Optimally functional fluorescein isothiocyanate‐labelled fibrinogen for quantitative studies of binding to activated platelets and platelet aggregation

Xia, Zhicheng; Wong, Truman; Liu, Q.; Kasirer-Friede, Ana; Brown, Elizabeth A.; Frojmovic, Mony M.

doi:10.1046/j.1365-2141.1996.445980.x

Cited by 84 publications

(61 citation statements)

References 20 publications

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“…Highly purified human fibrinogen (Enzyme Research Laboratories, South Bend, IN) was conjugated with fluorescein isothiocyanate (FITC) by using FITC-celite (Calbiochem-Novabiochem, Bad Soden, Germany) according to the method of Xia et al, 24 with the following modifications: incubation was carried out at pH 7.8 for 48 hours at 4°C, resulting in an FITC-toprotein ratio of 5.0 to 5.2. Type I collagen prepared for use in flow cytometry was described previously.…”

Section: Methodsmentioning

confidence: 99%

Sustained integrin ligation involves extracellular free sulfhydryls and enzymatically catalyzed disulfide exchange

Lahav

Jurk

Hess

et al. 2002

Blood

184

209

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Studies have suggested a pivotal role for free sulfhydryls in platelet integrin function, and enzyme-mediated reduction of disulfide bonds on platelets has been implicated. The platelet fibrinogen receptor ␣ IIb ␤ 3 is the best-studied platelet integrin and serves as a model system for studying the structure-function relation in this family of adhesion receptors. The demonstration of free sulfhydryls on the exofacial domain of purified ␣ IIb ␤ 3 , specifically in its activated conformation, prompted us to explore the potential for activation-dependent, enzymatically catalyzed thiol expression on intact platelets and the possible role of surface-associated protein disulfide isomerase (PDI) in ␣ IIb ␤ 3 ligation. Using the membrane-impermeant sulfhydryl blocker para-chloromercuriphenyl sulfonate, the inhibitor of disulfide exchange bacitracin, and the monoclonal anti-PDI antibody RL90, we examined fibrinogen binding to ␣ IIb ␤ 3 as well as ligation-induced allosteric changes in the conformation of ␣ IIb ␤ 3 . We sought to distinguish the possible involvement of disulfide exchange in agonist-induced platelet stimulation from its role in integrin ligation. Analysis of the role of free thiols in platelet aggregation suggested a thiolindependent initial ligation followed by a thiol-dependent stabilization of binding. IntroductionThe affinity of integrins for their ligands is tightly regulated, both by cellular events and subsequent to ligand binding. [1][2][3][4][5] Such cellular control over the interaction of extracellular proteins implies the existence of a mechanism to propagate information back and forth between the extracellular head and the cytoplasmic tail of the integrin receptor. In recent years, the posited mechanism has been the allosteric conformational changes occurring in the integrin receptor, both in response to cellular events that switch the receptor from low to high affinity (inside-out signaling) and external events that relay information about occupancy of the receptor (outside-in signaling). [6][7][8][9] Although information about the nature of the changes in conformation is slowly emerging (for review, see Woodside et al 10 ) and consequent changes in intracellular interactions are widely documented, 10-13 the molecular mechanism regulating such changes, particularly on the exofacial domain, has not been elucidated. We previously reported that extracellular sulfhydryls participate in conformational changes triggered by interaction of the platelet integrin ␣ 2 ␤ 1 with its natural ligand collagen. 14 We also found that disulfide exchange is required for platelet adhesion mediated by integrins ␣ 5 ␤ 1 and ␣ IIb ␤ 3 as well as ␣ 2 ␤ 1 and that surface-expressed protein disulfide isomerase (PDI) is involved in this exchange. 15 Furthermore, involvement of disulfide exchange in receptor function is a feature specific to the integrin-receptor family (J. L. et al, submitted manuscript, 2002). We therefore proposed that conformational changes subsequent to ligand interaction with the integrin lead to ...

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Section: Methodsmentioning

confidence: 99%

Sustained integrin ligation involves extracellular free sulfhydryls and enzymatically catalyzed disulfide exchange

Lahav

Jurk

Hess

et al. 2002

Blood

184

209

View full text Add to dashboard Cite

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“…To estimate the expression of GPVI on platelet surface, convulxin-FITC was used. Convulxin was incubated with FITC using the FITC-celite method according to Xia et al (20). The molar ratio of FITC to convulxin was 100:1.…”

Section: Analysis Of Annexin-v-fitc and Convulxin-fitc Binding Tomentioning

confidence: 99%

Impaired Platelet Activation in Familial High Density Lipoprotein Deficiency (Tangier Disease)

Nofer

Herminghaus

Brodde³

et al. 2004

Journal of Biological Chemistry

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ATP binding cassette transporter A1 (ABCA1) is involved in regulation of intracellular lipid trafficking and export of cholesterol from cells to high density lipoproteins. ABCA1 defects cause Tangier disease, a disorder characterized by absence of high density lipoprotein and thrombocytopenia. In the present study we have demonstrated that ABCA1 is expressed in human platelets and that fibrinogen binding and CD62 surface expression in response to collagen and low concentrations of thrombin, but not to ADP, are defective in platelets from Tangier patients and ABCA1-deficient animals. The expression of platelet membrane receptors such as GPVI, ␣ 2 ␤ 1 integrin, and GPIIb/IIIa, the collagen-induced changes in phosphatidylserine and cholesterol distribution, and the collagen-induced signal transduction examined by phosphorylation of LAT and p72 syk and by intracellular Ca 2؉ mobilization were unaltered in Tangier platelets. The electron microscopy of Tangier platelets revealed reduced numbers of dense bodies and the presence of giant granules typically encountered in platelets from Chediak-Higashi syndrome. Further studies demonstrated impaired release of dense body content in platelets from Tangier patients and ABCA1-deficient animals. In addition, Tangier platelets were characterized by defective surface exposure of dense body and lysosomal markers (CD63, LAMP-1, LAMP-2, CD68) during collagen-and thrombin-induced stimulation and by abnormally high lysosomal pH. We conclude that intact ABCA1 function is necessary for proper maturation of dense bodies in platelets. The impaired release of the content of dense bodies may explain the defective activation of Tangier platelets by collagen and low concentrations of thrombin, but not by ADP.

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“…To study the adhesive properties of the patient's GPIIb-IIIa, we incubated FITC-fibrinogen 50 and FITC-PAC-1 with platelets in Tyrode-HEPES buffer 51 To assess LIBS exposure on N.M.'s platelets, we incubated PRP supplemented with 10 Ϫ7 M PGE 1 with 2 mM RGDS or RGES peptide (Bachem, Voisins-le-Bretonneux, France) and AP5, D3, and 7D6. After washes, binding to platelets was detected using FITC-conjugated antimouse IgG F(abЈ) 2 and analyzed by flow cytometry.…”

Section: Platelet Glycoprotein Analysis By Flow Cytometrymentioning

confidence: 99%

A point mutation in the cysteine-rich domain of glycoprotein (GP) IIIa results in the expression of a GPIIb-IIIa (αIIbβ3) integrin receptor locked in a high-affinity state and a Glanzmann thrombasthenia–like phenotype

Ruiz

Liu

Sun

et al. 2001

Blood

104

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This article reports a Glanzmann thrombasthenia (GT) patient, N.M., with a point mutation in the third cysteine-rich repeat of ␤3-integrin or platelet glycoprotein (GP) IIIa, leading to the expression of a constitutively activated fibrinogen receptor. The diagnosis of GT was based on a severely reduced platelet-aggregation response to a series of agonists and approximately 20% of surface-expressed GPIIb-IIIa. The patient's GPIIb-IIIa constitutively expressed epitopes recognized by antibodies to ligandinduced binding sites (LIBS) and also spontaneously bound the fibrinogen-mimetic antibody, PAC-1. Furthermore, significant amounts of bound fibrinogen were detected on his platelets ex vivo. No signs of platelet activation were observed on sections of unstimulated platelets from N.M. by electron microscopy. Immunogold labeling highlighted the presence of surface-bound fibrinogen but revealed platelet heterogeneity with regard to the surface density. When the patient's platelets were stimulated by thrombin-receptor activating peptide, amounts of surface-expressed GPIIb-IIIa increased and the aggregation response improved, although it failed to normalize. Platelets from N.M. were able to adhere and spread on immobilized fibrinogen. Sequence analysis of genomic DNA from N.M. revealed a homozygous g1776T>C mutation in GPIIIa, leading to a Cys560Arg amino acid substitution. A stable Chinese hamster ovary (CHO) cell line was prepared expressing surface GPIIbArg560IIIa. Like platelets from the patient, GPIIb-Arg560IIIa-transfected CHO cells constitutively bound LIBS antibodies and PAC-1. They also showed an enhanced ability to adhere on surface-bound fibrinogen. Overall, these data demonstrate that a gain-of-function mutation can still be associated with a thrombasthenic phenotype even though platelets show spontaneous fibrinogen binding. IntroductionIntegrins are heterodimeric transmembrane proteins that mediate cell adhesion and cell migration. [1][2][3] They are involved in numerous fundamental biologic processes, including development, homing, inflammation, angiogenesis, and wound healing, all processes in which integrin function is subjected to a highly sophisticated regulation. [4][5][6][7][8] The platelet fibrinogen receptor, the ␣ IIb ␤ 3 integrin (glycoprotein [GP] IIb-IIIa), has constituted an ideal model for the study of an integrin's role in cell contact interactions. [9][10][11][12][13] This receptor has the capacity to shift through conformational changes from a low-affinity state unable to bind macromolecular ligands to a ligand-competent state on activated platelets. This process of affinity modulation is an essential part of integrin function. Furthermore, ligand binding to GPIIb-IIIa itself modifies the conformation of this integrin, exposing neoepitopes known as ligand-induced binding sites (LIBS) and leading to postreceptor occupancy events such as clustering of GPIIb-IIIa complexes, generation of secondary signals, and cytoskeletal rearrangement, all of which contribute to maximal platelet aggregatio...

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Optimally functional fluorescein isothiocyanate‐labelled fibrinogen for quantitative studies of binding to activated platelets and platelet aggregation

Cited by 84 publications

References 20 publications

Sustained integrin ligation involves extracellular free sulfhydryls and enzymatically catalyzed disulfide exchange

Sustained integrin ligation involves extracellular free sulfhydryls and enzymatically catalyzed disulfide exchange

Impaired Platelet Activation in Familial High Density Lipoprotein Deficiency (Tangier Disease)

A point mutation in the cysteine-rich domain of glycoprotein (GP) IIIa results in the expression of a GPIIb-IIIa (αIIbβ3) integrin receptor locked in a high-affinity state and a Glanzmann thrombasthenia–like phenotype

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