Comparison of soluble and placental transferrin receptors as standards for the determination of soluble transferrin receptor in humans.

Transferrin receptor is a transmembrane protein that mediates iron transport from blood into cells. The extracellular part of this receptor circulates in blood as soluble transferrin receptor (sTfR) and the immunological determination of this parameter is widely used in clinical practice. This study aimed at comparing the properties of sTfR and placental TfR (pTfR) and to evaluate the validity of pTfR as a standard for the determination of sTfR in human serum. sTfR and pTfR were studied by immunofluorescent assay and fast protein liquid chromatography (FPLC) gel filtration. Serum sTfR levels were calculated using sTfR or pTfR as a standard. The immunological activity of pTfR was lower than that of sTfR in all anti-TfR monoclonal antibody pairs. Upon FPLC gel filtration, pTfR eluted in a void volume of the column as a protein with a molecular weight (MW) of >1500 kDa, whereas the MW of sTfR corresponded to 237 kDa. This could be a result of micelle formation by pTfR because of its hydrophobic intracellular part. The serum sTfR levels calculated against sTfR were 2.5 times lower than those calculated against pTfR. Serum sTfR levels are overestimated when pTfR is used as the standard.

[Clinical significance of determining the soluble transferrin receptor]. / Klinicheskoe znachenie opredeleniia rastvorimogo transferrinovogo retseptora.

The aim of the study was to work out a method for determining the soluble transferrin receptor (TfR) on the basis of the direct immune-enzyme analysis, including its dynamic testing in patients with anemia of various etiologies. Reagents, needed for the above analysis, i.e. TfRs and antibodies to them, were obtained to implement the set goal. TfR was isolated by Kanevsky's affine chromatography from the homogenate of patients. The thus isolated TfR was used to immunize and to obtain the monoclonal antibodies. The conjugate of horseradish peroxidase (HP) and antibodies to TfR were made use of to determine the quantity of bound antibodies. One type of antibodies were immobilized in plates of the "Nunk" company (Denmark), and the other type of monoclonal antibodies were conjugated with HP. A reliably higher TfR in iron-deficiency anemia was shown during the determination of the TfR level in 36 donors and 266 patients. After a conducted ferrotherapy, the TfR level went down approaching the normal value. The TfR level was related with a disease stage and activity of the prolipherative process in cases of autoimmune hemolytic anemia, B12-folate-dependent anemia, lympho- and myeloprolipherative diseases and in oncology patients. The elaborated method opens up new possibilities for the differential diagnosis of anemia and provide for objective criteria of a conducted therapy.

Analysis of the activated partial thromboplastin time test using mathematical modeling.

Activated partial thromboplastin time (APTT) is a laboratory test for the diagnosis of blood coagulation disorders. The test consists of two stages: The first one is the preincubation of a plasma sample with negatively charged materials (kaolin, ellagic acid etc.) to activate factors XII and XI; the second stage begins after the addition of calcium ions that triggers a chain of calcium-dependent enzymatic reactions resulting in fibrinogen clotting. Mathematical modeling was used for the analysis of the APTT test. The process of coagulation was described by a set of coupled differential equations that were solved by the numerical method. It was found that as little as 2.3 x 10(-9) microM of factor XIIa (1/10000 of its plasma concentration) is enough to cause the complete activation of factor XII and prekallikrein (PK) during the first 20 s of the preincubation phase. By the end of this phase, kallikrein (K) is completely inhibited, residual activity of factor XIIa is 54%, and factor XI is activated by 26%. Once a clot is formed, factor II is activated by 4%, factor X by 5%, factor IX by 90%, and factor XI by 39%. Calculated clotting time using protein concentrations found in the blood of healthy people was 40.5 s. The most pronounced prolongation of APTT is caused by a decrease in factor X concentration.

Mathematical model for the blood coagulation prothrombin time test.

A mathematical model for the prothrombin time test is proposed. The time course of clotting factor activation during coagulation was calculated, and the sensitivity of the test to a decrease in the concentrations of coagulation proteins or their activities was studied. The model predicts that only severe coagulation disorders connected with a more than five-fold decrease in the concentrations or activities of the blood coagulation factors can be revealed by the test.

[The antithrombotic action of a protein C activator from the venom of Agkistrodon blomhoffi ussuriensis in thrombus formation in an extracorporeal shunt in rats]. / Antitromboticheskoe deistvie aktivatora proteina C iz iada Agkistrodon blomhoffi ussuriensis pri tromboobrazovanii v ékstrakorporal'nom shunte u krys.

An antithrombotic action of the protein C (PC) activator from the venom of Agkistrodon blomhoffi ussuriensis on the model of platelet-dependent thrombosis in the arteriovenous shunt in rats was under investigation. Administration of the PC activator to rats resulted in a dose-dependent prolongation of the thrombus formation time, in a decrease in PC and factor V levels in blood and in APTT prolongation. There were no changes in the tissue-type plasminogen activator level and in the ADP- or epinephrine-induced platelet aggregation, but platelet adhesion to glass decreased. The possible mechanism of the antithrombotic action of the PC activator appeared to be the factor V inactivation mediated by protein C activation and the decrease in platelet adhesion.

Protein C activator from the venom of Agkistrodon blomhoffi ussuriensis retards thrombus formation in the arterio-venous shunt in rats.

Protein C (PC) is an anticoagulant protein which, being activated by thrombin, degrades factors V/Va and VIII/VIIIa and releases a tissue-type plasminogen activator. Some Agkistrodon snake venoms contain PC activators which, in experiments, exert an anticoagulant action. An antithrombotic effect of the PC activator from the venom of A. blomhoffi ussuriensis on the model of thrombus formation in the arterio-venous shunt in rats was under investigation. Administration of the PC activator resulted in a dose-dependent prolongation of the thrombus formation time and a decrease in plasma PC activity, which were accompanied by a decrease in factor V activity and APTT prolongation. No reliable changes in the t-PA level, ADP- and epinephrine-induced platelet aggregation were observed. Platelet adhesion to glass beads diminished. We assume that the antithrombotic effect of the PC activator from the A. blomhoffi venom in the platelet-dependent thrombosis model is caused by PC activation and subsequent factor V inactivation as well as by platelet adhesiveness reduction.

[Protein C: mechanisms of activation and anticoagulant effect]. / Protein C: mekhanizmy aktivatsii i antikoaguliantnogo deistviia.

The mechanism of functioning of protein C can briefly be presented as follows. Thrombin and factor Xa formed in the blood via coagulation, interact with endothelial thrombomodulin and thereby activate protein C circulating in the blood. The activated protein C inhibits factors V and VIII and, in so doing, blocks the further production of thrombin. In this way protein C intermediates the loop of the negative feedback and prevents excessive thrombin generation. Besides, activated protein C enhances fibrinolysis, causing the release of the tissue plasminogen activator. Activated protein C is inhibited by a heparin-dependent inhibitor and alpha 1-antitrypsin and is then excreted from the organism. Congenital deficiency of protein C gives rise to thromboses; thrombotic diseases of various etiology are accompanied by protein C decline in the blood. Injection of exogenous protein C into the blood increases the anticoagulant activity of the blood and produces an antithrombotic effect.

[Participation of protein C in reaction of the anti-coagulant system to intravenous administration of thrombin and thromboplastin to rats]. / Uchastie proteina C v reaktsii protivosvertyvaiushchei sistemy na vnutrivennoe vvedenie krysam trombin i tromboplastina.

Dynamics of protein C concentration was studied in rat blood after administration of thrombin and thromboplastin. Administration of 0.5 ml 1% thromboplastin caused fast decrease of protein C concentration, down to 60% of the initial level, within 3 min, while activity of factor V reached the minimal rate (30%) within 5 min. Content of protein C returned to the initial level in blood within 2-2.5 hrs and of factor V--within 6 hrs. After administration of thrombin 3 NIH in content of protein C was decreased to 91.3% whereas heparin was released only after injection of 6 NIH. The data obtained suggest that the protein C system responded earlier to occurrence of thrombin in circulation as compared with the neurohumoral regulators of the anticoagulation system; the protein C system is one of primary mechanisms of the antithrombosis defence.

[The effect of heparin and thromboplastin on the half-life of 125I-protein C in the blood flow of rats]. / Vliianie geparina i tromboplastina na vremia poluzhizni 125I-proteina C v krovotoke krys.

The influence of heparin and thromboplastin on the halflife of 125I-protein C in rat blood was under investigation. It was found that t1/2 of protein C was of 2.3 h. The intravenous administration of heparin resulted in the prolongation of t1/2 to 6.5 h, that could be explained by inhibition of thrombin generation. Upon the 40-min infusion of thromboplastin the rate of 125I-protein C decay in blood enhanced. That could be explained by the generation of the endogenous thrombin and participation of thrombomodulin in the protein C activation as well as in the removal of the endogenous thrombin from blood.

[Regulation of thrombocyte stimulating and other activities of thrombin by modulators of the recognition site]. / Reguliatsiia trombotsitstimuliruiushchei i drugikh aktivnosti moduliatorami tsentra uznavaniia]

The structural and functional features of thrombin are under discussion: combination of restricted specificity and a central regulatory role in hemostasis. Thrombin specificity is mainly connected with special regions of the enzyme molecule--an additional recognition binding site for high molecular substrates. One can consider the additional site of thrombin as a kind of the allosteric centre changing thrombin-catalyzed functions at binding with modulator. Specific site of substrate (inhibitor or receptor) is used in the role of modulator. A computer search of that modulator was fulfilled by means of the program DOTHELIX. The peptides thymosin I and substance P which have regions similar to those of hirudin were shown to inhibit thrombin activity. The kinetic data point to the noncompetitive type of inhibition. The data on the high reactivity of the thrombin-activated protein C system confirm the idea of protein C to be the first defensive mechanism when thrombin is generated in blood and interacts with thrombomodulin.

[Antithrombotic effect of protein C activator from a snake venom]. / Protivotromboticheskii éffekt aktivatora proteina C iz zmeinogo iada.

Influence of the protein C activator from snake venom on blood coagulation was studied. Incubation of different concentrations of the activator with rat blood plasma resulted in a dose-dependent prolongation of the activated partial thromboplastin time (APTT). Cleavage of the protein C to the active form was detected by electrophoresis. Intravenous administration of the activator (100 mg/kg) into rats led to prolongation of APTT to 242 +/- 80%, to increase in the plasminogen activator level to 145 +/- 29% and to decrease in the factor V activity to 57 +/- 14%. When thrombosis was induced by means of administration of the thromboplastin lethal dose, pretreatment with the activator prevented animal death in 90% of cases. The effects of the activator observed appear to occur via transformation of the endogenous protein C into its active form.

[Generation of blood anticoagulant and fibrinolytic activity after intravenous administration of protein C-a in rats]. / Generatsiia antikaguliantnoi i fibrinoliticheskoi aktivnosti krovi pri vnutrivennom vvedenii proteina C-a krysam.

Protein C--vitamin K-dependent protein of the blood coagulation system possessing anticoagulant and fibrinolytic activities was under investigation. Activated partial thromboplastin time was shown to prolong to 214 +/- 8.9% from the first minute after intravenous administration of 0.51 mg per rat bovine protein Ca. After 5 minutes the activity of plasminogen activators increased to 339 +/- 52.8%. Both effects gradually diminished and came back to the starting level within 60-90 minutes. The factor V activity reduced two-fold and didn't return to basal level. We propose that protein Ca reveals its enzymatic activity within first minutes after administration and is blocked then with its inhibitor.