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Biomedical subjects

A V Mazurov

Publications and source records attributed to A V Mazurov.

At least 19 recordsLinked to original sources
PubMed1992

Identification of aspartic acid 514 through glutamic acid 542 as a glycoprotein Ib-IX complex receptor recognition sequence in von Willebrand factor. Mechanism of modulation of von Willebrand factor by ristocetin and botrocetin.

As the first step in hemostasis, the binding of von Willebrand factor (vWF) to the platelet membrane glycoprotein (GP) Ib-IX complex is essential for platelet adhesion at high-shear blood flow. This interaction in vivo requires the prior binding of vWF to the subendothelial matrix, a process which exposes a normally cryptic binding site on vWF for the GP Ib-IX complex. This process can be mimicked in vitro by modulators such as ristocetin or the snake venom protein botrocetin or by desialation of vWF. We have previously localized the GP Ib binding site on vWF to a monomeric dispase fragment which extends from Leu-480/Val-481 to Gly-718 in the primary sequence of mature vWF [Andrews, R. K., Gorman, J. J., Booth, W. J., Corino, G. L., Castaldi, P. A., & Berndt, M. C. (1989) Biochemistry 28, 8326-8336]. This fragment also contains a distinct binding site for botrocetin. Analysis of synthetic peptides corresponding to hydrophilic stretches of sequence within this fragment indicated that the sequence Asp-514-Glu-542 represents a major adhesive sequence involved in receptor recognition. This peptide inhibited both the ristocetin- and botrocetin-mediated binding of vWF to either platelets or purified GP Ib-IX complex (IC50 approximately 50-200 microM) as well as the asialo-vWF- and bovine vWF-dependent agglutination of platelets. Both the N- and C-terminal halves of the peptide were inhibitory but less so than the intact peptide. This peptide also inhibited botrocetin binding to vWF, suggesting that botrocetin modulates vWF-GP Ib interaction by binding in close proximity to the vWF adhesion sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Activation-dependent changes in human platelet PECAM-1: phosphorylation, cytoskeletal association, and surface membrane redistribution.

PECAM-1 is a recently described member of the immunoglobulin gene (Ig) superfamily that is expressed on the surface on platelets, several leukocyte subsets, and at the endothelial cell intracellular junction. Recent studies have shown that the extracellular domain of PECAM-1, which is comprised of 6 Ig-like homology units, participates in mediating cell-cell adhesion, plays a role in initiating endothelial cell contact, and may later serve to stabilize the endothelial cell monolayer. PECAM-1 also has a relatively large 108 amino acid cytoplasmic domain, with potential sites for phosphorylation, lipid modification, and other posttranslational events that could potentially modulate its adhesive function or regulate its subcellular distribution. Virtually nothing is known about the contribution of the intracellular region of the PECAM-1 molecule to either of these cellular processes. Using human platelets as a model, we now demonstrate that PECAM-1 becomes highly phosphorylated in response to cellular activation, and coincident with phosphorylation associates with the cytoskeleton of activated, but not resting, platelets. The engagement of PECAM-1 with the platelet cytoskeleton enables it to move large distances within the plane of the membrane of fully-spread, adherent platelets. This redistribution may similarly account for the ability of PECAM-1 to localize to the intracellular borders of endothelial cells once cell-cell contact has been achieved.

Antigens, Differentiation, Myelomonocytic↗
PubMed1992

Mechanisms of thrombocytopenia in patients with lymphoproliferative diseases.

Different factors are involved in the development of thrombocytopenia in patients with lymphoproliferative disorders. Significant correlation was detected between the number of megakaryocytes in bone marrow and platelet count (r = 0.485, p = 0.002, n = 37) and significant difference between the number of megakaryocyte in patients with normal platelet count (> 200,000/microliters) and patients with marked thrombocytopenia (platelet count < 100,000/microliters). All patients in the latter group (n = 15) had a relatively low number of megakaryocytes. Low but significant reverse correlation was found between the level of platelet-associated IgG (PA-IgG) and platelet count (r = -0.249, p = 0.024, n = 82) and significant difference between the mean levels of PA-IgG in the groups of patients with platelet count > 200,000/microliters and < 100,000/microliters. PA-IgG were increased in 46% of patients in the total group and in 65% of patients with platelet count < 100,000/microliters. The correlation between platelet count and PA-IgG was about 2 times higher in splenectomized (r = -0.549, p = 0.005, n = 24) than nonsplenectomized patients. All splenectomized patients with platelet count < 100,000/microliters (n = 8) had a significant increase in PA-IgG. Serum antibodies were detected in only 7% of tested patients. This group was characterized by severe thrombocytopenia (in 6 of 10 patients--platelet count < 50,000/microliters) and a high incidence of haemorrhages (in 5 of 10 patients). Thus the depression of platelet production was suggested to be the basic cause of thrombocytopenia in lymphoproliferative disorders. Involvement of immune mechanisms was revealed in a large number of patients and correlated with a deeper and more complicated thrombocytopenia.

Antibodies↗
PubMed1992

[The immunodiagnosis of thrombocyte membrane glycoprotein deficiencies: Glanzmann's thrombasthenia, Bernard-Soulier syndrome and GMP-140 protein deficiency].

Immunological methods were developed for the diagnosis of platelet membrane glycoprotein (GP) deficiencies. The number of membrane GP on platelet surface was determined as the binding of 125I-labeled monoclonal antibodies (mAB) directed against individual platelet GP. Total amount of GP in platelet lysate was assessed by immunoblotting with specific polyclonal antibodies. Methods were applied for patients with different thrombocytopathies. Binding of mAB VM16a, directed against GP IIb-IIIa was strongly decreased in patients with Glanzmann's thrombasthenia (0.5-14.5% of normal) and binding of anti-GP Ib mAB VM16d--in patient with Bernard-Soulier syndrome (0.5% of control) indicating the deficiencies of corresponding GP. In patient with gray platelet syndrome binding of both antibodies was not decreased but even increased. It was shown by immunoblotting that platelets from the patient with gray platelet syndrome contained normal amount of GP IIa, but strongly decreased amount of GMP-140 (14.5% of control)--membrane GP of platelet--granules.

Antibodies, Monoclonal↗
PubMed1992

[The radioisotope labelling of thrombocytes using a monoclonal antibody against membrane glycoproteins IIb-IIIa and the measurement of thrombocyte adhesion/aggregation on the substrate surface].

A new method for platelet labeling based on binding of monoclonal antibody to human platelets has been suggested in this study. Monoclonal antibody VM16a against membrane glycoproteins IIb-IIIa was labeled by 125I and then incubated with platelets. About 70% of added antibody was bound when it was used at the concentrations corresponding to the linear part of the concentration curve (0.5 and 1.0 micrograms/ml). Due to high efficiency of binding 125I-VM16a-labeled platelets were used for the measurement of adhesion/aggregation to the substrate in platelet-rich plasma without washing of the free label. Experiments with washed platelets double labeled with 51Cr and 125I-VM 6a showed high correlation between the data obtained with both labels. The method of platelet labeling has been applied for the assessment of drug action on platelet adhesion/aggregation. Measurements were performed in platelet-rich plasma and adhesion/aggregation was stimulated by ADP and analogue of thromboxane A2, U46619. It was shown/that antianginal drug trapidil strongly inhibited and antiatherogenic drug probucol did not affect platelet adhesion/aggregation stimulated by both agonists.

Adenosine Diphosphate↗
PubMed1991

A monoclonal antibody, VM64, reacts with a 130 kDa glycoprotein common to platelets and endothelial cells: heterogeneity in antibody binding to human aortic endothelial cells.

A new monoclonal antibody (mAb), VM64, reacts with a common antigen on the surface of human platelets and vascular endothelial cells (EC). Under nonreduced conditions it recognized in immunoblotting a protein of 130 kDa both in platelets and EC. VM64 precipitated the same 130 kDa protein from the lysate of surface radioiodinated platelets. Electrophoretic mobility of this protein was not altered by reduction and differed from the bands precipitated by reference mAb against platelet glycoproteins (GP) Ia-IIa, Ib, IIb-IIIa and GMP130. VM64 binding to platelets and EC was specific and saturable. The number of binding sites on platelets was 9.9 +/- 3.5 x 10(3) per platelet and on the surface of EC monolayer -2.40 +/- 0.32 x 10(6) per cell. VM64 also binds to platelets from Glanzmann's thrombasthenia patients which lack GPIIb-IIIa. VM64 did not affect platelet aggregation induced by ADP, collagen, thrombin and ristocetin. In the monolayers of EC from umbilical vein and human aorta, VM64 stained the area at the periphery of the cells adjacent to the cell-cell boundaries. In preconfluent cultures preferential staining was observed at the active leading margins of the cells. Unlike EC cultures from umbilical vein, where all cells were positively stained, in aortic EC cultures some unstained or poorly stained cells were constantly present, indicating a heterogeneity of EC population related to the expression of VM64 antigen. The biochemical characteristics of VM64 antigen, its presence both on platelets and EC and typical distribution on the surface of EC suggested that this antigen is identical to PECAM (CD31) protein.

Antibodies, Monoclonal↗
PubMed1991

Characterization of an antiglycoprotein Ib monoclonal antibody that specifically inhibits platelet-thrombin interaction.

Platelet glycoprotein Ib (GPIb) acts as a high-affinity thrombin binding site and as a receptor for von Willebrand Factor (vWF). A new anti-GPIb monoclonal antibody (mAB) VM16d was produced that specifically inhibited platelet-thrombin but not platelet-vWF interaction. The epitope for VM16d was located within the 45 kDa N-terminal region of the alpha-chain of GPIb. VM16d inhibited platelet aggregation induced by low dose thrombin (0.05 U/ml) but did not affect platelet aggregation induced by ristocetin, bovine vWF, ADP or collagen. The same inhibitory effects on thrombin-induced platelet aggregation were observed with the whole IgG molecule of VM16d and its F(ab')2 and F(ab') fragments. VM16d also inhibited 14C-serotonin secretion induced by low dose thrombin and binding of 125I-thrombin but not ristocetin-dependent binding of 125I-vWF to platelets. These data indicate that the high-affinity thrombin binding site is located on the N-terminal 45 kDa domain of GPIb and that it is topographically separated from the vWF binding site.

Adenosine Diphosphate↗
PubMed1991

[Inhibition of Fc-receptor dependent platelet aggregation by monoclonal antibodies against the glycoprotein IIb-IIIa complex].

A murine monoclonal antibody (MoAb) VM16a specifically binding to human platelets has been produced. Approximately 56,000 molecules of VM16a bound per platelet at saturation (Kd = 7.9 nM) but no binding to platelets from Glanzmann's thrombasthenia patients was detected. VM16a precipitated two proteins with molecular masses corresponding to those of glycoproteins (GP) IIb and IIIa from solubilized surface-labelled platelets. However, after dissociation of the GPIIb--IIIa complex with EDTA VM16a did not bind to platelets and precipitated nothing from their lysate, thus evidencing that its determinant is complex-dependent. VM16a had no effect on ADP-, thrombin- and ristocetin-induced platelet aggregation but inhibited the aggregation induced by collagen. This inhibitory effect was more pronounced in the presence of plasma. VM16a completely blocked the Fc-receptor-mediated aggregation induced by aggregated human IgG, aggregated murine IgG1 and the previously described MoAb VM58. F(ab')2 fragments of VM16a were also able to inhibit this aggregation by decreasing the rate of aggregation induced by aggregated IgG and by extending the lag phase of VM58-induced aggregation. These results suggest that the platelet Fc-receptor may be topographically associated with the GPIIb-IIIa complex.

Adenosine Diphosphate↗
PubMed1991

[Antithrombocyte antibodies in the serum and on the surface of the thrombocytes in patients with chronic lymphoproliferative diseases].

Antiplatelet antibodies were studied in patients with chronic lymphatic leukemia (CLL) using ELISA for detection of serum antibodies and RIA for determination of antibodies associated with platelet surface. Serum antibodies were identified in 1 out of 6 CLL patients with the platelet count 100,000-200,000 per microliter and in 7 out of 54 CLL patients with the platelet count lower than 100,000. Platelet-associated antibodies were not detected in patients with the normal platelet count, but were revealed in 14 out of 25 patients with the platelet count 100,000-200,000 per microliter and in 21 of 27 patients with the platelet count lower than 100,000 per microliter. The presence of a group of thrombocytopenic patients lacking serum or platelet-associated antibodies suggested that the development of thrombocytopenia in CLL could be mediated not only by antibodies but also by other pathogenic mechanisms, i.e. by the depression of megakaryocytes in bone marrow. Increase of the platelet count after steroid and cytostatic treatment in CLL patients correlated with decrease of antiplatelet antibodies. In splenectomized CLL patients, the increase of the platelet count and disappearance of hemorrhage occurred in patients with the low level as well as with the high level of antibodies after the operation.

Adrenal Cortex Hormones↗
PubMed1991

[Determination of antiplatelet antibodies on the surface of platelets by direct radioimmune method in patients with different types of immune thrombocytopenia].

Direct radioimmune assay (RIA) have been developed for detection of antibodies associated wild platelet membrane. Platelets from 12 patients with idiopathic thrombocytopenic purpura (ITP) and 27 patients with chronic lymphocytic leukemia (CLL) (platelet count (100,000 in 1 microliters) have been tested. Antibodies on platelets surface have been detected in all 12 patients with ITP and in 21 patients with CLL. In 6 CLL patients the number of immunoglobulins associated with platelets surface does not increase control level. It is possible, that in some CLL patients development of thrombocytopenia is mediated not only by platelet associated antibodies but by other mechanisms, one of which can be linked with the depression of megakaryocytes growth in bone marrow. Direct RIA for measurement of antibodies on platelet surface detect antiplatelet antibodies with higher frequency than indirect enzyme-linked-immunosorbent-assay (ELISA), developed earlier for assessment of antiplatelet antibodies in serum. Increase of platelet count in CLL patients after steroid and cytostatic treatment correlated with the decrease of platelet surface associated antibodies.

Autoantibodies↗
PubMed1991

[Adhesive activity of blood platelets in patients with stable or unstable angina pectoris and acute myocardial infarct].

Platelet adhesive activity was assayed in patients with stable angina (SA) or unstable angina (UA) and acute myocardial infarction (AMI). With scanning electron microscopy, platelet adhesions to Type IV collagen (C-IV) and plastic material, which had been stimulated by low-dose ADP, epinephrine and U-46 619, a stable thromboxane A2 analogue (agonist-induced adhesion) were determined. As compared with the control group, patients with SA or UA, showed significantly higher adhesion of platelets to the collagen substrate, whereas UA patients alone displayed a significantly increased agonist-induced adhesion when epinephrine was employed as an agonist. A sharp enhancement of platelet adhesion was characteristic of patients with AMI on days 1-3 of the onset. On days 4-5, the adhesive activity slightly dropped, and on day 7-10, it returned to the control level of healthy volunteers. The time course of adhesive activity decrease correlated with lower blood levels of creatine phosphokinase in patients with AMI. A high correlation was established between the alterations found in all the types of platelet adhesion during the course of AMI, which suggests there is an unspecific nature of increase and subsequent decrease platelet responses to all the inductors used. A comparison of patients taking and not taking aspirin (250 mg/day) revealed no differences both in the magnitude of an increase in platelet adhesion in the first 3 days and in that of its decrease on days 7-10 of AMI.

Adult↗
PubMed1990

Visualization of apo B, fibrinogen/fibrin, and fibronectin in the intima of normal human aorta and large arteries and during atherosclerosis.

Apolipoprotein B (apo B), fibrinogen/fibrin, blood platelets, factor VIII-related antigen of the blood coagulation system, and smooth muscle cells (SMC) were identified in the intima of normal and atherosclerotic human aorta and large arteries by the indirect immunofluorescence technique. Fibrinogen/fibrin was revealed by a monoclonal antibody (monAb) against the C-terminal region of human fibrinogen A alpha-chain. Fibronectin was visualized by monAb to the cellular form and against an epitope shared by different fibronectin subunit variants. In normal intima, fatty streaks, small amounts of fibrinogen/fibrin together with large amounts of apo B were observed. Fibronectin detected by two types of monAb was not found in extracellular matrix (ECM), whereas cellular fibronectin encircled SMC. According to the data obtained, fibrinogen/fibrin accumulates in plaques as a result of intramural thrombus incorporation, blood insudation, intramural haemorrhage, and in or around cells, apparently macrophages.

Aged↗
PubMed1990

[Agonist-induced platelet adhesion: use of the method in patients with myocardial infarction].

A new method of agonist-induced platelet adhesion has been developed for the evaluation of platelet activity. Platelet adhesion to plastic was stimulated by low doses of ADP, epinephrine and stable thromboxane analogue U46619. These inducers cause more than 3-fold increase of platelet adhesion in concentrations by 5-10 times lower than those necessary for stimulation of platelet aggregation in Born aggregometer. The method was applied for evaluation of platelet adhesive activity in patients with myocardial infarction. A dramatic increase of agonists-induced platelet adhesion was registered in patients with acute infarction, most significantly expressed when epinephrine was used as platelet agonist.

Adenosine Diphosphate↗
PubMed1989

Platelet interaction with [125I]-labeled collagen type III: requirement of fibrillar structure formation.

Interaction of collagen type III (CIII) with washed human platelets was studied using CIII preparation from human placenta. CIII was labeled with [125I] and [125I]-CIII in monomeric and fibrillar form [( 125I]-CIIIm and [125I]-CIIIf respectively) were incubated with platelets at room temperature. Platelet-associated and free labels were separated by centrifugation through 20% sucrose. Binding of [125I]-CIIIf was unsaturable, linearly dependent on the concentration of label and represented 28 +/- 3% of the added protein. In comparison with CIIIf, binding of [125I]-CIIIm was minimal and represents only 0.9 +/- 0.2% of the added protein. The binding of [125]-CIIIm was also nonsaturable and linearly depend on the concentration of the labeled protein. Platelet activation neither increases the CIIIf binding, nor stimulates the binding of CIIIm. The binding of [125I]-CIIIf was not inhibited by the excess of the unlabeled CIIIm. The data obtained suggests the absence of high-affinity collagen receptors in platelets and corroborates the hypothesis of multiple low-affinity interactions between collagen fibrils and platelet surface. Binding of CIIIf was very fast--the level of binding reached a plateau within 1 min, and was similar in the presence of Ca2+/Mg2+ and EDTA. Spectrophotometrically undetectable microfibril formation during the lag phase of fibrillogenesis was sufficient for nearly the same as with large fibrils binding of CIII to platelets. Unlike platelets red blood cells (RBC) fail to bind significant amounts of [125I]-CIIIf.

Autoradiography↗
PubMed1989

[Determination of antithrombocyte antibodies in the blood serum of patients with idiopathic thrombocytopenic purpura by an immunoenzyme method].

Enzyme-linked immunosorbent assay (ELISA) was developed for determination of serum antiplatelet antibodies. Platelets obtained from healthy donors of blood group 0(1) were washed off plasma and sedimented on the bottom of microtest wells. After washing off unattached platelets and blocking of plastic with albumin platelets were incubated with sera under investigation and binding of serum antibodies was detected using antihuman immunoglobulin antibodies conjugated with peroxidase. Ten patients with idiopathic thrombocytopenic purpura (ITP). 1 patient with systemic lupus erythematosus. 1 patient with red blood cell aplasia and 9 healthy donors (negative control) were studied by ELISA. Serum antibodies which effectively bound to platelets were detected in 5 patients with ITP, in patient with lupus erythematosus and in patient with red blood cell aplasia.

ABO Blood-Group System↗
PubMed1989

[Interaction of platelets with 125I-labelled collagen type III. The necessity for forming a fibrillary structure].

The interaction of type III collagen (CIII) with washed human platelets was studied, using a CIII preparation from human placenta. CIII was labeled with 125I, and the monomeric and fibrillar forms of 125I-CIII (125I-CIIIm and 125I-CIIIf, respectively) were incubated with the platelets at room temperature. The platelet-associated and free labels were separated by centrifugation through 20% sucrose. The binding of 125I-CIIIf was unsaturable, linearly dependent on the label concentration and made up to 28 +/- 3% of the added protein. In comparison with CIIIf, the binding of 125I-CIIIm was minimal, i.e., only 0.9 +/- 0.2% of the added protein; so it did not significantly increase the background level (label sedimented through 20% sucrose in the absence of platelets). Although the level of 125I-CIIIm was very low, the binding was also unsaturable and linearly dependent on the concentration of the labeled protein. Platelet activation did not influence the level of CIIIf binding, nor did it stimulate the binding of CIIIm. The binding of 125I-CIIIf was not inhibited by the unlabeled CIIIm. The data obtained testify to the absence of high affinity platelet collagen receptors and support the hypothesis on multiple low affinity interactions between collagen fibrils and platelet surface. The binding of CIIIf to platelets was characterized by very fast kinetics; the level of binding reached a plateau within the range of 1 min and was similar in the presence of Ca2+/Mg2+ and EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Platelets↗
PubMed1988

Potentiation of platelet interaction with collagen substrates by heparin is insensitive to aspirin.

The effects of two standard, unfractionated heparin preparations on collagen-induced adhesion and aggregation of platelets were studied by Born aggregometry and scanning electron microscopy (SEM). Heparin from porcine intestinal mucosa (HI) and from bovine lung (HL) added to human PRP in the concentration of 2.5 to 5.0 U/ml (1) did not induce platelet aggregation in suspension by itself but stimulated it in combination with the subthreshold doses of fibrillar human collagen type III (CIII); (2) increased by 1.5-2.0 fold the adhesion, but did not affect platelet spreading on a surface coated with human collagen type IV (CIV); and (3) enlarged by 2.5-3.0 fold the area of the CIII-coated surface covered with aggregates, increasing both the number and the size of surface-bound aggregates. Aspirin blocked platelet aggregation in suspension induced by low, near threshold doses, of fibrillar CIII and by subthreshold doses of CIII in combination with heparin, but had no effect on platelet aggregation induced by high (greater than 10 threshold) doses of CIII. Aspirin failed to decrease platelet adhesion to and spreading on CIV substrate, and formation of surface-bound aggregates on CIII substrate in the absence as well as in the presence of heparin.

Aspirin↗