Localization of Topically Applied TFPI Binding Sites in an Intimectomized Microvessel

Summary

Tissue factor pathway inhibitor, TFPI, has been shown to be highly effective as a topically applied antithrombotic in an arterial model of vascular thrombosis. To elucidate the mechanism and site of TFPI action, recombinant TFPI was conjugated to 30 nm diameter gold particles and used to localize the sites of TFPI binding in a traumatized microvessel by transmission electron microscopy. The model, the central artery of the rabbit ear, was transected, denuded of endothelial lining (intimectomized), and re-anastomosed. Prior to the restoration of blood flow, TFPI-gold or unconjugated gold particles in solution were applied by irrigation to the intimectomized vessel lumen. After 10 min of blood flow, the artery was harvested for electron microscopy. TFPI-gold binding was localized to the fine strands of fibrin that lined the lumen of the intimectomized section of the artery. Little or no binding was found on platelets, exposed smooth muscle, cell membrane fragments, or uninjured vessel segments. The TFPI-gold binding could be competed with native TFPI. TFPI-gold was inhibitory, although less potent than native TFPI, in a prothrombin time assay. Unconjugated gold exhibited very little binding in the vascular model. Hence, the TFPI-gold conjugate behaved like native TFPI. Our observations have identified the fibrin complex as an in vivo binding site for TFPI and suggest that this is an in vivo site of action for TFPI as a topical antithrombotic agent.

• References

• 1 Broze GJ, Girard T, Novotny WF. Regulation of coagulation by a multivalent Kunitz-type inhibitor. Biochemistry 1990; 29: 7539-7546
• 2 Rao LV M, Rapaport SI. Studies of a mechanism inhibiting the initiation of the extrinsic pathway of coagulation. Blood 1987; 69: 645-651
• 3 Broze GJ, Warren LA, Novotny WF, Higuchi DA, Girard JJ, Miletich JP. The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: insight into its possible mechanism of action. Blood 1988; 71: 335-343
• 4 Rapaport SI. The initiation of the tissue factor dependent pathway of blood coagulation. Adv Exp Med Biol 1990; 281: 97-103
• 5 Ryan J, Brett J, Tijburg P, Bach RR, Kisiel W, Stem D. Tumor necrosis factor-induced endothelial tissue factor is associated with subendothelial matrix vesicles but is not expressed on the apical surface. Blood 1992; 80: 966-974
• 6 Drake TA, Ruf W, Morrissey JH, Edgington TS. Function tissue factor is entirely cell surface expressed on lipopolysaccharide-stimulated human blood monocytes and a constitutively tissue factor producing neoplastic cell line. J Cell Biol 1989; 109: 389-395
• 7 Miletich JP, Jackson CM, Majems PW. Properties of the factor Xa binding site on platelets. J Biol Chem 1978; 253: 6908-6916
• 8 Eisenberg PR, Siegel JE, Abendschein DR, Miletich JP. Imnportance of factor Xa in determining the procoagulant activity of whole blood clots. J Clin Invest 1993; 91: 1877-1883
• 9 Callander NS, Rao LV M, Nordfang O, Sandset PM, Wam-Cramer B, Rapaport SI. Mechanism of binding of recombinant extrinsic pathway inhibitor (rEPI) to cultured cell surfaces. J Biol Chem 1992; 267: 876-882
• 10 Khouri RK, Koudsi B, Kaidling F, Omberg RL, Wun TC. Prevention of thrombosis by topical application of tissue factor pathway inhibitor in a rabbit model of vascular trauma. Ann Plast Surg 1993; 30: 398-404
• 11 Diaz-Collier JA, Palmier MO, Kretzmer KK, Bishop BF, Combs RG, Obu- kowicz MG, Frazier RB, Bild GS, Joy WD, Hill SR, Duffin KL, Gustafson ME, Junger KD, Grabner RW, Galluppi GR, Wun T-C. Refold and characterization of recombinant tissue factor pathway inhibitor expressed in Escherichia coli. Thromb Haemost 1994; 71: 339-346
• 12 Lui CY, Handley DA, Chien S. Gold labeling of thrombin and ultrastructural studies of thrombin-gold conjugate binding by fibrin. Anal Biochem 1985; 147: 49-56
• 13 Handley DA, Witte LD. Platelet-derived growth factor labeled to colloidal gold for use as a mitogenic receptor probe. Eur J Cell Biol 1984; 34: 281-288
• 14 Weitz JI, Huboda M, Massel D, Maraganore J, Hirsch J. Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors. J Clin Invest 1990; 86: 385-391
• 15 Broze GJ, Miletich JP. Characterization of the inhibition of tissue factor in serum. Blood 1987; 69: 150-157