Antiangiogenic Antithrombin Blocks the Heparan Sulfate-dependent Binding of Proangiogenic Growth Factors to Their Endothelial Cell Receptors

Weiqing Zhang,Benjamin Richard,Yan Xiong,Richard Swanson,Steven T Olson

doi:10.1074/jbc.m604905200

Weiqing Zhang, Benjamin Richard + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m604905200

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Abstract

The anticoagulant serpin antithrombin acquires a potent antiangiogenic activity upon undergoing conformational alterations to cleaved or latent forms. Here we show that antithrombin antiangiogenic activity is mediated at least in part through the ability of the conformationally altered serpin to block the proangiogenic growth factors fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF) from forming signaling competent ternary complexes with their protein receptors and heparan sulfate co-receptors on endothelial cells. Cleaved and latent but not native forms of antithrombin blocked the formation of FGF-2-FGF receptor-1 ectodomain-heparin ternary complexes, and the dimerization of these complexes in solution and similarly inhibited the formation of FGF-2-heparin binary complexes and their dimerization. Only antiangiogenic forms of antithrombin likewise inhibited (125)I-FGF-2 binding to its low affinity heparan sulfate co-receptor and blocked FGF receptor-1 autophosphorylation and p42/44 MAP kinase phosphorylation in cultured human umbilical vein endothelial cells (HUVECs). Moreover, treatment of HUVECs with heparinase III to specifically eliminate the FGF-2 heparan sulfate co-receptor suppressed the ability of antiangiogenic antithrombin to inhibit growth factor-stimulated proliferation. Antiangiogenic antithrombin inhibited full-length VEGF(165) stimulation of HUVEC proliferation but did not affect the stimulation of cells by the heparin-binding domain-deleted VEGF(121). Taken together, these results demonstrate that antiangiogenic forms of antithrombin block the proangiogenic effects of FGF-2 and VEGF on endothelial cells by competing with the growth factors for binding the heparan sulfate co-receptor, which mediates growth factor-receptor interactions. Moreover, the inability of native antithrombin to bind this co-receptor implies that native and conformationally altered forms of antithrombin differentially bind proangiogenic heparan sulfate domains.

Highlights

Angiogenesis, the growth of new capillaries from pre-existing vessels, is a key physiologic process whose dysregulation underlies many diseases [1]
We recently showed that the heparin-binding site of antithrombin is essential for mediating the antiangiogenic activity of cleaved and latent forms of the serpin [18], strongly implicating an endothelial cell heparan sulfate receptor or co-receptor in this activity
In the present study we have provided evidence to support the idea that cleaved and latent antithrombins produce their antiangiogenic effects by competing with the proangiogenic growth factors, fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF), for binding to low affinity heparan sulfate co-receptors and thereby attenuating the ability of the growth factors to bind their high affinity endothelial cell protein receptors and produce their mitogenic effects [21, 22]

Summary

Antithrombin Antiangiogenic Mechanism

We recently showed that the heparin-binding site of antithrombin, which mediates heparin binding and activation of the anticoagulant function of the serpin, is a critical mediator of the antiangiogenic activity of cleaved and latent forms of the protein [18] This finding in conjunction with reports that other endogeneous angiogenesis inhibitors such as endostatin and kallistatin bind heparin or heparan sulfate to produce their antiangiogenic effects [19, 20] suggested that antiangiogenic forms of antithrombin might act by binding endothelial cell-associated heparan sulfate molecules. Treatment of HUVECs with heparinase III to eliminate the HSPG co-receptor for FGF-2 is shown to abolish the inhibitory effect of latent antithrombin on growth factor-stimulated cell proliferation or capillary tube formation [26] Taken together, these findings demonstrate that the antiangiogenic function of antithrombin is mediated at least in part by blocking FGF-2 or VEGF binding to their HSPG coreceptors. This blockade results from the preferential binding of antiangiogenic forms of antithrombin to proangiogenic sequences or domains in heparin or heparan sulfate that are distinct from the anticoagulant sequence recognized by native antithrombin [27]

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