ALBERT

Description: Key Points The study describes a potential novel treatment of fetal alloimmune thrombocytopenia by dissecting the effector activities of an epitope-specific IgG antibody. Neither the in vivo transplacental transport nor the inhibiting properties of the blocking antibody are impaired by the N-glycan modification.

Description: Key Points The genome of iPSCs has been edited to encode antigenically-distinct human platelet alloantigens. The iPSC-derived megakaryocyte progenitor cells express the designed alloantigens for diagnostic, investigative, and future therapeutic use.

Description: Abstract 153 Outside-in signal transduction is one of several amplification pathways that platelets employ following their adhesion to components of the extracellular matrix or to multivalent ligands like fibrinogen and von Willebrand factor. Previous studies have shown that outside-in signal transduction initiated by the binding of fibrinogen to its receptor, the integrin αIIbβ3, results in the activation of β3-associated Src family kinases (J Cell Biol 157:265, 2002; J Cell Sci 121:1641, 2008) that phosphorylate key tyrosine residues within the cytoplasmic domain of the transmembrane immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor protein, Fcγ;RIIa (Blood 112:2780, 2008). “Activation” of FcγRIIa sets off a cascade of events that facilitate assembly and activation of other key signaling intermediates, including the tyrosine kinase Syk and phospholipase Cγ2. PLCγ2, through its lipase activity, generates lipid products that support a multitude of cellular activation responses, including cytoskeletal rearrangements leading to platelet shape change and spreading, secretion of platelet granules, and activation of additional cell surface integrins. Curiously, mice lack the gene encoding FcγRIIa, and murine platelets are notoriously poor at sending αIIbβ3-mediated outside-in signals into the cell. The purpose of the current investigation, therefore, was to determine whether enforced expression of FcγRIIa in mouse platelets might serve to amplify murine platelet activation downstream of ligand binding to αIIbβ3. We found that the spreading of platelets derived from FcγRIIa transgenic mice was more robust and extensive compared to that of their wild-type counterparts. ADP- and collagen related peptide-induced granule secretion, as measured by P-selectin exposure, was also enhanced, as was the degree of integrin of αIIbβ3 activation, as reported by the binding of the activation-dependent monoclonal antibody, JON/A. Taken together, these data provide further support for importance of FcγRIIa in transmitting αIIbβ3-mediated amplification signals into the cell, and help to explain the often-observed defect in αIIbβ3-mediated signal transduction in murine platelets. Disclosures: Newman: New York Blood Center: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy.

Description: The platelet membrane glycoprotein (GP)Ib-V-IX complex is the receptor for von Willebrand factor and is composed of four membrane-spanning polypeptides: GPIbα, GPIbβ, GPIX, and GPV. A qualitative or quantitative deficiency in the GPIb-V-IX complex on the platelet membrane is the cause of the congenital platelet disorder Bernard-Soulier syndrome (BSS). We describe the molecular basis of a novel variant BSS in a patient in which GPIbα was absent from the platelet surface but present in a soluble form in the plasma. DNA sequence analysis showed a homozygous dinucleotide deletion in the codon for Tyr 508 (TAT) in GPIbα. This mutation (GPIbαΔAT) causes a frame shift that alters the amino acid sequence of GPIbα within its transmembrane region. The hydrophobic nature of the predicted transmembrane region and the cytoplasmic tail at the COOH terminal are altered before reaching a new premature stop codon 38 amino acids short of the wild-type peptide. Although GPIbαΔAT was not detectable on the platelet surface, immunoprecipitation of plasma with specific monoclonal antibodies (MoAbs) identified circulating GPIbα. Transient expression of recombinant GPIbαΔAT in 293T cells also generated a soluble form of the protein. Moreover, when a plasmid encoding GPIbαΔAT was transiently transfected into Chinese hamster ovary (CHO) cells stably expressing the GPβ-IX complex, it failed to be expressed on the cell surface. Thus, a dinucleotide deletion in the codon for Tyr 508 causes a frameshift that alters the amino acid sequence of GPIbα starting within its transmembrane region, changes the hydrophobicity of the normal transmembrane region, and truncates the cytoplasmic domain affecting binding to the cytoskeleton and cytoplasmic proteins. This mutation affects anchoring of the GPIbα polypeptide in platelets and causes the observed BSS phenotype with circulating soluble GPIbα.

Description: Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kD member of the Ig gene superfamily that is expressed on the surface of circulating platelets, monocytes, neutrophils, and selective T-cell subsets. It is also a major component of the endothelial cell intercellular junction. Previous studies have shown that cross-linking PECAM-1 on the surface of leukocytes results in the activation of adhesion molecules of both the β1 and β2integrin family. In addition, the process of leukocyte transendothelial migration appears to be mediated, at least in part, by homophilic adhesive interactions that take place between leukocyte and endothelial cell junctional PECAM-1 molecules. However, little is known about the functional role of this membrane glycoprotein in human platelets. In the present study, we examined the effects of PECAM-1 engagement on integrin-mediated platelet-extracellular matrix or platelet-platelet interactions. Bivalent, but not monovalent, anti–PECAM-1 monoclonal antibodies (MoAbs) specific for membrane-proximal Ig-homology domain 6 significantly augmented platelet deposition (increased surface coverage) and aggregation (increased average size) onto extracellular matrix, under both oscillatory or defined low shear flow conditions (200 s−1) in a modified cone and plate viscometer. Moreover, bivalent anti-domain 6 MoAbs were capable of serving as costimulatory agonists to markedly enhance both adenosine diphosphate (ADP)- and platelet activating factor (PAF)-induced platelet aggregation responses. These antibodies appeared to act via outside-in signal transduction through PECAM-1, as evidenced by the fact that their binding (1) led to conformational changes in the αIIbβ3 integrin complex, (2) induced surface expression of P-selectin, and (3) resulted in the tyrosine phosphorylation of PECAM-1. Together, these data support a role for PECAM-1 in cellular activation and suggest that PECAM-1 may serve as a costimulatory agonist receptor capable of modulating integrin function in human platelets during adhesion and aggregation.

Description: Key Points PECAM-1 is enriched at endothelial cell intercellular junctions, where it regulates leukocyte trafficking and vascular permeability. An atomic-level model of junctional PECAM-1 has been built based on a 2.8-Å resolution structure of its homophilic-binding domain.

Description: Thrombocytopenia is a major side effect of cancer chemotherapy producing bleeding problems as well as cancer treatment delays. To preclude it, the platelets progenitors megakaryocytes need to be protected from drug-induced apoptosis. According to previous reports, anti-apoptotic molecules such as Bcl-2, Bcl-xL, and caspase inhibitors were found to interfere platelet formation by megakaryocytes, thus these apoptosis inhibitors are not appropriate therapeutic candidates for the prevention of drug-induced thrombocytopenia. Recently we developed a new type of cell permeable apoptosis inhibitor, Bax Inhibiting Peptides (BIPs), which are derived from the Bax binding domain of Ku70. Bax is a key mediator of apoptosis, and Ku70 is a multifunctional protein playing roles in cell survival and DNA repair. In the present study, we determined whether BIPs prevent chemotherapy-induced thrombocytopenia both in vitro and in vivo. Importantly, there are several cancer cell types that acquired mutations in genes of Bax and/or Bax activating factors. Therefore, we hypothesize that protection of non-tumorigenic essential cells (such as megakaryocytes) by BIPs is beneficial for patients suffering from Bax-negative cancer. For in vitro study, Dami cells (human pro-megakaryocyte cell line) and mouse primary cultured megakaryocytes were used. BIPs significantly inhibited apoptosis induction by anti-cancer drugs such as etoposide, cisplatin, doxorubicin, or paclitaxel. Importantly, BIPs did not interfere the activity of megakaryocytes to produce platelet-like particle in cell culture, suggesting that BIPs protect megakaryocytes from drug-induced apoptosis without suppressing their platelets production. For in vivo study, C57BL/6J mice and Bax-/- mice (backcrossed with C57BL/6J) were used. Etoposide (150 mg/kg, i.p.) induced a significant decrease of platelet count in whole blood within 2 days after the drug administration in wild type C57BL/6J mice. Pretreatment of mice with BIPs (166 mg/kg, i.p., 30 min before etoposide injection) blocked etoposide-induced platelet count decrease in these wild type mice. The same dose of etoposide did not induce significant platelet count decrease in Bax-/- mice. These results suggest that chemotherapy-induced thrombocytopenia, at least in part, is due to the stimulation of Bax-mediated cell death probably both in megakaryocytes and platelets. In our preliminary experiments, three times injection of caspase inhibitor (z-VAD-fmk, 166 mg/kg, i.p. every 24 hrs) caused prolongation of bleeding time from the wound (tail cut). Thus, as previously reported by other groups using in vitro system, caspase inhibitor may have its own side effect to inhibit platelet formation or/and activation in vivo. On the other hand, BIPs (166 mg/kg, 3 times every 24 hrs, i.p.) did not extend bleeding time from the tail. Taken together, BIP may become a useful supplemental therapeutic agent to block chemotherapy-induced thrombocytopenia.

Description: Interaction of resting platelets with exposed components of the subendothelial matrix is an important activating event that takes place early on at sites of vascular injury. Platelet responses to collagen are mediated both by the integrin α2β1 and the glycoprotein (GP)VI/Fc receptor (FcR) γ chain complex. Upon collagen binding, Src family kinases phosphorylate tyrosines within Immunoreceptor Tyrosine-based Inhibitory Motifs (ITAMs) that are present within the cytoplasmic domain of the FcRγ chain. These phosphorylated ITAMs serve as docking sites for the protein-tyrosine kinase Syk, which, when recruited and activated, initiates the rapid assembly of a series of adaptor proteins and protein-tyrosine kinases that are crucial for platelet activation responses. Recently, platelet activation by laminin, which binds the related integrin α6β1, was shown to also require signaling through the GPVI/FcRγ chain complex. Because the adhesion and signaling receptor, PECAM-1, serves as a negative regulator of collagen-induced platelet activation, we sought to determine whether PECAM-1 might similarly regulate platelet activation by laminin. We found that PECAM-1 became rapidly tyrosine phosphorylated on its cytoplasmic ITIMs following adhesion of either human or murine platelets to immobilized laminin, suggesting that it may be involved in modulating platelet responses to laminin. PECAM-1-deficient murine platelets exhibited both an increased rate and extent of cell spreading on immobilized laminin compared to their PECAM-1-positive, wild-type (wt) counterparts. Granule secretion, as determined by release of serotonin, was also augmented. Taken together, these data support the notion that PECAM-1 is a negative regulator of platelet responses to laminin.

Description: Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell surface glycoprotein receptor expressed on a range of blood cells, including platelets, and on vascular endothelial cells. PECAM-1 possesses adhesive and signaling properties, the latter being mediated by immunoreceptor tyrosine-based inhibitory motifs present on the cytoplasmic tail of the protein. Recent studies in vitro have demonstrated that PECAM-1 signaling inhibits the aggregation of platelets. In the present study we have used PECAM-1–deficient mice and radiation chimeras to investigate the function of this receptor in the regulation of thrombus formation. Using intravital microscopy and laser-induced injury to cremaster muscle arterioles, we show that thrombi formed in PECAM-1–deficient mice were larger, formed more rapidly than in control mice, and were more stable. Larger thrombi were also formed in control mice that received transplants of PECAM-1–deficient bone marrow, in comparison to mice that received control transplants. A ferric chloride model of thrombosis was used to investigate thrombus formation in carotid arteries. In PECAM-1–deficient mice the time to 75% vessel occlusion was significantly shorter than in control mice. These data provide evidence for the involvement of platelet PECAM-1 in the negative regulation of thrombus formation.

Description: The human neutrophil-specific adhesion antigen CD177 (NB1, HNA-2a or PRV-1) becomes upregulated on the cell surface in a number of inflammatory settings, and human alloantibodies specific for CD177 have been implicated in the development of transfusion related acute lung injury (TRALI). Recent studies have shown that CD177 functions as an important counter-receptor for the endothelial cell-cell junctional protein PECAM-1 (CD31) – a heterophilic adhesive interaction that appears to be mediated by Ig domain 6 of PECAM-1 and a still-to-be-identified site on CD177. Interestingly, a common single nucleotide polymorphism (SNP) exists within the PECAM-1 gene that produces an S536N amino acid substitution within Ig domain 6 that is proximal to the CD177 binding site. Whether this polymorphism influences PECAM-1/CD177 interactions and subsequent neutrophil transendothelial cell migration, however, is not known. To investigate the potential for the S536N PECAM-1 SNP to affect functionally important interactions between PECAM-1 and CD177, recombinant CD177 and PECAM-1 S536 or N536 allelic isoforms were produced in CHO mammalian cells, purified by affinity chromatography, and subjected to surface plasmon resonance analysis. We found that the S536 isoform of PECAM-1 bound with much higher affinity to CD177 than did its N536 counterpart (Ka for PECAM-1S536/CD177 = 5×10−7 versus a Ka for PECAM-1N536/CD177=1×10−7). To analyze the possible functional impact of the S536N polymorphism on neutrophil transendothelial migration, fMLP-stimulated neutrophils were allowed to transmigrate across human umbilical vein endothelial cell (HUVEC) monolayers that had been genotyped to be either PECAM-1 homozygous SS, heterozygous NS, or homozygous NN. We found that neutrophils migrated significantly faster across HUVECs expressing the PECAM-1S536, rather than the PECAM-1N536, allelic isoform. In addition, western blot analysis using a phospho-specific antibody revealed that cytoplasmic PECAM-1 ITIM tyrosines became phosphorylated to a greater extent in PECAM-1S536- versus PECAM-1N536-expressing HUVECs that had been incubated with either soluble recombinant CD177 or an anti-PECAM-1 mAb specific for Ig domain 6. Taken together, these data demonstrate that heterophilic interactions between neutrophil CD177 and endothelial cell PECAM-1 play an important role in neutrophil transendothelial migration, and the naturally-occurring S536N polymorphism within Ig domain 6 likely affects ligand binding-induced downstream PECAM-1 signaling. Allele-specific, PECAM-1-mediated effects on neutrophil transendothelial migration and inflammatory events such as TRALI may represent an important area for future investigation.