Thrombin–Fibrin(ogen) Interactions,Host Defense and Risk of Thrombosis

Fibrinogen is a well-known risk factor for arterial and venous thrombosis. Its function is not restricted to clot formation, however, as it partakes in a complex interplay between thrombin, soluble plasma fibrinogen, and deposited fibrin matrices. Fibrinogen, like thrombin, participates predominantly in hemostasis to maintain vascular integrity, but executes some important pleiotropic effects: firstly, as observed in thrombin generation experiments, fibrin removes thrombin from free solution by adsorption. The adsorbed thrombin is protected from antithrombins, notably α2-macroglobulin, and remains physiologically active as it can activate factors V, VIII, and platelets. Secondly, immobilized fibrinogen or fibrin matrices activate monocytes/macrophages and neutrophils via Mac-1 interactions. Immobilized fibrin(ogen) thereby elicits a pro-inflammatory response with a reciprocal stimulating effect of the immune system on coagulation. In contrast, soluble fibrinogen prohibits recruitment of these immune cells. Thus, while fibrin matrices elicit a procoagulant response, both directly by protecting thrombin and indirectly through the immune system, high soluble fibrinogen levels might protect patients due to its immune diminutive function. The in vivo influence of the ‘protective’ plasma fibrinogen versus the ‘pro-thrombotic’ fibrin matrices on thrombosis should be explored in future research.     

Block and Graft Copolymers of Poly(ethylene glycol) and Poly(dimethylsiloxane) for Blood Contacting Biomedical Materials Applications

The incorporation of both hydrophobic and hydrophilic groups into a synthetic polymer is a potent way of controlling its surface and interfacial properties. With this end result in mind, we describe herein the synthesis and characterization of poly(dimethylsiloxane-b-ethyleneoxide) block copolymers (PDMS-b-PEO) and poly(dimethylsiloxane-g-ethyleneoxide) grafted copolymers (PDMS-g-PEO). These amphiphillic copolymers were also investigated as surface modifying agents for passifying hydrophobic polymer surfaces in blood contacting applications. Specifically, the various (PDMS-b-PEO) and (PDMS-g-PEO) copolymers were coated onto poly(styrene-divinylbenzene) microspheres by their physical adsorption from solution. These surfaces were then evaluated for blood contacting applications utilizing a fibrinogen and thrombin protocol. In particular, the binding of fibrinogen and the functionality of the surface bound fibrinogen on an otherwise hydrophobic surface (polystyrene) was investigated. As the conversion of fibrinogen to fibrin is facilitated by thrombin, the aggregation of the copolymer-coated hydrophobic microspheres was followed using an optical method after the sequential exposure of the microspheres to fibrinogen and then to thrombin. We were able to determine how the adsorbed copolymers affected the functionality of the bound fibrinogen at an interface. Our hypothesis is that the hydrophobic siloxane units of the copolymers will be in close proximity to the polystyrene surface and that the PEO will extend out from the surface and therefore render the synthetic polymer system hemocompatible. Following the fibrinogen and thrombin protocol and determining the fibrinogen-dependent aggregation, the results show that the PDMS-b-PEO copolymers (having a PEO content from 4.6 to 11.5 weight %) were similar in terms of particle aggregation when compared to the pure polystyrene microspheres (blank) or to the microspheres that were coated with a linear PDMS homopolymer. By comparison, the PDMS-g-PEO copolymers (having a PEO content from 58 to 80 weight %) were seen to reduce the fibrinogen functionality on the microsphere system surface. Thus the data indicate that the PDMS-g-PEO copolymers can behave like molecular brushes that are able to pacify the surface of the hydrophobic polystyrene microspheres. A somewhat unexpected observation was that for the copolymer system having a low PEO content the fibrinogen-dependent aggregation of the otherwise hydrophobic microspheres was observed to increase relative to the pure microspheres (blank). It is clear from the findings of this investigation that the surface packing and molecular orientation of both the adsorbed copolymer and also of the fibrinogen are important factors that govern the properties and applications of blood contacting biomaterials.     

On the relationship between the adhesive properties and the structural features of fibrin sealants

The relationship between the adhesive properties and network architecture of fibrin sealants was studied at various fibrinogen, thrombin and calcium ion concentrations. Network features were characterized using Scanning Electron Microscopy at cryogenic temperature (cryo-SEM). This technique allows examination of the hydrated gel without drying. It was shown that by gradually increasing the calcium ion concentration, at constant thrombin and fibrinogen concentrations, the network appearance changed dramatically, from an almost isotropic structure at low concentration to highly oriented fibers at higher concentration of 7 mM CaCl₂. In addition, it was found that further increase in the calcium ion concentration induced fiber aggregation and formation of large clusters, co-existing with fine fibers. Changes in thrombin concentration at constant calcium and fibrinogen concentrations induced only slight changes in the gel appearance. Adhesive properties of fibrin sealants were measured using the shear-lap test and were correlated with structural features of the network. Holding fibrinogen and thrombin concentrations constant and varying the CaCl₂ concentration, maximal shear strength was found with 7 mM CaCl₂, corresponding to an oriented network. Holding fibrinogen and calcium chloride concentrations constant, a slight increase in shear strength was observed with increase in thrombin concentration, demonstrating again the correlation between the network structure and its adhesive properties.     

Aptamers Act as Activators for the Thrombin Mediated‐Hydrolysis of Peptide Substrates

Thrombin is the typical target in anticlotting therapy for many serious diseases such as heart attack and stroke. DNA aptamers are well‐known thrombin inhibitors that prevent fibrinogen hydrolysis. We have discovered that exosite‐targeting antithrombin aptamers enhance the activity of thrombin toward a small peptide substrate, Sar(N‐methylglycine)‐Pro‐Arg‐paranitroanilide, and that the activation of the enzyme by these aptamers is strongly inhibited by their complementary DNAs. Our study reveals that treatment with mixed aptamers or with a dual‐aptamer construct led to an 8.6‐ or 7.8‐fold enhancement in peptide hydrolysis relative to thrombin alone, a synergistic effect much higher than the activation observed with a monofunctional aptamer (1.5‐fold for Apt27 or 2.7‐fold for Apt15). In addition, we discovered that Apt27 is a biofunctional molecule for thrombin because of its activation effect. An enzyme kinetic study indicates that the binding of aptamers to exosites I and II significantly activates thrombin towards the peptide substrate, thus illustrating that binding of aptamers to exosites can allosterically regulate the active site of thrombin. Our study suggests the necessity of considering possible side effects when DNA aptamers are used for clinical applications involving the inhibition of thrombin‐mediated clotting.     

Binding of hirudin to meizothrombin

Prothrombin (coagulation factor II) is the inactive precursor molecule of thrombin (coagulation factor IIa). Proteolytic cleavage of the peptide bond Arg320-Ile321 converts prothrombin into the two-chain thrombin precursor meizothrombin. Meizothrombin hydrolyses peptidyl substrates, but cleavage of fibrinogen is poor. Unfortunately, meizothrombin exhibits a significant autocatalytic activity and thus is not structurally stable in solution. Hirudin, the 65-residue peptide anticoagulant from the salivary gland of the European leech Hirudo medicinalis, is a highly specific and effective thrombin inhibitor. To study the interactions of meizothrombin and hirudin, recombinant prothrombin with active site Asp419 replaced by Asn (D419N-prothrombin) was produced in CHO cells and transformed into D419N-meizothrombin in vitro. D419N-meizothrombin exhibited no proteolytic and autocatalytic activity. D419N-meizothrombin was affinity purified at an immobilized C- terminal hirudin-derived peptide demonstrating the presence and activity of the anion binding exosite. D419N-meizothrombin exhibited binding activity to hirudin immobilized at the solid phase in an ELISA. Incubation of D419N-meizothrombin with hirudin resulted in a significant increase of intrinsic fluorescence. Fluorescence titration of D419N-meizothrombin with hirudin produced a sharp break in the titration curve at the molar equivalence point and a total fluorescence enhancement of 24%. However, the titration curve did not reflect a simple binding mechanism. Incubation of D419N-meizothrombin with fibrinopeptide A and C-terminal hirudin peptide 54-65 did not change fluorescence emission. Trp468 located in the gamma-loop of thrombin was replaced by Phe in the double-mutant D419N/W468F-thrombin. Similar to D419N-thrombin and D419N-meizothrombin, formation of the D419N/W468F- thrombin/hirudin complex resulted a significant increase in intrinsic fluorescence. Apparently, the binding of hirudin induces similar structural changes in both meizothrombin and thrombin. The structural change does not involve the flexible gamma-loop. The results suggest that meizothrombin binds hirudin similar to thrombin.      

Bond Strength and Monomer Conversion of Bonding Agents Mixed with Restorative Composites Prior to Light Exposure

This study examined the effect of adhesive systems (either placed as directed or when mixed with composites prior to photocuring) on bond strength and monomer conversion. Occlusal surfaces of extracted human molars were ground flat and Scotchbond Multipurpose®, Single Bond®, or Clearfil SE Bond® adhesive systems were applied according to manufacturer's directions, mixed in situ with flowable or hybrid composites prior to photocuring. Specimens were prepared for microtensile bond-strength testing, and maximal bond strength at failure was recorded. Adhesives and composites were also placed on a diamond attenuated-total-reflectance unit, and infrared spectra were obtained kinetically. Addition of flowable Scotchbond prior to light-curing increased bond strength; however, no effect on Single Bond and Clearfil SE Bond was observed. The mixture of adhesives with composites resulted in lower monomer conversion for Scotchbond and Clearfil SE Bond.     

铝合金上锂盐转化膜的耐蚀性能

通过电化学测试和浸泡实验研究了铝及其合金上的锂盐转化膜、铬酸盐转化膜及自然氧化膜在氯化钠溶液中的耐蚀性能。结果表明：锂盐转化膜的耐蚀性能优于自然氧化膜的,而与铬酸盐转化膜的相当。     

Effect of polysaccharides on the hydration of cement paste at early ages

This work deals with the relative efficiency of polysaccharides and their influence on cement hydration. Several parameters such as the structure, concentration, average molecular weight, and soluble fraction value of polysaccharides were examined. Cement hydration was monitored by isothermal calorimetry, thermogravimetry (TGA), and Fourier transform infrared (FTIR) spectroscopy. Results clearly show that retardation increases with higher polysaccharide-to-cement weight ratio (P/C). Low-molecular-weight starch showed enhanced retarding effect on the hydration of cement. The retardation effect of polysaccharides is also dependent on the composition of cement.     

The combined effect of stirring and oxygen on the graft copolymerization of methyl methacrylate onto cellulose

Methyl methacrylate was grafted onto bleached kraft pulp using ceric ammonium nitrate as initiator. The effect of stirring on the conversion to polymer and copolymer was strongly influenced by the presence of oxygen. In an oxygen-free system, the conversion was found to first increase rapidly with rising stirrer speed and then level off. No decrease in conversion was observed even at very high stirrer speeds (up to 1000 rpm). Inclusion of oxygen resulted in the appearance of a maximum on the conversion versus stirring speed curve, followed by a decrease in conversion at more intense agitation. The presence of oxygen also adversely affected the grafting efficiency.     

分光光度法测定海参多糖含量方法的改进

对天青Ⅰ测定海参多糖的方法进行了改进,在前人研究的基础上优化了标准曲线,确定了该曲线的量程范围,使其具有专署性、精确性的同时,真正具备了可操作性。     

苯乙烯-丙烯酸丁酯体系超浓乳液的稳定性及共聚合的研究

本文探讨了乳化剂种类、乳化剂浓度及温度对苯乙烯－丙烯酸丁酯体系超浓乳液稳定性的影响。对其共聚合,主要探讨乳化剂浓度、单体体积分数、引发剂秀量对转化率的影响及它们和乳胶粒子径的关系,还 描绘了不同聚合条件下转化率随时间的变化曲线。     

氰酸酯/苯乙烯体系的反应性及固化树脂的性能研究

用傅里叶变换红外光谱(FT-IR)研究了氰酸酯单体的等温固化反应。获得了有机锡催化氰酸酯和苯乙烯体系在不同固化温度下的转化率和动力学参数。氰酸酯的转化率随着温度的升高和催化剂的使用而提高。不含催化剂时苯乙烯对氰酸酯的影响较小,而有机锡和苯乙烯同时存在时对氰酸酯有很强的催化作用。在所有的体系中,催化反应速率常数对氰酸酯单体浓度在动力学控制阶段表现为一级反应。改性体系的冲击强度和弯曲强度比未改性氰酸酯最高分别提高了45．3%和18．7%。     

Effect of non-ionic detergents on apparent enzyme mechanism: V121A mutant of Streptomyces cholesterol oxidase endowed with enhanced sensitivity towards detergents

One of the mutants of Streptomyces cholesterol oxidase with the Val121Ala mutation (V121A) was kinetically analysed. Although the reaction rate-substrate concentration curve of wild type follows a simple Michaelis-Menten equation, that of V121A is sigmoidal. The cooperativity was apparent and caused by non-ionic detergents that were used as a solvent of cholesterol. The concentration dependence of V121A on detergents was more significant than that of wild type, although the reaction rates of both enzymes decrease as the concentrations of detergents increase. Further experiments suggested that less hydrophobic interactions between V121A and detergents should be responsible for the apparent cooperativity. Since Val121 is in a hydrophobic loop located near the active site, the mutational effect is structurally discussed.      

Fibrinogen Replacement Therapy for Traumatic Coagulopathy: Does the Fibrinogen Source Matter?

Fibrinogen is the first coagulation protein to reach critically low levels during traumatic haemorrhage. There have been no differential effects on clinical outcomes between the two main sources of fibrinogen replacement: cryoprecipitate and fibrinogen concentrate (Fg-C). However, the constituents of these sources are very different. The aim of this study was to determine whether these give rise to any differences in clot stability that may occur during trauma haemorrhage. Fibrinogen deficient plasma (FDP) was spiked with fibrinogen from cryoprecipitate or Fg-C. A panel of coagulation factors, rotational thromboelastography (ROTEM), thrombin generation (TG), clot lysis and confocal microscopy were performed to measure clot strength and stability. Increasing concentrations of fibrinogen from Fg-C or cryoprecipitate added to FDP strongly correlated with Clauss fibrinogen, demonstrating good recovery of fibrinogen (r² = 0.99). A marked increase in Factor VIII, XIII and α₂-antiplasmin was observed in cryoprecipitate (p < 0.05). Increasing concentrations of fibrinogen from both sources were strongly correlated with ROTEM parameters (r² = 0.78–0.98). Cryoprecipitate therapy improved TG potential, increased fibrinolytic resistance and formed more homogeneous fibrin clots, compared to Fg-C. In summary, our data indicate that cryoprecipitate may be a superior source of fibrinogen to successfully control bleeding in trauma coagulopathy. However, these different products require evaluation in a clinical setting.     

Time-temperature and time-irradiation intensity superposition for photopolymerization of an epoxy based resin

In this paper the photoinitiated polymerization of a commercially available epoxy based resin for stereolithography (SL5170) was studied by means of photo differential scanning calorimetry (pDSC) at different temperature and irradiation intensities. Experimental results showed that temperature and light intensity have the same qualitative effects on the reaction kinetics. Time-temperature and time-intensity superposition principle was applied to the experimental curves. In both cases, it was shown that curves shifting on the time axis determines a single master curve. Individual curves significantly diverge from the master curve for high conversions, indicating that different regimes are encountered during photocuring of commercial stereolithography cationic resin. For low conversions, the reaction is kinetically controlled, whereas it becomes mainly diffusion controlled when the glass transition temperature approaches the isothermal cure temperature. At high conversion, master curves were also built in the diffusion-controlled regime, using different shift factors. The applicability of master curves to photopolymerization of epoxy based resin for stereolithography indicates that the mechanisms involved in network formation in the kinetic controlled regime are the same regardless of temperature and irradiation intensity. Consequently, time temperature transformation (TTT) and time intensity transformation (TIT) curves were built, as the time needed to reach polymer vitrification at each temperature and irradiation intensity.     

Effect of stirring on cellulose graft copolymerization. V. Influence of reaction parameters

Styrene was grafted onto dissolving pulp by the cellulose xanthate–Fe²⁺–H₂O₂ system. Reaction parameters were found to have strong influence on conversion to both copolymer and total polymer, as well as on the dependence of polymerization on stirring. The formation of polymer was almost completely inhibited by pure oxygen, while air only slowed down the reaction. Under inert atmosphere, the effect of agitator speed was found to be strongly dependent on monomer and substrate concentration as well as on the concentration of emulsifier. The location of the maximum on the conversion vs. agitator speed curve was strongly affected by the shape of the stirrer. The presence of emulsifier had a relatively small effect on copolymer formation in the case of acrylamide, a water-soluble monomer. Also the effect of stirring was less marked in the case of acrylamide. In all the systems investigated, the conversion to copolymer and total polymer was found to drop rapidly above a certain limiting agitator speed. The latter was different and characteristic for each system. No polymer formation was observed beyond 1000 rpm regardless of all other reaction conditions.     

Construction and structure-activity relationships of chimeric prourokinase derivatives with intrinsic thrombin-inhibitory potential

The blood clotting enzyme thrombin plays a central role in theaetiology of occlusive disorders such as stroke and acute myocardialinfarction. During fibrinolytic therapy with plasminogen activators,thrombin is neutralized by anticoagulative drugs. In order tocombine plasminogen-activating and thrombin-inhibitory activitieswe constructed chimeric derivatives of recombinant single-chain,urokinase-type plasminogen activator (rscu-PA) which comprisethe kringle and protease domain of rscu-PA fused via a linkersequence to a thrombin-inhibitory domain. The inhibitory domaincontains a sequence element directed to the active site of thrombinand a sequence taken from either hirudin or the human thrombinreceptor both binding to the fibrinogen recognition site ofthrombin. Analysing different sets of point mutants showed thatthe linker between the protease domain and the active sitedirectedsequence is contributing significantly to the thrombin-inhibitorypotential. Kinetic analysis of thrombin inhibition revealedthat most of the chimeras tested competitively inhibit the thrombin-mediatedcleavage of a peptide substrate in a concentration-dependentmanner; however, in two examples the insertion of one glycineresidue into the active site directed-sequence abolished theblockade of the active site. This supports the conclusion thatthe chimeras with high thrombin-inhibitory potential interactwith the active site and the fibrinogen recognition site ofthrombin.     

On the fine emulsion polymerization of styrene with non-ionic emulsifier

Summary The fine emulsion polymerizations of styrene initiated by a redox system ammonium peroxodisulfate/sodium thiosulfite stabilized by a non-ionic emulsifier were kinetically investigated. The dependence of the rate of polymerization on conversion or the emulsifier concentration was described by a curve with maximum at medium conversion. The maximum rate of polymerization is proportional to the − 0.45^th and 1.5^th power of initiator and emulsifier concentration, the number of particles to the 0.32^nd and 1.3^rd power of initiator and emulsifier concentration and the molecular weight to the − 0.62^th and −0.97^th power of initiator and emulsifier concentration, respectively. The results show a strong decrease in turbidity at around 20% conversion when emulsion turns into translucent latex. Deviation from the micellar nucleation model was attributed to the solubility of emulsifier in monomer, high level of nonmicellar aggregates, thick interfacial layer and transfer emulsion to microemulsion. The strong decrease of molecular weight with increasing emulsifier concentration is attributed to chain transfer events promoted by the high level of emulsifier at the reaction loci. Received: 16 November 1998/Revised version: 25 May, 10 October 1999/Accepted: 10 October 1999     

NaOH/AQ预处理对玉米秸秆脱木质素及酶解性能的影响

对玉米秸秆进行氢氧化钠/蒽醌（NaOH/AQ）去木质化预处理,考察了预处理温度、时间和NaOH用量对玉米秸秆脱木质素程度的影响,并探讨了脱木质素程度对提高预处理后物料酶解性能的影响。L₉（3⁴）正交试验得出较适宜预处理工艺条件为：温度160℃,时间60 min,NaOH用量（以绝干原料质量计）2.8%;其他条件为AQ用量0.05%,固液比1：5（g：mL）,此时木质素脱除率为75%,酶解后聚糖转化率达到73.79%。随着物料脱木质素程度的提高,其酶解效率相应增加;当木质素脱除率达到一定程度后,预处理后的聚糖转化率达到最大值,继续提高木质素脱除率,聚糖转化率反而降低。响应面优化的酶水解工艺条件为纤维素酶用量30 FPU/g,β-葡萄糖苷酶10 IU/g,反应时间72 h,温度50℃,底物质量分数2.5%,此时还原糖得率为85.62%。对酶解液进行HPLC分析,酶解液中的葡萄糖质量浓度为14.83 g/L,木糖质量浓度为4.83 g/L。XRD分析显示,预处理前后纤维素的晶型没有变化,而结晶度由31.40%提高至46.91%,表明物料中木质素和半纤维素发生了不同程度的溶出。     

能源植物菊芋制备生物基化合物研究进展

生物质作为工业填料在制备化学品的过程中具有可再生性、碳元素利用平衡等优点，但大部分能源植物存在来源于粮食必需品、与农作物争夺优质土地的问题。天然生物质菊芋因具有优良的生长特性、糖类组分含量高、单体官能团多样等特点，被认为是未来最重要的非粮能源植物之一。本文介绍了通过物理过程、生物过程及化学过程等不同途径高附加值化菊芋的研究进展，总结不同方式制备生物基化合物的特点。基于菊芋主要因其根茎中含有丰富的不易被人体消化的菊糖、果糖基多糖聚合度低、组成多糖和还原糖的碳源单体高度单一等优点，着重介绍目前菊芋根茎作为底物制备生物基化合物的过程，分析了通过化学催化法或发酵法制备多元醇、5-羟甲基糠醛、乳酸酯等产品的反应条件、催化剂或生物酶的类型等。基于菊芋秸秆中富含纤维素、半纤维素和木质素等木质纤维素的优势，简述了以纤维素和半纤维素类碳水化合物和木质素的主要研究现状，以及对菊芋秸秆直接催化转化的效果，突出菊芋秸秆转化的优势，提出菊芋秸秆作为底物高效制备目标产物的改进措施。对菊芋根茎和菊芋秸秆的高附加值化过程和效果的分析表明，加强对非粮能源植物菊芋的深加工与生物、化学转化技术的研究，配合生物法与化学法相结合的手段，能加快菊芋工业化应用取得实质性的进展。