Involvement of the heparanase procoagulant domain in bleeding and wound healing.

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.

PO-49 - Bone microinfarction and microcirculation thrombosis; is it a possible mechanism for bone pain among cancer patients?

INTRODUCTION: The blood supply of the bone and bone marrow are interconnected through a network of vessels. Arteries penetrating the bone flow into the marrow cavity while blood from marrow sinuses and veins leave the tissue via the bone. AIM: The current study was designed to search for thrombosis in the small blood vessels of bone and bone marrow. MATERIALS AND METHODS: Bone biopsies of 40 patients were studied using specific staining to fibrin with martin scarlet blue (MSB) and by immune-staining to tissue factor (TF), TF pathway inhibitor and heparanase that is a pro-angiogenic and pro-coagulant protein. Biopsies included: ten cases of bone metastasis from carcinoma origin, ten cases of avascular necrosis (AVN) of femur head and ten cases of osteoarthrosis of femur head. Ten cases with diffuse large cell lymphoma without bone or bone marrow involvement were the control group. RESULTS: Vessels density was higher in the bone and bone marrow of metastasis, AVN and osteoarthrosis biopsies compared to controls (P<0.001). Thrombi were documented in the bone and bone marrow of metastasis (5/10), AVN (6/10) and osteoarthrosis (6/10) biopsies and were absent in the controls. Staining of TF, TFPI and heparanase were more prominent, mainly in the blood vessels, in the biopsies of metastasis, AVN and osteoarthrosis compared to controls. CONCLUSIONS: The present study demonstrates for the first time thrombosis in the microcirculation of bone and bone marrow that can potentially contribute to patients bone pain. Increased level of TF and heparanase may contribute to the procoagulant and pro-angiogenic state. Intervention with anticoagulant drugs to prevent bone pain should be further evaluated.

Heparanase procoagulant activity is elevated and predicts survival in non-small cell lung cancer patients.

BACKGROUND: Heparanase is implicated in angiogenesis and tumor progression. We had earlier demonstrated that heparanase may also affect the hemostatic system in a non-enzymatic manner. It forms a complex and enhances the activity of the blood coagulation initiator- tissue factor (TF). Although increased heparanase antigen level in the plasma and biopsies of cancer patients was previously demonstrated, in the present study we evaluated, for the first time, the heparanase procoagulant activity in the plasma of patients with lung cancer. MATERIALS AND METHODS: Sixty five patients with non-small cell lung cancer at presentation and twenty controls were recruited. Plasma was studied for TF / heparanase procoagulant activity, TF activity and heparanase procoagulant activity using chromogenic assay and heparanase antigen levels by ELISA. RESULTS: Heparanase antigen levels were higher in the study group compared to control (P=0.05). TF / heparanase activity, and even more apparent, heparanase procoagulant activity were significantly higher in the study group compared to controls (P=0.008, P<0.0001, respectively). No significant difference was observed in the TF activity between the groups. Survival of patients with heparanase procoagulant activity higher than 31 ng/ml predicted a mean survival of 9 ± 1.3 months while heparanase procoagulant activity of 31 ng/ml or lower predicted a mean survival of 24 ± 4 months (P=0.001). Heparanase procoagulant activity was higher than 31 ng/ml in the four cases of thrombosis detected during the follow-up period. CONCLUSIONS: Elevated heparanase procoagulant activity in patients with lung cancer reveals a new mechanism of coagulation system activation in malignancy. Heparanase procoagulant activity can potentially be used as a predictor for survival.

Novel peptides that inhibit heparanase activation of the coagulation system.

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.

Association of homocysteine, vitamin B12, folic acid, and MTHFR C677T in patients with a thrombotic event or recurrent fetal loss.

Hyperhomocysteinemia (HHC) is a known risk factor for venous and arterial thrombosis. Thrombophilia workup includes the level of homocysteine and other related parameters such as: vitamin B(12), folic acid, and methylenetetrahydrofolate reductase (MTHFR) C677T genotype. As the levels of homocysteine, vitamin B(12), folic acid, and MTHFR C677T genotype are linked biochemically, we hypothesized that a statistical association will be found between them. The purpose of the present study was to assess the association between the four parameters in patients with a thrombotic event or recurrent fetal loss. The potential study population included 326 patients who were referred to the Thrombosis and Hemostasis Unit; 125 of these patients had at least one pathological test result of the four parameters. The correlations between homocysteine and vitamin B(12) as well as between homocysteine and folic acid were found to be weak (r = -0.236 and r = -0.209, respectively). No significant difference was revealed between the mean homocysteine level and the CC, CT, and TT MTHFR genotypes (p = 0.246). In conclusion, in the population studied, the association between homocysteine, vitamin B(12), folic acid, and MTHFR C677T is weak. The results raise doubt as to whether the current routine evaluation of HHC, as part of thrombophilia workup, truly reflects the increased risk of thrombosis.

Heparanase induces tissue factor expression in vascular endothelial and cancer cells.

BACKGROUND: Over-expression of tissue factor (TF) and activation of the coagulation system are common in cancer patients. Heparanase is an endo-beta-D-glucuronidase that cleaves heparan sulfate chains on cell surfaces and in the extracellular matrix, activity that closely correlates with cell invasion, angiogenesis and tumor metastasis. The study was undertaken to investigate the involvement of heparanase in TF expression. METHODS: Tumor-derived cell lines were transfected with heparanase cDNA and TF expression was examined. The effect of exogenous addition of active and inactive heparanase on TF expression and activity was studied in tumor cell lines and primary human umbilical vein endothelial cells. TF expression was also explored in heparanase over-expressing transgenic (Tg) mice. Blast cells were collected from acute leukemia patients and TF and heparanase expression levels were analyzed. RESULTS: Over-expression of heparanase in tumor-derived cell lines resulted in a 2-fold increase in TF expression levels, and a similar trend was observed in heparanase Tg mice in vivo. Likewise, exogenous addition of heparanase to endothelial or tumor-derived cells resulted in enhanced TF expression and activity. Interestingly, TF expression was also induced in response to enzymatically inactive heparanase, suggesting that this effect was independent of heparanase enzymatic activity. The regulatory effect of heparanase on TF expression involved activation of the p38 signaling pathway. A positive correlation between TF expression levels and heparanase activity was found in blasts collected from 22 acute leukemia patients. CONCLUSIONS: Our results indicate that in addition to its well-known function as an enzyme paving a way for invading cells, heparanase also participates in the regulation of TF gene expression and its related coagulation pathways.