Mouse Platelets Research Articles

Canalicular system reorganization during mouse platelet activation as revealed by 3D ultrastructural analysis

The canalicular system (CS) has been defined as: 1) an inward, invaginated membrane connector that supports entry into and exit from the platelet; 2) a static structure stable during platelet isolation; and 3) the major source of plasma membrane (PM) for surface area expansion during activation. Recent analysis from STEM tomography and serial block face electron microscopy has challenged the relative importance of CS as the route for granule secretion. Here, We used 3D ultrastructural imaging to reexamine the CS in mouse platelets by generating high-resolution 3D reconstructions to test assumptions 2 and 3. Qualitative and quantitative analysis of whole platelet reconstructions, obtained from immediately fixed or washed platelets fixed post-washing, indicated that CS, even in the presence of activation inhibitors, reorganized during platelet isolation to generate a more interconnected network. Further, CS redistribution into the PM at different times, post-activation, appeared to account for only about half the PM expansion seen in thrombin-activated platelets, in vitro, suggesting that CS reorganization is not sufficient to serve as a dominant membrane reservoir for activated platelets. In sum, our analysis highlights the need to revisit past assumptions about the platelet CS to better understand how this membrane system contributes to platelet function.

Low-dose Btk inhibitors selectively block platelet activation by CLEC-2.

Inhibitors of Bruton tyrosine kinase (Btk) have been proposed as novel antiplatelet agents. In this study we show that low concentrations of the Btk inhibitor ibrutinib block CLEC-2-mediated activation and tyrosine phosphorylation including Syk and PLCγ2 in human platelets. Activation is also blocked in patients with X-linked agammaglobulinemia (XLA) caused by a deficiency or absence of Btk. In contrast, the response to GPVI is delayed in the presence of low concentrations of ibrutinib or in patients with XLA, and tyrosine phosphorylation of Syk is preserved. A similar set of results is seen with the second-generation inhibitor, acalabrutinib. The differential effect of Btk inhibition in CLEC- 2 relative to GPVI signaling is explained by the positive feedback role involving Btk itself, as well as ADP- and thromboxane A2-mediated activation of P2Y12 and TP receptors, respectively. This feedback role is not seen in mouse platelets and, consistent with this, CLEC-2-mediated activation is blocked by high but not by low concentrations of ibrutinib. Nevertheless, thrombosis was absent in eight out of 13 mice treated with ibrutinib. These results show that Btk inhibitors selectively block activation of human platelets by CLEC-2 relative to GPVI suggesting that they can be used at low doses in patients to target CLEC-2 in thrombo-inflammatory disease.

Catalytic dysregulation of SHP2 leading to Noonan syndromes affects platelet signaling and functions

AbstractSrc homology 2 domain–containing phosphatase 2 (SHP2), encoded by the PTPN11 gene, is a ubiquitous protein tyrosine phosphatase that is a critical regulator of signal transduction. Germ line mutations in the PTPN11 gene responsible for catalytic gain or loss of function of SHP2 cause 2 disorders with multiple organ defects: Noonan syndrome (NS) and NS with multiple lentigines (NSML), respectively. Bleeding anomalies have been frequently reported in NS, but causes remain unclear. This study investigates platelet activation in patients with NS and NSML and in 2 mouse models carrying PTPN11 mutations responsible for these 2 syndromes. Platelets from NS mice and patients displayed a significant reduction in aggregation induced by low concentrations of GPVI and CLEC-2 agonists and a decrease in thrombus growth on a collagen surface under arterial shear stress. This was associated with deficiencies in GPVI and αIIbβ3 integrin signaling, platelet secretion, and thromboxane A2 generation. Similarly, arterial thrombus formation was significantly reduced in response to a local carotid injury in NS mice, associated with a significant increase in tail bleeding time. In contrast, NSML mouse platelets exhibited increased platelet activation after GPVI and CLEC-2 stimulation and enhanced platelet thrombotic phenotype on collagen matrix under shear stress. Blood samples from NSML patients also showed a shear stress–dependent elevation of platelet responses on collagen matrix. This study brings new insights into the understanding of SHP2 function in platelets, points to new thrombopathies linked to platelet signaling defects, and provides important information for the medical care of patients with NS in situations involving risk of bleeding.

Evaluation of blood collection methods and anticoagulants for platelet function analyses on C57BL/6J laboratory mice

The exploration of thrombotic mechanisms relies on the application of blood collection methods from laboratory mice with a minimal pre-activation of platelets and the clotting system. So far, very little is known on how the blood collection method and the anticoagulant used influence pre-activation of mouse platelets and coagulation. To determine the most suitable blood collection method, we systematically compared blood collection by heart puncture, Vena cava puncture, and puncture of the retro-orbital vein plexus and the use of citrate, heparin, and EDTA as frequently used anticoagulants with regard to platelet activation and whole blood clotting parameters. The activation of platelet-rich plasma diluted in Tyrode’s buffer was analyzed by flow cytometry, analyzing the exposure of P-selectin and activated integrin αIIbβ3. Clotting of whole blood was profiled by thrombelastometry. Puncture of the retro-orbital vein plexus by plastic capillaries is not superior in terms of blood volume and platelet pre-activation, whereas heart puncture and Vena cava puncture resulted in similarly high blood volumes. Cardiac puncture and Vena cava puncture did not result in pre-activated platelets with citrate as an anticoagulant, but the use of EDTA resulted in increased levels of integrin αIIbβ3 activation. Puncture of the retro-orbital vein plexus by plastic capillaries resulted in increased platelet integrin αIIbβ3 activation, which could be prevented by soaking with citrate or coating with heparin. Further, activation of coagulation in citrated whole blood by puncture of the retro-orbital vein plexus using a blunt plastic capillary was observed by thromboelastometry. The use of citrate is the optimal anticoagulant in mouse platelet assays. Blood collections from the heart or Vena cava represent reliable alternatives to retro-orbital puncture of the vein plexus to avoid pre-activation of platelets and coagulation.

Platelets and cancer-associated thrombosis: focusing on the platelet activation receptor CLEC-2 and podoplanin.

Patients with cancer have an increased risk of thromboembolism, which is the second leading cause of death in these patients. Several mechanisms of the prothrombotic state in these patients have been proposed. Among them are a platelet activation receptor, C-type lectin-like receptor 2 (CLEC-2), and its endogenous ligand podoplanin, which are the focus of this review. CLEC-2 is almost specifically expressed in platelets/megakaryocytes in humans. A membrane protein, podoplanin is expressed in certain types of cancer cells, including squamous cell carcinoma, brain tumor, and osteosarcoma, in addition to several normal tissues, including kidney podocytes and lymphatic endothelial cells but not vascular endothelial cells. In the bloodstream, podoplanin induces platelet activation by binding to CLEC-2 and facilitates hematogenous cancer metastasis and cancer-associated thrombosis. In an experimental lung metastasis model, the pharmacological depletion of CLEC-2 from platelets in mice resulted in a marked reduction of lung metastasis of podoplanin-expressing B16F10 cells. Control mice with B16F10 orthotopically inoculated in the back skin showed massive thrombus formation in the lungs, but the cancer-associated thrombus formation in CLEC-2-depleted mice was significantly inhibited, suggesting that CLEC-2-podoplanin interaction stimulates cancer-associated thrombosis. Thromboinflammation induced ectopic podoplanin expression in vascular endothelial cells or macrophages, which may also contribute to cancer-associated thrombosis. CLEC-2 depletion in cancer-bearing mice resulted in not only reduced cancer-associated thrombosis but also reduced levels of plasma inflammatory cytokines, anemia, and sarcopenia, suggesting that cancer-associated thrombosis may cause thromboinflammation and cancer cachexia. Blocking CLEC-2-podoplanin interaction may be a novel therapeutic strategy in patients with podoplanin-expressing cancer.

Biochemical and immunocytochemical characterization of coronins in platelets

Rapid reorganization of the actin cytoskeleton in response to receptor-mediated signaling cascades allows platelets to transition from a discoid shape to a flat spread shape upon adhesion to damaged vessel walls. Coronins are conserved regulators of the actin cytoskeleton turnover but they also participate in signaling events. To gain a better picture of their functions in platelets we have undertaken a biochemical and immunocytochemical investigation with a focus on Coro1. We found that class I coronins Coro1, 2 and 3 are abundant in human and mouse platelets whereas little Coro7 can be detected. Coro1 is mainly cytosolic, but a significant amount associates with membranes in an actin-independent manner and does not translocate from or to the membrane fraction upon exposure to thrombin, collagen or prostacyclin. Coro1 rapidly translocates to the Triton insoluble cytoskeleton upon platelet stimulation with thrombin or collagen. Coro1, 2 and 3 show a diffuse cytoplasmic localization with discontinuous accumulation at the cell cortex and actin nodules of human platelets, where all three coronins colocalize. Our data are consistent with a role of coronins as integrators of extracellular signals with actin remodeling and suggests a high extent of functional overlap among class I coronins in platelets.

VAS2870 and VAS3947 attenuate platelet activation and thrombus formation via a NOX-independent pathway downstream of PKC

NADPH oxidase (NOX) enzymes are involved in a various physiological and pathological processes such as platelet activation and inflammation. Interestingly, we found that the pan-NOX inhibitors VAS compounds (VAS2870 and its analog VAS3947) exerted a highly potent antiplatelet effect. Unlike VAS compounds, concurrent inhibition of NOX1, 2, and 4 by treatment with ML171, GSK2795039, and GKT136901/GKT137831 did not affect thrombin and U46619-induced platelet aggregation. These findings suggest that VAS compounds may inhibit platelet aggregation via a NOX-independent manner. Thus, we aimed to investigate the detailed antiplatelet mechanisms of VAS compounds. The data revealed that VAS compounds blocked various agonist-induced platelet aggregation, possibly via blocking PKC downstream signaling, including IKKβ and p38 MAPK, eventually reducing platelet granule release, calcium mobilization, and GPIIbIIIa activation. In addition, VAS compounds inhibited mouse platelet aggregation-induced by collagen and thrombin. The in vivo study also showed that VAS compounds delayed thrombus formation without affecting normal hemostasis. This study is the first to demonstrate that, in addition to inhibiting NOX activity, VAS compounds reduced platelet activation and thrombus formation through a NOX-independent pathway downstream of PKC. These findings also indicate that VAS compounds may be safe and potentially therapeutic agents for treating patients with cardiovascular diseases.

IPLA2β Contributes to Activation of Human and Mouse Platelets Via ITAM Receptors Fcγ RIIA and GPVI

Platelet activation by ITAM receptors contributes to hemostasis, thrombosis, vascular integrity and host defense. In the course of our studies of FcγRIIA-mediated platelet activation, we became interested in those mechanisms that require neither full Syk activation nor changes in intracellular calcium. Calcium-independent phospholipase family member iPLA2β has been observed to modulate phospholipid remodeling and second messenger generation in human platelets (Beckett, Thromb Res 2007; Duvernay, Biochem 2015), while iPLA2γ has been studied in knockout mouse platelets (Yoda, JTH 2014) with modest effects noted on thrombosis and hemostasis. These enzymes do not require increased cytoplasmic calcium for their activity in cleaving the acyl group from the sn2 position of phospholipids to yield a free fatty acid and a lysophospholipid. However, the precise role of iPLA2β in human and mouse platelet activation has not been elucidated. Neither has the contribution of iPLA2β in the response to FcγRIIA-mediated activation been reported. We identified the presence of iPLA2β protein in western blots of human and FcγRIIA transgenic mouse platelets. Of interest, multiple isoforms arising from proteolytic cleavage were detected. We treated washed human and FcγRIIA transgenic mouse platelets with agonists to FcγRIIA (IV.3 + GAM) and to GPVI (collagen or collagen-related peptide) in the absence and presence of pharmacologic inhibitors of iPLA2β. At a range of agonist doses up to 3X threshold, we observed significant inhibition of aggregation, dense granule secretion and alpha granule secretion (p<0.05 vs. vehicle only, n = 3 to 4 each). Inhibition occurred with either S-BEL (bromo-enolactone) or with FKGK18 (a fluoroketone), two chemically distinct iPLA2β inhibitor molecules with different modes of action. The IC50 for S-BEL was found to be 1.02 uM for human FcγRIIA, 2.04 uM for human GPVI, and 2.76 uM for transgenic mouse FcγRIIA activated platelets. FKGK18 was less potent, with IC50s at 7.88 uM for human FcγRIIA. In contrast, iPLA2γ inhibitor R-BEL was able to inhibit FcγRIIA -mediated activation, but at an IC50 of 2.62 uM. Notably, iPLA2β inhibition could eliminate ATP secretion from dense granules downstream of FcγRIIA and GPVI activation. When we added ADP to FcγRIIA stimulation in the presence of inhibitory doses of S-BEL, we overcame the inhibition. We have identified for the first time that iPLA2β contributes to aggregation and secretion of both human and FcγRIIA transgenic mouse platelets. The platelets were slightly more sensitive to FcγRIIA than to GPVI inhibition. In other activatable secretory cells, iPLA2β plays both a homeostatic and signaling role. The mechanisms of iPLA2β action in platelets merit further study. Studies are in progress with genetic knockdown and knockout of the enzyme, to complement the findings with inhibitors. Disclosures No relevant conflicts of interest to declare.

Modulation of Thrombin-Induced Platelet Activation By Defibrotide

Defibrotide is a mixture of single-stranded phosphodiester oligonucleotides 9-80 bases in length derived from mammalian tissue and it is the only medication that is FDA approved for the prevention and treatment of post-transplant veno-occlusive disease, also known as sinusoidal obstructive syndrome (VOD/SOS). The mechanism of action of defibrotide is not well understood. A better understanding of the molecular mechanism by which defibrotide prevents and reverses the microvascular occlusion seen in VOD/SOS will be critical in the development of novel therapeutics for VOD/SOS and other related disorders. Our central hypothesis is that single stranded phosphodiester oligonucleotide mixtures such as defibrotide alter thrombin-induced platelet and endothelial cell activation primarily via altered signaling through protease activated receptors (PARs). To determine the effect of defibrotide on thrombin-induced platelet activation we performed flow cytometry on washed platelets from humans and mice. Washed platelets were incubated with agonist (1 nM thrombin, 100 μM synthetic PAR1 agonist TRAP6, or 100 μM synthetic PAR4 agonist PAR4AP) in the presence or absence of 1 mg/mL of defibrotide. Fluorescent antibodies against P-selectin (anti-CD62P antibody) and against the high affinity conformation of the αIIβ3 receptor (PAC1 antibody for human samples and JON/A antibody for murine samples) were added to assess for inhibition of platelet activation by defibrotide. Fluorescent anti-PAR1 antibodies SPAN12 (which spans the canonical thrombin cleavage site) and WEDE15 (which binds at the hirudin-like domain) were also added to separate samples to assess whether defibrotide inhibits cleavage of the PAR1 N-terminal domain by thrombin. After twenty minutes of incubation at room temperature the samples were analyzed by flow cytometry. We calculated the geometric mean of the fluorescence intensity (MFI) for three replicate samples and data are shown as the fold change compared to the washed platelet control without addition of agonist or defibrotide. For thrombin-stimulated human platelets, both anti-CD62P binding (MFI fold over control 5.7 vs. 1.2) and PAC1 antibody binding (MFI fold over control 7.1 vs. 1.7) are significantly reduced in the presence of defibrotide (See Figure 1a). For thrombin-stimulated murine platelets there is a small decrease in anti-CD62P antibody binding (MFI fold over control 8.4 vs. 6.6), but no significant change in JON/A antibody binding (See Figure 1b). Binding of both anti-CD62P and PAC1 antibodies to human platelets stimulated with synthetic PAR agonists was unaffected by the presence of defibrotide (See Figure 1c). There is no significant difference in SPAN12 or WEDE15 binding in thrombin-stimulated platelets in the absence or presence of defibrotide. However, defibrotide alone does appear to interfere with WEDE15 binding in unstimulated platelets (See Figure 1d). In conclusion, we showed that defibrotide inhibits thrombin-induced human platelet activation at low concentrations of thrombin, as shown by a decrease in surface platelet activation markers measured by flow cytometry. This effect is much less pronounced under the same conditions if mouse platelets are used, supporting the hypothesis that defibrotide inhibits platelet activation by alteration of PAR1 receptor signaling, since PAR1 receptors are not present on mouse platelets. Defibrotide does not interfere with TRAP6-induced platelet activation, evidence against interference with the intracellular portion of the signaling pathway, since TRAP6 is a synthetic PAR1 agonist that binds directly into the ligand-binding pocket of the receptor, eliminating the need for cleavage of the PAR1 extracellular domain. The decrease in WEDE15 antibody binding to washed platelets seen with defibrotide alone suggests that the mechanism may involve alterations in the hirudin-like domain of the PAR1 receptor which is crucial for thrombin binding and cleavage site specificity. Ongoing studies including investigating the effect of defibrotide on peptide mimics of the extracellular N-terminal domain of the PAR1 receptor and the effect of defibrotide on the activation of endothelial cells will likely shed light on the exact mechanism of platelet response modulation by defibrotide and may provide new targets for treatment of the microvascular occlusion that characterizes VOD/SOS. Disclosures No relevant conflicts of interest to declare.

Human PAR4 Is a More Potent Receptor for Activating Platelets Than Mouse PAR4

PAR4 is a protease-activated receptor with major roles in both platelet aggregation and platelet procoagulant function, contributing to both hemostasis and thrombosis in vivo. It is a major target for anti-thrombotic agents in current development. There are notable differences in the amino acid sequences between human (hu) PAR4 and mouse (mu) PAR4 in domains associated with the mechanisms of receptor action. These include the second transmembrane domain, which has a Valine at position 120 in muPAR4 while it is Alanine or Threonine in huPAR4 (Edelstein, Nature Med 2014). Other differences include 4 non-conservative amino acid changes in extracellular loop 2 and a major non-conservative change (mu = Cysteine, hu = Glutamine) in helix 8 in the cytosolic carboxy terminal (Ramachandran, Mol Pharm 2017). We generated a unique set of mice which enable us to compare for the first time the potential differences in platelet activation between huPAR4 and muPAR4 in the platelet context, rather than in heterologous cells. We generated and characterized 5 independent lines of mice transgenic for human PAR4, using an approach with a large genomic clone which we have implemented successfully in the past. Each of these huPAR4 transgenic lines has been bred to the mouse PAR4 knockout mice generated by Coughlin and colleagues (generously provided by S. Kunapuli, Temple University). The mice are referred to as PAR4 tgKO mice; 3 express the hu Thr120 allele and 2 the Ala120 allele. The level of huPAR4 expression in the tgKO platelets is equivalent to that of muPAR4 in wild-type mouse platelets. Washed platelets from wild-type mice, PAR4 tgKO mice, and muPAR4 KO mice were stimulated with a range of concentrations of PAR4 activating peptide (PAR4-AP, AYPGKF) and the activation of αIIbβ3 and expression of P-selectin on the surface were determined with flow cytometry. As expected, muPAR4 knockout mice showed no response to the treatment, but reacted normally to other agonists. While we observed small differences between the hu Ala120 and Thr120 tgKO mice, consistent with prior reports by Bray, Edelstein and colleagues for human platelets, we observed large and statistically significant differences between all tgKO mouse platelets tested and wild-type mouse platelets. In summary, all other things being equal (i.e. the same platelet context), human PAR4 is a more potent receptor for platelet activation than mouse PAR4. Studies are in progress to elucidate the contribution of the different functional domains and the roles of heterotrimeric G proteins, calcium and other signaling intermediates. Figure Disclosures No relevant conflicts of interest to declare.

IL-1β Secreted from Mutant Cells Carrying JAK2-V617Ffavors Early Clonal Expansion and Promotes MPN Disease Initiation and Progression

JAK2-V617F is the most frequently recurring somatic mutation in patients with myeloproliferative neoplasm (MPN), but it can also be found in healthy individuals with clonal hematopoiesis of indeterminate potential (CHIP) with a frequency much higher than the incidence of MPN. This suggests that the acquisition of the JAK2-V617F is not the rate-limiting step and other factors might be required for the expansion of the JAK2 mutated clone and initiation of MPN disease. Chronic inflammation is a hallmark of advanced MPN and is associated with progression to myelofibrosis and AML. Interleukin-1β (IL-1β) is one of the master regulators of the inflammatory state and its aberrant activity has been implicated in various pathological diseases including MPN. Here we focused on the early stages of MPN disease initiation and examined the role of IL-1β in this context. We hypothesized that IL-1β mediated inflammation may promote early expansion of the JAK2 mutant clone to reach a critical clone size capable of initiating MPN. We used a genetic approach and crossed IL-1β knockout (IL1β-/-) mice with our tamoxifen inducible SclCreER;JAK2-V617F (VF) mice, generating a triple mutant SclCreER;JAK2-V617F;IL-1β-/-(VF;IL1β-/-) line. We then transplanted two million bone marrow (BM) cells from VF and VF;IL1β-/- mice into lethally irradiated wildtype (WT) orIL1β-/- recipients. Complete blood counts monitored every 4 weeks for up to 32 weeks post transplantation showed reduced platelet, neutrophil, leukocyte and monocyte counts in mice transplanted with VF;IL1β-/-as compared to VF. Furthermore, terminal analysis at week 16 and 32 revealed reduced splenomegaly and bone marrow fibrosis in the mice receiving VF cells lacking IL1β. This experiment shows that IL1β plays an important role in MPN pathogenesis in this mouse model. To test the hypothesis that IL1β favors clonal expansion during MPN disease initiation, we performed competitive dilution assays by mixing BM cells from VF or VF;IL1β-/-mice that also co-express the GFP protein as a reporter (VF;GFP or VF;IL1β-/-;GFP) with BM cells from IL1β-/- mice in 1:100 ratio and transplanted into lethally irradiated WT recipients (Figure 1A). Successful engraftment was defined by presence of >1% GFP+ cells within Gr-1+ granulocytes in peripheral blood (PB) at week 18 after transplantation. In mice transplanted with VF;GFP, we found engraftment in 25 of 29 (86%) recipients whereas in mice transplanted with VF;IL1β-/-;GFP, only 18 of 29 (62%) recipients showed engraftment. Moreover, 10 of 25 (40%) mice engrafted with VF;GFP developed MPN at 24 weeks after transplantation as compared to only 2 of 18 (11%) mice engrafted with VF;IL1β-/-;GFP cells. GFP chimerism measured every 6 weeks in peripheral blood (PB) from erythroid (Ter119+), megakaryocytic (CD61+) and granulocytic lineages (Gr-1+) was significantly reduced in mice transplanted with VF;IL1β-/-;GFP compared to mice transplanted with VF;GFP cells (Figure 1A), suggesting the capacity to produce IL-1β protein by the VF cells was promoting the expansion of the clone and MPN manifestation.To define the relative contributions of hematopoietic and non-hematopoietic cell derived IL-1β in promoting MPN initiation, we performed competitive dilution assays in IL1β-/-recipients (Figure 1B). We detected engraftment in 27 of 30 (90%) IL1β-/-recipients transplanted with VF;GFP and 27 of 33 (82%) mice transplanted with VF;IL1β-/-;GFP. Furthermore, 9 of 27 (33%) mice engrafted with either VF;GFP or VF;IL1β-/-;GFP developed MPN at 24 weeks after transplantation. However GFP chimerism in Ter119, Gr-1 and CD61 was lower in mice transplanted with VF;IL1β-/-;GFP compared to mice transplanted with VF;GFP (Figure 1B). We further looked at plasma IL-1β protein levels by ELISA (Figure 1C). Interestingly, we found that IL-1β protein levels were also reduced in WT mice transplanted with VF;IL1β-/-;GFP donor cells, indicating that the non-hematopoietic WT cell cannot compensate for the deficiency of IL-1β in the VF clone. Overall, our results demonstrate that IL-1β favors early clonal expansion and show that IL-1β produced by the JAK2 mutant cells is required for optimal MPN disease initiation. Disclosures No relevant conflicts of interest to declare.

Acute Influenza A Virus Infection Affects Cell Cycle and Lineage Output of Hematopoietic Stem Cells

Long-term hematopoietic stem cells (LT-HSC) persist in quiescence to maintain their hematopoietic potential throughout life. In the case of need LT-HSC can be activated to replenish the pool of blood cells. We investigated the impact of acute influenza A virus (IAV) infection on hematopoiesis in C57Bl/6N mice, focusing on the most immature HSC and progenitors. Mice were infected with a lethal dose of IAV PR/8/1934 H1N1 (humane endpoints reached within 6 days post infection (dpi)). In two further groups, mice were treated daily with oseltamivir (antiviral neuraminidase inhibitor, dpi 0-4) or were vaccinated with single-cycle vesicular stomatitis virus replicon particles expressing a miss-matched neuraminidase from influenza A virus Yamaguchi/7/2004 H5N1 four weeks prior to infection. Both treatments rescued mice from infection-induced mortality. Every day 6-9 mice were analyzed for differences in the bone marrow (BM) and blood by flow cytometry and multiplex cytokine assays as well as in the lung to determine viral tissue titers and histopathology. HSC functionality was analyzed in a competitive BM transplantation of infected and non-infected mice. Irrespective of the treatment, high IAV lung tissue titers (≥5x106 tissue culture infectious dose 50) in the first days post infection (dpi 1-5) were associated with activation of HSC into the cell cycle. LT-HSCs (LSK, CD150+, CD34- and CD48-) were 50% less quiescent and shifted into the G1/S-G2-M phase (dpi 2-6) and returned to quiescence state after virus clearance (dpi 10). Furthermore, we detected 1.5-fold increase in proliferation of phenotypic LT-HSC. Differentiation was increased towards lymphoid progenitors (≥3-fold more compared to non-infected mice) during the acute phase of infection in untreated and oseltamivir treated mice and myeloid progenitors were reduced ~50% in all groups (dpi 4-8). We found the inflammatory cytokines IFNγ, IL-1α, IL-6, and TNFα to be significantly upregulated in the BM of untreated and oseltamivir treated mice but less in vaccinated animals (dpi 2-4). IL-1α or IL-6 stimulation of LT-HSCs was sufficient to initiate proliferation in cell culture. In all groups the initial drop of the peripheral platelet count (~30% lower compared to non-infected mice, dpi 2) was replenished with an excessive production of platelets (~45% increased, dpi 8-15). Histopathology and electron microscopy revealed the sequestration and accumulation of platelets in pulmonary capillaries and vessels. Subsequently, we detected twice as many mature megakaryocytes in the BM (dpi 2-4) and elevated CD41 expression on HSCs (LSK, CD150+ and CD34-; 3-fold more compared to non-infected mice dpi 2-6) indicating a myeloid/platelet-biased HSC compartment in response to infection. Competitive whole BM transplantation with activated LT-HSCs from the acute phase of infection vs non-infected mice showed delayed reconstitution of T-cells but a preferential differentiation towards platelets in recipient mice. Taken together, local IAV infection in the lung substantially affected LT-HSC quiescence and differentiation by inflammatory cytokines with systemic consequences and a myeloid/platelet-biased lineage output. Disclosures No relevant conflicts of interest to declare.

A Novel Approach for Generating Platelet-Delivered FVIII: Role of Transient LRP1 Expression during Megakaryopoiesis

Human platelets endocytose coagulation factor (F) V into their α-granules, in part, via low-density lipoprotein (LDL) receptor-related protein 1 (LRP1). In contrast to humans, mouse FV is endogenously expressed during megakaryopoiesis and stored in α-granules. The FV-related coagulation factor FVIII is not endogenously expressed in megakaryocytes (Mks) and thought not to be endocytosed by human or murine Mks/platelets. The lack of FVIII endocytosis is surprising as LRP1 on multiple cell types is involved in FVIII uptake and clearance. We and others have shown that FVIII can be ectopically expressed during human or murine megakaryopoiesis and stored in α-granules. These platelets are effective in the delivery of FVIII to sites of vascular injury and improve outcome in hemophilia A (HA) mice even in the presence of circulating inhibitors. Thus, it has been proposed that lentiviral bone marrow gene therapy to ectopically express FVIII during megakaryopoiesis may be a curative strategy especially for patients with HA and intractable inhibitors. However, we have shown that pFVIII has a toxic effect on Mks during intracellular processing in the endoplasmic reticulum/Golgi, limiting platelet yield and pFVIII levels. We have recently shown that exogenous FV can be taken up by in vitro differentiated CD34+- and by induced pluripotent stem cell (iPSC)-derived Mks and asked if the same can be done with FVIII. We found that Mks can take up full-length FVIII (Advate) (Fig. 1A), B-domainless (BD) FVIII (Xyntha, not shown) and BD FVIIIR1645H (not shown), a FVIII mutant we have shown is particularly effective when released by platelets. Uptake is half maximum at 0.25 IU/ml following overnight incubation. Endocytosed FVIII colocalizes with labeled fibrinogen uptake (Fig. 1A), supporting its localization to α-granules, and this FVIII uptake can be blocked by receptor-associated protein (RAP), a blocker of LDL receptor family members, including LRP1 (Fig. 1A) or by including FVIII's carrier protein von Willebrand factor (VWF) (Fig. 1B). To test the biological efficacy of endocytosed pFVIII, we took advantage of our previous studies showing that infused Mks into mice release highly functional platelets after becoming entrapped in the lungs. To focus our studies on the released human platelets and the hemostatic efficacy of the endocytosed pFVIII, we used immunodeficient NOD-scid IL2rgnull (NSG) mice that were also FVIII-deficient and that only expressed mutant VWF that binds human, but not mouse, platelet glycoprotein Ib/IX (VWFR1326H) (N/8/V mice). We infused 1x106 human FVIII-endocytosed Mks into these N/8/V mice resulting in ~1-10% being human platelets in recipient mice. We studied hemostatic efficacy by rotational thromboelastography (ROTEM) and demonstrated that the in vivo-released FVIII from released human platelets within the N/8/V blood corrected hemostasis in this system (Fig. 1C). These FVIII-containing platelets fully corrected clotting as well in a FeCl3 carotid artery injury model (Fig. 1D). In summary, we found that in vitro-grown human Mks can endocytose FVIII from the media into their α-granules in sufficient amounts to have potential clinical application in the care of HA patients. This endocytosis is likely via LRP1. FVIII is not found normally in platelets likely because of a combination of the following: 1) in plasma, FVIII it is bound to VWF, 2) circulating platelets also lack LRP1, and 3) in the marrow, there is little or no free FVIII for endocytosis by LRP1-positive Mks. We propose that endocytosed FVIII by Mks can be an important clinical application of commercial, in vitro-grown Mks for patients with HA and inhibitors who need supplemental long-lasting, hemostatic support to their emicizumab without the thrombotic risks of FVIII-bypassing agents. Disclosures Sabatino: Spark Therapeutics: Patents & Royalties. Camire:Pfizer: Research Funding.

Understanding the Mechanisms Underlying Thrombocytopenia in Visceral Leishmaniasis

Visceral leishmaniasis (VL) is a neglected tropical parasitic disease caused by Leishmania parasites and only second to malaria in terms of worldwide morbidity and mortality. According to recent WHO report, there are 500,000 cases of VL worldwide leading to ~30,000 deaths per year. VL is endemic in 98 countries but the major disease burden is contributed by Brazil, India and Sudan. Disease manifestations include fever, weight loss, hepatosplenomegaly, immune dysregulations and extensive hematological complications. We have shown previously using experimental models of infection that the infiltration of CD4+ T cells results in disruption to the bone marrow environment, resulting in dysfunctional hematopoietic stem and progenitor cells self-renewal (Pinto et al, PLOS Pathogens, 2017) and aberrant medullary erythropoiesis causing pathological anemia (Preham et al, Frontiers in Immunology, 2018). Thrombocytopenia is also dominant hematological feature seen in both human and experimental models that may reflect either reduced platelet production or enhanced clearance. However, the mechanisms of VL-driven thrombocytopenia remain poorly understood. The aim of this study is to explore the possible underlying mechanisms from platelet production to phagocytic cells dependent clearance. Using a murine experimental model of VL, we demonstrate a steady decrease in the platelet count from d14 onwards in infected mice culminating in severe thrombocytopenia on d28 of infection (infected: 225.9 ±35.7 vs naïve: 1005 ±90.6, x 106/µl). Critically, thrombocytopenia is completely reversible after a single dose of liposomal amphotericin B (Ambisome @ 8mg/kg bodyweight, IV) which clears parasites by delivering the drug directly to parasite harbouring tissue macrophages, thereby improving parasite clearance and reducing toxicity. Despite significant thrombocytopenia, the number and gross morphology of bone marrow megakaryocytes (MKs) were not altered, but MK ultrastructure studies using transmission electron microscopy identified significantly reduced demarcation membranes in infected mice compared to naïve. Levels of plasma thrombopoietin (TPO), the key regulator of MK differentiation and platelet production, were decreased in infected vs naïve mice (1254 ± 95.49 vs 3249 ± 125.1 pg/ml) and administration of exogenous TPO resulted in complete recovery of platelet counts. Given that the majority of TPO is produced by the liver, reduction in the levels of circulating TPO during infection is likely due to destruction of liver architecture by parasite loaded hepatic granulomas. Together, these data suggest that despite some changes in MK cytoplasmic maturation, the bone marrow microenvironment remains supportive of MK differentiation capacity during VL. As platelet production is not significantly altered by VL, we next determined effects on platelet clearance. Large number of highly active splenic macrophages are common in VL and are known for their phagocytic properties. Experiments conducted on VL-infected splenectomised mice demonstrated a reduction in thrombocytopenia compared to sham-operated infected mice (685 ±32 vs 297± 16, x 106/µl) and showed a great response to exogenous TPO, implying splenic clearance may be involved in thrombocytopenia. Partial depletion of splenic macrophages in infected mice using clodronate liposomes did not alter platelet count, whereas neutrophil deletion (anti-Gr1 mAb @ 250ug/g IP) in infected mice resulted in a near 2-fold increase in platelet counts. Furthermore, circulating platelets in VL infected mice were IgG coated compared to naive which is likely to further enhance autoimmune platelet clearance. Severe thrombocytopenia and bleeding are important clinical manifestations of VL. Our findings clearly demonstrate that the mechanisms of thrombocytopenia in VL are multifactorial but do not cause permanent long term damage to the BM microenvironment. Critically, these changes could be reversed rapidly by clearing parasitemia, using TPO agonists to increase numbers of circulating platelets and/or by reducing platelet clearance. This highlights the possibility of re-evaluating the current treatment regimen in VL endemic countries by including therapeutic interventions aimed at reversing severe thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

RhoA Targeting Suppresses Cold-Induced Platelet Lesion and Allows Refrigerated Storage of Functional Human Platelets to Fourteen Days

Platelet transfusion is required for the support therapy of patients with hematological disorders and cancer. Current practice of donor-derived platelet storage at room temperature (RT) associates with an inherent risk of microbial contamination and a limit of 5-7 day shelf life. Refrigerated platelets are hemostatically superior than RT platelets but their survival in circulation is severely reduced. Storage in cold temperature induces loss of galactosylation/sialylation of platelet GPIb with exposure of b-N-acetyl glucosamine of N-linked glycans, which clusters on the platelet plasma membrane. Upon transfusion, these clusters are recognized by macrophages and hepatocytes resulting in lectin-mediated platelet clearance. We hypothesized that the long-term refrigerated storage lesion depends on the mislocalization of membrane-bound glycosyl- and syalyl-transferases in lipid rafts and endocytotic intermediates of cold-stored platelets. By using a combination of genetic and pharmacological approaches, we investigated whether the three major members of the Rho GTPase family, RHOA, RAC1 and/or CDC42, which are molecular switches regulating actomyosin dynamics and signaling in platelets. We found that cooling of platelets induces a sustained activation of RhoA and Rac1, but not Cdc42 in mouse and human platelets. Inducible genetic deletion of platelet RhoA in mice prevents clearance of cold-stored platelets upon transfusion in wild-type congenic animals (WT: 2±0.5 hrs x fraction of infusate; RhoAΔ/Δ: 8± 0.7 hrs x fraction of infusate; p≤0.05). When human platelets were stored in either plasma or PAS-IIIM (PAS-E) for up to 14 days, the inclusion in the storage medium of a rationally designed RHOA inhibitor G04 can prevent the phagocytosis of 14-day refrigerated platelets by activated macrophages in vitro (RT-stored, vehicle-treated platelets: 20%±4; cold stored, vehicle-treated 70±5%; cold-treated, G04(10μM)- treated: 25±3%; p≤0.001) or in vivo clearance as assessed by the area under the curve of surviving human platelets (hrs x fraction of infusate) transfused into clodronate-treated, sub-lethally irradiated non-obese diabetic, γc-/- (NSG) mice (RT-stored, vehicle-treated platelets: 16±1.0; cold-stored, vehicle-treated: 6±0.5; cold-stored, G04(10μM)-treated: 15±0.6; p≤0.001). This inhibition is reversible by either a wash or a 3-fold dilution that resulted in a reversion of the inhibitory effect on aggregation and in vivo correction of the bleeding time of mice pre-treated with aspirin (RT-stored, vehicle-treated: 45±4 sec.; cold-stored, vehicle-treated: 150±10 sec.; cold-stored, G04(10μM)-treated: 76±3.8 sec.; p≤0.001). Mechanistically, RHOA inhibition prevents the cold-induced cytoskeleton/shape change and spreading on fibrinogen through the prevention of the formation of lipid rafts enriched in glycosyl- and syalyl-transferase activities and the endocytosis of vacuoles enriched in GPIbα. Addition of the lipid raft disruptor b-cyclodextrin, but not of the RHOA downstream effector ROCK inhibitor Fasudil, phenocopies the effect of G04 on the prevention of cold-induced platelet damage. Thus, RHOA is the key mediator of the platelet storage lesion through a ROCK independent mechanism and its reversible inhibition allows the functional maintenance of cold-stored platelets in both survival and hemostatic properties. Our pre-clinical data support the concept that a platelet additive solution containing a low-affinity RHOA inhibitor is useful in preventing platelet storage lesion while fully maintaining their hemostatic function after 14 days of storage. A rationally designed cold storage regimen is highly feasible, which could resolve the platelet clearance problem and meet an urgent need in transfusion medicine for the support therapy of patients with thrombocytopenia or thrombocytopathy. Disclosures Cancelas: Cellphire: Research Funding; Velico: Consultancy, Research Funding; Hemanext: Consultancy, Research Funding; Fresenius-Kabi: Research Funding; Cerus Co.: Research Funding; TerumoBCT: Consultancy, Research Funding; Macopharma Inc: Research Funding; Cytosorbents: Research Funding.

Targeting Metabolic Enzyme Pyruvate Kinase M2: A Novel Strategy to Inhibit Platelet Function and Arterial Thrombosis

Background: The cellular responses initiated upon platelet activation are energy consuming. Activated platelets, in comparison to their resting state, exhibit a high level of aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen) relative to oxidative phosphorylation (OXPHOS), suggesting that metabolic plasticity exists in platelets. Although aerobic glycolysis yields less total ATP when compared to OXPHOS, the rate of ATP generation is faster in aerobic glycolysis compared to OXPHOS, which we hypothesize is well suited for high-energy requirement during platelet activation. The glycolytic enzyme pyruvate kinases (PKs) catalyzes the final step of glycolysis and contributes to net ATP production. Four PK isoforms (L, R, M1 and M2) exist in mammals: L and R isoforms are expressed in the liver and red blood cells; the M1 isoform is expressed in most adult tissues that have high catabolic demands including muscle and brain; M2 is expressed in cells including activated platelets and leukocytes. Unlike other isoforms of PK that function only as tetramers, PKM2 can exist in either a tetrameric state or a dimeric state. PKM2 is allosterically regulated by the upstream metabolite fructose-1, 6 biphosphate. While PKM1 and tetrameric PKM2 favor ATP production from OXPHOS through the TCA cycle, dimeric PKM2 drives aerobic glycolysis. The glycolytic and non-glycolytic functions of PKM2 in platelets have not investigated yet. Objective: We tested an innovative concept that whether targeting metabolic enzyme PKM2 will inhibit platelet function and arterial thrombosis. Methods: Using a specific inhibitor of PKM2 (that prevents PKM2 dimerization and stabilizes tetramers) and a range of standardized platelet in vitro assays, we determined the mechanistic role of PKM2 in modulating platelet function in human and mice. To provide definitive evidence, we generated a megakaryocyte or platelet-specific PKM2-/- mouse (PKM2fl/flPF4Cre). Susceptibility to thrombosis was evaluated in vitro (microfluidics flow chamber) and in vivo (FeCl3-induced carotid and laser-injury induced mesenteric artery thrombosis models) by utilizing intravital microscopy. Susceptibility to hemostasis was evaluated in tail bleeding assay. Results: Human and mouse platelets pretreated with PKM2 inhibitor significantly decreased platelet aggregation to sub-optimal doses of collagen, convulxin, thrombin, and ADP. Consistent with this, inhibiting PKM2 dimerization reduced αIIbβ3 activation, alpha and dense granule secretion, clot retraction that was concomitant with decreased glucose uptake. Furthermore, treatment with PKM2 inhibitor reduced Akt and GSK3β phosphorylation, that are predominantly involved in PI3K/Akt signaling, suggesting a non-glycolytic role of the PKM2 in regulating platelet function. In microfluidics flow chamber assay, human and whole mouse blood pretreated with PKM2 inhibitor formed small thrombi when perfused over collagen for 5 minutes at an arterial shear rate of 1500s-1 (P<0.05 vs. vehicle). In agreement with PKM2 inhibitor studies, platelets from PKM2fl/flPF4Cre mice exhibited decreased agonist-induced platelet aggregation, which was in agreement with decreased alpha and dense granule secretion, αIIbβ3 activation, clot retraction, lactate production, and Akt and GSK3β phosphorylation (P<0.05 vs. PKM2fl/fl littermate controls). Wild-type mice-treated with PKM2 inhibitor and/or PKM2fl/flPF4Cre were less susceptible to thrombosis in the FeCl3-induced carotid and laser injury-induced mesenteric artery thrombosis models. Lack of effect on tail bleeding time suggested normal hemostasis in PKM2fl/flPF4Cre mice and PKM2 inhibitor-treated wild-type mice. No sex-based differences were observed. Currently, we are performing platelet metabolomics to determine the effect of targeting PKM2 on metabolic pathways. Conclusions: Our results suggest that manipulating metabolic plasticity by targeting dimeric PKM2 may be explored as a novel strategy to inhibit platelet function and arterial thrombosis. Disclosures Lentz: Novo Nordisk Inc.: Consultancy, Honoraria, Research Funding.

Alpha-Dystroglycan Supports Platelet Aggregation and Thrombus Formation

No relevant conflicts of interest to declare.

Coactosin-like 1 integrates signaling critical for shear-dependent thrombus formation in mouse platelets.

Platelet aggregate formation is a multistep process involving receptor-mediated, as well as biomechanical, signaling cascades, which are highly dependent on actin dynamics. We have previously shown that actin depolymerizing factor (ADF)/n-cofilin and Twinfilin 2a, members of the ADF homology (ADF-H) protein family, have distinct roles in platelet formation and function. Coactosin-like 1 (Cotl1) is another ADF-H protein that binds actin and was also shown to enhance biosynthesis of pro-inflammatory leukotrienes (LT) in granulocytes. Here, we generated mice lacking Cotl1 in the megakaryocyte lineage (Cotl1−/−) to investigate its role in platelet production and function. Absence of Cotl1 had no impact on platelet counts, platelet activation or cytoskeletal reorganization under static conditions in vitro. In contrast, Cotl1 deficiency markedly affected platelet aggregate formation on collagen and adhesion to immobilized von Willebrand factor at high shear rates in vitro, pointing to an impaired function of the platelet mechanoreceptor glycoprotein (GP) Ib. Furthermore, Cotl1−/−platelets exhibited increased deformability at high shear rates, indicating that the GPIb defect may be linked to altered biomechanical properties of the deficient cells. In addition, we found that Cotl1 deficiency markedly affected platelet LT biosynthesis. Strikingly, exogenous LT addition restored defective aggregate formation of Cotl1−/− platelets at high shear in vitro, indicating a critical role of platelet-derived LT in thrombus formation. In vivo, Cotl1 deficiency translated into prolonged tail bleeding times and protection from occlusive arterial thrombus formation. Together, our results show that Cotl1 in platelets is an integrator of biomechanical and LT signaling in hemostasis and thrombosis.

4323Detection of early inflammation in myocarditis by molecular magnetic resonance imaging of activated platelets

Abstract Introduction A noninvasive imaging strategy for diagnosis and localization of early myocarditis would be of great clinical interest. However, resolution of current imaging techniques is limited. Platelets play an important role in inflammatory processes but the role in myocarditis is unknown. Therefore, the aim of this project was to examine the role of platelets in myocarditis and establish a sensitive non-invasive molecular MRI in-vivo imaging strategy for diagnosis of myocarditis with a contrast agent against activated platelets in mice. Methods Myocarditis was induced by subcutaneous injection of an emulsion of porcine cardiac myosin and complete freud's adjuvant (CFA) in Balb/c mice. Inflammatory activity was targeted with a contrast agent against activated platelets consisting of microparticles of iron oxide (MPIO) conjugated to a single chain antibody directed against ligand-induced binding sites (LIBS) on activated glycoprotein IIb/IIIa (=LIBS-MPIO). In comparison, we applied an unspecific control antibody linked to microparticles of iron oxide (control-MPIO) and injected LIBS-MPIO to mice subjected to incomplete freud's adjuvant (iCFA). All imaging results were correlated to immunohistochemistry findings. Results Histological evaluation showed significantly higher binding of LIBS-MPIOs to platelet enriched, CD41-positive inflamed myocardium two days after induction of myocarditis in comparison to later time points (7d, 14d, 21d) (p<0.05) and control-MPIO (p<0.05) injection. In iCFA injected mice no significant LIBS-MPIO binding was found (p<0.05). In 3D in-vivo MRI we could specifically detect focal signal effects in LIBS-MPIO injected mice 2 days after induction of myocarditis, whereas in control-MPIO injected mice no signal effect was visible. Quantification of the myocardial MRI signal confirmed a signal decrease after LIBS-MPIO injection and significant fewer signals in comparison to control-MPIO injection (p<0.05). As a perspective, we also found CD41 positive areas in histology of human myocarditis specimens. Conclusions Platelets are involved in the inflammation of myocarditis. Molecular MRI with LIBS-MPIO can image them at an early time point. This noninvasive imaging strategy is of clinical interest for both diagnostic and prognostic purposes, and highlights the potential of molecular MRI for characterization of cardiovascular pathologies such as myocardial inflammation.

Chemical constituents from heartwoods of Caesalpinia sappan with antiplatelet aggregation activities

ObjectiveTo clarify the active constituents of the heartwoods of Caesalpinia sappan, a traditional Chinese medicine with the functions of promoting blood circulation (Huoxue in Chinese) and removing blood stasis (Quyu in Chinese). MethodsThe chemical constituents were isolated and purified by combination of silica gel and Sephadex LH-20 column chromatography, along with semipreparative HPLC. Their chemical structures were established by multiple spectroscopic methods and comparison with literature data. The in vitro antiplatelet aggregation activities were evaluated using mouse platelet induced by AYPGKF-NH2, a gold agonist of protease-activated receptor 4 (PAR4). ResultsTwo new phenols, methyl 2-(4,4′,5′-trihydroxy-2′-(methoxymethyl) biphenyl-2-yloxy) acetate (1) and 1′-methylcaesalpin J (2), together with 24 known compounds (3–26), were isolated from the heartwoods of C. sappan. Among them, sappanchalcone (16) and brazilin (20) showed inhibitory activities against mouse platelet aggregation with IC50 values of 114.8 µmol/L and 100.8 µmol/L, respectively. ConclusionAntiplatelet compounds from C. sappan targeting at PAR4 are reported for the first time.