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Background: While it is known that obesity increases the risk for cardiovascular disease (CVD), there has been no clear investigation into the effect an obesogenic diet exerts onto circulating platelets. As platelets are the critical actors in the formation of occlusive clots that manifest as a myocardial infarction or stroke, it is pertinent to establish any potential link between obesity and platelet activity. Methods: To examine the effect of a Western diet on baseline platelet activity, male C57BL6/J mice were fed either a control (10% fat, 7% sucrose, 0.3% salt Research Diets Inc. D12450J) or Western (40% fat, 30% sucrose, 8% salt Research Diets Inc. D06111701) diet ad libitum for 20 weeks beginning at the time of weaning. At 12, 16 and 20 weeks after beginning diet administration whole blood was collected via the retro-orbital sinus. Blood was used to generate platelet rich plasma (PRP) which was stimulated with collagen (20mg/ml) prior to staining for flow cytometry analysis. Flow cytometry staining utilized antibodies to recognize the platelet receptors GPIbα, P-Selectin and the activated form of integrin αIIbβ3 (JON/A). Additionally, body composition was likewise determined at these time points using Echo-MRI while bodyweight was measured weekly. Results: Western diet fed mice gained slightly more weight than control mice and had a higher percentage of body fat (measured by EchoMRI). Surprisingly, the two groups differed in GPIbα and activated αIIbβ3 expression on circulating platelets at all three observed time points. Specifically, platelets from control-fed mice displayed significantly higher GPIbα expression throughout the study while Western diet fed animals had higher levels of activated αIIbβ3. There was no difference in platelet expression of P-Selectin between the groups at any time point. Conclusions: Platelet activation is not a binary process where platelets display a definitive “active” or “inactive” phenotype. Though terminal platelet activation elicits de-granulation and an increase in P-Selectin expression, the spectrum of platelet stimulation preceding this is marked by variable incidences of integrin activation and GPIbα shedding. Here we demonstrate proof of principle that dietary factors, such as the consumption of a Western diet, can facilitate early changes to the stimulation pattern of circulating platelets which is then maintained long-term. While the consequence of this altered platelet stimulation profile is unknown with regards to the pathways of hemostasis and thrombosis, further investigations of downstream signaling and amplification pathways are needed. Funding: Arkansas Children's Research Institute/Arkansas Bioscience Institute Postgraduate Fellowship: AWD-037176 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BACKGROUND AND PURPOSE: Platelet membrane glycoprotein Ibα (GPIbα), the major ligand-binding subunit of the GPIb-IX-V complex, binds to a number of ligands contributing to hemostasis, thrombosis, and inflammation. Binding to von Willebrand factor (VWF) initiates the process of hemostasis/thrombosis, while binding to the leukocyte receptor Macrophage-1 antigen (Mac-1) has been implicated in modulating the inflammatory response. Thus as GPIbα resides at the nexus of thrombosis and inflammation, we investigated the impact of GPIbα on radiation injury outcomes as this injury triggers both the thrombotic and inflammatory pathways. MATERIALS AND METHODS: We used wild-type (WT) C57BL/6J mice and a dysfunctional GPIbα mouse model, in which endogenous GPIbα is replaced with a non-functional α-subunit (hIL-4R/Ibα), to determine whether the impairment of platelet GPIbα alters radiation response. Following exposure to 8.5 Gy total body irradiation (TBI), a series of parameters including radiation lethality, platelet-neutrophil/monocyte interactions, neutrophil/monocyte activation, serum cytokine levels and intestinal injury, were compared between the strains. RESULTS: The lack of functional GPIbα resulted in higher radiation lethality, greater monocyte activation, increased levels of serum pro-inflammatory cytokines, heightened intestinal damage, and a reduction of intestinal neutrophil recovery. CONCLUSION: These data suggest that loss of platelet GPIbα enhances radiation toxicity and that GPIbα-mediated interactions may play a crucial role in limiting radiation damage. Thus, a mechanistic understanding of the biological impact of GPIbα following TBI could provide crucial insights for improving the safety of radiotherapy and minimizing the deleterious effects of accidental or occupational exposure to high-dose radiation.

Platelet-dependent mechanisms for excessive clotting and bleeding in CKD remain undefined. Moreover, the platelet's contributions to inflammation, and specifically to CKD, are equally elusive. To date, descriptions of changes in the functional properties of circulating platelets during CKD have provided confusing interpretations. Experimental approaches that can advance our understanding of platelet dysfunction in CKD are needed, and studies that provide mechanistic insights into the dynamic relationships between thrombosis, bleeding, and inflammation associated with CKD will be essential to improve clinical management and outcomes for this vulnerable population. This article summarizes existing literature characterizing platelets in CKD and identifies areas that need further investigation.

Platelets are best known for their vasoprotective responses to injury and inflammation. Here, we have asked whether they also support vascular integrity when neither injury nor inflammation is present. Changes in vascular barrier function in dermal and meningeal vessels were measured in real time in mouse models using the differential extravasation of fluorescent tracers as a biomarker. Severe thrombocytopenia produced by two distinct methods caused increased extravasation of 40-kDa dextran from capillaries and postcapillary venules but had no effect on extravasation of 70-kDa dextran or albumin. This reduction in barrier function required more than 4 h to emerge after thrombocytopenia was established, reverting to normal as the platelet count recovered. Barrier dysfunction was also observed in mice that lacked platelet-dense granules, dense granule secretion machinery, glycoprotein (GP) VI, or the GPVI signaling effector phospholipase C (PLC) γ2. It did not occur in mice lacking α-granules, C type lectin receptor-2 (CLEC-2), or protease activated receptor 4 (PAR4). Notably, although both meningeal and dermal vessels were affected, intracerebral vessels, which are known for their tighter junctions between endothelial cells, were not. Collectively, these observations 1) highlight a role for platelets in maintaining vascular homeostasis in the absence of injury or inflammation, 2) provide a sensitive biomarker for detecting changes in platelet-dependent barrier function, 3) identify which platelet processes are required, and 4) suggest that the absence of competent platelets causes changes in the vessel wall itself, accounting for the time required for dysfunction to emerge.

// Caleb K. Stein 1 , Charlotte Pawlyn 2 , Shweta Chavan 1 , Leo Rasche 1 , Niels Weinhold 1 , Adam Corken 1 , Amy Buros 1 , Pieter Sonneveld 3 , Graham H. Jackson 4 , Ola Landgren 5 , Tariq Mughal 6, 7 , Jie He 6 , Bart Barlogie 1 , P. Leif Bergsagel 8 , Faith E. Davies 1 , Brian A. Walker 1 , Gareth J. Morgan 1 1 The Myeloma Institute, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA 2 The Institute of Cancer Research, London, UK 3 Eramus University, Rotterdam, NL 4 Department of Haematology, Newcastle University, Newcastle-upon-Tyne, UK 5 Memorial Sloan Kettering Cancer Center, New York, New York, USA 6 FoundationOne Medicine, Cambridge, Massachusetts, USA 7 Tufts Medical Center, Boston, Massachusetts, USA 8 Mayo Clinic, Scottsdale, Arizona, USA Correspondence to: Caleb K. Stein, email: CKStein@uams.edu Keywords: multiple myeloma, gene expression profiling, mutational analysis, translocation cyclin D (TC) Received: January 13, 2017 Accepted: February 15, 2017 Published: February 24, 2017 ABSTRACT We examined a set of 805 cases that underwent DNA sequencing using the FoundationOne Heme (F1H) targeted sequencing panel and gene expression profiling. Known and likely variant calls from the mutational data were analyzed for significant associations with gene expression defined translocation cyclin D (TC) molecular subgroups. The spectrum of KRAS, NRAS, and BRAF codon mutations varied across subgroups with NRAS mutations at Q61 codon being common in hyperdiploid (HRD) and t(11;14) myeloma while being rare in MMSET and MAF. In addition, the presence of RAS-RAF mutations was inversely associated with NFκB pathway activation in all subgroups excluding MAF. In the MMSET subgroup, cases with low FGFR3 expression frequently had RAS-RAF mutations. Conditional inference tree analysis determined that mutation and homozygous deletion of TP53, CDKN2C, and RB1 were key prognostic factors associated with adverse outcome in a non-relapse clinical setting. In conclusion, this study highlights the heterogeneity in the distribution and clinical outcomes of RAS codon and other mutations in multiple myeloma dependent upon primary molecular subgroup.

Sepsis triggers a complex series of pathophysiologic events involving inflammatory responses and coagulation abnormalities. While circulating blood platelets are well-characterized for their contributions to coagulation, increasingly platelet-dependent effects on inflammation are being recognized. Here, we focus on the platelet membrane receptor, glycoprotein VI (GPVI), and its role in platelet microparticle (pMP) release. The GPVI receptor is a platelet-specific collagen membrane receptor that, upon ligand binding, facilitates the release of pMPs. As membrane-bound platelet fragments of less than 1 μm, pMPs are known to have both pro-inflammatory and pro-coagulant properties. Thus, pMPs are potentially impacting sepsis at multiple stages of the inflammatory response. Studies are presented documenting the impact of the most common GPVI haplotypes, GPVIa and GPVIb, on pMP levels and release in healthy individuals (n = 49). The GPVIa haplotype corresponds to an approximately twofold increase in circulating pMPs as a percentage of total microparticles in healthy individuals along with a heightened in vitro release of pMPs. Additionally, patients admitted to a paediatric intensive care unit (ICU) (n = 73) with an initial diagnosis of sepsis were recruited and their GPVI haplotypes determined. Septic patients of the GPVIa haplotype (n = 59) were statistically more likely to present with a diagnosis of severe sepsis or septic shock, as compared with GPVIb individuals (n = 14). Independent disease classification via PELOD-2 and Pediatric Risk of Mortality III scores confirmed individuals with the GPVIa haplotype were more likely to have significant organ failure. Thus, GPVI haplotypes influence pMP levels in the circulation and are predictive of sepsis severity when presenting to the ICU.

Although once primarily recognized for its roles in hemostasis and thrombosis, the platelet has been increasingly recognized as a multipurpose cell. Indeed, circulating platelets have the ability to influence a wide range of seemingly unrelated pathophysiologic events. Here, we highlight some of the notable observations that link platelets to inflammation, reinforcing the platelet’s origin from a lower vertebrate cell type with both hemostatic and immunologic roles. In addition, we consider the relevance of platelets in cancer biology by focusing on the hallmarks of cancer and the ways platelets can influence multistep development of tumors. Beyond its traditional role in hemostasis and thrombosis, the platelet’s involvement in the interplay between hemostasis, thrombosis, inflammation, and cancer is likely complex, yet extremely important in each disease process. The existence of animal models of platelet dysfunction and currently used antiplatelet therapies provide a framework for understanding mechanistic insights into a wide range of pathophysiologic events. Thus, the basic scientist studying platelet function can think beyond the traditional hemostasis and thrombosis paradigms, while the practicing hematologist must appreciate platelet relevance in a wide range of disease processes.

The platelet paradigm that is well established in hemostasis and thrombosis can be extended to other disease states. A consideration for some major health issues, such as inflammation, cancer, infection, and neuroscience, and how platelet function impacts the pathophysiology of each clinical situation is provided.Decades of research and knowledge of platelet function exist and the same is true for inflammation and cancer. The literature is full of platelet biology overlapping into other, nonthrombotic disease states. However, major gaps exist that prevent a complete mechanistic understanding of platelet function in these other diseases. Although much of the overlap provides antidotal relationships, future studies will likely uncover novel pathophysiological pathways that are highly relevant to human diseases.Recent findings in four major disease areas, inflammation, cancer, infection, and neuroscience, are described, with current literature linking the disease to platelet function. The availability of antiplatelet therapies, such as aspirin, exists and future consideration can be given as to whether antiplatelet therapy is potentially beneficial or harmful as the mechanisms of platelet involvement are better defined.

A dynamic reciprocating crosstalk between cancer cells and platelets presents both opportunities and challenges for an increased understanding of tumorigenesis and improved patient therapies (1). The importance of understanding this relationship finds merit in the widespread use of cardiovascular therapies that target normal platelet function but impact diseases seemingly unrelated to hemostasis or thrombosis (2). In the case of cancer, there exists expanding databases to support anti-cancer effects for common medications, such as aspirin (3). However, major questions remain for “how, where, and when” aspirin exerts its effect on the progression of cancer. A systematic approach for understanding the “how, when and where” question can be based on defining the platelet’s relevance within each component of the “hallmarks of cancer” (4-6).

Single-scorpionates of nickel(II), Tp(R)NiX or Tpm(R)NiX, are kinetic products whose preparation has generally required considerable steric constraints on the ligands (i.e., R = phenyl, tert-butyl, or isopropyl) to prevent formation of intractable two-ligand products like (Tp(R))(2)Ni. It is well established that the facial tridentate chelates hydrotris(3,5-dimethylpyrazolyl)borate (Tp*(-)), tris(3,5-dimethylpyrazolyl)methane (Tpm*), and trispyrazolylmethane (Tpm), all readily form two-ligand complexes as thermodynamic products. For the first time we report a route to the single-ligand complex TpmNiX(2)(OH(2))(n) (X = Cl and Br). We also report a novel method for making single-ligand nickel(II) scorpionate complexes using preformed tetrahalonickelate(II) ion in nitromethane. The complex Tpm*NiCl(2)(OH(2))(n) was also prepared here for the first time utilizing an alternative method first reported by Zargarian and co-workers (Inorg. Chim. Acta 2006, 2592). TpmNiX(2)(OH(2))(n) are kinetic products, and although they are stable indefinitely in the solid state, they readily convert to the thermodynamic product (Tpm)(2)Ni(2+) in solution over the course of several hours at room temperature and in a matter of minutes at 100 degrees C. The new nitromethane/NiX(4)(2-) method offers an alternative route to monoscorpionates of first row transition metals, for which tetrahalometallate ions are common. HOCH(2)Tpm (2,2,2-tris(pyrazolyl)ethanol) was covalently attached to polystyrene synthesis beads and found to bind nickel(II) (from NiX(4)(2-)) in a manner similar to Tpm. Solid state electronic spectra of supported-TpmNiCl(2) are comparable to those measured for their homogeneous complexes. Covalently supported scorpionates are expected to further extend the utility of this rich ligand class in areas of heterogeneous catalysis and metal-protein interactions.

Objective— The platelet glycoprotein Ib-IX (GP Ib-IX) receptor is a well-characterized adhesion receptor supporting hemostasis and thrombosis via interactions with von Willebrand factor. We examine the GP Ib-IX/von Willebrand factor axis in murine polymicrobial sepsis, as modeled by cecal ligation and puncture (CLP). Approach and Results— Genetic absence of the GP Ib-IX ligand, von Willebrand factor, prolongs survival after CLP, but absence of the receptor, GP Ib-IX, does not. Because absence of either von Willebrand factor or GP Ib-IX significantly impairs hemostasis and thrombosis, we sought to define additional GP Ib-IX–dependent pathways impacting survival in the CLP model. We document that the absence of GP Ib-IX leads to reduced platelet–neutrophil and platelet–monocyte interactions. Twenty-four hours after CLP, absence of GP Ib-IX coincides with an alteration in cytokine levels, such as tumor necrosis factor-α secreted by monocytes, and increased macrophage-1 antigen expression by neutrophils. Conclusions— In contrast to the well-characterized proinflammatory properties of platelets, we describe in the CLP model an anti-inflammatory property associated with platelet GP Ib-IX. Thus, a single platelet receptor displays a dual modulatory role in both the thrombotic and inflammatory pathways associated with polymicrobial sepsis. In sharing leucine-rich motifs with toll-like receptors, platelet GP Ib-IX can be considered a multifunctional participant in hemostasis, thrombosis, and the inflammatory cascade. The results highlight a dynamic role for platelets in systemic inflammation and add to the complex pathophysiologic events that occur during the dysregulated coagulation and inflammation associated with sepsis.

Objective— Poor prognosis of sepsis is associated with bacterial lipopolysaccharide (LPS)-induced intravascular inflammation, microvascular thrombosis, thrombocytopenia, and disseminated intravascular coagulation. Platelets are critical for thrombosis, and there has been increasing evidence of the importance of platelets in endotoxemia. The platelet adhesion receptor, the glycoprotein Ib-IX complex (GPIb-IX), mediates platelet adhesion to inflammatory vascular endothelium and exposed subendothelium. Thus, we have investigated the role of GPIb-IX in LPS-induced platelet adhesion, thrombosis, and thrombocytopenia. Approach and Results— LPS-induced mortality is significantly decreased in mice expressing a functionally deficient mutant of GPIbα. Furthermore, we have developed a micellar peptide inhibitor, MPαC (C13H27CONH-SIRYSGHpSL), which selectively inhibits the von Willebrand factor -binding function of GPIb-IX and GPIb-IX–mediated platelet adhesion under flow without affecting GPIb-IX–independent platelet activation. MPαC inhibits platelet adhesion to LPS-stimulated endothelial cells in vitro and alleviates LPS-induced thrombosis in glomeruli in mice. Importantly, MPαC reduces mortality in LPS-challenged mice, suggesting a protective effect of this inhibitor during endotoxemia. Interestingly, MPαC, but not the integrin antagonist, Integrilin, alleviated LPS-induced thrombocytopenia. Conclusions— These data indicate an important role for the platelet adhesion receptor GPIb-IX in LPS-induced thrombosis and thrombocytopenia, and suggest the potential of targeting GPIb as an antiplatelet strategy in managing endotoxemia.

The preparation of a resin-supported boron-scorpionate ligand and its nickel(II) coordination complexes are reported. The supported ligand is prepared as its potassium salt, making it a general reagent suitable for chelation of any transition metal ion. Resin-immobilized benzotriazole (Bead-btz) reacted cleanly with KTp* (Tp* = hydrotris(3,5-dimethylpyrazolyl)borate) by heterocycle metathesis in warm dimethylformamide (DMF) to yield bead-Tp'K, {resin-btz(H)B(pz*)(2)}K. Significantly, bead-Tp'K readily bound nickel(II) from simple salts with minimal leaching of the nickel ion. Bead-Tp'NiNO(3) reacts further with cysteine thiolate (ethyl ester), imparting the deep green color to the beads characteristic of a Tp(R)NiCysEt coordination sphere. Bead-Tp'NiCysEt exhibited an oxygen sensitivity similar to Tp*NiCysEt in solution (Inorg. Chem. 1999, p 5690) and also independently verified for a selenocystamine analogue, Tp*NiSeCysAm. Addition of fresh cysteine thiolate ethyl ester to oxidized bead-Tp'NiCysEt reproduced the original green color. Heterocycle metathesis was also used to prepare KTp' as a white solid. Reaction with nickel(II) gave (Tp')(2)Ni, separable into two different isomers. The air-sensitive molybdenum(0) complex, [PPh(4)][Tp'Mo(CO)(3)], was also prepared and the C(s) complex symmetry demonstrated by infrared and (13)C NMR spectroscopies. Immobilized TpmMo(CO)(3) was prepared from the previously reported resin-supported tris(pyrazolyl)methane. In contrast to its weak coordination of nickel(II) (Inorg. Chem. 2009, p 3535), bead-Tpm proved a strong chelate toward this second row metal. The supported scorpionates described here should find use in studies of selective metal-protein binding, metalloprotein modeling, and heterogeneous catalysis, and render such scorpionate applications amenable to combinatorial methods.

The platelet glycoprotein (GP) Ib-IX receptor complex is expressed exclusively on the surface of platelets and is well characterized as a primary adhesion receptor supporting normal hemostasis and pathologic thrombosis. Beyond hemostasis and thrombosis, platelets can also participate in the innate immune response and inflammation. While the platelet as a contributor to the immune continuum is recognized, many aspects of the molecular mechanisms whereby platelets influence the immune response are still undefined. Here, we report studies using a murine model of GP Ib-IX deficiency linking GP Ib-IX to the immune response associated with polymicrobial sepsis, as modeled by cecal ligation and puncture (CLP). In the CLP model, genetic absence of the major GP Ib-IX hemostatic ligand, von Willebrand factor (VWF), improves survival following CLP when compared to control wild-type animals (p= 0.003, Logrank analysis). This suggests a VWF role in thrombosis contributes to survival outcome following CLP. In contrast, genetic absence of the VWF platelet receptor, GP Ib-IX, does not improve survival with no statistical difference comparing wild-type animals to GPIb-IX deficient animals. The molecular basis to explain improved survival in VWF-deficient (ligand deficient) but not GPIb-IX deficient (receptor deficient) animals was pursued. We tested the hypothesis GPIb-IX has normal physiologic and pathophysiologic functions beyond platelet adhesion influencing infection and an inflammatory response. Indeed, GPIb-IX influencing the innate immune response is not completely unexpected since a hallmark structural feature of each subunit of the GPIb-IX receptor is leucine rich repeats, the common motif to all members of the toll like receptor family (TLRs). Whether structural similarities are a consequence of ancestral origins for GPIb-IX and TLRs is unknown. We first documented in the absence of murine platelet GP Ib-IX there are reduced platelet-neutrophil and platelet-monocyte interactions under normal conditions and following CLP in whole blood. Whether there are physiologic consequences for disrupting a platelet/monocyte and/or platelet/neutrophil axis was determined via multianalyte profiling of circulating cytokine levels on a Luminex analyzer following CLP. In the absence of GP Ib-IX there is a robust and statistically significant increase 24 hrs following CLP in some of the major proinflammatory cytokines produced by monocytes and macrophages, including TNFα, MCP-1, MIP-β, IL-6, and IL-15. Increases in cytokines, such as IL-5 and IL-13, associated with other immune cells were also observed. These results highlight a coagulation/inflammation interface where the platelet, and specifically GP Ib-IX, contributes to the pathophysiology of CLP. On the one hand, absence of platelet GPIb-IX reduces thrombotic potential, but it occurs at the expense of upregulation of inflammatory cytokine release leading to a reduced survival in CLP. Clearly, survival outcomes in CLP reflect a complex dysregulation of coagulation and inflammation where platelet GPIb-IX likely contributes to both processes with physiologic consequences. Understanding dysregulation of the coagulation/ inflammation interface and identifying a platelet receptor (GPIb-IX) critical to both adds new information to this complex set of pathophysiologic events Sharing the common structural motifs, leucine rich repeats, with the well characterized family of toll-like receptors, platelet GPIb-IX should now be considered an active participant in the inflammatory cascade. Disclosures: No relevant conflicts of interest to declare.