Thrombin Cleaves Prolactin Into a Potent 5.6-kDa Vasoinhibin: Implication for Tissue Repair

Abstract

Vasoinhibin is an endogenous prolactin (PRL) fragment with profibrinolytic, antivasopermeability, and antiangiogenic effects. The fact that blood clotting, vascular permeability, and angiogenesis are functionally linked during the wound-healing process led us to investigate whether thrombin, a major protease in tissue repair, generates vasoinhibin. Here, we have incubated human PRL with thrombin and analyzed the resulting proteolytic products by Western blot, mass spectrometry, high-performance liquid chromatography purification, recombinant production, and bioactivity. We unveil a main thrombin cleavage site at R48-G49 that rapidly (< 10 minutes) generates a 5.6-kDa fragment (residues 1-48) with full vasoinhibin activity, that is, it inhibited the proliferation, invasion, and permeability of cultured endothelial cells and promoted the lysis of a fibrin clot in plasma with a similar potency to that of a conventional 14-kDa vasoinhibin (residues 1-123). The R48-G49 cleavage site is highly conserved throughout evolution and precedes the intramolecular disulfide bond (C58-C174), thereby allowing the 5.6-kDa vasoinhibin to be released without a reduction step. Furthermore, the 5.6-kDa vasoinhibin is produced by endogenous thrombin during the clotting process. These findings uncover the smallest vasoinhibin known, add thrombin to the list of PRL-cleaving proteases generating vasoinhibin, and introduce vasoinhibin as a thrombin-activated mechanism for the regulation of hemostasis, vasopermeability, and angiogenesis in response to tissue injury.

Sulpiride‐induced hyperprolactinaemia increases retinal vasoinhibin and protects against diabetic retinopathy in rats

“…The protein has the molecular mass (~16 kDa) of a common vasoinhibin and contains the N-terminal region of PRL that defines vasoinhibin. 31 This result is consistent with the clinical trial showing that levosulpiride increases bioactive vasoinhibin in the vitreous of patients with DR 21 and suggests that sulpiride-induced hyperprolactinaemia favours the incorporation of PRL into the eye of diabetic rats where it gets cleaved to vasoinhibin.…”

“…This vasoinhibin-like protein has the molecular mass of a vasoinhibin and contains the N-terminal region of PRL that defines vasoinhibin. 31 Consistent with the increase in vasoinhibin, hypervasopermeability was prevented in the retina of hyperprolactinaemic diabetic rats, whereas hyperprolactinaemic nondiabetic animals showed no change in retinal vasopermeability (Figure 5C).…”

“…Only the retina from diabetic rats made hyperprolactinaemic by the delivery of ovine PRL (lane 8) contained an immunoreactive 16 kDa band that may, thereby, correspond to ovine vasoinhibin (oVi). This vasoinhibin‐like protein has the molecular mass of a vasoinhibin and contains the N‐terminal region of PRL that defines vasoinhibin 31 . Consistent with the increase in vasoinhibin, hypervasopermeability was prevented in the retina of hyperprolactinaemic diabetic rats, whereas hyperprolactinaemic nondiabetic animals showed no change in retinal vasopermeability (Figure 5C).…”

“…However, sulpiride did enhance the Western blot detection of a vasoinhibin‐like protein in the retina of diabetic rats. The protein has the molecular mass (~16 kDa) of a common vasoinhibin and contains the N‐terminal region of PRL that defines vasoinhibin 31 21 and suggests that sulpiride‐induced hyperprolactinaemia favours the incorporation of PRL into the eye of diabetic rats where it gets cleaved to vasoinhibin.…”

Human Placental Tissue Contains A Placental Lactogen–Derived Vasoinhibin

“…1B ). Because vasoinhibin corresponds to the N-terminal end of PL and is known to aggregate [ 13 , 14 ], it is tempting to speculate that the 28-kDa protein could correspond to a vasoinhibin trimer. However, because the 28-kDa protein was not detected by anti–full-length PL polyclonal antibodies (see Fig.…”