scholarly journals Identification of a Binding Site for Glycoprotein Ibα in the Apple 3 Domain of Factor XI

2004 ◽  
Vol 279 (44) ◽  
pp. 45470-45476 ◽  
Author(s):  
Frank A. Baglia ◽  
David Gailani ◽  
José A. López ◽  
Peter N. Walsh
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Thrombus Formation ◽  
Platelet Glycoprotein ◽  
Heparin Binding ◽  
Factor Xi ◽  
Platelet Binding ◽  
Factor Xia ◽  
Consolidation Phase ◽  
Platelet Thrombus

Factor XI (FXI) is a homodimeric plasma zymogen that is cleaved at two internal Arg369–Ile370bonds by thrombin, factor XIIa, or factor XIa. FXI circulates as a complex with the glycoprotein high molecular weight kininogen (HK). FXI binds to specific sites (Kd= ∼10 nm,Bmax= ∼1,500/platelet) on the surface of stimulated platelets, where it is efficiently activated by thrombin. The FXI Apple 3 (A3) domain mediates binding to platelets in the presence of HK and zinc ions (Zn2+) or prothrombin and calcium ions. The platelet glycoprotein (GP) Ib-IX-V complex is the receptor for FXI (Baglia, F. A., Badellino, K. O., Li, C. Q., Lopez, J. A., and Walsh, P. N. (2002)J. Biol. Chem.277, 1662–1668). Using surface plasmon resonance, we determined that FXI binds specifically to glycocalicin, the extracellular domain of GPIbα, in a Zn2+-dependent fashion (Kd= ∼52 nm). We now show that recombinant FXI A3 domain inhibits FXI inbinding to glycocalicin in the presence of Zn2+, whereas the recombinant FXI A1, A2, or A4 domains have no effect. Experiments with full-length recombinant FXI mutants show that, in the presence of Zn2+, glycocalicin binds FXI at a heparin-binding site in A3 (Lys252and Lys253) and not by amino acids previously shown to be required for platelet binding (Ser248, Arg250, Lys255, Phe260, and Gln263). However, binding in the presence of HK and Zn2+requires Ser248, Arg250, Lys255, Phe260, and GLn263and not Lys252and Lys253. Thus, binding of FXI to GPIbα is mediated by amino acids in the A3 domain in the presence or absence of HK. This interaction is important for the initiation of the consolidation phase of blood coagulation and the generation of thrombin at sites of platelet thrombus formation.

scholarly journals Antithrombotic effect of a monoclonal antibody to the platelet glycoprotein IIb/IIIa receptor in an experimental animal model

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 783-786 ◽  
Author(s):  
BS Coller ◽  
JD Folts ◽  
LE Scudder ◽  
SR Smith
Keyword(s):  
Monoclonal Antibody ◽  
Animal Model ◽  
Platelet Aggregation ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Platelet Glycoprotein ◽  
Antithrombotic Effect ◽  
Antithrombotic Agent ◽  
Antithrombotic Effects ◽  
Platelet Thrombus

A murine monoclonal antibody directed at the platelet glycoprotein IIb/IIIa complex, which blocks platelet aggregation ex vivo, was tested for its antithrombotic effects in an established animal model of acute platelet thrombus formation in partially stenosed arteries. Infusion of 0.7 to 0.8 mg/kg of the F(ab')2 fragment of the antibody completely blocked new thrombus formation despite multiple provocations, making it the most potent antithrombotic agent tested in this model.

scholarly journals Biomechanical thrombosis: the dark side of force and dawn of mechano-medicine

2019 ◽  
Vol 5 (2) ◽  
pp. 185-197 ◽  
Author(s):  
Yunfeng Chen ◽  
Lining Arnold Ju
Keyword(s):  
Platelet Aggregation ◽  
Blood Clotting ◽  
Thrombus Formation ◽  
Dark Side ◽  
Atherosclerotic Plaques ◽  
Excessive Bleeding ◽  
Glycoprotein Vi ◽  
Device Implantation ◽  
Platelet Binding ◽  
Platelet Thrombus

Arterial thrombosis is in part contributed by excessive platelet aggregation, which can lead to blood clotting and subsequent heart attack and stroke. Platelets are sensitive to the haemodynamic environment. Rapid haemodynamcis and disturbed blood flow, which occur in vessels with growing thrombi and atherosclerotic plaques or is caused by medical device implantation and intervention, promotes platelet aggregation and thrombus formation. In such situations, conventional antiplatelet drugs often have suboptimal efficacy and a serious side effect of excessive bleeding. Investigating the mechanisms of platelet biomechanical activation provides insights distinct from the classic views of agonist-stimulated platelet thrombus formation. In this work, we review the recent discoveries underlying haemodynamic force-reinforced platelet binding and mechanosensing primarily mediated by three platelet receptors: glycoprotein Ib (GPIb), glycoprotein IIb/IIIa (GPIIb/IIIa) and glycoprotein VI (GPVI), and their implications for development of antithrombotic ‘mechano-medicine’ .

scholarly journals Discovery of Allosteric Modulators of Factor XIa by Targeting Hydrophobic Domains Adjacent to Its Heparin-Binding Site

2013 ◽  
Vol 56 (6) ◽  
pp. 2415-2428 ◽  
Author(s):  
Rajesh Karuturi ◽  
Rami A. Al-Horani ◽  
Shrenik C. Mehta ◽  
David Gailani ◽  
Umesh R. Desai
Keyword(s):  
Binding Site ◽  
Allosteric Modulators ◽  
Heparin Binding ◽  
Heparin Binding Site ◽  
Factor Xia

Identification of a Binding Site for Integrin αIIbβ3 in the von Willebrand factor (VWF) A1 Domain: Dual Roles for the A1 Domain in Platelet Thrombus Formation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3658-3658
Author(s):  
Junmei Chen ◽  
Miguel A. Cruz ◽  
José A. López
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Shear Stresses ◽  
Platelet Surface ◽  
Rgd Sequence ◽  
Platelet Thrombus ◽  
A1 Domain ◽  
Von Willebrand

Abstract In 1999, Wu et al found that blood from patients with type 3 von Willebrand disease (lacking VWF in both plasma and platelets) could not form thrombi on a collagen surface (Arterioscler. Thromb. Vasc Biol2000, 201661–1667). This suggested that VWF was absolutely required for the accumulation of platelets in thrombi under flow, even in the presence of fibrinogen. Platelets have two VWF receptors, the GP Ib-IX-V complexes and αIIbβ3 , the former mediating the initial tethering and attachment of platelets onto VWF and the latter being involved in platelet-platelet contacts. GP Ib-IX-V binds VWF within the A1 domain and αIIbβ3 is known to bind an Arg-Gly-Asp (RGD) sequence in the C1 domain. In the study of Wu et al, reconstitution of the VWF-deficient plasma with recombinant VWF missing the A1 domain failed to restore thrombus formation, even when the collagen surface was first coated with wild-type VWF to allow platelet attachment. The A1 domain is thus important not only for initial platelet adhesion but also for thrombus accumulation, possibly by binding another platelet receptor. Consistent with this, the number of binding sites for the isolated A1 domain on the platelet surface is more than twice the number of GP Ibα polypeptides. The receptor responsible for these binding sites is unknown but αIIbβ3 is a good candidate given its high copy number and the marked defect seen in platelet thrombus formation in its absence or blockade. Of interest, while deletion of A1 prevented thrombus formation in the studies of Wu et al, mutation of the VWF RGD sequence did not. We therefore examined whether αIIbβ3 also binds within the VWF A1 domain. We found the following. 1) Purified, unactivated αIIbβ3 binds to immobilized A1 domain, binding blocked by antibodies to either αIIbβ3 or A1. 2) Unactivated αIIbβ3 does not interact with immobilized full-length VWF, but binds VWF in the presence of ristocetin. The binding of αIIbβ3 to both VWF and isolated A1 is blocked by the αIIbβ3 antibody c7E3 but not by RGD peptides, and by the A1 antibody 6G1. This suggests that the αIIbβ3 binding site in the A1 domain may overlap the 6G1 epitope (residues 700-709), which is distinct from the GPIbα binding site. 3) 6G1 inhibits shear-induced platelet aggregation—a process that requires both GP Ibα and αIIbβ3—without blocking GP Ibα binding. 4) Platelets firmly adhere on the surface containing A1 and cross-linked collagen-related peptide (CRP), a potent GP VI agonist, at high shear stresses. The CRP-GP VI interaction is not strong enough to arrest platelets under flow, suggesting that GP VI signals could activate αIIbβ3, and αIIbβ3 could mediate firm adhesion. Consistent with this, the αIIbβ3 antibody c7E3 prevented firm platelet adhesion. In summary, we find that αIIbβ3 binds to the A1 domain, in or near the sequence of Glu700-Asp709. In addition to its apparent role in platelet-platelet interactions during thrombus growth, the binding of αIIbβ3 to the VWF A1 domain may also facilitate the binding of GP Ibα to a distinct region of A1, as the site of αIIbβ3 overlaps the binding site of ristocetin and 6G1, both which induce VWF to bind GP Ibα. Therefore, by binding to the same site as 6G1 and ristocetin in the C-terminal peptide of A1, αIIbβ3 may regulate the affinity of A1 for GP Ibα in flowing blood.

Factor XI Deficient Mice Have Reduced Platelet Accumulation and Fibrin Deposition after Laser Injury.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 218-218
Author(s):  
T. Regan Baird ◽  
David Gailani ◽  
Bruce Furie ◽  
Barbara C. Furie
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Thrombus Formation ◽  
Factor Xa ◽  
Fibrin Deposition ◽  
Wild Type ◽  
Factor Xi ◽  
Factor Xia ◽  
Tissue Factor Pathway ◽  
Platelet Accumulation

Abstract Tissue factor exposure at sites of vascular injury results in the generation of factor Xa and thrombin. A current model of blood coagulation suggests that the amount of thrombin generated through this pathway is limited by the inhibition of the factor VIIa-tissue factor complex by tissue factor pathway inhibitor in the presence of factor Xa. The initial thrombin activates a number of hemostatic proteins including factor XI. Factor XIa then activates factor IX leading to generation of the tenase complex to maintain the thrombin flux. While in vitro studies support this hypothesis the importance of factor XI for thrombus formation in vivo remains unclear. We have examined thrombus formation upon laser injury to the arterioles (30–50 μm diameter) of the cremaster muscle in living mice lacking factor XI using digital multi-channel fluorescence intravital microscopy. Platelets were labeled with Alexa 488 conjugated murine CD41 Fab fragments by systemic infusion of the antibody. Maximum platelet accumulation in factor XI null mice (median of 35 thrombi in 4 mice) is only 25% of that of wild type mice (median of 40 thrombi in 4 mice) after injury (p<0.03). The time course of platelet accumulation is similar between both genotypes. Maximum platelet accumulation occurs in approximately 90 seconds (p<0.2). Fibrin deposition was observed simultaneously using an Alexa 660 conjugated anti-fibrin antibody that does not recognize fibrinogen. Maximum fibrin deposition in factor XI null mice is 50% that of wild type mice (p<0.001) and the rate of fibrin generation is slower in factor XI null mice. However, the time to achieve half maximal fibrin deposition is approximately the same in factor XI null mice (77 sec) compared to wild type mice (63.5 sec, p<0.09). These data suggest that the primary difference in response to laser induced injury between the factor XI null mice and wild type mice is the level of thrombin generated and supports the hypothesis that factor XI participates in maintaining thrombin flux after inhibition of the factor VII-tissue factor. The model above postulates a single source of tissue factor, the vessel wall, and further, that the tissue factor-factor VIIa complex formed from the exposed tissue factor is rapidly inactivated by tissue factor pathway inhibitor after the appearance of the initial factor Xa formed. In addition it has been suggested that a rapidly growing thrombus blocks access to vascular wall tissue factor. However we have recently observed that there is a P-selectin and P-selectin glycoprotein ligand 1 dependent pathway of blood coagulation that recruits blood borne tissue factor into a growing thrombus at sites of laser-induced vessel injury. Both vessel wall and blood borne tissue factor are required for normal thrombus formation. Our data suggest that although tissue factor is continuously recruited to the growing thrombus, factor XIa plays a significant role in thrombin generation.

scholarly journals Characterization of a Heparin Binding Site on the Heavy Chain of Factor XI

1998 ◽  
Vol 273 (47) ◽  
pp. 31153-31159 ◽  
Author(s):  
Mingming Zhao ◽  
Tarek Abdel-Razek ◽  
Mao-Fu Sun ◽  
David Gailani
Keyword(s):  
Binding Site ◽  
Heavy Chain ◽  
Heparin Binding ◽  
Factor Xi ◽  
Heparin Binding Site

scholarly journals Functional domains in the heavy-chain region of factor XI: a high molecular weight kininogen-binding site and a substrate-binding site for factor IX

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 244-251
Author(s):  
FA Baglia ◽  
D Sinha ◽  
PN Walsh
Keyword(s):  
Binding Site ◽  
Heavy Chain ◽  
Solid Phase ◽  
Competitive Inhibitor ◽  
Factor Ix ◽  
Functional Domains ◽  
Binding Studies ◽  
Factor Xi ◽  
Proteolytic Activation ◽  
Factor Xia

To probe the molecular interactions of factor XI we have prepared two monoclonal antibodies (MoAbs; 5F7 and 3C1), each of which binds the heavy chain of reduced and alkylated factor XIa. Competitive solid phase radioimmunoassay (RIA) binding studies revealed that 5F7 and 3C1 are directed against different epitopes within factor XI. One antibody (5F7) blocked the surface-mediated proteolytic activation of factor XI and its binding to HMW kininogen, but had no effect on factor-XIa- catalyzed factor IX activation. The other antibody (3C1) is a competitive inhibitor of factor-IX activation by factor XIa, but blocked factor-XI binding to HMW kininogen only at 1,000-fold higher concentration than 5F7. Moreover, HMW kininogen had no effect on the kinetics of factor-XIa-catalyzed factor-IX activation. Furthermore, factor XI CNBr peptide fragments that bind to the 5F7 and 3C1 antibodies were isolated. The peptides that bound to the 5F7 antibody blocked the binding of HMW kininogen to factor XI but did not inhibit factor-XIa-catalyzed factor-IX activation. However, the peptides isolated by the 3C1 antibody inhibited factor-XIa-catalyzed factor-IX activation and had no effect on factor-XI binding to HMW kininogen. Our results indicate that distinct functional domains within the heavy chain region of factor XI are important for the binding of factor XI to HMW kininogen and for activation of factor IX by factor XIa.

Dimeric Glycoprotein VI Binds to Collagen but Not to Fibrin

2018 ◽  
Vol 118 (02) ◽  
pp. 351-361 ◽  
Author(s):  
Mariam Ebrahim ◽  
Janina Jamasbi ◽  
Kristin Adler ◽  
Remco Megens ◽  
Yacine M'Bengue ◽  
...  
Keyword(s):  
Atherosclerotic Plaque ◽  
Fusion Proteins ◽  
Thrombus Formation ◽  
Platelet Glycoprotein ◽  
Binding Studies ◽  
Glycoprotein Vi ◽  
Fibrin Formation ◽  
Platelet Thrombus ◽  
Flowing Blood ◽  
Coated Surfaces

AbstractPlatelet glycoprotein VI (GPVI) acts as a decisive collagen receptor in atherothrombosis. Besides collagen, injured atherosclerotic plaques expose tissue factor (TF) that triggers fibrin formation. Two recent studies reported that platelet GPVI also functions as fibrin receptor, which would importantly widen the mode of action of GPVI-targeted antithrombotic drugs. We studied the binding of two GPVI fusion proteins to fibrin under static and arterial flow conditions. Fibrin was prepared from purified fibrinogen or generated more physiologically from endogenous fibrinogen by coagulating plasma with thrombin. Fibrin formation was also triggered by exposing TF-coated surfaces or human atherosclerotic plaque slices to arterially flowing blood. By binding studies and advanced optical imaging, we found that recombinant dimeric GPVI-Fc fusion proteins with Fc from either IgG1 (GPVI-Fc1) or IgG2 (GPVI-Fc2) bound to collagen fibres, but neither to fibrin prepared from purified fibrinogen obtained from three suppliers, nor to physiological fibrin formed by thrombin in plasma or triggered by exposing TF or atherosclerotic plaque slices to arterially flowing blood. Our findings do not support a role of dimeric platelet GPVI as receptor for fibrin. This is important for the understanding of plaque-triggered platelet thrombus formation and is clinically relevant for future GPVI-targeting therapies with recombinant GPVI-Fc and anti-GPVI antibodies.

Determinants of von Willebrand Factor Function

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-17-SCI-17
Author(s):  
Cécile V. Denis ◽  
Olivier D. Christophe ◽  
Peter J. Lenting
Keyword(s):  
Von Willebrand Factor ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Active Form ◽  
Regulatory Pathways ◽  
Spontaneous Interaction ◽  
Platelet Binding ◽  
Platelet Thrombus ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract SCI-17 Platelet thrombus formation is a multistep process involving a number of molecular players, including von Willebrand factor (vWF). vWF is an adhesive multimeric protein that acts as a molecular bridge between the subendothelium and the glycoprotein Ib/IX/V receptor complex on platelets. Furthermore, vWF promotes the expansion of the platelet plug by cross-linking platelets via binding to integrin αIIbβ3. It is important to keep in mind that before participating in the formation of platelet-rich thrombi, vWF and platelets coexist in the circulation without interacting with each other. For optimal function, it is essential that vWF-platelet interactions occur in a timely way, that is, not too early and not too late. In the former case, spontaneous interaction may lead to intravascular thrombosis, while in the latter, hemorrhagic complications may arise. In order to reach this fine balance of regulation, a number of mechanisms are in place that contribute to control vWF function. In the last few years, considerable progress has been made in either revealing or better understanding such determinants. Physiologically, most of these mechanisms are dedicated to the prevention of excessive vWF-platelet interactions. These include shear-stress-mediated vWF conformational changes that lead to exposure or nonexposure of the platelet-binding site and cleavage sites on the vWF molecule. Intramolecular shielding of the vWF-platelet binding domain by adjacent domains also contributes to vWF reactivity. A major determinant of vWF function is related to its multimeric size, which can be controlled by proteolysis by ADAMTS13 and by other proteases, such as granzyme B or neutrophil elastase. The thiol reductase activity of ADAMTS13 toward vWF also contributes to multimer regulation. Finally, interaction of vWF with plasma proteins such as β2-glycoprotein I, or with endothelial proteins such as osteoprotegerin and galectins, can also participate in keeping vWF from binding excessively to platelets. Pathologically, dysregulations of the above-mentioned mechanisms may lead to either an overly active form of vWF or, in contrast, to an inactive protein. Additional determinants can also become prominent, such as the presence of mutations in the vWF sequence, leading to the genetic bleeding disorder known as von Willebrand disease. Determinants affecting vWF-platelet function have been studied extensively, as vWF participation in platelet thrombus formation is its best known and most important role. However, rather fascinating mechanisms have been identified that can modulate other functions of vWF. An example thereof is the recent identification of vWF cleavage by ADAM28 expressed by carcinoma cells in order to escape the proapoptotic action of vWF on such cells. Another example is the regulation of the Factor VIII binding capacity of vWF that can be controlled by cleavage by granzyme M. Identification of these various regulatory pathways now opens new avenues to act upon in order to better control the fine balance between the prohemostatic and the prothrombotic roles of vWF. Disclosures: No relevant conflicts of interest to declare.

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