The recombinant two kringle domain of human tissue-type plasminogen activator (TK1-2) has been shown to inhibit endothelial cell proliferation, angiogenesis, and tumor cell growth despite of sharing a low amino acid sequence homology with angiostatin. Here, we explored a possible inhibitory mechanism of action of TK1-2 by focusing on antimigratory effect. TK1-2 effectively inhibited endothelial cell migration induced by basic fibroblast growth factor or vascular endothelial growth factor in a dose-dependent manner and tube formation on Matrigel. It blocked basic fibroblast growth factor -induced or vascular endothelial growth factor -induced phosphorylation of extracellular signal-regulated kinase 1/2 and formation of actin stress fibers and focal adhesions. Interestingly, TK1-2 alone induced the weak phosphorylation of focal adhesion kinase, whereas it inhibited focal adhesion kinase phosphorylation induced by growth factors. When immobilized, TK1-2 promoted adhesion and spreading of endothelial cells compared with bovine serum albumin. However, treatment with anti-A 2 B 1 blocking antibody markedly diminished endothelial cell adhesion to immobilized TK1-2 compared with anti-A v B 3 or anti-A 5 B 1 antibody. Pretreatment of soluble TK1-2 also altered the binding level of anti-A 2 B 1 antibody to endothelial cells in fluorescence-activated cell sorting analysis. Indeed, a blocking antibody against integrin A 2 B 1 or knocking down of integrin A 2 expression prevented the inhibitory effect of TK1-2 in cell migration. Therefore, these results suggest that TK1-2 inhibits endothelial cell migration through inhibition of signaling and cytoskeleton rearrangement in part by interfering with integrin A

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<div>Abstract<p>The recombinant two kringle domain of human tissue-type plasminogen activator (TK1-2) has been shown to inhibit endothelial cell proliferation, angiogenesis, and tumor cell growth despite of sharing a low amino acid sequence homology with angiostatin. Here, we explored a possible inhibitory mechanism of action of TK1-2 by focusing on antimigratory effect. TK1-2 effectively inhibited endothelial cell migration induced by basic fibroblast growth factor or vascular endothelial growth factor in a dose-dependent manner and tube formation on Matrigel. It blocked basic fibroblast growth factor–induced or vascular endothelial growth factor–induced phosphorylation of extracellular signal-regulated kinase 1/2 and formation of actin stress fibers and focal adhesions. Interestingly, TK1-2 alone induced the weak phosphorylation of focal adhesion kinase, whereas it inhibited focal adhesion kinase phosphorylation induced by growth factors. When immobilized, TK1-2 promoted adhesion and spreading of endothelial cells compared with bovine serum albumin. However, treatment with anti-α₂β₁ blocking antibody markedly diminished endothelial cell adhesion to immobilized TK1-2 compared with anti-α_vβ₃ or anti-α₅β₁ antibody. Pretreatment of soluble TK1-2 also altered the binding level of anti-α₂β₁ antibody to endothelial cells in fluorescence-activated cell sorting analysis. Indeed, a blocking antibody against integrin α₂β₁ or knocking down of integrin α₂ expression prevented the inhibitory effect of TK1-2 in cell migration. Therefore, these results suggest that TK1-2 inhibits endothelial cell migration through inhibition of signaling and cytoskeleton rearrangement in part by interfering with integrin α₂β₁. [Mol Cancer Ther 2008;7(7):2133–41]</p></div>

<div>Abstract<p>The recombinant two kringle domain of human tissue-type plasminogen activator (TK1-2) has been shown to inhibit endothelial cell proliferation, angiogenesis, and tumor cell growth despite of sharing a low amino acid sequence homology with angiostatin. Here, we explored a possible inhibitory mechanism of action of TK1-2 by focusing on antimigratory effect. TK1-2 effectively inhibited endothelial cell migration induced by basic fibroblast growth factor or vascular endothelial growth factor in a dose-dependent manner and tube formation on Matrigel. It blocked basic fibroblast growth factor–induced or vascular endothelial growth factor–induced phosphorylation of extracellular signal-regulated kinase 1/2 and formation of actin stress fibers and focal adhesions. Interestingly, TK1-2 alone induced the weak phosphorylation of focal adhesion kinase, whereas it inhibited focal adhesion kinase phosphorylation induced by growth factors. When immobilized, TK1-2 promoted adhesion and spreading of endothelial cells compared with bovine serum albumin. However, treatment with anti-α₂β₁ blocking antibody markedly diminished endothelial cell adhesion to immobilized TK1-2 compared with anti-α_vβ₃ or anti-α₅β₁ antibody. Pretreatment of soluble TK1-2 also altered the binding level of anti-α₂β₁ antibody to endothelial cells in fluorescence-activated cell sorting analysis. Indeed, a blocking antibody against integrin α₂β₁ or knocking down of integrin α₂ expression prevented the inhibitory effect of TK1-2 in cell migration. Therefore, these results suggest that TK1-2 inhibits endothelial cell migration through inhibition of signaling and cytoskeleton rearrangement in part by interfering with integrin α₂β₁. [Mol Cancer Ther 2008;7(7):2133–41]</p></div>

Supplementary Fig from Antimigratory effect of TK1-2 is mediated in part by interfering with integrin α₂β₁