GPIb? regulates platelet size by controlling the subcellular localization of filamin.
ABSTRACT: Interaction between the cytoplasmic domain of GPIb? with its cytoskeletal binding partner, filamin, is a major determinant of platelet size, and deficiency of either protein results in macrothrombocytopenia. To clarify the mechanism by which GPIb?-filamin interactions regulate platelet production, we manipulated the expression levels of filamin and GPIb in cultured embryonic stem cells (ESCs) that were subsequently differentiated into platelets. Knocking down filamins A and B resulted in the production of ESC-derived proplatelets with abnormally large swellings and proplatelet shafts that generated giant platelets in culture. Large platelets could also be generated by overexpressing GPIb? in ESCs, or by overexpressing in vivo a transgene encoding a chimeric protein containing the cytoplasmic domain of GPIb?. To identify the mechanism by which the GPIb:filamin ratio regulates platelet size, we manipulated filamin and GPIb? levels in HEK293T cells and examined the effects of overexpressing either protein on their ability to traffic to the cell periphery. Accumulation of either protein within the endoplasmic reticulum resulted in trapping of the other. Taken together, these data demonstrate that coordinated expression of GPIb? and filamin is required for efficient trafficking of either protein to the cell surface, and for production of normal-sized platelets.
Project description:Platelets have evolved a highly specialized membrane skeleton that provides stability to the plasma membrane and facilitates adhesion under high shear stress. The cytoskeletal anchorage of glycoprotein (GP) Ib? plays an important role in regulating the membrane skeleton. However, its role in regulating membrane stability remains unknown. To investigate this role, we have developed a new mouse model that expresses wild-type human GPIb? (hGPIb?(WT)), or a mutant form of human GPIb? that has a selective defect in its ability to bind filamin A and anchor to the membrane skeleton (hGPIb?(FW)-Phe568Ala and Trp570Ala substitutions). Our study demonstrates that the link between platelet GPIb and the cytoskeleton does not alter the intrinsic ligand binding function of GPIb? or the ability of the receptor to stimulate integrin ?(IIb)?(3)-dependent spreading. However, exposure of hGPIb?(FW) platelets to pathologic shear rate levels (5000 to 40,000 s(-1)) leads to the development of unstable membrane tethers, defective platelet adhesion, and loss of membrane integrity, leading to complete disintegration of the platelet cell body. These outcomes suggest that the GPIb?-filamin A interaction not only regulates the architecture of the membrane skeleton, but also maintains the mechanical stability of the plasma membrane under conditions of high shear.
Project description:The platelet storage lesion accelerates platelet clearance after transfusion, but the underlying molecular mechanism remains elusive. Although inhibiting sheddase activity hampers clearance of platelets with storage lesion, the target platelet protein responsible for ectodomain shedding-induced clearance is not definitively identified. Monoclonal antibody 5G6 was developed recently to bind specifically human platelet receptor glycoprotein (GP)Ib? and inhibit its shedding but not shedding of other receptors. Here, the role of GPIb? shedding in platelet clearance after transfusion was addressed.Both human leukoreduced apheresis-derived platelets and transgenic mouse platelets expressing human GPIb? were stored at room temperature in the presence and absence of 5G6 Fab fragment. At various time points, aliquots of stored platelets were analyzed and compared. 5G6 Fab inhibited GPIb? shedding in both platelets during storage and preserved higher level of GPIb? on the platelet surface. Compared with age-matched control platelets, 5G6 Fab-stored platelets exhibited similar levels of platelet activation, degranulation, and agonist-induced aggregation. 5G6 Fab-stored human GPIb? platelets exhibited significantly higher post-transfusion recovery and in vivo hemostatic function in recipient mice than control platelets. Consistently, 5G6 Fab-stored, 8-day-old human platelets produced similar improvement in post-transfusion recovery in immunodeficient mice and in ex vivo thrombus formation over collagen under shear flow.Specific inhibition of GPIb? shedding in the stored platelets improves post-transfusion platelet recovery and hemostatic function, providing clear evidence for GPIb? shedding as a cause of platelet clearance. These results suggest that specific inhibition of GPIb? shedding may be used to optimize platelet storage conditions.
Project description:Filamin A (FLNa), a dimeric actin cross-linking and scaffold protein with numerous intracellular binding partners, anchors the platelet adhesion glycoprotein (GP) Ib-IX-V receptor to actin cytoskeleton. We mapped the GPIbalpha binding site to a single domain of FLNa and resolved the structure of this domain and its interaction complex with the corresponding GPIbalpha cytoplasmic domain. This is the first atomic structure of this class of membrane glycoprotein-cytoskeleton connection. GPIbalpha binds in a groove formed between the C and D beta strands of FLNa domain 17. The interaction is strikingly similar to that between the beta7 integrin tail and a different FLNa domain, potentially defining a conserved motif for FLNa binding. Nevertheless, the structures also reveal specificity of the interfaces, which explains different regulatory mechanisms. To verify the topology of GPIb-FLNa interaction we also purified the native complex from platelets and showed that GPIb interacts with the C-terminus of FLNa, which is in accordance with our biochemical and structural data.
Project description:BACKGROUND:The glycoprotein (GP) Ib-IX-V complex is a unique platelet plasma membrane receptor, which is essential for platelet adhesion and thrombus formation. GPIb?, part of the GPIb-IX-V complex, has several physiological ligands such as von Willebrand factor (vWF), thrombospondin and distinct coagulation factors, which trigger platelet activation. Despite having an important role, intracellular GPIb-IX-V signaling and its regulation by other pathways are not well defined. Our aim was to establish the intracellular signaling response of selective GPIb? activation in human platelets, in particular the role of the tyrosine kinase Syk and its regulation by cAMP/PKA and cGMP/PKG pathways, respectively. We addressed this using echicetin beads (EB), which selectively bind to GPIb? and induce platelet aggregation. METHODS:Purified echicetin from snake Echis carinatus venom was validated by mass spectrometry. Washed human platelets were incubated with EB, in the presence or absence of echicetin monomers (EM), Src family kinase (SFK) inhibitors, Syk inhibitors and the cAMP- and cGMP-elevating agents iloprost and riociguat, respectively. Platelet aggregation was analyzed by light transmission aggregometry, protein phosphorylation by immunoblotting. Intracellular messengers inositolmonophosphate (InsP1) and Ca2+i were measured by ELISA and Fluo-3?AM/FACS, respectively. RESULTS:EB-induced platelet aggregation was dependent on integrin ?IIb?3 and secondary mediators ADP and TxA2, and was antagonized by EM. EB stimulated Syk tyrosine phosphorylation at Y352, which was SFK-dependent and Syk-independent, whereas Y525/526 phosphorylation was SFK-dependent and partially Syk-dependent. Furthermore, phosphorylation of both Syk Y352 and Y525/526 was completely integrin ?IIb?3-independent but, in the case of Y525/526, was partially ADP/TxA2-dependent. Syk activation, observed as Y352/ Y525/Y526 phosphorylation, led to the phosphorylation of direct substrates (LAT Y191, PLC?2 Y759) and additional targets (Akt S473). PKA/PKG pathways inhibited EB-induced platelet aggregation and Akt phosphorylation but, surprisingly, enhanced Syk and LAT/PLC?2 tyrosine phosphorylation. A similar PKA/PKG effect was confirmed with convulxin-/GPVI-stimulated platelets. EB-induced InsP1 accumulation/InsP3 production and Ca2+-release were Syk-dependent, but only partially inhibited by PKA/PKG pathways. CONCLUSION:EB and EM are specific agonists and antagonists, respectively, of GPIb?-mediated Syk activation leading to platelet aggregation. The cAMP/PKA and cGMP/PKG pathways do not inhibit but enhance GPIb?-/GPVI-initiated, SFK-dependent Syk activation, but strongly inhibit further downstream responses including aggregation. These data establish an important intracellular regulatory network induced by GPIb?.
Project description:BACKGROUND:Platelet glycoprotein Ib? (GPIb?) extracellular domain, which is part of the receptor complex GPIb-IX-V, plays an important role in tumor metastasis. However, the mechanism through which GPIb? participates in the metastatic process remains unclear. In addition, potential bleeding complication remains an obstacle for the clinical use of anti-platelet agents in cancer therapy. METHODS:We established a series of screening models and obtained rat anti-mouse GPIb? monoclonal antibodies (mAb) 1D12 and 2B4 that demonstrated potential value in suppressing cancer metastasis. To validate our findings, we further obtained mouse anti-human GPIb? monoclonal antibody YQ3 through the same approach. RESULTS:1D12 and 2B4 affected the von Willebrand factor (vWF)-GPIb? interaction via binding to GPIb? aa 41-50 and aa 277-290 respectively, which markedly inhibited the interaction among platelets, tumor cells, and endothelial cells in vitro, and reduced the mean number of surface nodules in the experimental and spontaneous metastasis models in vivo. As expected, YQ3 inhibited lung cancer adhesion and demonstrated similar value in metastasis. More importantly, for all three mAbs in our study, none of their Fabs induced thrombocytopenia. CONCLUSION:Our results therefore supported the hypothesis that GPIb? contributes to tumor metastasis and suggested potential value of using anti-GPIb? mAb to suppress cancer metastasis.
Project description:When refrigerated platelets are rewarmed, they secrete active sialidases, including the lysosomal sialidase Neu1, and express surface Neu3 that remove sialic acid from platelet von Willebrand factor receptor (VWFR), specifically the GPIb? subunit. The recovery and circulation of refrigerated platelets is greatly improved by storage in the presence of inhibitors of sialidases. Desialylated VWFR is also a target for metalloproteinases (MPs), because GPIb? and GPV are cleaved from the surface of refrigerated platelets. Receptor shedding is inhibited by the MP inhibitor GM6001 and does not occur in Adam17(?Zn/?Zn) platelets expressing inactive ADAM17. Critically, desialylation in the absence of MP-mediated receptor shedding is sufficient to cause the rapid clearance of platelets from circulation. Desialylation of platelet VWFR therefore triggers platelet clearance and primes GPIb? and GPV for MP-dependent cleavage.
Project description:Fucoidans are sulfated fucose-based polysaccharides that activate platelets and have pro- and anticoagulant effects; thus, they may have therapeutic value. In the present study, we show that 2 synthetic sulfated ?-l-fucoside-pendant glycopolymers (with average monomeric units of 13 and 329) and natural fucoidans activate human platelets through a Src- and phosphatidylinositol 3-kinase (PI3K)-dependent and Syk-independent signaling cascade downstream of the platelet endothelial aggregation receptor 1 (PEAR1). Synthetic glycopolymers and natural fucoidan stimulate marked phosphorylation of PEAR1 and Akt, but not Syk. Platelet aggregation and Akt phosphorylation induced by natural fucoidan and synthetic glycopolymers are blocked by a monoclonal antibody to PEAR1. Direct binding of sulfated glycopolymers to epidermal like growth factor (EGF)-like repeat 13 of PEAR1 was shown by avidity-based extracellular protein interaction screen technology. In contrast, synthetic glycopolymers and natural fucoidans activate mouse platelets through a Src- and Syk-dependent pathway regulated by C-type lectin-like receptor 2 (CLEC-2) with only a minor role for PEAR1. Mouse platelets lacking the extracellular domain of GPIb? and human platelets treated with GPIb?-blocking antibodies display a reduced aggregation response to synthetic glycopolymers. We found that synthetic sulfated glycopolymers bind directly to GPIb?, substantiating that GPIb? facilitates the interaction of synthetic glycopolymers with CLEC-2 or PEAR1. Our results establish PEAR1 as the major signaling receptor for natural fucose-based polysaccharides and synthetic glycopolymers in human, but not in mouse, platelets. Sulfated ?-l-fucoside-pendant glycopolymers are unique tools for further investigation of the physiological role of PEAR1 in platelets and beyond.
Project description:The interaction of platelet glycoprotein Ib? (GPIb?) with von Willebrand factor (VWF) initiates hemostasis after vascular injury and also contributes to pathological thrombosis. GPIb? binding to the VWF A1 domain (VWFA1) is a target for antithrombotic intervention, but attempts to develop pharmacologic inhibitors have been hindered by the lack of animal models because of the species specificity of the interaction. To address this problem, we generated a knockin mouse with Vwf exon 28-encoding domains A1 and A2 replaced by the human homolog (VWFh28). VWFh28 mice (M1HA) were crossbred with a transgenic mouse strain expressing human GPIb? on platelets (mGPIb?null;hGPIb?Tg; H1MA) to generate a new strain (H1HA) with humanized GPIb?-VWFA1 binding. Plasma VWF levels in the latter 3 strains were similar to those of wild-type mice (M1MA). Compared with the strains that had homospecific GPIb?-VWF pairing (M1MA and H1HA), M1HA mice of those with heterospecific pairing had a markedly greater prolongation of tail bleeding time and attenuation of thrombogenesis after injury to the carotid artery than H1MA mice. Measurements of GPIb?-VWFA1 binding affinity by surface plasmon resonance agreed with the extent of observed functional defects. Ristocetin-induced platelet aggregation was similar in H1HA mouse and human platelet-rich plasma, and it was comparably inhibited by monoclonal antibody NMC-4, which is known to block human GPIb?-VWFA1 binding, which also inhibited FeCl3-induced mouse carotid artery thrombosis. Thus, the H1HA mouse strain is a fully humanized model of platelet GPIb?-VWFA1 binding that provides mechanistic and pharmacologic information relevant to human hemostatic and thrombotic disorders.
Project description:Platelets are essential for maintaining haemostasis and play a key role in the pathogenesis of cardiovascular disease. Upon ligation of platelet receptors through subendothelial matrix proteins, intracellular reactive oxygen species (ROS) are generated, further amplifying the platelet activation response. Thrombin, a potent platelet activator, can signal through GPIb? and protease-activated receptor (PAR) 1 and PAR4 on human platelets, and recently has been implicated in the generation of ROS. While ROS are known to have key roles in intra-platelet signalling and subsequent platelet activation, the precise receptors and signalling pathways involved in thrombin-induced ROS generation have yet to be fully elucidated.To investigate the relative contribution of platelet GPIb? and PARs to thrombin-induced reactive oxygen species (ROS) generation.Highly specific antagonists targeting PAR1 and PAR4, and the GPIb?-cleaving enzyme, Naja kaouthia (Nk) protease, were used in quantitative flow cytometry assays of thrombin-induced ROS production. Antagonists of PAR4 but not PAR1, inhibited thrombin-derived ROS generation. Removal of the GPIb? ligand binding region attenuated PAR4-induced and completely inhibited thrombin-induced ROS formation. Similarly, PAR4 deficiency in mice abolished thrombin-induced ROS generation. Additionally, GPIb? and PAR4-dependent ROS formation were shown to be mediated through focal adhesion kinase (FAK) and NADPH oxidase 1 (NOX1) proteins.Both GPIb? and PAR4 are required for thrombin-induced ROS formation, suggesting a novel functional cooperation between GPIb? and PAR4. Our study identifies a novel role for PAR4 in mediating thrombin-induced ROS production that was not shared by PAR1. This suggests an independent signalling pathway in platelet activation that may be targeted therapeutically.
Project description:Trauma-induced coagulopathy (TIC) is a complex, multifactorial failure of hemostasis that occurs in 25% of severely injured patients and results in a fourfold higher mortality. However, the role of platelets in this state remains poorly understood. We set out to identify molecular changes that may underpin platelet dysfunction after major injury and to determine how they relate to coagulopathy and outcome. We performed a range of hemostatic and platelet-specific studies in blood samples obtained from critically injured patients within 2 hours of injury and collected prospective data on patient characteristics and clinical outcomes. We observed that, although platelet counts were preserved above critical levels, circulating platelets sampled from trauma patients exhibited a profoundly reduced response to both collagen and the selective glycoprotein VI (GPVI) agonist collagen-related peptide, compared with those from healthy volunteers. These responses correlated closely with overall clot strength and mortality. Surface expression of the collagen receptors GPIb? and GPVI was reduced on circulating platelets in trauma patients, with increased levels of the shed ectodomain fragment of GPVI detectable in plasma. Levels of shed GPVI were highest in patients with more severe injuries and TIC. Collectively, these observations demonstrate that platelets experience a loss of GPVI and GPIb? after severe injury and translate into a reduction in the responsiveness of platelets during active hemorrhage. In turn, they are associated with reduced hemostatic competence and increased mortality. Targeting proteolytic shedding of platelet receptors is a potential therapeutic strategy for maintaining hemostatic competence in bleeding and improving the efficacy of platelet transfusions.