Prothrombin Time Of Human Plasma Research Articles

Effect of in-vitro addition of sulfated xylans, glucosans or chondroitins on the prothrombin time of human plasma and on the enhancement of activation of glutamic plasminogen by tissue plasminogen activator

Three each of sulfated xylans, glucosans or chondroitins were investigated. A comparison of the infrared analysis of the native and sulfated amylopectin showed the presence of peaks corresponding to 800/cm for S-O and 1200-1300/cm for S=O only in the sulfated amylopectin and earlier results using C NMR showed sulfation of hyroxymethyl groups (C-6) of amylopectin. Based upon % sulfate the three classes of compounds following sulfation contained more than one sulfate group per sugar unit. All of them exhibited significant in-vitro anticoagulant property by inhibiting thrombin generation at very low concentrations. In general sulfated xylans or glucosans were better anticoagulants than the chondroitins. The results of in-vitro studies of the activation of glutamic plasminogen (Glu-Plg) by tissue plasminogen activator (t-PA) using 0.05 mol/l Tris buffer (pH 7.35) containing physiological concentration of NaCl (0.9%), showed that oat spelts xylan sulfate and amylopectin sulfate gave a 20-fold enhancement of the activation in a synergistic manner when used in combination with 32.4 mmol/l of lysine. The mechanism of enhancement was investigated by dilution studies. Sulfated amylopectin interacted with Glu-Plg but not with t-PA and lysine which interacted with both Glu-Plg and t-PA enhanced the activation in a synergistic manner using low concentrations of t-PA.

Blocking the Initiation of Coagulation by RNA Aptamers to Factor VIIa

The tissue factor/factor VIIa complex is thought to be the primary initiator of most physiologic blood coagulation events. Because of its proximal role in this process, we sought to generate new inhibitors of tissue factor/factor VIIa activity by targeting factor VIIa. We employed a combinatorial RNA library and in vitro selection methods to isolate a high affinity, nuclease-resistant RNA ligand that binds specifically to coagulation factor VII/VIIa. This RNA inhibits the tissue factor-dependent activation of factor X by factor VIIa. Kinetic analyses of the mechanism of action of this RNA suggest that it antagonizes factor VIIa activity by preventing formation of a functional factor VII/tissue factor complex. Furthermore, this RNA significantly prolongs the prothrombin time of human plasma in a dose dependent manner, and has an in vitro half-life of approximately 15 h in human plasma. Thus, this RNA ligand represents a novel class of anticoagulant agents directed against factor VIIa.

Inhibition of Serine Proteases of the Blood Coagulation System by Squash Family Protease Inhibitors

Squash family inhibitors are the smallest protein serine protease inhibitors, being composed of approximately 30 amino acid residues. We isolated 8 squash family inhibitors from the seeds of bitter gourd, squash, gourd and luffa and examined their effect on serine proteases of the blood coagulation system. Five of them prolonged the activated partial thromboplastin time of human plasma to various extents, but three did not. Only Momordica charantia (bitter gourd) trypsin inhibitor-II prolonged the prothrombin time of human plasma. All inhibitors inhibited the amidolytic activities of factor XIIa, plasma kallikrein, factor Xa, but did not inhibit significantly those of factor XIa, factor IXa, factor VIIa, and thrombin. Ki values for factor XIIa, plasma kallikrein, and factor Xa were in the order of 10(-6)-10(-9), 10(-4)-10(-5), and 10(-4)-10(-6)M, respectively. The prolongation of the activated partial thromboplastin time by inhibitors appeared to correspond to their inhibitory potencies for factor XIIa. Momordica charantia trypsin inhibitor-II, which has the strongest inhibitory potency toward the amidolytic activity of factor Xa, with a Ki value 10-100 times smaller than those of other inhibitors, inhibited the activation of factor X by factor VIIa-tissue factor complex or factor IXa, while others did not.

Amino Acid Sequence and Inhibitory Activity of Rhesus Monkey Tissue Factor Pathway Inhibitor (TFPI): Comparison with Human TFPI1

Rhesus monkey cDNA for tissue factor pathway inhibitor (TFPI) was cloned by means of the reverse transcriptase-polymerase chain reaction, using liver mRNA, and its nucleotide sequence was determined by sequencing five independent clones. Monkey TFPI was found to have a signal peptide of 28 amino acid residues and to be a mature protein of 276 amino acid residues, in which three and seventeen amino acid residue substitutions compared to human TFPI were found, respectively. All the cysteine residues, three putative carbohydrate-linked asparagine residues, and the P1 amino acid residues of each of the three Kunitz inhibitor domains were conserved in the two species. Recombinant monkey TFPI (rTFPI) was isolated from the culture medium of transformed Chinese hamster ovary cells. Amino acid sequence analysis and immunoblotting analysis, using polyclonal and monoclonal antibodies, showed that the carboxyl-terminal basic part of Rhesus monkey rTFPI had been truncated. The inhibitory activity of monkey rTFPI was compared with that of human rTFPI without the carboxyl-terminal basic part. The prothrombin time of human plasma was slightly more prolonged by the addition of monkey rTFPI than by that of human rTFPI. However, no significant differences were found between the potencies of human and monkey rTFPI as to the inhibition of factor Xa and tissue factor-factor VIIa complex.

Immunotoxicology of brown recluse spider ( Loxosceles reclusa) venom

A purified toxic protein from Loxosceles reclusa venom was assayed for its in vitro effects on the human immunological and blood clotting systems. The toxin caused inhibition of neutrophil chemotaxis, depletion of serum hemolytic complement, prolongation of the activated partial thromboplastin time and depletion of clotting factors XII, XI, IX and VIII by an average of 44% in human plasma. The prothrombin time of human plasma was also prolonged by 1.5 – 2.0 sec. No effect of the purified toxin was observed on microbicidal ability of neutrophils, the release of enzymes from neutrophils or the adherence of neutrophils to glass beads.

Cationic antibiotics and phospholipase C as tools in the study of phospholipid structure and function. I. Inhibition of the in vitro clotting system by cationic antibiotics.

The one-stage prothrombin time of human plasma was prolonged by the addition to the thromboplastic mixture of either colistin sulfate or polymyxin B. Colistimethate sodium, a derivative of colistin in which the amino groups of the cyclic polypeptide antibiotic are substituted, did not show this effect. The inhibition was greater if the antibiotics were incubated with thromboplastin in the absence of Ca++. In the Hicks–Pitney modification of the thromboplastin generation time test, the two polyamine antibiotics reduced the amount of thromboplastic activity generated. Generation, however, appeared to be initiated sooner. It is postulated that these actions by the antibiotics are the results of modifications in the zeta potentials of the thromboplastic micelles.

Erythrocyte extract (hemolysate) and tissue thromboplastin: their action on heparin activity.

SummaryThe inhibitory action of heparin on prothrombin consumption of normal plate-let-poor human plasma in a silicone-coated test tube can be partially overcome by increasing amounts of hemolysate. Likewise, prothrombin time of human plasma to which a fixed amount of heparin has been added can be shortened and brought to a constant but higher value than normal by adding increasing amounts of tissue thromboplastin.

THE INHIBITION OF BLOOD CLOTTING BY PHOSPHATE ESTERS OF VITAMINS K AND E

Vitamin K1phosphate was incubated with thromboplastin and the mixture used for the determination of the one-stage prothrombin time of human plasma. No change in thromboplastic activity was observed. The thrombin times of an artificial clotting system composed of a 1% fibrinogen solution were prolonged following incubation with vitamin K1phosphate. The inhibitory activity of vitamin K1phosphate, alpha-tocopherol phosphate, and menadiol sodium diphosphate on the one-stage prothrombin and the clotting times of rat and human plasmas was followed. Vitamin K1phosphate and alpha-tocopherol phosphate showed greatest activity while Synkavite showed only slight activity. At low concentrations of the phosphate derivatives the inhibition was found to be a linear function of the log of the prothrombin and clotting time. It is suggested that the phosphorylated derivatives inhibit the clotting process by interfering with the polymerization of fibrin.

THE INHIBITION OF BLOOD CLOTTING BY PHOSPHATE ESTERS OF VITAMINS K AND E

Vitamin K1phosphate was incubated with thromboplastin and the mixture used for the determination of the one-stage prothrombin time of human plasma. No change in thromboplastic activity was observed. The thrombin times of an artificial clotting system composed of a 1% fibrinogen solution were prolonged following incubation with vitamin K1phosphate. The inhibitory activity of vitamin K1phosphate, alpha-tocopherol phosphate, and menadiol sodium diphosphate on the one-stage prothrombin and the clotting times of rat and human plasmas was followed. Vitamin K1phosphate and alpha-tocopherol phosphate showed greatest activity while Synkavite showed only slight activity. At low concentrations of the phosphate derivatives the inhibition was found to be a linear function of the log of the prothrombin and clotting time. It is suggested that the phosphorylated derivatives inhibit the clotting process by interfering with the polymerization of fibrin.

ANTICOAGULANT EFFECT OF CHARONINSULFURIC ACID

The anticoagulant effect of charoninsulfuric acid, a polysaccharide sulfuric acid obtained from the mucus of [Charonia lampas] was investigated both in vivo and in vitro. The results obtained were as follows:1) Charoninsulfuric acid is a strong anticoagulant. The clotting time of whole blood was prolonged slightly by adding only 1γ charoninsulfuric acid to 1ml. of rabbit blood or 0.1γ to 1ml. of human blood. The more its concentration increased, the longer the clotting time became, and in concentration of over 50γ in 1ml. of rabbit or human blood the clot was not detected over 24 hours.when charoninsulfuric acid was intravenously injected (1 mg per Kg of body weight), the clotting time of rabbit whole blood was prolonged and reached the maximal level after 15 minutes. Then, the clotting time decreased gradually to normal level within 1 hour or so.2) In experiments in vitro, charoninsulfuric acid produced no remarkable prolongation of prothrombin time of rabbit plasma by adding charoninsulfuric acid up to 20γ per ml. of blood, while prothrombin time of human plasma was prolonged by adding only 0.05γ per ml. of blood and the more the concentration increased, the more the prothrombin time was prolonged.Prothrombin time of rabbit plasma measured before and after the intravenous injection of charoninsulfuric acid (1mg. per Kg. of body weight) revealed no remarkable change.3) Stable factor (SPCA) was not affected by charoninsulfuric acid both in vitro and in vivo.4) Charoninsulfuric acid has not only anti-labile factor activity but also anti-prothrombin activity.5) Antithromboplastic activity of charoninsulfuric acid was demonstrated by thromboplastin generation test both of Matsuoka-Yamanaka and of Biggs-Douglas.6) Thromboplastin dilution curve of rabbit plasma, which means the prothrombin time measured with serial twofold diluted rabbit brain extract, was also prolonged after the intravenous injection of charoninsulfuric acid (1mg. per Kg. of body weight).7) When charoninsulfuric acid was intravenously injected (1mg. per Kg. of body weight), leucocyte and platelet count of rabbit were temporarily decreased, while the erythrocyte count showed no remarkable change.