Platelet Glycoprotein Ib-IX Research Articles

Essential role of zyxin in platelet biogenesis and glycoprotein Ib-IX surface expression

Platelets are generated from the cytoplasm of megakaryocytes (MKs) via actin cytoskeleton reorganization. Zyxin is a focal adhesion protein and wildly expressed in eukaryotes to regulate actin remodeling. Zyxin is upregulated during megakaryocytic differentiation; however, the role of zyxin in thrombopoiesis is unknown. Here we show that zyxin ablation results in profound macrothrombocytopenia. Platelet lifespan and thrombopoietin level were comparable between wild-type and zyxin-deficient mice, but MK maturation, demarcation membrane system formation, and proplatelet generation were obviously impaired in the absence of zyxin. Differential proteomic analysis of proteins associated with macrothrombocytopenia revealed that glycoprotein (GP) Ib-IX was significantly reduced in zyxin-deficient platelets. Moreover, GPIb-IX surface level was decreased in zyxin-deficient MKs. Knockdown of zyxin in a human megakaryocytic cell line resulted in GPIbα degradation by lysosomes leading to the reduction of GPIb-IX surface level. We further found that zyxin was colocalized with vasodilator-stimulated phosphoprotein (VASP), and loss of zyxin caused diffuse distribution of VASP and actin cytoskeleton disorganization in both platelets and MKs. Reconstitution of zyxin with VASP binding site in zyxin-deficient hematopoietic progenitor cell-derived MKs restored GPIb-IX surface expression and proplatelet generation. Taken together, our findings identify zyxin as a regulator of platelet biogenesis and GPIb-IX surface expression through VASP-mediated cytoskeleton reorganization, suggesting possible pathogenesis of macrothrombocytopenia.

Force-Regulated Refolding of the Mechanosensory Domain in the Platelet Glycoprotein Ib-IX Complex

In platelets, the glycoprotein (GP) Ib-IX receptor complex senses blood shear flow and transmits the mechanical signals into platelets. Recently, we have discovered a juxtamembrane mechanosensory domain (MSD) within the GPIbα subunit of GPIb-IX. Mechanical unfolding of the MSD activates GPIb-IX signaling into platelets, leading to their activation and clearance. Using optical tweezer-based single-molecule force measurement, we herein report a systematic biomechanical characterization of the MSD in its native, full-length receptor complex and a recombinant, unglycosylated MSD in isolation. The native MSD unfolds at a resting rate of 9 × 10−3 s−1. Upon exposure to pulling forces, MSD unfolding accelerates exponentially over a force scale of 2.0 pN. Importantly, the unfolded MSD can refold with or without applied forces. The unstressed refolding rate of MSD is ∼17 s−1 and slows exponentially over a force scale of 3.7 pN. Our measurements confirm that the MSD is relatively unstable, with a folding free energy of 7.5 kBT. Because MSD refolding may turn off GPIb-IX’s mechanosensory signals, our results provide a mechanism for the requirement of a continuous pulling force of >15 pN to fully activate GPIb-IX.

Force-Induced Unfolding of a Mechanosensory Domain in Platelet Glycoprotein (Gp)Ib-IX under Solution and Adherent Conditions

Force-Induced Unfolding of a Mechanosensory Domain in Platelet Glycoprotein (Gp)Ib-IX under Solution and Adherent Conditions

Extracellular signal‐regulated kinase 5 associates with casein kinase II to regulate GPIb‐IX‐mediated platelet activation via the PTEN/PI3K/Akt pathway

Background The platelet glycoprotein (GP) Ib-IX complex plays essential roles in thrombosis and hemostasis. The mitogen-activated protein kinases (MAPKs) ERK1/2 and p38 have been shown to be important in the GPIb-IX-mediated signaling leading to integrin activation. However, the roles of the MAPK extracellular signal-regulated kinase 5 (ERK5) in GPIb-IX-mediated platelet activation are unknown. Objective To reveal the function and mechanisms of ERK5 in GPIb-IX-mediated platelet activation. Methods The functions of ERK5 in GPIb-IX-mediated human platelet activation were assessed using botrocetin/VWF, ristocetin/VWF, or platelet adhesion to von Willebrand factor (VWF) under shear stress in the presence of a specific inhibitor of ERK5. ERK5-associated proteins were pulled down from Chinese hamster ovary (CHO) cells transfected with HA-tagged-ERK5, identified by mass spectrometry, and confirmed in human platelets. Roles of ERK5-associated proteins in GPIb-IX-mediated platelet activation were clarified using specific inhibitors. Results The phosphorylation levels of ERK5 were significantly enhanced in human platelets stimulated with botrocetin/VWF or ristocetin/VWF. The ERK5 inhibitor XMD8-92 suppressed the second wave of human platelet aggregation induced by botrocetin/VWF or ristocetin/VWF and inhibited human platelet adhesion on immobilized VWF under shear stress. Casein kinase II (CKII) was identified as an ERK5-associated protein in human platelets. The CKII inhibitor TBB, similar to the ERK5 inhibitor XMD8-92, specifically restrained PTEN phosphorylation, therefore suppressing Akt phosphorylation in human platelets treated with botrocetin/VWF. Conclusion ERK5 associates with CKII to play essential roles in GPIb-IX-mediated platelet activation via the PTEN/PI3K/Akt pathway.

Membrane skeleton orchestrates the platelet glycoprotein (GP) Ib-IX complex clustering and signaling.

SummaryPlatelet glycoprotein Ib‐IX complex is affixed to the membrane skeleton through interaction with actin binding protein 280 (ABP‐280). We find that removal of the ABP‐280 binding sites in GP Ibα cytoplasmic tail has little impact on the complex clustering induced by antibody crosslinking. However, large truncation of the GP Ibα cytoplasmic tail allows the formation of larger patches of the complex, suggesting that an ABP‐280 independent force may exist. Besides, we observe that the signaling upon GP Ib‐IX clustering is elicited in both membrane lipid domain dependent and independent manner, a choice that relies on how the membrane skeleton interacts with the complex. Our findings suggest a more complex mechanism for how the membrane skeleton regulates the GP Ib‐IX function. © 2016 The Authors IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 68(10):823–829, 2016

Biophysical Characterization of Mechanosensors within the Plasma Protein von Willebrand Factor and its Receptor Platelet Glycoprotein Ib-IX

The large, multimeric plasma protein von Willebrand Factor (VWF) plays an essential role in capturing platelets onto the damaged vascular wall, allowing the initiation of blood clotting. Platelet binding is achieved by interaction between the A1 domain of the Iba chain of platelet surface receptor Glycoprotein (GP) Ib-IX. Both GPIB-IX and VWF are capable of sensing and reacting to tensile forces. Using the single-molecule force measurement approach, we have recently identified a quasi-stable mechano-sensitive domain (MSD) of ∼60 residues between the macroglycopeptide region and the transmembrane helix of the GPIba subunit. The MSD unfolds at 5-20 pN when subjected to mechanical stretch by the engaged A1. Unfolding of the MSD triggers receptor signaling and stimulates platelets. Similarly, earlier studies by the Springer group have identified the mechanosensor within VWF, the A2 domain (i.e., the domain adjacent to A1). Tensile force at the level of 10-20 pN unfolds the A2 structure, leading to A2's subsequent cleavage by plasma enzyme ADAMTS-13 (Zhang, X. et al. Science, 324:1330-4). Since both MSD and A2 unfold at a similar force level, simultaneous activation of both mechanosensors may occur given that the interacting VWF and GPIb-IX are stretched to provide sufficient force. Herein, we show that optical tweezer-controlled pulling of the full-length of VWF on GPIb-IX indeed induced unfolding of both MSD and A2. Additional studies using recombinant wildtype and mutant VWF suggest that the unfolding of MSD and A2 is dependent on the mechanical strengths of the VWF-GPIba and VWF-collagen interactions, as well as on the activation state of the VWF. Together, the study has revealed the biomechanical properties of VWF and GPIb-IX, with significant implications for the pathogenesis of Von Willebrand Disease and GPIb-IX- related blood diseases.

The Transmembrane Domains of β and IX Subunits Mediate the Localization of the Platelet Glycoprotein Ib-IX Complex to the Glycosphingolipid-enriched Membrane Domain

We have previously reported that the structural elements of the GP Ib-IX complex required for its localization to glycosphingolipid-enriched membranes (GEMs) reside in the Ibβ and IX subunits. To identify them, we generated a series of cell lines expressing mutant GP Ibβ and GP IX where 1) the cytoplasmic tails (CTs) of either or both GP Ibβ and IX are truncated, and 2) the transmembrane domains (TMDs) of GP Ibβ and GP IX were swapped with the TMD of a non-GEMs associating molecule, human transferrin receptor. Sucrose density fractionation analysis showed that the removal of either or both of the CTs from GP Ibβ and GP IX does not alter GP Ibα-GEMs association when compared with the wild type. In contrast, swapping of the TMDs of either GP Ibβ or GP IX with that of transferrin receptor results in a significant loss (∼ 50%) of GP Ibα from the low density GEMs fractions, with the largest effect seen in the dual TMD-replaced cells (> 80% loss) when compared with the wild type cells (100% of GP Ibα present in the GEMs fractions). Under high shear flow, the TMD-swapped cells adhere poorly to a von Willebrand factor-immobilized surface to a much lesser extent than the previously reported disulfide linkage dysfunctional GP Ibα-expressing cells. Thus, our data demonstrate that the bundle of GP Ibβ and GP IX TMDs instead of their individual CTs is the structural element that mediates the β/IX complex localization to the membrane GEMs, which through the α/β disulfide linkage brings GP Ibα into the GEMs.

Abstract 598: A Platelet Glycoprotein Ib-IX-specific 14-3-3/Rac1/LIMK1 Signaling Pathway Promotes Thrombin-Induced Platelet Activation

Rationale: Thrombin-induced platelet activation requires protease-activated receptors. However, the platelet glycoprotein (GP) Ib-IX complex (GPIb-IX) binds to thrombin and is important for low dose thrombin-induced platelet activation. It is unclear how GPIb-IX promotes thrombin-induced platelet activation. Objective: To clearly elucidate the mechanism by which GPIb-IX promotes thrombin-induced platelet activation. Methods and Results: We reconstituted GPIb-IX (GPIb) /Protease-activated receptors (PARs) cooperativity in response to thrombin in Chinese Hamster Ovary (CHO) cells expressing PAR1. Thrombin-induced PAR1-dependent calcium signaling was significantly enhanced by GPIb expression. This effect of GPIb appears to require GPIb signaling, as mutation of a cytoplasmic binding site for an intracellular signaling molecule, 14-3-3, in GPIbα abolished the stimulatory effect of GPIb. The importance of GPIb-IX-14-3-3 interaction in promoting thrombin-induced platelet activation was also shown by pretreating human platelets with MPαC, an inhibitory peptide based on a critical 14-3-3 binding site in the C-terminus of GPIbα, which inhibited thrombin-induced platelet activation, but did not affect thrombin binding to platelets. Furthermore, 14-3-3 binding site deletion in GPIbα or MPαC-pretreatment inhibited thrombin-induced activation of Rac1 and phosphorylation of LIMK1. To determine the role of the Rac1/LIMK1 signaling pathway in mediating thrombin-induced GPIb signaling and platelet activation, we examined the effects of Rac1 knockout, LIMK1 knockout and Rac1-inhibitor on low dose thrombin-induced calcium response and platelet activation. Rac1 inhibitor, NSC23766, abolished the GPIb-dependent cell response in a reconstituted CHO cell model. Rac1 knockout platelets showed diminished platelet response to thrombin and were not different from wild type platelets in the presence of MPαC. Importantly, LIMK1-/- platelets display defective thrombin-induced platelet activation but enhanced PAR4-activating peptide induced platelet activation. Conclusions: The stimulatory role of GPIb in thrombin-induced platelet activation requires a thrombin-induced GPIb-specific 14-3-3/Rac1/LIMK1 signaling pathway.

A Signaling Mechanism By Which Platelet Glycoprotein Ib-IX Promotes Thrombin-Induced Platelet Activation

Numerous reports indicate that the platelet glycoprotein (GP) Ib-IX complex (GPIb-IX) binds directly to the potent platelet agonist thrombin and is important for promoting thrombin-induced platelet activation. However, how GPIb-IX contributes to thrombin-induced platelet activation is unclear. It has been suggested that thrombin binding to GPIb facilitates the cleavage, and thus activation, of the protease-activated receptors (PAR). Our data indicate that GPIb-IX promotes thrombin signaling through a GPIb-IX signaling mechanism. We reconstituted GPIb-IX (GPIb) /Protease-activated receptor (PAR) cooperativity in response to thrombin in Chinese Hamster Ovary (CHO) cells expressing PAR1. Thrombin-induced PAR1-dependent calcium signaling was significantly enhanced by GPIb expression, and this effect of GPIb appears to require GPIb signaling, as deletion of the cytoplasmic binding site for an intracellular signaling molecule, 14-3-3, in GPIbα abolished the stimulatory effect of GPIb. The importance of GPIb-14-3-3 interaction in promoting thrombin-induced platelet activation was also shown in human platelets, in which pretreatment with MPαC, an inhibitory peptide based on a critical 14-3-3 binding site in the C-terminus of the GPIbα, inhibited thrombin-induced platelet activation. Furthermore, 14-3-3 binding site deletion in GPIba or MPαC-pretreatment inhibited thrombin-induced activation of Rac1 and phosphorylation of LIMK1, both of which have been shown to mediate von Willebrand factor-induced GPIb signaling, and the role of GPIb in promoting thrombin signaling was abolished with a Rac-inhibitor, NSC23766 or in Rac1-/- platelets. Importantly, LIMK1-/- platelets display defective thrombin-induced platelet activation but enhanced PAR4-activating peptide induced platelet activation. DisclosuresNo relevant conflicts of interest to declare.

Inherited Giant Platelet Disorder, Kashmiri Thrombocytopenia, a Common Syndrome Found in Srinagar, India

The causative genetic mutations of inherited giant platelet disorders (IGPD) encompass genes coding for the platelet glycoprotein Ib-IX complex (Bernard Soulier syndrome and its variants), myosin heavy chain 9 (MYH9 gene mutated in May-Hegglin anomaly and other IGPDs), GATA-01 (GATA-related thrombocytopenia), TUBB-1, ITGA2, ITGAB3, FLNA and some others. IGPDs are frequently associated with other disorders including renal disease, sensorineural deafness, and leukocyte inclusion bodies. Most are accompanied with variable degrees of bleeding diathesis, while others, like TUBB1 IGPD, do not have any bleeding manifestations. Harris platelet syndrome (HPS), previously called asymptomatic constitutional macrothrombocytopenia, is an autosomal dominant disorder characterized by low-normal to severe thrombocytopenia IGPD and absence of bleeding. HPS has also been observed in healthy blood donors from the northeastern part of India (Bengal) and some areas of Bangladesh, Bhutan and Nepal.We describe a high prevalence of an autosomal dominantly inherited form of IGPD with mild to severe thrombocytopenia in the Muslim population in Kashmir Valley in the northern Indian subcontinent. 830 voluntary, healthy, male blood donors from Kashmir Valley were included in the study. They were aged 15-55 years (median 31 years) and underwent ancillary screening as follows; CBC, peripheral smear, HBV, HCV, HIV, ANA and Anti-H pylori antibodies. 15% of the donors had thrombocytopenia (mean platelet count 109.6 compared to 189.9 in controls; p=<0.0001). No differences were noted in age between the 2 groups. The mean platelet volume (MPV) in thrombocytopenic subjects was higher (12.53 + 0.78 vs 9.52 + 1.03 fl). The red cell distribution width (RDW) in thrombocytopenic subjects was higher than in those with normal counts (15.6 + 1.61 Vs 13. 22 + 1.36, p=<0.001). Hematocrit and other red cell indices were not different in the 2 groups. None of the participants had a history of bleeding, renal disease, sensorineural deafness, or leukocyte inclusion bodies. Peripheral blood platelet morphology revealed large platelets in all subjects. In a pilot study of 7 families, Kashmiri thrombocytopenia was compatible with autosomal dominant inheritance affecting both genders. The congenital nature of Kashmiri thrombocytopenia was demonstrated by analyses of 34 consecutive neonates born in Sher-i-Kashmir Institute Hospital; among 20 girls and 19 boys, we found 18% (2 male and 5 female) to have low platelet count, the mean platelet count of the affected group when compared to unaffected group were 102.6 vs 234 (p=<0.001) respectively.We then searched for a causative mutation using the following approaches. We sequenced the MYH9 gene and no mutation was found. We then employed SNP array analyses using Shared Genome Segment (SGS) and Whole Genome Association Study (WGAS). We were able to exclude all previously reported IGPD-causing genes. SGS that overlapped with WGAS narrowed the target into 3 chromosome regions in Chr. 5 (rs6872531-rs100072476), Chr. 9 (rs11999541-rs12682912) and Chr. 10 (rs11013452-rs7083349). The performed SNP analyses included large genomic segments as candidates for a Kashmiri thrombocytopenia-causative gene. To further narrow down the cause of this disorder, we recruited 3 TRIO families (an affected parent and a child) for stronger linkage analysis and next-generation sequencing to continue the search for the cause of the Kashmiri thrombocytopenia. DisclosuresNo relevant conflicts of interest to declare.

Abstract 11947: Unraveling the Mechanism for the Stimulatory Role of Platelet GPIb-IX-V in Thrombin-Induced Platelet Activation

Numerous reports indicate that the platelet glycoprotein (GP) Ib-IX complex (GPIb-IX) binds directly to the potent platelet agonist thrombin and is important for promoting thrombin-induced platelet activation. However, how GPIb-IX contributes to thrombin-induced platelet activation is unclear. It has been suggested that thrombin binding to GPIb facilitates the cleavage, and thus activation, of the protease-activated receptors (PAR). Our data indicate that GPIb-IX promotes thrombin signaling through a GPIb-IX signaling mechanism. Pretreatment of human platelets with MPalphaC, an inhibitory peptide based on a critical 14-3-3 signaling protein binding site on the cytoplasmic domain of the GPIb alpha chain, inhibited thrombin-induced platelet activation. MPalphaC-treatment inhibited thrombin-induced activation of Rac1 and LIMK1, both of which are known to play essential roles in GPIb signaling. To more specifically determine the role of GPIb-IX, we reconstituted GPIb-IX-facilitated thrombin signaling in Chinese Hamster Ovary cells expressing PAR1. Thrombin induced signaling was significantly enhanced by GPIb-expression, and deletion of the cytoplasmic 14-3-3-binding domain of GPIb alpha abolished the stimulatory effect of GPIb on thrombin signaling. Furthermore, the role of GPIb-IX in promoting thrombin signaling requires Rac1, and GPIb-IX-dependent Rac1 activation and LIMK phosphorylation are abolished in delta 605 cells expressing a 14-3-3-binding defective mutant GPIb alpha. Taken together, these data suggest that the stimulatory role of GPIb in thrombin signaling requires a C-terminal 14-3-3-binding region which mediates activation of a Rac1/LIMK1 pathway that promotes thrombin signaling leading to platelet activation.

Platelet Glycoprotein Ib-IX as a Regulator of Systemic Inflammation

The platelet glycoprotein Ib-IX (GP Ib-IX) receptor is a well-characterized adhesion receptor supporting hemostasis and thrombosis via interactions with von Willebrand factor. We examine the GP Ib-IX/von Willebrand factor axis in murine polymicrobial sepsis, as modeled by cecal ligation and puncture (CLP). Genetic absence of the GP Ib-IX ligand, von Willebrand factor, prolongs survival after CLP, but absence of the receptor, GP Ib-IX, does not. Because absence of either von Willebrand factor or GP Ib-IX significantly impairs hemostasis and thrombosis, we sought to define additional GP Ib-IX-dependent pathways impacting survival in the CLP model. We document that the absence of GP Ib-IX leads to reduced platelet-neutrophil and platelet-monocyte interactions. Twenty-four hours after CLP, absence of GP Ib-IX coincides with an alteration in cytokine levels, such as tumor necrosis factor-α secreted by monocytes, and increased macrophage-1 antigen expression by neutrophils. In contrast to the well-characterized proinflammatory properties of platelets, we describe in the CLP model an anti-inflammatory property associated with platelet GP Ib-IX. Thus, a single platelet receptor displays a dual modulatory role in both the thrombotic and inflammatory pathways associated with polymicrobial sepsis. In sharing leucine-rich motifs with toll-like receptors, platelet GP Ib-IX can be considered a multifunctional participant in hemostasis, thrombosis, and the inflammatory cascade. The results highlight a dynamic role for platelets in systemic inflammation and add to the complex pathophysiologic events that occur during the dysregulated coagulation and inflammation associated with sepsis.

The Platelet Glycoprotein Ib-IX Axis In Murine Polymicrobial Sepsis

The platelet glycoprotein (GP) Ib-IX receptor complex is expressed exclusively on the surface of platelets and is well characterized as a primary adhesion receptor supporting normal hemostasis and pathologic thrombosis. Beyond hemostasis and thrombosis, platelets can also participate in the innate immune response and inflammation. While the platelet as a contributor to the immune continuum is recognized, many aspects of the molecular mechanisms whereby platelets influence the immune response are still undefined. Here, we report studies using a murine model of GP Ib-IX deficiency linking GP Ib-IX to the immune response associated with polymicrobial sepsis, as modeled by cecal ligation and puncture (CLP). In the CLP model, genetic absence of the major GP Ib-IX hemostatic ligand, von Willebrand factor (VWF), improves survival following CLP when compared to control wild-type animals (p= 0.003, Logrank analysis). This suggests a VWF role in thrombosis contributes to survival outcome following CLP. In contrast, genetic absence of the VWF platelet receptor, GP Ib-IX, does not improve survival with no statistical difference comparing wild-type animals to GPIb-IX deficient animals. The molecular basis to explain improved survival in VWF-deficient (ligand deficient) but not GPIb-IX deficient (receptor deficient) animals was pursued. We tested the hypothesis GPIb-IX has normal physiologic and pathophysiologic functions beyond platelet adhesion influencing infection and an inflammatory response. Indeed, GPIb-IX influencing the innate immune response is not completely unexpected since a hallmark structural feature of each subunit of the GPIb-IX receptor is leucine rich repeats, the common motif to all members of the toll like receptor family (TLRs). Whether structural similarities are a consequence of ancestral origins for GPIb-IX and TLRs is unknown. We first documented in the absence of murine platelet GP Ib-IX there are reduced platelet-neutrophil and platelet-monocyte interactions under normal conditions and following CLP in whole blood. Whether there are physiologic consequences for disrupting a platelet/monocyte and/or platelet/neutrophil axis was determined via multianalyte profiling of circulating cytokine levels on a Luminex analyzer following CLP. In the absence of GP Ib-IX there is a robust and statistically significant increase 24 hrs following CLP in some of the major proinflammatory cytokines produced by monocytes and macrophages, including TNFα, MCP-1, MIP-β, IL-6, and IL-15. Increases in cytokines, such as IL-5 and IL-13, associated with other immune cells were also observed. These results highlight a coagulation/inflammation interface where the platelet, and specifically GP Ib-IX, contributes to the pathophysiology of CLP. On the one hand, absence of platelet GPIb-IX reduces thrombotic potential, but it occurs at the expense of upregulation of inflammatory cytokine release leading to a reduced survival in CLP. Clearly, survival outcomes in CLP reflect a complex dysregulation of coagulation and inflammation where platelet GPIb-IX likely contributes to both processes with physiologic consequences. Understanding dysregulation of the coagulation/ inflammation interface and identifying a platelet receptor (GPIb-IX) critical to both adds new information to this complex set of pathophysiologic events Sharing the common structural motifs, leucine rich repeats, with the well characterized family of toll-like receptors, platelet GPIb-IX should now be considered an active participant in the inflammatory cascade. Disclosures:No relevant conflicts of interest to declare.

Role for Platelet Glycoprotein Ib-IX and Effects of its Inhibition in Endotoxemia-Induced Thrombosis, Thrombocytopenia, and Mortality

Poor prognosis of sepsis is associated with bacterial lipopolysaccharide (LPS)-induced intravascular inflammation, microvascular thrombosis, thrombocytopenia, and disseminated intravascular coagulation. Platelets are critical for thrombosis, and there has been increasing evidence of the importance of platelets in endotoxemia. The platelet adhesion receptor, the glycoprotein Ib-IX complex (GPIb-IX), mediates platelet adhesion to inflammatory vascular endothelium and exposed subendothelium. Thus, we have investigated the role of GPIb-IX in LPS-induced platelet adhesion, thrombosis, and thrombocytopenia. LPS-induced mortality is significantly decreased in mice expressing a functionally deficient mutant of GPIbα. Furthermore, we have developed a micellar peptide inhibitor, MPαC (C13H27CONH-SIRYSGHpSL), which selectively inhibits the von Willebrand factor -binding function of GPIb-IX and GPIb-IX-mediated platelet adhesion under flow without affecting GPIb-IX-independent platelet activation. MPαC inhibits platelet adhesion to LPS-stimulated endothelial cells in vitro and alleviates LPS-induced thrombosis in glomeruli in mice. Importantly, MPαC reduces mortality in LPS-challenged mice, suggesting a protective effect of this inhibitor during endotoxemia. Interestingly, MPαC, but not the integrin antagonist, Integrilin, alleviated LPS-induced thrombocytopenia. These data indicate an important role for the platelet adhesion receptor GPIb-IX in LPS-induced thrombosis and thrombocytopenia, and suggest the potential of targeting GPIb as an antiplatelet strategy in managing endotoxemia.

Role of 14-3-3ζ in Platelet Glycoprotein Ibα-von Willebrand Factor Interaction-Induced Signaling

The interaction of platelet glycoprotein (GP) Ib-IX with von Willebrand factor (VWF) exposed at the injured vessel wall or atherosclerotic plaque rupture initiates platelet transient adhesion to the injured vessel wall, which triggers intracellular signaling cascades leading to platelet activation and thrombus formation. 14-3-3ζ has been verified to regulate the VWF binding function of GPIb-IX by interacting with the cytoplasmic domains of GPIb-IX. However, the data regarding the role of 14-3-3ζ in GPIb-IX-VWF interaction-induced signaling still remain controversial. In the present study, the data indicate that the S609A mutation replacing Ser609 of GPIbα with alanine (S609A) significantly prevented the association of 14-3-3ζ with GPIbα before and after the VWF binding to GPIbα. GPIb-IX-VWF interaction-induced activations of Src family kinases and protein kinase C were clearly reduced in S609A mutation. Furthermore, S609A mutation significantly inhibited GPIb-IX-VWF interaction-induced elevation of cytoplasmic Ca2+ levels in flow cytometry analysis. Taken together, these data indicate that the association of 14-3-3ζ with the cytoplasmic domain of GPIbα plays an important role in GPIb-IX-VWF interaction-induced signaling.

The Association of 14-3-3ζ and Filamin A with the Central Cytoplasmic Domain of GPIbα Involves in Regulation of the Von Willebrand Factor Binding and Signaling Functions of Platelet Glycoprotein Ib-IX.

Abstract 5073 IntroductionConformational changes in von Willebrand factor (VWF) exposed at the injured vessel wall initiate platelet tethering and simultaneously trigger signaling cascades leading to platelet adhesion, spreading, and thrombus formation. Although the intracellular signaling protein 14-3-3ζ and the membrane skeleton protein filamin A have been confirmed to interact with the cytoplasmic domain of GPIbα and involve in the regulation of VWF binding function, whereas, the mechanisms still remain unclear. We have recently identified a novel 14-3-3ζ binding site with partial phosphorylation of Ser559 in resting platelets located in the cytoplasmic filamin A binding domain of GPIbα. Disruption of the central cytoplasmic 14-3-3ζ binding site of GPIbα did not abolish the interaction between 14-3-3ζ and GPIb-IX, whereas obviously diminished VWF binding function. Here we show that disruption of 14-3-3ζ binding to GPIbα by GPIbα mutants or cell permeable peptides enhances the association of filamin A with GPIb-IX and the expression of GPIb-IX in the plasma membrane. In contrast, disruption of 14-3-3ζ binding to GPIbβ, which would enhance the opportunity for 14-3-3ζ monomer to bind to GPIbα, reduces filamin A-GPIb-IX interaction and GPIb-IX membrane expression. Compared with a GPIb-IX mutant truncating the whole cytoplasmic domain of GPIbα (Δ551), GPIb-IX mutant retaining only the central 14-3-3ζ binding site of the cytoplasmic domain of GPIbα (Δ565) showed an enhanced cell adhesion to VWF under flow and ristocetin-induced VWF binding. Furthermore, the two cell permeable peptides, MP-P and MPαC, which had been confirmed to block the central 14-3-3ζ binding site of GPIbα (MP-P) or diminished the association of 14-3-3ζ with GPIb-IX complex (MPαC), obviously inhibited ristocetin induced wild-type GPIb-IX cell aggregation. In addition, MP-P and MPαC inhibited botrocetin-induced platelet spreading and lamellipodia formation on VWF matrix, and GPIb-IX-VWF interaction induced co-immunoprecipitation of Src with GPIb-IX. Taken together, these data indicate that the binding of 14-3-3ζ and filamin A to the central cytoplasmic domain of GPIbα involves in regulation of surface expression, VWF binding and signaling functions of glycoprotein Ib-IX. DisclosuresNo relevant conflicts of interest to declare.

Identification of a Novel 14-3-3ζ Binding Site Within the Cytoplasmic Domain of Platelet Glycoprotein Ibα That Plays a Key Role in Regulating the von Willebrand Factor Binding Function of Glycoprotein Ib-IX

The interaction between platelet glycoprotein (GP) Ib-IX and von Willebrand factor (VWF) is initiated by conformational changes in immobilized VWF and is also regulated by the intraplatelet proteins 14-3-3zeta and filamin A. Both 14-3-3zeta and filamin A associate with the cytoplasmic domain of GPIbalpha, whereas little is known about their relationship in regulating the VWF binding function of GPIb-IX. To explore the mechanism underlying the roles of 14-3-3zeta and filamin A in regulating the VWF binding function of GPIb-IX. A truncation mutant of GPIbalpha (Delta565) deleting the C-terminal 14-3-3zeta binding sites retained 14-3-3zeta binding function, in contrast, deletion of the C-terminal residues 551 to 610 of GPIbalpha totally abolished 14-3-3zeta binding, indicating that the residues 551 to 564 of GPIbalpha are important in the interaction between 14-3-3zeta and GPIb-IX. An antibody recognizing phosphorylated R557GpSLP561 sequence reacted with GPIbalpha suggesting phosphorylation of a population of GPIbalpha molecules at Ser559, and a membrane permeable phosphopeptide (MP-P), R557GpSLP561 corresponding to residues 557 to 561 of GPIbalpha eliminated the association of 14-3-3zeta with Delta565. MP-P also promoted GPIb-IX association with the membrane skeleton, and inhibited ristocetin-induced platelet agglutination, VWF binding to platelets and platelet adhesion to immobilized VWF. Furthermore, a GPIb-IX mutant replacing Ser559 of GPIbalpha with alanine showed an enhanced association with the membrane skeleton, reduced ristocetin-induced VWF binding, and diminished ability to mediate cell adhesion to VWF under flow conditions. These data suggest a phosphorylation-dependent binding of 14-3-3zeta to central filamin A binding site of GPIbalpha, and the dimeric 14-3-3zeta binding to both the C-terminal site and central RGpSLP site inhibits GPIb-IX association with the membrane skeleton and promotes the VWF binding function of GPIb-IX.

Binding of platelet glycoprotein Ibbeta through the convex surface of leucine-rich repeats domain of glycoprotein IX.

The mechanism of assembly of the platelet glycoprotein (GP) Ib-IX complex from GPIbalpha, GPIbbeta and GPIX subunits is not entirely clear. In this complex, ectodomains of both GPIbbeta and GPIX subunits contain two leucine-rich repeats (LRR) and share high sequence similarity. However, they differ noticeably in stability, hampering further analysis of their interaction. Guided by analysis of the LRR structure, we report a well-folded Ibbeta/IX chimera and its usage in dissecting GPIX function. In this chimera, three non-contiguous sequences that may constitute the putative convex surface of the GPIbbeta ectodomain are replaced by their GPIX counterparts. Like GPIbbeta but unlike GPIX ectodomain, it can secrete from transfected Chinese hamster ovary cells and fold into a stable conformation. Furthermore, replacing the ectodomain in GPIX with the Ibbeta/IX chimera, but not the GPIbbeta ectodomain, preserved its interaction with GPIbbeta as demonstrated by its native-like GPIbbeta-induced increase in surface expression and coimmunoprecipitation. The putative convex surface of the LRR domain in GPIX is sufficient, in the context of full-length subunit, to mediate its association with GPIbbeta.

Identification of five novel 14-3-3 isoforms interacting with the GPIb-IX complex in platelets.

Binding of von Willebrand factor to the platelet glycoprotein (GP)Ib-IX complex initiates a signaling cascade leading to integrin alpha(IIb)beta(3) activation, a key process in hemostasis and thrombosis. Interaction of 14-3-3zeta with the intracytoplasmic domain of GPIb appears to be a major effector of this activation pathway. The aim of our study was to determine whether other members of the 14-3-3 family bind to GPIb-IX. In this study, western blot analyses showed that platelets also contain the 14-3-3beta, 14-3-3gamma, 14-3-3epsilon, 14-3-3eta and 14-3-3theta isoforms, but lack 14-3-3sigma. Coimmunoprecipitation studies in platelets and CHO transfectants demonstrated that all six 14-3-3 isoforms expressed in platelets, including, as previously reported, 14-3-3zeta, bind to GPIb-IX. In addition, their interaction was found to critically require the same GPIbalpha domains (580-590 and 605-610) already identified as essential for 14-3-3zeta binding, in agreement with the conservation of the sequence of the I-helix among these different isoforms. Pull-down experiments indicated that all six 14-3-3 isoforms present in platelets bind to GPIbbeta. In contrast, deletion or mutation of the GPIbbeta intracytoplasmic tail did not affect the interaction of GPIb-IX with the 14-3-3 isoforms, questioning the importance of this domain. Our study suggests that, to inhibit GPIb-induced integrin alpha(IIb)beta(3) activation, a more appropriate strategy than inhibiting individual 14-3-3 isoforms would be to target the 14-3-3-binding motif on GPIb or, alternatively, the conserved 14-3-3 I-helix.

Clinical benefit of recombinant factor VIIa in management of bleeds and surgery in two brothers suffering from the Bernard–Soulier syndrome

Bernard-Soulier syndrome (BSS) is a rare congenital bleeding disorder characterized by thrombocytopenia and giant platelets that display impaired or absent platelet agglutination with ristocetin. The disease is caused by mutations in genes controlling the expression of the platelet glycoprotein Ib-IX complex. The most severely affected patients suffer from profound muco-cutaneous bleeding tendency. Patients suffering from BSS may require platelet transfusion in prevention and management of severe bleeding. However, recent publications have suggested that recombinant factor VIIa (rFVIIa) infusion may support haemostasis in BSS. The present report describes two brothers who received treatment with rFVIIa together with tranexamic acid on a total of six occasions in management of haemostasis in minor surgery and for a serious spontaneous upper gastrointestinal tract bleed. Although platelet transfusion was omitted, haemostasis was regarded excellent in all of these treatment episodes.