PEPTIDES FROM FIBRINOGENAND FIBRONECTIN CHANGE THE CONFORMATIONOF PURIFIED PLATELET GLYCOPROTEIN IIb-IIIa

1987 ◽  
Author(s):  
L V Parise ◽  
B Steiner ◽  
L Nannizzi ◽  
D A Phillips
Keyword(s):  
Conformational Changes ◽  
Amino Acid Sequences ◽  
Platelet Glycoprotein ◽  
Maximal Effect ◽  
Hydrodynamic Properties ◽  
Platelet Surface ◽  
Specific Amino Acid ◽  
Fibrinogen Binding ◽  
A Chain ◽  
And Function

Specific amino acid sequences in fibrinogen and fibronectin appear to mediate the binding of these ligands to the glycoprotein (GP) IIb-IIIacomplex in platelets. Thesesequences include LGGAKQAGDV from the y chain of fibrinogen, and RGD(S) from the a chain of fibrinogenand the cell-binding domain of fibronectin. Several recent reports suggest thatfibrinogen and/or peptides with these sequences cause clustering of GPIIb-IIIa on the platelet surface and Na+/H+ exchange in epinephrine-stimulated platelets. Thus, it is possible that occupancy of specific sites on GP Ilb-IIIa affects its conformation, initiating such events. In this study,we determined whether LGGAKQAGDV, RGDS, and related peptides affect the conformation of purified platelet GP IIb-IIIa. Conformational changes in GP IIb-IIIa were evaluated bychanges in proteolytic susceptibility and hydrodynamic properties. Thepurified GP IIb-IIIa complex was fund to be resistant to proteolysis bythrombin. However, pretreatment of GP IIb-IIIa with various peptidesincreased the susceptibility ofGP libα to thrombin-induced proteolysis,as quantitated onpolyacryfamide gels.The order of potency of these peptides was RGDS<LGGAKQAGDV < KGDS < RGES. This order of potency agrees with that for the abilityof these peptides to inhibit 125I-fibrinogen binding to platelets. The effect of the peptides on proteolysis was time-, temperature-, and concentration-dependent; RGDS Induced a half-maximal effect at ˜60μM. Evaluation of the hydrodynamic properties of GP IIb-IIIa showed that LGGAKQAGDV orRGDS, but not RGES, decreased thesedimentation coefficient of GP IIb-IIIa from 8.5S to 7.7 S or7.4, S,respectively. This changewas accompanied by an increase in theStoke’s radius from 73 A to 84 A. These results suggestthat LGGAKQAGDV andRGDS alterthe conformationof the purified GPIIb-IIIa heterodimer complex by causing it to unfold.This change in conformation may be related to changesin the distribution and function of GP IIb-IIIaon the platelet surface that occurwith occupancy ofligand binding sites.

THROMBOSPONDIN SPECIFICALLY INTERACTS WITH AMINO ACID SEQUENCES WITHIN THE A α- AND B β- CHAINS OF FIBRINOGEN

1987 ◽  
Author(s):  
Theresa Bacon-Baguley ◽  
Suzanne Kendra-Franczak ◽  
Daniel Walz
Keyword(s):  
Platelet Aggregation ◽  
Cyanogen Bromide ◽  
Gel Filtration ◽  
Amino Acid Sequences ◽  
Reverse Phase ◽  
Platelet Surface ◽  
Chain Component ◽  
Fibrinogen Binding ◽  
Platelet Aggregate ◽  
Single Peptide

Thrombospondin (TSP) is responsible for the secretion-dependent phase of platelet aggregation. The mechanism of this action is believed to be through the binding of TSP to fibrinogen, resulting in the stabilization of the platelet aggregate. It has been established that the binding of fibrinogen to the platelet surface is dependent upon peptide sequences present, respectively, in the Aa- and y-chains. We have hypothesized that the binding of TSP to fibrinogen is also dependent upon unique fibrinogen peptide sequences. To test this hypothesis we have examined the interaction of TSP and f.ih.r.inogen. using..a.-blat-b.inding assaLy of reduced fibrinogen, the separated fibrinogen chains, selected fibrinogen domains or peptides generated from cyanogen bromide cleaved chains. Iodinated TSP bound specifically to the Aα - and Bβ - chains. Binding to these chains was calcium independent, mutually exclusive and could be blocked either by preincubation of TSP with 9.4 μ M fibrinogen or by preincubation of fibrinogen with 1.1 nM thrombospondin. TSP bound to the D and DD plasmin fragment of fibrinogen; TSP interacted exclusively with the B-chain component of the DD fragment. The cyanogen bromide fragments of the separated Aα - and Bβ -chains were resolved through a combination of gel filtration and reverse-phase chromatography. TSP was found to bind to a single peptide within these fibrinogen chains. These studies demonstrate that thrombospondin interacts with at least two distinct sites on fibrinogen, and these sites differ from those already described for fibrinogen binding to platelets.

scholarly journals Factor XIIIa binding to activated platelets is mediated through activation of glycoprotein IIb-IIIa

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 1006-1016 ◽  
Author(s):  
AD Cox ◽  
DV Devine
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Activation ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
A Chain ◽  
Functional Receptor

Abstract Stabilization of a clot is dependent on fibrin cross-linking mediated by the transglutaminase, factor XIIIa (FXIIIa). In addition to fibrin stabilization, FXIIIa acts on a number of platelet-reactive proteins, including fibronectin and vitronectin, as well as the platelet proteins, glycoprotein (GP) IIb-IIIa, myosin, and actin. However, conditions inducing the platelet-activation dependent binding of FXIIIa have not been characterized nor have the sites mediating FXIIIa binding been identified. The generation of FXIIIa and consequent detection of FXIIIa on the platelet surface were compared with other thrombin- induced activation events; the rate at which FXIIIa bound to activated platelets was much slower than platelet degranulation or fibrin(ogen) binding. Whereas platelets could be rapidly induced to express a functional receptor for FXIIIa, the rate of FXIIIa binding to platelets is limited by the rate of conversion of FXIII to FXIIIa. Immunoprecipitation of radiolabeled platelets using polyclonal anti- FXIII A-chain antibody identified two proteins corresponding to GPIIb and GPIIIa. Preincubation of intact platelets with 7E3, a monoclonal antibody that blocks the fibrinogen binding site, or GRGDSP peptide inhibited FXIIIa binding by about 95% when measured by flow cytometry; FXIIIa binding to purified GPIIb-IIIa was also inhibited by 7E3. The binding of FXIIIa to purified GPIIb-IIIa was enhanced by the addition of fibrinogen, but not by that of fibronectin or thrombospondin, suggesting that FXIIIa also binds to fibrinogen associated with the complex. These observations suggest that activated platelets bearing FXIIIa may enhance stabilization of platelet-rich thrombi through surface-localized cross-linking events.

scholarly journals Increased binding of fibrinogen to platelets in diabetes: the role of prostaglandins and thromboxane

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 156-162 ◽  
Author(s):  
G DiMinno ◽  
MJ Silver ◽  
AM Cerbone ◽  
G Riccardi ◽  
A Rivellese ◽  
...  
Keyword(s):  
Binding Sites ◽  
Thromboxane A2 ◽  
Adenosine Diphosphate ◽  
Normal Subjects ◽  
Dienoic Acid ◽  
Platelet Glycoprotein ◽  
Platelet Surface ◽  
Prostaglandin Endoperoxide ◽  
Fibrinogen Binding ◽  
Before And After

Previous studies suggested a role for prostaglandins or thromboxane A2, or both in the exposure of fibrinogen receptors on normal platelets in response to several aggregating agents. Platelets from diabetics are known to be more sensitive to aggregating agents and to produce more prostaglandins and thromboxane than platelets from normal subjects. We compared fibrinogen binding to platelets from diabetic subjects with binding to platelets from normal subjects and determined whether aspirin (which inhibits the formation of prostaglandins and thromboxane) would inhibit the binding of fibrinogen to platelets from diabetic subjects and whether this correlated with its effects on platelet aggregation. We found the following: Aspirin suppressed thromboxane formation and rendered the platelets less sensitive to the induction of aggregation by adenosine diphosphate (ADP) or collagen. The amount of U-46619 [( 15s]-hydroxy-11-alpha, 9-alpha [epoxy-methano]- prosta[5Z,13E]-dienoic acid, a stable analog of prostaglandin endoperoxide/thromboxane A2) necessary to induce aggregation, was similar in normal and diabetic subjects and was unchanged after ingestion of aspirin. Binding of 125I-fibrinogen following stimulation of platelets by ADP or collagen was greater in diabetic (because more binding sites were exposed) than in normal subjects. However, following stimulation by U-46619, binding was similar in diabetic and normal subjects. Aspirin caused a reduction in the exposure of binding sites on both platelets from diabetic and normal subjects, so that (in this respect) platelets from diabetic subjects became more like those from normal subjects. Effects of the monoclonal antibody B59.2, which is specific for the platelet glycoprotein IIb-IIIa complex (the presumed receptor for fibrinogen on the platelet surface) were also studied. The amount of this antibody that bound to platelets was the same for normal and diabetic subjects both before and after aspirin and with or without stimulation by ADP or collagen. In addition, B59.2 inhibited aggregation and fibrinogen binding in both platelets from diabetic and normal subjects. The combined data suggest that the glycoprotein IIb- IIIa complex of platelets from diabetic subjects is similar to that of platelets from normal subjects and that the increased fibrinogen binding and aggregation of platelets from diabetic subjects in response to ADP or collagen is mediated by increased formation of prostaglandin endoperoxide or thromboxane A2, or both.

The GPIb thrombin-binding site is essential for thrombin-induced platelet procoagulant activity

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2469-2478 ◽  
Author(s):  
Dagmar Dörmann ◽  
Kenneth J. Clemetson ◽  
Beate E. Kehrel
Keyword(s):  
Binding Site ◽  
Critical Role ◽  
Good Evidence ◽  
Procoagulant Activity ◽  
Platelet Glycoprotein ◽  
Platelet Surface ◽  
Fibrinogen Binding ◽  
Thrombin Activation ◽  
Thrombotic Diseases ◽  
Von Willebrand

Abstract The role of the platelet glycoprotein (GP) Ib-V-IX receptor in thrombin activation of platelets has remained controversial although good evidence suggests that blocking this receptor affects platelet responses to this agonist. The mechanism of expression of procoagulant activity in response to platelet agonists is also still obscure. Here, the binding site for thrombin on GPIb is shown to have a key role in the exposure of negatively charged phospholipids on the platelet surface and thrombin generation, in response to thrombin, which also requires protease-activated receptor-1, GPIIb-IIIa, and platelet-platelet contact. Von Willebrand factor binding to GPIb is not essential to initiate development of platelet procoagulant activity. Inhibition of fibrinogen binding to GPIIb-IIIa also failed to block platelet procoagulant activity. Both heparin and low molecular weight heparin block thrombin-induced platelet procoagulant activity, which may account for part of their clinical efficacy. This study demonstrates a new, critical role for platelet GPIb in hemostasis, showing that platelet activation and coagulation are tightly interwoven, which may have implications for alternative therapies for thrombotic diseases.

scholarly journals Tetrafibricin, a novel non-peptide fibrinogen receptor antagonist, induces conformational changes in glycoprotein IIb/IIIa

1994 ◽  
Vol 301 (3) ◽  
pp. 785-791 ◽  
Author(s):  
T Satoh ◽  
W C Kouns ◽  
Y Yamashita ◽  
T Kamiyama ◽  
B Steiner
Keyword(s):  
Conformational Changes ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Rgd Peptides ◽  
High Affinity ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Binding Function ◽  
High Affinity Receptor ◽  
Affinity Receptor

Arg-Gly-Asp (RGD) is an amino acid sequence in fibrinogen recognized by platelet glycoprotein (GP) IIb/IIIa. Recently, it was found that RGD peptide binding to GPIIb/IIIa leads to conformational changes in the complex that are associated with the acquisition of high-affinity fibrinogen-binding function. In this study, we found that tetrafibricin, a novel non-peptidic GPIIb/IIIa antagonist, induced similar conformational changes in GPIIb/IIIa as did RGD peptides. Tetrafibricin increased the binding of purified inactive GPIIb/IIIa to immobilized pl-80, a monoclonal antibody that preferentially recognizes ligand-occupied GPIIb/IIIa. Exposure of the pl-80 epitope by tetrafibricin was also observed on resting human platelets by flow cytometry. On intact platelets, the conformational changes transformed GPIIb/IIIa into a high-affinity receptor for fibrinogen and triggered subsequent platelet aggregation. Tetrafibricin is the first non-peptidic GPIIb/IIIa antagonist reported that has the capacity to induce conformational changes in GPIIb/IIIa.

scholarly journals Does Covera-19 know ‘when to hold ‘em or ‘when to fold ‘em? A translational thought experiment

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Gerald Dieter Griffin
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Structural Integrity ◽  
Thought Experiment ◽  
Amino Acid Sequences ◽  
Acid And Base ◽  
Effects Of Ph ◽  
Base Balance ◽  
Environmental Milieu ◽  
And Function

AbstractThe function of proteins depends on their structure. The structural integrity of proteins is dynamic and depends on interacting nearby neighboring moieties that influence their properties and induce folding and structural changes. The conformational changes induced by these nearby neighbors in the micro-environmental milieu at that moment are guided by chemical or electrical bonding attractions.There are few literature references that describe the potential for environmental milieu changes to disfavor SARS-CoV-2 attachment to a receptor for survival outside of a host. There are many studies on the effects of pH (acid and base balance) supporting its importance for protein structure and function, but few focus on pH role in extracellular or intracellular protein or actionable requirements of Covera-19.‘Fold ‘em or Hold ‘em’ is seen by the various functions and effects of furin as it seeks an acidic milieu for action or compatible amino acid sequences which is currently aided by its histidine component and the structural changes of proteins as they enter or exit the host. Questions throughout the text are posed to focus on current thoughts as reviewing applicable COVID-19 translational research science in order to understand the complexities of Covid-19.The pH needs of COVID-19 players and its journey through the human host and environment as well as some efficacious readily available repurposed drugs and out-of-the box and easily available treatments are reviewed.

scholarly journals Factor XIIIa binding to activated platelets is mediated through activation of glycoprotein IIb-IIIa

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 1006-1016 ◽  
Author(s):  
AD Cox ◽  
DV Devine
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Activation ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
A Chain ◽  
Functional Receptor

Stabilization of a clot is dependent on fibrin cross-linking mediated by the transglutaminase, factor XIIIa (FXIIIa). In addition to fibrin stabilization, FXIIIa acts on a number of platelet-reactive proteins, including fibronectin and vitronectin, as well as the platelet proteins, glycoprotein (GP) IIb-IIIa, myosin, and actin. However, conditions inducing the platelet-activation dependent binding of FXIIIa have not been characterized nor have the sites mediating FXIIIa binding been identified. The generation of FXIIIa and consequent detection of FXIIIa on the platelet surface were compared with other thrombin- induced activation events; the rate at which FXIIIa bound to activated platelets was much slower than platelet degranulation or fibrin(ogen) binding. Whereas platelets could be rapidly induced to express a functional receptor for FXIIIa, the rate of FXIIIa binding to platelets is limited by the rate of conversion of FXIII to FXIIIa. Immunoprecipitation of radiolabeled platelets using polyclonal anti- FXIII A-chain antibody identified two proteins corresponding to GPIIb and GPIIIa. Preincubation of intact platelets with 7E3, a monoclonal antibody that blocks the fibrinogen binding site, or GRGDSP peptide inhibited FXIIIa binding by about 95% when measured by flow cytometry; FXIIIa binding to purified GPIIb-IIIa was also inhibited by 7E3. The binding of FXIIIa to purified GPIIb-IIIa was enhanced by the addition of fibrinogen, but not by that of fibronectin or thrombospondin, suggesting that FXIIIa also binds to fibrinogen associated with the complex. These observations suggest that activated platelets bearing FXIIIa may enhance stabilization of platelet-rich thrombi through surface-localized cross-linking events.

Assessment of the effects of Syk and BTK inhibitors on GPVI-mediated platelet signaling and function

2021 ◽  
Author(s):  
Tony J. Zheng ◽  
Elizabeth R. Lofurno ◽  
Alexander R. Melrose ◽  
Hari Hara Sudhan Lakshmanan ◽  
Jiaqing Pang ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Platelet Glycoprotein ◽  
Critical Systems ◽  
Functional Responses ◽  
Platelet Surface ◽  
Glycoprotein Vi ◽  
Platelet Signaling ◽  
Granule Secretion ◽  
And Function ◽  
Btk Inhibitors

Spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (BTK) play critical roles in platelet physiology, facilitating ITAM-mediated signaling downstream of platelet glycoprotein VI (GPVI) and GPIIb/IIIa receptors. Small molecule tyrosine kinase inhibitors (TKIs) targeting Syk and BTK have been developed as anti-neoplastic and anti-inflammatory therapeutics and have also gained interest as anti-platelet agents. Here, we investigate the effects of 12 different Syk and BTK inhibitors on GPVI-mediated platelet signaling and function. These inhibitors include, four Syk inhibitors, Bay 61-3606, R406 (fostamatinib), entospletinib, TAK-659, four irreversible BTK inhibitors, ibrutinib, acalabrutinib, ONO-4059 (tirabrutinib), AVL-292 (spebrutinib), and four reversible BTK inhibitors, CG-806, BMS-935177, BMS-986195, and fenebrutinib. In vitro, TKIs targeting Syk or BTK reduced platelet adhesion to collagen, dense granule secretion, and alpha granule secretion in response to the GPVI agonist CRP-XL. Similarly, these TKIs reduced the percentage of activated integrin αIIbβ3 on the platelet surface in response to CRP-XL, as determined by PAC-1 binding. While all TKIs tested inhibited PLCγ2 phosphorylation following GPVI-mediated activation, other downstream signaling events proximal to PI3K and PKC were differentially affected. In addition, reversible BTK inhibitors had less pronounced effects on GPIIb/IIIa-mediated platelet spreading on fibrinogen and differentially altered the organization of PI3K around microtubules during platelets spreading on fibrinogen. Select TKIs also inhibited platelet aggregate formation on collagen under physiological flow conditions. Together, our results suggest that TKIs targeting Syk or BTK inhibit central platelet functional responses but may differentially affect protein activities and organization in critical systems downstream of Syk and BTK in platelets.

scholarly journals Increased binding of fibrinogen to platelets in diabetes: the role of prostaglandins and thromboxane

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 156-162 ◽  
Author(s):  
G DiMinno ◽  
MJ Silver ◽  
AM Cerbone ◽  
G Riccardi ◽  
A Rivellese ◽  
...  
Keyword(s):  
Binding Sites ◽  
Thromboxane A2 ◽  
Adenosine Diphosphate ◽  
Normal Subjects ◽  
Dienoic Acid ◽  
Platelet Glycoprotein ◽  
Platelet Surface ◽  
Prostaglandin Endoperoxide ◽  
Fibrinogen Binding ◽  
Before And After

Abstract Previous studies suggested a role for prostaglandins or thromboxane A2, or both in the exposure of fibrinogen receptors on normal platelets in response to several aggregating agents. Platelets from diabetics are known to be more sensitive to aggregating agents and to produce more prostaglandins and thromboxane than platelets from normal subjects. We compared fibrinogen binding to platelets from diabetic subjects with binding to platelets from normal subjects and determined whether aspirin (which inhibits the formation of prostaglandins and thromboxane) would inhibit the binding of fibrinogen to platelets from diabetic subjects and whether this correlated with its effects on platelet aggregation. We found the following: Aspirin suppressed thromboxane formation and rendered the platelets less sensitive to the induction of aggregation by adenosine diphosphate (ADP) or collagen. The amount of U-46619 [( 15s]-hydroxy-11-alpha, 9-alpha [epoxy-methano]- prosta[5Z,13E]-dienoic acid, a stable analog of prostaglandin endoperoxide/thromboxane A2) necessary to induce aggregation, was similar in normal and diabetic subjects and was unchanged after ingestion of aspirin. Binding of 125I-fibrinogen following stimulation of platelets by ADP or collagen was greater in diabetic (because more binding sites were exposed) than in normal subjects. However, following stimulation by U-46619, binding was similar in diabetic and normal subjects. Aspirin caused a reduction in the exposure of binding sites on both platelets from diabetic and normal subjects, so that (in this respect) platelets from diabetic subjects became more like those from normal subjects. Effects of the monoclonal antibody B59.2, which is specific for the platelet glycoprotein IIb-IIIa complex (the presumed receptor for fibrinogen on the platelet surface) were also studied. The amount of this antibody that bound to platelets was the same for normal and diabetic subjects both before and after aspirin and with or without stimulation by ADP or collagen. In addition, B59.2 inhibited aggregation and fibrinogen binding in both platelets from diabetic and normal subjects. The combined data suggest that the glycoprotein IIb- IIIa complex of platelets from diabetic subjects is similar to that of platelets from normal subjects and that the increased fibrinogen binding and aggregation of platelets from diabetic subjects in response to ADP or collagen is mediated by increased formation of prostaglandin endoperoxide or thromboxane A2, or both.

The Platelet Glycoprotein IIb/IIIa Complex Is lnvolved in the Adhesion of Activated Platelets to Leukocytes

1993 ◽  
Vol 70 (03) ◽  
pp. 514-521 ◽  
Author(s):  
Peter Spangenberg ◽  
Helge Redlich ◽  
Iris Bergmann ◽  
Wolfgang Lösche ◽  
Matthias Götzrath ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Polymorphonuclear Leukocytes ◽  
Surface Membrane ◽  
Rosette Formation ◽  
Platelet Glycoprotein ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Normal Expression ◽  
Adhesion Of Platelets

SummaryThe adhesion of activated platelets to leukocytes (rosette formation) seems to be mediated by CD62 on platelets and its counterreceptor (CD 15 or a sialic acid-containing glycoprotein) on polymorphonuclear leukocytes (PMNL). However, neither treatment of platelets with an anti-CD62 antibody or fucoidan nor treatment of PMNL with anti-CD15 antibody or neuraminidase are able to inhibit completely the adhesion. Therefore, we have studied the platelet GPIIb/IIIa complex (CD41a) for its involvement in the adhesion of activated platelets to PMNL. The following evidences point to a participation of CD41a in the adhesion of activated platelets to leukocytes: a) inhibition of adhesion by monoclonal antibodies (mab) raised toward CD41a, b) inhibition of adhesion by peptides such as RGDS and echistatin, c) inhibition of adhesion by dissociation of the CD41a complex with EGTA, and d) inhibition of rosette formation using platelets from a thrombasthenic patient which have almost no CD41a in the surface membrane but a normal expression of CD62. It is likely that fibrinogen is involved in the adhesion of platelets to PMNL via CD41a, since fibrinogen increases the rosette formation of ADP-stimulated platelets. Furthermore, the incubation of unstimulated platelets with fibrinogen and an antibody raised against glycoprotein III a which stimulates fibrinogen binding to the platelet surface results in an enlarged rosette formation.

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