Predictive value of four items of new thrombus markers combined with conventional coagulation tests for thrombosis in antiphospholipid syndrome / 北京大学学报(医学版)

OBJECTIVE@#To explore the predictive value of four items of new thrombus markers combined with conventional coagulation tests for thrombosis in antiphospholipid syndrome.@*METHODS@#A total of 121 antiphospholipid syndrome (APS) patients who hospitalized at Peking University People's Hospital from March 2022 to January 2023 were selected and divided into thrombus group (50 cases) and nonthrombus group (71 cases) according to whether thrombosis occurred. The differences of laboratory characteristics including antiphospholipid antibodies were compared between the thrombotic and non-thrombotic groups. Chemiluminescent immunoassay was used to detect thrombomodulin (TM), thrombin-antithrombin complex (TAT), Plasmin-α2 plasmin inhibitor complex (PIC), and tissue plasminogen activator inhibitor complex (t-PAIC) in plasma from venous. The independent risk factors of thrombosis in patients with APS were determined using binary Logistic regression. Receiver operating characteristic (ROC) curve analysis was applied to evaluate the efficacy of each index on the prediction of thrombosis.@*RESULTS@#Compared with the patients without thrombosis, the patients with thrombosis were older [49 (32, 64) years vs. 36 (32, 39) years, P < 0.05]. The percentages of male, smoking, hypertension, and global antiphospholipid syndrome score (GAPSS)≥10 in the patients with thrombosis were significantly higher than those in the patients without thrombosis (P < 0.05). The positive rates of anticardiolipin antibody (aCL) and lupus anticoagulant (LA) in the thrombotic group were significantly higher than those in the non-thrombotic group (P < 0.05), and the levels of prothrombin time, activated partial thromboplastin time, fibrinogen, fibrin degradation product in the thrombotic group were significantly higher than those in the non-thrombotic group (P < 0.05).Among the thrombosis group, venous thrombosis accounted for 19 (38.00%), including deep vein thrombosis (16, 84.21%) and pulmonary embolism accounted (5, 26.32%); Arterial thrombosis accounted for 35 (70.00%), including myocardial infarction (6, 17.14%) cerebral infarction (30, 85.71%). The patients in the thrombotic group had significantly greater TM levels than those in the non-thrombotic group (P < 0.05).There were no significant dif-ferences between the two groups in TAT (Z=-1.420, P=0.156), PIC (Z=-0.064, P=0.949), and t-PAIC (Z=-1.487, P=0.137). Univariate and binary Logistic regression analysis of relevant variables showed that advanced age [OR=1.126, P=0.002], elevated TM [OR=1.325, P=0.048], prolonged prothrombin time (PT) [OR=4.127, P=0.008] were independent risk factors for thrombosis in the patients with APS. ROC curve analysis of the above three independent risk factors showed that the combined detection of age, PT and TM had the highest Yoden index (0.727) and sensitivity (83.0%), with a specificity of 89.7%.@*CONCLUSION@#TAT, PIC, TM, and t-PAIC may reflect thrombus formation from the coagulation system, fibrinolysis system, and endothelial system. The combined of age TM and PT is superior to the application of a single marker, which has diagnostic value for the early identification of APS thrombosis.

Plasmin-α2-plasmin inhibitor complex and thrombin-antithrombin complex in risk stratification of massive transfusion in patients with postpartum hemorrhage / 中国输血杂志

【Objective】 To analyze the value of plasmin-α2-plasmin inhibitor complex (PIC) and thrombin-antithrombin complex (TAT) for risk stratification of massive transfusion (MT) in patients with postpartum hemorrhage (PPH). 【Methods】 Clinical data and blood samples of patients with PPH in our hospital from January 2019 to December 2022 were retrospectively analyzed. MT (MT group, n=60) was defined as transfusion of red blood cells≥10 U within 24 h after delivery, and 3.25 ng/mL and PIC level>1.04 μg/mL were independent risk factors for MT after PPH. 【Conclusion】 Elevated TAT and PIC levels are independent predictors of MT in patients with PPH, and their combined predictive efficacy is better.

Changes of new coagulation markers in healthy pregnant women and establishment of reference intervals in Changsha / 中南大学学报(医学版)

OBJECTIVES@#There is a high coagulation state in pregnant women, which is prone to coagulation and fibrinolysis system dysfunction. This study aims to explore the latest coagulation markers-thrombomodulin (TM), thrombin-antithrombin complex (TAT), plasmin-α2 plasmin inhibitor complex (PIC), and tissue plasminogen activator/plasminogen activator inhibitor compound (tPAI-C) in different stages of pregnancy, establish reference intervals (RIs) for healthy pregnant women of Chinese population, and to provide an effective and reliable reference for clinicians.@*METHODS@#A total of 492 healthy pregnant women, who underwent pregnancy examination and delivery in the Department of Obstetrics, Second Xiangya Hospital of Central South University from October 2019 to October 2020, were enrolled for this study. They were assigned into the first trimester group, the second trimester group, the third trimester group, and the puerperium group according to the pregnancy period, and 123 healthy non-pregnant women were selected as the controls. Plasma levels of TM, TAT, PIC and tPAI-C were analyzed by automatic chemiluminescence immunoassay analyzer. The RIs for TM, TAT, PIC, and tPAI-C were defined using non-parametric 95% intervals, determined following Clinical and Laboratory Standards Institute Document C28-A3c (CLSI C28-A3c), and Formulation of Reference Intervals for the Clinical Laboratory Test Items (WS/T402-2012).@*RESULTS@#TM and TAT levels increased gradually in the first, second, and third trimester women and decreased in the puerperium women (P<0.05 or P<0.01). PIC level of healthy non-pregnant women was lower than that of pregnant women (P<0.05 or P<0.01), but PIC level of pregnant and puerperium women did not differ significantly (P>0.05). tPAI-C level in healthy non-pregnant women was lower than that of pregnant women (P<0.05 or P<0.01), and tPAI-C level was significantly decreases in the puerperium women (P<0.01). The RIs for TM were as follows: Healthy non-pregnant women at 3.20-4.60 TU/mL, the first and second trimester at 3.12-7.90 TU/mL, the third trimester at 3.42-8.29 TU/mL, puerperium at 2.70-6.40 TU/mL. The RIs for TAT were as follows: Healthy non-pregnant women at 0.50-1.64 ng/mL, the first and second trimester at 0.52-6.91 ng/mL, the third trimester at 0.96-12.92 ng/mL, puerperium at 0.82-3.75 ng/mL. The RIs for PIC were as follows: Healthy non-pregnant women at 0.160-0.519 ng/mL, pregnant women at 0.162-0.770 μg/mL. The RIs for tPAI-C were as follows: Healthy non-pregnant women at 1.90-4.80 ng/mL, the first and second trimester at 2.03-9.33 ng/mL, the third trimester at 2.80-14.20 ng/mL, puerperium at 1.10-8.40 ng/mL.@*CONCLUSIONS@#The levels of 4 new coagulation markers TM, TAT, PIC, and tPAI-C in pregnant women are increased significantly during pregnancy and gradually return to normal after delivery. The RIs for TM, TAT, PIC, and tPAI-C in pregnant women by trimester are established according to CLSI C28-A3c, thus providing a clinical reference for clinician in judgement of thrombotic risk.

Detection and Clinical Significance of Thrombus Molecular Markers in Diffuse Large B-cell Lymphoma / 肿瘤防治研究

Objective To investigate the detection and clinical significance of thrombus molecular markers in diffuse large B-cell lymphoma (DLBCL). Methods We collected the blood specimens of 60 patients with DLBCL, involving 23 cases in the initial treatment group, 24 cases in the remission group and 13 cases in the non-remission group, 23 cases in the thrombus group and 37 cases in the non-thrombus group. We selected 46 healthy people in the same period as the control group. The levels of thrombomodulin (TM), plasmin-α2 plasmin inhibitor complex (PIC), tissue plasminogen activator-plasminogen activator inhibitor-1 complex (t-PAIC) and thrombin-antithrombin Ⅲ complex (TAT) in plasma were detected by chemical immunoassay, and the levels of lactate dehydrogenase (LDH) in serum was detected by automatic biochemical analyzer. We analyzed the differences of thrombus molecular markers among groups and prognostic factors. Results The levels of TM and PIC in plasma of lymphoma patients were higher than those in health control group (P < 0.05). The levels of TM and PIC in the initial treatment and non-remission groups were significantly higher than those in the remission group (P < 0.05). The levels of TM, PIC and TAT in thrombus group were higher than those in non-thrombus group (P < 0.05). TM and PIC levels in plasma were closely related to the prognosis of DLBCL patients. PIC was an independent prognostic factor (P < 0.001). TM and PIC levels were correlated with LDH prognostic indicators in lymphoma patients. Conclusion TM and PIC levels in plasma are significantly increased in DLBCL patients. They are expected to be the indicators for effectiveness and prognosis of DLBCL patients.

A evaluation of coagulation biomarkers for diagnosis and therapeutic monitoring of deep venous thrombosis of lower extremities / 临床检验杂志

Objective@#To evaluate the changes of molecular markers of thrombosis in patients with deep venous thrombosis of lower extremities and analyze their value in the detection of venous thrombosis and evaluate the therapeutic effects. @*Methods@#In case-control study, we selected traumatic patients after surgery from Beijing Jishuitan Hospital during December 2018 to May 2019. A total of 64 patients with thrombosis were in DVT group, 39 patients without thrombosis were in non-DVT group, and 28 healthy subjects in the same period were in healthy control group. Venous blood samples were taken from all these people. Coagulation parameters thrombin-antithrombin complexes (TAT), plasmin-α2-plasmin inhibitor complexes (PIC) and tissue-type plasminogen activator-inhibitor complexes (t-PAIC) were detected at first diagnosis and one month after rivaroxaban anticoagulation therapy beginning. The differences of the markers between these groups were compared. @*Results@#The coagulation markers of the patients with lower extremity deep venous thrombosis increased significantly at diagnosis. The levels of plasma TAT, PIC, t-PAIC and sTM in DVT group were significantly higher than those in non-DVT group (P＜0.05). The levels of plasma TAT, PIC and t-PAIC in DVT group were higher than those in healthy control group (P＜0.05). There was no significant difference in sTM level between DVT group and healthy control group (P＞0.05). The results and changes of TAT, PIC, t-PAIC in the patients before and after one month of anticoagulation therapy were statistically different (P＜0.05) in comparison. @*Conclusion@#The molecular markers of thrombosis, TAT, PIC and t-PAIC, could effectively detect deep venous thrombosis of lower extremities and showed significant efficacy in evaluating the efficacy of anticoagulation therapy.

Clinical study on the diagnostic value of thrombomodulin combined with thrombin-antithrombin complex in disseminated intravascular coagulation / 医学研究生学报

Objective According to the cell-based coagulation theory, antithrombin complex (TAT) reflecting the activation of coagulation system, plasmin-α2 anti-plasmin complex (PIC) reflecting the activation of fibrinolytic system, thrombomodulin (TM) and tissue plasminogen activator-plasminogen activator inhibitor-1 complex (t-PAIC) reflecting vascular endothelial function were selected to explore their diagnostic values for disseminated intravascular coagulation. Methods A prospective study was conducted on 154 patients in the Department of Critical Care Medicine of the 908th Hospital from May to December 2018. The subjects were divided into non-overt DIC group (n=134) and overt DIC group (n=20) according to the diagnostic criteria of International Thrombus and Hemostatic Association. The differences among groups of TM, t-PAIC, TAT and PIC were compared along with statistical analysis. Results Compared with TM [10.5 (8.0~14.3) TU/mL], TAT [9.6 (4.9~21.8) ng/mL], PIC [1.253 (0.789~2.802) μg/mL] and t-PAIC [ 11.2 (7.1~22.1) ng/mL] in non-overt DIC group, TM [16.8 (11.8~21.5) TU/mL], TAT [33.6 (10.3~120.0) ng/mL], PIC [4.080 (0.814~8.651) μg/mL] and t-PAIC [19.4 (10.0~30.1)ng/mL] ) in overt DIC group were significantly increased (P<0.05). The area under the curve of TM>14.85 TU/mL combined with TAT>23.05 ng/mL as the standard diagnostic overt DIC was 0.835 (P=0.000), and the sensitivity, specificity, positive predictive value and negative predictive value were 0.85, 0.761, 0.592, 0.925 respectively. Conclusion TM combined with TAT has a higher diagnostic efficacy for overt DIC.

Application of four items of thrombosis detection in venous thrombosis of malignant tumor patients / 中国医师进修杂志

Objective@#To evaluate the clinical value of four items of thrombosis detection, including thrombin-antithrombin complex (TAT), α2-plasmin inhibitor-plasmin complex (PIC), thrombomodulin (TM) and tissue plasminogen activator-inhibitor complex (t-PAIC), combined with D-dimer (D-D) and fibrin degradation products (FDP) in venous thrombosis in patients with malignant tumor.@*Methods@#A total of 904 patients with malignant tumor from October 2017 to March 2019 in General Hospital of Heilongjiang Province Land Reclamation Bureau were selected (malignant tumor group), and 200 healthy physical examination patients were selected as healthy control group. Among 904 patients with malignant tumor, 92 patients had venous thrombosis (thrombosis group), and 812 patients had not venous thrombosis (non-thrombosis group). The TAT, PIC, TM, t-PAIC, FDP and D-D were detected. The relationship between TAT, PIC, TM, t-PAIC, D-D, FDP and venous thrombosis was analyzed by binary Logistic regression. The receiver operating characteristic (ROC) curve was used to determine the diagnostic performance of each index, and the maximum value of the Youden index was the optimal cut-off value.@*Results@#The TAT, PIC, TM, t-PAIC, D-D and FDP in malignant tumor group were significantly higher than those in healthy control group, and there were statistical differences (P<0.01). The TAT, PIC, TM, t-PAIC, D-D and FDP in thrombosis group were significantly higher than those in non-thrombosis group: 20.20 (12.30, 59.45) μg/L vs. 8.60 (4.87, 15.15) μg/L, 1.23 (0.69, 2.84) mg/L vs. 0.70 (0.37, 1.45) mg/L, 14.55 (8.12, 21.10) kU/L vs. 10.05 (7.975, 13.90) kU/L, 10.20 (7.30, 15.17) μg/L vs. 7.40 (5.20, 12.65) μg/L, 3.42 (1.38, 7.07) μg/L vs. 1.69 (0.53, 4.64) μg/L, 6.41 (3.21, 17.05) mg/L vs. 5.15 (2.26, 10.01) mg/L, and there were statistical differences (P<0.01 or <0.05). Binary Logistic regression analysis result showed that TAT, PIC, TM, t-PAIC, D-D and FDP were correlated with venous thrombosis in patients with malignant tumor (OR = 1.277, 1.209, 1.107, 1.089, 1.260, 1.078 and 0.002; P<0.01 or <0.05). ROC curve result showed that the optimal cut-off values of TAT, PIC, TM, t-PAIC, D-D and FDP in the diagnosis of venous thrombosis in patients with malignant tumor were 24.450 μg/L, 2.624 mg/L, 17.750 kU/L, 13.250 μg/L, 5.290 μg/L and 22.435 mg/L; and the area under curve (AUC) were 0.788, 0.659, 0.621, 0.597, 0.626 and 0.598, respectively. The AUC of TAT + PIC + TM + t-PAIC and TAT + PIC + TM + t-PAIC + D-D + FDP in the diagnosis of venous thrombosis in patients with malignant tumor were significantly higher than D-D + FDP (0.808 and 0.796 vs. 0.633). Ninety patients with TAT>24.450 μg/L or PIC>2.624 mg/L were selected. Fourty-five cases of them were injected with low molecular weight heparin (experimental group) for 6 weeks, and another 45 cases were not treated with low molecular weight heparin (control group). Both groups were followed up for 1 year. The incidence of venous thrombosis in the experimental group was significantly lower than that in control group: 2.22% (1/45) vs. 15.56% (7/45), the survival time was significantly longer than that in control group: (10.6 ± 3.1) months vs. (8.5 ± 2.8) months, and there were statistical differences (P<0.05), and no bleeding occurred in experimental group.@*Conclusions@#Four items of thrombosis detection combined with D-D and FDP is better than single detection. It is the best non-invasive method to detect venous thrombosis. It can predict the possibility of venous thrombosis in patients with malignant tumor at an early stage, and help patients actively use preventing drug, determine the best and most reasonable treatment time, improve the prognosis of patients, and prolong survival time.

Avances en el conocimiento de las proteínas de la leche materna / Advances in the knowledge about human milk proteins

La glándula mamaria y la leche materna son el resultado de millones de años de una evolución que llevó a una composición óptima para el crecimiento y desarrollo de recién nacidos y lactantes; la leche materna favorece el crecimiento, la adaptación y la supervivencia de su organismo y de sus órganos inmaduros. Análisis recientes han demostrado en ella la presencia de 1606 proteínas que en su mayoría son sintetizadas en los acinos de la glándula mamaria aunque otras proteínas y péptidos provienen de órganos como el sistema linfático y el aparato digestivo. La composición de la leche materna incluye enzimas que modifican sus proteínas y originan péptidos antimicrobianos, antihipertensivos y estimuladores del metabolismo. Esta actividad proteolítica actúa en sitios específicos de las cadenas peptídicas de la proteína de la leche. La activación extemporánea de estos enzimas en los acinos es regulada por péptidos inhibidores y activadores que previenen procesos inflamatorios. Algunos enzimas de la leche actúan en el tubo digestivo de recién nacidos y lactantes y complemen tan la menor concentración y actividad de sus propios enzimas digestivos. Así, la enteroquinasa de la leche estimula la liberación de enzimas pancreáticos (mediada por el estímulo de la colecistoquinina-pancreozimina); la lipasa activada por las sales biliares complementa la baja producción de lipasa pancreática. Estas actividades probablemente facilitan la nutrición de los prematuros, cuyo tubo di gestivo es más permeable a las proteínas parcialmente hidrolizadas y cuyas actividades enzimáticas y factores defensivos locales no han alcanzado su plena madurez. Esto también puede estimular en ellos la tolerancia inmunológica. En este artículo se presentan los aspectos fisiológicos relevantes de la leche materna, y los avances en el conocimiento de su composición, para el cabal conocimiento del pediatra de esta importante materia.

The mammary gland and maternal milk are the product of millions of years of evolution that resul ted in an optimal composition that sustains the growth and development of newborns and infants. Maternal milk supports the growth, adaptation and survival of this immature organism. Recent studies have detected 1606 different proteins in human milk, most of them synthesized in the acini of the glandular tissue while others originate from distant organs such as the lymphoid tissue and the digestive tract. Maternal milk enzymes modify its proteins and liberate peptides with antimicrobial, antihypertensive or stimulatory activities. This proteolytic activity occurs at specific sites in peptide chains. To prevent the extemporaneous activation of these proteolytic enzymes, that would result in inflammatory processes, maternal milk also contains inhibitory peptides that together with the stimulatory peptides conform a complex regulatory system. Some enzymes in maternal milk main tain their activity in the gastrointestinal tract of infants and compensate for the decreased activity of digestive tract enzymes in newborns. Thus, the milk enterokynase stimulates the release of pancreatic proteases as it induces the liberation of cholecystokynin/pancreozymin. The bile salt-activated lipase of human milk is activated in the duodenum by the infants' bile salts and partially compensates for the low levels of pancreatic lipase in newborns. These milk enzymes probably contribute to the nutrition of premature infants as they increase the availability of amino acids and peptides in their upper gastrointestinal tract; furthermore, as their intestinal epithelium is more permeable to peptides and partially digested protein this may help induce immune tolerance. The most relevant issues in the physiology and composition of the maternal milk are presented in this review.

Clinical efficacy of combination therapy with Butylphthalide and fibrinolytic enzyme for acute cerebral infarction / 中华老年医学杂志

Objective To evaluate the clinical efficacy of Butylphthalide combined with fibrinolytic enzyme in the treatment of acute cerebral infarction. Methods A total of 60 elderly patients with acute cerebral infarction who had missed the opportunity of thrombolytic therapy were enrolled in this study from January 2014 to December 2016 at Zhengzhou University Second Affiliated Hospital.Patients were randomly divided into an observation group(n= 30)and a control group(n= 30).Patients in the control group were treated with fibrinolytic enzyme alone,and those in the observation group received Butylphthalide combined with fibrinolytic enzyme.Peak flow velocity and mean flow velocity of the middle cerebral artery were analyzed and compared between the two groups before and after treatment.Data on clinical safety,efficacy,the national institutes of health stroke scale(NIHSS)and the Barthel score were also compared between the two groups. Results After treatment,the peak velocity 〔(74.60 ± 4.31)cm/s vs.(69.19 ± 3.36)cm/s〕 and average velocity 〔(38.71 ± 2.29)cm/s vs.(34.01 ± 2.01)cm/s〕 of the middle cerebral artery were significantly increased in the treatment group compared with those in the observation group (both P< 0.05).Meanwhile,the clinical efficacy was significantly higher in the observation group (96.7% vs.73.3%,P< 0.05).Moreover,NIHSS scores 〔(5.91 ± 3.51)vs.(8.31 ± 3.09)〕and Barthel scores 〔(71.29 ± 3.81)vs.(53.60 ± 4.33)〕 were significantly improved in the treatment group compared with those in the observation group(both P< 0.05).In addition,there were no obvious adverse reactions during treatment in the two groups. Conclusions Butylphthalide combined with plasmin can safely and effectively improve cerebral hemodynamics and prognosis in patients with acute cerebral infarction.

Effect of factors on pepsin and trypsin fibrinolytic activity and deactivation of fibrinolytic activity / 中草药

Objective: To investigate the method for study on the effect of factors on pepsin and trypsin fibrinolytic activity and deactivation of fibrinolytic activity and to eliminate the interference of pepsin and trypsin on the detection of crude protein fibrinolytic activity of Armadillidium vulgare (porcellio plasmin) in order to obtain the proteins or peptides which have the smaller molecular weight but higher titer during the pepsin and trypsin degradation. Methods: To study the effect of pepsin and trypsin deactivation on pH value, temperature, metal ions, enzyme inhibitor, surfactant, and responsing fibrinolytic by fiber fibrin plate assay. The better enzyme deactivation process was obtained and used for studying the effect on the fibrinolytic activity of urokinase, lumbrokinase, and porcellio plasmin. Results: All the pH value, temperature, metal ions, enzyme inhibitor, and surfactant have had an impact on pepsin and trypsin fibrinolytic activity. Among them the optimum deactivation of pepsin was pH 6.0-8.0, while the optimum deactivation of trypsin was mixed preparation with TLCK at the concentration of 25 mg/mL and EDTA at the concentration of 1 mmol/L. Conclusion: This study has obtained the better enzyme deactivation process which could be used for the detection of fibrinolytic activity of pepsin and trypsin degradation product by fiber fibrin plate assay, the operation is simple, and the repeatability and stability are good.

Determinación del tiempo de lisis del coágulo humano (in vitro) con el plasminógeno/plasmina de cuatro especies: humano, bovino, caprino y porcino / Determining Human Clot Lysis Time (in vitro) with Plasminogen/Plasmin from Four Species (Human, Bovine, Goat, and Swine): humano, bovino, caprino e suíno / Determinação do tempo de lise do coágulo humano (in vitro) com o plasminogênio/plasmina de quatro espécies

Las enfermedades cardiovasculares son la primera causa de muerte en todo el mundo, entre las cuales las anomalías del sistema del plasminógeno/plasmina son un factor importante en la deficiente lisis de los coágulos sanguíneos. En esta investigación se estudió el sistema fibrinolítico en cuatro especies de mamíferos, entre las que se identificó el plasminógeno humano como el más eficiente en cuanto a su poder trombolítico. Se investigó y se identificó el plasminógeno entre cuatro especies (humano, bovino, caprino y porcino) más eficiente en la lisis del coágulo humano in vitro. Los plasminógenos fueron purificados de forma idéntica por cromatografía de afinidad. El fibrinógeno humano se purificó por fraccionamiento con etanol. Tanto la purificación del plasminógeno como la del fibrinógeno se caracterizaron por electroforesis unidimensional SDS-PAGE al 10 %. La formación del coágulo humano, in vitro, así como su disolución por el plasminógeno/plasmina consistió en la determinación del tiempo de lisis desde la formación del coágulo hasta su dilución. La purificación de las proteínas arrojó una pureza mayor al 95 %; el del plasminógeno humano demostró mayor capacidad de lisis del coágulo que los plasminógenos de los animales. Se determinó que la mayor catálisis y eficiencia corresponden al plasminógeno/plasmina humano, que disuelve el coágulo humano hasta tres veces más rápido que las especies irracionales.

Cardiovascular disease is the leading cause of death worldwide, including failures in the plasminogen/plasmin system which is an important factor in poor lysis of blood clots. This article studies the fibrinolytic system in four species of mammals, and it identifies human plasminogen with highest thrombolysis efficiency. It examines plasminogen from four species (human, bovine, goat, and swine) and identifies the most efficient one in human clot lysis in vitro. All plasminogens were identically purified by affinity chromatography. Human fibrinogen was purified by fractionation with ethanol. The purification of both plasminogen and fibrinogen was characterized by one-dimensional SDS-PAGE (10%). Human clot formation in vitro and its dissolution by plasminogen/plasmin consisted of determining lysis time from clot formation to its dilution. Purification of proteins showed greater than 95% purity, human plasminogen showed greater ability to lyse clot than animal plasminogen. The article concludes that human plasminogen/plasmin has the greatest catalysis and efficiency, as it dissolves human clot up to three times faster than that of irrational species.

As doenças cardiovasculares são a primeira causa de morte em todo o mundo, entre as quais as anomalias do sistema do plasminogênio/plasmina são um fator importante na deficiente lise dos coágulos sanguíneos. Nesta pesquisa se estudou o sistema fibrinolítico em quatro espécies de mamíferos, entre as que se identificou o plasminogênio humano como o mais eficiente em quanto ao seu poder trombolítico. Realizou-se uma pesquisa e se identificou o plasminogênio entre quatro espécies, (humano, bovino, caprino e suíno) mais eficiente na lise do coágulo humano in vitro. Os plasminogênios foram purificados de forma idêntica por cromatografía de afinidade. O fibrinogênio humano se purificou por fracionamento com etanol. Tanto a purificação do plasminogênio como a do fibrinogênio se caracterizaram por eletroforese unidimensional SDS-PAGE a 10 %. A formação do coágulo humano, in vitro, assim como sua dissolução pelo plasminogênio/plasmina consistiu na determinação do tempo de lise desde a formação do coágulo até a sua diluição. A purificação das proteínas mostrou uma pureza maior a 95 %, o do plasminogênio humano demonstrou maior capacidade de lise do coágulo que os plasminogênios dos animais. Determinou-se que a maior catálise e eficiência correspondem ao plasminogênio/plasmina humana, que dissolve o coágulo humano até três vezes mais rápido do que as espécies irracionais.

Thrombolysis of plasmin and its derivatives:research advances / 国际药学研究杂志

All of the thrombolytic agents currently approved for use in humans are plasminogen activators, the application of which is limited by bleeding complications at vascular injury sites and plasminogen content in the thrombus. Plasmin is rapidly neutral-ized in the circulation by α2-antiplasmin and tolerated without bleeding. With the application of catheter-based delivery, the unique bio-chemical properties of plasmin make it a safe and effective direct fibrinolytics. Plasmin derivatives, including miniplasmin,Δ-plasmin and microplsmin, display more thrombolysis efficacy and better hemostatic safety in preclinical study and clinical trials. This review sum-marizes the current information on plasmin and its derivatives, including the advances on biochemical properties, preclinical and clinical trials.

Thrombolysis of plasmin and its derivatives: Research advances / 国际药学研究杂志

All of the thrombolytic agents currently approved for use in humans are plasminogen activators, the application of which is limited by bleeding complications at vascular injury sites and plasminogen content in the thrombus. Plasmin is rapidly neutralized in the circulation by α2-antiplasmin and tolerated without bleeding. With the application of catheter-based delivery, the unique biochemical properties of plasmin make it a safe and effective direct fibrinolytics. Plasmin derivatives, including miniplasmin, {increment}-plasmin and microplsmin, display more thrombolysis efficacy and better hemostatic safety in preclinical study and clinical trials. This review summarizes the current information on plasmin and its derivatives, including the advances on biochemical properties, preclinical and clinical trials.

Diferencias cinéticas de las plasminas de ocho especies mamíferas: activaciones y secuencias de los terminales-N / Kinetic Differences of Plasmins from Eight Mammalian Species: N-Terminal Activations and Sequences / Diferenças cinéticas das plasminas de oito espécies mamíferas: ativações e sequencias dos terminais-N

El artículo determina los parámetros cinéticos de las plasminas de ocho especies de mamíferos y sus terminales-N. Los ocho plasminógenos fueron purificados por los mismos métodos (cromatografías de afinidad e intercambio iónico), y activados con urocinasa, partiendo de una concentración común y su cinética valorada según las coordenadas de Lineweaver-Burk. Para esto se utilizó la cinética enzimática de Michaelis Menten, ampliamente usada en el estudio de enzimas. Todos los plasminógenos mostraron una pureza superior al 95% y una banda de 92 kDa en la electroforesis, al comparar con el estándar de peso molecular utilizado. Las plasminas del equino y canino mostraron la misma K M por el sustrato cromogénico (0,438 mM), siendo esta la de mayor afinidad en este estudio y la humana la de menor afinidad (5,3 mM). También fueron determinadas la constante catalítica y la velocidad de conversión del sustrato cromogénico a producto. Los terminales-N de los plasminógenos de las ocho especies fueron determinados, y se encontraron diferencias entre el humano y los animales; así mismo entre algunos animales. Solo el porcino y el ovino no mostraron diferencia alguna en su terminal-N (DPPDDY). Se demostró la unificación del método de purificación de los plasminógenos para cualquier especie, las diferencias cinéticas de las ocho plasminas estudiadas, y las similitudes y diferencias en la secuencia de los terminales-N de las ocho especies.

The paper determines the kinetic parameters of plasmins from eight species of mammals and their N-terminals. They were purified by the same methods (affinity chromatography and ion exchange) and activated with urokinase, starting from a common concentration and its kinetics judged according to Lineweaver-Burk coordinates. For such purpose, MichaelisMenten enzyme kinetics, which is widely used in the study of enzymes, was implemented. When compared with the molecular weight standard used, all plasminogen showed a purity exceeding 95% and a 92 kDa band on electrophoresis. Equine and canine plasmins showed the same K M due to the chromogenic substrate (0.438 mM), this being the one with the highest affinity in this study, and the human being the one with the lower affinity (5.3 mM). The catalytic constant and the conversion rate of the chromogenic substrate to product were also determined. The N-terminals of the plasminogens of the eight species were determined, and differences were found between humans and animals, as well as between some animals. Only pigs and sheep showed no differences in their N-terminal (DPPDDY). The unification of the method for purification of plasminogens for any species was demonstrated, as well as the kinetic differences of the eight plasmins studied and the similarities and differences in the sequence the N-terminals of the eight species.

El artigo determina os parâmetros cinéticos das plasminas de oito espécies de mamíferos e seus terminais-N. Estes foram purificados pelos mesmos métodos (cromatografias de afinidade e intercambio iônico) e ativados com uroquinase, partindo de uma concentração comum e sua cinética avaliada segundo as coordenadas de Lineweaver-Burk. Para isto se utilizou a cinética enzimática de Michaelis-Menten, amplamente usada no estudo de enzimas. Todos os plasminogênios mostraram uma pureza superior a 95 % e uma banda de 92 kDa na eletroforese, ao comparar com o padrão de peso molecular utilizado. As plasminas do equino e canino mostraram a mesma KM pelo substrato cromogênico (0,438 mM), sendo esta a de maior afinidade neste estudo e a humana a de menor afinidade (5,3 mM). Também foram determinadas a constante catalítica e a velocidade de conversão do substrato cromogênico a produto. Os terminais-N dos plasminogênios das oito espécies foram determinados, e se encontraram diferenças entre o humano e os animais, da mesma forma entre alguns animais. Somente o suíno e o ovino não mostraram diferença alguma em seu terminal-N (DPPDDY). Demonstrou-se a unificação do método de purificação de os plasminogênios para qualquer espécie, as diferenças cinéticas das oito plasminas estudadas e as similitudes e diferencias na sequencia dos terminais-N das oito espécies.

Plasmin degradation of the alpha chain of fibrinogen/fibrin: improved activation constant and activity determination in assays for tissue plasminogen activator / Degradación por la plasmina de la cadena alfa del fibrinógeno/fibrina: mejoría de la constante de activación y determinación de la actividad en ensayos para el activador del plasminógeno tisular

Objectives. The aim of this investigation was to increase the efficiency of ternary complex formation (fibrin-plasminogen-tissue-plasminogen activator) in the degradation process of the three-dimensional soluble fibrin monomer. Materials and methods. Fibrinogen was purified from human plasma by repeating precipitation six times, using different concentrations of cold ethanol. Fibrinogen was converted to DesAAfibrinogen by degradation with bathroxobin. Human plasminogen was purified by affinity and ion-exchange chromatography, and activated to plasmin by incubation with urokinase. Digested DesAAfibrinogen was prepared by controlled digestion with plasmin. Results. This study demonstrates that the α-chains of DesAAfibrinogen sterically hinder the formation of the ternary complex and are first degraded by plasmin. The degradation of fibrin(ogen) facilitates the in vitro determination of tissue plasminogen activator activity. Finally, release of fibrinopeptide A from bathroxobin-cleaved fibrinogen was confirmed, optimized and evaluated by various methods. Conclusions. Use of digested desAAfibrinogen with plasmin yielded a more stable activation constant of the ternary complex than that of undigested DesAAfibrinogen.

Objetivos. El propósito de la presente investigación fue incrementar la eficacia de la formación del complejo terciario (fibrina-plasminógeno-activador tisular del plasminógeno) en el proceso de degradación de la estructura tridimensional del monómero de fibrina soluble. Materiales y métodos. El fibrinógeno fue purificado de plasma humano, por seis precipitaciones repetidas, con diferentes concentraciones de etanol frío. El fibrinógeno fue convertido a desAAfibrinógeno por degradación con batroxobina. El plasminógeno humano fue purificado por cromatografías de afinidad e intercambio iónico y activado a plasmina con uroquinasa. El desAAfibrinogeno digerido fue preparado por digestión controlada con plasmina. Resultados. Este estudio demuestra que la cadena α del desAAfibrinógeno, dificulta la formación del complejo terciario, por impedimentos estéricos, por lo cual la cadena α se sometió a hidrólisis controlada con plasmina, facilitando así la determinación in vitro de la actividad del activador tisular del plasminógeno. Finalmente, la liberación del fibrinopéptido A por hidrólisis del fibrinógeno con batroxobina, fue confirmada, optimizada y evaluada por varios métodos. Conclusiones. El uso de desAAfibrinogeno digerido con plasmina da una constante de activación más estable en la formación del complejo terciario que el desAAfibrinógeno no digerido (fibrina-plasminogeno- activador tisular del plasminógeno).

Observation on the effect of plasmin combined with aspirin in the treatment of acute cerebral infarction / 中国基层医药

Objective To observe the clinical effect of plasmin combined with aspirin in treating acute cerebral infarction and to explore the influence to blood rheology and nerve function.Methods 148 cases with acute cerebral infarction were randomly divided into two groups.The control group (n =74) was treated by honghua injection.The study group (n =74) was treated by plasmin combined with aspirin.After treatment,the clinical effect,blood rheology and neurological recovery were observed.Results The total effective rate of the study group was 97.2％,which was significantly higher than that of the control group(74.3％) (x2 =15.88,P ＜ 0.05).The blood rheology indexes of the two groups had improved and neural function of the two groups was restored,but those in the study group were obviously better than control group (t =3.56,3.42,4.26,3.22,4.16,all P ＜ 0.05).Conclusion Plasmin combined with aspirin in treating acute cerebral infarction has accurate clinical curative effect,can markedly improve the blood rheology and promote nerve functional recovery,is worth of clinical spreading.

Efficacy of Autologous Plasmin-Assisted Vitrectomy for Rhegmatogenous Retinal Detachment

PURPOSE: To evaluate the efficacy and complication of autologous plasmin (AP) injected before vitrectomy for rhegmatogenous retinal detachment (RRD). METHODS: Intravitreal AP injection (0.2 ml) was performed on the eyes without posterior vitreous detachment (PVD) 20 minutes before the vitrectomy for RRD. The extent of PVD was evaluated intraoperatively. Surgical PVD induction was performed and the ease of the procedure was graded. The extent of PVD, ease of PVD induction, and complications (including incidence of iatrogenic retinal break) were compared to those of the control eyes. In order to evaluate complications and measure activated partial thromboplastin time, a microbial culture of injected AP was performed and the rate of postoperative intraocular hemorrhage was investigated. Change in visual acuity and the rate of retinal reattachment were compared in order to evaluate the long-term surgical outcome. RESULTS: The extent of PVD was greater in the AP group than in the control group, and vitreal separation was facilitated by intravitreal AP injection. However, ease of PVD induction and frequency of iatrogenic retinal break found were not significantly different between cases and controls. Neither postoperative intraocular hemorrhage nor systemic coagulation abnormality occurred. Postoperative endophthalmitis and positive microbial culture of the AP solution were also not reported. There was no significant difference in the change in visual acuity and the rate of retinal reattachment between the two groups. CONCLUSIONS: Intravitreal AP injection can facilitate vitrectomy for RRD and has no effect on the rate of retinal reattachment.

Comparación de la activación y la cinética de laplasmina bufalina con la humana / Ativação e cinética comparativa da plasmina bufalina e a humana / Comparative activation and kinetic of plasmin bufaline with the human one

El plasminógeno es el zimógeno de la plasmina, enzima activada a nivel fisiológico por el activadortisular del plasminógeno y la urokinasa, la plasmina es la enzima encargada de disolver el coágulosanguíneo. En este estudio se compararon la plasmina humana con la bufalina en su forma de activación dezimógeno a enzima y en la afinidad hacia el sustrato cromogénico. Los plasminógenos fueron purificadospor el mismo método de cromatografías de afinidad y cambio iónico. De igual manera las activaciones sehicieron utilizando urokinasa humana en ambos casos. La plasmina bufalina demostró mayor activacióny afinidad (1.35mM) que la plasmina humana (2.16 mM), siendo la bufalina 1.5 veces mas afin al sustratocromogénico que la humana. Este estudio demuestra que el método de purificación de los plasminógenospuede ser el mismo para muchas especies, se demuestra una vez más que las plasminas animales al parecerson más eficientes en la disolución del coágulo o degradación de sustratos, que la plasmina humana.Este estudio indica que la plasmina bufalina puede ser utilizada en los parámetros que se determinanclínicamente en pacientes con problemas cardiovasculares, reduciendo el tiempo de determinación de estosparámetros fibrinolíticos, que pueden dar al médico un margen de tiempo superior para actuar.

The Plasminogen is the zymogene of the Plasmin, enzyme which physiologically is activated by twodifferent enzymes, the tissue plasminogen activator and the urokinase, the plasmin is the enzyme that dissolves blood clots. In this study the human plasmin was compared to the bufaline plasmin, in theactivation from the zymogene to the enzyme form as well as in the affinity to the chromogenic substrate.The two plasminogens were purified by the same chromatographies methods: affinity and ion-exchange.Furthermore, both plasminogens were activated by human urokinase. The bufaline plasmin showed moreactivation and affinity (1.35 mM) that the human plasmin (2.16 mM), in addition, the bufaline plasmindemonstrated a 1.5 times more affinity to the chromogenic substrate that the human plasmin. This studydemonstrated that the plasminogens of several species can be purified by this method. Besides, one moretime the animal’s plasmins probably to be more efficient in the dissolution of blood clots or degradation ofsubstrates than the human plasmin. More over this study indicated that the bufaline plasmin can be usedin clinical determinations of patients with cardiovascular diseases. This also reduces the determinationtime of fibrinolytic parameters that physicians can give, having more time to take appropriate treatment.

O plasminogênio é o zymogen da plasmina, enzima ativada a nivel fisiológico pelo ativador tissulardo plasminogênio e uroquinase, plasmina é a enzima responsável de dissolver o coágulo de sanguíneo.neste estudo foi comparada a plasmina humana com a plasmina búbalina em seu modo de ativaçãode zymogen a enzima e na afinidade substrato cromogênico. Os plasminogênio foram purificados como mesmo método de cromatografia de afinidade e de troca iônica, e as ativações foram feitas usandouroquinase humana nos dois casos. A Búfalo plasmina mostrou maior ativação e afinidade (1.35 mM)que a plasmina humana (2.16 mM), sendo a bufalina 1.5 vezes mais afim ao substrato Cromogênico quea humana. Este estudo mostrou que o método de purificação do plasminogênios pode ser o mesmo paramuitas espécies, alem disso, que as plasminas animais são mais eficientes na dissolução do coáguloo degradação de substratos que a plasmina humana. Este estudo indicou que a plasmina búfalo podeser utilizada nos parâmetros determinados clínicamente em pacientes com problemas cardiovasculares,diminuindo o tempo de determinação destes parâmetros fibrinolíticos, que podem dar ao médico umintervalo de maior tempo para atuar.

The effect mechanism of pharmacological vitreolysis with plasmin and hyaluronidase / 眼科研究

Background Many ophthalmologists have proved that the intravitreal injection of plasmin can safely induce posterior vitreous detachment(PVD),but if it can generate the complete PVD need further to seek confirmation.Researches showed that the safe dose and toxicity dose of dispase are very near,so its application is limited.Whether hyaluronidase can induce PVD is still in controversy.Objective This study is to clarity the mechanism of pharmacological vitreolysis with plasmin and hyaluronidase.Methods Plasmin 4μmol/L,2μmol/L and 1μmol/L,plasmin 1μmol/L+ hyaluronidase 20μmol/L,hyaluronidase alone were intravitreally injected in lateral eye of 4 clean New Zealand white rabbits respectively,and 0.1mL BSS was injected as control group.Electron immunocytochemical technique was used to detect the laminin and fibronectin of interface between vitreous and retina in 7 days after intravitreal injection.Other 14 eyes of 7 clean New Zealand white rabbits were used in this study.Plasmin 1μmol/L + hyaluronidase 20μmol/L was intravitreally injected in the lateral eyes,and only plasmin 1μmol/L was injected in the fellow eyes.Plasmin activity in vitreous was evaluated in 15 and 30 minutes,1 hour,2,3,6,12 hours after intravitreal injection.The use of animals followed the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results The amounts of laminin and fibronectin in the vitreoretinal interface were decreased in 4μmol/L plasmin group,2μmol/L plasmin group,1μmol/L plasmin group,1μmol/L plasmin+20μmol/L hyaluronidase group compared with control group(P＜0.01).No significant difference was seen in the density of gold particles of anti FN between 20μmol/L hyaluronidase group and control group (P＞0.05).The change of amounts of fibronectin in the vitreoretinal interface was similar to that of laminin.Plasmin activity remained the highest level 1 hour after injection and thereafter gradually decreased and extincted in 12 hours and presented the same trend between plasmin 1μmol/L+hyaluronidase 20μmol/L group and only plasmin 1μmol/L group.Conclusion The mechanism of pharmacological vitreolysis is to dissolve laminin and fibronectin in the interface between vitreous and retina and therefore induce PVD.Combination of plasmin with hyaluronidase can increase the efficiency of pharmacological vitreolysis.The optimum selection of drug in inducing PVD should consider not only its role of lysis laminin and fibronectin but also the role of liquefying the vitreous.

The effects of plasmin Kringle domains deficiency mutant on the interface of vitreous and retina in rabbit eyes / 眼科研究

Background The vitreoretinal traction plays a critical role in the formation of macular hole and cystoid macular edema.Enzymatic vitreolysis has potential in relieving vitreoretinal traction as a simple and less invasive method in comparison with pars plane vitrectomy.ObjectiveThis study is to investigate the effects of plasmin mutant with kringle domains deficiency(Plm△K)on vitreoretinal interface in new Zealand white rabbits.Methods Plm△K was prepared through activating plasminogen mutant with Kringle domains deficiency (Plg△K) by tissue plasminogen activator (tPA).100μL of Plm△K at the dose of 0.5,1.0 and 1.5μmol/min was injected respectively into the vitreous of 48 New Zealand white rabbits and 16 eyes for each dose.B-scan and optical coherence tomography (OCT) were performed to detect the structure variety at the vitreoretinal interface in 1 day and 7 days after injection.The gross anatomy analysis with triamcinolone acetonide fine particle suspension,as well as histopathological examinations by scanning electron microscopy,was performed in the different time points mentioned above.Results Two peptide chains were determined with the relative molecular weight about 26000 and 5000 by the gel imaging analysis of reduced SDS-PAGE.Separation of the posterior vitreous cortex from retina was found after intravitreous injection under the B-scan and OCT.The ultrastructure change of vitreoretinal interface as well as the examination of fine particle suspension by triamcinolone acetonide demonstrated the same outcome.The degree of remnants of vitreous cortex showed the negative correlation with the dosage of Plm△K (r=-0.9516,P=0.048).No significant correlation was found between the degree of remnants of vitreous cortex and the action time(r=-0.720,P=0.470).There was no obvious morphological difference in outer layer of retina between control eyes and Plm△K-treated eyes.No drug-related adverse event was found after intravitreous injection of Plm△K.Conclusion Intravitreous injection of Plm△K alone can induce complete separation of vitreous from retina.This procedure is safe and effective.