Factor V C2 domain contains a major thrombin-binding site responsible for thrombin-catalyzed factor V activation.

Factor (F)V is converted into its active form, FVa, by limited proteolysis. Thrombin-catalyzed activation of FV is essential for its full cofactor activation. Previously, we reported that thrombin was bound to the C2 domain in the light chain of FVIII. As FV has a similar domain structure to FVIII, we focused on the FV C2 domain as a possible binding region for thrombin. Kinetic parameters, measured by surface plasmon resonance, revealed that the K(d) values of anhydro-thrombin for FV, FVa, and the FV C2 domain were 66, 240, and 670 nmol L(-1), respectively. FV activation was increased by approximately 9-fold by the addition of thrombin. In the presence of the FV C2 domain, this increase of the FV activation was inhibited. However, FV activation was not inhibited by the addition of the FVIII C2 domain. FV was cleaved into a 105-kDa heavy chain and a 71/74-kDa light chain by thrombin-catalyzed proteolysis at Arg709, Arg1018 and Arg1545. In the presence of the FV C2 domain, the cleavage was inhibited at all sites. Proteolysis was not affected by the addition of the FVIII C2 domain. These results indicated that the FV C2 domain contains a major binding site for thrombin and that this domain is necessary for the proteolysis at all cleavage sites. Furthermore, the present results also suggested that thrombin has an independent binding site for FV different from that for FVIII.

Initiation and propagation of coagulation from tissue factor-bearing cell monolayers to plasma: initiator cells do not regulate spatial growth rate.

Exposure of tissue factor (TF)-bearing cells to blood is the initial event in coagulation and intravascular thrombus formation. However, the mechanisms which determine thrombus growth remain poorly understood. To explore whether the procoagulant activity of vessel wall-bound cells regulates thrombus expansion, we studied in vitro spatial clot growth initiated by cultured human cells of different types in contact pathway-inhibited, non-flowing human plasma. Human aortic endothelial cells, smooth muscle cells, macrophages and lung fibroblasts differed in their ability to support thrombin generation in microplate assay with peaks of generated thrombin of 60 +/- 53 nmol L(-1), 135 +/- 57 nmol L(-1), 218 +/- 55 nmol L(-1) and 407 +/- 59 nmol L(-1) (mean +/- SD), respectively. Real-time videomicroscopy revealed the initiation and spatial growth phases of clot formation. Different procoagulant activity of cell monolayers was manifested as up to 4-fold difference in the lag times of clot formation. In contrast, the clot growth rate, which characterized propagation of clotting from the cell surface to plasma, was largely independent of cell type (< or = 30% difference). Experiments with factor VII (FVII)-, FVIII-, FX- or FXI-deficient plasmas and annexin V revealed that (i) cell surface-associated extrinsic Xase was critical for initiation of clotting; (ii) intrinsic Xase regulated only the growth phase; and (iii) the contribution of plasma phospholipid surfaces in the growth phase was predominant. We conclude that the role of TF-bearing initiator cells is limited to the initial stage of clot formation. The functioning of intrinsic Xase in plasma provides the primary mechanism of sustained and far-ranging propagation of coagulation leading to the physical expansion of a fibrin clot.

Two subpopulations of thrombin-activated platelets differ in their binding of the components of the intrinsic factor X-activating complex.

Binding of fluorescein-labeled coagulation factors IXa, VIII, X, and allophycocyanin-labeled annexin V to thrombin-activated platelets was studied using flow cytometry. Upon activation, two platelet subpopulations were detected, which differed by 1-2 orders of magnitude in the binding of the coagulation factors and by 2-3 orders of magnitude in the binding of annexin V. The percentage of the high-binding platelets increased dose dependently of thrombin concentration. At 100 nm of thrombin, platelets with elevated binding capability constituted approximately 4% of total platelets and were responsible for the binding of approximately 50% of the total bound factor. Binding of factors to the high-binding subpopulation was calcium-dependent and specific as evidenced by experiments in the presence of excess unlabeled factor. The percentage of the high-binding platelets was not affected by echistatin, a potent aggregation inhibitor, confirming that the high-binding platelets were not platelet aggregates. Despite the difference in the coagulation factors binding, the subpopulations were indistinguishable by the expression of general platelet marker CD42b and activation markers PAC1 (an epitope of glycoprotein IIb/IIIa) and CD62P (P-selectin). Dual-labeling binding studies involving coagulation factors (IXa, VIII, or X) and annexin V demonstrated that the high-binding platelet subpopulation was identical for all coagulation factors and for annexin V. The high-binding subpopulation had lower mean forward and side scatters compared with the low-binding subpopulation ( approximately 80% and approximately 60%, respectively). In its turn, the high-binding subpopulation was not homogeneous and included two subpopulations with different scatter values. We conclude that activation by thrombin induces the formation of two distinct subpopulations of platelets different in their binding of the components of the intrinsic fX-activating complex, which may have certain physiological or pathological significance.

Role of activation of the coagulation factor VIII in interaction with vWf, phospholipid, and functioning within the factor Xase complex.

Blood coagulation factor VIII (fVIII) in its nonactivated form circulates in plasma in a complex with von Willebrand factor (vWf). Upon activation by thrombin- or factor Xa-mediated site-specific proteolysis, activated fVIII (fVIIIa) serves as a cofactor for factor IXa. This protein complex assembled on a phospholipid surface (factor Xase) activates factor X. This complex plays the key role in the intrinsic pathway of blood coagulation. We reviewed the molecular events triggered by fVIII activation, which are required for the assembly and functioning of the Xase complex, including fVIIIa dissociation from vWf and a significant increase of fVIII affinity for binding to the phospholipid surface. Both events are mediated by activation-related cleavage within fVIII light chain (LCh), releasing the 40 amino-acid N-terminal LCh peptide, which is followed by a conformational change within the C2 domain. The conformational change within LCh is also required for the optimal fVIII cofactor functioning within the factor Xase complex, exerted via fVIIIa interactions with phospholipid, factor IXa, and factor X. Since factor IXa not only stabilizes but also proteolytically inactivates fVIIIa within the factor Xase complex, the stability of the membrane-bound fVIIIa in the presence and absence of factor IXa is discussed. In conclusion, we outline some new possible directions of the research. One of them arises from the recently demonstrated ability of plasma lipoproteins to provide a phospholipid surface for the assembly of the factor Xase complex in vitro. This finding raises a possibility that lipoproteins participate in factor Xase functioning in vivo and suggests a direct link between elevated levels of lipoproteins associated with atherosclerosis and increased thrombogenicity associated with this disease.

Catabolism of the coagulation factor VIII: can we prolong lifetime of f VIII in circulation?

The coagulation factor VIII is required for normal haemostasis, because deficiency or genetic defects in this molecule cause a life-threatening coagulation disorder known as hemophilia A. While the role of f VIII in the intrinsic pathway of blood coagulation has been extensively studied, the mechanisms responsible for f VIII turnover in circulation have not been characterized until recently. This review focuses on the finding that f VIII catabolism in vitro and in vivo is mediated by low-density lipoprotein receptor-related protein (LRP), representing a hepatic clearance receptor. FVIII interaction with LRP involves two distinct sites localized within the C2 and A2 domains of f VIII. We discuss the contribution of the A2 site (residues 484-509) and the C2 site in f VIII catabolism in the presence and absence of vWf. We present the evidence that LRP-mediated f VIII catabolism is facilitated by cell-surface heparan sulfate proteoglycans (HSPGs), which bind to the A2 residues 558-565 of f VIII. Because both LRP- and HSPGs-binding sites within the A2 domain are potentially exposed in the circulating f VIII/vWf complex, we discuss the possibility of prolongation of the f VIII lifetime in circulation by disrupting these sites employing site-directed mutagenesis. In its turn, generation of a novel recombinant f VIII may be prospective for more efficient hemophilia A therapy.

The future of recombinant coagulation factors.

Hemophilias A and B are X chromosome-linked bleeding disorders, which are mainly treated by repeated infusions of factor (F)VIII or FIX, respectively. In the present review, we specify the limitations in expression of recombinant (r)FVIII and summarize the bioengineering strategies that are currently being explored for constructing novel rFVIII molecules characterized by high efficiency expression and improved functional properties. We present the strategy to prolong FVIII lifetime by disrupting FVIII interaction with its clearance receptors and demonstrate how construction of human-porcine FVIII hybrid molecules can reduce their reactivity towards inhibitory antibodies. While the progress in improving rFIX is impeded by low recovery rates, the authors are optimistic that the efforts of basic science may ultimately lead to higher efficiency of replacement therapy of both hemophilias A and B.

Dominant C2 domain epitope specificity of inhibitor antibodies elicited by a heat pasteurized product, factor VIII CPS-P, in previously treated hemophilia A patients without inhibitors.

From June, 1990, to November, 1991, in The Netherlands and Belgium, 16 previously treated severe hemophilia A patients (PTP) developed inhibitors after exposure to factor VIII CPS-P, a new heat pasteurized product. A previously untreated patient (PUP) also developed an inhibitor to CPS-P. In inhibitor neutralization assays with recombinant fVIII C2 and A2 domain polypeptides, plasmas from 14 PTPs were > or = 79% neutralized by C2 and < 10% by A2, but the PUP plasma was partially neutralized by C2 (48%) and A2 (28%). Immunoprecipitation assays of the PTP and PUP plasmas with the fVIII heavy chain and with recombinant C2 and A3-C1 polypeptides confirmed that the C2 dominant immune response to CPS-P was found only in the PTPs. Competition of the binding of 2 inhibitors to 125I-CPS-P by unlabeled CPS-P and another plasma fVIII was similar, demonstrating that the antibody response was not directed to epitopes only present in CPS-P. We propose that the immunogenicity of the CPS-P C2 domain was altered by heat pasteurization.

Inhibitors in German hemophilia A patients treated with a double virus inactivated factor VIII concentrate bind to the C2 domain of FVIII light chain.

To reduce the risk of transmission of hepatitis A virus, an Octapharma produced factor VIII (fVIII) concentrate treated with solvent detergent (FVIII-SD) was further pasteurized after purification. This product, Octavi SDPlus (FVIII-SDP), was marketed in Europe in 1993 to 1995. Inhibitors appeared from September to October, 1995, in 12 of 109 previously treated German hemophilia A patients. A study of similarly treated Belgian patients, who also developed inhibitors, had shown antibodies to the fVIII light chain (domains A3-C1-C2) only. In the present study, the epitope specificity of 8 German inhibitor plasmas was also found to be restricted to the light chain. In radioimmunoprecipitation assays to localize the light chain epitope(s), antibody binding to heavy chain (domains A1-A2-B) was 11-148 fold lower than to the C2 domain, and binding to recombinant A3-C1 was barely detectable. These results were supported by >95% neutralization of a high responder inhibitor titer by the C2 domain.

An alloantibody recognizing the FVIII A1 domain in a patient with CRM reduced haemophilia A due to deletion of a large portion of the A1 domain DNA sequence.

We report the development of a FVIII inhibitor in a patient with severe, cross reacting material reduced (CRM(R)) haemophilia A. The level of Factor VIII antigen (FVIII:Ag) measured by ELISA using anti-C2 monoclonal and alloantibodies was 1.9 U/dl. This baseline FVIII:Ag level was increased to 8.3 U/dl after administration of DDAVP. The anti-FVIII inhibitor titer was 2.9 Bethesda U/ml. DNA analysis showed a large deletion of the FVIII gene from exon 4 to 7, corresponding to amino acid residues 111-317 included within the A1 domain. The size of the gene deletion was approximately 28 kb. 5' and 3' breakpoints were identified by sequencing in intron 3 and intron 7, respectively. FVIII mRNA was detected in the patient's peripheral lymphocytes and the deletion spanning exon 4 to 7 was confirmed at the RNA level. Immunoprecipitation experiments using 125I labeled A1, A2 and light chain demonstrated that the inhibitor reacted only with the 54 kDa A1 domain. The inhibitor activity was more than 95% neutralized by A1 domain polypeptide. Our findings suggest a close relationship between the inhibitor epitope and the specific gene deletion with regard to the pathogenesis of the inhibitor in this patient.

Effect of von Willebrand Factor and its proteolytic fragments on kinetics of interaction between the light and heavy chains of human factor VIII.

It was previously shown that vWF increases the rate of divalent cation-mediated fVIII reconstitution from isolated light chain (LCh) and heavy chain (HCh) subunits. We examined the effect of vWF on kinetic parameters for interaction between LCh and HCh in the presence of Ca2+ and Mn2+ ions, the most effective mediators of fVIII reconstitution from isolated subunits, and determined the minimal structural portion of vWF able to enhance fVIII formation. We found that affinity (Kd) for LCh/HCh binding mediated by Ca2+ and Mn2+ was 91 and 34.9 nM in the absence of vWF and 15.5 and 5.6 nM in its presence. This decrease of Kd resulted from a sixfold increase of the association rate constant (k(on)) for this interaction. The value of the dissociation rate constant (k(off)) for LCh/HCh complex was lower in the presence of Mn2+ (k(off) 4.6x 10(-6) s(-1)) than Ca2+ (k(off) 8.4 x 10(-6) s(-1)) but in both cases vWF had no effect on k(off). This indicates that at physiological concentration of 1 nM the rate of fVIII inactivation via dissociation to subunits would be entirely determined by the k(off) value, and it should not depend on the presence of vWF. Indeed, our experiments demonstrated that vWF did not have any effect on the rate of fVIII inactivation resulting from its dissociation to subunits at the physiological concentrations of the fVIII and vWF proteins. We identified the minimal portion of the vWF molecule, able to enhance reconstitution of fVIII from isolated subunits. Only vWF large proteolytic N-terminal homodimeric fragment SPIII (vWF residues 1-1365), but not small monomeric N-terminal fragment SPIII-T4 (1-272), both of which are known to contain a major fVIII binding site, was able to support reconstitution of fVIII activity from isolated LCh and HCh subunits in the presence of Mn2+ or Ca2+. The effect of SPIII on the LCh/HCh association was similar to that of vWF, because both proteins identically increased of the value of k(on) and did not alter the k(off) value.

In hemophilia A and autoantibody inhibitor patients: the factor VIII A2 domain and light chain are most immunogenic.

Factor VIII (fVIII) is a protein cofactor essential for blood coagulation, and it binds in the factor Xase complex to factors IXa, X, and phospholipid. In about 30% of severe hemophilia A patients, treatment with fVIII leads to production of anti-fVIII antibodies. Anti-fVIII autoantibodies also rarely appear in normal individuals. Those antibodies that inactivate fVIII (inhibitors) prevent optimal fVIII therapy. Inhibitor epitopes were previously localized to the fVIII A2, A3, and C2 domains and to an acidic amino acid region between A1 and A2. Such anti-fVIII antibodies interfere with fVIII binding to components of the factor Xase complex and prevent blood coagulation. When total anti-fVIII titers were determined for each fVIII domain in 43 inhibitor plasmas by immunoprecipitation (IP) and inhibitor neutralization assays, the anti-light chain (LCh) antibody titer was highest, anti-A2 was intermediate, and anti-A1 and anti-B were low. The relative immunogenicity of the fVIII domains in hemophilic and autoantibody inhibitor patients was similar.

Efficient factor VIII affinity purification using a small synthetic ligand.

BACKGROUND: Hemophilia A is currently treated by infusions of the coagulation factor (F) VIII, of which production and purification remain a challenging task. Current purification procedures using immunoaffinity chromatography are cumbersome, expensive, and suffer from the instability of the applied antibody ligands, which elute along with the product and contaminate it. Recently, FVIII was purified using octapeptide ligands, but their use is limited due to the low resistance to proteases. OBJECTIVE: Our goal was to develop and evaluate a novel ligand for FVIII purification, overcoming the drawbacks of current procedures. METHODS: Peptide ligands were screened for binding of (125)I-plasma-derived-FVIII (pdFVIII) in a microbead assay. A selected ligand-coated Toyopearl resin was then used for pdFVIII purification from cell-conditioned Delbucco's modified Eagle's medium (DMEM) containing fetal bovine serum. The proteolytic stability of ligand was measured by incubating with human serum and proteinase K, and its cytotoxicity towards human OV-MZ-6 cells was assayed. RESULTS: A high-affinity octapeptidic FVIII ligand was modified into the small, highly stable and non-toxic peptidomimetic ligand L4 by rational and combinatorial design without affecting its affinity for FVIII. Using ligand L4-coated Toyopearl resin, pdFVIII was isolated from cell-conditioned medium with high purity and 89% column retention after elution with a mild buffer containing 0.6 m NaCl at pH 6.8. CONCLUSIONS: Ligand L4 offers a valuable alternative to antibody-based procedures for laboratory and industrial production. Its synthesis by established solid-phase procedures is straightforward and considerably cheaper than the biotechnological production of antibodies, and safety concerns associated with the use of biological material are overcome.

Mathematical models of blood coagulation and platelet adhesion: clinical applications.

At present, computer-assisted molecular modeling and virtual screening have become effective and widely-used tools for drug design. However, a prerequisite for design and synthesis of a therapeutic agent is determination of a correct target in the metabolic system, which should be either inhibited or stimulated. Solution of this extremely complicated problem can also be assisted by computational methods. This review discusses the use of mathematical models of blood coagulation and platelet-mediated primary hemostasis and thrombosis as cost-effective and time-saving tools in research, clinical practice, and development of new therapeutic agents and biomaterials. We focus on four aspects of their application: 1) efficient diagnostics, i.e. theoretical interpretation of diagnostic data, including sensitivity of various clotting assays to the changes in the coagulation system; 2) elucidation of mechanisms of coagulation disorders (e.g. hemophilias and thrombophilias); 3) exploration of mechanisms of action of therapeutic agents (e.g. recombinant activated factor VII) and planning rational therapeutic strategy; 4) development of biomaterials with non-thrombogenic properties in the design of artificial organs and implantable devices. Accumulation of experimental knowledge about the blood coagulation system and about platelets, combined with impressive increase of computational power, promises rapid development of this field.

Strategies towards a longer acting factor VIII.

The reduced mortality, improved joint outcomes and enhanced quality of life, which have been witnessed in the developed world for patients with haemophilia, have been an outstanding achievement. Advancements in biotechnology contributed significantly through the development of improved pathogen screening, viral inactivation techniques and the development of recombinant clotting factors. These were partnered with enhanced delivery of care through comprehensive haemophilia centres, adoption of home therapy and most recently effective prophylaxis. This came at great costs to governments, medical insurers and patients' families. In addition, barriers persist limiting the adoption and adherence of effective prophylactic therapy. Biotechnology has been successful at overcoming similar barriers in other disease states. Long-acting biological therapeutics are an incremental advance towards overcoming some of these barriers. Strategies that have been successful for other therapeutic proteins are now being applied to factor VIII (FVIII) and include modifications such as the addition of polyethylene glycol (PEG) polymers and polysialic acids and alternative formulation with PEG-modified liposomes. In addition, insight into FVIII structure and function has allowed targeted modifications of the protein to increase the duration of its cofactor activity and reduce its clearance in vivo. The potential advantages and disadvantages of these approaches will be discussed.

A mechanism for inhibition of factor VIII binding to phospholipid by von Willebrand factor.

von Willebrand factor (vWf) acts as a carrier for blood coagulation factor VIII (fVIII) in the circulation. The amino-terminal 272 residues of mature vWf contain a high affinity fVIII binding site. Upon thrombin activation, fVIII is released from vWf, thereby allowing its binding to phospholipid which is required for its procoagulant activity. Although phospholipid and vWf compete for fVIII binding, it was previously suggested that their binding sites are not closely juxtaposed within the fVIII protein because only amino-terminal vWf proteolytic fragments larger than SPIII-T4 (1-272) were able to block the binding of fVIII to phospholipid. We have demonstrated, however, that SPIII-T4 is able to inhibit fVIII binding to phosphatidylserine (PS) in a dose-dependent fashion, but only at concentrations higher than those used in previous experiments. Our demonstration that the Kd values for vWf and SPIII-T4 for fVIII are 0.52 nM and 48 nM, respectively, explain this discrepancy. Inhibition (> 95%) of SPIII-T4 binding to fVIII by a purified recombinant fVIII C2 domain polypeptide demonstrated that SPIII-T4 binds directly to C2, as we had previously shown for vWf. The similarity of the C2 binding sites for vWf and SPIII-T4 was further confirmed by the identical inhibitory effects of synthetic peptides and monoclonal antibodies (mAbs) on vWf-fVIII or SPIII-T4 fVIII binding. In both cases, binding was inhibited by synthetic peptide 2303-2332, containing a PS binding site, and by mAb NMC-VIII/5 Fab' (epitope within C2 residues 2170-2327). We propose that vWf, via residues 1-272, and PS compete for fVIII binding because they recognize overlapping sites within fVIII C2 domain residues 2303-2332.

The acidic region of the factor VIII light chain and the C2 domain together form the high affinity binding site for von willebrand factor.

A binding site for von Willebrand factor (vWf) was previously localized to the carboxyl terminus of the C2 domain of the light chain (LCh) of factor VIII (fVIII). The acidic region of the LCh, residues 1649-1689, also controls fVIII.vWf binding by an unknown mechanism. Although anti-acidic region monoclonal antibodies prevent formation of the fVIII.vWf complex, the direct involvement of the acidic region in this binding has not been demonstrated. By limited proteolysis of LCh with Staphylococcus aureus V8 protease, we prepared 14- and 63-kDa LCh fragments, which begin with fVIII residues 1672 and 1795, respectively. Using surface plasmon resonance to measure binding interactions, we demonstrated that the 14-kDa fragment binds to vWf, but its affinity for vWf (Kd 72 nM) was 19-fold lower than that of LCh. This was not due to an altered conformation of the acidic region within the 14-kDa fragment, since its affinity for an anti-acidic region monoclonal antibody was similar to that of LCh. All LCh derivatives lacking the acidic region (thrombin-cleaved LCh, recombinant C2, and 63-kDa fragment) had also greatly reduced affinities for vWf (Kd 564-660 nM) compared with LCh (Kd 3.8 nM). In addition, the similar affinities of these derivatives for vWf indicated that apart from its acidic region, the LCh contains no vWf binding site other than the one within C2. The reduced affinities of the LCh derivatives lacking the acidic region for monoclonal antibody NMC-VIII/5 (epitope, C2 residues 2170-2327) indicated that removal of the acidic region leads to a conformational change within C2. This change is likely to affect the conformation of the vWf binding site in C2, which overlaps the epitope of NMC-VIII/5; therefore, the acidic region also appears to be required to maintain the optimal conformation of this vWf binding site. Our results demonstrate that the acidic region and the C2 domain are both directly involved in forming a high affinity binding site for vWf.

[Protective effect of various forms of human ceruloplasmin in copper-induced erythrocyte lysis]. / Zashchitnoe deistvie raznykh form tseruloplazmina cheloveka pri med'- indutsirovannom lizise éritrotsitov.

It is known that human ceruloplasmin (CP) is made up of several isoforms which differ by the structure of their carbohydrate fragment. One of these isoforms, CP1, which makes up to approximately 40% of the native CP molecule and which contains a carbohydrate fragment, [formula: see text] is specifically bound to human erythrocyte (ER) receptors. This isoform was isolated by using lectin affinity chromatography. It was found that CP1 produces a much stronger protective effect on ER during Cu(2+)-induced lysis as compared with CP. A kinetic analysis of Cu2+ accumulation and reduced glutathione (GSH) decline in ER revealed that the lack of correlation between these two processes. It was found that in the presence of CP and CP1 the GSH concentration is not critical for the hemolytic resistance of ER. In the presence of CP1 ER hemolysis occurs at a slower rate whereas the GSH decline at a much faster rate than in the presence of CP.

The protective effect of different forms of human ceruloplasmin in copper-induced lysis of red blood cells.

The comparison of protective effects of native ceruloplasmin (CP) and of preparation CP1 containing carbohydrate fragment GlcNAc(beta(1,4]GlcNAc which specifically binds on RBC (alpha(1,6)Fuc receptors showed that CP1 exhibits much more powerful protective effect on RBC in copper-induced lysis. It was found, however, that CP2 (native CP devoided of CP1) protected RBC as well as CP despite its inability of binding to RBC membrane. CP and CP1 in a similar way decrease copper concentration in RBC. It was shown that copper accumulation and GSH decrease in RBC are two independent and concurrent processes; the copper and GSH concentrations are not the factors determining RBC resistance to hemolysis. CP inhibits the reaction of superoxide radicals generation as a result of Cu interaction with -SH groups of RBC membrane; the effect is more pronounced than the effect of catalase or superoxide dismutase. CP and CP1 preparations equally inhibit this reaction. Apparently CP reception on RBC leads not only to membrane protection from superoxide and hydroxyl radicals but represents a more complex process.

Studies on receptor interaction of ceruloplasmin with human red blood cells.

The interaction of CP with the red blood cell (RBC) receptor was shown to be a Ca2+ dependent process and be limited by CP binding on RBC membrane which is not followed by CP transport through the membrane into RBC. The nature of receptor interaction was determined. It was shown that receptors are formed by glycoproteins of PAS1 and PAS2 (glycoforin dimer and monomer, respectively) and terminal residues of sialic acid of these glycoproteins are important for CP reception. Receptor carbohydrate specificity was determined. Biantennary structure of CP molecule carbohydrate moiety which is bound to the receptor owing to 2 structural fragments: sialic acid terminal residues and the fragment including acetylglucosamine dimer and fucose, plays the main role in CP reception.

Immunoenzyme determination of total serum ceruloplasmin. Application to Wilson disease.

Enzyme immunoassay for ceruloplasmin (CP)*, employing monospecific CP antibodies labeled with horse radish peroxidase was developed. This method permits to determine total content of CP, which is present in Wilson disease patients' blood in enzymatically active and enzymatically inactive forms. The evidence is presented that the method can be used for a direct determination of CP in blood serum. The minimal CP concentration which may be determined by enzyme immunoassay (IEA) is 5.10(-9) g/ml. The method was used for determination of CP concentrations in Wilson disease patients' blood with different disease severity. Analysis of blood samples taken from 6 Wilson disease patients with the use of IEA method revealed similar total CP concentrations. At the same time, the oxidase activities of CP in the blood of different patients varied more than sevenfold.