Platelet antithrombin defect in malignancy: platelet protein alterations

Abstract Sixty-eight patients with malignant disease were divided into two groups based on the results of the platelet antithrombin test (PAT). The normal group had a PAT clotting time ranging from 21.4 to 29.8 seconds, which was equivalent to 25% to 65% inactivation of the 2 U of thrombin added to the test system. The other group showed abnormal PAT clotting time, less than 21.4 seconds or less than 25% thrombin inactivation. The polypeptide composition of platelets from the two patient groups was analyzed by sodium dodecyl sulfate (SDS)- electrophoresis on 7.5% polyacrylamide gels. A polypeptide of 180,000 apparent mol wt was decreased or absent in both Coomassie blue- and Alcian blue-stained gels of the platelets from patients whose PAT was abnormal; this polypeptide comigrated with purified platelet thrombospondin. Tritium labeling of platelet surface glycoproteins by the periodate-borohydride method followed by two-dimensional electrophoresis was performed on platelets of seven patients with abnormal PAT. When they were compared with ten patients with normal PAT, a glycoprotein of 140,000 apparent mol wt with a pl of 4.5 to 5.2 was decreased in platelets of all seven patients with abnormal PAT. Nitrocellulose replicas of one-dimensional gels of platelets from 13 of 14 patients with abnormal PAT showed decreased reaction with an anti- human platelet glycocalicin antiserum. Platelets of these same patients also showed a decreased or absent platelet agglutination induced by ristocetin. Patients with normal PAT had a mean agglutination slope of 1.25 +/- 0.6 (n = 26) as compared with 0.37 +/- 0.34 (n = 26) for the abnormal PAT group (P less than .001). Results indicate that platelets from a subpopulation of tumor patients characterized by decreased platelet antithrombin activity have alterations in two platelet glycoproteins, identified as GPIb and thrombospondin.

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Platelet antithrombin defect in malignancy: platelet protein alterations

Blood ◽

10.1182/blood.v69.2.479.bloodjournal692479 ◽

1987 ◽

Vol 69 (2) ◽

pp. 479-485

Author(s):

EM Sloand ◽

DM Kenney ◽

FC Chao ◽

J Lawler ◽

JL Tullis

Keyword(s):

Sodium Dodecyl ◽

Test System ◽

Clotting Time ◽

Platelet Surface ◽

Platelet Protein ◽

Coomassie Blue ◽

Patient Groups ◽

Protein Alterations ◽

Sds Electrophoresis ◽

Tritium Labeling

Sixty-eight patients with malignant disease were divided into two groups based on the results of the platelet antithrombin test (PAT). The normal group had a PAT clotting time ranging from 21.4 to 29.8 seconds, which was equivalent to 25% to 65% inactivation of the 2 U of thrombin added to the test system. The other group showed abnormal PAT clotting time, less than 21.4 seconds or less than 25% thrombin inactivation. The polypeptide composition of platelets from the two patient groups was analyzed by sodium dodecyl sulfate (SDS)- electrophoresis on 7.5% polyacrylamide gels. A polypeptide of 180,000 apparent mol wt was decreased or absent in both Coomassie blue- and Alcian blue-stained gels of the platelets from patients whose PAT was abnormal; this polypeptide comigrated with purified platelet thrombospondin. Tritium labeling of platelet surface glycoproteins by the periodate-borohydride method followed by two-dimensional electrophoresis was performed on platelets of seven patients with abnormal PAT. When they were compared with ten patients with normal PAT, a glycoprotein of 140,000 apparent mol wt with a pl of 4.5 to 5.2 was decreased in platelets of all seven patients with abnormal PAT. Nitrocellulose replicas of one-dimensional gels of platelets from 13 of 14 patients with abnormal PAT showed decreased reaction with an anti- human platelet glycocalicin antiserum. Platelets of these same patients also showed a decreased or absent platelet agglutination induced by ristocetin. Patients with normal PAT had a mean agglutination slope of 1.25 +/- 0.6 (n = 26) as compared with 0.37 +/- 0.34 (n = 26) for the abnormal PAT group (P less than .001). Results indicate that platelets from a subpopulation of tumor patients characterized by decreased platelet antithrombin activity have alterations in two platelet glycoproteins, identified as GPIb and thrombospondin.

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Studies on The Function of Platelet Surface Antigens

10.1055/s-0039-1687065 ◽

1979 ◽

Author(s):

J Connellan ◽

I. Smith ◽

B. Barlow ◽

P.A. Castaldi

Keyword(s):

Gel Electrophoresis ◽

Antiplatelet Antibody ◽

Platelet Surface ◽

Platelet Membranes ◽

Coomassie Blue ◽

Rabbit Igg ◽

Centrifuge Tube ◽

Agar Gel Electrophoresis ◽

Membrane Components ◽

Sds Electrophoresis

Heterologous antibodies to platelets and isolated membranes have influences on the response of intact platelets to aggregating stimuli. This study is concerned with identification of the active antigens in an effort to assign a functional role to specified membrane components. Antisera were produced in rabbits to membranes obtained from washed platelets. Platelets were labelled with 125I by using lactoperoxidase and DD125ISA. 4 major labelled proteins were obtained on SDS gel electrophoresis. Several of these labelled proteins reacted with the antiplatelet membrane antibody. These proteins were isolated by treating a mixture of platelet membranes with antiplatelet antibody and subsequently incubated with goat anti-rabbit IgG. Two proteins were detected in this fraction on SDS electrophoresis. IgG was purified from antisera by QAE sephadex chromatography and insolubilized on CNBr-Sepharose. Disrupted platelets or platelet membranes were exposed to the immobilized IgG in a centrifuge tube. The Sepharoae-IgG-antigen complex was sedimented, washed with Tris ph 7.5, 3M NaCl and finally Guanidine HCl. Three proteins were detected on SDS electrophoresis of the Guanidine-HCl wash. Crossover Agar gel electrophoresis was performed on platelet membranes and whole platelets solubilized in triton. Two protein reactants, with a line of partial identity, were evident when the gels were stained with Coomassie Blue. since F (ab)2 fragments isolated from the antiserum have inhibitory effects in the response to ristocetin and collagen, it is possible that the proteins under study may be involved in some platelet surface reactions.

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dRVVT Is more Sensitive than KCT or TTI for Detecting Lupus Anticoagulant Activity of Anti-β2-glycoprotein I Autoantibodies

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614453 ◽

1999 ◽

Vol 81 (02) ◽

pp. 256-258 ◽

Cited By ~ 53

Author(s):

A. Biasiolo ◽

P. Rampazzo ◽

T. Brocco ◽

V. Pengo

Keyword(s):

Lupus Anticoagulant ◽

Anticoagulant Activity ◽

Test System ◽

Clotting Time ◽

Lupus Anticoagulants ◽

Glycoprotein I ◽

Tissue Thromboplastin ◽

The Mean ◽

Kaolin Clotting Time ◽

Antibody Preparations

SummaryAnti-β2-glycoprotein I (β2-GPI) antibodies behave as classical Lupus Anticoagulants (LA), as they inhibit phospholipid-dependent coagulation reactions and their activity disappears in the presence of excess exogenous phospholipids (PLs). We have recently shown that a certain amount of PLs in the dilute Russell Viper Venom Time (dRVVT) test system is required to express LA activity of anti β2-GPI antibodies. We have now extended this observation to two other tests, i.e., Kaolin Clotting Time (KCT) in which PLs are not added, and Tissue Thromboplastin Inhibition test (TTI) in which PLs are extremely diluted. In fact, affinity-purified antibody preparations from 5 patients with antiphospholipid syndrome did not express or only weakly expressed anticoagulant activity in both tests; the mean ratios of coagulation times obtained with purified antibodies and that of control buffer were 1.11 and 1.0 for KCT and TTI, respectively. On the contrary, the mean ratios in dRVVT were 1.31 and 1.49 at a PLs dilution of 1:8 and 1:64, respectively. Therefore, the presence of LA activity due to autoantibodies to β2-GPI is characterized by a positive dRVVT and negative or only weakly positive KCT and TTI.

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Development and characterization of monoclonal antiplatelet autoantibodies from autoimmune thrombocytopenic purpura-prone (NZW x BXSB)F1 mice

Blood ◽

10.1182/blood.v82.3.837.bloodjournal823837 ◽

1993 ◽

Vol 82 (3) ◽

pp. 837-844 ◽

Cited By ~ 1

Author(s):

H Mizutani ◽

RW Engelman ◽

Y Kurata ◽

S Ikehara ◽

RA Good

Keyword(s):

Nude Mice ◽

Reticuloendothelial System ◽

Hybridoma Cells ◽

Enzyme Linked Immunosorbent Assay ◽

Thrombocytopenic Purpura ◽

Platelet Surface ◽

Autoimmune Thrombocytopenic Purpura ◽

Platelet Protein ◽

Double Stranded Dna ◽

Systemic Autoimmunity

Male (NZW x BXSB)F1 (W/BF1) mice develop systemic autoimmunity involving autoantibodies, progressive thrombocytopenia, lupus nephritis, and degenerative coronary vascular disease with myocardial infarction. Platelet-associated IgG (PAIgG) on the platelet surface mediates platelet destruction by the reticuloendothelial system in the autoimmune thrombocytopenic purpura (ATP) of W/BF1 mice. Because the epitopes targeted in ATP by PAIgG have not been identifiable using serum from thrombocytopenic W/BF1 mice, we developed seven hybridomas secreting antiplatelet monoclonal antibodies (MoAbs) using splenocytes of thrombocytopenic W/BF1 mice. Epitopes recognized by three MoAbs were similar to those recognized by PAIgG, because eluted IgG from platelets of thrombocytopenic W/BF1 mice inhibited platelet binding by MoAbs in competitive micro-enzyme-linked immunosorbent assay. Hybridoma cells or purified Ig from the ascites of two clones (2A12 and 6A6), when injected into nude mice produced acute thrombocytopenia, elevated the levels of PAIgG, purpura, and megakaryocytosis. MoAbs of two clones also reacted with single-stranded DNA or double-stranded DNA, and one of these clones (4–13) bound to cardiolipin (CL) but was nonpathogenic in nude mice, suggesting that anti-CL and antiplatelet autoantibodies can be distinct. On immunoblotting analysis, antiplatelet MoAbs frequently bound a 100-Kd platelet protein. These MoAbs contribute to an understanding of the etiopathogenesis of ATP and the several antigens and autoantibodies involved.

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Equine synovial fluid protein equalization via combinatorial peptide ligand libraries

Turkey Diseases, Production and Management - German Journal of Veterinary Research ◽

10.51585/gjvr.2021.4.0026 ◽

2021 ◽

Vol 1 (4) ◽

pp. 18-22

Author(s):

Pablo Fueyo ◽

Marco Galleguillos ◽

Cristóbal Dörner ◽

Pedro A. Smith ◽

Francisca Godoy ◽

...

Keyword(s):

Synovial Fluid ◽

Sodium Dodecyl ◽

Electrophoretic Pattern ◽

Peptide Ligand ◽

Joint Pathology ◽

Coomassie Blue ◽

Standard Procedures ◽

Sds Page ◽

New Biomarkers ◽

Synovial Fluid Protein

To gain further knowledge of the equine synovial fluid (SF) proteome, we propose a protocol based on the equalization of the relative concentrations of its proteins, which leads to the modification of the standard electrophoretic pattern revealing low-abundance proteins that otherwise remain undetected. Fresh SF samples were collected from ten macroscopically normal metacarpophalangeal joints of crossbred horses. The samples were processed using standard procedures as the control and via combinatorial peptide ligand libraries (CPLL) using low ionic forces (NaH2PO4 10 mM) at different pHs (4.0, 7.0, and 9.3) with 10% sodium dodecyl sulfate (SDS) and 25 mM DTT for protein resolubilization. Proteins were then separated by conventional 8% SDS-PAGE and stained with coomassie blue. After separation of the equalized proteins, there was a significant reduction in the albumin (the most abundant protein in the SF) and, at the same time, other protein bands arise that were not visible without CPLL processing. In addition, there was variation in the protein profiles at different pHs. The results suggest that protein equalization of the equine SF by CPLL could be a useful tool to better understand the articular homeostasis and/or for the detection of new biomarkers of joint pathology.

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Additional Glycoprotein Defects In Glanzmann’s Thrombasthenia Platelets

10.1055/s-0038-1652008 ◽

1981 ◽

Author(s):

J L McGregor ◽

K J Clemetson ◽

E James ◽

A Capitanio ◽

M Dechavanne ◽

...

Keyword(s):

Sialic Acid ◽

Gel Electrophoresis ◽

Lens Culinaris ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Platelet Surface ◽

Glanzmann’S Thrombasthenia ◽

Healthy Donors ◽

Glanzmann's Thrombasthenia ◽

Dimensional Electrophoresis

Glanzmann’s thrombasthenia (G.T.) platelets are deficient in 2 major membrane GP (IIb and IIIa). In order to investigate if these are the only defects in this disorder, platelets from G.T. patients and from healthy donors were isolated, washed and surface-labelled by techniques specific for protein or for sugars (sialic acid or penultimate galactose/N-acetylgalactosamine residues). Labelled or unlabelled platelets were solubilized in sodium dodecyl sulphate (SDS) and separated by 2-dimensional polyacrylamide gel electrophoresis, first according to isoelectric point and then according to molecular weight. Glycoproteins from unlabelled platelets separated by 2-dimensional electrophoresis were identified by binding of 125I-labelled Lens culinaris lectin (mannose, glucose specific) GPIIbA1 and IIIaA1 were absent in one G.T. patient while in others lower amounts of 2 GP were found in positions similar to these GP. Major membrane GP (IbA1, IbA2, IbB1 and IIIbA1) had more intensely labelled terminal sialic acid moieties in G.T. platelets than in normals. A major membrane GP designated Ic had an altered pi and its penultimate galactose/N-acetyl galactosamine residues labelled more intensely in G.T. platelets than in controls. One high M.Wt. GP and a number of lower M.Wt. GP (IVa, IVb and VII) normally found in platelets of healthy donors were absent in G.T. platelets. These results indicate strongly that there is a major perturbation of the platelet surface in G.T.

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Immunochemical characterization of the platelet-specific alloantigen Leka: a comparative study with the PlA1 alloantigen

Blood ◽

10.1182/blood.v64.6.1212.1212 ◽

1984 ◽

Vol 64 (6) ◽

pp. 1212-1219 ◽

Cited By ~ 56

Author(s):

N Kieffer ◽

B Boizard ◽

D Didry ◽

JL Wautier ◽

AT Nurden

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Human Platelets ◽

Blue Staining ◽

Igg Antibody ◽

Immune Disorder ◽

Immunochemical Characterization ◽

Coomassie Blue Staining ◽

Coomassie Blue

Abstract We report the immunochemical characterization of a new platelet- specific alloantigen detected using an IgG antibody isolated from the serum of a patient with posttransfusion purpura (PTP). In indirect immunoprecipitation experiments, the antibody, termed anti-Leka, predominantly precipitated glycoprotein (GP) IIb from Triton X-100 lysates of normal human platelets. In an immunoblot procedure, which involved the transfer of platelet polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to nitrocellulose membrane, anti-Leka bound exclusively to GP IIb. Under identical conditions, four anti-PlA1 antibodies each reacted with GP IIIa. No binding of anti-Leka IgG occurred to Leka (-) platelets or to their separated polypeptides although GP IIb was normally detected by Coomassie blue staining. After electrophoresis of reduced platelet proteins, the Leka determinant was localized to the IIb alpha chain. Thus, unlike the PlA1 antigen, the Leka determinant was not destroyed by disulfide reduction. Analysis of platelets from a patient with Glanzmann's thrombasthenia revealed little or no binding in the GP IIb position. Anti-Leka permitted the identification of 76,000 and 60,000 dalton fragments of GP IIb retained by the platelet following chymotrypsin treatment. Our results further highlight the immunogenicity of the GP IIb-IIIa complex. They also suggest that antibodies against GP IIb can cause the thrombocytopenia observed in PTP and that anti-PlA1 antibodies do not account exclusively for the pathophysiology of this immune disorder.

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Antivinculin antibodies in sera of patients with immune thrombocytopenia and in sera of normal subjects

Blood ◽

10.1182/blood.v79.1.161.161 ◽

1992 ◽

Vol 79 (1) ◽

pp. 161-168 ◽

Cited By ~ 23

Author(s):

Y Tomiyama ◽

R Kekomaki ◽

J McFarland ◽

TJ Kunicki

Keyword(s):

Immune Thrombocytopenia ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Surface Protein ◽

Normal Subjects ◽

Enzyme Linked Immunosorbent Assay ◽

Two Dimensional ◽

Platelet Protein ◽

Sds Page ◽

Significant Difference

Abstract We have characterized a 120-Kd antigen that frequently reacts with serum antibodies from patients with immune thrombocytopenia or normal subjects. Immunoblots made after two-dimensional nonreduced/reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional isoelectric focusing/SDS-PAGE demonstrated that this 120-Kd protein has the same molecular weight under nonreduced or reduced conditions, is not a surface protein, and has an isoelectric point (pl) of 6.4 to 6.5. From these data, one likely candidate is the intracellular platelet protein, vinculin. Monoclonal antivinculin antibody reacts with this 120-Kd protein, and purified human platelet vinculin is bound by antibodies that recognize the 120-Kd protein. Therefore, we conclude that this 120-Kd protein is identical to vinculin. Data obtained from a sensitive enzyme-linked immunosorbent assay demonstrate the presence of naturally occurring antivinculin antibodies in many normal sera. However, the incidence of antivinculin antibodies in patient sera (67%; 55 of 82 sera) is significantly (P less than .01) higher than that in normal sera (40%; 32 of 80 sera), and there is a significant difference (P less than .05) between the mean levels of antivinculin antibodies in patient and normal sera. Whereas the levels of these antibodies in patient and normal sera overlap, 2 of 82 sera from patients with thrombocytopenia express unusually high levels of such antibodies. The pathologic significance of these antibodies remains to be determined.

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Phosphorylation Of Platelet Actin-Binding Protein During Platelet Activation

10.1055/s-0038-1652791 ◽

1981 ◽

Author(s):

Roger C Carroll ◽

Jonathan M Gerrard

Keyword(s):

Molecular Weight ◽

Platelet Activation ◽

Time Course ◽

Binding Protein ◽

Sodium Dodecyl ◽

Apparent Molecular Weight ◽

Actin Binding ◽

Actin Binding Protein ◽

Platelet Protein ◽

External Calcium

We have followed the 32P-labelling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples we found 2 to 3 fold labelling increases in actin-binding protein 30 to 60 seconds after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis of purified actin-binding protein as well as cytoskeletal cores prepared by detergent extraction of activated 32p-iabelled platelets. In addition, these cytoskeletal cores indicated that the 32P-labelled actin-binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labelling of actin-binding protein over an 8 minute time course revealed that in aggregating platelet samples rapid desphosphorylation to almost initial levels occurred between 3 and 5 minutes. A similar curve was obtained for the 20,000 apparent molecular weight peptide. This rapid dephosphorylation was shown to be dependent on platelet aggregation in the absence of external calcium or in thrombastenic platelets lacking the aggregation response to activation. These results suggest that phosphorylation of actin-binding protein initiates its association with the platelet cytoskeleton during activation.

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Investigation of the mechanisms of monoclonal antibody-induced platelet activation

Blood ◽

10.1182/blood.v78.4.1019.1019 ◽

1991 ◽

Vol 78 (4) ◽

pp. 1019-1026 ◽

Cited By ~ 78

Author(s):

P Horsewood ◽

CP Hayward ◽

TE Warkentin ◽

JG Kelton

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Binding Sites ◽

Fc Receptors ◽

Secondary Antibody ◽

Igg Antibodies ◽

Platelet Surface ◽

Platelet Protein ◽

Platelet Binding ◽

Protein Components

Abstract Antiplatelet antibodies can activate platelets causing platelet aggregation and the release reaction. However, the pathway of activation by these antibodies is unknown and several potential mechanisms are possible. In this report, we describe studies investigating potential pathways of platelet activation by IgG antibodies. We tested 16 different IgG monoclonal antibodies (MoAbs) against a variety of platelet surface components and found that six antibodies were capable of causing platelet aggregation and release. These included MoAbs against glycoprotein (GP) IIb/IIIa, CD9, GPIV, and two other not well-characterized platelet components. There was no relationship between the number of platelet binding sites and the ability of an MoAb to activate the platelets. By adding intact and F(ab')2 preparations of the MoAb to control or Fc receptor-blocked platelets, we found that in all instances the MoAbs initiated platelet activation via interacting with the platelet Fc receptors. Clustering of the platelet protein components using a secondary antibody did not cause activation. Studies into the pathway of Fc-dependent activation demonstrated that the MoAbs were capable of activating platelets by occupying Fc receptors on adjacent platelets (interplatelet activation), as well as on the same platelet (intraplatelet activation).

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