Individual phosphatidylinositol transfer proteins have distinct functions that do not involve lipid transfer activity.

Platelets use signal transduction pathways facilitated by class I phosphatidylinositol transfer proteins (PITPs). The 2 mammalian class I PITPs, PITPα and PITPß, are single PITP domain soluble proteins that are encoded by different genes and share 77% sequence identity, although their individual roles in mammalian biology remain uncharacterized. These proteins are believed to shuttle phosphatidylinositol and phosphatidylcholine between separate intracellular membrane compartments, thereby regulating phosphoinositide synthesis and second messenger formation. Previously, we observed that platelet-specific deletion of PITPα, the predominantly expressed murine PITP isoform, had no effect on hemostasis but impaired tumor metastasis formation and disrupted phosphoinositide signaling. Here, we found that mice lacking the less expressed PITPß in their platelets exhibited a similar phenotype. However, in contrast to PITPα-null platelet lysates, which have impaired lipid transfer activity, PITPß-null platelet lysates have essentially normal lipid transfer activity, although both isoforms contribute to phosphoinositide synthesis in vitro. Moreover, we found that platelet-specific deletion of both PITPs led to ex vivo platelet aggregation/secretion and spreading defects, impaired tail bleeding, and profound tumor dissemination. Our study also demonstrated that PITP isoforms are required to maintain endogenous phosphoinositide PtdInsP2 levels and agonist-stimulated second messenger formation. The data shown here demonstrate that the 2 isoforms are functionally overlapping and that a single isoform is able to maintain the homeostasis of platelets. However, both class I PITP isoforms contribute to phosphoinositide signaling in platelets through distinct biochemical mechanisms or different subcellular domains.

Signaling Through FcγRIIA and the C5a-C5aR Pathway Mediate Platelet Hyperactivation in COVID-19.

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibited higher basal levels of activation measured by P-selectin surface expression and had poor functional reserve upon in vitro stimulation. To investigate this question in more detail, we developed an assay to assess the capacity of plasma from COVID-19 patients to activate platelets from healthy donors. Platelet activation was a common feature of plasma from COVID-19 patients and correlated with key measures of clinical outcome including kidney and liver injury, and APACHEIII scores. Further, we identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions. These data identified these potentially actionable pathways as central for platelet activation and/or vascular complications and clinical outcomes in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect.

Tmprss6-ASO as a tool for the treatment of Polycythemia Vera mice.

Polycythemia Vera (PV) is a chronic myeloproliferative neoplasm resulting from an acquired driver mutation in the JAK2 gene of hematopoietic stem and progenitor cells resulting in the overproduction of mature erythrocytes and abnormally high hematocrit, in turn leading to thromboembolic complications. Therapeutic phlebotomy is the most common treatment to reduce the hematocrit levels and consequently decrease thromboembolic risk. Here we demonstrate that, by using the iron restrictive properties of the antisense oligonucleotides against Tmprss6 mRNA, we can increase hepcidin to achieve effects equivalent to therapeutic phlebotomy. We provide evidence that this less invasive approach could represent an additional therapeutic tool for the treatment of PV patients.

Signaling through FcγRIIA and the C5a-C5aR pathway mediates platelet hyperactivation in COVID-19.

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibit higher basal levels of activation measured by P-selectin surface expression, and have a poor functional reserve upon in vitro stimulation. Correlating clinical features to the ability of plasma from COVID-19 patients to stimulate control platelets identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions, thus identifying these potentially actionable pathways as central for platelet activation and/or vascular complications in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect. These studies have implications for the role of platelet hyperactivation in complications associated with SARS-CoV-2 infection. ONE-SENTENCE SUMMARY: The FcγRIIA and C5a-C5aR pathways mediate platelet hyperactivation in COVID-19.

PIKfyve Deficiency in Myeloid Cells Impairs Lysosomal Homeostasis in Macrophages and Promotes Systemic Inflammation in Mice.

Macrophages are professional phagocytes that are essential for host defense and tissue homeostasis. Proper membrane trafficking and degradative functions of the endolysosomal system are known to be critical for the function of these cells. We have found that PIKfyve, the kinase that synthesizes the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate, is an essential regulator of lysosomal biogenesis and degradative functions in macrophages. Genetically engineered mice lacking PIKfyve in their myeloid cells (PIKfyvefl/fl LysM-Cre) develop diffuse tissue infiltration of foamy macrophages, hepatosplenomegaly, and systemic inflammation. PIKfyve loss in macrophages causes enlarged endolysosomal compartments and impairs the lysosomal degradative function. Moreover, PIKfyve deficiency increases the cellular levels of lysosomal proteins. Although PIKfyve deficiency reduced the activation of mTORC1 pathway and was associated with increased cleavage of TFEB proteins, this does not translate into transcriptional activation of lysosomal genes, suggesting that PIKfyve modulates the abundance of lysosomal proteins by affecting the degradation of these proteins. Our study shows that PIKfyve modulation of lysosomal degradative activity and protein expression is essential to maintain lysosomal homeostasis in macrophages.

Phosphatidylinositol transfer proteins regulate megakaryocyte TGF-ß1 secretion and hematopoiesis in mice.

We hypothesized that megakaryocyte (MK) phosphoinositide signaling mediated by phosphatidylinositol transfer proteins (PITPs) contributes to hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) regulation. Conditional knockout mice lacking PITPs specifically in MKs and platelets (pitpα-/- and pitpα-/-/ß-/-) bone marrow (BM) manifested decreased numbers of HSCs, MK-erythrocyte progenitors, and cycling HPCs. Further, pitpα-/-/ß-/- BM had significantly reduced engrafting capability in competitive transplantation and limiting dilution analysis. Conditioned media (CM) from cultured pitpα-/- and pitpα-/-/ß-/- BM MKs contained higher levels of transforming growth factor ß1 (TGF-ß1) and interleukin-4 (IL-4), among other myelosuppressive cytokines, than wild-type BM MKs. Correspondingly, BM flush fluid from pitpα-/- and pitpα-/-/ß-/- mice had higher concentrations of TGF-ß1. CM from pitpα-/- and pitpα-/-/ß-/- MKs significantly suppressed HPC colony formation, which was completely extinguished in vitro by neutralizing anti-TGF-ß antibody, and treatment of pitpα-/-/ß-/- mice in vivo with anti-TGF-ß antibodies completely reverted their defects in BM HSC and HPC numbers. TGF-ß and IL-4 synergized to inhibit HPC colony formation in vitro. Electron microscopy analysis of pitpα-/-/ß-/- MKs revealed ultrastructural defects with depleted α-granules and large, misshaped multivesicular bodies. Von Willebrand factor and thrombospondin-1, like TGF-ß, are stored in MK α-granules and were also elevated in CM of cultured pitpα-/-/ß-/- MKs. Altogether, these data show that ablating PITPs in MKs indirectly dysregulates hematopoiesis in the BM by disrupting α-granule physiology and secretion of TGF-ß1.

Phosphatidylinositol transfer protein-α in platelets is inconsequential for thrombosis yet is utilized for tumor metastasis.

Platelets are increasingly recognized for their contributions to tumor metastasis. Here, we show that the phosphoinositide signaling modulated by phosphatidylinositol transfer protein type α (PITPα), a protein which shuttles phosphatidylinositol between organelles, is essential for platelet-mediated tumor metastasis. PITPα-deficient platelets have reduced intracellular pools of phosphoinositides and an 80% reduction in IP3 generation upon platelet activation. Unexpectedly, mice lacking platelet PITPα form thrombi normally at sites of intravascular injuries. However, following intravenous injection of tumor cells, mice lacking PITPα develop fewer lung metastases due to a reduction of fibrin formation surrounding the tumor cells, rendering the metastases susceptible to mucosal immunity. These findings demonstrate that platelet PITPα-mediated phosphoinositide signaling is inconsequential for in vivo hemostasis, yet is critical for in vivo dissemination. Moreover, this demonstrates that signaling pathways within platelets may be segregated into pathways that are essential for thrombosis formation and pathways that are important for non-hemostatic functions.

Loss of PIKfyve in platelets causes a lysosomal disease leading to inflammation and thrombosis in mice.

PIKfyve is essential for the synthesis of phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] and for the regulation of endolysosomal membrane dynamics in mammals. PtdIns(3,5)P2 deficiency causes neurodegeneration in mice and humans, but the role of PtdIns(3,5)P2 in non-neural tissues is poorly understood. Here we show that platelet-specific ablation of PIKfyve in mice leads to accelerated arterial thrombosis, and, unexpectedly, also to inappropriate inflammatory responses characterized by macrophage accumulation in multiple tissues. These multiorgan defects are attenuated by platelet depletion in vivo, confirming that they reflect a platelet-specific process. PIKfyve ablation in platelets induces defective maturation and excessive storage of lysosomal enzymes that are released upon platelet activation. Impairing lysosome secretion from PIKfyve-null platelets in vivo markedly attenuates the multiorgan defects, suggesting that platelet lysosome secretion contributes to pathogenesis. Our findings identify PIKfyve as an essential regulator for platelet lysosome homeostasis, and demonstrate the contributions of platelet lysosomes to inflammation, arterial thrombosis and macrophage biology.

Loss of ATE1-mediated arginylation leads to impaired platelet myosin phosphorylation, clot retraction, and in vivo thrombosis formation.

Protein arginylation by arginyl-transfer RNA protein transferase (ATE1) is emerging as a regulator protein function that is reminiscent of phosphorylation. For example, arginylation of ß-actin has been found to regulate lamellipodial formation at the leading edge in fibroblasts. This finding suggests that similar functions of ß-actin in other cell types may also require arginylation. Here, we have tested the hypothesis that ATE1 regulates the cytoskeletal dynamics essential for in vivo platelet adhesion and thrombus formation. To test this hypothesis, we generated conditional knockout mice specifically lacking ATE1 in their platelets and in their megakaryocytes and analyzed the role of arginylation during platelet activation. Surprisingly, rather than finding an impairment of the actin cytoskeleton structure and its rearrangement during platelet activation, we observed that the platelet-specific ATE1 knockout led to enhanced clot retraction and in vivo thrombus formation. This effect might be regulated by myosin II contractility since it was accompanied by enhanced phosphorylation of the myosin regulatory light chain on Ser19, which is an event that activates myosin in vivo. Furthermore, ATE1 and myosin co-immunoprecipitate from platelet lysates. This finding suggests that these proteins directly interact within platelets. These results provide the first evidence that arginylation is involved in phosphorylation-dependent protein regulation, and that arginylation affects myosin function in platelets during clot retraction.

RhoA is essential for maintaining normal megakaryocyte ploidy and platelet generation.

RhoA plays a multifaceted role in platelet biology. During platelet development, RhoA has been proposed to regulate endomitosis, proplatelet formation, and platelet release, in addition to having a role in platelet activation. These processes were previously studied using pharmacological inhibitors in vitro, which have potential drawbacks, such as non-specific inhibition or incomplete disruption of the intended target proteins. Therefore, we developed a conditional knockout mouse model utilizing the CRE-LOX strategy to ablate RhoA, specifically in megakaryocytes and in platelets to determine its role in platelet development. We demonstrated that deleting RhoA in megakaryocytes in vivo resulted in significant macrothrombocytopenia. RhoA-null megakaryocytes were larger, had higher mean ploidy, and exhibited stiff membranes with micropipette aspiration. However, in contrast to the results observed in experiments relying upon pharmacologic inhibitors, we did not observe any defects in proplatelet formation in megakaryocytes lacking RhoA. Infused RhoA-null megakaryocytes rapidly released platelets, but platelet levels rapidly plummeted within several hours. Our evidence supports the hypothesis that changes in membrane rheology caused infused RhoA-null megakaryocytes to prematurely release aberrant platelets that were unstable. These platelets were cleared quickly from circulation, which led to the macrothrombocytopenia. These observations demonstrate that RhoA is critical for maintaining normal megakaryocyte development and the production of normal platelets.

Platelets lacking PIP5KIγ have normal integrin activation but impaired cytoskeletal-membrane integrity and adhesion.

Three isoforms of phosphatidylinositol-4-phosphate 5-kinase (PIP5KIα, PIP5KIß, and PIP5KIγ) can each catalyze the final step in the synthesis of phosphatidylinositol-4,5-bisphosphate (PIP2), which in turn can be either converted to second messengers or bind directly to and thereby regulate proteins such as talin. A widely quoted model speculates that only p90, a longer splice form of platelet-specific PIP5KIγ, but not the shorter p87 PIP5KIγ, regulates the ligand-binding activity of integrins via talin. However, when we used mice genetically engineered to lack only p90 PIP5KIγ, we found that p90 PIP5KIγ is not critical for integrin activation or platelet adhesion on collagen. However, p90 PIP5KIγ-null platelets do have impaired anchoring of their integrins to the underlying cytoskeleton. Platelets lacking both the p90 and p87 PIP5KIγ isoforms had normal integrin activation and actin dynamics, but impaired anchoring of their integrins to the cytoskeleton. Most importantly, they formed weak shear-resistant adhesions ex vivo and unstable vascular occlusions in vivo. Together, our studies demonstrate that, although PIP5KIγ is essential for normal platelet function, individual isoforms of PIP5KIγ fulfill unique roles for the integrin-dependent integrity of the membrane cytoskeleton and for the stabilization of platelet adhesion.

Structural and signaling requirements of the human melanocortin 4 receptor for MAP kinase activation.

In addition to its well known stimulation of cAMP production, the human melanocortin type 4 (hMC4) receptor recently has been shown to mediate p44/42 MAPK activation. This finding opens new questions about the structural and signaling mechanisms that connect the receptor to this alternate cell signaling pathway. Point mutants in the hMC4 receptor that have been associated with obesity were constructed and transfected into HEK 293 cells. Functional analyses then were done to determine if these mutations would similarly impact cAMP formation and p44/42 MAPK signaling. Whereas a D90N mutation in the second transmembrane domain and a D298A mutation in the seventh transmembrane domain impaired both cAMP formation and p44/42 MAPK activation, a more conservative D298N mutation retained cAMP formation but abolished p44/42 MAPK activation. The D298N mutation identified, for the first time, differential structural requirements of the hMC4 receptor for activation of the cAMP and p44/42 MAPK pathways. Furthermore, functional characterizations of a series of chimeric receptors combining the hMC4 receptor and the hMC3 subtype, a receptor that does not couple to p44/42 MAPK activation despite stimulating adenylyl cyclase, indicate that the hMC4 cytoplasmic tail is a necessary structural element for p44/42 MAPK signaling. Subsequent investigation of the signaling requirements for p44/42 MAPK activation demonstrated that the adenylyl cyclase inhibitor 2', 5'-dideoxyadenosine blocked agonist-induced p44/42 MAPK activation, but the PKA inhibitor Rp cAMPS did not. Taken together, these data indicate that cAMP is required, but not sufficient for p44/42 MAPK activation and suggest structural elements required for hMC4 receptor signaling.

Identification of structural determinants for G protein-independent activation of mitogen-activated protein kinases in the seventh transmembrane domain of the angiotensin II type 1 receptor.

Although the intrareceptor mechanisms whereby the angiotensin II (AngII) type 1 receptor activates phospholipase C (PLC) have been extensively investigated, analogous studies of signaling through mitogen-activated protein kinases (MAPK) have been lacking. We investigated MAPK activation and traditional G(q)/PLC signaling in transfected cells using AngII and the signaling selective agonist [Sar(1),Ile(4),Ile(8)] AngII (SII). SII stimulated MAPK without inositol trisphosphate (IP(3)) production and thereby stabilizes an activated receptor state linked to G protein-independent MAPK signaling. Using receptor mutagenesis, we focused on the seventh transmembrane domain and identified three key residues-Tyr(292), Phe(293), and Thr(287). At least three distinct activated states were revealed: 1) an AngII-stabilized state linked to G(q)/PLC signaling, 2) an AngII-stabilized state connected to G protein-independent MAPK activation, and 3) a SII-stabilized state associated with G protein-independent MAPK signaling. The mutant Y292F failed to exhibit AngII-induced IP(3) turnover yet remained capable of AngII-induced MAPK activation. SII failed to stimulate MAPK in Y292F-transfected cells. Thus, Tyr(292) is a key epitope for activated states 1 and 3 but not required for activated state 2. Although the F293L mutant retained normal AngII responses, it also showed an IP(3) response to SII, indicating that Phe(293) may be involved in constraining the receptor to its inactive state. Mutations of Thr(287) abolished all SII-induced signaling without affecting any AngII responses. Thr(287) therefore represents a key residue for a SII-stabilized activated state. Taken together, the data identified a novel structural requirement (Thr(287)) for the SII-stabilized activated state and redefined the mechanistic roles for Tyr(292) and Phe(293).

Rattus norvegicus melanocortin 3 receptor: a corrected sequence.

Examination of the Rattus norvegicus genome reveals differences in the melanocortin 3 receptor (MC3R) compared with the published sequence (accession X70667). To clarify these differences, we used RT-PCR to clone MC3R from Sprague Dawley rats. These efforts revealed a sequence for the rat MC3R consistent with that predicted by the rat genome, but different from the published receptor by three amino acids, all of which were located in the predicted second transmembrane domain (TM2). Analysis of these residues revealed that TM2 of the rat MC3R is more homologous with other species than previously considered. The presently described sequence maps onto chromosome 3 of the rat genome, which shows highly conserved synteny with the mouse chromosome 2 and the human chromosome 20. Transient expression revealed high affinity binding of [125I]-NDP-MSH and a concentration-dependent cAMP response to the synthetic agonist MTII. These data both clarify the sequence of the MC3R and demonstrate the great utility of genomic information recently made available.

Temporal encoding for auditory computation: physiology of primary afferent neurons in sound-producing fish.

Many fish rely on sounds for communication, yet the peripheral structures containing the hair cells are simple, without the morphological specializations that facilitate frequency analysis in the mammalian cochlea. Despite this, neurons in the midbrain of sound-producing fish (Pollimyrus) have complex receptive fields, extracting features from courtship sounds. Here we present an analysis of the initial encoding of sounds by the primary afferents and demonstrate that the representation of sound undergoes a substantial transformation as it ascends to the midbrain. Afferents were isolated as they coursed from the sacculus through the medulla. Tones (100 Hz-1.2 kHz) elicited synchronized spikes [vector strength (VS) >0.9] on each stimulus cycle [coefficient of variation (CV) <1.1], with little spike rate adaptation. Most afferents (67%) were spontaneously active and began synchronizing 10 dB below rate threshold. Rate thresholds for the most sensitive afferents (65 dB) were close to behavioral thresholds. The distribution of characteristic frequencies and best sensitivities was matched to the spectrum of sounds of this species and to its audiogram. Three clusters of afferents were identified, one including afferents that generated spike bursts and had v-shaped response areas (bursters), and two others that included entrained afferents with broad response areas (entrained types I and II). All afferents encoded the timing of clicks within click trains with time-locked spikes, and none showed selectivity for interclick intervals. Understanding the computations that yield complex receptive fields is an essential goal for auditory neuroscience, and these data on primary encoding advance this goal, allowing a comparison of inputs with feature-extracting midbrain neurons.