scholarly journals Fibrinogen Hershey IV: A novel dysfibrinogen with a γV411I mutation in the integrin αIIbβ3 binding site

2008 ◽  
Vol 99 (06) ◽  
pp. 1008-1012 ◽  
Author(s):  
Veronica Flood ◽  
Hamid Al-Mondhiry ◽  
Chantelle Rein ◽  
Kristine Alexander ◽  
Rehana Lovely ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Biological Effects ◽  
Terminal Segment ◽  
Wild Type ◽  
Binding Assays ◽  
Normal Platelet ◽  
Platelet Receptor ◽  
Direct Binding ◽  
Aggregation Inhibition ◽  
Carboxyl Terminal

SummaryThe carboxyl terminal segment of the fibrinogen γ chain from γ408–4ll plays a crucial role in platelet aggregation via interactions with the platelet receptor αIIbβ3. We describe here the first naturally-occurring fibrinogen point mutation affecting this region and demonstrate its effects on platelet interactions. DNA sequencing was used to sequence the proband DNA, and platelet aggregation and direct binding assays were used to quantitate the biological effects of fibrinogen Hershey IV. The Hershey IV proband was found to be heterozygous for two mutations, γV411I and γR275C. Little difference in aggregation was seen when fibrinogen Hershey IV was compared to normal fibrinogen. However, less aggregation inhibition was observed using a competing synthetic dodecapeptide containing the V411I mutation as compared to the wild-type dodecapeptide. Purified fibrinogen Hershey IV also bound to purified platelet αIIbβ3 with a lower affinity than wild-type fibrinogen. These findings show that the γV411I mutation results in a decreased ability to bind platelets. In the heterozygous state, however, the available wild-type fibrinogen appears to be sufficient to support normal platelet aggregation.

Fibrinogen Hershey IV: A Novel Dysfibrinogen with a γ V411I Mutation in the Integrin αIibβ3 Binding Site.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2133-2133
Author(s):  
Veronica H. Flood ◽  
Hamid A. Al-Mondhiry ◽  
Antony C. Bakke ◽  
David H. Farrell
Keyword(s):  
Platelet Aggregation ◽  
Novel Mutation ◽  
Terminal Segment ◽  
Normal Donor ◽  
Thrombin Time ◽  
Wild Type ◽  
Mutant Peptide ◽  
Fibrinogen Binding ◽  
Platelet Receptor ◽  
Bleeding History

Abstract The C-terminal segment of the fibrinogen γ chain plays a crucial role in platelet aggregation via its interaction with the platelet receptor αIIbβ3. It is well established that the last four amino acids of the γ chain (408 to 411) are critical for this function, as mutations or deletions of this region abrogate fibrinogen’s ability to bind αIIbβ3. We describe here the first naturally occurring fibrinogen mutation affecting the C-terminal region of the γ chain and investigate its effects on platelet interactions. The proband, a 49-year-old woman, was diagnosed with dysfibrinogenemia based on a prolonged thrombin time and low fibrinogen activity (55 mg/dL). Her bleeding history was significant for menorrhagia and one episode of post-operative hemorrhage. DNA sequencing of the fibrinogen genes demonstrated heterozygosity for two mutations, γ R275C and γ V411I. The latter γ V411I mutation represents a novel mutation affecting the C-terminal amino acid of the γ chain. We hypothesized that this mutation would decrease fibrinogen’s affinity for the platelet receptor αIIbβ3. In order to isolate the effects of this mutation on fibrinogen-platelet binding, γ 400-411 dodecapeptides were synthesized to mimic the C-terminal γ chain sequence. One peptide contained the wild-type sequence ending in valine (γ 400-V411), and the second peptide incorporated the isoleucine mutation (γ 400-I411). Previous studies have demonstrated that the wild type γ 400-V411 dodecapeptide inhibits platelet aggregation by competing for fibrinogen binding to αIIbβ3. We performed platelet aggregation studies comparing inhibition of aggregation with the wild-type γ 400-V411 and the mutant γ 400-I411 peptides. Washed platelets were obtained from a normal donor, and platelet aggregation monitored using the agonist ADP. The IC50 for the initial rate of aggregation with the γ 400-I411 peptide was 214 μM, compared to 133 μM with the wild-type peptide. We then examined the extent of aggregation in the presence of either wild-type or mutant peptide. Consistent with the previous results, total aggregation was lower with the wild-type peptide compared to the mutant peptide. The IC50 for the γ 400-I411 peptide was 450 μM compared to 250 μM with the γ 400-V411 peptide. Overall, these findings suggest that the γ I411 mutation results in a decreased ability to bind platelets. In the heterozygous state, however, the available amount of wild-type fibrinogen may be sufficient to support platelet aggregation. The bleeding diathesis observed for the proband could therefore reflect other factors, especially the γ R275C mutation.

Disparities in estimates of IgG bound to normal platelets

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 200-202 ◽  
Author(s):  
N Blumberg ◽  
D Masel ◽  
M Stoler
Keyword(s):  
Direct Measurement ◽  
Immunosorbent Assay ◽  
Competitive Binding ◽  
Enzyme Linked Immunosorbent Assay ◽  
Binding Assays ◽  
Competitive Assay ◽  
Standard Curve ◽  
Normal Platelet ◽  
Direct Binding ◽  
Competitive Binding Assays

Abstract Estimates of the number of IgG molecules bound to normal platelets have ranged from several hundred to several tens of thousands. The lower estimates were generated from direct binding assays and stoichiometric assumptions. The higher values derive from competitive binding assays, in which platelet-associated IgG (PAIgG) is calculated from a standard curve using soluble IgG standards. Using a kinetic-ELISA (enzyme-linked immunosorbent assay) antiglobulin assay, we measured normal platelet IgG to be 21,200 +/- 9,400 molecules per platelet when a competitive assay and soluble IgG standards were used. Direct measurement of bound antiglobulin by kinetic-ELISA and stoichiometric assumptions yielded a measurement of 259 +/- 117 IgG molecules per platelet. Soluble IgG and PAIgG are not comparable in their ability to bind anti-IgG. Disparities in estimates of normal PAIgG are probably due to methodological differences. The estimate most likely to be correct is several hundred IgG or less per normal platelet.

scholarly journals Human Platelets Recognize a Novel Surface Protein, PadA, on Streptococcus gordonii through a Unique Interaction Involving Fibrinogen Receptor GPIIbIIIa

2009 ◽  
Vol 78 (1) ◽  
pp. 413-422 ◽  
Author(s):  
Helen J. Petersen ◽  
Ciara Keane ◽  
Howard F. Jenkinson ◽  
M. Margaret Vickerman ◽  
Amy Jesionowski ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Surface Protein ◽  
Dependent Manner ◽  
Streptococcus Gordonii ◽  
Wild Type ◽  
Fibrinogen Receptor ◽  
Platelet Receptor

ABSTRACT The concept of an infectious agent playing a role in cardiovascular disease is slowly gaining attention. Among several pathogens identified, the oral bacterium Streptococcus gordonii has been implicated as a plausible agent. Platelet adhesion and subsequent aggregation are critical events in the pathogenesis and dissemination of the infective process. Here we describe the identification and characterization of a novel cell wall-anchored surface protein, PadA (397 kDa), of S. gordonii DL1 that binds to the platelet fibrinogen receptor GPIIbIIIa. Wild-type S. gordonii cells induced platelet aggregation and supported platelet adhesion in a GPIIbIIIa-dependent manner. Deletion of the padA gene had no effect on platelet aggregation by S. gordonii but significantly reduced (>75%) platelet adhesion to S. gordonii. Purified N-terminal PadA recombinant polypeptide adhered to platelets. The padA mutant was unaffected in production of other platelet-interactive surface proteins (Hsa, SspA, and SspB), and levels of adherence of the mutant to fetuin or platelet receptor GPIb were unaffected. Wild-type S. gordonii, but not the padA mutant, bound to Chinese hamster ovary cells stably transfected with GPIIbIIIa, and this interaction was ablated by addition of GPIIbIIIa inhibitor Abciximab. These results highlight the growing complexity of interactions between S. gordonii and platelets and demonstrate a new mechanism by which the bacterium could contribute to unwanted thrombosis.

scholarly journals Uptake and metabolic fate of [HisA8,HisB4,GluB10,HisB27]insulin in rat liver in vivo

1998 ◽  
Vol 332 (2) ◽  
pp. 421-430 ◽  
Author(s):  
François AUTHIER ◽  
Gianni M. Di GUGLIELMO ◽  
Gillian M. DANIELSEN ◽  
John J. M. BERGERON
Keyword(s):  
Insulin Receptor ◽  
Subcellular Fractionation ◽  
Wild Type ◽  
Binding Assays ◽  
Insulin Degrading Enzyme ◽  
Direct Binding ◽  
Temporal Interaction ◽  
Kinetics Of Uptake

Receptor-mediated endocytosis and subsequent endosomal proteolysis of [125I]TyrA14-[HisA8,HisB4,GluB10,HisB27]insulin ([125I]TyrA14-H2 analogue), an insulin analogue exhibiting a high affinity for the insulin receptor, has been studied in liver parenchymal cells by quantitative subcellular fractionation and compared with that of wild-type [125I]TyrA14-insulin. Whereas the kinetics of uptake of the H2 analogue by liver was not different from that of insulin, the H2 analogue radioactivity after the 2 min peak declined significantly more slowly. A significant retention of the H2 analogue compared with insulin in both plasma membrane and endosomal fractions was observed and corresponded to decreased processing and dissociation of the H2 analogue. Cell-free endosomes preloaded in vivo with radiolabelled ligands and incubated in vitro processed insulin and extraluminally released insulin intermediates at a 2–3-fold higher rate than the H2 analogue. In vitro proteolysis of both non-radiolabelled and monoiodinated molecules by endosomal lysates showed a decreased response to the endosomal proteolytic machinery for the H2 analogue. However, in cross-linking and competition studies the H2 analogue exhibited an affinity for insulin-degrading enzyme identical with that of wild-type insulin. Brij-35-permeabilized endosomes revealed a 2-fold higher rate of dissociation of insulin from internalized receptors compared with the H2 analogue. After the administration of a saturating dose of both ligands, a rapid and reversible ligand-induced translocation of insulin receptor was observed, but without receptor loss. The H2 analogue induced a higher receptor concentration and tyrosine autophosphorylation of the receptor β subunit in endosomes. Moreover, a prolonged temporal interaction of the in vivo injected H2 analogue with receptor was observed by direct binding assays performed on freshly prepared subcellular fractions. These results indicate that endosomal proteolysis for the H2 analogue is slowed as a result of an increased residence time of the analogue on the insulin receptor and a low affinity of endosomal acidic insulinase for the dissociated H2 molecule.

Disparities in estimates of IgG bound to normal platelets

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 200-202
Author(s):  
N Blumberg ◽  
D Masel ◽  
M Stoler
Keyword(s):  
Direct Measurement ◽  
Immunosorbent Assay ◽  
Competitive Binding ◽  
Enzyme Linked Immunosorbent Assay ◽  
Binding Assays ◽  
Competitive Assay ◽  
Standard Curve ◽  
Normal Platelet ◽  
Direct Binding ◽  
Competitive Binding Assays

Estimates of the number of IgG molecules bound to normal platelets have ranged from several hundred to several tens of thousands. The lower estimates were generated from direct binding assays and stoichiometric assumptions. The higher values derive from competitive binding assays, in which platelet-associated IgG (PAIgG) is calculated from a standard curve using soluble IgG standards. Using a kinetic-ELISA (enzyme-linked immunosorbent assay) antiglobulin assay, we measured normal platelet IgG to be 21,200 +/- 9,400 molecules per platelet when a competitive assay and soluble IgG standards were used. Direct measurement of bound antiglobulin by kinetic-ELISA and stoichiometric assumptions yielded a measurement of 259 +/- 117 IgG molecules per platelet. Soluble IgG and PAIgG are not comparable in their ability to bind anti-IgG. Disparities in estimates of normal PAIgG are probably due to methodological differences. The estimate most likely to be correct is several hundred IgG or less per normal platelet.

scholarly journals Platelet Receptor Activity for Predicting Survival in Patients with Intracranial Bleeding

2021 ◽  
Vol 10 (10) ◽  
pp. 2205
Author(s):  
Barbara Dragan ◽  
Barbara Adamik ◽  
Malgorzata Burzynska ◽  
Szymon Lukasz Dragan ◽  
Waldemar Gozdzik
Keyword(s):  
Platelet Aggregation ◽  
Function Analysis ◽  
Strong Predictor ◽  
Adenosine Diphosphate ◽  
Intracranial Bleeding ◽  
Rank Test ◽  
Blood Coagulation Disorders ◽  
Platelet Response ◽  
Normal Platelet ◽  
Platelet Receptor

Blood coagulation disorders in patients with intracranial bleeding as a result of head injuries or ruptured aneurysms are a diagnostic and therapeutic problem and appropriate assessments are needed to limit CNS damage and to implement preventive measures. The aim of the study was to monitor changes in platelet aggregation and to assess the importance of platelet dysfunction for predicting survival. Platelet receptor function analysis was performed using the agonists arachidonic acid (ASPI), adenosine diphosphate (ADP), collagen (COL), thrombin receptor activating protein (TRAP), ristocetin (RISTO) upon admission to the ICU and on days 2, 3, and 5. On admission, the ASPI, ADP, COL, TRAP, and RISTO tests indicated there was reduced platelet aggregation, despite there being a normal platelet count. In ‘Non-survivors’, the platelet response to all agonists was suppressed throughout the study period, while in ‘Survivors’ it improved. Measuring platelet function in ICU patients with intracranial bleeding is a strong predictor related to outcome: patients with impaired platelet aggregation had a lower 28-day survival rate compared to patients with normal platelet aggregation (log-rank test p = 0.014). The results indicated that measuring platelet aggregation can be helpful in the early detection, diagnosis, and treatment of bleeding disorders.

Platelet Activation and Aggregation Induced by Recombinant von Willebrand Factors Reproducing Four Type 2B von Willebrand Disease Missense Mutations

1998 ◽  
Vol 79 (01) ◽  
pp. 211-216 ◽  
Author(s):  
Lysiane Hilbert ◽  
Claudine Mazurier ◽  
Christophe de Romeuf
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Inhibit Platelet Aggregation ◽  
Wild Type ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryType 2B of von Willebrand disease (vWD) refers to qualitative variants with increased affinity of von Willebrand factor (vWF) for platelet glycoprotein Ib (GPIb). All the mutations responsible for type 2B vWD have been located in the A1 domain of vWF. In this study, various recombinant von Willebrand factors (rvWF) reproducing four type 2B vWD missense mutations were compared to wild-type rvWF (WT-rvWF) for their spontaneous binding to platelets and their capacity to induce platelet activation and aggregation. Our data show that the multimeric pattern of each mutated rvWF is similar to that of WT-rvWF but the extent of spontaneous binding and the capacity to induce platelet activation and aggregation are more important for the R543Q and V553M mutations than for the L697V and A698V mutations. Both the binding of mutated rvWFs to platelets and platelet aggregation induced by type 2B rvWFs are inhibited by monoclonal anti-GPIb and anti-vWF antibodies, inhibitors of vWF binding to platelets in the presence of ristocetin, as well as by aurin tricarboxylic acid. On the other hand, EDTA and a monoclonal antibody directed against GPIIb/IIIa only inhibit platelet aggregation. Furthermore, the incubation of type 2B rvWFs with platelets, under stirring conditions, results in the decrease in high molecular weight vWF multimers in solution, the extent of which appears correlated with that of plasma vWF from type 2B vWD patients harboring the corresponding missense mutation. This study supports that the binding of different mutated type 2B vWFs onto platelet GPIb induces various degrees of platelet activation and aggregation and thus suggests that the phenotypic heterogeneity of type 2B vWD may be related to the nature and/or location of the causative point mutation.

Essential Athrombia

1975 ◽  
Vol 33 (02) ◽  
pp. 278-285 ◽  
Author(s):  
Şeref Inceman ◽  
Yücel Tangün
Keyword(s):  
Platelet Aggregation ◽  
Bleeding Time ◽  
Bleeding Tendency ◽  
Clot Retraction ◽  
Platelet Factor ◽  
Prolonged Bleeding Time ◽  
Normal Platelet ◽  
Aggregation Response ◽  
Platelet Disorder ◽  
Fibrinogen Content

SummaryA constitutional platelet function disorder in a twelve-year-old girl characterized by a lifelong bleeding tendency, prolonged bleeding time, normal platelet count, normal clot retraction, normal platelet factor 3 activity and impaired platelet aggregation was reported.Platelet aggregation, studied turbidimetrically, was absent in the presence of usual doses of ADP (1–4 μM), although a small wave of primary aggregation was obtained by very large ADP concentrations (25–50 μM). The platelets were also unresponsive to epinephrine, thrombin and diluted collagen suspensions. But an almost normal aggregation response occurred with strong collagen suspensions. The platelets responded to Ristocetin. Pelease of platelet ADP was found to be normal by collagen and thrombin, but impaired by kaolin. Platelet fibrinogen content was normal.The present case, investigated with recent methods, confirms the existence of a type of primary functional platelet disorder characterized solely by an aggregation defect, described in 1955 and 1962 under the name of “essential athrombia.”

Modulation of Platelet Activation by Native DNA – Initial Description of Platelet Receptor Type, Number and Discrimination for Native DNA

1981 ◽  
Vol 45 (03) ◽  
pp. 263-266 ◽  
Author(s):  
B A Fiedel ◽  
M E Frenzke
Keyword(s):  
Platelet Aggregation ◽  
Human Platelets ◽  
Scatchard Analysis ◽  
Type Number ◽  
Single Class ◽  
Receptor Ligand ◽  
Platelet Receptor ◽  
Platelet Binding ◽  
Ligand Interactions ◽  
Initial Description

SummaryNative DNA (dsDNA) induces the aggregation of isolated human platelets. Using isotopically labeled dsDNA (125I-dsDNA) and Scatchard analysis, a single class of platelet receptor was detected with a KD = 190 pM and numbering ~275/platelet. This receptor was discriminatory in that heat denatured dsDNA, poly A, poly C, poly C · I and poly C · poly I failed to substantially inhibit either the platelet binding of, or platelet aggregation induced by, dsDNA; by themselves, these polynucleotides were ineffective as platelet agonists. However, poly G, poly I and poly G · I effectively and competitively inhibited platelet binding of the radioligand, independently activated the platelet and when used at a sub-activating concentration decreased the extent of dsDNA stimulated platelet aggregation. These data depict a receptor on human platelets for dsDNA and perhaps certain additional polynucleotides and relate receptor-ligand interactions to a physiologic platelet function.

A Physiologic Regulator of Collagen-Induced Platelet Aggregation: Inhibition by Clq

1981 ◽  
Vol 45 (02) ◽  
pp. 110-115 ◽  
Author(s):  
György Csákó ◽  
Eva A Suba
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Platelet Reactivity ◽  
High Specificity ◽  
Turbidimetric Method ◽  
Specific Manner ◽  
Aggregation Inhibition ◽  
Different Tissues

SummaryPlatelet aggregations were studied by a turbidimetric method in citrated human platelet-rich plasmas (PRP) in vitro. Human Clq inhibited the aggregations caused by collagens derived from different tissues and species. Clq was needed by weight in comparable quantities to collagen for neutralizing the aggregating effect. The dependence of the inhibitory reaction on the preincubation of platelets with Clq and the differences in the occurrence of aggregating substances in supernatants of PRP triggered with collagen in the presence or absence of Clq, confirmed that Clq exerts its effect by preventing fixation of collagen to platelets. In addition, the high specificity of the inhibitory action of Clq for collagen-induced platelet aggregation was demonstrated by results obtained for testing a variety of aggregating agents in combination with Clq and/or collagen.Since normal concentrations of Clq in the blood are in the range of inhibitory doses of Clq for collagen-induced platelet aggregations in vitro and upon activation of complement Clq is known to dissociate from Cl, it is proposed that Clq may participate in a highly specific manner in regulating platelet reactivity to collagen in vivo.

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