Heparinases can produce biologically active oligosaccharides by specifically cleaving the α-(1,4) glycosidic linkages of heparin and heparan sulphate. Heparinases are divided into heparinase and heparanase. Because heparinase is an effective biocatalyst, more and more researchers pay attention to the application of heparinase in medical field in the recent years. Combined with the related research work in our group, the application value of heparinase in the medical field was summarized, such as the determination of the structure of heparin, the preparation of low-molecular-weight heparin and ultra-low-molecular-weight heparin, tumor therapy and as a heparin antagonist. In addition, we summarized the definition, source of heparinase and its application in the medicine field. Heparinases have a great application prospect in the field of medicine.
Heparin ; Heparin Lyase ; metabolism ; Heparitin Sulfate ; Oligosaccharides ; Polysaccharide-Lyases

OBJECTIVE: To investigate the expression of HPA, CK2beta and HIF-1alpha gene in nasopharyngeal carcinoma (NPC) tissues, and the correlation between their expression with the clinical characteristics of NPC and the relativity of HPA, CK2beta and HIF-1alpha gene in NPC tissues. METHOD: HPA, CK2beta and HIF-1alpha were detected with Super-Vision immunohistochemical method using antibody in 49 NPC specimens and 30 specimens with chronic nasopharyngitis tissue (CNT). RESULT: The expression of HPA, CK2beta and HIF-1alpha in NPC tissue were significantly higher than those in CNT tissue (P<0.05, separately). The expression of HPA, CK2beta and HIF-1alpha were significantly related to the TNM stage and whether recurrence or metastasis occur after treatment (P<0.05, separate ly), but there was no obvious correlation between its expression and the sex of NPC patient (P>0.05). The expression of HIF-1alpha was significantly related to the age of NPC patient (P<0.05), while HPA, CK2beta were not. The expression of HPA, CK2beta and HIF-1alpha in NPC tissue was positively correlated with each other (P<0.05, separately). CONCLUSION HPA, CK2beta and HIF-1alpha play synergetic role in development of NPC, which plays an important role in invasiveness,recurrence and metastasis of NPC. There could be a positive cooperation among HPA, CK2beta and HIF-1alpha in the carcinogenesis and development of NPC.
Carcinoma ; Casein Kinase II ; metabolism ; Female ; Heparin Lyase ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Male ; Middle Aged ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Neoplasm Staging

OBJECTIVETo investigate the effect of low-molecular-weight heparin (LMWH) combined with paclitaxel (PTX) on the invasiveness and migration of nasopharyngeal carcinoma cells and explore the molecular mechanisms.

METHODSMTT assay was used to detect the growth inhibition induced by LMWH and PTX in CNE1 and CNE2 cells. Wound healing assay and Transwell migration assay were employed to assess the effects of the drugs on the cell migration, and Transwell invasion assay was used to evaluate the cell invasiveness. The cellular expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) were analyzed by Western blotting. ELISA was used to determine the expression of heparanase (HPA) in the culture medium of the cells.

RESULTSMTT assay showed an obvious suppression of CNE1 and CNE2 cell proliferation in response to LMWH and PTX treatments. Treatment with 200 U·ml LMWH combined with 0.1 µmol·L PTX for 24 h resulted in the inhibition rates of migration of 66.70% and 70.53% in CNE1 and CNE2 cells, respectively significantly higher than the rates in cells with PTX treatment alone. The combined treatment with LMWH and PTX for 24 h also caused a significantly higher inhibition rate of cell invasion than LMWH and PTX alone. LMWH enhanced the down-regulation of MMP-9 and HPA induced by PTX.

CONCLUSIONLMWH can enhance the inhibitory effect of PTX on the migration and invasion of nasopharyngeal carcinoma cells, the mechanism of which may involve the down-regulation of MMP-9 and HPA expressions.

Carcinoma ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Glucuronidase ; metabolism ; Heparin Lyase ; metabolism ; Heparin, Low-Molecular-Weight ; administration & dosage ; pharmacology ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; Nasopharyngeal Neoplasms ; pathology ; Paclitaxel ; administration & dosage ; pharmacology ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism

Heparinase III is an enzyme that specifically cleaves certain sequences of heparan sulfate. Previous reports showed that this enzyme expressed in Escherichia coli was highly prone to aggregation in inclusion bodies and lacks detectable biological activity. In this paper, we fused a glutathione-S-transferase (GST) tag to the N-terminus of heparinase III gene and expressed the fusion protein in Escherichia coli to develop an expression system of soluble heparinase III. As a result, approximately 80% of the fusion protein was soluble. The protein was then purified to near homogeneity via one-step affinity chromatography. A 199.4-fold purification was achieved and the purified enzyme had a specific activity of 101.7 IU/mg protein. This represented 32.3% recovery of the total activity of recombinant GST-heparinase III. The maximum enzyme production was achieved when bacteria were induced with 0.5 mmol/L isopropyl-beta-D-thiogalactoside at 15 degrees C for 12 h. The enzyme showed maximum activity at 30 degrees C and pH 7.5. And the enzyme activity was stimulated by 1 mmol/L Ca2+ and 150 mmol/L NaCl.
Escherichia coli ; genetics ; metabolism ; Flavobacterium ; enzymology ; genetics ; growth & development ; Glutathione Transferase ; biosynthesis ; genetics ; Heparin Lyase ; biosynthesis ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

OBJECTIVETo study the expression of CD138 and heparinase in hepatocellular carcinoma (HCC) and its relationship with tumor development, progression, metastasis and recurrence.

METHODSTissue microarray and immunohistochemical study (EnVision method) for CD138 and heparinase was performed on tissue microarray which consisted of 197 cases of HCC, including adjacent non-neoplastic liver tissues, and 66 cases of HCC metastases.

RESULTSThe rates of CD138 expression in HCC and adjacent non-neoplastic liver tissues were 48.7% (96/197) and 65.0% (128/197, P < 0.05) respectively. In early-stage and late-stage tumors, the expression rates were 61.7% (29/47) and 44.7% (67/150, P < 0.05) respectively. The rate in patients with metastasis was 33.3% (22/66), as compared with 53.6% (45/84, P < 0.05) in patients without metastasis. In patients with tumor recurrence occurring within or after 1 post-operative year, the expression rates were 23.3% (7/30) and 61.1% (11/18, P < 0.05) respectively. On the other hand, the rates of expression of heparinase in HCC and adjacent non-neoplastic liver tissues were 35.5% (70/197) and 12.7% (25/197, P < 0.05) respectively. In early-stage and late-stage tumors, the expression rates were 29.8% (14/47) and 37.3% (56/150, P > 0.05) respectively. The rate in patients with metastasis was 48.5% (32/66), as compared with 28.6% (24/84, P < 0.05) in patients without metastasis. In patients with tumor recurrence occurring within or after 1 post-operative year, the expression rates were 50.0% (15/30) and 44.4% (8/18, P > 0.05) respectively. In the 66 cases of metastatic HCC studied, the expression rate of CD138 was lower in the heparinase-positive subgroup (P < 0.05).

CONCLUSIONSLoss of CD138 expression is related to HCC development, progression, metastasis and recurrence. Overexpression of heparinase, when coupled with loss of CD138 expression, may take part in tumor metastasis of HCC.

Adult ; Aged ; Aged, 80 and over ; Carcinoma, Hepatocellular ; metabolism ; secondary ; Female ; Follow-Up Studies ; Heparin Lyase ; metabolism ; Humans ; Liver ; metabolism ; Liver Neoplasms ; metabolism ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Recurrence, Local ; Neoplasm Staging ; Neoplastic Cells, Circulating ; metabolism ; Peritoneal Neoplasms ; metabolism ; secondary ; Portal Vein ; Syndecan-1 ; metabolism ; Tissue Array Analysis

BACKGROUND: Endogenous heparinoid substances have been mentioned as one of the causes of coagulopathy during liver transplantation. Some reported that heparin effects after reperfusion increase with decreasing liver function, as assessed by the Child-Pugh classification. Comparisons of native and heparinase TEG can assess the quantity of heparin effects and distinguish the cause of coagulopathy. We investigated the heparin effects before reperfusion by heparinase-guided TEG and the correlation between heparin effects and the UNOS and Child-Pugh score. METHODS: 67 liver transplanted patients were studied and divided two groups. Two groups were control group that exist heparin effect and experimental group that does not exist heparin effect during preanhepatic period. Native and heparinase TEG are performed simultaneously after anesthetic induction. Present heparin effects were defined as coagulation time (gamma + kappa) differs more than 20% between native and heparinase TEG showing the native TEG's index is out of the normal range. RESULTS: Heparin effects were present before reperfusion in 29.8% of liver transplantation cases and these were related more with the Child-Pugh classification than UNOS (gamma = 0.31, P = 0.012). There were many transfusions of packed red cells and a large infusion amount through RIS in the group with heparin effects but there was no statistical significance. CONCLUSIONS: We could confirm that heparin effects appear already before reperfusion in 29.8% of the cases using heparinase-guided TEG and this correlates with the Child-Pugh classification
Classification ; Heparin Lyase ; Heparin* ; Heparinoids ; Humans ; Liver Transplantation* ; Liver* ; Reference Values ; Reperfusion ; Thrombelastography

Orthotopic liver transplantation is frequently associated with severe bleeding, especially after reperfusion of the grafted liver. Heparin or heparinoids are released from the grafted liver and cause additional blood coagulation disorders. Recently many investigators have used a heparinase guided thromboelastogram (TEG) to control and confirm heparin effects not only on liver transplantation but also cardiac surgery. We reported a clinical case using a heparinase guided TEG to observe the duration of postreperfusion heparin effects.
Blood Coagulation Disorders ; Hemorrhage ; Heparin Lyase* ; Heparin* ; Heparinoids ; Humans ; Liver Transplantation ; Liver* ; Reperfusion ; Research Personnel ; Thoracic Surgery ; Transplants

BACKGROUND: Heparin released from grafted liver immediately after declamping is one of causes of coagulopathy, and its presence has been diagnosed by comparing thromboelastography(TEG) of blood treated with 0.01% of protamine and untreated blood. However, protamine may affect coagulation if the amount of protamine is not optimal to heparin in the blood sample. Heparinase, an enzyme isolated from Flavobacterium Heparinum, neutralizes heparin without adversely affecting coagulation. Therefore we compared the TEGs of blood treated with heparinase and protamine to clarify the sensitivity and reliability of heparinase in reversing the heparin effect. METHODS: Differences in Reaction time(R time), Alpha angle, Maximal Amplitude(MA) between native and heparinase treated TEG on reperfusion in 8 cases of orthotopic liver transplantations were compared with those between native and protamine in 14 cases of OLT. RESULTS: On reperfusion, all of TEGs treated with heparinase showed more improved data rather than native one in R time, Alpha angle and MA. But, in protamine treated blood, R time and Alpha angle in 6 patients and MA in 3 patients were more depressed. The scattergram show that TEGs treated with heparinase on reperfusion have almost positive difference, but TEGs treated with protamine did not have positive results consistently. CONCLUSIONS: Heparinase is a more reliable reagent and activator than protamine on TEG for detecting heparin effects on reperfusion without showing in-vitro anticoagulation. Those results suggest that heparinase on TEGs can make diagnosis of coagulopathy developed immediately after reperfusion efficiently.
Diagnosis ; Flavobacterium ; Heparin Lyase* ; Heparin* ; Humans ; Liver Transplantation* ; Liver* ; Reperfusion* ; Thrombelastography* ; Transplantation ; Transplants

BACKGROUND: Heparin released from grafted liver immediately after declamping is one of causes of coagulopathy, and its presence has been diagnosed by comparing thromboelastography(TEG) of blood treated with 0.01% of protamine and untreated blood. However, protamine may affect coagulation if the amount of protamine is not optimal to heparin in the blood sample. Heparinase, an enzyme isolated from Flavobacterium Heparinum, neutralizes heparin without adversely affecting coagulation. Therefore we compared the TEGs of blood treated with heparinase and protamine to clarify the sensitivity and reliability of heparinase in reversing the heparin effect. METHODS: Differences in Reaction time(R time), Alpha angle, Maximal Amplitude(MA) between native and heparinase treated TEG on reperfusion in 8 cases of orthotopic liver transplantations were compared with those between native and protamine in 14 cases of OLT. RESULTS: On reperfusion, all of TEGs treated with heparinase showed more improved data rather than native one in R time, Alpha angle and MA. But, in protamine treated blood, R time and Alpha angle in 6 patients and MA in 3 patients were more depressed. The scattergram show that TEGs treated with heparinase on reperfusion have almost positive difference, but TEGs treated with protamine did not have positive results consistently. CONCLUSIONS: Heparinase is a more reliable reagent and activator than protamine on TEG for detecting heparin effects on reperfusion without showing in-vitro anticoagulation. Those results suggest that heparinase on TEGs can make diagnosis of coagulopathy developed immediately after reperfusion efficiently.
Diagnosis ; Flavobacterium ; Heparin Lyase* ; Heparin* ; Humans ; Liver Transplantation* ; Liver* ; Reperfusion* ; Thrombelastography* ; Transplantation ; Transplants

Residual heparin effects after protamine reversal is a potential bleeding disorder associated with cardiopulmonary bypass(CPB). To differentiate this from the other multiple factors causing coagulopathy should be initialized in the setting of management. The purpose of this study was to compare simple activated clotting time(ACT) and thromboelastography(TEG) with heparinase treated ACT and TEG for detecting residual heparin effects to distinguish rapidly the presence of heparin from the effects of other factors because the enzyme heparinase specifically neutralized heparin. After institution approval, 20 patients who required open heart surgery were studied. Baseline kaoline ACT, heparinase ACT, TEG and heparinase TEG(Haemoscope) were obtained before CPB on the same blood sample. The repeated tests were performed on the same blood samples 20 minutes after protamine reversal following CPB. Differences between heparinase treated tests and untreated tests were also evaluated at the same time. Wilcoxon signed ranked test was used to compare the results between before and after bypass. None of patients had significant postoperative bleeding complication. All tests before bypass were normal. Twenty minutes after protamine reversal, 3 patients showed kaoline ACT were extended above 10% of the value of heparinase ACT but all of them remained within normal range. However, nearly all patients showed heparin effects on TEG. The heparin effects on TEG were defined as significant differences in all of parameters, especially in alpha angle and R+K time between simple TEG and heparinase TEG. In Conclusion, heparinase treated ACT and native ACT are not sensitive to residual heparin effects after CPB. Their normal results did not preclude residual heparin effects on heparinase modified TEG. However, it might be further investigated to need additional protamine in the case of residual heparin effects on TEG.
Cardiopulmonary Bypass* ; Hemorrhage ; Heparin Lyase* ; Heparin* ; Humans ; Kaolin ; Reference Values ; Thoracic Surgery ; Thrombelastography*