ABSTRACT
Essentials Heparanase forms a complex with tissue factor and enhances the generation of factor Xa. The present study was aimed to identify the procoagulant domain of heparanase. Procoagulant peptides significantly shortened bleeding time and enhanced wound healing. Tissue factor pathway inhibitor (TFPI)-2 derived peptides inhibited the procoagulant peptides. SUMMARY: Background Heparanase, which is known to be involved in angiogenesis and metastasis, was shown to form a complex with tissue factor (TF) and to enhance the generation of activated factor X (FXa). Our study demonstrated that peptides derived from TF pathway inhibitor (TFPI)-2 impeded the procoagulant effect of heparanase, and attenuated inflammation, tumor growth, and vascularization. Aims To identify the procoagulant domain in the heparanase molecule, and to evaluate its effects in a model of wound healing that involves inflammation and angiogenesis. Methods Twenty-four potential peptides derived from heparanase were generated, and their effect was studied in an assay of FXa generation. Peptides 14 and 16, which showed the best procoagulant effect, were studied in a bleeding mouse model and in a wound-healing mouse model. Results Peptides 14 and 16 increased FXa levels by two-fold to three-fold, and, at high levels, caused consumption coagulopathy. The TFPI-2-derived peptides explored in our previous study were found to inhibit the procoagulant effect induced by peptides 14 and 16. In the bleeding model, time to clot formation was shortened by 50% when peptide 14 or peptide 16 was topically applied or injected subcutaneously. In the wound-healing model, the wound became more vascular, and its size was reduced to one-fifth as compared with controls, upon 1 week of exposure to peptide 14 or peptide 16 applied topically or injected subcutaneously. Conclusions The putative heparanase procoagulant domain was identified. Peptides derived from this domain significantly shortened bleeding time and enhanced wound healing.
Subject(s)
Coagulants/chemistry , Glucuronidase/chemistry , Hemorrhage/metabolism , Wound Healing , Animals , Blood Coagulation/drug effects , Factor Xa/chemistry , Fibrin Fibrinogen Degradation Products/chemistry , Fibrinogen/chemistry , Glycoproteins/metabolism , Hematologic Agents/pharmacology , Humans , Inflammation , Male , Mice , Neoplasm Metastasis , Neovascularization, Pathologic , Partial Thromboplastin Time , Peptides/chemistry , Protein Domains , Prothrombin Time , Thrombelastography , Thromboplastin/metabolism , ThrombosisABSTRACT
Heparanase is implicated in cell invasion, tumour metastasis and angiogenesis. It forms a complex and enhances the activity of the blood coagulation initiator - tissue factor (TF). We describe new peptides derived from the solvent accessible surface of TF pathway inhibitor 2 (TFPI-2) that inhibit the heparanase procoagulant activity. Peptides were evaluated in vitro by measuring activated coagulation factor X levels and co-immunoprecipitation. Heparanase protein and/or lipopolysaccharide (LPS) were injected intra-peritoneally and inhibitory peptides were injected subcutaneously in mouse models. Plasma was analysed by ELISA for thrombin-antithrombin complex (TAT), D-dimer as markers of coagulation activation, and interleukin 6 as marker of sepsis severity. Peptides 5, 6, 7, 21 and 22, at the length of 11-14 amino acids, inhibited heparanase procoagulant activity but did not affect TF activity. Injection of newly identified peptides 5, 6 and 7 significantly decreased or abolished TAT plasma levels when heparanase or LPS were pre-injected, and inhibited clot formation in an inferior vena cava thrombosis model. To conclude, the solvent accessible surface of TFPI-2 first Kunitz domain is involved in TF/heparanase complex inhibition. The newly identified peptides potentially attenuate activation of the coagulation system induced by heparanase or LPS without predisposing to significant bleeding tendency.
