Evaluation and analysis of bleeding risk of anticoagulation therapy in severe non-ICU patients with COVID-19 based on heparinase-modified TEG / 中国输血杂志

【Objective】 To investigate the clinical application of heparinase-modified TEG (hmTEG) in evaluating coagulation status and monitoring anticoagulant therapy in severe non-ICU patients with COVID-19. 【Methods】 The clinical data of severe non-ICU patients with COVID-19 confirmed to be infected with novel coronary disease (SARS-CoV-2) from December 2022 to May 2023 were analyzed retrospectively. The patients were divided into therapeutic dose group and prophylactic dose group according to the initial dose of enoxaparin. The changes of platelet count, activated partial thromboplastin time, prothrombin time, thrombin time, fibrinogen, D-dimer, TEG and hmTEG before and after heparin treatment were compared between the two groups, so as to evaluate the changes of coagulation function and bleeding risk of COVID-19 severe non-ICU patients after anticoagulation with different doses of heparin. 【Results】 A total of 179 severe non-ICU patients with COVID-19 were enrolled in this study, including 102 patients in therapeutic dose group and 77 patients in prophylactic dose group. Before receiving heparin anticoagulation, except for age(63.4±11.6 vs 59.8±9.1) D-dimer(678 ng/mL vs 621 ng/mL) and MA values [(69.1±10.2)mm vs (65.6±8.5)mm], there were no statistical differences in platelet count, activated partial thromboplastin time, prothrombin time, thrombin time, fibrinogen, R value, K time, α angle and coagulation index (CI) between the therapeutic dose group and the prophylactic dose group (P>0.05). After receiving heparin anticoagulation, there were significant differences in CKR value [(12.2±4.1)min vs (10.2±3.3)min] and CKHR value [(8.1±3.2)min vs (7.1±2.6)min] between therapeutic dose group and prophylactic dose group (P0.05). Meanwhile, the proportion of heparin overdose in the therapeutic dose group was significantly higher than that in the prophylactic dose group 15.69%(16/102) vs 5.19%(4/77)(P0.05). 【Conclusion】 In the current epidemic trend of COVID-19, in order to reduce the occurrence of bleeding events, the heparin dose should be selected more carefully in the prevention of thrombosis in severe non-ICU patients with COVID-19. The individualized assessment of bleeding risk by hmTEG is more conducive to the adjustment and control of heparin dose.

Morphologic and molecular study on the lens anterior capsule in systemic sclerosis / Relato sobre estudo morfológico e molecular da cápsula anterior do cristalino na esclerose sistêmica

ABSTRACT This study aimed to analyze the anterior lens capsule specimens from both eyes of a patient with systemic sclerosis and compare them to the eyes of a control patient. No significant differences between systemic sclerosis and control eyes were observed in the results from the hematoxylin-eosin and picrosirius staining. In the samples obtained from both systemic sclerosis and control eyes, there were expressions of caspase, a molecule expressed in cell death by apoptosis. Heparanase was overexpressed in the systemic sclerosis sample compared to the control sample. Therefore, the anterior lens capsule of the patient with systemic sclerosis is probably affected by the disease since it showed marked expression of heparanase 1.(AU)

RESUMO Analisamos as amostras das cápsulas anteriores do cristalino de uma paciente com esclerose sistêmica e comparamos com as de um paciente controle. Não foram observadas diferenças significativas entre esclerose sistêmica e controle nos resultados da coloração com hematoxilina-eosina e picrosirius. Nas amostras obtidas da esclerose sistêmica e do controle, obtivemos expressão de caspase, uma molécula expressa na morte celular por apoptose. A heparinase foi expressa de forma mais marcante na amostra de esclerose sistêmica quando comparada ao controle. Portanto, a cápsula anterior do cristalino da paciente com esclerose sistêmica provavelmente foi afetada pela doença, uma vez que mostrou expressão aumentada de heparinase 1.(AU)

Activating effect of extracellular histones on heparinase in chlorine-induced acute lung injury / 中国职业医学

OBJECTIVE: To investigate the induction and activation of heparinase by extracellular histones in acute respiratory distress syndrome(ARDS) induced by chlorine in mice.METHODS: The specific pathogen free adult male C57 BL/6 mice were randomly divided into control group, chlorine injured group, histone injured group, anti-histone antibody group and heparinase inhibitor group, with six mice in each group.The mice in the control group and histone injured group were exposed to clean air, and the mice in the other three groups were exposed to chlorine gas at a dose of 580.0 mg/m~3 for 30 minutes by systemic dynamic inhalation.Mice in the histone injured group were injected with 50 mg/kg body weight calf thymus histone by tail vein.One hour before exposure, mice in the anti-histone antibody group were pretreated with 20 mg/kg body weight anti-histone H4 antibody by tail vein injection, and mice in the heparinase inhibitor group were injected with 2 mg/kg body weight OGT2115(heparinase inhibitor). The other three groups were given equal volume of 0.9% sodium chloride solution by tail vein injection. After 24 hours of exposure, arterial blood was collected for blood gas analysis and the lung tissue was collected for histopathological examination. The protein level of heparinase in lung tissue were detected using enzyme-linked immunosorbent assay, and the activity of heparinase were detected by measuring the product of heparan degradation. The protein expression of pro-heparinase and active heparinase were detected by Western blotting.RESULTS: The dyspnea developed of mice in the chlorine injured group and histone injured group, diffuse inflammation occurred in lung tissue, the oxygenation index in arterial blood decreased(all P<0.05), and the protein level and activity of heparinase in lung tissue, as well as the relative expression of pro-heparinase and active heparinase were increased compared with the control group(all P<0.05). The dyspnea, hypoxemia and acute lung injury of mice in the anti-histone antibody group were alleviated, and the protein level of heparinase in lung tissue, as well as the relative expression levels of pro-heparinase and active heparinase were decreased(all P<0.05), compared with chlorine injury group and histone injury group.The dyspnea, hypoxemia and acute lung injury were alleviated in the heparinase inhibitor group, and the activity of heparinase and the relative expression of pro-heparinase in the lung tissue were decreased compared with the chlorine injury group(all P<0.05). CONCLUSION: During the occurrence and development of chlorine-induced ARDS in mice, extracellular histones aggravate lung injury by inducing the expression and activation of heparinase. Acute lung injury can be alleviated by inhibiting the expression and activation of heparinase.

Research progress of heparinase in the field of medicine / 生物工程学报

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.

Comparison of crude heparin pretreatment methods and identification of crude heparin from various species origins by quantitative PCR / 中国生化药物杂志

Objective To compare magnetic beads kit,agrose gel recovery kit and heparinase I three methods to purify the micro DNA from crude heparin, then use q-PCR to identify the species origins and select the best method.Methods Using magnetic beads kit,agrose gel recovery kit and heparinase I to purify micro DNA from crude heparin and combined the porcine,bovine and ovine identification kits to identify the species origins and conformed the minimum detection limit of different percentage of ovine crude heparin in porcine crude heparin.Results Three pretreatment methods all can solve the pretreatment difficulties and we found that the haparinase was the best method; the minimum detection limit was 0.01%of ovine crude heparin in porcine crude heparin.Conclusion The heparinase method is the best pretreatment method and can successfully solve the pretreatment difficulties.Heparinase combine the porcine, bovine and ovine identification kits can identify the species origins from crude heparin.

Heparinase Digestion-based Disaccharide Analysis of Clinical Heparin and Heparinoids Drug / 分析化学

Heparin and low molecular weight heparin have been widely used in clinical therapy as anticoagulants in cardiovascular disease and in hemodialysis. Crude heparin is usually prepared from porcine intestinal mucosa. Purified heparin is a mixture of polysaccharides consisting mainly of repeating GlcNS(6S)-IdoA2S disaccharides and other disaccharides with different GlcNAc/GlcNS±3S±6S-GlcA/IdoA±2S residues. Heparin injections are drugs prepared from heparin active pharmaceutical ingredient ( API ) that is prepared from crude heparin. Low molecular weight heparins are dominant heparin-based drugs used clinically, which are prepared by degrading heparin into smaller sizes. As a result, low molecular weight heparins are sharing the same major disaccharides but have different reducing and non-reducing ends. In current study, we focused on the disaccharide compositional analysis of clinically used heparin and heparin-based drugs. HeparinaseⅠ,II, and Ⅲ were used to degrade all heparin and heparin-based drugs including heparin sodium injection, Enoxaparin sodium injection, Nadroparin calcium injection, Dalteparin sodium injection, Fondaparinux sodium into disaccharides. All the degraded products were analyzed by strong anion high perforance liquid chromatography ( SAX-HPLC) coupled with an UV-detector. Commercially available unsaturated disaccharide standards were then used for structral identification. Furthermore, unusual disaccharides present in Nadroparin, Dalteparin, and Fondaparinux were confirmed by reversed-phase ion pair HPLC coupled with mass spectrometry. The developed method produced detailed structural information, which should be useful for quality control of heparin and heparin-based drugs.

Preparation and structural sequence analysis of heparan sulfate dodecasaccharide / 中国药学杂志

OBJECITVE: To prepare a heparan sulfate dodecasaccharide and analyze its structural sequence. METHODS: HS-derived oligosaccharide was prepared by heparinase II depolymerization. Size-uniform dodecasaccharide mixture was acquired by low pressure gel permeation chromatography on Bio-Gel P-10 column. The dodecasaccharides were further separated by strong anion-ex-change high-performance liquid chromatography (SAX-HPLC) to get a charge-uniform dodecasaccharide. The dodecasaccharide was completely hydrolyzed by heparinases I, II, and III, its disaccharides composition was analyzed by HPLC, and its structural sequence was confirmed by electrospray collision-induced dissociation mass spectrometry (ES-CID-MSn). RESULTS: One size- and charge-uniform pure dodecasaccharide was separated from HS enzymatic hydrolyzed mixture, and it was composed by δUA-GlcNAc (IV-A) and AUA-GlcNS (IV-S) with the relative ratio of 5-1. Based on the ES-CID-MSn analysis, its structural sequence was δUA-GlcNAc-GlcA-GlcNS-GlcA-GlcNAc-GlcA-GlcNAc- GlcA-GlcNAc-GlcA-GlcNAc. CONCLUSION: The dodecasaccharide was prepared from HS, its detailed structure is analyzed, and it will offer a basis for the structure-activity relationship study of HS oligosaccharide.

SAX-HPLC method for quantification of 1,6-anhydro derivatives of enoxaparin sodium / 中国药学杂志

OBJECTIVE: To establish an HPLC method to determine the levels of the characteristic 1, 6-anhydro derivatives of enoxaparin sodium.

Expression of microRNAs targeting heparinase in gastric cancer / 第二军医大学学报

Objective To investigate the expression of heparinase (HPA) and miRNAs that may regulate HPA expression in the gastric cancer tissue. Methods The expression levels of HPA were determined by real-time PCR in the gastric cancer tissues. Based on the binding of miRNAs with the 3′ untranslated region (3′-UTR) of HPA, we screened for miRNAs targeting HPA using bioinformatics method and determined the expression levels of these miRNAs in the gastric cancer tissues using real-time PCR. Results The expression of HPA was higher in the gastric cancer tissues compared with those in the tumor-adjacent and normal tissues. Bioinformatics analysis showed that miR-18b, miR-137, miR-502, and miR-299-3p might inhibit HPA expression by binding to its 3′-UTR. Real-time PCR analysis revealed lower expression levels of miR-18b, miR- 137, miR-502, and miR-299-3p in the gastric cancer tissues compared with in the adjacent and normal tissues. Conclusion HPA is highly expressed in the gastric cancer tissues; miR-18b, miR-137, miR-502 and miR-299-3p, which may target HPA, are lowly expressed, indicating miRNAs targeting HPA may participate in the carcinogenesis of gastric cancer.

Inhibition of Sanggenon G Isolated from Morus alba on the Metastasis of Cancer Cell / 中草药·英文版

Objective An organic layer prepared from the cortex of Morus alba (Moraceae) was studied in order to identify the active compounds for heparinase. Methods Bioassay-guided fractionation resulted in the isolation of sanggenon G Results The compound showed inhibitory activity with IC50 of 3.7 μmol/L on heparinase in vitro as well as 24 μmol/L in invasion assay using MDA-MB231 cells. Sanggenon G also had the moderate cytotoxicity at SW 620 (colon) and ACHN (kidney) cancer cell lines with IC50 of 10.96 and 13.44 μmol/L, respectively. Conclusion This is the first time that prenylated flavonoid sanggenon G is described as heparinase inhibitor. Besides, this flavonoid would be expected to be a metastasis inhibitor of cancer cells and also a valuable reagent to explore the mechanism of heparinase/heparanase-mediated metastasis.

Role of low-molecule-weight heparin in cancer metastasis / 国际外科学杂志

Cancer metastasis is multi-step process that requires cancer cells to jump through many hoops.The low-molecule-weight beparin (LMWH) has been reported effective for the treatment of metastasis to some extent, the mechanisms are proposed as anticoagulation, inhibition of heparanase, selectins, adhesion, angiogenesis mediated by the tumor cells, and the effects on cell cycle and apoptosis.

Progress in the Study of Heparinases / 中国生物工程杂志

Heparinases,a kind of polysaccharide lyases,can degrade heparin and heparin sulfate to low molecular weight polysaccharides.It has been noted that many bacteria have heparinases although only few of them have been purified and characterized.Heparinases I,II and III from Flavobacterium heparinum have been extensively studied for many years and been commercialized recently.Heparinases have some important applications in the industry and clinic as well as in the determination of heparin structure,which is a very important anticoagulant drug used world-widely.The recent progresses in isolation of heparinase-producing bacteria,genome mapping of heparinase homologs in sequenced bacteria and archaea genomes,purification of heparinases and the study of their biochemistry and regulation were reviewed.The recombinant expression of these enzymes as well as important applications of heparinases and their potential applications in the future will also be highlighted.

Evaluation of Heparin Effects in Liver Transplantation Using Heparinase-Guided Thromboelastogram / 대한마취과학회지

BACKGROUND:Complex coagulopathy during liver transplantation is influenced by many factors. A thromboelastogram (TEG) provides rapid information concerning coagulopathy and provides an aid for treatment. Heparin has been reported of a transplanted liver influence the pathogenic status of coagulation after reperfusion. Heparinase-guided TEG (hgTEG) can be easily used to determine the effects of heparin by comparing it with native TEG (nTEG), and provide a guide of the effect of heparin treatment. METHODS: We evaluated the effects of heparin before and after reperfusion during liver transplantation, using nTEG and hgTEG. Seventy three patients underwent liver transplantation during the study period. They were evaluated for the effect of heparin after anesthetic induction and divided into two groups according to the presence (Heparin Group) or the absence (Non-heparin Group) of heparin induced effects. The presence of heparin effects were defined as a difference of over 20% in the coagulation time (r + k) on both TEGs, and an TEG beyond the normal range. Duration of the effect of heparin was defined up to the time when the normal nTEG index range was reached or when the difference in coagulation time using of the TEGs was < 20%. All patients were placed under general anesthesia and managed with routine supportive measurement including Rapid Infusion System (RIS). Both nTEG and hgTEG were measured after anesthetic induction and 10, 60, 120, 180, 240 and 300 minutes after reperfusion. RESULTS: The results were as follows; Incidence of heparin effects before reperfusion was 27.4%. Heparin effects after reperfusion were prolonged in the Heparin Group versus the Non-heparin Group, but without statistical significance. Blood products in the two groups were not significantly different. CONCLUSIONS: Heparin effects before reperfusion were not found to be correlated with heparin effects after reperfusion or with the amounts of transfused blood products. According to these results, we speculate that the two effects of heparin have separate roles in liver transplantation.

Evaluation of the Relationship between the Patient's Severity and the Presence of Heparin Effects with the Use of Heparinase-Guided TEG in Liver Transplantation / 대한마취과학회지

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

Clinical Observation of Postreperfusion Heparine Effects with a Heparinase Guided Thromboelastogram in a Liver Transplanted Patient / 대한마취과학회지

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.

Heparinase is More Reliable than Protamine for Detecting Heparin Effects on Thromboelastography on Reperfusion of Liver Transplantation / 대한마취과학회지

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.

Heparinase is More Reliable than Protamine for Detecting Heparin Effects on Thromboelastography on Reperfusion of Liver Transplantation / 대한마취과학회지

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.

Comparative Study of Heparinase Treated Activated Clotting Time with Hephrinase Treated Thromboelastography for Detecting Residual Heparin Effects Following Cardiopulmonary Bypass / 대한마취과학회지

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.

Effect of Addition Nucleotides on Heparinase Production by Flavobacterium heparinum / 微生物学通报

The effect of addition nucleotides on heparinase production by Flavobacterium heparinum was reseached. The result indicated that addition of nucleotides cause different effects on heparinase production depend on its addi ng mode, single kind of nucleotide would restrain, but multiplex promote. The s trongest promotion happened when the propotion of nucleotides was consistent wit h the nucleic acids of heparinase mRNA. HPLC result confined the entrance of nu cleoides to the bactera cell. And it also suggested the synthesis metabolic of nucleic acids in F.heparinum was imbalance, there always be higher lever of purin nucleoides. L-Asp was added to medium culture to enhance pyridine nucle oides thynesis in order to improve the enzyme production ,which has been affecte d by the imbalance of nucleoides.

ULTRASTRUCTURAL OBSERVATIONS OF THE ENZYMATICALLY DIGESTED HUMAN AMNIOTIC BASEMENT MEMBRANE / 解剖学报

Using enzymatic digestions on human amniotic basement membrane (HABM), it was observed ultrastructurally that heparinase Ⅱ digestion resulted in formation of microvilli on the surface of HABM and thinning and vesiculation of lamina densa and lamina lucida layers. This suggested that the heparinase Ⅱ-digested HABM had increased its surface area for more efficient cell attachment. The main action of heparinase Ⅱ is to digest heparan sulphate on HABM surface, which leads to exposure of more sites of laminin for cell attachment and neurite outgrowth; therefore heparinase Ⅱ-digested HABM can increase neuronal growth to 150%. For those HABM digested by heparinase Ⅰ, collagenase Ⅰ, and chondroitinase AC, they lost most of the sites for attachment and neurite outgrowth and as a consequence, the growth of neurons decreased. This morphological study supports heparinase Ⅱ digested HABM as an ideal substratum for neuronal attachment and growth.