Simultaneous Addition Of Calcium Research Articles

Platelet-Based Factor VIII Activity Is Inhibited By Anti-C2 Domain Antibodies That Don’t Significantly Inhibit Factor VIII Activity in a Standard Assay

The major complication of hemophilia A treatment is the development of inhibitory antibodies against factor VIII (fVIII), causing severe bleeding in spite of infused fVIII. Most antibodies are targeted to the A2 or the C2 domain of fVIII and the degree of inhibition by these antibodies measured in clinical fVIII assays fails to predict the severity of bleeding risk. We investigated whether the degree of inhibition of platelet dependent fVIII activity by a panel of monoclonal anti-C2 domain mAbs deviated from the degree of inhibition in a commercial one-stage assay.Methods: Platelet dependent fVIII activity was measured in a defined activated platelet time (APT) assay containing fresh, density gradient-purified human platelets and fVIII deficient plasma supplemented with fVIII. Coagulation was triggered by simultaneous addition of calcium, thrombin receptor activation peptides PAR-1 and PAR-4, and factor XIa without other exogenous activators or vesicles. Results were compared to a commercial aPTT assay (Helena Laboratories, TX) and to results of a novel factor Xase assay developed to recapitulate the conditions in an APT. In the novel assay fresh, density-gradient purified platelets were activated by thrombin followed by inactivation with hirudin and incubated with fVIII and VWF, fIXa, and fX. In a second step, phospholipid vesicles, fVa and prothrombin were added to produce thrombin in proportion to the fXa generated in the first step. For comparison to the aPTT this assay was also done using purified sonicated phospholipid vesicles instead of activated platelets in the first step. The reaction was quenched with EDTA and thrombin generation was determined immediately in a thermosttated kinetics microtiter plate reader (Molecular Devices) at 250C using the chromogenic substrate S-2238 (Diapharma). A panel of 10 defined, epitope mapped mAbs against the fVIIIC2 domain was chosen in order to survey the relationship between inhibition and binding of anti-C2 antibodies.Results: The APT exhibited a log-linear relationship between fVIII and time to fibrin strand formation over a 4-log range, encompassing 0.01% - 100% plasma fVIII. Nine of 10 mAbs inhibited fVIII activity 89-96% while mAb F85 did not inhibit. There was excellent correlation for the degree of inhibition in the platelet-based factor Xase assay for 8 of 10 antibodies with F85 inhibiting the platelet Xase but not the APT and I109 inhibiting the platelet Xase assay less than the APT. In contrast, there was no correlation between inhibition of the aPTT-based assay and the platelet assay. Four mAbs did not inhibit the aPTT assay yet inhibited approximately 90% of platelet-based activity. The novel factor Xase assay, using phospholipid vesicles instead of activated platelets, showed inhibitory results that correlated to the degree of inhibition in the aPTT assay (which also utilizes purified phospholipids). Thus, the novel assay indicates that the degree of inhibition in both platelet-based and vesicle-based clotting assays is a consequence of the membrane type rather than unidentified clotting modifiers in plasma.Conclusions: We have developed a novel factor Xase assay to measure the low levels of residual fVIII activity, at physiologic concentrations, in the presence of inhibitory antibodies. Antibody inhibition of fVIII activity correlated with inhibition measured in the aPTT and APT clotting assays,. Our results confirm that the anti-C2 inhibitory antibodies have substantially different inhibition on a platelet membrane than in an aPTT-based assay and that the differing degree of inhibition is primarily due to fVIII susceptibility to inhibition when functioning on platelet membranes vs. phospholipid vesicles. These results suggest that current inhibitor assays might underestimate the frequency of significant inhibitory antibodies. DisclosuresNo relevant conflicts of interest to declare.

Effect of Ca2+ and Mg2+ ions on the sintering and spectroscopic properties of cr‐doped yttrium aluminum garnet ceramics

AbstractIn this work, we investigated the effects of Ca2+ and Mg2+ ions and annealing temperature on the spectroscopic parameters of chromium‐doped yttrium aluminum garnet ceramics (Cr:YAG). Samples were obtained with either a separate or a simultaneous addition of calcium and magnesium oxides. To achieve this, aqueous suspensions were prepared using Y2O3, Al2O3, Cr2O3, MgO, and CaO high‐purity powders as raw materials. The obtained suspensions were freeze‐granulated, pressed into pellets, debinded, and subjected to reactive sintering in vacuum at 1715°C for 6 h. Each material was annealed in air with temperatures between 1300 and 1700°C. Samples were also compared to Cr:YAG ceramics with the addition of silica as a sintering aid. All the materials obtained were then exposed to 445 nm excitation, and emission spectra in the visible and infrared wavelengths were recorded. The results showed that the emission spectra of Cr:YAG ceramics varied according to the annealing conditions: as‐sintered samples exhibited strong emissions of around 680 nm and, after air annealing, of around 1400 nm. This phenomenon is attributed to the Cr3+→Cr4+ transition. Samples doped solely with MgO exhibited the highest emission intensity in the infrared region. Thus, Mg2+ ions provided the best conversion efficiency of chromium ions.

Platelet Membranes and Activation Pathway Determine Inhibitory Activity of Anti-Factor VIII C2 Domain Antibodies

Inhibitory antibodies against factor VIII (fVIII) are the major complication of hemophilia A treatment, causing severe bleeding in spite of infused fVIII. Most antibodies are directed against the A2 or the C2 domain of fVIII and the degree of inhibition in clinical fVIII assays does not predict the severity of bleeding risk. We recently discovered that fVIII binds to a macromolecular complex including low molecular weight fibrin and the αIIbβ3integrin on membranes of thrombin-stimulated platelets rather than exposed phosphatidylserine (PS). Because clinical fVIII assays utilize PS-rich vesicles to support fVIII activity we asked whether the degree of inhibition of platelet-dependent activity by anti-C2 domain mAb’s differs from inhibition on PS-rich phospholipid vesicles.Methods: Platelet dependent fVIII activity was measured in a defined activated platelet time (APT) assay containing fresh, density gradient-purified human platelets and fVIII deficient plasma supplemented with fVIII. Coagulation was triggered by simultaneous addition of calcium, thrombin receptor activation peptides for PAR-1 and PAR-4, and either factor XIa (intrinsic pathway) or factor fXa (extrinsic pathway) without standard exogenous activators or vesicles. aPTT assays were performed on the same plasma-preparations using a commercial preparation of activator and phospholipid vesicles. Assays were performed using BBL fibrometers, electromechanical devices. A panel of 12 defined, epitope-mapped mAb’s against the fVIII-C2 domain was chosen in order to survey the relationship between inhibition and binding of anti-C2 antibodies. mAb’s at 10 µg/ml were pre-incubated with fVIII for 60 min in the absence of VWF before mixing the fVIII-antibody mixture with plasma.Results: Platelets from nine donors supported a similar, near log-linear relationship between fVIII concentration and fibrin strand formation spanning at least 10,000-fold (0.0001 - 1 unit/ml). Time to fibrin strand formation was inversely related to platelet concentration over a range of at least 1 x 106 - 2 x 108/ml. Factor XIa and factor Xa concentrations of 20 pM and 0.01 pM, respectively were chosen emulating the intrinsic and extrinsic pathways.Four mAb’s inhibited fVIII activity <10% in the aPTT assay, four others by 50-80%, with the final four in the 90-99% range. fVIII activity in the intrinsic APT had little correlation to inhibition in the aPTT assay with four mAb’s causing less inhibition of the APT and the remaining 8 antibodies having 5-10 fold more inhibition. No mAb caused inhibition that was within 2-fold for the aPTT and APT assays.For the extrinsic APT, triggered by fXa, all antibodies produced greater than 99% inhibition of fVIII activity with 10 antibodies having 2-10 fold greater inhibition than with the intrinsic pathway.Conclusions: These results show that both platelet membranes and the pathway to fVIII activation determine the degree of inhibition by anti-C2 antibodies. The inhibition of platelet-dependent fVIII activity has little correlation to inhibition of the aPTT fVIII activity. Further, anti-C2 antibodies show greater inhibition of platelet-based activity when the fVIII activation pathway is via factor Xa vs. factor XIa. Additional studies will be needed to determine whether residual fVIII activity from the intrinsic APT or extrinsic APT better correlates to bleeding risk and whether the basis for activation pathway-dependent inhibition correlates to different proteolytic cleavage sites for factor VIII. DisclosuresMeeks:Genentech: Membership on an entity’s Board of Directors or advisory committees; Grifols: Membership on an entity’s Board of Directors or advisory committees; CSL Behring: Membership on an entity’s Board of Directors or advisory committees; Bayer Healthcare: Membership on an entity’s Board of Directors or advisory committees; Biogen: Membership on an entity’s Board of Directors or advisory committees; Shire: Membership on an entity’s Board of Directors or advisory committees. Novakovic:Harvard Medical School: Patents & Royalties: Methods and Assays for Factor VIII Activity. Gilbert:Baxalta/Shire: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Harvard Medical School: Patents & Royalties: Methods and Assays for Factor VIII Activity; Grifols: Membership on an entity’s Board of Directors or advisory committees; Bayer: Membership on an entity’s Board of Directors or advisory committees, Research Funding.

EDTA-dependent pseudothrombocytopenia: further insights and recommendations for prevention of a clinically threatening artifact

Ethylenediaminetetra-acetic acid (EDTA) is widely used as anticoagulant in laboratory medicine. EDTA-dependent pseudothrombocytopenia is a rare phenomenon (i.e., around 0.1% in the general population), which is mostly due to the presence of EDTA-dependent antiplatelet antibodies that react optimally between 0°C and 4°C, recognize the cytoadhesive receptors gpIIb-IIIa, stimulate the expression of activation antigens, trigger activation of tyrosine kinase, platelet agglutination and clumping in vitro, which finally lead to a spuriously decreased platelet count. The reliable and timely identification of this artifact is essential, since there a high chance that it may be confused with other life-threatening platelet disorders, or otherwise lead to inappropriate clinical and therapeutic decision-making. Five basic criteria should be fulfilled to raise the clinical suspicion of EDTA-dependent pseudothrombocytopenia, i.e., (i) abnormal platelet count, typically <100×10(9)/L; (ii) occurrence of thrombocytopenia in EDTA-anticoagulated samples at room temperature, but to a much lesser extent in samples collected with other anticoagulants and/or kept warmed at ~37°C; (iii) time-dependent fall of platelet count in the EDTA specimen; (iv) evidence of platelet aggregates and clumps in EDTA-anticoagulated samples with either automated cell counting or microscopic analysis; (v) lack of signs or symptoms of platelet disorders. Several remedies have been proposed, such as warming the sample to 37°C or using additives or specific formulations of anticoagulants including buffered sodium citrate, heparin, ammonium oxalate, β-hydroxyethyltheophylline, sodium fluoride, CPT (trisodium citrate, pyridoxal 5'-phosphate and Tris), antiplatelet agents, potassium azide, amikacin, kanamycin or other aminoglycosides, and calcium replacement with the simultaneous addition of calcium chloride/heparin. According to available evidences, the most suitable and practical approach so far for most clinical laboratories seems, however, the recollection of blood samples using sodium citrate, CPT or calcium chloride/heparin as additives, maintaining the specimen at 37°C until the platelet count has been completed.

Microstructure development of sand-cast AZ-type magnesium alloys modified by simultaneous addition of calcium and neodymium

In this paper, microstructure of AZ-type magnesium alloys was modified by simultaneous addition of Ca and Nd. AZ alloys with added 1 wt%Ca and 4.5 wt%Nd were prepared via sand-casting. Al content of the alloy was varied in a range of 3.6–9 wt%. Microstructure observation of these alloys shows that Mg grains refine with increase in the Al content. In the microstructure, globular Al 2Nd particles are found inside and outside the Mg grains. At grain boundaries, Al 2Ca and Mg 17Al 12 are observed and their amount increases with increase in the Al content. Eutectic Mg/Mg 17Al 12 was not identified in low Al alloy, which is commonly observed in commercial AZ alloys. Thermal analyses show that the melting point is higher than AZ alloy but decreases with increase in the Al content. Quenching experiments were carried out to clarify its microstructure development characteristics. Additionally, thermal properties were measured and they are well related to microstructure characteristics.

Ignition prevention of magnesium by simultaneous addition of calcium and zirconium

The influence of simultaneous addition of calcium and zirconium on the ignition of magnesium was investigated. The ignition behaviour and ignition prevention of magnesium by calcium and zirconium were also clarified. As soon as pure magnesium melts in the air, it ignites. However, magnesium alloy containing 0.5 mass% Ca ignites continuously at a temperature around 40 K higher than the ignition temperature of pure magnesium. The continuous ignition temperature of Mg-0.5 mass% Ca alloy rises to 1020 K by the addition of 1.6 mass %Zr. The prevention of ignition is obtained by the simultaneous addition of 1.3 mass% calcium and 1.4 mass% zirconium, even at a temperature higher than 1083 K. The oxide on the surface of Mg-Ca and Mg-Ca-Zr alloys was detected as calcium oxide by AES and X-ray diffraction analyses. The content of hydrogen in molten magnesium decreases with the addition of calcium and zirconium. The rise of the ignition temperature in magnesium is due to the prevention of reaction of magnesium with oxygen by the calcium oxide film and the decrease of hydrogen in solution by the addition of calcium and zirconium.

Extracellular magnesium decreases the secretory response of rat peritoneal mast cells to compound 48/80 in vitro

Exposure of rat peritoneal must cells to magnesium in the absence of extracellular calcium resulted in a time- and dose-dependent decrease in the secretory response induced by compound 48/80. The decrease was prevented by a low extracellular concentration of calcium. Furthermore, the decreased secretory responsiveness was dose-dependently restored by the addition of calcium to the cells simultaneously with compound 48/80. Preincubation with magnesium also inhibited antigen-induced histamine secretion in a dose-dependent manner. This was reversed by the simultaneous addition of calcium and the secretory stimulus. A dose-dependent decrease in antigen induced histamine secretion that was reversed by calcium was also observed. Exposure of the mast cells to magnesium for 15 min resulted in a parallel decrease in histamine secretion and in the cellular content of 45Ca 2+. These observations suggest that magnesium may decrease the secretory response by displacing the cellular calcium which is utilized in stimulus-secreton coupling.

Activation of chitin synthetase from Phycomyces blakesleeanus by calcium and calmodulin

Levels of basal chitin synthetase in cell-free extracts from Phycomyces blakesleeanus were reduced by breakage of cells in the presence of EDTA or EGTA. Addition of Ca2+ to these extracts activated chitin synthetase. Maximal activation was obtained after 2 h at a Ca2+ concentration of 2–5 mM. Activation by calcium was not reduced by any protease inhibitor tested but benzamidine, whereas the weak proteolytic activity of the extracts was inhibited by antipain. Larger levels of chitin synthetase activation were obtained by the simultaneous addition of calcium and calmodulin in most, but not all extracts. This further activation by calmodulin was prevented by TFP. ATP or cAMP did not stimulate activation by calcium or calcium-calmodulin.

Effect of magnesium on the calcium-dependent transient kinetics of sarcoplasmic reticulum ATPase, studied by stopped flow fluorescence and phosphorylation.

At pH 7 and 20 degrees C and in the absence of potassium and magnesium, the intrinsic fluorescence rise after addition of calcium to a calcium-deprived enzyme was monoexponential. On the other hand, when the calcium-deprived enzyme was preincubated with magnesium, this fluorescence rise was clearly biphasic at high calcium concentrations. For a constant magnesium concentration (higher than millimolar), the rate constant of the slow phase and the amplitude of the fast phase rose for the same range of calcium concentrations, between pCa 5 and 4. At pH 6, fluorescence signals were monophasic even with 20 mM Mg2+. We also found that at pH 7, phosphorylation of the enzyme after simultaneous addition of calcium, magnesium, and ATP was faster when sarcoplasmic reticulum was originally calcium-deprived in the presence of magnesium. At pH 6, on the contrary, preincubation with magnesium did not influence the phosphorylation time course. It is concluded that (a) magnesium produces an enzyme conformation which is able to react with calcium and ATP as required by the kinetics of the ATPase reaction; (b) the magnesium-enzyme complex presents one readily accessible site of relatively low affinity (Kd congruent to 25 microM) for calcium; (c) occupancy of this site by calcium triggers a relatively slow (4-6 s-1) conformational change unmasking a second high affinity site; (d) interaction between and occupancy of these two calcium sites occur with positive cooperativity and yield enzyme activation; and (e) the kinetic difference between the low time constant of the second component of fluorescence rise following addition of calcium (in the absence of ATP) and the relatively fast phosphorylation obtained in the same conditions (but in the presence of ATP) is attributed to an Mg2+-dependent accelerating effect of ATP on enzyme isomerization.

Evidence for the regulation of the branched chain alpha-keto acid dehydrogenase multienzyme complex by a phosphorylation/dephosphorylation mechanism.

The regulation of the branched chain alpha-keto acid dehydrogenase complex by covalent modification was investigated in rat liver mitochondria. Depletion of intramitochondrial calcium and magnesium caused an inactivation of the branched chain alpha-keto acid dehydrogenase complex. Following inactivation of the branched chain complex, addition of calcium or magnesium ions separately to incubations of mitochondria only partially reactivated the enzyme complex. However, simultaneous addition of calcium and magnesium activated the branched chain enzyme complex rapidly and nearly completely. Mitochondrial incubations were performed in the presence of [32P]phosphate under conditions known to activate or to inactivate the branched chain alpha-keto acid dehydrogenase complex. Evidence demonstrating that [32P]-phosphate was incorporated into two major protein bands separated in sodium dodecyl sulfate-polyacrylamide gels of the mitochondrial incubations is presented. Migration of the labeled mitochondrial protein bands in the gel system corresponded exactly to the migration of the alpha subunit of the purified heart-derived pyruvate dehydrogenase (decarboxylase, E1) and the alpha subunit of the purified kidney-derived branched chain alpha-keto acid dehydrogenase (decarboxylase, E1). Furthermore, when the measured activity of the branched chain complex was minimized, the amount of [32P]phosphate incorporated into the alpha chain of the branched chain enzyme was maximal. Conversely, incubation conditions which activated maximally the enzyme complex minimized the [32P]phosphate incorporation into the alpha subunit of the branched chain dehydrogenase.

Effects of extracellular calcium ions on the elevation of guanosine 3′, 5′-monophosphate level in neuroblastoma cells

The levels of guanosine 3′,5′-monophosphate in neuroblastoma N1E115 cells were increased 10- to 50-fold by depolarizing agents (110 mM KCl and 1 mM ouabain), 10 μM ionophore A23187, 1 mM sodium azide, 1 mM N-methyl- N′-nitro- N-nitrosoguanidine, 2 mM carbamylcholine and 10 μM prostaglandin E 1 in the medium containing 1.8 mM calcium. Removal of calcium from the incubation medium and addition of 3 mM ethyleneglycol-bis( ß-aminoethyl ether)- N, N, N′, N′-tetraacetic acid abolished responses of guanosine 3′,5′-monophosphate to depolarizing agents and reduced the basal level of guanosine 3′,5′-monophosphate to 10%. In contrast, guanosine 3′,5′-monophosphate levels still increased in the calcium-free medium in response to carbamylcholine, prostaglandin E 1 and ionophore A23187, although the absolute responses to these agents were reduced by 60–80%. The simultaneous addition of calcium with depolarizing agents to the calcium-free medium restored the responses to these agents, while the addition of calcium with carbamylcholine, prostaglandin E 1 or ionophore A23187 did not restore the responses. The combination of carbamylcholine with ionophore A23187 induced the same amount of increase of guanosine 3′,5′-monophosphate as that elicited by ionophore A23187 alone. In contrast, the combination of 110 mM KCl with carbamylcholine or ionophore A23187 resulted in a greater increase of guanosine 3′,5′-monophosphate than that induced by carbamylcholine, ionophore A23187 or 110 mM KCl alone, but less than additive.

Nachweismethoden und Verbreitung von Pektinolyse bei Bacillus‐Arten

AbstractUsing 4 different methods, a total of 126 Bacillus strains representing 33 different species, was examined for their ability to degrade methoxylated and demethylated pectin. The highest number of pectinolytic strains (35) was found with a newly developed, tubed polypectate‐gel, which gave complete liquefaction upon pectinolysis. The pectinolytic activity appeared to be restricted to 8 recognized species belonging to 5 groups: The B. maceranspolymyxa group, the B. licheniformis‐pumilius‐subtilis group, B. pantothenticus, B. alvei, and B. lentus. However, not all strains of a single species revealed a positive pectinolytic activity. Thus, only a part of the strains of the second and fifth group were found to be pectinolytic. Pectinolysis with B. alvei (ILM 21, JO 42) and B. lentus (CCM 35, CCM 2214) was recorded not until a prolonged incubation in the tubed pectate‐gel was used.Pectinolysis of selected B. licheniformis and B. pantothenticus strains was stimulated markedly by ammonium‐ and phosphat‐ions at various pH levels. The simultaneous addition of calcium and phosphate to the medium decreased the intensity of pectinolysis clearly. Calcium, if added solely, stimulated pectinolytic activity of B. pantothenticus strains only at a reduced concentration.