Signaling through GP Ib-IX-V activates αIIbβ3 independently of other receptors

Blood ◽  
2004 ◽  
Vol 103 (9) ◽  
pp. 3403-3411 ◽  
Author(s):  
Ana Kasirer-Friede ◽  
Maria Rita Cozzi ◽  
Mario Mazzucato ◽  
Luigi De Marco ◽  
Zaverio M. Ruggeri ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Signaling Proteins ◽  
Phosphatidylinositol 3 Kinase ◽  
Kinase C ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Pharmacologic Inhibition

Abstract Platelet adhesion to von Willebrand factor (VWF) activates αIIbβ3, a prerequisite for thrombus formation. However, it is unclear whether the primary VWF receptor, glycoprotein (GP) Ib-IX-V, mediates αIIbβ3 activation directly or through other signaling proteins physically associated with it (eg, FcR γ-chain), possibly with the contribution of other agonist receptors and of VWF signaling through αIIbβ3. To resolve this question, human and GP Ibα transgenic mouse platelets were plated on dimeric VWF A1 domain (dA1VWF), which engages only GP Ib-IX-V, in the presence of inhibitors of other agonist receptors. Platelet adhesion to dA1VWF induced Src kinase-dependent tyrosine phosphorylation of the FcR γ-chain and the adapter molecule, ADAP, and triggered intracellular Ca2+ oscillations and αIIbβ3 activation. Inhibition of Ca2+ oscillations with BAPTA-AM prevented αIIbβ3 activation but not tyrosine phosphorylation. Pharmacologic inhibition of protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI 3-kinase) prevented αIIbβ3 activation but not Ca2+ oscillations. Inhibition of Src with 2 distinct compounds blocked all responses downstream of GP Ib-IX-V under static or flow conditions. However, dA1VWF-induced responses were reduced only slightly in GP Ibα transgenic platelets lacking FcR γ-chain. These data establish that GP Ib-IX-V itself can signal to activate αIIbβ3, through sequential actions of Src kinases, Ca2+ oscillations, and PI 3-kinase/PKC. (Blood. 2004;103:3403-3411)

Inside-Out Signaling through ADAP Is Required for VWF-GPIb-IX-V Mediated Integrin AIibb3 Activation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 379-379
Author(s):  
Ana Kasirer-Friede ◽  
Barry P. Moran ◽  
Jennifer Nagrampa-Orje ◽  
Ken Swanson ◽  
Burkhart Schraven ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Platelet Adhesion ◽  
Binding Partner ◽  
Protein Levels ◽  
Normal Platelet ◽  
Static Conditions ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Inside Out

Abstract The interaction between von Willebrand factor (VWF) and platelet GPIb-IX-V is a key biological mechanism in hemostasis. In a previous study of signal transduction between GPIb-IX-V and integrin α Iibβ 3, we observed that platelet adhesion to a dimeric VWF A1 domain fragment via GP Ib-IX-V induced the tyrosine phosphorylation of the adapter protein, ADAP (Slap-130, FYB). While this protein has been implicated in cell proliferation and integrin clustering in leukocytes, its role in platelet function has not been determined. Here we used ADAP knockout mice to address this issue. Initiation of signaling in platelets adherent to murine dimeric VWF A1 domain caused α Iibβ 3 activation in wild-type, ADAP+/+ platelets, as determined by the binding of FITC-fibrinogen or POW-2, an anti-α Iibβ 3 antibody Fab specific for high-affinity α Iibβ 3. However, α Iibβ 3 activation through GP Ib-IX-V was diminished by 70–80 % in ADAP−/− platelets (P <0.01), despite normal platelet adhesion under these static conditions. α Iibβ 3 activation by other agonist pathways, such as those stimulated through ADP or Par4 thrombin receptors was also decreased in ADAP−/− platelets (P < 0.05 and 0.01 respectively). In addition, following adhesion of ADAP−/− platelets to VWF A1, tyrosine phosphorylation of numerous proteins, including the ADAP binding partner, SLP-76, was markedly reduced. Surprisingly, deletion of ADAP also affected the expression of another ADAP binding partner, Skap-HOM, and transient transfection of CHO cells confirmed that ADAP expression regulated Skap-HOM protein levels. Platelets from Skap-HOM−/− mice, which have normal ADAP levels, showed normal α Iibβ 3 activation, indicating that the inside-out signaling defect in ADAP−/− platelets was specifically due to deficiency of ADAP and not Skap-HOM. ADAP−/− mice displayed more re-bleeding from tail wounds compared to ADAP+/+ mice (64% vs. 16%, p < 0.01). Under shear flow conditions over a combined matrix of VWF A1A2 and fibronectin to test interactions involving GP Ib-IX-V and α Iibβ 3, respectively, ADAP−/− platelets displayed reduced α IIbβ 3-dependent adhesion. These studies establish that ADAP is a necessary component of the inside-out signaling pathways downstream of GP Ib-IX-V and other receptors in platelets that regulate α IIbβ 3 affinity.

A G-quartet oligonucleotide blocks glycoprotein Ib-mediated platelet adhesion and aggregation under flow conditions

2009 ◽  
Vol 102 (09) ◽  
pp. 529-537 ◽  
Author(s):  
Zhou Zhou ◽  
Aubrey Bernardo ◽  
Qiqing Zhu ◽  
Yongli Guan ◽  
Wensheng Sun ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Flow Conditions ◽  
Β Sheets ◽  
A1 Domain ◽  
Von Willebrand ◽  
Potential Interactions ◽  
Willebrand Factor ◽  
Binding Subunit

SummaryPlatelets arrest bleeding by adhering to and aggregating on the subendothelium exposed at the site of vessel injury.This process is initiated by the interaction between the subendothelium von Willebrand factor (VWF) and the glycoprotein (GP) Ib-IX-V complex on platelets.However,the same interaction also results in thrombosis at the site of a ruptured atherosclerotic plaque. Reagents regulating the GP Ib-VWF interaction will therefore have direct impact on haemostasis and thrombosis. We have characterised an oligonucleotide G-quartet (T30923) that specifically blocks VWF binding to GP Ibα, theVWF-binding subunit of the GP Ib-IX-V complex.We evaluated the potential interactions of T30923 with GP Ibα and VWF A1 domain by computer simulated molecular dockings, which identified four T30923 docking sites in the β-sheets of the N-terminal region of GP Ibα (E14-D18, S39, D63-S64, and D83-S85). Experimentally,T30923 bound GP Ibα and dose-dependently blocked platelet aggregation induced by ristocetin and thrombin, but not by botrocetin, collagen, TRAP, and ADP. It also blocked shear-induced platelet aggregation and thrombus formation on immobilized VWF under arterial shear stress. These results demonstrate that T30923 may have therapeutic potentials to regulate the GP IbαVWF interaction.

Levels of von Willebrand factor and ADAMTS13 determine clinical outcome after cardioversion for atrial fibrillation

2011 ◽  
Vol 105 (03) ◽  
pp. 435-443 ◽  
Author(s):  
Veronika Bruno ◽  
Rudolf Jarai ◽  
Susanne Gruber ◽  
Thomas Höchtl ◽  
Ivan Brozovic ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Independent Predictor ◽  
Thrombus Formation ◽  
Critical Role ◽  
Inverse Correlation ◽  
Von Willebrand ◽  
Rhythm Stability ◽  
Willebrand Factor

SummaryVon Willebrand factor (vWF) plays an essential role in platelet adhesion and thrombus formation. Patients with atrial fibrillation (AF) exhibit higher plasma vWF and lower ADAMTS13 antigen levels compared to controls. Little is known about vWF and ADAMTS13 in AF patients treated with cardioversion (CV). Thus we investigated the alterations of plasma vWF and ADAMTS13 after CV and evaluated the predictive value of these parameters for recurrence of AF. In this observational study we determined plasma levels of vWF and ADAMTS13 in 77 patients before and immediately after CV, as well as 24 hours (h) and six weeks thereafter, by means of commercially available assays. The vWF/ ADAMTS13-ratio was significantly elevated immediately after CV (p=0.02) and 24 h after CV (p=0.002) as compared to baseline levels. ADAMTS13, 24 h after CV, exhibited a significant association with recurrence of AF (HR: 0.97; p=0.037). Accordingly, tertiles of ADAMTS13 showed a stepwise inverse correlation with the risk of recurrent AF (HR: 0.50; p=0.009). After adjustment for confounders, ADAMTS13 remained significant as an independent predictor of recurrent AF (HR: 0.61; p=0.047). Similarly, the vWF/ADAMTS13-ratio, 24 h after CV, was associated with rhythm stability and remained an independent predictor of recurrent AF (HR: 1.88; p=0.028). The regulation of vWF and its cleaving protease ADAMTS13 after CV might play a critical role in producing a pro-thrombotic milieu immediately after CV for AF. Since ADAMTS13 plasma concentration and the vWF/ADAMTS13-ratio are independently associated with rhythm stability, these indexes might be used for prediction of recurrence of AF.

Simulation of platelet adhesion and aggregation regulated by fibrinogen and von Willebrand factor

2008 ◽  
Vol 99 (01) ◽  
pp. 108-115 ◽  
Author(s):  
Koichiro Yano ◽  
Ken-ichi Tsubota ◽  
Takuji Ishikawa ◽  
Shigeo Wada ◽  
Takami Yamaguchi ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Simple Shear Flow ◽  
Stokesian Dynamics ◽  
General Observation ◽  
Significant Development ◽  
Platelet Adhesion And Aggregation ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryWe propose a method to analyze platelet adhesion and aggregation computationally, taking into account the distinct properties of two plasma proteins, vonWillebrand factor (vWF) and fibrinogen (Fbg). In this method, the hydrodynamic interactions between platelet particles under simple shear flow were simulated using Stokesian dynamics based on the additivity of velocities. The binding force between particles mediated by vWF and Fbg was modeled using the Voigt model. Two Voigt models with different properties were introduced to consider the distinct behaviors of vWF and Fbg. Our results qualitatively agreed with the general observation of a previous in-vitro experiment, thus demonstrating that the significant development of thrombus formation in height requires not only vWF, but also Fbg. This agreement of simulation and experimental results qualitatively validates our model and suggests that consideration of the distinct roles of vWF and Fbg is essential to investigate the physiological and pathophysiological mechanisms of thrombus formation using a computational approach.

Endothelial CD40 Mediates Microvascular von Willebrand Factor-Dependent Platelet Adhesion Inducing Inflammatory Venothrombosis in ADAMTS13 Knockout Mice

2020 ◽  
Vol 120 (03) ◽  
pp. 466-476
Author(s):  
Sibgha Tahir ◽  
Andreas H. Wagner ◽  
Steffen Dietzel ◽  
Hanna Mannell ◽  
Joachim Pircher ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Knockout Mice ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Cd40 Ligand ◽  
Inflammatory Conditions ◽  
String Formation ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Background von Willebrand factor (vWF) plays an important role in platelet activation. CD40–CD40 ligand (CD40L) induced vWF release has been described in large vessels and cultured endothelium, but its role in the microcirculation is not known. Here, we studied whether CD40 is expressed in murine microvessels in vivo, whether CD40L induces platelet adhesion and leukocyte activation, and how deficiency of the vWF cleaving enzyme ADAMTS13 affects these processes. Methods and Results The role of CD40L in the formation of beaded platelet strings reflecting their adhesion to ultralarge vWF fibers (ULVWF) was analyzed in the murine cremaster microcirculation in vivo. Expression of CD40 and vWF was studied by immunohistochemistry in isolated and fixed cremasters. Microvascular CD40 was only expressed under inflammatory conditions and exclusively in venous endothelium. We demonstrate that CD40L treatment augmented the number of platelet strings, reflecting ULVWF multimer formation exclusively in venules and small veins. In ADAMTS13 knockout mice, the number of platelet strings further increased to a significant extent. As a consequence extensive thrombus formation was induced in venules of ADAMTS13 knockout mice. In addition, circulating leukocytes showed primary and rapid adherence to these platelet strings followed by preferential extravasation in these areas. Conclusion CD40L is an important stimulus of microvascular endothelial ULVWF release, subsequent platelet string formation and leukocyte extravasation but only in venous vessels under inflammatory conditions. Here, the lack of ADAMTS13 leads to severe thrombus formation. The results identify CD40 expression and ADAMTS13 activity as important targets to prevent microvascular inflammatory thrombosis.

scholarly journals Novel aptamer to von Willebrand factor A1 domain (TAGX-0004) shows total inhibition of thrombus formation superior to ARC1779 and comparable to caplacizumab

Haematologica ◽  
2019 ◽  
Vol 105 (11) ◽  
pp. 2631-2638 ◽  
Author(s):  
Kazuya Sakai ◽  
Tatsuhiko Someya ◽  
Kaori Harada ◽  
Hideo Yagi ◽  
Taei Matsui ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Von Willebrand Factor ◽  
Plasmon Resonance ◽  
Binding Sites ◽  
Thrombus Formation ◽  
Static Conditions ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand factor (VWF) is a blood glycoprotein that plays an important role in platelet thrombus formation through interaction between its A1 domain and platelet glycoprotein Ib. ARC1779, an aptamer to the VWF A1 domain, was evaluated in a clinical trial for acquired thrombotic thrombocytopenic purpura (aTTP). Subsequently, caplacizumab, an anti-VWF A1 domain nanobody, was approved for aTTP in Europe and the United States. We recently developed a novel DNA aptamer, TAGX-0004, to the VWF A1 domain; it contains an artificial base and demonstrates high affinity for VWF. To compare the effects of these three agents on VWF A1, their ability to inhibit ristocetin- or botrocetin-induced platelet aggregation under static conditions was analyzed, and the inhibition of thrombus formation under high shear stress was investigated in a microchip flow chamber system. In both assays, TAGX-0004 showed stronger inhibition than ARC1779, and had comparable inhibitory effects to caplacizumab. The binding sites of TAGX-0004 and ARC1779 were analyzed with surface plasmon resonance performed using alanine scanning mutagenesis of the VWF A1 domain. An electrophoretic mobility shift assay showed that R1395 and R1399 in the A1 domain bound to both aptamers. R1287, K1362, and R1392 contributed to ARC1779 binding, and F1366 was essential for TAGX-0004 binding. Surface plasmon resonance analysis of the binding sites of caplacizumab identified five amino acids in the VWF A1 domain (K1362, R1392, R1395, R1399, and K1406). These results suggested that TAGX-0004 possessed better pharmacological properties than caplacizumab in vitro and might be similarly promising for aTTP treatment.

The role of platelet von Willebrand factor in platelet adhesion and thrombus formation: a study of 34 patients with various subtypes of type I von Willebrand disease

1994 ◽  
Vol 86 (2) ◽  
pp. 327-332 ◽  
Author(s):  
Edith Fressinaud ◽  
Augusto B. Federici ◽  
Giancarlo Castaman ◽  
Chantal Rothschild ◽  
Francesco Rodeghiero ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Von Willebrand Disease ◽  
Type I ◽  
Von Willebrand ◽  
Willebrand Factor

scholarly journals Recognition of PF4-VWF complexes by heparin-induced thrombocytopenia antibodies contributes to thrombus propagation

Blood ◽  
2020 ◽  
Vol 135 (15) ◽  
pp. 1270-1280 ◽  
Author(s):  
Ian Johnston ◽  
Amrita Sarkar ◽  
Vincent Hayes ◽  
Gavin T. Koma ◽  
Gowthami M. Arepally ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Complex Formation ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Microfluidic System ◽  
Microfluidic Channels ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by complexes between platelet factor 4 (PF4) and heparin or other polyanions, but the risk of thrombosis extends beyond exposure to heparin implicating other PF4 partners. We recently reported that peri-thrombus endothelium is targeted by HIT antibodies, but the binding site(s) has not been identified. We now show that PF4 binds at multiple discrete sites along the surface of extended strings of von Willebrand factor (VWF) released from the endothelium following photochemical injury in an endothelialized microfluidic system under flow. The HIT-like monoclonal antibody KKO and HIT patient antibodies recognize PF4-VWF complexes, promoting platelet adhesion and enlargement of thrombi within the microfluidic channels. Platelet adhesion to the PF4-VWF-HIT antibody complexes is inhibited by antibodies that block FcγRIIA or the glycoprotein Ib-IX complex on platelets. Disruption of PF4-VWF-HIT antibody complexes by drugs that prevent or block VWF oligomerization attenuate thrombus formation in a murine model of HIT. Together, these studies demonstrate assembly of HIT immune complexes along VWF strings released by injured endothelium that might propagate the risk of thrombosis in HIT. Disruption of PF4-VWF complex formation may provide a new therapeutic approach to HIT.

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.

Glycoprotein Ibα and von Willebrand factor in primary platelet adhesion and thrombus formation: Lessons from mutant mice

2008 ◽  
Vol 99 (02) ◽  
pp. 264-270 ◽  
Author(s):  
Anil K. Chauhan ◽  
Denisa D. Wagner ◽  
Wolfgang Bergmeier
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Mutant Mice ◽  
Receptor Complex ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Primary Platelet ◽  
Adhesion Process ◽  
Main Ligand

SummaryThe von Willebrand factor (VWF) receptor complex, glycoprotein (GP)Ib-V-IX, and its main ligand VWF play a key role in the adhesion process of platelets to sites of vascular injury. Recent studies in mutant mice have shed new light on the importance of either molecule for the development of arterial and venous thrombosis. In this review, we summarize the most important aspects from these studies.

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