The C-terminal fragment of axon guidance molecule Slit3 binds heparin and neutralizes heparin's anticoagulant activity

Abstract

Slit3 is a large molecule with multiple domains and belongs to axon guidance families. To date, the biological functions of Slit3 are still largely unknown. Our recent study demonstrated that the N-terminal fragment of Slit3 is a novel angiogenic factor. In this study, we examined the biological function of the C-terminal fragment of human Slit3 (HSCF). The HSCF showed a high-affinity binding to heparin. The binding appeared to be heparin/heparan sulfate-specific and depends on the size, the degree of sulfation, the presence of N- and 6-O-sulfates and carboxyl moiety of the polysaccharide. Functional studies observed that HSCF inhibited antithrombin binding to heparin and neutralized the antifactor IIa and Xa activities of heparin and the antifactor IIa activity of low-molecular-weight heparin (LMWH). Thromboelastography analysis observed that HSCF reversed heparin's anticoagulation in global plasma coagulation. Taken together, these observations demonstrate that HSCF is a novel heparin-binding protein that potently neutralizes heparin's anticoagulation activity. This study reveals a potential for HSCF to be developed as a new antidote to treat overdosing of both heparin and LMWH in clinical applications.

Heparan sulfate deficiency disrupts developmental angiogenesis and causes congenital diaphragmatic hernia

“…Moreover, fully desulfated heparin (Des-Hep), N-desulfated heparin (N-Des-Hep), and 6-O-desulfated heparin (6-Des-Hep) lost their ability to compete with heparin for SLIT3 binding, whereas 2-O-desulfated heparin (2-Des-Hep) did not ( Figure 6G). Similar binding characteristics have been observed for SLIT3-C (27). These binding measurements were consistent with the effect of Ndst1 deficiency on SLIT-ROBO signaling, reducing both N-and 6-O-sulfation of HS ( Figure 6, B-D).…”

“…Recombinant human SLIT3 N-terminal fragment (aa 34-919) was purchased from R&D Systems or was expressed in house. The N-terminal fragment was cloned into a modified pcDNA 3.1 vector (Invitrogen) with an HPC4 tag at the N terminus and a 6x-His tag at the C terminus (27). The construct was transfected into mouse neuroblastoma N2A cells (ATCC), and stably expressing clones were obtained.…”

“…23,[41][42][43]. SPR measurements were performed with biotinylated heparin immobilized on streptavidin-coated Sensor SA chips on a BIAcore 3000 using BIA evaluation software (version 4.0.1) (23,27,41). For direct binding analysis, SLIT3 protein samples were diluted in buffer containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, and 0.005% surfactant P20, at pH 7.4.…”

Probing the impact of GFP tagging on Robo1-heparin interaction

“…It was reported that Robo1 interacts with heparin/HS, and the interaction is important for Slit2-Robo1 signaling since heparin/HS binds to both Slit2 and Robo1 facilitating the ligand-receptor interaction [24-27]. In our previous report, we used SPR to characterize the binding of Robo1 to heparin immobilized on SA sensor chips [35].…”

“…In the present study, we used Robo1 (Roundabout homolog 1, with or without GFP tagging) in a heparin binding study. Robo1 is the receptor for SLIT1-3 which are thought to act as molecular guidance cue in cellular migration, including axonal navigation at the ventral midline of the neural tube and projection of axons to different regions during neuronal development, and in neovascularization [24-27]. We used surface plasmon resonance (SPR) spectroscopy to study the effect of the GFP tagging on the heparin-binding interactions with Robo1.…”