Clues for Understanding the Structure and Function of a Prototypic Human Integrin: The Platelet Glycoprotein IIb/IIIa Complex

1994 ◽  
Vol 72 (01) ◽  
pp. 001-015 ◽  
Author(s):  
Juan J Calvete
Keyword(s):  
Structure Function ◽  
Thrombus Formation ◽  
Platelet Glycoprotein ◽  
Primary Role ◽  
Clot Retraction ◽  
Inhibitory Peptide ◽  
Rgd Sequence ◽  
Platelet Receptor ◽  
Adhesive Proteins ◽  
And Function

SummaryThe glycoprotein (GP) IIb/IIIa, a Ca2+-dependent heterodimer, is the major integrin on the platelet plasma membrane. On resting platelets GPIIb/IIIa is maintained in an inactive conformation and serves as a low affinity adhesion receptor for surface-coated fibrinogen, whereas upon platelet activation signals within the cytoplasma alter the receptor function of GPIIb/IIIa (inside-out signalling), which undergoes a measurable conformational change within its exoplasmic domains, and becomes a competent receptor for soluble fibrinogen and some other RGD sequence-containing plasma adhesive proteins. Upon ligand binding, further structural alterations trigger the association of receptor-occupied GPIIb/IIIa complexes with themselves within the plane of the membrane. The simultaneous binding of dimeric fibrinogen molecules to GPIIb/IIIa clusters on adjacent platelets leads to platelet aggregation, which promotes attachment of fibrinogen-GPIIb/IIIa clusters to the cytoskeleton (outside-in signalling). This, in turn, provides the necessary physical link for clot retraction to occur, and generates a cascade of intracellular biochemical reactions which result in the formation of a multiprotein signalling complex at the cytoplasmic domains of GPIIb/IIIa. Glycoprotein IMIIa, also called αIIbβ3 in the integrin nomenclature, plays thus a primary role in both platelet adhesion and thrombus formation at the site of vascular injury. In addition, the human glycoprotein Ilb/IIIa complex is the most thoroughly studied integrin receptor, its molecular biology and major features of its primary structure having been elucidated mainly during the last six years. Furthermore, localization of functionally relevant monoclonal antibody epitopes, determination of the cross-linking sites of inhibitory peptide ligands, proteolytic dissection of the isolated integrin, and analysis of natural and artificial GPIIb/IIIa mutants have recently provided a wealth of information regarding structure-function relationships of human GPIIb/IIIa. The aim of this review is to summarize these many structural and functional data in the perspective of an emerging model. Although most of the interpretations based on structural elements of this initial biochemical model require independent confirmation, they may help us to understand the structure-function relationship of this major platelet receptor, and of other members of the integrin superfamily, as well as to perform further investigations in order to test current hypotheses.

scholarly journals Epitope mapping by cDNA expression of a monoclonal antibody which inhibits the binding of von Willebrand factor to platelet glycoprotein IIb/IIIa

1992 ◽  
Vol 284 (3) ◽  
pp. 711-715 ◽  
Author(s):  
G Piétu ◽  
A S Ribba ◽  
G Chérel ◽  
D Meyer
Keyword(s):  
Monoclonal Antibody ◽  
Von Willebrand Factor ◽  
Fusion Proteins ◽  
Solid Phase ◽  
Platelet Glycoprotein ◽  
Rgd Sequence ◽  
Adhesive Proteins ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Beta Galactosidase

In order to study the structure-function relationship of von Willebrand Factor (vWF), we have located the epitope of a well-characterized monoclonal antibody (MAb) to vWF (MAb 9). This MAb reacts with the C-terminal portion of the vWF subunit, SPII fragment [amino acids (aa) 1366-2050], which includes an Arg-Gly-Asp (RGD) sequence at positions 1744-1746, and totally inhibits vWF and SPII binding to platelet membrane glycoprotein IIb/IIIa (GPIIb/IIIa). A recombinant DNA library was constructed by cloning small (250-500 nucleotides) vWF cDNA fragments into the lambda gt11 vector and these inserts were expressed as fusion proteins with beta-galactosidase. Immunological screening of the library with 125I-MAb 9 identified three immunoreactive clones. vWF inserts were amplified by the PCR and their sequences demonstrated overlapping nucleotides from positions 7630 to 7855 of vWF cDNA, coding for aa residues 1698-1773 of the mature subunit, indicating that this is the epitope of MAb 9. vWF-beta-galactosidase fusion protein reacted with 125I-MAb 9 by Western blotting. In a solid-phase radioimmunoassay, the purified fusion proteins decreased the binding of vWF to 125I-MAb 9 by 50%, and this inhibition was dose-dependent between 3.5 and 120 nM. Therefore the epitope of MAb 9 is located within aa 1698-1773 of the vWF subunit, which includes the RGD sequence implicated in the binding of adhesive proteins of GPIIb/IIIa.

Platelet Cytoskeletal Force Transmission Through The GPIb-IX-V Complex

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 197-197
Author(s):  
Shirin Feghhi ◽  
Adam D. Munday ◽  
Wes Tooley ◽  
Rajsekar Shreya ◽  
José A. López ◽  
...  
Keyword(s):  
Cytoplasmic Domain ◽  
Force Generation ◽  
Platelet Glycoprotein ◽  
Force Transmission ◽  
Cho Cell ◽  
Integrin Αiibβ3 ◽  
Platelet Receptor ◽  
Adhesive Proteins ◽  
Contractile Forces ◽  
A1 Domain

Abstract Platelets are the primary cellular components of the hemostatic plug that forms during primary hemostasis. The first step in this process is platelet adhesion from the flowing blood to a surface, carried out by the platelet glycoprotein (GP) Ib-IX-V binding to immobilized von Willebrand factor (VWF). Adhesion is followed by activation of integrin αIIbβ3, which mediates the attachment of platelets to each other by binding multivalent ligands such as VWF or fibrinogen. To stabilize the hemostatic plug and strengthen its attachment to the wound site, platelets must transmit contractile forces from actin and myosin proteins in their cytoskeleton to extracellular matrix proteins within the vessel wall or to the adhesive proteins between adjacent platelets. Integrin αIIbβ3 is one of the membrane proteins capable of transmitting these forces, having a direct link to the platelet cytoskeleton through talin and other focal adhesion related proteins. In the current study, we investigated whether the GPIb-IX-V complex is also capable of force transmission after binding ligand. The GPIb-IX-V complex contains 4 polypeptides, GPIbα, GPIbβ, GPIX and GPV. Only GPIbα binds VWF, which it does through VWF's A1 domain. GPIbα also attaches the complex to the actin and membrane skeletons through its cytoplasmic domain, with the large skeletal protein filamin functioning as the intermediary. There is strong evidence that the GPIbα-A1 bond is force sensitive, becoming stronger as force is applied to it, a property that defines it as a “catch bond”. For this reason, we investigated the role of GPIbα in transmitting platelet forces using a new tool that we have developed to measure contractile forces generated by platelets. This tool, composed of arrays of nanoposts separated by 2 μm (Figure 1), was fabricated using e-beam lithography. VWF was adsorbed to the tips of the nanoposts and platelets were allowed to adhere, spread, and contract. To assess the contribution of αIIbβ3 and GPIbα to force generation, we blocked these receptors with the antibodies 7E3 and AK2, respectively. Treatment with 7E3 significantly lowered the force generated, but did not eliminate it completely (57% reduction). AK2 had a smaller effect (20% reduction), and the combination of the two usually abolished force generation. We observed a similar force reduction (30%) as AK2 treatment when we blocked the VWF A1 domain with recombinant GPIbα N-terminus. Because VWF contains binding sites for more than one platelet receptor, and although purified, could have trace amounts of other plasma proteins, we also evaluated force generation on nanoposts coated with recombinant VWF A1 domain, which should only bind GPIbα. In this case, the platelets generated forces similar to those observed when αIIbβ3 was blocked by 7E3, providing further evidence that GPIbα can transmit forces by binding the A1 domain. Figure 1.Platelet bending nanoposts.Figure 1. Platelet bending nanoposts. As a final test of the ability of GPIbα to support force generation, we examined whether Chinese hamster ovary (CHO) cells expressing the GPIb-IX complex (CHOαβIX, fully functional but lacking GPV) could generate force on VWF or A1 domain (Figure 2). CHOαβIX cells adhered, spread and generated forces of similar magnitude on microposts (larger because of the larger cell size) coated with either substrate. CHOβIX cells, lacking GPIbα, did not adhere to either substrate.Figure 2.CHO cell bending microposts.Figure 2. CHO cell bending microposts. To investigate the requirement for cytoskeletal attachment of the complex in force generation, we studied a CHOαβIX line containing GPIbα truncated after residue 518 and therefore lacking almost the entire cytoplasmic domain. These cells adhered and spread on VWF-coated microposts, but generated minimal contractile force. Together, these results indicate that the GPIb-IX-V complex is able to transmit cytoskeletal contractile forces to its ligand, VWF, in a process requiring the cytoplasmic domain of GPIbα. This is the first example of a non-integrin transmitting force to an external substrate. Disclosures: Sniadecki: Stasys Medical Corporation: Equity Ownership, Founder Other.

The humanized anti-glycoprotein Ib monoclonal antibody h6B4-Fab is a potent and safe antithrombotic in a high shear arterial thrombosis model in baboons

2008 ◽  
Vol 100 (10) ◽  
pp. 670-677 ◽  
Author(s):  
Alexandre Fontayne ◽  
Muriel Meiring ◽  
Seb Lamprecht ◽  
Jan Roodt ◽  
Eddy Demarsin ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Side Effects ◽  
Blood Loss ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Plasma Concentrations ◽  
Platelet Glycoprotein ◽  
Fab Fragment ◽  
Platelet Receptor

SummaryThe Fab-fragment of 6B4, a murine monoclonal antibody targeting the human platelet glycoprotein (GP) Ibα and blocking the binding of von Willebrand factor (VWF), is a powerful antithrombotic. In baboons, this was without side effects such as bleeding or thrombocytopenia. Recently, we developed a fully recombinant and humanized version of 6B4-Fab-fragment, h6B4-Fab, which maintains its inhibitory capacities in vitro and ex vivo after injection in baboons. We here investigated the antithrombotic properties, the effect on bleeding time and blood loss and initial pharmacokinetics of h6B4-Fab in baboons. The antithrombotic effect of h6B4-Fab on acute platelet-mediated thrombosis was studied in baboons where thrombus formation is induced at an injured and stenosed site of the femoral artery, allowing for cyclic flow reductions (CFRs) which are measured on an extracorporeal femoral arteriovenous shunt. Injection of 0.5 mg/kg h6B4-Fab significantly reduced the CFRs by 80%, whereas two extra injections, resulting in cumulative doses of 1.5 and 2.5 mg/kg, completely inhibited the CFRs. Platelet receptor occupancy, plasma concentrations and effects ex vivo were consistent with what was previously observed. Finally, minimal effects on bleeding time and blood loss, no spontaneous bleeding and no thrombocytopenia were observed. We therefore conclude that h6B4-Fab maintains the antithrombotic capacities of the murine 6B4-Fab, without causing side effects and therefore can be used for further development.

scholarly journals Thymosin β4 is essential for thrombus formation by controlling the G-actin/F-actin equilibrium in platelets

Haematologica ◽  
2021 ◽  
Author(s):  
Inga Scheller ◽  
Sarah Beck ◽  
Vanessa Göb ◽  
Carina Gross ◽  
Raluca A. I. Neagoe ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Critical Role ◽  
Actin Dynamics ◽  
Thymosin Β4 ◽  
Clot Retraction ◽  
And Function ◽  
External Signals

Coordinated rearrangements of the actin cytoskeleton are pivotal for platelet biogenesis from megakaryocytes (MKs) but also orchestrate key functions of peripheral platelets in hemostasis and thrombosis, such as granule release, the formation of filopodia and lamellipodia, or clot retraction. Along with profilin (Pfn) 1, thymosin β4 (encoded by Tmsb4x) is one of the two main G-actin sequestering proteins within cells of higher eukaryotes, and its intracellular concentration is particularly high in cells that rapidly respond to external signals by increased motility, such as platelets. Here, we analyzed constitutive Tmsb4x knockout (KO) mice to investigate the functional role of the protein in platelet production and function. Thymosin β4 deficiency resulted in a macrothrombocytopenia with only mildly increased platelet volume and an unaltered platelet life span. MK numbers in the bone marrow (BM) and spleen were unaltered, however, Tmsb4x KO MKs showed defective proplatelet formation in vitro and in vivo. Thymosin β4 deficient platelets displayed markedly decreased G-actin levels and concomitantly increased F-actin levels resulting in accelerated spreading on fibrinogen and clot retraction. Moreover, Tmsb4x KO platelets showed activation defects and an impaired immunoreceptor tyrosine-based activation motif (ITAM) signaling downstream of the activating collagen receptor glycoprotein (GP) VI. These defects translated into impaired aggregate formation under flow, protection from occlusive arterial thrombus formation in vivo and increased tail bleeding times. In summary, these findings point to a critical role of thymosin β4 for actin dynamics during platelet biogenesis, platelet activation downstream of GPVI and thrombus stability.

Polymorphism of the Platelet Glycoprotein IIIa Gene in Patients with Coronary Stenosis

1998 ◽  
Vol 79 (06) ◽  
pp. 1126-1129 ◽  
Author(s):  
Domingo Gonzalez-Lamuño ◽  
Rafael Hernandez-Estefania ◽  
Thierry Colman ◽  
Miguel Pocovi ◽  
Miguel Delgado-Rodriguez ◽  
...  
Keyword(s):  
Coronary Stenosis ◽  
Coronary Revascularization ◽  
Platelet Glycoprotein ◽  
Old Patients ◽  
A Value ◽  
Percutaneous Coronary Revascularization ◽  
Platelet Receptor ◽  
Glycoprotein Iiia ◽  
Artery Disease ◽  
Adhesive Proteins

SummaryBased on genetic variability, structural differences in the glycoprotein IIb/IIIa platelet receptor for adhesive proteins result in individual differences in the thrombogenicity of platelets. Recent studies suggest a controversial association between a genetic polymorphism of the glycoprotein IIIa gene (PlA2) and the risk of coronary artery disease. In our study, the prevalence of the PlA2 allele in a group of patients undergoing percutaneous coronary revascularization was 37%, a value significantly higher than in controls [13%, odds ratio (OR) = 3.93, 95% CI, 1.84 to 8.53] suggesting a significant association between this polymorphism and documented coronary stenosis, which is strongest among <60 years old patients (OR = 12.30, 95% CI, 2.98 to 70.93). This polymorphism represents an inherited risk factor for severe cardiovascular disease due to coronary occlusion.

PLATELET GLYCOPROTEIN IIb-IIIa-LIKE PROTEINS MEDIATE ENDOTHELIAL CELL ATTACHMENT TO ADHESIVE MATRIX PROTEINS AND ARE UP-REGULATED BY PHORBOL ESTERS

1987 ◽  
Author(s):  
I F Charo ◽  
L A Fitzgerald ◽  
D Meyer ◽  
L S Bekeart ◽  
D R Phillips
Keyword(s):  
Monoclonal Antibody ◽  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Phorbol Esters ◽  
Matrix Proteins ◽  
Platelet Glycoprotein ◽  
Mrna Levels ◽  
Platelet Receptor ◽  
Adhesive Proteins

Human endothelial cells (EC) express glycoproteins that are similar to the platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa), the platelet receptor for adhesive proteins. Although GP IIb—IIIa is abundant in both platelets and EC, its only known function is to mediate platelet aggregation. The present study tests the hypotheses that EC attachment to adhesive proteins in the extracellular matrix is mediated by the GP IIb-IIIa-1ike proteins. Endothelial cells attached well to glass slides that were previously coated with adhesive proteins, but not albumin. To determine whether GP IIb-IIIa was involved, EC adherence was measured in the presence and absence of a GP IIb-IIIa monoclonal antibody (7E3) which inhibits fibrinogen (Fg) binding to platelets. The attachment of EC to Fg and von Willebrand factor (vWf), but not fibronectin (Fn) coated slides, was completely inhibited by 7E3. Attachment to vitronectin was partially inhibited. In contrast, EC attachment to Fn was specifically inhibited by a Fn-receptor antibody. Endothelial cell adherence to vWf was also inhibited by a monoclonal antibody (Mab9) against the GP IIb-IIIa binding domain of vWf, but not by antibodies agains.t other portions of vWf. We have further found that 7E3 disrupts monolayers of endothelial cells by detaching the cells from their extracellular matrix. EC incubated in phorbol myris-tate.acetate (PMA) increase in size and appear more tightly adherent to their extracellular matrix. To determine if PMA increases synthesis of cellular receptors for matrix proteins, we have used cDNA probes to measure the mRNA levels of the large subunit of the Fn-receptor (FnRα) and GP IIIa in EC. After a 4 hour incubation in the presence of PMA (10 nM), there was a 2-fold increase in the mRNA levels of both FnRα and GP IIIa, as well as increased cell spreading on the matrix. We conclude: i) the GP Ilb-IIIa complex in EC is a surface receptor for specific adhesive proteins, and is distinct from the FnR, and ii) both GP IIIa and FnRα synthesis are increased by PMA, which causes a concomittant change in cell morphology.

Antiplatelet Effect of FK633, a Platelet Glycoprotein Ilb/IIIa Antagonist, on Thrombus Formation and Vascular Patency after Thrombolysis in the Injured Hamster Carotid Artery

1997 ◽  
Vol 77 (03) ◽  
pp. 562-567 ◽  
Author(s):  
Takehiro Kaida ◽  
Hiroyuki Matsuno ◽  
Masayuki Niwa ◽  
Osamu Kozawa ◽  
Hideo Miyata ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Carotid Artery ◽  
Vascular Injury ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Separate Experiment ◽  
Platelet Glycoprotein ◽  
Neointima Formation ◽  
Vascular Patency ◽  
Injured Artery

SummaryThe antithrombotic and restenosis-preventing effects of FK633, an inhibitor of platelet aggregation via binding to the glycoprotein (GP) Ilb/IIIa receptor, were studied. IC50 value of FK633 against platelet aggregation ex vivo induced by 2.5 |iM adenosine diphosphate (ADP) was 5.4 X 10"7 M as determined using hamster platelet rich plasma. The inhibitory effect was also investigated in vivo on thrombus formation at the carotid arterial wall injured by a modified catheter. As a control, the left carotid artery was injured and the time required to develop a thrombotic occlusion (3.9 ±1.1 min, mean ± S.E.M., n = 18) was determined. Then, the right carotid artery of the same animal was injured while a continuous intravenous (i.v.) infusion of FK633 was administered at doses of 0 (saline), 0.1,0.3 or 1.0 mg/kg/h. The time to occlusion was dose-dependently prolonged. In a separate experiment, 10% of the total tPA dose (0.52 mg/kg) was injected into the injured artery as a bolus and the remaining was infused i.v. at a constant rate for 30 min. When FK633 (0.3 or 1.0 mg/kg/h) was infused together with tPA, late patency of the reperfused artery was much improved as compared with that of treatment with tPA alone. Bleeding time, measured at the end of the tPA infusion, was markedly prolonged when the higher dose of FK633 (1.0 mg/kg/h) was coadministered, however coadministration of the lower dose of FK633 (0.3 mg/kg/h) was almost without prolongation on the bleeding time, despite a significant effect on the vascular patency after thrombolysis. Next, neointima formation was evaluated 2 weeks after the vascular injury. When FK633 (0.3 mg/kg/h) was continuously infused i. v. by an implanted osmotic pump for 3,7 or 14 days after the vascular injury, the neointimal area formation was significantly suppressed in the treatment groups for 7 or 14 days. These findings suggest that FK633 inhibits platelet activation in the injured artery and improves vascular patency after thrombolysis with tPA with a concomitant suppression of neointima formation.

Platelet Suruival and Function in Rats with Enhanced Thrombotic Tendency

1985 ◽  
Vol 54 (04) ◽  
pp. 739-743 ◽  
Author(s):  
Federica Delaini ◽  
Elisabetta Dejana ◽  
Ine Reyers ◽  
Elisa Vicenzi ◽  
Germana De Bellis Vitti ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Nephrotic Syndrome ◽  
Platelet Function ◽  
Laboratory Tests ◽  
Thrombus Formation ◽  
The Other ◽  
Mean Survival Time ◽  
The Mean ◽  
And Function ◽  
Thrombotic Tendency

SummaryWe have investigated the relevance of some laboratory tests of platelet function in predicting conditions of thrombotic tendency. For this purpose, we studied platelet survival, platelet aggregation in response to different stimuli, TxB2 and 6-keto-PGFlα production in serum of rats bearing a nephrotic syndrome induced by adriamycin. These animals show a heavy predisposition to the development of both arterial and venous thrombosis. The mean survival time was normal in nephrotic rats in comparison to controls. As to aggregation tests, a lower aggregating response was found in ADR-treated rats using ADP or collagen as stimulating agents. With arachidonic acid (AA) we observed similar aggregating responses at lower A A concentrations, whereas at higher AA concentrations a significantly lower response was found in nephrotic rats, despite their higher TxB2 production. Also TxB2 and 6-keto-PGFlα levels in serum of nephrotic rats were significantly higher than in controls. No consistent differences were found in PGI2-activity generated by vessels of control or nephrotic rats.These data show that platelet function may appear normal or even impaired in rats with a markedly increased thrombotic tendency. On the other hand, the significance of high TxB2 levels in connection with mechanisms leading to thrombus formation remains a controversial issue.

scholarly journals Platelet Shape Changes during Thrombus Formation: Role of Actin-Based Protrusions

2021 ◽  
Vol 41 (01) ◽  
pp. 014-021
Author(s):  
Markus Bender ◽  
Raghavendra Palankar
Keyword(s):  
Blood Loss ◽  
Actin Filaments ◽  
Thrombus Formation ◽  
Short Review ◽  
Critical Component ◽  
Clot Retraction ◽  
Shape Changes ◽  
Platelet Shape

AbstractPlatelet activation and aggregation are essential to limit blood loss at sites of vascular injury but may also lead to occlusion of diseased vessels. The platelet cytoskeleton is a critical component for proper hemostatic function. Platelets change their shape after activation and their contractile machinery mediates thrombus stabilization and clot retraction. In vitro studies have shown that platelets, which come into contact with proteins such as fibrinogen, spread and first form filopodia and then lamellipodia, the latter being plate-like protrusions with branched actin filaments. However, the role of platelet lamellipodia in hemostasis and thrombus formation has been unclear until recently. This short review will briefly summarize the recent findings on the contribution of the actin cytoskeleton and lamellipodial structures to platelet function.

Structure, Function, and Tort Law

2020 ◽  
Vol 13 (1) ◽  
pp. 31-79
Author(s):  
Dan Priel
Keyword(s):  
Structure Function ◽  
Tort Law ◽  
Structure And Function ◽  
Vicarious Liability ◽  
Causal Uncertainty ◽  
Popular View ◽  
And Function

AbstractA popular view among tort theorists is that an explanation of tort law must take account its “structure,” since this structure constitutes the law’s “self-understanding.” This view is used to both criticize competing functional accounts of tort law, especially economic ones, that are said to ignore tort law’s structure, and, more constructively, as a basis for explaining various tort doctrines. In this essay, I consider this argument closely and conclude that it is faulty. To be valid, one needs a non-question begging way of identifying the essence of tort law. I argue that law’s “self-understanding” can only make sense if it means the understanding of certain people. Examining those, I conclude that the claim of structuralists is false, for there are many people who take its function to be central. I then further show that if one wishes to understand the development of tort law’s doctrine one must take both structure and function into account. I demonstrate this claim by examining the development of the doctrine dealing with causal uncertainty and vicarious liability.

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