Regulation of G protein-coupled receptor trafficking by inefficient plasma membrane expression: molecular basis of an evolved strategy

Despite the prevalence of G protein-coupled receptors as transducers of signals from hormones, neurotransmitters, odorants, and light, little is known about mechanisms that regulate their plasma membrane expression (PME), although misfolded receptors are recognized and retained by a cellular quality control system (QCS). Convergent evolution of the gonadotropin-releasing hormone (GnRH) receptor (GnRHR) progressively decreases inositol phosphate production in response to agonist, validated as a measure of PME of receptor. A pharmacological chaperone that optimizes folding also increases PME of human, but not of rat or mouse, GnRHR because a higher percentage of human GnRHRs are misfolded structures due to their failure to form an apparent sulfhydryl bridge, and they are retained by the QCS. Bridge formation is increased by deleting (primate-specific) Lys191. In rat or mouse GnRHR that lacks Lys191, the bridge is non-essential and receptor is efficiently routed to the plasma membrane. Addition of Lys191 alone to the rat sequence did not diminish PME, indicating that other changes are required for its effects. A strategy, based on identification of amino acids that both 1) co-evolved with the Lys191 and 2) were thermodynamically unfavorable substitutions, identified motifs in multiple domains of the human receptor that control the destabilizing influence of Lys191 on a particular Cys bridge, resulting in diminished PME. The data show a novel and underappreciated means of posttranslational control of a G protein-coupled receptor by altering its interaction with the QCS and provide a biochemical explanation of the basis of disease-causing mutations of this receptor.     

Surface plasmon resonance and free oscillation rheometry in combination: a useful approach for studies on haemostasis and interactions between whole blood and artificial surfaces

In haemostatic and biomaterial research biological processes at surfaces and in the bulk phase of the surface-contacting medium are important. The present work demonstrates the usefulness of the combination of surface plasmon resonance (SPR), sensitive to changes in refractive index at surfaces, and free oscillation rheometry (FOR), sensitive to rheological properties of the bulk, for simultaneous real-time measurements on coagulation and fibrinolysis of blood plasma and coagulation of whole blood. SFLLRN stimulated coagulation of native whole blood presented a higher SPR signal with different appearance than plasma coagulation, while the FOR signals corresponding to plasma and whole blood coagulation were similar. This indicated that the SPR technique was more sensitive to cell-surface interactions than to fibrin formation in whole blood during coagulation, while the FOR technique were equally sensitive to coagulation in whole blood and plasma. Spontaneous coagulation of native whole blood in contact with methyl- and hydroxyl-terminated self-assembled monolayers (SAM) on gold and gold surfaces regenerated after coagulation were also studied. The regenerated gold surfaces displayed the shortest coagulation times, although the contact-activation of blood coagulation for these surfaces was low. The methylated and hydroxylated surfaces were comparable in terms of coagulation activation, while the hydroxylated surfaces presented FOR signals that indicated detaching of the coagulum from the surface. The combination of SPR and FOR is well suited for studies of cell- and protein-surface interactions and simultaneous bulk processes. Possible applications are investigations of blood cell defects in patients and monitoring of native whole blood interactions with artificial surfaces.     

Surface plasmon resonance (SPR) analysis of coagulation in whole blood with application in prothrombin time assay

It is previously shown that surface plasmon resonance (SPR) can be used to study blood plasma coagulation. This work explores the use of this technique for the analysis of tissue factor induced coagulation, i.e. prothrombin time (PT) analysis, of whole blood and plasma. The reference method was nephelometry. The prothrombin time analysis by SPR was performed by mixing two volumes of blood/plasma, one volume of thromboplastin, and one volume of CaCl2 solution directly on a sensor surface. The measurements show good agreement between nephelometry and SPR plasma analysis and also between SPR plasma and whole blood analysis. The effect of anticoagulant treatment on the clotting times was significant both quantitatively and qualitatively. The impact on the SPR signal of different physiological events in the coagulation process is discussed, and tentative interpretations of the sensorgram features are given. The major advantage of the SPR method compared to nephelometry is the possibility to perform analysis on whole blood instead of plasma. In conclusion, SPR is a promising method for whole blood coagulation analysis.     

Rheological approach to the analysis of blood coagulation in endothelial cell-coated tubes: Activation of the intrinsic reaction on the erythrocyte surface

Coagulation of blood in cultured endothelial cell-coated tubes was examined using a theological technique. Coagulation of recalcified, platelet-free plasma in contact with an endothelial cell monolayer did not occur within the experimental time period (more than 150 min). The endothelial cell surface did not activate the intrinsic coagulation reaction or the extrinsic coagulation reaction initiated by tissue factor. The time of onset of coagulation in platelet-free plasma supplemented with erythrocytes was nearly the same as that of whole blood (31.2 ± 5.5 min), which was shorter than that for platelet-rich plasma (54.3 ± 14.3 min) and platelet-free plasma supplemented with granulocytes (58.3 ± 6.3 min). In factor VII-, XI- or XII- deficient, platelet-free plasma supplemented with erythrocytes, the time of onset of coagulation was about 30 min. The coagulation of factor IX-deficient, platelet-free plasma supplemented with erythrocytes, however, did not occur within the experimental time period. These data suggest that activation of factor IX on the erythrocyte surface is capable of activating the intrinsic coagulation system.     

Surface plasmon resonance detection of blood coagulation and platelet adhesion under venous and arterial shear conditions

A surface plasmon resonance (SPR) based flow chamber device was designed for real time detection of blood coagulation and platelet adhesion in platelet rich plasma (PRP) and whole blood. The system allowed the detection of surface interactions throughout the 6mm length of the flow chamber. After deposition of thromboplastin onto a section of the sensor surface near the inlet of the flow chamber, coagulation was detected downstream of this position corresponding to a SPR signal of 7 to 8 mRIU (7 to 8 ng/mm2). A nonmodified control surface induced coagulation 3.5 times slower. Platelet adhesion to gold and fibrinogen coated surfaces in the magnitude of 1.25 and 1.66 mRIU was also shown with platelets in buffer, respectively. SPR responses obtained with PRP and whole blood on surfaces that were methylated or coated with von Willebrand factor (vWF), fibrinogen, or collagen, coincided well with platelet adhesion as observed with fluorescence microscopy in parallel experiments. The present SPR detection equipped flow chamber system is a promising tool for studies on coagulation events and blood cell adhesion under physiological flow conditions, and allows monitoring of short-range surface processes in whole blood.     

Control of Heparin Therapy

Heparin therapy in 114 patients was controlled by daily blood tests—the whole blood coagulation time, kaolin-activated partial thromboplastin time of plasma, and plasma heparin assay. Bleeding episodes occurred in 7 out of 92 patients (7·6%) who had normal haemostatic mechanisms before therapy and in 11 out of 22 patients (50%) with defective haemostasis, mostly due to intravascular coagulation or renal failure. The dose of heparin ranged from 20,000 to 60,000 units in each 24-hour period. In some patients bleeding was related to overdosage, but in others the laboratory tests indicated satisfactory or suboptimal dosage at the time of bleeding. Though there were positive correlations between the results of the three tests, these were not close, and no one test was preferable. Hence laboratory control of heparin therapy is unsatisfactory and patients may bleed despite careful control of the dose by all three methods.     

血栓弹力图指导食管癌患者临床输血的价值及其与常规凝血实验检测指标的相关性分析

目的:探讨血栓弹力图(TEG)指导食管癌患者临床输血的价值及其与常规凝血实验检测指标的相关性。方法:选取2017年1月-2019年3月在我院收治的食管癌手术治疗需输血的99例患者作为研究对象,将99例患者随机分为常规凝血功能检测组和TEG组,常规凝血功能检测组采用常规凝血实验检查结果指导输血,TEG组采用TEG检查结果指导输血,对比两组输血前后的常规凝血实验检测指标以及临床用血量,对比TEG组输血前后的TEG指标,分析TEG指标与常规凝血实验检测指标的相关性。结果:两组患者输血前凝血四项和血小板计数(PLT)差异无统计学意义(P>0.05),输血后两组活化凝血酶时间(APTT)、凝血酶原时间(PT)、凝血酶时间(TT)、纤维蛋白原(FIB)均有不同程度的改善(P<0.05),TEG组PT、TT较常规凝血功能检测组低(P<0.05);输血后,TEG组患者凝血反应时间(R值)、血凝块形成时间(K值)较输血前降低,最大血凝块强度(MA值)、凝血综合指数(CI值)升高,凝血形成速率(Angle角)增大,差异有统计学意义(P<0.05);Pearson相关性分析结果显示,R值与APTT呈正相关(P<0.05),K值与PLT呈负相关,与FIB呈正相关(P<0.05),Angle角、MA值、CI值与FIB、PLT呈正相关(P<0.05);TEG组新鲜冰冻血浆、冷沉淀输注量少于常规凝血功能检测组,差异有统计学意义(P<0.05)。结论:TEG能更好地指导食管癌手术患者各种血液成分的合理输注,有效改善凝血异常情况,减少输血用量,TEG指标与常规凝血实验检测指标存在一定的相关性。     

Blood coagulation and fibrinolysis of rats fed fish oil: reduced coagulation factors especially involved in intrinsic pathway and increased activity of plasminogen activator inhibitor

Differences in the coagulation and fibrinolytic system of rats fed a fish oil based diet (fish oil diet) and fed a soybean oil based diet (control diet) were determined. Concentrations of plasma lipids were depressed in rats fed the fish oil diet. Prothrombin time (PT) and activated partial thromboplastin time (APTT) of rats fed the fish oil diet were longer than for the rats fed the control diet. Fish oil intake lowered the activities of most of the blood coagulation factors, and strongly depressed the factors involved in the intrinsic pathway. Fish oil also affected the fibrinolysis of rats. Plasminogen activator inhibitor (PAI) activity was elevated in rats fed the fish oil diet. In this study, both blood coagulation and fibrinolysis were down-regulated by feeding the fish oil diet.     

细菌性败血症引起鲫凝血障碍的研究

以嗜水气单胞菌感染体重７０ｇ左右的鲫并检测了感染鱼和对照鱼的血液学及播散性血管内凝血判定指标等参数。结果表明细菌性败血症病鱼严重贫血,存在明显的凝血障碍,在疾病发展过程中存在播散性血管内凝血现象。这一些象导致了病鱼全身性出血,并在疾病的病理生理过程中起了重要作用。     

Quaternized chitosan (QCS)/poly (aspartic acid) nanoparticles as a protein drug-delivery system

The aim of this study was to generate a new type of nanoparticles made of quaternized chitosan (QCS) and poly (aspartic acid) and to evaluate their potential for the association and delivery of protein drugs. QCS and poly (aspartic acid) were processed to nanoparticles via the ionotropic gelation technique. The size, polydispersity, zeta potential, and morphology of the nanoparticles were characterized. Entrapment studies of the nanoparticles were conducted using bovine serum albumin (BSA) as a model protein. The effects of the pH value of nanoparticles with different QCS/poly (aspartic acid) ratios, QCS molecular weight (MW), poly (aspartic acid) concentration, and BSA concentration on the nanoparticle size, the nanoparticle yield, and BSA encapsulation were studied in detail. Suitably pH value of nanoparticles with different QCS/poly (aspartic acid) ratios, moderate QCS MW, optimal concentration ratio of poly (aspartic acid), and QCS favored more nanoparticles formed and higher BSA encapsulation efficiency. The release of BSA from nanoparticles was pH-dependent. Fast release occurred in 0.1 M phosphate buffer solution (PBS, pH 7.4), while the release was slow in 0.1 M HCl (pH 1.2). The results showed that the new QCS/poly (aspartic acid) nanoparticles have a promising potential in protein delivery system.     

Effect of soluble fibrin on the blood coagulation process and platelets aggregation

The accumulation of soluble fibrin (SF) in the blood plasma causes acceleration of the final stage of blood coagulation. It increases functional activity of a hemostasis system platelet link, that is the precondition of thrombotic complication. Accumulation of SF in the blood plasma is accompanied by proportional reduction of coagulation time in ancistron and thrombin time tests, and also the intensification of platelets aggregation process. A conclusion was drawn that for early diagnostics of the DIC-syndrom it is expedient to carry out complex estimation of the hemostasis system with obligatory definition of the blood SF content, performance of ancistron and thrombin time tests, and also study of platelets aggregation.     

Comparison of surface plasmon resonance and quartz crystal microbalance in the study of whole blood and plasma coagulation

The coagulation of blood plasma and whole blood was studied with a surface plasmon resonance (SPR) based device and a quartz crystal microbalance instrument with energy dissipation detection (QCM-D). The SPR and QCM-D response signals were similar in shape but differing in time scales, reflecting differences in detection mechanisms. The QCM-D response time was longer than SPR, as a physical coupling of the sample to the substrate is required for molecules to be detected by the QCM-method. Change of sample properties within the evanescent field is sufficient for detection with SPR. Both the SPR signals and the QCM-D frequency and dissipation shifts showed dependency on concentrations of coagulation activator and sensitivity to heparin additions. The ratio of dissipation to frequency shifts, commonly considered to reflect viscoelastic properties of the sample, varied with the concentration of activator in blood plasma but not in whole blood. Additions of heparin to the thromboplastin activated whole blood sample, however, made the ratio variation reoccur. Implications of these observations for the understanding of the blood coagulation processes as well as the potential of the two methods in the clinic and in research are discussed.     

Simultaneous determination of fentanyl and midazolam using high-performance liquid chromatography with ultraviolet detection

When measuring fentanyl and midazolam simultaneously in the same plasma sample with standard high-performance liquid chromatography–ultraviolet (HPLC–UV) detection, overlap of the fentanyl peak by the midazolam peak occurs, which makes fentanyl determination impossible. We tested the hypothesis that by acidifying the methanol mobile phase with 0.02% perchloric acid, 70%, it would be possible to separate both peaks. The UV detector was set at 200 nm. Calibration curves for fentanyl (range 0–2000 pg/ml) and midazolam (range 0–400 ng/ml) were linear (r>0.99). The detection limits were 200 pg/ml (fentanyl) and 10 ng/ml (midazolam). Precision and accuracy for intra- and inter-assay variability as well as in-line validation with quality control samples (QCS) were acceptable (< 15 and 20%, respectively), except for fentanyl QCS of 200 pg/ml (17.8% precision). Although less sensitive than gas chromatography–mass spectrometry (GC–MS), reliable measurements of fentanyl, simultaneously with midazolam, can be performed with this HPLC–UV system.     

蝮蛇抗栓酶对冻伤家兔血凝系统某些指标的影响

本研究观察了家兔双后足重度冻伤后用蝮蛇抗栓酶（０．２５Ｕ／ｋｇ体重加入２０ｍｌ／ｋｇ体重生理盐水中,耳缘静脉点滴）进行治疗后其血凝系统某些指标的改变。结果表明,重度冻伤后家兔的出血时间及凝血时间明显缩短,血小板功能呈不同程度的改变,血浆纤维蛋白原含量增加,血液处于高凝状态;用蝮蛇抗栓酶治疗后,上述各项指标均有不同程度的改善,结果提示,蝮蛇抗栓酶可明显缓解冻伤所致的血凝增强的病变过程,这对重度冻伤的治疗将起到重要作用     

The human plasma fibrinolytic system: Regulation and control

Summary The factors involved in the regulation and control of the human plasma fibrinolytic system at the cellular level are unknown at this time. The physiological regulation of plasmin formation in plasma depends primarily on the nature of the circulating zymogen, plasminogen, the physiological activators formed both in the blood and in the vascular endothelium, and the specific plasmin inhibitors found both in plasma and in certain of the cellular elements of the blood. The biosynthesis of the zymogen must be under genetic control, and the activators are probably released, after thrombus and clot formation, from components involved in the surface-mediated initiation of the coagulation system, and from the vascular endothelium. Activation of plasminogen can occur both in the fluid phase surrounding the thrombus and probably at thrombus surfaces, involving both the fibrin clot and the platelet membrane. The plasmin inhibitors act to control the system in order to prevent proteolytic degradation of important physiological trace proteins of the coagulation, complement and kallikrein-kinin systems by the enzyme.     

Coagulation of dust grains in the plasma of an RF discharge in argon

Results are presented from experimental studies of coagulation of dust grains of different sizes injected into a low-temperature plasma of an RF discharge in argon. A theoretical model describing the formation of dust clusters in a low-temperature plasma is developed and applied to interpret the results of experiments on the coagulation of dust grains having large negative charges. The grain size at which coagulation under the given plasma conditions is possible is estimated using the developed theory. The theoretical results are compared with the experimental data.     

Measuring the mechanical properties of blood clots formed via the tissue factor pathway of coagulation

Thrombelastography (TEG) is a method that is used to conduct global assays that monitor fibrin formation and fibrinolysis and platelet aggregation in whole blood. The purpose of this study was to use a well-characterized tissue factor (Tf) reagent and contact pathway inhibitor (corn trypsin inhibitor, CTI) to develop a reproducible thrombelastography assay. In this study, blood was collected from 5 male subjects (three times). Clot formation was initiated in whole blood with 5 pM Tf in the presence of CTI, and fibrinolysis was induced by adding tissue plasminogen activator (tPA). Changes in viscoelasticity were then monitored by TEG. In quality control assays, our Tf reagent, when used at 5 pM, induced coagulation in whole blood in 3.93 ± 0.23 min and in plasma in 5.12 ± 0.23 min (n=3). In TEG assays, tPA significantly decreased clot strength (maximum amplitude, MA) in all individuals but had no effect on clot time (R time). The intraassay variability (CVa<10%) for R time, angle, and MA suggests that these parameters reliably describe the dynamics of fibrin formation and degradation in whole blood. Our Tf reagent reproducibly induces coagulation, making it an ideal tool to quantify the processes that contribute to mechanical clot strength in whole blood.     

Quartz crystal microbalance-with dissipation monitoring (QCM-D) for real time measurements of blood coagulation density and immune complement activation on artificial surfaces

A recently developed variant of quartz crystal microbalance (QCM) called QCM-with dissipation monitoring (QCM-D) allows simultaneous and simple measurements of changes in adsorbed mass as well as the viscoelastic property (D-factor) of deposited protein layers on the sensor surface. We have taken the QCM-D technology a step further and demonstrated its advantages in the study of protein assembly as a consequence of surface induced immune complement activation, or contact activated blood coagulation. In the present study we have continued our QCM-D investigations of surface assembly of fibrin clot formation and complement activation and incubated differently modified quartz sensor surfaces in blood plasma and sera. Polymer surfaces used were spin-coated polyethylene, poly(ethylene terephtalate), poly(methylmetacrylate) and poly(dimethylsiloxane). Also used were sputtered titanium and heparin grafted surfaces. In this investigation we found that we could describe the surface induced coagulation with four independent parameters: (1) Time of onset of coagulation, (2) fibrin deposition rate, (3) total frequency shift at stable plateau, and (4) fibrin clot density. The most important finding was that the blood plasma clot density can be assessed with the use of D determinations and that the clot density varied significantly with the chemical composition of the surface. However, the D-factor did not give any new analytical information about the possible complement activation mechanisms. Nevertheless, the QCM-D was found to be a reliable tool for the analysis of surface induced complement activation. We also compared the QCM-D technique with traditional enzyme immuno assay (EIA) measurements of soluble products from the surface activation of the complement and coagulation systems. We found that the results from EIA and QCM-D measurements corresponded well for the complement activation but not for the coagulation, probably due to the biological complexity of the coagulation system.     

缩宫素联合卡前列素氨丁三醇对产后出血患者凝血功能及血流动力学的影响

目的:探讨缩宫素联合卡前列素氨丁三醇对产后出血患者凝血功能及血流动力学的影响。方法:选取我院于2014年1月-2017年4月期间收治的产后出血患者126例,根据乱数表法分为对照组(n=63)与研究组(n=63),其中对照组给予缩宫素治疗,研究组则给予缩宫素联合卡前列素氨丁三醇治疗。观察并比较两组患者产后出血情况、凝血功能以及血流动力学各项指标,同时观察两组患者的生活质量及不良反应发生情况。结果:研究组患者产后出血发生率、产后2 h出血量以及产后24 h出血量均明显低于对照组(P0.05)。两组患者活化部分凝血活酶时间(APTT)、血浆凝血酶原时间(PT)、血浆凝血酶时间(TT)、纤维蛋白原(Fg)相比无统计学差异(P0.05)。治疗2 h后两组患者心率(HR)较治疗前升高,且对照组高于研究组,收缩压(SBP)、舒张压(DBP)较治疗前降低,且对照组低于研究组(P0.05);治疗24 h后研究组患者SBP高于对照组(P0.05);两组患者不同时间的血氧饱和度(SPO_2)比较均无统计学差异(P0.05)。研究组患者的躯体功能、精神健康、情感职能、社会活动以及社会功能得分均高于对照组(P0.05)。对照组患者不良反应发生率为7.94%,与研究组的6.35%比较无统计学差异(P0.05)。结论:缩宫素联合卡前列素氨丁三醇可有效减少患者产后出血发生率,维持患者血流动力学稳定,对患者凝血功能无影响,生活质量评分较高,值得临床推广。     

Impact of blood sampling technique on blood quality and animal welfare in haemophilic mice

Blood collection in mice can be a challenge, in particular for samples used for coagulation analysis as initiation of coagulation during the procedures can influence the results. Blood collection from the retrobulbar venous plexus is commonly used but the method remains controversial. Several alternatives exist but not all are applicable to mice with a compromised coagulation system because of subsequently excessive bleeding. We therefore wanted to explore whether blood collection by puncture of the submandibular vein could replace blood collected from the retrobulbar venous plexus during pharmacokinetic and pharmacodynamic studies in mice lacking coagulation factor VIII (FVIII). The plasma concentrations of recombinant activated factor VII were independent of the blood collection method in a pharmacokinetic study. The same applied to the thromboelastographic profile of mice with normal coagulation in a pharmacodynamic study. However, excessive haemorrhages were observed in all FVIII knockout mice after a single puncture of the submandibular vein and 60% of the mice were euthanized 2-4 h after the blood collection. In contrast, no or only slight haemorrhage was observed in animals subjected to blood collection from the retrobulbar venous plexus. No signs of distress determined by blood glucose level or clinical abnormalities of the eye were observed after puncture of the retrobulbar venous plexus. In conclusion, blood collected by puncture of the submandibular vein and retrobulbar venous plexus has a quality which allows it to be used in coagulation assays. However, because of excessive bleedings, puncture of the submandibular vein is not recommended in mice lacking FVIII.