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.

Regulatory strategies limiting endosomal Toll-like receptor activation in B cells.

The recognition of pathogen-associated nucleic acid (NA) promotes effective immunity against invading pathogens. However, endosomal Toll-like receptor (TLR) activation by self-NA also underlies the pathogenesis of systemic autoimmune diseases, such as systemic lupus erythematosus (SLE). For this reason, the activation thresholds of NA-sensing TLRs must be tightly regulated to balance protective and pathogenic immune responses. In this study, we will provide an overview of the evolutionary mechanisms designed to limit the aberrant activation of endosomal TLRs by self-ligands, focusing on four broad strategies. These include the following: 1) the production of nucleases able to degrade self-DNA and RNA; 2) the cell-specific regulation of endosomal TLR expression; 3) the spatial and temporal control of TLR positioning at a sub-cellular level; and 4) the modulation of downstream TLR signaling cascades. Given the critical role of B cells in lupus pathogenesis, where possible, we will describe evidence for B cell-specific induction of these regulatory mechanisms. We will also highlight our own work showing how modulation of B cell endolysosomal flux tunes NA-sensing TLR activation signals. In the face of inevitable generation of self-NA during normal cellular turnover, these parallel mechanisms are vital to protect against pathogenic inflammation.

Trypanosoma cruzi trans-sialidase initiates a program independent of the transcription factors RORγt and Ahr that leads to IL-17 production by activated B cells.

Here we identified B cells as a major source of rapid, innate-like production of interleukin 17 (IL-17) in vivo in response to infection with Trypanosoma cruzi. IL-17(+) B cells had a plasmablast phenotype, outnumbered cells of the TH17 subset of helper T cells and were required for an optimal response to this pathogen. With both mouse and human primary B cells, we found that exposure to parasite-derived trans-sialidase in vitro was sufficient to trigger modification of the cell-surface mucin CD45, which led to signaling dependent on the kinase Btk and production of IL-17A or IL-17F via a transcriptional program independent of the transcription factors RORγt and Ahr. Our combined data suggest that the generation of IL-17(+) B cells may be a previously unappreciated feature of innate immune responses required for pathogen control or IL-17-mediated autoimmunity.

Cutting Edge: Systemic Autoimmunity in Murine STAT3 Gain-of-Function Syndrome Is Characterized by Effector T Cell Expansion in the Absence of Overt Regulatory T Cell Dysfunction.

Germline gain-of-function mutations in the transcriptional factor STAT3 promote early-onset multisystemic autoimmunity. To investigate how increased STAT3 promotes systemic inflammation, we generated a transgenic knock-in strain expressing a pathogenic human mutation STAT3K392R within the endogenous murine locus. As predicted, STAT3K392R mice develop progressive lymphoid hyperplasia and systemic inflammation, mirroring the human disease. However, whereas the prevailing model holds that increased STAT3 activity drives human autoimmunity by dysregulating the balance between regulatory T cells and Th17 cell differentiation, we observed increased Th17 cells in the absence of major defects in regulatory T cell differentiation or function. In addition, STAT3K392R animals exhibited a prominent accumulation of IFN-γ-producing CD4+ and CD8+ T cells. Together, these data provide new insights into this complex human genetic syndrome and highlight the diverse cellular mechanisms by which dysregulated STAT3 activity promotes breaks in immune tolerance.

Anti-HLA antibodies in recipients of CD19 versus BCMA-targeted CAR T-cell therapy.

Antibodies against foreign human leukocyte antigen (HLA) molecules are barriers to successful organ transplantation. B cell-depleting treatments are used to reduce anti-HLA antibodies but have limited efficacy. We hypothesized that the primary source for anti-HLA antibodies is long-lived plasma cells, which are ineffectively targeted by B cell depletion. To study this, we screened for anti-HLA antibodies in a prospectively enrolled cohort of 49 patients who received chimeric antigen receptor T-cell therapy (CARTx), targeting naïve and memory B cells (CD19-targeted, n = 21) or plasma cells (BCMA-targeted, n = 28) for hematologic malignancies. Longitudinal samples were collected before and up to 1 year after CARTx. All individuals were in sustained remission. We identified 4 participants with anti-HLA antibodies before CD19-CARTx. Despite B cell depletion, anti-HLA antibodies and calculated panel reactive antibody scores were stable for 1 year after CD19-CARTx. Only 1 BCMA-CARTx recipient had pre-CARTx low-level anti-HLA antibodies, with no follow-up samples available. These data implicate CD19neg long-lived plasma cells as an important source for anti-HLA antibodies, a model supported by infrequent HLA sensitization in BCMA-CARTx subjects receiving previous plasma cell-targeted therapies. Thus, plasma cell-targeted therapies may be more effective against HLA antibodies, thereby enabling improved access to organ transplantation and rejection management.

Cutting Edge: A Threshold of B Cell Costimulatory Signals Is Required for Spontaneous Germinal Center Formation in Autoimmunity.

Cognate interactions between autoreactive B and T cells promote systemic lupus erythematosus pathogenesis by inter alia facilitating spontaneous germinal center (GC) formation. Whereas both myeloid and B cell APCs express B7 ligands (CD80 and CD86), the prevailing model holds that dendritic cell costimulation is sufficient for CD28-dependent T cell activation. In this study, we report that B cell-intrinsic CD80/CD86 deletion unexpectedly abrogates GCs in murine lupus. Interestingly, absent GCs differentially impacted serum autoantibodies. In keeping with distinct extrafollicular and GC activation pathways driving lupus autoantibodies, lack of GCs correlated with loss of RNA-associated autoantibodies but preserved anti-dsDNA and connective tissue autoantibody titers. Strikingly, even heterozygous B cell CD80/CD86 deletion was sufficient to prevent autoimmune GCs and RNA-associated autoantibodies. Together, these findings identify a key mechanism whereby B cells promote lupus pathogenesis by providing a threshold of costimulatory signals required for autoreactive T cell activation.

Recent advances in immunotherapies for lupus nephritis.

Childhood-onset systemic lupus erythematosus (SLE) is characterized by increased rates of kidney involvement, termed lupus nephritis. Despite the significant morbidity and mortality associated with this disease, lupus nephritis trials have been plagued by repeated failures to meet clinical endpoints. However, improvements in trial design and the development of targeted approaches have begun to yield promising results, including two new FDA-approved lupus nephritis treatments since 2020. These include belimumab, a monoclonal antibody targeting the B cell survival cytokine BAFF (B cell activating factor), and voclosporin, a cyclosporin analog with improved pharmacokinetic characteristics. In this review, we will summarize the data supporting regulatory approval for these agents in lupus nephritis and highlight ongoing clinical trials targeting the diverse immunologic drivers of renal inflammation in SLE. While pediatric patients remain underrepresented in lupus clinical trials, given the increased severity of childhood-onset SLE and need for long-term protection from kidney damage, we anticipate the need for off-label use of these targeted therapies in the pediatric population. Future studies are needed to define optimal patient selection, drug combinations, and treatment duration in pediatric lupus nephritis.

BAFF inhibition in SLE-Is tolerance restored?

The B cell activating factor (BAFF) inhibitor, belimumab, is the first biologic drug approved for the treatment of SLE, and exhibits modest, but durable, efficacy in decreasing disease flares and organ damage. BAFF and its homolog APRIL are TNF-like cytokines that support the survival and differentiation of B cells at distinct developmental stages. BAFF is a crucial survival factor for transitional and mature B cells that acts as rheostat for the maturation of low-affinity autoreactive cells. In addition, BAFF augments innate B cell responses via complex interactions with the B cell receptor (BCR) and Toll like receptor (TLR) pathways. In this manner, BAFF impacts autoreactive B cell activation via extrafollicular pathways and fine tunes affinity selection within germinal centers (GC). Finally, BAFF and APRIL support plasma cell survival, with differential impacts on IgM- and IgG-producing populations. Therapeutically, BAFF and combined BAFF/APRIL inhibition delays disease onset in diverse murine lupus strains, although responsiveness to BAFF inhibition is model dependent, in keeping with heterogeneity in clinical responses to belimumab treatment in humans. In this review, we discuss the mechanisms whereby BAFF/APRIL signals promote autoreactive B cell activation, discuss whether altered selection accounts for therapeutic benefits of BAFF inhibition, and address whether new insights into BAFF/APRIL family complexity can be exploited to improve human lupus treatments.

Lupus nephritis transcriptomics across space and time.

Current immunosuppression regimens for lupus nephritis are incompletely effective, placing patients at risk for poor long-term outcomes. This emphasizes the need to dissect pathogenic mechanisms in lupus nephritis, to inform the development of targeted therapies. In this issue of Kidney International, Parikh et al. performed transcriptomic analysis of pretreatment and posttreatment protocol kidney biopsies, segregated into glomerular and tubulointerstitial compartments, to identify candidate molecular pathways distinguishing treatment responders and nonresponders.

TACI haploinsufficiency protects against BAFF-driven humoral autoimmunity in mice.

Polymorphisms in TACI, a BAFF family cytokine receptor, are linked to diverse human immune disorders including common variable immunodeficiency (CVID) and systemic lupus erythematosus (SLE). Functional studies of individual variants show modest impacts on surface TACI expression and/or downstream signal transduction, indicating that relatively subtle variation in TACI activity can impact human B-cell biology. However, significant complexity underlies TACI biology, including both positive and negative regulation of physiologic and pathogenic B-cell responses. To model these contradictory events, we compared the functional impact of TACI deletion on separate models of murine SLE driven by T cell-independent and -dependent breaks in B-cell tolerance. First, we studied whether reduced surface TACI expression was sufficient to protect against progressive BAFF-mediated systemic autoimmunity. Strikingly, despite a relatively modest impact on surface TACI levels, TACI haploinsufficiency markedly reduced pathogenic RNA-associated autoantibody titers and conferred long-term protection from BAFF-driven lupus nephritis. In contrast, B cell-intrinsic TACI deletion exerted a limited impact of autoantibody generation in murine lupus characterized by spontaneous germinal center formation and T cell-dependent humoral autoimmunity. Together, these combined data provide new insights into TACI biology and highlight how TACI signals must be tightly regulated during protective and pathogenic B-cell responses.

Microfluidic post method for 3-dimensional modeling of platelet-leukocyte interactions.

Microvascular thrombosis and inflammation (thromboinflammation) are major causes of morbidity and mortality in critically ill patients with limited therapeutic options. Platelets are central to thromboinflammation, and microvascular platelet thrombi are highly effective at recruiting and activating leukocytes at sites of endothelial injury. Whilst parallel-plate flow chambers, microslides and straight microchannel assays have been widely used to recapitulate leukocyte adhesive behavior on 2-dimensional (2D) surfaces, none of these methods achieve high fidelity 3-dimensional (3D) geometries emulating microvascular platelet thrombi. As a result, the role of hydrodynamic factors in regulating leukocyte interactions with platelet thrombi remains ill-defined. Here, we report a microfluidic post model that allows visualization and analysis of neutrophil-platelet interactions in a 3D flow field. We have utilized the unique mechanosensitive features of platelets to enable selective micropatterning of the 3D posts with human or mouse platelets. By modulating the activation status of platelets, our method enables precise control of platelet surface reactivity and neutrophil recruitment. In addition, our microfluidic post assay accurately recapitulated the rolling versus stationary adhesion behavior of single neutrophils and demonstrated the efficacy of the P-selectin and Mac-1 blocking antibodies to reduce neutrophil recruitment and stationary adhesion, respectively. Moreover, the geometry of posts had a major influence on the efficiency of neutrophil recruitment and adhesion stability. This new post method highlights the importance of platelet 3D geometries in facilitating efficient, localized neutrophil recruitment. These findings have potentially important implications for the potent proinflammatory function of microvascular platelet thrombi.

Rapid assembly and profiling of an anticoagulant sulfoprotein library.

Hematophagous organisms produce a suite of salivary proteins which interact with the host's coagulation machinery to facilitate the acquisition and digestion of a bloodmeal. Many of these biomolecules inhibit the central blood-clotting serine proteinase thrombin that is also the target of several clinically approved anticoagulants. Here a bioinformatics approach is used to identify seven tick proteins with putative thrombin inhibitory activity that we predict to be posttranslationally sulfated at two conserved tyrosine residues. To corroborate the biological role of these molecules and investigate the effects of amino acid sequence and sulfation modifications on thrombin inhibition and anticoagulant activity, a library of 34 homogeneously sulfated protein variants were rapidly assembled using one-pot diselenide-selenoester ligation (DSL)-deselenization chemistry. Downstream functional characterization validated the thrombin-directed activity of all target molecules and revealed that posttranslational sulfation of specific tyrosine residues crucially modulates potency. Importantly, access to this homogeneously modified protein library not only enabled the determination of key structure-activity relationships and the identification of potent anticoagulant leads, but also revealed subtleties in the mechanism of thrombin inhibition, between and within the families, that would be impossible to predict from the amino acid sequence alone. The synthetic platform described here therefore serves as a highly valuable tool for the generation and thorough characterization of libraries of related peptide and/or protein molecules (with or without modifications) for the identification of lead candidates for medicinal chemistry programs.

Thromboinflammation: challenges of therapeutically targeting coagulation and other host defense mechanisms.

Thrombosis with associated inflammation (thromboinflammation) occurs commonly in a broad range of human disorders. It is well recognized clinically in the context of superficial thrombophlebitis (thrombosis and inflammation of superficial veins); however, it is more dangerous when it develops in the microvasculature of injured tissues and organs. Microvascular thrombosis with associated inflammation is well recognized in the context of sepsis and ischemia-reperfusion injury; however, it also occurs in organ transplant rejection, major trauma, severe burns, the antiphospholipid syndrome, preeclampsia, sickle cell disease, and biomaterial-induced thromboinflammation. Central to thromboinflammation is the loss of the normal antithrombotic and anti-inflammatory functions of endothelial cells, leading to dysregulation of coagulation, complement, platelet activation, and leukocyte recruitment in the microvasculature. α-Thrombin plays a critical role in coordinating thrombotic and inflammatory responses and has long been considered an attractive therapeutic target to reduce thromboinflammatory complications. This review focuses on the role of basic aspects of coagulation and α-thrombin in promoting thromboinflammatory responses and discusses insights gained from clinical trials on the effects of various inhibitors of coagulation on thromboinflammatory disorders. Studies in sepsis patients have been particularly informative because, despite using anticoagulant approaches with different pharmacological profiles, which act at distinct points in the coagulation cascade, bleeding complications continue to undermine clinical benefit. Future advances may require the development of therapeutics with primary anti-inflammatory and cytoprotective properties, which have less impact on hemostasis. This may be possible with the growing recognition that components of blood coagulation and platelets have prothrombotic and proinflammatory functions independent of their hemostatic effects.

Exploring the experiences of changes to support access to primary health care services and the impact on the quality and safety of care for homeless people during the COVID-19 pandemic: a study protocol for a qualitative mixed methods approach.

BACKGROUND: Despite high level of health care need amongst people experiencing homelessness, poor access is a major concern. This is sometimes due to organisational and bureaucratic barriers, but also because they often feel stigmatised and treated badly when they do seek health care. The COVID-19 pandemic and the required social distancing measures have caused unprecedented disruption and change for the organisation of primary care, particularly for people experiencing homelessness. Against this backdrop there are many questions to address regarding whether the recent changes required to deliver services to people experiencing homelessness in the context of COVID-19 will help to address or compound problems in accessing care and inequalities in health outcomes. METHODS: An action led and participatory research methodology will be employed to address the study objectives. Interviews with people experiencing homelessness were will be conducted by a researcher with lived experience of homelessness. Researchers with lived experience are able to engage with vulnerable communities in an empathetic, non-judgemental way as their shared experience promotes a sense of trust and integrity, which in turn encourages participation in research and may help people speak more openly about their experience. The experiences of health professionals and stakeholders delivering and facilitating care for people experiencing homelessness during the pandemic will also be explored. DISCUSSION: It is important to explore whether recent changes to the delivery of primary care in response to the COVID-19 pandemic compromise the safety of people experiencing homelessness and exacerbate health inequalities. This could have implications for how primary healthcare is delivered to those experiencing homelessness not only for the duration of the pandemic but in the future.

Functional Characterization of CD11c+ Age-Associated B Cells as Memory B Cells.

Age-associated B cells (ABCs) are a unique subset of B cells defined by surface CD11b and CD11c expression. Although ABC expansion has been observed in both human and animal studies in the setting of advanced age, during humoral autoimmunity and following viral infection, the functional properties of this cellular subset remain incompletely defined. In the current study, we demonstrate that ABCs fulfill the criteria for memory B cells (MBCs), based on evidence of Ag-dependent expansion and persistence in a state poised for rapid differentiation into Ab-secreting plasma cells during secondary responses. First, we show that a majority of ABCs are not actively cycling but exhibit an extensive replication history consistent with prior Ag engagement. Second, despite unswitched surface IgM expression, ABCs show evidence of activation-induced cytidine deaminase (AID)-dependent somatic hypermutation. Third, BCRs cloned from sorted ABCs exhibit broad autoreactivity and polyreactivity. Although the overall level of ABC self-reactivity was not increased relative to naive B cells, ABCs lacked features of functional anergy characteristic of autoreactive B cells. Fourth, ABCs express MBC surface markers consistent with being poised for rapid plasma cell differentiation during recall responses. Finally, in a murine model of viral infection, adoptively transferred CD11c+ B cells rapidly differentiated into class-switched Ab-secreting cells upon Ag rechallenge. In summary, we phenotypically and functionally characterize ABCs as IgM-expressing MBCs, findings that together implicate ABCs in the pathogenesis of systemic autoimmunity.

Potent Trivalent Inhibitors of Thrombin through Hybridization of Salivary Sulfopeptides from Hematophagous Arthropods.

Blood feeding arthropods, such as leeches, ticks, flies and mosquitoes, provide a privileged source of peptidic anticoagulant molecules. These primarily operate through inhibition of the central coagulation protease thrombin by binding to the active site and either exosite I or exosite II. Herein, we describe the rational design of a novel class of trivalent thrombin inhibitors that simultaneously block both exosites as well as the active site. These engineered hybrids were synthesized using tandem diselenide-selenoester ligation (DSL) and native chemical ligation (NCL) reactions in one-pot. The most potent trivalent inhibitors possessed femtomolar inhibition constants against α-thrombin and were selective over related coagulation proteases. A lead hybrid inhibitor possessed potent anticoagulant activity, blockade of both thrombin generation and platelet aggregation in vitro and efficacy in a murine thrombosis model at 1 mg kg-1 . The rational engineering approach described here lays the foundation for the development of potent and selective inhibitors for a range of other enzymatic targets that possess multiple sites for the disruption of protein-protein interactions, in addition to an active site.

Generation of functional murine CD11c+ age-associated B cells in the absence of B cell T-bet expression.

Age-associated B cells (ABC), a novel subset of activated B cells defined by CD11b and CD11c expression, have been linked with both protective anti-viral responses and the pathogenesis of systemic autoimmunity. Expression of the TH 1 lineage transcription factor T-bet has been identified as a defining feature of ABC biology, with B cell-intrinsic expression of this transcription factor proposed to be required for ABC formation. In contrast to this model, we report that Tbx21 (encoding T-bet)-deficient B cells upregulate CD11b and CD11c surface expression in vitro in response to integrated TLR and cytokine signals. Moreover, B cell-intrinsic T-bet deletion in a murine lupus model exerted no impact of ABC generation in vivo, with Tbx21-/- ABCs exhibiting an identical surface phenotype to wild-type (WT) ABCs. Importantly, WT and Tbx21-/- ABCs sorted from autoimmune mice produced equivalent amounts of IgM and IgG ex vivo following TLR stimulation, indicating that T-bet-deficient ABCs are likely functional in vivo. In summary, our data contradict the established literature by demonstrating that T-bet expression is not uniformly required for ABC generation.

Partial loss of actin nucleator actin-related protein 2/3 activity triggers blebbing in primary T lymphocytes.

T lymphocytes utilize amoeboid migration to navigate effectively within complex microenvironments. The precise rearrangement of the actin cytoskeleton required for cellular forward propulsion is mediated by actin regulators, including the actin-related protein 2/3 (Arp2/3) complex, a macromolecular machine that nucleates branched actin filaments at the leading edge. The consequences of modulating Arp2/3 activity on the biophysical properties of the actomyosin cortex and downstream T cell function are incompletely understood. We report that even a moderate decrease of Arp3 levels in T cells profoundly affects actin cortex integrity. Reduction in total F-actin content leads to reduced cortical tension and disrupted lamellipodia formation. Instead, in Arp3-knockdown cells, the motility mode is dominated by blebbing migration characterized by transient, balloon-like protrusions at the leading edge. Although this migration mode seems to be compatible with interstitial migration in three-dimensional environments, diminished locomotion kinetics and impaired cytotoxicity interfere with optimal T cell function. These findings define the importance of finely tuned, Arp2/3-dependent mechanophysical membrane integrity in cytotoxic effector T lymphocyte activities.

An integrin αIIbß3 intermediate affinity state mediates biomechanical platelet aggregation.

Integrins are membrane receptors that mediate cell adhesion and mechanosensing. The structure-function relationship of integrins remains incompletely understood, despite the extensive studies carried out because of its importance to basic cell biology and translational medicine. Using a fluorescence dual biomembrane force probe, microfluidics and cone-and-plate rheometry, we applied precisely controlled mechanical stimulations to platelets and identified an intermediate state of integrin αIIbß3 that is characterized by an ectodomain conformation, ligand affinity and bond lifetimes that are all intermediate between the well-known inactive and active states. This intermediate state is induced by ligand engagement of glycoprotein (GP) Ibα via a mechanosignalling pathway and potentiates the outside-in mechanosignalling of αIIbß3 for further transition to the active state during integrin mechanical affinity maturation. Our work reveals distinct αIIbß3 state transitions in response to biomechanical and biochemical stimuli, and identifies a role for the αIIbß3 intermediate state in promoting biomechanical platelet aggregation.

Intrinsic apoptosis circumvents the functional decline of circulating platelets but does not cause the storage lesion.

The circulating life span of blood platelets is regulated by the prosurvival protein BCL-XL It restrains the activity of BAK and BAX, the essential prodeath mediators of intrinsic apoptosis. Disabling the platelet intrinsic apoptotic pathway in mice by deleting BAK and BAX results in a doubling of platelet life span and concomitant thrombocytosis. Apoptotic platelets expose phosphatidylserine (PS) via a mechanism that is distinct from that driven by classical agonists. Whether there is any role for apoptotic PS in platelet function in vivo, however, is unclear. Apoptosis has also been associated with the platelet storage lesion (PSL), the constellation of biochemical deteriorations that occur during blood bank storage. In this study, we investigated the role of BAK/BAX-mediated apoptosis in hemostasis and thrombosis and in the development of the PSL. We show that although intrinsic apoptosis is rapidly induced during storage at 37°C, it is not detected when platelets are kept at the standard storage temperature of 22°C. Remarkably, loss of BAK and BAX did not prevent the development of the PSL at either temperature. BAK/BAX-deficient mice exhibited increased bleeding times and unstable thrombus formation. This phenotype was not caused by impaired PS exposure, but was associated with a defect in granule release from aged platelets. Strikingly, rejuvenation of BAK/BAX-deficient platelets in vivo completely rescued the observed hemostatic defects. Thus, apoptotic culling of old platelets from the bloodstream is essential to maintain a functional, hemostatically reactive platelet population. Inhibiting intrinsic apoptosis in blood banked platelets is unlikely to yield significant benefit.