb Haemost. 2003;  90(2) 309-316
• 31 Linhardt R J, Gunay N S. Production and chemical processing of low molecular weight heparins.  Semin Thromb Hemost. 1999;  25(suppl 3) 5-16