The Dual Regulatory Role of Amino Acids Leu480 and Gln481 of Prothrombin

Prothrombin (FII) is activated to α-thrombin (IIa) by prothrombinase. Prothrombinase is composed of a catalytic subunit, factor Xa (fXa), and a regulatory subunit, factor Va (fVa), assembled on a membrane surface in the presence of divalent metal ions. We constructed, expressed, and purified several mutated recombinant FII (rFII) molecules within the previously determined fVa-dependent binding site for fXa (amino acid region 473–487 of FII). rFII molecules bearing overlapping deletions within this significant region first established the minimal stretch of amino acids required for the fVa-dependent recognition exosite for fXa in prothrombinase within the amino acid sequence Ser⁴⁷⁸–Val⁴⁷⁹–Leu⁴⁸⁰–Gln⁴⁸¹–Val⁴⁸². Single, double, and triple point mutations within this stretch of rFII allowed for the identification of Leu⁴⁸⁰ and Gln⁴⁸¹ as the two essential amino acids responsible for the enhanced activation of FII by prothrombinase. Unanticipated results demonstrated that although recombinant wild type α-thrombin and rIIa^S478A were able to induce clotting and activate factor V and factor VIII with rates similar to the plasma-derived molecule, rIIa^SLQ→AAA with mutations S478A/L480A/Q481A was deficient in clotting activity and unable to efficiently activate the pro-cofactors. This molecule was also impaired in protein C activation. Similar results were obtained with rIIa^ΔSLQ (where rIIa^ΔSLQ is recombinant human α-thrombin with amino acids Ser⁴⁷⁸/Leu⁴⁸⁰/Gln⁴⁸¹ deleted). These data provide new evidence demonstrating that amino acid sequence Leu⁴⁸⁰–Gln⁴⁸¹: 1) is crucial for proper recognition of the fVa-dependent site(s) for fXa within prothrombinase on FII, required for efficient initial cleavage of FII at Arg³²⁰; and 2) is compulsory for appropriate tethering of fV, fVIII, and protein C required for their timely activation by IIa.     

Autologous bone marrow–derived cells for venous leg ulcers treatment: a pilot study

Background

Chronic venous leg ulcers (VLUs) are a common problem in clinical practice and available treatments are not satisfactory. The use of adjuvant therapies in combination with lower limb compression may lead to improved healing rates. Chronic wounds are candidates for new strategies in the emergent field of regenerative medicine. Bone marrow–derived cells (BMDCs) contain cells and secrete cytokines known to participate in wound healing. Thus, BMDC therapy seems a logical strategy for the treatment of chronic wounds. Our objective was to evaluate feasibility, safety and initial clinical outcome of autologous BMDC therapy associated with standard treatment in patients with VLUs.

两种介入途径治疗急性、亚急性门静脉及肠系膜上静脉血栓的临床疗效分析

目的：评价介入技术治疗急性和亚急性门静脉（portal vein,PV）及肠系膜上静脉（superior mesenterie vein, SMV）血栓形成的临床疗效。方法：对28例治疗急性和亚急性（发病一周至一月）的PV及SMV血栓患者进行介入治疗。按介入治疗途径不同分为下述两组：选择经颈静脉穿刺门静脉（transjugular intrahepatic portosystemic shunt , TIPS途径）置管溶栓（19例）和经皮穿肝内门脉（percutaneous transhepatic）置管溶栓治疗（9例）。结果：所有患者随访一周至三个月,其中治疗成功24例,临床症状明显改善,无严重并发症。经TIPS途径治疗的患者组中,16例随访显示大部分血栓被清除,门静脉系统有血流通过,临床症状缓解。3例SMV及PV恢复部分血流,但临床症状无明显改善。经皮穿肝内门脉直接置管溶栓治疗组中,6例患者的PV及SMV内血栓大部分清除,血流基本改善,2例患者PV及SMV血流部分好转,临床症状无明显改善,严重并发症1例（术后两天死于腹腔出血）。结论：经TIPS途径介入技术和经皮穿肝内门脉直接置管溶栓治疗是治疗急性和亚急性PV及SMV血栓形成的有效方法,前者的疗效及安全性均好于后者。     

Prothrombin in Normal Human Cerebrospinal Fluid Originates from the Blood

In spite of the fact that prothrombin is produced by cells within the central nervous system, its presence in the cerebrospinal fluid (CSF) has not been investigated. We determined the concentration of prothrombin in CSF with reference to the concentration in plasma in paired samples from 18 normal control patients and 4 patients with relapsing-remitting type of multiple sclerosis (MS). The newly developed ELISA was very specific (no cross-reactivity with thrombin) and sensitive (detection limit—0.7 ng/ml) with an imprecision of CV = 8.3% (intraseries) and 7.0% (interassay). The mean prothrombin concentration in normal CSF was 0.55 mg/l (CV ± 33%, range: 0.28–0.93 mg/l), in normal plasma 121.8 mg/l ± 21%, resulting in a mean CSF/plasma concentration quotient (Q_Proth)—4.5 · 10^–3 (CV ± 35%, range: 2.1–8.3 · 10^–3) corresponding to a mean albumin quotient in this group of subjects of Q_Alb = 5.8 · 10^–3. Due to the Q_Proth and the molecular weight of prothrombin (72 kDa)—similar to that of albumin—we conclude that prothrombin in normal human CSF originates predominantly (>95%) from blood. The enzymatic activity in CSF is conserved. Comparable results obtained in MS patients with only few small MRI lesions suggest that local chronic inflammatory disease of the central nervous system does not influence prothrombin concentration in the CSF if the blood-CSF barrier function is normal.     

A pathway of coagulation on endothelial cells

Although the endothelial cell is considered antithrombogenic, endothelium has recently been shown to participate in procoagulant reactions. Factor IX bound to specific endothelial cell sites can be activated by the intrinsic and extrinsic pathways of coagulation. Perturbation of endothelium results in induction of tissue factor which promotes factor VIIa-mediated activation of factors IX and X, thus initiating procoagulant events on the endothelial surface. Cell bound factor IXa, in the presence of factor VIII, promotes activation of factor X. The factor Xa formed can interact with endothelial cell factor V/Va, resulting in prothrombin activation. Thrombin then cleaves fibrinogen and a fibrin clot closely associated with the endothelial cell forms. The perturbed endothelial cell thus provides a focus of localized procoagulant events. This model suggests a simple endothelial-cell-dependent mechanism for initiation of coagulation at the site of an injured or pathological vessel.     

The influence of Annexin32, a new Ca2+-dependent phospholipid-binding protein, on coagulation time and thrombosis

The pharmacodynamics of Annexin32, a new Ca2+-dependent phospholipid-binding protein, was studied by measuring coagulation time in rabbits and venous thrombosis in rabbits and rats. Rabbits and rats were given Annexin32 by intravenous administration. Then Kaolin partial thromboplastin time (KPTT), thrombosis in vitro and in vivo were assayed. The results showed that KPTT of rabbits was prolonged (p < 0.01), and the length and weight of thrombus in vitro were reduced (p < 0.01) after administration of Annexin32 at 1 mg/kg. It also inhibited thrombosis in vivo and reduced the weight of venous thrombus significantly in rats (p < 0.01). All these results suggested that Annexin32 possesses the characteristic of antithrombotic effect and fewer side effects on coagulation time.     

THE BOOK CORNER

In order to increase the yield of prothrombin complex concentrates (PCCs) and to reduce their associated thrombotic risks, the influence of washing conditions on the yield, purity, and balance of coagulation factors (FII, FVII, FIX, and FX), and inhibitor proteins (PC, PS, PZ, and AT [antithrombin]) in PCCs was investigated by orthogonal testing, in which three variables (sodium citrate, NaCl, and pH) and their three levels were selected. It was found that AT yield and purity were extraordinarily low, and at lower NaCl content, the general yield, purity, and balance were higher, lower, and better, respectively; however, the results became contrary at higher NaCl. Moreover, within the investigated levels, NaCl was the first determinant for the yield except AT and the purity except FVII, PC, PS, and AT. Sodium citrate was the first determinant for AT yield and FVII, PS, and AT purity. The yield except FII, PS, and AT decreased and the purity except PC increased with increase of sodium citrate content. Just for PC purity, pH was the first determinant. The effect with pH fluctuation on the yield and purity was characteristically unobvious. The outcome undoubtedly supplies the guidance to further improve PCCs.     

Proteomic analysis of venous thromboembolism

Venous thrombosis has a multicausal basis, and is characterized by a multifaceted combination of inherent phenotypic complexity and genetic predisposition, with acquired and triggering factors further and acutely influencing the likelihood of an individual experiencing a clinically significant thrombotic event. Traditional coagulation tests, especially clot-based assays, are useful for describing major abnormalities in the hemostatic response, but fail in their application to assessing thrombotic risk in the healthy population. Recent evidence also attests that the analysis of a vast array of genes can only explain part of the individual thrombotic risk. Thus, proteomic analysis may hold promise for characterizing or understanding biological pathways and pathophysiological interactions, for improving the diagnosis and identifying novel therapeutic approaches to this prevalent and life-threatening disorder. Herein, we present and discuss available data on proteomic analysis of venous thromboembolism, concluding that further studies supported by high-throughput techniques should be undertaken to elucidate or understand biological pathways and pathophysiological interactions.     

Mouse Complete Stasis Model of Inferior Vena Cava Thrombosis

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE). In the United States (U.S.), the high morbidity and mortality rates make VTE a serious health concern ^1-2. After heart disease and stroke, VTE is the third most common vascular disease ³. In the U.S. alone, there is an estimated 900,000 people affected each year, with 300,000 deaths occurring annually ³. A reliable in vivo animal model to study the mechanisms of this disease is necessary.The advantages of using the mouse complete stasis model of inferior vena cava thrombosis are several. The mouse model allows for the administration of very small volumes of limited availability test agents, reducing costs dramatically. Most promising is the potential for mice with gene knockouts that allow specific inflammatory and coagulation factor functions to be delineated. Current molecular assays allow for the quantitation of vein wall, thrombus, whole blood, and plasma for assays. However, a major concern involving this model is the operative size constraints and the friability of the vessels. Also, due to the small IVC sample weight (mean 0.005 grams) it is necessary to increase animal numbers for accurate statistical analysis for tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent (ELISA), zymography, vein wall and thrombus cellular analysis, and whole blood and plasma assays ^4-8.The major disadvantage with the stasis model is that the lack of blood flow inhibits the maximal effect of administered systemic therapeutic agents on the thrombus and vein wall.     

Assessment of genetic mutations in the XRCC2 coding region by high resolution melting curve analysis and the risk of differentiated thyroid carcinoma in Iran

Homologous recombination (HR) is the major pathway for repairing double strand breaks (DSBs) in eukaryotes and XRCC2 is an essential component of the HR repair machinery. To evaluate the potential role of mutations in gene repair by HR in individuals susceptible to differentiated thyroid carcinoma (DTC) we used high resolution melting (HRM) analysis, a recently introduced method for detecting mutations, to examine the entire XRCC2 coding region in an Iranian population. HRM analysis was used to screen for mutations in three XRCC2 coding regions in 50 patients and 50 controls. There was no variation in the HRM curves obtained from the analysis of exons 1 and 2 in the case and control groups. In exon 3, an Arg(188)His polymorphism (rs3218536) was detected as a new melting curve group (OR: 1.46; 95%CI: 0.432-4.969; p = 0.38) compared with the normal melting curve. We also found a new Ser(150)Arg polymorphism in exon 3 of the control group. These findings suggest that genetic variations in the XRCC2 coding region have no potential effects on susceptibility to DTC. However, further studies with larger populations are required to confirm this conclusion.     

血栓通注射液联合利伐沙班预防下肢骨折术后下肢深静脉血栓形成疗效及对患者血液流变学的影响

目的:探讨血栓通注射液联合利伐沙班预防下肢骨折术后下肢深静脉血栓(DVT)形成的疗效及对患者血液流变学的影响。方法:选取2012年4月到2015年7月西安医学院第一附属医院收治的125例下肢骨折术后患者,随机分为对照组63例和观察组62例,对照组患者给予血栓通治疗,观察组患者在对照组的基础上给予利伐沙班治疗。观察两组治疗前后DVT发生率、血清D-二聚体(DD)、同型半胱氨酸(Hcy)以及高敏C-反应蛋白(hs-CRP)、血液流变学指标包括红细胞压积(HTC)、全血黏度(WBV)和纤维蛋白原的变化以及用药安全性。结果:术后3周,观察组DVT的总发生率为9.7%(6例),显著低于对照组的44.4%(28例),差异具有统计学意义(P0.05)。治疗后,两组患者的血清DD、Hcy、hs-CRP、HTC、WBV以及纤维蛋白原水平均较治疗前明显降低(P0.05),且观察组该6个指标显著低于对照组,差异均具有统计学意义(P0.05)。此外,观察组的不良反应发生率为9.68%,对照组为7.94%,两组间比较无明显差异(P0.05)。结论:血栓通注射液联合利伐沙班可有效减少DVT的发生并改善患者的血液高凝状态,是一种预防下肢骨折术后DVT形成的有效方案。     

综合护理对脑卒中患者下肢深静脉血栓形成的预防效果

目的:分析综合护理干预在脑卒中偏瘫患者下肢深静脉血栓形成中的预防效果及临床效应。方法:将120例经颅部CT或MRI确诊的脑卒中患者分为观察组和对照组,每组各60例。对照组给予常规护理干预,观察组进行综合护理干预,比较不同干预措施患者下肢深静脉血栓形成情况。结果:观察组DVT发生率为3.33%,对照组为18.33%,观察组干预效果明显优于对照组,差异有统计学意义(P0.05)。结论:综合护理干预可有效降低脑卒中偏瘫患者DVT的形成,在脑卒中偏瘫患者DVT方面具有显著的预防作用与临床效应,值得借鉴。     

Expression of a cGMP compatible Lucilia sericata insect serine proteinase debridement enzyme

Previously, we demonstrated the effectiveness of a research grade recombinant chymotrypsin, derived from the larvae of Lucilia sericata, in "debriding" slough/eschar from venous leg ulcers ex vivo. Furthermore, we were able to formulate this enzyme for successful delivery to in vitro wound healing assays, from a prototype hydrogel wound dressing, and showed that enzyme delivered in this way could degrade wound tissue ex vivo. Recently, to progress biotechnological development of the enzyme as a potential therapeutic product, we explored expression using current good manufacturing practice (cGMP) guidelines, and now report that a recombinant chymotrypsin I zymogen from L. sericata can be expressed in the cGMP acceptable strain of Escherichia coli (BLR-DE3). In addition, the conditions required for purification, refolding and activation of the chymotrypsinogen have been determined. The activated enzyme was stable, and effective in digesting wound slough/eschar tissue. To summarise, we have successfully initiated the production and characterisation of a novel cGMP compatible product for use in future clinical trials.