GPIbα-filamin A interaction regulates megakaryocyte localization and budding during platelet biogenesis.

ABSTRACT: Glycoprotein Ibα (GPIbα) is expressed on the surface of platelets and megakaryocytes (MKs) and anchored to the membrane skeleton by filamin A (flnA). Although GPIb and flnA have fundamental roles in platelet biogenesis, the nature of this interaction in megakaryocyte biology remains ill-defined. We generated a mouse model expressing either human wild-type (WT) GPIbα (hGPIbαWT) or a flnA-binding mutant (hGPIbαFW) and lacking endogenous mouse GPIbα. Mice expressing the mutant GPIbα transgene exhibited macrothrombocytopenia with preserved GPIb surface expression. Platelet clearance was normal and differentiation of MKs to proplatelets was unimpaired in hGPIbαFW mice. The most striking abnormalities in hGPIbαFW MKs were the defective formation of the demarcation membrane system (DMS) and the redistribution of flnA from the cytoplasm to the peripheral margin of MKs. These abnormalities led to disorganized internal MK membranes and the generation of enlarged megakaryocyte membrane buds. The defective flnA-GPIbα interaction also resulted in misdirected release of buds away from the vasculature into bone marrow interstitium. Restoring the linkage between flnA and GPIbα corrected the flnA redistribution within MKs and DMS ultrastructural defects as well as restored normal bud size and release into sinusoids. These studies define a new mechanism of macrothrombocytopenia resulting from dysregulated MK budding. The link between flnA and GPIbα is not essential for the MK budding process, however, it plays a major role in regulating the structure of the DMS, bud morphogenesis, and the localized release of buds into the circulation.

Corrigendum: 14-3-3ζ regulates the mitochondrial respiratory reserve linked to platelet phosphatidylserine exposure and procoagulant function.

This corrects the article DOI: 10.1038/ncomms12862.

14-3-3ζ regulates the mitochondrial respiratory reserve linked to platelet phosphatidylserine exposure and procoagulant function.

The 14-3-3 family of adaptor proteins regulate diverse cellular functions including cell proliferation, metabolism, adhesion and apoptosis. Platelets express numerous 14-3-3 isoforms, including 14-3-3ζ, which has previously been implicated in regulating GPIbα function. Here we show an important role for 14-3-3ζ in regulating arterial thrombosis. Interestingly, this thrombosis defect is not related to alterations in von Willebrand factor (VWF)-GPIb adhesive function or platelet activation, but instead associated with reduced platelet phosphatidylserine (PS) exposure and procoagulant function. Decreased PS exposure in 14-3-3ζ-deficient platelets is associated with more sustained levels of metabolic ATP and increased mitochondrial respiratory reserve, independent of alterations in cytosolic calcium flux. Reduced platelet PS exposure in 14-3-3ζ-deficient mice does not increase bleeding risk, but results in decreased thrombin generation and protection from pulmonary embolism, leading to prolonged survival. Our studies define an important role for 14-3-3ζ in regulating platelet bioenergetics, leading to decreased platelet PS exposure and procoagulant function.

High shear-dependent loss of membrane integrity and defective platelet adhesion following disruption of the GPIbα-filamin interaction.

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) Ibα 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 GPIbα (hGPIbα(WT)), or a mutant form of human GPIbα that has a selective defect in its ability to bind filamin A and anchor to the membrane skeleton (hGPIbα(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 GPIbα or the ability of the receptor to stimulate integrin α(IIb)ß(3)-dependent spreading. However, exposure of hGPIbα(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 GPIbα-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.

Dissecting isoform selectivity of PI3K inhibitors: the role of non-conserved residues in the catalytic pocket.

The last few years have seen the identification of numerous small molecules that selectively inhibit specific class I isoforms of PI3K (phosphoinositide 3-kinase), yet little has been revealed about the molecular basis for the observed selectivities. Using site-directed mutagenesis, we have investigated one of the areas postulated as being critical to the observed selectivity. The residues Thr(886) and Lys(890) of the PI3Kgamma isoform project towards the ATP-binding pocket at the entrance to the catalytic site, but are not conserved. We have made reciprocal mutations between those residues in the beta isoform (Glu(858) and Asp(862)) and those in the alpha isoform (His(855) and Gln(859)) and evaluated the potency of a range of reported PI3K inhibitors. The results show that the potencies of beta-selective inhibitors TGX221 and TGX286 are unaffected by this change. In contrast, close analogues of these compounds, particularly the alpha-isoform-selective compound (III), are markedly influenced by the point mutations. The collected data suggests two distinct binding poses for these inhibitor classes, one of which is associated with potent PI3Kbeta activity and is not associated with the mutated residues, and a second that, in accord with earlier hypotheses, does involve this pair of non-conserved amino acids at the catalytic site entrance and contributes to the alpha-isoform-selectivity of the compounds studied.

A functional 14-3-3zeta-independent association of PI3-kinase with glycoprotein Ib alpha, the major ligand-binding subunit of the platelet glycoprotein Ib-IX-V complex.

Engagement of the adhesion receptor glycoprotein (GP) Ib-IX-V by von Willebrand factor (VWF) mediates platelet adhesion to damaged vessels and triggers platelet activation and thrombus formation in heart attack and stroke. GPIb-IX-V contains distinct 14-3-3zeta-binding sites at the GPIb alpha C-terminus involving phosphorylation of Ser609, an upstream site involving phosphorylated Ser587/Ser590, and a protein kinase A (PKA)-dependent site on GPIb beta involving Ser166. 14-3-3zeta regulates the VWF-binding affinity of GPIb-IX-V and inhibiting 14-3-3zeta association blocks receptor signaling, suggesting a key functional role for 14-3-3zeta. We used deletion mutants of GPIb alpha expressed in Chinese hamster ovary (CHO) cells to define the relationship of 14-3-3zeta binding to another GPIb-IX-V-associated signaling protein, phosphoinositide 3-kinase (PI3-kinase). Pull-down experiments involving glutathione S-transferase (GST)-PI3-kinase/p85-subunit and GST-14-3-3zeta indicated that both proteins interacted with contiguous GPIb alpha sequences 580 to 590/591 to 610. Deleting these, but not upstream sequences of GPIb alpha expressed in CHO cells, inhibited VWF/ristocetin-dependent Akt phosphorylation, relative to wild-type receptor, confirming this region encompassed a functional PI3-kinase-binding site. Pull-down experiments with GST-p85 truncates indicated the GPIb alpha-binding region involved the p85 breakpoint cluster region (BCR) domain, containing RSXSXP. However, pull-down of GPIb-IX was unaltered by mutation/deletion/phosphorylation of this potential 14-3-3zeta-binding sequence in mutant constructs of GST-p85, suggesting PI3-kinase bound GPIb alpha independently of 14-3-3zeta; 14-3-3zeta inhibitor peptide R18 also blocked pull-down of receptor by GST-14-3-3zeta but not GST-p85, and GST-p85 pull-downs were unaffected by excess 14-3-3zeta. Together, these data suggest the GPIb alpha C-terminus regulates signaling through independent association of 14-3-3zeta and PI3-kinase.

Leucine-rich repeats 2-4 (Leu60-Glu128) of platelet glycoprotein Ibalpha regulate shear-dependent cell adhesion to von Willebrand factor.

Glycoprotein (GP) Ib-IX-V binds von Willebrand factor (VWF), initiating thrombosis at high shear stress. The VWF-A1 domain binds the N-terminal domain of GPIbalpha (His1-Glu282); this region contains seven leucine-rich repeats (LRR) plus N- and C-terminal flanking sequences and an anionic sequence containing three sulfated tyrosines. Our previous analysis of canine/human and human/canine chimeras of GPIbalpha expressed on Chinese hamster ovary (CHO) cells demonstrated that LRR2-4 (Leu60-Glu128) were crucial for GPIbalpha-dependent adhesion to VWF. Paradoxically, co-crystal structures of the GPIbalpha N-terminal domain and GPIbalpha-binding VWF-A1 under static conditions revealed that the LRR2-4 sequence made minimal contact with VWF-A1. To resolve the specific functional role of LRR2-4, we compared wild-type human GPIbalpha with human GPIbalpha containing a homology domain swap of canine for human sequence within Leu60-Glu128 and a reverse swap (canine GPIbalpha with human Leu60-Glu128) for the ability to support adhesion to VWF under flow. Binding of conformation-specific anti-GPIbalpha antibodies and VWF binding in the presence of botrocetin (which does not discriminate between species) confirmed equivalent expression of wild-type and mutant receptors in a functional form competent to bind ligand. Compared with CHO cells expressing wild-type GPIbalpha, cells expressing GPIbalpha, where human Leu60-Glu128 sequence was replaced by canine sequence, supported adhesion to VWF at low shear rates but became increasingly ineffective as shear increased from 50 to 2000 s(-1). Together, these data demonstrate that LRR2-4, encompassing a pronounced negative charge patch on human GPIbalpha, is essential for GPIbalpha.VWF-dependent adhesion as hydrodynamic shear increases.

Identification of a unique filamin A binding region within the cytoplasmic domain of glycoprotein Ibalpha.

Binding of the platelet GPIb/V/IX (glycoprotein Ib/V/IX) receptor to von Willebrand factor is critical for platelet adhesion and aggregation under conditions of rapid blood flow. The adhesive function of GPIbalpha is regulated by its anchorage to the membrane skeleton through a specific interaction with filamin A. In the present study, we examined the amino acid residues within the cytoplasmic tail of GPIbalpha, which are critical for association with filamin A, using a series of 25-mer synthetic peptides that mimic the cytoplasmic tail sequences of wild-type and mutant forms of GPIbalpha. Peptide binding studies of purified human filamin A have demonstrated a major role for the conserved hydrophobic stretch L567FLWV571 in mediating this interaction. Progressive alanine substitutions of triple, double and single amino acid residues within the Pro561-Arg572 region suggested an important role for Trp570 and Phe568 in promoting GPIbalpha binding to filamin A. The importance of these two residues in promoting filamin A binding to GPIbalpha in vivo was confirmed from the study of Chinese-hamster ovary cells expressing GPIbalpha Trp570-->Ala and Phe568-->Ala substitutions. Phenotypic analysis of these cell lines in flow-based adhesion studies revealed a critical role for these residues in maintaining receptor anchorage to the membrane skeleton and in maintaining cell adhesion to a von Willebrand factor matrix under high-shear conditions. These studies demonstrate a novel filamin A binding motif in the cytoplasmic tail of GPIbalpha, which is critically dependent on both Trp570 and Phe568.

Identification of a novel 14-3-3zeta binding site within the cytoplasmic tail of platelet glycoprotein Ibalpha.

The glycoprotein Ib-V-IX (GPIb-V-IX) complex interacts with subendothelial von Willebrand factor (VWF) to ensure recruitment of platelets at sites of vascular injury, a process that culminates in integrin alpha(IIb)beta(3)-dependent stable adhesion and spreading. Interaction of the 14-3-3zeta adaptor protein with the C-terminal 606-610 phosphoserine motif of the GPIbalpha subunit has been implicated in the control of alpha(IIb)beta(3) activation and cell spreading. In this study, we have examined potentially novel 14-3-3zeta binding sites by expressing mutant forms of GPIbalpha in Chinese-hamster-ovary (CHO) cells. Analysis of a series of neighboring 11-12 residue deletions identified a critical role for the 580-LVAGRRPSALS-590 sequence in promoting GPIbalpha-14-3-3zeta interaction. Development of a phosphospecific antibody demonstrated high levels of phosphorylation of the Ser587 and Ser590 residues in resting platelets (which became dephosphorylated during platelet spreading on VWF), and peptides containing these phosphorylated residues effectively displaced 14-3-3zeta from GPIbalpha. Analysis of single and double alanine substitutions of Ser587 and Ser590 demonstrated a major role for these residues in promoting GPIbalpha-14-3-3zeta binding. Moreover, these cell lines exhibited a defect in cell spreading on immobilized VWF. These studies demonstrate the existence of a second major 14-3-3zeta binding site within the cytoplasmic tail of GPIbalpha that has an important functional role in regulating integrin-dependent cell spreading.

Role of the intracellular domains of GPIb in controlling the adhesive properties of the platelet GPIb/V/IX complex.

Glycoprotein (GP) Ib/V/IX complex-dependent platelet adhesion to von Willebrand factor (VWF) is supported by the 45-kd N-terminal extracellular domain of the GPIb alpha subunit. Recent results with an adhesion blocking antibody (RAM.1) against GPIb beta, which is disulfide linked to GPIb alpha, have suggested a novel function of this subunit in regulating VWF-mediated platelet adhesion, possibly involving its intracellular face. A putative cooperation between the GPIb alpha and GPIb beta cytoplasmic domains was investigated by measuring the adhesion under flow to immobilized VWF of K562 and Chinese hamster ovary (CHO) cells transfected with GPIb/(V)/IX containing mutations in this region. Adhesion of cells carrying a glycine substitution of the GPIb beta Ser166 phosphorylation site was 50% lower than normal and became insensitive to inhibition by RAM.1. In contrast, forskolin or PGE(1) treatment increased both the phosphorylation of GPIb beta and adhesion of control cells, both effects being reversed by RAM.1, but had no influence on cells expressing the Ser166Gly mutation. A role of the GPIb alpha intracellular domain was also apparent as the VWF-dependent adhesion of cells containing deletions of the entire (Delta 518-610) or portions (Delta 535-568, Delta 569-610) of the GPIb alpha cytoplasmic tail was insensitive to RAM.1 inhibition. Cells carrying progressive 11 amino acid deletions spanning the GPIb alpha 535-590 region were equally unresponsive to RAM.1, with the exception of those containing GPIb alpha Delta 569-579, which behaved like control cells. These findings support a role of the GPIb beta intracellular domain in controlling the adhesive properties of the GPIb/V/IX complex through phosphorylation of GPIb beta Ser166 and point to the existence of cross-talk between the GPIb beta and GPIb alpha intracellular domains.

Interaction between platelet glycoprotein Ibalpha and filamin-1 is essential for glycoprotein Ib/IX receptor anchorage at high shear.

The interaction of the glycoprotein (GP) Ib-V-IX receptor complex with the membrane skeleton of platelets is dependent on a specific interaction between the cytoplasmic tail of GPIbalpha and filamin-1. This interaction has been proposed to regulate key aspects of platelet function, including the ligand binding of GPIb-V-IX and the ability of the cells to sustain adhesion to von Willebrand factor (vWf) under high shear. In this study we have examined sequences in the GPIbalpha intracellular domain necessary for interaction of the receptor with filamin-1. We have identified two adjacent sequences involving amino acids 557-568 and 569-579 of the GPIbalpha cytoplasmic domain that are critical for normal association between the receptor complex and filamin-1. Under flow conditions, Chinese hamster ovary (CHO) cells expressing these two mutant receptors exhibited an increase in translocation velocity that was associated with increased cell detachment from the vWf matrix at high shear. The shear-dependent acceleration in velocity of mutant Delta557-568 and Delta569-579 CHO cells was associated with a critical defect in receptor anchorage, evident from significant extraction of GPIb-IX from the CHO cell membrane at high shear. These studies define a critical role for amino acids within the 557-579 sequence of GPIbalpha for interaction with filamin-1.