Designed polyanionic coiled-coil proteins: acceleration of heparin cofactor II inhibition of thrombin

Novel polyanionic proteins were designed to increase the rate of heparin cofactor II (HC) inhibition of α-thrombin, an essential protease in the coagulation cascade. Two α-helical coiled-coil proteins, a 62-residue dimer containing 8 Glu residues (E8C) and a 104-residue dimer containing 14 Glu residues (E14C), plus two 31-residue control peptides containing 8 Glu residues each (E8A and E8B), were chemically synthesized, structurally characterized and enzymatically assayed. Circular dichroic spectrophotometry indicated that both E8C and E14C formed stable two-chain α-helical coiled coils at pH 7 and 25 °C. The control peptides were only partially α-helical. E14C remained folded at 90 °C but E8C was half unfolded at 49 °C. Coiled-coil proteins E8C and E14C maximally accelerated by 35- and 33-fold, respectively, the rate of HC inhibition of α-thrombin. None of these compounds accelerated antithrombin inhibition of α-thrombin, and neither control peptide accelerated HC inhibition of α-thrombin. Acceleration of the HC inhibition of α-thrombin showed bimodal dependence on the concentration of the polyanionic protein, which is consistent with formation of a HC-coiled-coil-thrombin ternary complex. The results suggest that antithrombotic polyanionic α-helical coiled-coil proteins can be designed and synthesized and that the occurrence of secondary structure can be correlated with biologcal activity. © Munksgaard 1995.     

中药混乱品种鉴别研究——7. 蕲蛇及其混淆品鉴别之二

作者前已报道蕲蛇及其伪品滑鼠蛇的鉴别,又据报道有些省发现蕲蛇商品药材中混有烙铁头、山烙铁头两种蛇的干燥体。作者对这两种混淆品与蕲蛇在体表花纹、鳞片以及骨骼的形态与组织特征等方面进行了鉴别研究。     

Interaction of antithrombin III with preadsorbed albumin-heparin conjugates

The adsorption of antithrombin III (AT III) onto polystyrene surfaces preadsorbed with albumin or albuminheparin conjugates was studied using a two step enzyme immuno assay. When AT III-buffer solutions were used, the highest adsorption values were measured on high affinity albumin-heparin conjugate pretreated surfaces. Less AT III adsorption was found on nonfractionated albumin-heparin conjugate preadsorbed surfaces. AT III adsorption could also be detected on low affinity conjugate and albumin coated surfaces. When AT III was adsorbed from plasma or plasma dilutions with buffer, only AT III on surfaces preadsorbed with high affinity or nonfractionated albumin-heparin conjugate was found. These results demonstrate that the heparin moiety of the conjugate is directed to the solution phase whereas the albumin moiety contacts the polystyrene surfaca     

Native and activated antithrombin inhibits TMPRSS2 activity and SARS-CoV-2 infection

Host cell proteases such as TMPRSS2 are critical determinants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tropism and pathogenesis. Here, we show that antithrombin (AT), an endogenous serine protease inhibitor regulating coagulation, is a broad-spectrum inhibitor of coronavirus infection. Molecular docking and enzyme activity assays demonstrate that AT binds and inhibits TMPRSS2, a serine protease that primes the Spike proteins of coronaviruses for subsequent fusion. Consequently, AT blocks entry driven by the Spikes of SARS-CoV, MERS-CoV, hCoV-229E, SARS-CoV-2 and its variants of concern including Omicron, and suppresses lung cell infection with genuine SARS-CoV-2. Thus, AT is an endogenous inhibitor of SARS-CoV-2 that may be involved in COVID-19 pathogenesis. We further demonstrate that activation of AT by anticoagulants, such as heparin or fondaparinux, increases the anti-TMPRSS2 and anti-SARS-CoV-2 activity of AT, suggesting that repurposing of native and activated AT for COVID-19 treatment should be explored.     

血浆纤维蛋白原与冠心病患者胆固醇、血液流变学的相关性及药物干预的研究

目的：研究血浆纤维蛋白原（ｆｉｂｒｉｎｏｇｅｎ,ＦＧ）与冠心病（ＣＨＤ）患者胆固醇、血液流变学的相关性及腹蛇抗栓酶（ＡＨＶＡＴ）的治疗效果。方法：观察６６ 例ＣＨＤ 患者血浆ＦＧ、胆固醇（ＣＨＯ）及血浆粘度、低切变血粘度、红细胞聚集指数等血液流变学指标与６０ 例正常人对照。将６６例ＣＨＤ患者随机分为ＡＨＶＡＴ治疗组３２ 例及肝素治疗组３４ 例,观察治疗前后上述指标的变化。结果：ＣＨＤ组血浆ＦＧ、ＣＨＯ、血浆粘度、低切变血粘度、红细胞聚集指数均明显高于正常对照组（Ｐ＜ ０．０１）。ＣＨＤ组患者血浆ＦＧ 水平与ＣＨＯ、血浆粘度、低切变血粘度、红细胞聚集指数呈直线相关（Ｐ＜ ０．０５, Ｐ＜ ０．０１）。用ＡＨＶＡＴ治疗后上述指标明显下降（Ｐ＜ ０．０５）。结论：血浆ＦＧ 升高与ＣＨＯ 升高有关,血浆ＦＧ 升高可导致ＣＨＤ患者血浆粘度、低切变血粘度及红细胞聚集指数的升高,用ＡＨＶＡＴ 治疗可降低ＣＨＤ患者血浆ＦＧ及ＣＨＯ水平,从多方面改善血液流变学指标     

Comparison of immunochemical and biological properties of human anti thrombin during blood clotting and upon interaction with exogenous thrombin

Modification of immunological and biological properties of human antithrombin were studied in plasma-serum pairs and in defibrinated plasma supplemented with human thrombin. Modified antithrombin obtained through whole-blood clotting or upon addition of exogenous thrombin appeared the same with regards to its electrophoretic or biological properties. However, amounts of thrombin higher than that physiologically available, had to be used to obtain a "serum-like" antithrombin in thrombin supplemented plasma suggesting different pathways for this transformation. This was in agreement with the observation in plasma of a modification of antithrombin antigenic properties upon thrombin addition whereas no difference was demonstrated when comparing serum to normal plasma. It may be concluded that the inactivation of antithrombin and the appearance of electrophoretically modified forms in normal serum is not mainly due to the formation of enzyme-inhibitor complexes and therefore that proteolytically modified, enzyme-free forms of antithrombin demonstrated in purified systems (Fish et al. 1979) could be of physiological relevance.     

江浙蝮蛇毒镇痛组分的中枢作用机制研究

 目的　探讨江浙蝮蛇毒镇痛C4组分的可能作用途径。方法　通过C4组分与阿托品、利血平、纳络酮药物的相互作用 ,探索其与乙酰胆碱受体、囊泡递质、阿片受体间可能的作用 ;通过脊髓化鼠实验、侧脑室给药和足跖定压实验揭示其中枢性作用 ;通过不同脑区的组织匀浆测定神经肽含量 ,考察C4组分对不同中枢部位的作用大小。结果C4组分不通过乙酰胆碱受体作用 ,而与阿片受体相关 ,它通过中枢起效 ,主要作用部位集中在丘脑、尾状核和红核等 ,对亮氨酸脑啡肽的含量有刺激增高作用。结论 C4组分与中枢亮氨酸脑啡肽关系密切。     

危重新生儿弥散性血管内凝血前期的监测与治疗

目的探讨监测危重新生儿血浆凝血酶原片段F1＋2、凝血酶抗凝血酶复合物（TAT）及D-二聚体等凝血及纤溶因子水平对弥散性血管内凝血前期（pre-DIC）的诊断及治疗意义。方法对NICU收治的96例危重症及36例正常新生儿检测F1＋2、TAT及D-二聚体水平，对30例诊断pre-DIC的患儿予抗凝治疗，并监测治疗后F1＋2、TAT及D-二聚体水平。结果危重症组与对照组相比，F1＋2、TAT、D-二聚体水平均增高，差异有极显著性（P均〈0．01）。TAT对pre-DIC诊断的敏感性较高，F1＋2特异性强；三者在抗凝治疗后均明显下降。F1＋2与TAT、D-二聚体三者之间均呈直线正相关，F1＋2与TAT、D-二聚体的相关系数分别为r=0．70P〈0．01；r=0.42 P〈005；TAT与D-二聚体呈正相关（r=0．35 P〈0．05）；F1＋2、TAT、D-二聚体与危重评分呈直线负相关（r=-0．68、-072、-054P均〈0.01）。结论检测TAT、F1＋2、D-二聚体水平监测危重新生儿的血液高凝状态，有助于pre—DIC的诊断，并可协助判断抗凝治疗的效果。     

蕲蛇药材及其市售混淆品的Cyt b基因序列与分析

目的分析蕲蛇药材正品及其市场收集样品的Cyt b基因序列,为蕲蛇药材分子鉴别提供依据。方法利用Cyt b基因,对蕲蛇药材及其市场收集样品进行了序列测定和分析。结果蕲蛇药材存在着混杂现象;蕲蛇药材正品与其市售混淆品的Cyt b基因序列有着显著差异,该序列在蕲蛇种内差异率为0%~0.91%,与混淆品间的差异率为18.57%~23.78%。结论Cyt b基因序列是一种鉴别蕲蛇药材与其混淆品较好的分子遗传标记。     

尖吻蝮蛇毒中一种新类凝血酶的分离纯化

用阴离子交换色谱和凝胶过滤色谱技术,从尖吻蝮蛇毒中纯化得到一个具有凝血活性的组分.经Superdex 75 HR10/30预装柱检测,纯度达99.91%.SDS-PAGE显示为单一条带,还原、非还原条件下分子量分别为59.25k、52.58k.该组分的凝血酶比活为41.5u/mg,精氨酸酯酶比活为14.29u/mg,但不激活血浆凝血因子ⅩⅢ.EDTA不能抑制其凝血活性,而苯甲磺酰氟则产生不可逆抑制作用.结果表明该酶是一种新尖吻蝮蛇毒类凝血酶.