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Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal alloantibodies directed against paternally inherited human platelet alloantigens (HPAs) present on the surface of fetal and neonatal platelets. There are currently no approved therapies for the prevention of FNAIT. We report herein the ability of 2 human HPA-1a-specific therapeutic candidates, one a polyclonal, and the other a monoclonal antibody, to prevent alloimmunization in a novel preclinical mouse model of FNAIT. Both antibody preparations effected the rapid and complete elimination of HPA-1a+ platelets from circulation and prevented the development of HPA-1a alloantibodies. HPA-1a- female mice treated prophylactically with anti-HPA-1a antibody prior to exposure to HPA-1a+ platelets gave birth to HPA-1a+/- pups with significantly improved platelet counts and no bleeding symptoms. These preclinical data establish both the potential and threshold exposure targets for prophylactic treatment with HPA-1a-specific antibodies for the prevention of FNAIT in humans., (© 2022 by The American Society of Hematology.)

Competing Interests: Conflict-of-interest disclosure: The author declares no competing financial interests.

Aims: PECAM-1 is an abundant endothelial cell surface receptor that becomes highly enriched at endothelial cell-cell junctions, where it functions to mediate leukocyte transendothelial migration, sense changes in shear and flow, and maintain the vascular permeability barrier. Homophilic interactions mediated by the PECAM-1 extracellular domain are known to be required for PECAM-1 to perform these functions; however, much less is understood about the role of its cytoplasmic domain in these processes., Main Methods: CRISPR/Cas9 gene editing technology was employed to generate human endothelial cell lines that either lack PECAM-1 entirely, or express mutated PECAM-1 missing the majority of its cytoplasmic domain (∆CD-PECAM-1). The endothelial barrier function was evaluated by Electric Cell-substrate Impedance Sensing, and molecular mobility was assessed by fluorescence recovery after photobleaching., Key Findings: We found that ∆CD-PECAM-1 concentrates normally at endothelial cell junctions, but has the unexpected property of conferring increased baseline barrier resistance, as well as a more rapid rate of recovery of vascular integrity following thrombin-induced disruption of the endothelial barrier. Fluorescence recovery after photobleaching analysis revealed that ∆CD-PECAM-1 exhibits increased mobility within the plane of the plasma membrane, thus allowing it to redistribute more rapidly back to endothelial cell-cell borders to reform the vascular permeability barrier., Significance: The PECAM-1 cytoplasmic domain plays a novel role in regulating the rate and extent of vascular permeability following thrombotic or inflammatory challenge., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Immune complexes consisting of heparin, platelet factor 4 (PF4), and PF4/heparin-reactive antibodies are central to the pathogenesis of heparin-induced thrombocytopenia (HIT). It is as yet unclear what triggers the initial induction of pathogenic antibodies. We identified B cells in peripheral blood of healthy adults that produce PF4/heparin-specific antibodies following in vitro stimulation with proinflammatory molecules containing deoxycytosine-deoxyguanosine (CpG). Similarly, B cells from unmanipulated wild-type mice produced PF4/heparin-specific antibodies following in vitro or in vivo CpG stimulation. Thus, both healthy humans and mice possess preexisting inactive/tolerant PF4/heparin-specific B cells. The findings suggest that breakdown of tolerance leads to PF4/heparin-specific B-cell activation and antibody production in patients developing HIT. Consistent with this concept, mice lacking protein kinase Cδ (PKCδ) that are prone to breakdown of B-cell tolerance produced anti-PF4/heparin antibodies spontaneously. Therefore, breakdown of tolerance can lead to PF4/heparin-specific antibody production, and B-cell tolerance may play an important role in HIT pathogenesis.

Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that can cause fatal arterial or venous thrombosis/thromboembolism. Immune complexes consisting of platelet factor 4 (PF4), heparin, and PF4/heparin-reactive antibodies are central to the pathogenesis of HIT. However, the B-cell origin of HIT antibody production is not known. Here, we show that anti-PF4/heparin antibodies are readily generated in wild-type mice on challenge with PF4/heparin complexes, and that antibody production is severely impaired in B-cell-specific Notch2-deficient mice that lack marginal zone (MZ) B cells. As expected, Notch2-deficient mice responded normally to challenge with T-cell-dependent antigen nitrophenyl-chicken γ globulin but not to the T-cell-independent antigen trinitrophenyl-Ficoll. In addition, wild-type, but not Notch2-deficient, B cells plus B-cell-depleted wild-type splenocytes adoptively transferred into B-cell-deficient μMT mice responded to PF4/heparin complex challenge. PF4/heparin-specific antibodies produced by wild-type mice were IgG2b and IgG3 isotypes. An in vitro class-switching assay showed that MZ B cells were capable of producing antibodies of IgG2b and IgG3 isotypes. Lastly, MZ, but not follicular, B cells adoptively transferred into B-cell-deficient μMT mice responded to PF4/heparin complex challenge by producing PF4/heparin-specific antibodies of IgG2b and IgG3 isotypes. Taken together, these data demonstrate that MZ B cells are critical for PF4/heparin-specific antibody production.

The integrin family is composed of a series of 24 αβ heterodimer transmembrane adhesion receptors that mediate cell-cell and cell-extracellular matrix interactions. Adaptor molecules bearing immunoreceptor tyrosine-based activation motifs (ITAMs) have recently been shown to cooperate with specific integrins to increase the efficiency of transmitting ligand-binding-induced signals into cells. In human platelets, Fc receptor γ-chain IIa (FcγRIIa) has been identified as an ITAM-bearing transmembrane receptor responsible for mediating "outside-in" signaling through αIIbβ3, the major adhesion receptor on the platelet surface. To explore the importance of FcγRIIa in thrombosis and hemostasis, we subjected FcγRIIa-negative and FcγRIIa-positive murine platelets to a number of well-accepted models of platelet function. Compared with their FcγRIIa-negative counterparts, FcγRIIa-positive platelets exhibited increased tyrosine phosphorylation of Syk and phospholipase Cγ2 and increased spreading upon interaction with immobilized fibrinogen, retracted a fibrin clot faster, and showed markedly enhanced thrombus formation when perfused over a collagen-coated flow chamber under conditions of arterial and venous shear. They also displayed increased thrombus formation and fibrin deposition in in vivo models of vascular injury. Taken together, these data establish FcγRIIa as a physiologically important functional conduit for αIIbβ3-mediated outside-in signaling, and suggest that modulating the activity of this novel integrin/ITAM pair might be effective in controlling thrombosis.

Platelets are essential for normal hemostasis, but close regulation is required to avoid the destructive effects of either inappropriate platelet activation or excessive responses to injury. Here, we describe a novel complex comprising the scaffold protein, spinophilin (SPL), and the tyrosine phosphatase, SHP-1, and show that it can modulate platelet activation by sequestering RGS10 and RGS18, 2 members of the regulator of G protein signaling family. We also show that SPL/RGS/SHP1 complexes are present in resting platelets where constitutive phosphorylation of SPL(Y398) creates an atypical binding site for SHP-1. Activation of the SHP-1 occurs on agonist-induced phosphorylation of SHP-1(Y536), triggering dephosphorylation and decay of the SPL/RGS/SHP1 complex. Preventing SHP-1 activation blocks decay of the complex and produces a gain of function. Conversely, deleting spinophilin in mice inhibits platelet activation. It also attenuates the rise in platelet cAMP normally caused by endothelial prostacyclin (PGI(2)). Thus, we propose that the role of the SPL/RGS/SHP1 complex in platelets is time and context dependent. Before injury, the complex helps maintain the quiescence of circulating platelets by maximizing the impact of PGI(2). After injury, the complex gradually releases RGS proteins, limiting platelet activation and providing a mechanism for temporal coordination of pro thrombotic and antithrombotic inputs.

Aims: To investigate the mechanism by which platelet endothelial cell adhesion molecule 1 (PECAM-1/CD31), an immunoglobulin (Ig)-superfamily cell adhesion and signaling receptor, regulates pro-inflammatory cytokine levels. The purpose of the present investigation was to test the hypothesis that PECAM-1 influences circulating cytokine levels by regulating the trafficking of activated, cytokine-producing leukocytes to sites of inflammation., Main Methods: PECAM-1+/+ and PECAM-1-/- mice were subjected to lipopolysaccharide (LPS)-induced endotoxemia, and systemic cytokine levels were measured by Bioplex multiplex cytokine assays. Flow cytometry was employed to enumerate leukocytes at inflammatory sites and to measure cytokine synthesis in leukocyte sub-populations. Enzyme-linked immunosorbent assay (ELISA) was used to measure cytokine levels in tissue samples and in supernatants of in vitro-stimulated leukocytes., Key Findings: We confirmed earlier reports that mice deficient in PECAM-1 had greater systemic levels of pro-inflammatory cytokines following intraperitoneal (IP) LPS administration. Interestingly, expression of PECAM-1, in mice, had negligible effects on the level of cytokine synthesis by leukocytes stimulated in vitro with LPS and in peritoneal macrophages isolated from LPS-injected mice. There was, however, excessive accumulation of macrophages and neutrophils in the lungs of PECAM-1-deficient, compared with wild-type, mice--an event that correlated with a prolonged increase in lung pro-inflammatory cytokine levels., Significance: Our results demonstrate that PECAM-1 normally functions to dampen systemic cytokine levels during LPS-induced endotoxemia by diminishing the accumulation of cytokine-producing leukocytes at sites of inflammation, rather than by modulating cytokine synthesis by leukocytes., (Copyright © 2011. Published by Elsevier Inc.)

Immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptors inhibit cellular responsiveness to immunoreceptor tyrosine-based activation motif (ITAM)-linked receptors. Although tyrosine phosphorylation is central to the initiation of both inhibitory ITIM and stimulatory ITAM signaling, the events that regulate receptor phosphorylation are incompletely understood. Previous studies have shown that ITAM tyrosines engage in structure-inducing interactions with the plasma membrane that must be relieved for phosphorylation to occur. Whether ITIM phosphorylation is similarly regulated and the mechanisms responsible for release from plasma membrane interactions to enable phosphorylation, however, have not been defined. PECAM-1 is a dual ITIM-containing receptor that inhibits ITAM-dependent responses in hematopoietic cells. We found that the PECAM-1 cytoplasmic domain is unstructured in an aqueous environment but adopts an α-helical conformation within a localized region on interaction with lipid vesicles that mimic the plasma membrane. The lipid-interacting segment contains the C-terminal ITIM tyrosine and a serine residue that undergo activation-dependent phosphorylation. The N-terminal ITIM is excluded from the lipid-interacting segment, and its phosphorylation is secondary to phosphorylation of the membrane-interacting C-terminal ITIM. On the basis of these findings, we propose a novel model for regulation of inhibitory signaling by ITIM-containing receptors that relies on reversible plasma membrane interactions and sequential ITIM phosphorylation.

Unfractionated heparin (UFH) is a widely used anticoagulant that has long been known to potentiate platelet responses to subthreshold doses of platelet agonists. UFH has been reported to bind and induce modest conformational changes in the major platelet integrin, αIIbβ3, and induce minor changes in platelet morphology. The mechanism by which UFH elicits these platelet-activating effects, however, is not well understood. We found that both human and murine platelets exposed to UFH, either in solution or immobilized onto artificial surfaces, underwent biochemical and morphologic changes indicative of a potentiated state, including phosphorylation of key cytosolic signaling molecules and cytoskeletal changes leading to cell spreading. Low molecular weight heparin and the synthetic pentasaccharide, fondaparinux, had similar platelet-potentiating effects. Human or mouse platelets lacking functional integrin αIIbβ3 complexes and human platelets pretreated with the fibrinogen receptor antagonists eptifibatide or abciximab failed to become potentiated by heparin, demonstrating that heparin promotes platelet responsiveness via its ability to initiate αIIbβ3-mediated outside-in signaling. Taken together, these data provide novel insights into the mechanism by which platelets become activated after exposure to heparin and heparin-coated surfaces, and suggest that currently used glycoprotein IIb-IIIa inhibitors may be effective inhibitors of nonimmune forms of heparin-induced platelet activation.

Inhibition of platelet responsiveness is important to control pathologic thrombus formation. Platelet-endothelial cell adhesion molecule-1 (PECAM-1) and the Src family kinase Lyn inhibit platelet activation by the glycoprotein VI (GPVI) collagen receptor; however, it is not known whether PECAM-1 and Lyn function in the same or different inhibitory pathways. In these studies, we found that, relative to wild-type platelets, platelets derived from PECAM-1-deficient, Lyn-deficient, or PECAM-1/Lyn double-deficient mice were equally hyperresponsive to stimulation with a GPVI-specific agonist, indicating that PECAM-1 and Lyn participate in the same inhibitory pathway. Lyn was required for PECAM-1 tyrosine phosphorylation and subsequent binding of the Src homology 2 domain-containing phosphatase-2, SHP-2. These results support a model in which PECAM-1/SHP-2 complexes, formed in a Lyn-dependent manner, suppress GPVI signaling.

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a cell adhesion and signaling receptor that is expressed on hematopoietic and endothelial cells. PECAM-1 is vital to the regulation of inflammatory responses, as it has been shown to serve a variety of pro-inflammatory and anti-inflammatory functions. Pro-inflammatory functions of PECAM-1 include the facilitation of leukocyte transendothelial migration and the transduction of mechanical signals in endothelial cells emanating from fluid shear stress. Anti-inflammatory functions include the dampening of leukocyte activation, suppression of pro-inflammatory cytokine production, and the maintenance of vascular barrier integrity. Although PECAM-1 has been well-characterized and studied, the mechanisms through which PECAM-1 regulates these seemingly opposing functions, and how they influence each other, are still not completely understood. The purpose of this review, therefore, is to provide an overview of the pro- and anti-inflammatory functions of PECAM-1 with special attention paid to mechanistic insights that have thus far been revealed in the literature in hopes of gaining a clearer picture of how these opposing functions might be integrated in a temporal and spatial manner on the whole organism level. A better understanding of how inflammatory responses are regulated should enable the development of new therapeutics that can be used in the treatment of acute and chronic inflammatory disorders., (Copyright 2010 Elsevier Inc. All rights reserved.)

In this issue of Blood, Canobbio and colleagues report on the unique and redundant contributions of 2 isoforms of PI3K, PI3Kbeta and PI3Kgamma, to platelet activation by GPCRs and ITAM-coupled receptors.

Immunoreceptor tyrosine-based activation motif (ITAM)-containing proteins have recently been demonstrated in macrophages and neutrophils to be required for cell surface integrins to transmit activation signals into the cell. To identify ITAM-bearing proteins that mediate signaling via the platelet-specific integrin alphaIIbbeta3, fibrinogen binding was induced by (1) allowing platelets to spread directly on immobilized fibrinogen, or (2) activating the PAR1 thrombin receptor on platelets in suspension. Both initiated strong, ligand binding-dependent tyrosine phosphorylation of the ITAM-bearing platelet Fc receptor, FcgammaRIIa, as well as downstream phosphorylation of the protein tyrosine kinase Syk and activation of phospholipase Cgamma2 (PLCgamma2). Addition of Fab fragments of an FcgammaRIIa-specific monoclonal antibody strongly inhibited platelet spreading on immobilized fibrinogen, as well as downstream tyrosine phosphorylation of FcgammaRIIa, Syk, and PLCgamma2, and platelets from a patient whose platelets express reduced levels of FcgammaRIIa exhibited markedly reduced spreading on immobilized fibrinogen. Finally, fibrinogen binding-induced FcgammaRIIa phosphorylation did not occur in human platelets expressing a truncated beta3 cytoplasmic domain. Taken together, these data suggest that ligand binding to platelet alphaIIbbeta3 induces integrin cytoplasmic domain-dependent phosphorylation of FcgammaRIIa, which then enlists selected components of the immunoreceptor signaling cascade to transmit amplification signals into the cell.

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.

Platelet glycoprotein (GP) VI is a 62-kDa membrane glycoprotein that exists on both human and murine platelets in a noncovalent complex with the Fc receptor (FcR) gamma chain. The GPVI/FcRgamma-chain complex serves as the major activating receptor for collagen, as evidenced by observations that platelets genetically deficient in GPVI or the FcRgamma chain are highly refractory to collagen-induced platelet activation. Recently, several different rat anti-murine GPVI monoclonal antibodies, termed JAQs 1, 2, and 3, were produced that had the unique property of "immunodepleting" GPVI from the murine platelet surface and rendering it unresponsive to collagen or GPVI-specific agonists like convulxin or collagen-related peptide (CRP). Herein, we describe a patient with a mild bleeding disorder and a moderately reduced platelet count whose platelets fail to become activated in response to collagen or CRP and inefficiently adhere to and form thrombi on immobilized collagen under conditions of arterial shear. Although the amount of GPVI platelet mRNA and the nucleotide sequence of the GPVI gene were found to be normal, both GPVI and the FcRgamma chain were nearly absent from the platelet surface and were markedly reduced in wholeplatelet detergent lysates. Patient plasma contained an autoantibody that bound specifically to GPVI-positive, normal platelets, and cleared soluble GPVI from the plasma, suggesting that the patient suffers from a rare form of idiopathic thrombocytopenic purpura caused by a GPVI-specific autoantibody that mediates clearance of the GPVI/FcRgamma-chain complex from the platelet surface. Since antibody-induced GPVI shedding now has been demonstrated in both humans and mice, these studies may provide a rationale for developing therapeutic reagents that induce temporary depletion of GPVI for the treatment of clinical thrombosis.

Platelet adhesion at sites of vascular injury is mediated, in part, by interaction of the platelet plasma membrane glycoprotein (GP) Ib/V/IX complex with von Willebrand Factor (VWF) presented on collagen-exposed surfaces. Recent studies indicate that GPIb/V/IX may be functionally coupled with the Fc receptor gamma (FcR gamma)-chain, which, by virtue of its cytoplasmic immunoreceptor tyrosine-based activation motif, sends activation signals into the cell. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is an inhibitory receptor that has previously been shown to negatively regulate platelet responses to collagen, which transduces activation signals via the GPVI/FcR gamma-chain complex. To determine whether PECAM-1 might similarly regulate signals emanating from GPIb/FcR gamma, we compared activation and aggregation responses to VWF of PECAM-1-positive and PECAM-1-deficient murine platelets. PECAM-1 and the FcR gamma-chain became rapidly tyrosine phosphorylated in platelets following botrocetin-induced VWF binding, but FcR gamma-chain tyrosine phosphorylation was delayed in PECAM-1-positive, versus PECAM-1-deficient, platelets. PECAM-1-deficient platelets were hyperaggregable to VWF, exhibited enhanced spreading and, under conditions of arterial flow, formed markedly larger thrombi on immobilized VWF than did wild-type platelets. Taken together, these data support the notion that engagement of the GPIb complex, in addition to sending activation signals, also initiates a negative feedback loop involving PECAM-1 that controls the rate and extent of platelet activation.

Programmed cell death, or apoptosis, is a tightly regulated, naturally occurring process by which damaged or unwanted cells are removed. Dysregulated apoptosis has been implicated in a variety of pathophysiological conditions, including degenerative diseases, tissue remodeling, and tumorigenesis. The decision to live or die results from integration of numerous environmental signals transmitted by specific classes of cell surface receptors that bind hormones, growth factors, or components of the extracellular matrix. Here we show that platelet endothelial cell adhesion molecule-1 (PECAM-1), a homophilic-binding member of the immunoreceptor tyrosine-based inhibitory motif (ITIM) family of inhibitory receptors, functions prominently to inhibit apoptosis in naturally occurring vascular cells subjected to apoptotic stimuli. Murine endothelial cells and human T lymphocytes lacking PECAM-1 were found to be far more sensitive than their PECAM-1-expressing counterparts to multiple death signals that stimulate Bax, a multidomain, proapoptotic member of the Bcl-2 family that plays a central role in mitochondrial dysfunction-dependent apoptosis. In addition, PECAM-1 markedly suppressed Bax overexpression-induced cytochrome c release, caspase activation, and nuclear fragmentation. Amino acid substitutions within PECAM-1's extracellular homophilic binding domain, or within its cytoplasmic ITIM, completely abolished PECAM-1-mediated cytoprotection. Taken together, these data implicate PECAM-1 as a novel and potent suppressor of Bax-mediated apoptosis and suggest that members of the immunoglobulin gene (Ig) superfamily, like cell surface integrins, may also transmit survival signals into blood and vascular cells.

Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a cell adhesion molecule with a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when phosphorylated, binds Src homology 2 domain-containing protein-tyrosine phosphatase (SHP-2). PECAM-1 is expressed at endothelial cell junctions where exposure to inflammatory intermediates may result in post-translational amino acid modifications that affect protein structure and function. Reactive nitrogen species (RNS), which are produced at sites of inflammation, nitrate tyrosine residues, and several proteins modified by tyrosine nitration have been found in diseased tissue. We show here that the RNS, peroxynitrite, induced nitration of both full-length cellular PECAM-1 and a purified recombinant PECAM-1 cytoplasmic domain. Mass spectrometric analysis of tryptic fragments revealed quantitative nitration of ITIM tyrosine 686. A synthetic peptide containing 3-nitrotyrosine at position 686 could not be phosphorylated nor bind SHP-2. These data suggest that ITIM tyrosine nitration may represent a mechanism for modulating phosphotyrosine-dependent signal transduction pathways.