Structural Requirements for TFPI-Mediated Inhibition of Neointimal Thickening After Balloon Injury in the Rat

Abstract

Abstract-The intimal thickening that follows vascular injury is inhibited by periprocedural tissue factor pathway inhibitor (TFPI) treatment in animal models. TFPI is a multivalent Kunitz-type protease inhibitor that inhibits factor Xa via its second Kunitz domain and the factor VIIa/tissue factor (TF) complex via its first Kunitz domain. The basic C-terminus of TFPI is required for the binding of TFPI to cell surfaces and cell-bound TFPI mediates the internalization and degradation of factor X and the down regulation of surface factor VIIa/TF activity. The C-terminus of TFPI is also required for its reported direct inhibition of smooth muscle cell proliferation in vitro. To examine the structural requirements for the inhibition of neointimal formation by TFPI, several TFPI-related proteins were tested in the rat carotid angioplasty model: 1) XK 1 , a hybrid protein containing the N-terminal portion of factor X and the first Kunitz domain of TFPI that directly inhibits factor VIIa/TF; 2) TFPI WT , the full-length TFPI molecule that inhibits factor Xa and factor VIIa/TF and binds cell surfaces; 3) TFPI K36I , an altered form of TFPI that inhibits factor Xa, but not factor VIIa/TF, and binds cell surfaces; 4) TFPI 13-161 , a truncated form of TFPI that inhibits factor VIIa/TF but interacts with factor Xa poorly and does not bind to cell surfaces. Seven day infusions of XK 1 , TFPI WT , and high levels of TFPI K36I begun the day before balloon-induced vascular injury produced a significant reduction in the intimal hyperplasia measured 28 days after angioplasty. The infusion of high concentrations of TFPI was ineffective in this model. These in vivo results directly mirror the ability of each TFPI-related protein to inhibit tissue thromboplastin-induced coagulation in rat plasma: XK 1 ϷTFPI WT ϾTFPI K36I Ͼ ϾTFPI . The studies confirm the important role of TF-mediated coagulation in the smooth muscle proliferation and neointimal thickening that follows vascular injury and suggest that the anticoagulant effect alone of TFPI and TFPI-related proteins is sufficient to explain their therapeutic action. Key Words: tissue factor pathway inhibitor Ⅲ angioplasty Ⅲ rat T he mechanisms underlying the intimal thickening and associated restenosis that follow arterial injury are complex, but recent data suggest that activation of the coagulation cascade plays an important role in the process. 1-6 Tissue factor (TF) is an integral membrane protein that serves as an essential cofactor for plasma factor VII(a) in the initiation of coagulation. TF is present at high levels in atherosclerotic plaques, and TF expression is induced in vascular smooth muscle cells after balloon injury. [7][8][9] The factor VIIa/TF catalytic complex activates factor X, which in turn produces thrombin generation. Both factor Xa and thrombin have been shown to be potent mitogens for vascular smooth cells in vitro. 4 -6 Moreover, anticoagulant agents that inhibit thrombin (hirudin), factor Xa (tick anticoagulant protein, TAP), or factor VIIa/TF (inactivat...

Lipoprotein (a) binds and inactivates tissue factor pathway inhibitor: a novel link between lipoproteins and thrombosis

“…All antibiotics were obtained from Gibco BRL (Rockville, MD). Synthesized c-terminus (cTFPI 37 ) and scrambled peptides (cTFPIs) were obtained from the Mayo Protein Core facility.…”

“…Further experiments on apo(a) binding were performed after immobilization of a 37-amino acid peptide spanning the c-terminus region of TFPI (cTFPI 37 ). To determine whether apo(a)-cTFPI 37 binding was lysine dependent, experiments were performed in the presence and absence of excess EACA (0.2 M).…”

“…To determine whether apo(a)-cTFPI 37 binding was lysine dependent, experiments were performed in the presence and absence of excess EACA (0.2 M). Lp(a) binding to cTFPI 37 and a scrambled form of that peptide, cTFPIs, was also performed to examine whether the specific TFPI c-terminus sequence was required for Lp(a) binding.…”

Local gene transfer of tissue factor pathway inhibitor regulates intimal hyperplasia in atherosclerotic arteries

“…In healthy vessels, TF is present in the adventitia and in the deep tunica media (9,10) but increases in the vascular intima and media after arterial injury (11) and in atherosclerotic states, where TF accumulates especially in lipid-laden plaques (10,12,13). In animal models of vascular injury, systemic administration of the inhibitor of TF (TF pathway inhibitor, or TFPI) (14)(15)(16), or of inhibitors of factor Xa (17) or thrombin (18), has suggested a role for the TF pathway of blood coagulation and thrombosis in the pathogenesis of postinjury intimal hyperplasia. However, these studies have not distinguished between the local influence of TF inhibition in the injured vessel wall and the systemic anticoagulant effects achieved with i.v.…”

HIV envelope gp120 activates human arterial smooth muscle cells

“…TF is a primary determinant of the thrombogenicity of human atherosclerotic plaques and has been implicated in SMC proliferation and neointimal thickening following vascular injury (44) and in the development of cardiac vasculopathy (45). Factor Xa, the product of proteolytic cleavage of factor X by the TF-FVIIa complex, can cleave and thus activate protease-activated receptor 2 (PAR2) (46).…”