Biology subjects

Whiteheart, S. W.

Publications and source records attributed to Whiteheart, S. W..

7 recordsLinked to original sources

bioRxiv2025

VAMP8 Deficiency Attenuates AngII-Induced Abdominal Aortic Aneurysm Formation via Platelet Reprogramming and Enhanced Extracellular Matrix Stability

BACKGROUNDAs vascular sentries, platelets, and their ability to release a host of bioactive molecules, are critical for vascular homeostasis as well as hemostasis. Despite data linking platelet activation to abdominal aortic aneurysms (AAA) and rupture, the underlying mechanisms remain poorly understood. This study addresses the hypothesis that VAMP8, the primary v-SNARE controlling platelet exocytosis, contributes to AAA formation. METHODS AND RESULTSIn an AngII-infused hypercholesterolemic mouse model, we observed significant platelet consumption, indicated by decreased platelet counts at both acute (5-day) and chronic (28-day) time points. Platelets accumulated at sites of elastin degradation and within false lumens of the abdominal aorta after 28 days of AngII infusion. Bulk RNA sequencing analysis of washed platelets and their releasates after 5 days of AngII infusion revealed significant transcriptomic changes, suggesting rapid reprogramming of platelet function. Parallel RNA-seq analysis of suprarenal aortic tissue highlighted changes in genes associated with extracellular matrix (ECM) organization, inflammation, and platelet signaling, linking platelets to vascular remodeling suggesting a "platelet-aorta axis". Laser speckle imaging in a FeCl injury model confirmed that VAMP8 deficiency impaired platelet function, resulting in delayed thrombosis. In vivo experiments demonstrated that VAMP8-/- mice were protected against AngII-induced AAA and aortic rupture. Aortic diameter analysis further revealed that VAMP8 deficiency significantly attenuated AngII-driven aortic pathology. RNA-seq analysis of platelets and aortic tissue suggests that loss of VAMP8 affects expression of genes controlling ECM degradation and aortic wall stability consistent with the protective effect of VAMP8 loss on AAA. CONCLUSIONShort-term AngII infusion appears to reprogram the platelet transcriptome, which may affect the aorta and contribute to AAA formation. Controlling cargo release from platelets via VAMP8 deficiency results in profound attenuation of aortic aneurysms. This introduces a novel paradigm for understanding the impact of reprogrammed platelet cargo secretion and function in aortopathies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/635525v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@22bcecorg.highwire.dtl.DTLVardef@a15207org.highwire.dtl.DTLVardef@1106fd8org.highwire.dtl.DTLVardef@1ee0ea_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPlatelet transcriptome is altered at early aneurysmal stage. C_LIO_LIVAMP8 deficiency attenuates aortic aneurysms, potentially via enhanced ECM stability. C_LIO_LIVAMP8 deficiency significantly alters various genes contributing to aortic wall structure and stability in both platelets and suprarenal aortic tissue. C_LI

physiology↗

bioRxiv2025

Platelet endocytosis and α-granule cargo packaging are essential for normal skin wound healing

The high prevalence of chronic wounds, i.e., 2.5-3% of the US population, causes a large social and financial burden. Physiological wound healing is a multi-step process that involves different cell types and growth factors. Platelet-rich plasma or platelet-derived factors have been used to accelerate wound repair, but their use has been controversial with mixed results. Thus, a detailed functional understanding of platelet functions in wound healing beyond hemostasis is needed. This study investigated the importance of platelet -granule cargo packaging and endocytosis in a dorsal full-thickness excisional skin wound model using mice with defects in -granule cargo packaging (Nbeal2-/- mice) and endocytosis (platelet-specific Arf6-/- and VAMP2/3{Delta} mice). We found that proper kinetic and morphological healing of dorsal skin wounds in mice requires both de novo as well as endocytosed platelet -granule cargo. Histological and morphometric analyses of cross-sectional wound sections illustrated that mice with defects in -granule cargo packaging or platelet endocytosis had delayed (epi)dermal regeneration in both earlier and advanced healing. This was reflected by reductions in wound collagen and muscle/keratin content, delayed scab formation and/or resolution, re-epithelialization, and cell migration and proliferation. Molecular profiling analysis of wound extracts showed that the impact of platelet function extends beyond hemostasis to the inflammation, proliferation, and tissue remodeling phases via altered expression of several bioactive molecules, including IL-1{beta}, VEGF, MMP-9, and TIMP-1. These findings provide a basis for advances in clinical wound care through a better understanding of key mechanistic processes and cellular interactions in (patho)physiological wound healing. Key pointsO_LIDe novo and endocytosed platelet -granule cargo support physiological skin wound healing C_LIO_LIPlatelet function in wound healing extends to the inflammation, proliferation, and tissue remodeling phases C_LI

cell biology↗

bioRxiv2024

Inhibition of RhoA-mediated secretory autophagy in megakaryocytes mitigates myelofibrosis in mice

Megakaryocytes (MKs) are large, polyploid cells that contribute to bone marrow homeostasis through the secretion of cytokines such as transforming growth factor {beta}1 (TGF{beta}1). During neoplastic transformation, immature MKs accumulate in the bone marrow where they induce fibrotic remodeling ultimately resulting in myelofibrosis. Current treatment strategies aim to prevent MK hyperproliferation, however, little is understood about the potential of targeting dysregulated cytokine secretion from neoplastic MKs as a novel therapeutic avenue. Unconventional secretion of TGF{beta}1 as well as interleukin 1{beta} (IL1{beta}) via secretory autophagy occurs in cells other than MKs, which prompted us to investigate whether similar mechanisms are utilized by MKs. Here, we identified that TGF{beta}1 strongly co-localized with the autophagy marker light chain 3B in native MKs. Disrupting secretory autophagy by inhibiting the small GTPase RhoA or its downstream effector Rho kinase (ROCK) markedly reduced TGF{beta}1 and IL1{beta} secretion in vitro. In vivo, conditional deletion of the essential autophagy gene Atg5 from the hematopoietic system limited megakaryocytosis and aberrant cytokine secretion in an MPLW515L-driven transplant model. Similarly, mice with a selective deletion of Rhoa from the MK and platelet lineage were protected from progressive fibrosis. Finally, disease hallmarks in MPLW515L-transplanted mice were attenuated upon treatment with the autophagy inhibitor hydroxychloroquine or the ROCK inhibitor Y27632, either as monotherapy or in combination with the JAK2 inhibitor ruxolitinib. Overall, our data indicate that aberrant cytokine secretion is dependent on secretory autophagy downstream of RhoA, targeting of which represents a novel therapeutic avenue in the treatment of myelofibrosis. One Sentence SummaryTGF{beta}1 is released from megakaryocytes via RhoA-mediated secretory autophagy, and targeting this process can alleviate fibrosis progression in a preclinical mouse model of myelofibrosis.

cell biology↗

bioRxiv2024

Single-Platelet Mapping of Jugular, Puncture-Wound Thrombi Reveals the Spatial Evolution of Platelet Activation

BackgroundThe contributions of platelet activation to thrombus formation during hemostatic bleeding cessation likely involve multiple activation states. However, the spatial and temporal distribution of platelets in these states has not been defined in a clot. ObjectivesTo use single-platelet mapping of activation states within jugular vein puncture thrombi to determine how the spatial distribution of platelet state evolves during hemostasis. MethodsMontaged, wide-area electron micrographs (EM) were taken at various time points, post-puncture, and annotated for platelet activation state. These classifications were mapped onto the images to identify regions of platelet activation and calculate neighbor associations. The importance of -granule secretion was tested using VAMP8-/- mice. Resultsmapping of platelet activation states at 1 min post-puncture showed extensive spatial intermixing of most platelet activation classes. No high-activation-state, platelet-rich core was observed in 5-min post-puncture thrombi, rather such platelets tended to be localized on the interior surfaces of thrombus vaults open to the circulation. Only at a later stage, 20 min post-puncture, was distinct clustering of high activation, degranulated, cytosol-rich platelets observed. These clusters localized to the central portion of the intravascular platelet-rich crown, and they were now inaccessible to the circulation. Counterintuitively, deletion of the primary platelet v-SNARE, VAMP8, increased the frequency and spatial clustering of highly activated, degranulated platelets in association with intra-thrombus vault surfaces at 5 min post puncture. ConclusionsWe conclude recent multi-activation state models can provide a realistic thrombus formation framework if linked together to encompass the dynamics of puncture wound formation.

systems biology↗

bioRxiv2024

Unfolded Von Willebrand Factor Binds Protein S and Reduces Anticoagulant Activity

Protein S (PS), the critical plasma cofactor for the anticoagulants tissue factor (TF) pathway inhibitor (TFPI) and activated protein C (APC), circulates in two functionally distinct pools: free (anticoagulant) or bound to complement component 4b-binding protein (C4BP) (anti-inflammatory). Acquired free PS deficiency is detected in several viral infections, but its cause is unclear. Here, we identified a shear-dependent interaction between PS and von Willebrand Factor (VWF) by mass spectrometry. Consistently, plasma PS and VWF comigrated in both native and agarose gel electrophoresis. The PS/VWF interaction was blocked by TFPI but not APC, suggesting an interaction with the C-terminal sex hormone binding globulin (SHBG) region of PS. Microfluidic systems, mimicking arterial laminar flow or disrupted turbulent flow, demonstrated that PS stably binds VWF as VWF unfolds under turbulent flow. PS/VWF complexes also localized to platelet thrombi under laminar arterial flow. In thrombin generation-based assays, shearing plasma decreased PS activity, an effect not seen in the absence of VWF. Finally, free PS deficiency in COVID-19 patients, measured using an antibody that binds near the C4BP binding site in SHBG, correlated with changes in VWF, but not C4BP, and with thrombin generation. Our data suggest that PS binds to a shear-exposed site on VWF, thus sequestering free PS and decreasing its anticoagulant activity, which would account for the increased thrombin generation potential. As many viral infections present with free PS deficiency, elevated circulating VWF, and increased vascular shear, we propose that the PS/VWF interaction reported here is a likely contributor to virus-associated thrombotic risk. Key PointsO_LIVon Willebrand Factor (VWF) binds Protein S (PS) in a shear-dependent manner, reducing the free PS pool and its anticoagulant activity. C_LIO_LIThe PS/VWF complex forms under turbulent flow conditions, is stable in whole blood, and localizes to growing platelet thrombi. C_LI

biochemistry↗

bioRxiv2023

Platelet-Localized ST6Gal1 Does Not Impact IgG Sialylation

The IgG antibody class forms an important basis of the humoral immune response, conferring reciprocal protection from both pathogens and autoimmunity. IgG function is determined by the IgG subclass, as defined by the heavy chain, as well as the glycan composition at N297, the conserved site of N-glycosylation within the Fc domain. For example, lack of core fucose promotes increased antibody-dependent cellular cytotoxicity, whereas 2,6-linked sialylation by the enzyme ST6Gal1 helps to drive immune quiescence. Despite the immunological significance of these carbohydrates, little is known about how IgG glycan composition is regulated. We previously reported that mice with ST6Gal1-deficient B cells have unaltered IgG sialylation. Likewise, ST6Gal1 released into the plasma by hepatocytes does not significantly impact overall IgG sialylation. Since IgG and ST6Gal1 have independently been shown to exist in platelet granules, it was possible that platelet granules could serve as a B cell-extrinsic site for IgG sialylation. To address this hypothesis, we used a platelet factor 4 (Pf4)-Cre mouse to delete ST6Gal1 in megakaryocytes and platelets alone or in combination with an albumin-Cre mouse to also remove it from hepatocytes and the plasma. The resulting mouse strains were viable and had no overt pathological phenotype. We also found that despite targeted ablation of ST6Gal1, no change in IgG sialylation was apparent. Together with our prior findings, we can conclude that in mice, neither B cells, the plasma, nor platelets have a substantial role in homeostatic IgG sialylation.

immunology↗

bioRxiv2020

Calcium ion chelation preserves platelet function during cold storage

ObjectivePlatelet transfusion is a life-saving therapy to prevent or treat bleeding in patients with thrombocytopenia or platelet dysfunction. However, for more than six decades, safe and effective strategies for platelet storage have been an impediment to widespread use of platelet transfusion. Refrigerated platelets are cleared rapidly from circulation, precluding cold storage of platelets for transfusion. Consequently, platelets are stored at room temperature (RT) with an upper limit of 5 days due to risks of bacterial contamination and loss of platelet function. This practice severely limits platelet availability for transfusion. This study is to identify the mechanism of platelet clearance after cold storage and develop a method for platelet cold storage. Approach and ResultsWe found that rapid clearance of cold-stored platelets was largely due to integrin activation and apoptosis. Deficiency of integrin {beta}3 or caspase-3 prolonged cold-stored platelets in circulation. Pre-treatment of platelets with EGTA, a cell impermeable calcium ion chelator, reversely inhibited cold storage-induced platelet activation and consequently prolonged circulation of cold-stored platelets. Moreover, transfusion of EGTA-treated, cold-stored platelets, but not RT-stored platelets, into the mice deficient in glycoprotein Ib significantly shortened tail-bleeding times and diminished blood loss. ConclusionIntegrin activation and apoptosis is the underlying mechanism of rapid clearance of platelets after cold storage. Addition of a cell impermeable calcium ion chelator to platelet products is potentially a simple and effective method to enable cold storage of platelets for transfusion.

cell biology↗