Principles of dielectric blood coagulometry as a comprehensive coagulation test.

Dielectric blood coagulometry (DBCM) is intended to support hemostasis management by providing comprehensive information on blood coagulation from automated, time-dependent measurements of whole blood dielectric spectra. We discuss the relationship between the series of blood coagulation reactions, especially the aggregation and deformation of erythrocytes, and the dielectric response with the help of clot structure electron microscope observations. Dielectric response to the spontaneous coagulation after recalcification presented three distinct phases that correspond to (P1) rouleau formation before the onset of clotting, (P2) erythrocyte aggregation and reconstitution of aggregates accompanying early fibrin formation, and (P3) erythrocyte shape transformation and/or structure changes within aggregates after the stable fibrin network is formed and platelet contraction occurs. Disappearance of the second phase was observed upon addition of tissue factor and ellagic acid for activation of extrinsic and intrinsic pathways, respectively, which is attributable to accelerated thrombin generation. A series of control experiments revealed that the amplitude and/or quickness of dielectric response reflect platelet function, fibrin polymerization, fibrinolysis activity, and heparin activity. Therefore, DBCM sensitively measures blood coagulation via erythrocytes aggregation and shape changes and their impact on the dielectric permittivity, making possible the development of the battery of assays needed for comprehensive coagulation testing.

Site-Selective RNA Activation by Acridine-Modified Oligodeoxynucleotides in Metal-Ion Catalyzed Hydrolysis: A Comprehensive Study.

Various types of acridine were conjugated to DNA and used for site-selective RNA scission together with another unmodified DNA and a Lu(III) ion. The target phosphodiester linkage in the substrate RNA was selectively and efficiently activated, and was hydrolyzed by the free Lu(III) ion. Among the investigated 14 conjugates, the conjugate bearing 9-amino-2-isopropoxy-6-nitroacridine was the best RNA-activator. Systematic evaluation of the RNA-activating ability of the acridines showed that (1) the acridines act as an acid catalyst within the RNA activation, (2) the amino-group at the 9-position of acridine is essential to modulate the acidity of acridine, (3) the electron-withdrawing group at the 3-position further enhances the acid catalysis, and (4) the substituent at the 2-position sterically modulates the orientation of acridine-intercalation favorably for the catalysis. Moreover, it is revealed that the opposite base of acridine does not inhibit direct interaction of acridine with the target phosphodiester linkage.

Simultaneous assessment of blood coagulation and hematocrit levels in dielectric blood coagulometry.

BACKGROUND: In a whole blood coagulation test, the concentration of any in vitro diagnostic agent in plasma is dependent on the hematocrit level but its impact on the test result is unknown. OBJECTIVE: The aim of this work was to clarify the effects of reagent concentration, particularly Ca2+, and to find a method for hematocrit estimation compatible with the coagulation test. METHODS: Whole blood coagulation tests by dielectric blood coagulometry (DBCM) and rotational thromboelastometry were performed with various concentrations of Ca2+ or on samples with different hematocrit levels. DBCM data from a previous clinical study of patients who underwent total knee arthroplasty were re-analyzed. RESULTS: Clear Ca2+ concentration and hematocrit level dependences of the characteristic times of blood coagulation were observed. Rouleau formation made hematocrit estimation difficult in DBCM, but use of permittivity at around 3 MHz made it possible. The re-analyzed clinical data showed a good correlation between permittivity at 3 MHz and hematocrit level (R2=0.83). CONCLUSIONS: Changes in the hematocrit level may affect whole blood coagulation tests. DBCM has the potential to overcome this effect with some automated correction using results from simultaneous evaluations of the hematocrit level and blood coagulability.

Design of phosphoramidite monomer for optimal incorporation of functional intercalator to main chain of oligonucleotide.

Chirally pure phosphoramidite monomers bearing 9-amino-6-chloro-2-methoxyacridine were synthesized from D- or L-threoninol and omega-aminocarboxylic acid, and incorporated into oligonucleotides. These acridine-DNA conjugates formed stable duplexes with complementary RNA because of intercalation of the acridine to DNA/RNA heteroduplexes. The stability of duplexes was not very dependent on either the chirality of the central carbon bearing the acridine or the length of the side chain. However, the ability for site-selective activation of the phosphodiester linkage in front of the acridine, which induced Lu(III)-promoted RNA scission, was strongly dependent on these two factors. The largest activation was achieved when the monomer unit was prepared from L-threoninol and 4-aminobutyric acid and the acridine was bound to the amino group. By attaching the more acidic 9-amino-2-methoxy-6-nitroacridine to this optimized scaffold, a quite effective acridine-DNA conjugate for site-selective RNA scission was obtained.

Simultaneous use of highly acidic acridine and rigid chiral linker for efficient site-selective RNA scission.

To the middle of oligonucleotide, 9-amino-2-methoxy-6-nitroacridine (pKa = 8.8) and 9-amino-6-chloro-2-methoxyacridine (pKa = 10.5) were tethered through three linkers, and the abilities of these conjugates for site-selective activation of RNA (inducing site-selective scission by Lu(III)) were compared. The RNA-activating ability was strongly dependent on both the acidity of acridine and the structure of linker. Combination of highly acidic acridine and rigid chiral linker leads to unprecedented efficient site-selective RNA activation.

Conjugation of various acridines to DNA for site-selective RNA scission by lanthanide ion.

Three types of DNA conjugates having 9-acridinecarboxamide, 9-aminoacridine, and 9-amino-6-chloro-2-methoxyacridine at the 5'-ends were synthesized and used for site-selective RNA scission together with another unmodified DNA and Lu(III) ion. The target phosphodiester linkages in the substrate RNA were selectively and efficiently activated and were hydrolyzed by free Lu(III) ion. The conjugate bearing 9-amino-6-chloro-2-methoxyacridine was the most active. However, its duplex with the substrate RNA was almost as stable as that of the 9-aminoacridine-bearing conjugate, which was much less active for the RNA activation. The 9-acridinecarboxamide-bearing conjugate was only marginally active. The substituents on the acridine groups in these conjugates positively participate in the present RNA activation, probably by fixing the orientation of the acridine rings.

Metal ion-induced site-selective RNA hydrolysis by use of acridine-bearing oligonucleotide as cofactor.

New types of noncovalent ribozyme-mimics for site-selective RNA scission are prepared by combining metal ions with oligonucleotides bearing an acridine. Lanthanide(III) ions and various divalent metal ions (Zn(II), Mn(II), Cu(II), Ni(II), Co(II), Mg(II), and Ca(II)) are employed without being bound to any sequence-recognizing moiety. The modified oligonucleotide forms a heteroduplex with the substrate RNA, and selectively activates the phosphodiester linkages in front of the acridine. As a result, these linkages are preferentially hydrolyzed over the others, even though the metal ions are not fixed anywhere. The scission is efficient under physiological conditions, irrespective of the sequence at the target site. Site-selective RNA scission is also successful with the combination of an oligonucleotide bearing an acridine at its terminus, another unmodified oligonucleotide, and the metal ion. In a proposed mechanism, the acridine pushes the unpaired ribonucleotide out of the heteroduplex and changes the conformation of RNA at the target site for the sequence-selective activation.