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.     

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.     

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.     

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。     

Type II antithrombin deficiency caused by a large in‐frame insertion: structural,functional and pathological relevance

Summary. Background: The metastable native conformation of serpins is required for their protease inhibition mechanism, but also renders them vulnerable to missense mutations that promote protein misfolding with pathological consequences. Objective: To characterize the first antithrombin deficiency caused by a large in‐frame insertion. Patients/Methods: Functional, biochemical and molecular analysis of the proband and relatives was performed. Recombinant antithrombin was expressed in HEK‐EBNA cells. Plasma and recombinant antithrombins were purified and sequenced by Edman degradation. The stability was evaluated by calorimetry. Reactive centre loop (RCL) exposure was determined by thrombin cleavage. Mutant antithrombin was crystallized as a dimer with latent plasma antithrombin. Results: The patient, with a spontaneous pulmonary embolism, belongs to a family with significant thrombotic history. We identified a complex heterozygous in‐frame insertion of 24 bp in SERPINC1, affecting strand 3 of β‐sheet A, a region highly conserved in serpins. Surprisingly, the insertion resulted in a type II antithrombin deficiency with heparin binding defect. The mutant antithrombin, with a molecular weight of 59 kDa, had a proteolytic cleavage at W49 but maintained the N‐terminal disulphide bonds, and was conformationally sensitive. The variant was non‐inhibitory. Analysis of the crystal structure of the hyperstable recombinant protein showed that the inserted sequence annealed into β‐sheet A as the fourth strand, and maintained a native RCL. Conclusions: This is the first case of a large in frame‐insertion that allows correct folding, glycosylation, and secretion of a serpin, resulting in a conformationally sensitive non‐inhibitory variant, which acquires a hyperstable conformation with a native RCL.     

Successful therapy with argatroban for superior mesenteric vein thrombosis in a patient with congenital antithrombin deficiency

A 38-year-old woman was admitted with superior mesenteric vein (SMV) thrombosis, which was refractory to anticoagulation therapy. The plasma antithrombin activity was decreased and hardly compensated by concentrated antithrombin preparation due to high consumption rate. However, successful anticoagulation was achieved by administration of direct thrombin inhibitor, argatroban. Family studies of antithrombin activity revealed that she had type I congenital antithrombin deficiency. A novel heterozygous mutation in the gene for antithrombin (single nucleotide T insertion at 7916 and 7917, Glu 272 to stop in exon 4) was identified. Argatroban administration would be effective in the treatment of congenital antithrombin deficiency with SMV thrombosis.     

Inherited antithrombin deficiency: a review

Summary. Antithrombin (AT) is a potent inactivator of thrombin and factor Xa and the major inhibitor of blood coagulation. Inherited AT deficiencies are uncommon, with prevalences in the general population between 1 in 500 and 1 in 5000. They are either quantitative (type I) or qualitative (type II). Type II is subdivided into the more common, but less thrombogenic, type IIb deficiency caused by a defect in the heparin‐binding region of AT and the less common, but more thrombophilic, type IIa variant caused by mutations in the thrombin‐binding site. A pleiotropic type IIc deficiency also exists. In the evaluation of a thrombophilic individual, a functional AT assay (AT activity) should be used and the diagnosis of AT deficiency only established after acquired causes have been ruled out and repeat AT testing on an additional sample has been performed. A subsequent antigenic AT assay result leads to differentiation between type I and type II deficiency. Further specialized tests help subclassify the type II deficiencies, but this is typically not carried out for clinical purposes, even though it might be helpful to assess thrombosis risk. AT deficiency is associated with an increased risk for venous thromboembolism (VTE) and pregnancy loss. The association with arterial thrombosis is only weak. VTE prophylaxis and treatment management will be discussed in this article and existing treatment guidelines presented. The lack of data surrounding the use of AT concentrates and the resulting ambiguity as to when to use such concentrates will be discussed.     

Molecular bases of antithrombin deficiency in French families: identification of seven novel mutations in the antithrombin gene

We have investigated the molecular bases of familial antithrombin deficiency in eight French families. Eight mutations in the antithrombin coding exons were identified, seven of which were novel mutations. In all cases, individuals were heterozygous for the mutation. We found two small frameshift deletions in exon 3a, leading to type I deficiency. Five missense mutations in exons 3b or 5 also caused type I deficiency and their potential consequences on the antithrombin three-dimensional structure were analysed. The last mutation in exon 4 was associated with a type II 'reactive site' deficiency: a dysfunctional antithrombin that is affected in its interaction with thrombin was present in circulation.