Simultaneous measurement of blood coagulation and erythrocyte sedimentation by means of a rheological technique

With a damped-oscillation rheometer, changes in the rheological properties, i.e., logarithmic damping factor (LDF) and period, as obtained from a damped-oscillation curve, were monitored during the coagulation of blood. In our earlier studies, the time of onset of coagulation (Ti) of the blood sample was only determined from the change in LDF. When coagulation of the blood and sedimentation of erythrocytes occurred together, the Ti value could not be determined from the change in LDF. In this paper, a method for determining the Ti value from the change in the period of the damped-oscillation curve was investigated. It was found that the period increased and leveled off as blood coagulation progressed, and the Ti value was determined from the middle point between the minimum and maximum values of the period. In addition, it was suggested that the level of erythrocyte sedimentation could be estimated from the initial decrease in LDF. In blood obtained from diabetic patients, a good correlation between the initial decrease in the LDF and the concentration of fibrinogen was observed. Our study demonstrates that when erythrocyte sedimentation and blood coagulation occur simultaneously, this rheological technique makes it possible to measure the Ti value and erythrocyte sedimentation.     

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.     

The role of aggregation kinetics in the sedimentation of erythtocytes

The well-known S-shaped settling curves are obtained as solutions of an autonomic dynamical system deduced mathematically from the generalized Stokes formula, the blood volume conservation law, and the Smoluchowski theory of particle coagulation. Numerical computations and parametric analysis of the deduced two nonlinear differential equations for the plasma zone thickness and aggregate size are given. It is shown that the model presented makes it possible, on the basis of experimentally recorded sedimentation curves and aggregate size growth, to identify quantitatively the values of the essential physical parameters of the coupled processes of erythrocyte aggregation and sedimentation. This method of identification could be used as a diagnostic test in hematological laboratories.     

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.     

Versican G3 domain promotes blood coagulation through suppressing the activity of tissue factor pathway inhibitor-1

We have detected versican, a member of the large chondroitin sulfate proteoglycans, and its degraded C-terminal G3 fragments in human plasma and observed that the versican G3 domain promoted blood coagulation. Silencing G3 expression with small interfering RNA reduced the effect of G3 on coagulation. Plasma coagulation assays suggest that G3 enhances coagulation irrespective of its actions on platelets and white blood cells. To examine how versican affected blood coagulation, we used normal human plasma and different types of coagulation factor-deficient plasmas. The experiments indicated that versican enhanced coagulation through the extrinsic pathway, and that Factor VII was the target molecule. FVII activity assays showed that G3 activated FVII in the presence of plasma but not with purified FVII directly. Yeast two-hybrid, immunoprecipitation, and gel co-migration assays showed that G3 interacted with the tissue factor pathway inhibitor-1 (TFPI-1). TFPI-1 activity assays suggested that G3 inhibited TFPI-1 activity, allowing FVIIa and FXa to facilitate the coagulation process. G3-induced blood coagulation was further confirmed with a mouse model in a real-time manner. Taken together, these results indicate that versican may represent a new target for the development of therapies against atherosclerosis.     

Determination of erythrocyte aggregation

One of the most common health criteria--erythrocyte sedimentation rate (ESR)--is considered in the paper. It is shown that the simple model presented, based on the generalized Stokes formula, the blood volume conservation law, and the Smoluchowski theory of particles coagulation, makes it possible, on the basis of experimentally recorded sedimentation curves, to identify quantitatively the values of the essential physical parameters of the coupled processes of erythrocyte aggregation and sedimentation. The analytical solution of Smoluchowski equation is used to evaluate the sedimentation and aggregation rate constants. The problem of determining the erythrocyte aggregation rate (EAR) is transformed to a minimization task in which only the experimental results for ESR are needed. Experimentally ESR is measured accurately enough by using an equipment set up just for the purpose. This method of identification could be used as a diagnostic test in hematological laboratories.     

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。     

Real time monitoring of the effects of Heparan Sulfate Proteoglycan (HSPG) and surface charge on the cell adhesion process using thickness shear mode (TSM) sensor

The effects of Heparan Sulfate Proteoglycan (HSPG) and surface charge on the cellular interactions of the cell membrane with different substrates to determine the kinetics of cell adhesion was studied using thickness shear mode (TSM) sensor. The TSM sensor was operated at its first, third, fifth and seventh harmonics. Since the penetration depth of the shear wave decreases with increases in frequency, the multi-resonance operation of the TSM sensor was used to monitor the changes in the kinetics of the cell-substrate interaction at different distances from the sensor surface. During the sedimentation and the initial attachment of the cells on the sensor surface, the changes in the sensor resonant frequency and the magnitude response were monitored. First, HSPGs were partially digested with the enzyme Heparinase III to evaluate the effect of HSPG on the cell adhesion process. The results indicated that HSPG did not have any effect on the kinetics of the initial attachment, but it did reduce the strength of steady-state cell adhesion. Next, we investigated the effect of the electrostatic interactions of the cell membrane with the substrate on the cell adhesion. In this case, the sensor surface was coated with positively charged Poly-D-Lysine (PDL). It was observed that electrostatic interaction of the negatively charged cell membrane with the PDL surface promoted the initial cell adhesion but did not support long-term cell adhesion. The multi-resonant TSM technique was shown to be a very promising method for monitoring specific interfacial effects involving in cell adhesion process in real-time.     

Leukocyte isolation by sedimentation: the effect of rouleau-promoting agents on leukocyte differential count.

The rouleau-promoting agents dextran and polyvinylpyrrolidone (PVP) were used to accelerate erythrocyte sedimentation in order to harvest the leukocyte rich plasma (LRP). The objective of the work was to determine if agent concentration or blood: agent ratio had any effect on the leukocyte differential count and if so at what agent concentration and agent:blood ratio did the LRP leukocyte differential count most closely match the whole blood leukocyte differential count. With both sedimentation agents the data clearly indicate that both parameters effect LRP differential counts and that low concentrations of sedimentation agents are most important in obtaining LRP differential counts which most closely match the whole blood differential counts.     

Cells, proteins, and certain physical-chemical properties of brook trout (Salvelinus fontinalis) blood

Laboratory brook trout were used to evaluate, refine, or develop biochemical procedures for the analysis of fish blood. Analytical values were obtained for the following blood properties: total and differential leucocytes and erythrocytes; erythrocyte and plasma proteins (by electrophoresis); plasma refractive index; erythrocyte sedimentation rate; erythrocyte osmotic fragility; blood surface tension and density; haemoglobin; and packed cell volume. These blood factors are discussed with reference to fish health and disease and to changes caused by deleterious quantities of water pollutants.     

Experiences with a statistical quality control system (QCS) for coagulation diagnostics parameters

With the Quality-Control-Service (QCS) for blood coagulation a system for the statistical quality control of blood coagulation methods is presented. The system is based on the universal control plasma PreciClot which contains target values in the normal and abnormal range. As the control plasma is used daily from the participants for quality control exercises and datas are statistically analyzed each month this programme of quality assessment can be compared with a monthly ring trial. For the methods prothrombin time (PT/Quick), activated partial prothrombin time (APTT), fibrinogen assay (Fibrinogen) and thrombin time (Thrombin) datas of a survey period (January-December 1985) with 75 labs were evaluated. Calculated results for the methods are given and accuracy and precision of the methods are compared with the results of former ring trials. Based on the results the interlaboratory reliability of the methods is discussed and the advantages of QCS for blood Coagulation for a better information about quality of coagulation tests are presented.     

Subcellular localization of cyclic ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities in porcine airway smooth muscle

Recent studies have provided evidence for a role of cyclic ADP-ribose (cADPR) in the regulation of intracellular calcium in smooth muscles of the intestine, blood vessels and airways. We investigated the presence and subcellular localization of ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of beta-NAD(+) to cADPR, and cADPR hydrolase, the enzyme that degrades cADPR to ADPR, in tracheal smooth muscle (TSM). Sucrose density fractionation of TSM crude membranes provided evidence that ADP-ribosyl cyclase and cADPR hydrolase activities were associated with a fraction enriched in 5'-nucleotidase activity, a plasma membrane marker enzyme, but not in a fraction enriched in either sarcoplasmic endoplasmic reticulum calcium ATPase or ryanodine receptor channels, both sarcoplasmic reticulum markers. The ADP-ribosyl cyclase and cADPR hydrolase activities comigrated at a molecular weight of approximately 40 kDa on SDS-PAGE. This comigration was confirmed by gel filtration chromatography. Investigation of kinetics yielded K(m) values of 30.4+/-1.5 and 695. 3+/-171.2 microM and V(max) values of 330.4+/-90 and 102.8+/-17.1 nmol/mg/h for ADP-ribosyl cyclase and cADPR hydrolase, respectively. These results suggest a possible role for cADPR as an endogenous modulator of [Ca(2+)](i) in porcine TSM cells.     

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.     

Factors affecting sedimentational separation of bacteria from blood

Rapid diagnosis of blood infections requires fast and efficient separation of bacteria from blood. We have developed spinning hollow disks that separate bacteria from blood cells via the differences in sedimentation velocities of these particles. Factors affecting separation included the spinning speed and duration, and disk size. These factors were varied in dozens of experiments for which the volume of separated plasma, and the concentration of bacteria and red blood cells (RBCs) in separated plasma were measured. Data were correlated by a parameter of characteristic sedimentation length, which is the distance that an idealized RBC would travel during the entire spin. Results show that characteristic sedimentation length of 20 to 25 mm produces an optimal separation and collection of bacteria in plasma. This corresponds to spinning a 12-cm-diameter disk at 3,000 rpm for 13 s. Following the spin, a careful deceleration preserves the separation of cells from plasma and provides a bacterial recovery of about 61 ± 5%.     

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.     

The isolation and characterisation of a platelet-specific beta-globulin (beta-thromboglobulin) and the detection of antiurokinase and antiplasmin released from thrombin-aggregated washed human platelets.

A protein fraction was isolated from the supernatant of thrombin-aggregated washed human platelets and was shown, by immunodiffusion techniques, to contain a platelet-specific beta-globulin (beta-thromboglobulin) as the major component. A molecular weight of 35 800 was determined for beta-thromboglobin from the measured sedimentation coefficient of3.0 S and Stokes radius of 2.85 nm. Beta-Thromboglobin was detected in the serum from whole blood and the supernatant of 48-h-old platelet-rich plasma and 28-day-old citrated whole blood, but not in platelet-poor plasma. The fraction containing beta-thromboglobulin was shown to possess an antiurokinase activity but was devoid of antiplasmin activity. A further fraction of approximate molecular weight 70 000 was also isolated which contained an antiplasmin but was devoid of antiurokinase activity.     

The use of fluorogenic substrates to monitor thrombin generation for the analysis of plasma and whole blood coagulation

Thrombin is central to the process of coagulation and monitoring its activity is a reliable indicator of the rate and extent of coagulation. I have employed a range of fluorogenic peptide substrates as indicators of coagulation via the formation of active thrombin. This system enabled coagulation to be monitored in a kinetic fashion, and the use of fluorescence enabled a wide range of samples to be analyzed including lyophilized plasma containing fibrin, fresh platelet-poor plasma, platelet-rich plasma, and even whole blood. Coagulation could be monitored following triggering by tissue factor, ellagic acid, or each of the proteases preceding thrombin in the coagulation network. Using this assay procedure I have investigated the anticoagulant activities of a number of compounds and the results indicate that this assay would be useful for the kinetic analysis of coagulation in various plasma preparations, or even whole blood.     

Pathogen Reduction in Human Plasma Using an Ultrashort Pulsed Laser

Pathogen reduction is a viable approach to ensure the continued safety of the blood supply against emerging pathogens. However, the currently licensed pathogen reduction techniques are ineffective against non-enveloped viruses such as hepatitis A virus, and they introduce chemicals with concerns of side effects which prevent their widespread use. In this report, we demonstrate the inactivation of both enveloped and non-enveloped viruses in human plasma using a novel chemical-free method, a visible ultrashort pulsed laser. We found that laser treatment resulted in 2-log, 1-log, and 3-log reductions in human immunodeficiency virus, hepatitis A virus, and murine cytomegalovirus in human plasma, respectively. Laser-treated plasma showed ≥70% retention for most coagulation factors tested. Furthermore, laser treatment did not alter the structure of a model coagulation factor, fibrinogen. Ultrashort pulsed lasers are a promising new method for chemical-free, broad-spectrum pathogen reduction in human plasma.     

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.     

Enzymes of snake venoms

Snakes' venom is a mixture of biologically active substances, containing proteins and peptides. A number of these proteins interact with haemostasis system components. Activators and inhibitors affecting blood coagulation and fibrinolysis systems are of special interest. Venom components can be classified into three main groups, such as procoagulants, anticoagulants and fibrinolytic enzymes according to their action. This review is focused on enzymes from Agkistrodon halys halys venom. They are thrombine-like enzyme, named Ancystron-H, flbrinogenolytic enzyme, protein C activator and platelet aggregation inhibitor. Ancystron-H is used for determination of fibrinogen level in blood plasma of patients undergoing heparin treatment and blood coagulation inhibitors accumulation. The fibrinogenolytic enzyme can be used as the instrument for protein-protein interactions in fibrinogen-fibrin system. The protein C activator is used for protein C level determination in blood plasma with different pathologies. Functions of the platelet aggregation inhibitor, belonging to disintegrins group, can be used for development of antithrombotic preparations. Information about the use of snake venoms in science and medicine is presented.