A new rheological method to measure fluidity change of blood during coagulation : application to in vitro evaluation of anticoagulability of artificial materials

In order to attempt in vitro evaluation of antithrombogenecity for materials of artificial blood vessel tube, a new type of rheometer was developed. The rheometer originally consists of a cylindrical tube suspended from a torsion wire and filled with blood. The tube is excited in torsional oscillation and subsequent damped oscillation is observed. The apparatus can sensitively follow the change of fluidity during coagulation of blood. The damped oscillation curves during coagulation for fibrinogen - thrombin solution and blood put in a cylindrical tube made of the artificial material were measured. For fibrinogen - thrombin solution with lower fibrinogen and thrombin concentrations, the values of logarithmic damping factor (LDF) during coagulation increased and then decreased through a maximum. For blood and fibrinogen-thrombin solution with the higher concentrations of fibrinogen and thrombin, LDF monotonously decreased with the progress of coagulation. With a glass tube, the decrease of LDF for whole blood taken without anticoagulant rapidly occurred within about 15 min after sampling, while, with expanded polytetrafluoroethylene (EPTFE; Goar tex) and polydimethylsiloxane (Silastic) tubes, the decrease of LDF proceeds over 40-60 min. The present method is probably available for in vitro evaluation of anticoagulability or antithrombogenecity of artificial materials.     

Procoagulant activity of collagen. Effect of difference in type and structure of collagen

Procoagulant activities of different types and structures of collagen were examined. Collagens used were types I (including its methylated and succinylated forms), II, III, IV and V. Each collagen was coated on an inner surface of a glass tube. The change of fluidity during coagulation of blood in the tube was measured by means of a new rheological technique. For monomeric collagen, the procoagulant activity of the succinylated form (negatively charged) was higher than that of the methylated form (positively charged). The procoagulant activity of type IV (dry) was lower than that of other types of collagen. For fibrillar collagens, the initiation of coagulation for type V (non-banded) was fairly delayed compared to those for types I, II and III (banded). An increase in water content in both monomeric and fibrillar forms promoted procoagulant activity. For most of the collagen forms, the addition of factor XII inhibitor (Polybrene) to blood brought about a remarkable delay of the initiation of coagulation, suggesting that the activation of factor XII on the collagen surface is one of main factors governing procoagulant activity. In addition, our data suggest that large numbers of adherent platelets to the collagen surface promote activation of the intrinsic coagulation system.     

Influence of age and hematocrit on the coagulation of blood

To examine the effects of age, hematocrit and the daily variation in hematocrit on coagulation of blood, the time of onset of coagulation (Ti) of whole blood obtained from donors including normal subjects and patients was measured by means of a rheological technique. The Ti value of recalcified blood decreased with an increase in age, but in donors aged 65 years or more (the elderly), the Ti value was almost independent of age. The Ti value for blood obtained from the elderly was significantly lower at lower hematocrit levels, but that for blood obtained from young donors was almost independent of hematocrit. The daily variation in hematocrit in individuals was small (maximum variation: about 4%), and the variation had little effect on the Ti value. However, a slight increase in hematocrit was considered to bring about a significant increase in viscosity at lower shear rates. Therefore, it is suggested that a slight increase in hematocrit under stagnant flow conditions is one of major risk factors for venous thrombogenesis, especially in the elderly.     

Microscopic velocimetry with a scaled-up model for evaluating a flow field over cultured endothelial cells

A microscopic velocimetry technique for evaluating the flow field over cultured endothelial cells was developed. Flow around a cell model scaled up by a factor of 100 was visualized by using an optical microscope and was quantified by using particle-tracking velocimetry. Wall shear stress on the model surface was determined from a two-dimensional velocity field interpolated from measured velocity vectors. Accuracy of the velocimetry was verified by measuring the flow over a sinusoidal cell model that had a wall shear stress profile analytically determined with linear perturbation theory. Comparison of the experimental results with the analytical solution revealed that the total error of the measured wall shear stress was 6 percent.     

Role of Synaptophysin in Exocytotic Release of Dopamine from Xenopus Oocytes Injected with Rat Brain mRNA

1. The role of synaptophysin in the exocytotic release of dopamine (DA) was examined in Xenopus laevis oocytes injected with rat brain mRNA.2. The mRNA-injected oocytes showed DA uptake which depended on the incubation time and external DA concentrations.3. Stimulation with KCl (10–50 mM) of mRNA-injected oocytes preloaded with DA evoked external Ca²⁺-dependent release of DA. The noninjected and water-injected oocytes did not produce uptake of DA and stimulation-evoked release of DA.4. The high-KCl (50 mM)-stimulated release of DA decreased in the oocytes injected with rat brain mRNA together with antibody to synaptophysin.5. Immunoblot analysis demonstrated that synaptophysin was expressed in the brain mRNA-injected oocytes but not in the noninjected and water-injected oocytes.6. Thus, uptake and release machinery similar to native dopaminergic nerve terminals was expressed in Xenopus oocytes by injecting mRNA-extracted from the rat brain, and synaptophysin may play a role in the exocytotic release of DA.     

Characterizations of critical processes in liquid-liquid phase separation of the elastomeric protein-water system: microscopic observations and light scattering measurements

Biological self-assembly process of tropoelastin in an extracellular space, viewed as a key step of the elastogenesis, can be mimicked by the temperature-dependent coacervation of the elastin-related polypeptide-water system. Early and late stages of the phase separation behavior of the bovine neck ligamental alpha-elastin-water system were examined respectively by the laser light scattering photometry and phase contrast microscopy. Changes in the hydrodynamic size of molecular assemblies and visible microcoacervate droplet size were traced as a function of the concentration of alpha-elastin and temperature. Near the critical point, alpha-elastin concentration of 0.11 mg/mL and temperature of 21.5 degrees C, the phase separation was initiated after fast increase of the hydrodynamic size of primary aggregates as scattering particles and followed by the appearance of larger microcoacervate droplets with a broad size distribution. Whereas in the off-critical region, slow decrease of the hydrodynamic size of primary particles induced phase separation with smaller droplets of a narrow size distribution. Observation of the phase separation processes in the alpha-elastin-water system with metal chlorides and hydrophobic synthetic model polypeptide-water system indicated that the fast and slow molecular assembly processes were based on the fundamental hydrophobic interactions and involvements of electrostatic interactions between charged amino acid residues, respectively.     

Molecular characterization of a novel yeast cell-wall acid phosphatase cloned from Kluyveromyces marxianus

A novel Kluyveromyces marxianus gene that encodes an acid phosphatase, Pho610, was cloned in Saccharomyces cerevisiae. The deduced amino acid sequence was distinct from S. cerevisiae phosphatases but similar to some fungal enzymes. A peculiar feature of the sequence is that it has hydrophobic stretches both at the N- and C-termini, which is a characteristic of the precursors of glycosylphosphatidylinositol(GPI)-anchored proteins. When the gene was expressed in S. cerevisiae, the active enzyme was recovered in the periplasmic fraction by glucanase digestion. The Pho610 polypeptide was highly glycosylated and a significant portion was covalently linked to the cell-wall glucan. The enzyme was secreted when the C-terminal region was truncated to remove the GPI signal. Therefore, Pho610 is a novel cell-wall protein having an enzyme activity.     

Characteristic interaction of Ca2+ ions with elastin coacervate: Ion transport study across coacervate layers of α-elastin and elastin model polypeptide, (Val-Pro-Gly-Val-Gly)n

Ion transport characteristics across a macrocoacervate layer membrane composed of aqueous elastin model polypeptides with a specific repeating pentapeptide sequence, H-(Val-Pro-Gly-Val-Gly)_n-Val-OMe (n ≥ 40), were investigated. Transmembrane potential responses for NaCl, MgCl₂, and CaCl₂ concentration-cell systems were measured and examined systematically by comparing with those across a coacervate membrane composed of bovine neck ligamental α-elastin. In the case of the NaCl and MgCl₂ systems, potential responses across these protein liquid membranes were different noticeably from each other depending upon the molecular structure with and without charged peptide side chains, whereas in the CaCl₂ systems the transmembrane potential responses across the noncharged polypentapeptide coacervate membrane were comparable with those across the α-elastin coacervate membrane carrying both the positively and negatively charged amino acid residues as an amphoteric ion-exchange membrane. These results indicated that mechanisms of major Ca²⁺ ion transport are based on the specific and selective interactions with electrically neutral sites of elastin, such as the polypentapeptide backbone chain. © 1996 John Wiley & Sons, Inc.     

Treatment with cyclophosphamide reverses acquired resistance to collagen arthritis subsequent to recovery from passive arthritis

Passive arthritis induced by anticollagen antibody was a mild, transient disease from which the animals normally recovered and the rats that had recovered from passive arthritis were resistant to develop a second phase of arthritis following a second administration of anticollagen antibody or the subsequent challenge with type II collagen. However, treatment of the recovered rats with cyclophosphamide (CY) shortly before a second administration of a serum concentrate induced a second episode of acute polyarthritis in 82% of the rats. In addition, CY treatment without further administration of a serum concentrate induced a recurrence of arthritis in 25% of the recovered rats. Similarly, treatment of the recovered rats with CY shortly before the challenge with type II collagen induced a second episode of arthritis in 83% of the rats. However, treatment with CY shortly before the challenge was unable to restore the suppressed humoral response to type II collagen. These results provide evidence that the resistant state is mediated, at least in part, by CY-sensitive events.