Volume electron microscopy reveals heterogeneity of the hemostatic response in veins and arteries

Blood Advances · 2026-02-12 · 1 citations

ABSTRACT: Intravital imaging studies have provided insights into the spatial and temporal variations of platelet activation and thrombin generation that occur during hemostasis; however, these studies are generally limited to small vessels due to the practical limitations of imaging in thicker tissues. Recent advances in cleared tissue fluorescence imaging as well as volume electron microscopy (vEM) coupled with machine learning-based image segmentation provide an opportunity for analysis of the 3-dimensional structure of complex tissues. We utilized these technologies to examine hemostatic plugs from murine jugular veins and carotid arteries to investigate the spatial distribution of platelet activation and biochemical responses in these disparate physiologic contexts. Both venous and arterial hemostatic plugs had a heterogeneous structure with regions of sparsely and densely packed platelets. Despite similar injury sizes, arterial hemostatic plugs were at least an order of magnitude larger than venous plugs. The difference in plug size was primarily due to a 19-fold increase in the population of densely packed platelets in the extravascular compartment. Venous plugs displayed significant platelet aggregation extending into the vessel lumen and developed distinctive fibrin and red blood cell-filled cavities. Complementary fluorescence microscopy revealed that platelet activation was spatially heterogeneous in both contexts, with α-granule secretion and phosphatidylserine exposure confined to specific microenvironments, highlighting tightly regulated thrombin activity. Overall, our findings reveal both conserved and distinct mechanisms of hemostatic thrombus formation in different physiologic contexts. They also demonstrate the power of vEM coupled with machine learning-based image segmentation for the quantitative analysis of large imaging data sets from complex tissues.

Understanding how a highly prevalent <i>GRK5</i> polymorphism affects platelets and enhances thrombotic risk

Blood · 2026-01-20 · 4 citations

ABSTRACT: Inherited genetic variants that modulate platelet function contribute significantly to thrombotic disorders, yet their mechanisms and clinical implications remain underexplored. Two genome-wide association studies identified an A→G variant (rs10886430) in the first intron of G protein-coupled receptor kinase 5 (GRK5), found in homozygosity in ∼5 million Americans. The homozygous GRK5 GG genotype is associated with an increased risk of stroke and venous thromboembolism, but the mechanistic link between this variant and thrombotic risk has remained unclear. To investigate this, we identified 3 GG individuals. GRK5 protein levels in GG platelets were 90% lower than in AA controls. The significant reduction in GRK5 levels in GG platelets led to elevated platelet responsiveness to thrombin and a protease-activated receptor 1 (PAR1) agonist but not a PAR4 agonist. These findings were corroborated in GRK5-/- induced pluripotent stem cell-derived megakaryocytes, transgenic Grk5-deficient murine platelets, and AA platelets exposed to a GRK5 inhibitor. We demonstrated that PAR1 internalization was reduced in GG platelets, leading to enhanced PAR1 signaling. Under venous shear in an endothelialized microfluidic system, GG platelets exhibited increased accumulation, which was reversed by PAR1 inhibition with vorapaxar. In an arterial murine thrombosis model following human platelet infusion, GG platelets also showed enhanced thrombus formation in vivo. This study provides, to our knowledge, the first experimental evidence directly linking a highly prevalent human GRK5 variant to defective PAR1 regulation and increased thrombotic risk. Together, these findings establish that the GRK5 GG genotype confers increased thrombotic potential through impaired PAR1 desensitization, providing mechanistic insight that connects human genetics, thrombin receptor signaling, and thrombotic disease.

Phosphatidylserine-rich platelet blebs enhance hemostasis after mucous membrane injury but not after skin or vascular injury: Studies with ANO6-deficient mice

Blood · 2025-11-03

Abstract Platelets expose high levels of phosphatidylserine (PS) on large membrane blebs in response to strong agonists. These annexin A5-positive platelets are termed “procoagulant platelets.” Formation of PS-rich platelet blebs is impaired in people and mice deficient in functional TMEM16F/ANO6, a seven transmembrane domain protein that acts as a channel for PS and other phospholipids. Deficiency of ANO6 is the molecular cause of Scott Syndrome, a disorder characterized by mucous membrane bleeding and decreased platelet-supported thrombin generation but normal bleeding time. We wished to further probe the impact of PS-rich platelet blebs on hemostasis utilizing an ANO6 deficient mouse model. ANO6 heterozygous mice were bred on a pure C57Bl/6J genetic background with a gene trap between exons one and two (ANO6-/+). ANO6-/+ intercross produced no viable null pups at weaning. Timed matings demonstrated the expected mendelian ratio of ANO6-/- mice at E18.5 indicating perinatal lethality. This was partially rescued by outcrossing mice onto 129/SvJ mice. ANO6-/- mice on a 50/50 mixed genetic background (C57BL/6J and 129/SvJ) exhibited decreased PS exposure on platelets in response to TRAP4, thrombin (IIa), convulxin (CVX), TRAP4 + CVX, and A23187, and prolonged tail bleeding times compared to WT littermate controls. These data are consistent with published data for mice on mixed genetic background. However, after mice were backcrossed more than 4 generations into the C57BL/6 background the phenotype changed. To preserve the rescue genes, ANO6-/- mice were backcrossed with C57BL6 to produce the next ANO6+/- generation. The resulting ANO6+/- mice were then crossbred to produce the next generation of ANO6-/- mice that could be further backcrossed to C57BL6. This alternating mating pattern was repeated to produce ANO6-/- mice on the C57Bl6 genetic background with the rescue genes. Subsequent experiments on these mice, demonstrated no difference in PS exposure on resting platelets or in response to IIa. PS exposure was impaired on the “high PS” fraction of platelets following IIa + CVX or A23187 stimulation. The fraction of platelets in the high PS gate was not different but the quantity of PS exposed was 50-75% lower. In a novel 3-stage assay, where platelets support factor Xa generation followed by IIa generation, we found approximately 4x more IIa generated by +/+ platelets vs. -/- platelets with platelet stimulation by TRAP4 + CVX. ANO6-/- mice exhibited normal tail bleeding times using 3 different techniques. Likewise, bleeding times and blood loss from jugular vein puncture were comparable to wild-type littermate controls. Similarly, IIa generation and plasma clotting times were the same when supported by -/- mice vs. +/+ mice. These data demonstrate that ANO6 deficiency does not result in an overt bleeding phenotype and suggest strain-specific modifiers of these assays on a mixed background. We hypothesized that the hemostatic deficit of -/- mice would be uncovered when coagulation was triggered by higher concentrations of initiators that overwhelm intrinsic anticoagulation pathways. Therefore, we evaluated plasma clotting with increasing concentrations of factor XIa (intrinsic) or factor Xa (extrinsic pathway). Under these conditions, clotting was faster and +/+ platelets accelerated coagulation more than -/- platelets after TRAP4 + CVX and A23187 stimulation. To evaluate hemostasis when coagulation pathways are plausibly upregulated, we developed a mucous membrane bleeding assay in mice. Gastric ulcers were induced by indomethacin via gastric gavage. Gastric bleeding was evaluated over the next 64 hr using a chemiluminescence assay for heme in mouse feces. ANO6-/- mice lost twice as much blood as littermate controls (p &amp;lt; 0.05). Together these results indicate that the mouse model emulates Scott syndrome with excess mucous membrane bleeding and decreased platelet thrombin generation but normal bleeding time. However, there is no perinatal lethality reported in humans. Our data suggest that formation of PS-rich platelet blebs and the quantity of PS on the blebs is not rate-limiting for ordinary hemostasis. These data suggest the presence of an uncharacterized mechanism of coagulation suppression on PS-rich platelet membranes when the level of coagulation initiation is low or moderate.

Mechanistic insights into how g protein-coupled receptor kinases (GRKs) regulate thrombopoiesis

Blood · 2025-11-03

Abstract Platelet disorders encompass abnormalities in platelet function as well as alterations in platelet counts, including thrombocytopenia and thrombocythemia. Patients with these disorders are at an increased risk of hemorrhage or thrombosis. Megakaryocytes (MKs), the precursors of platelets, reside in the bone marrow and undergo a tightly regulated maturation program known as megakaryopoiesis, accompanied by cytoskeletal changes that drive thrombopoiesis (proplatelet formation). However, the mechanisms regulating megakaryopoiesis and thrombopoiesis remain poorly understood. G protein-coupled receptor kinases (GRKs) are serine/threonine kinases that attenuate signaling from agonist-occupied G protein-coupled receptors (GPCRs) in a phosphorylation-dependent manner. In addition, GRKs also phosphorylate non-GPCR substrates, thereby influencing various cellular processes. We previously demonstrated that GRKs are critical negative regulators of platelet activation and thrombus formation through thrombin and/or ADP receptors. Importantly, GRK5 and GRK6 expression is upregulated during hematopoietic stem cell differentiation into MKs, leading us to hypothesize that GRKs play essential roles in MK development and platelet biogenesis. To test this hypothesis, we generated MK- and platelet-specific double knockouts (DKO) for GRK5 and GRK6 in both human and mouse cells. Compared to Grk5 and Grk6 individual KOs,DKO mice displayed severe thrombocytopenia with platelet counts reduced to ~5% of normal. DKO platelets showed a significantly reduced survival (t1/2=36 hours), clearing faster than either single knockouts or controls (t1/2=48 hours). Furthermore, DKO platelets displayed increased integrin activation and granule secretion in both the resting and activated states, which may contribute to their enhanced clearance. However, splenectomy failed to elevate platelet counts in these DKO mice. Bone marrow analysis of DKO mice revealed abnormally shaped MKs clustered near blood vessels, along with myelofibrosis, osteosclerosis, and increased angiogenesis. Notably, plasma levels of TPO and TGF-β1 were 2-3-fold higher in DKO mice than in WT. Additionally, transmission electron microscopy demonstrated pronounced alterations in the demarcation membrane system in DKO bone marrow MKs. Ex vivo cultured DKO MKs displayed normal size, ploidy, and CD41a+CD42d+ surface expression. Importantly, machine learning-assisted image segmentation revealed that DKO MKs had approximately 64% fewer proplatelet-forming MKs, 60% fewer proplatelet tips per cell, and proplatelet tips that were 40% larger compared to WT MKs. To delineate the molecular mechanisms, we performed proteomics and phosphoproteomics using WT and DKO induced pluripotent stem cell-derived MKs (iMKs) and mouse bone marrow-derived MKs. After normalization, five proteins were consistently dephosphorylated in DKO samples compared to WT samples in both iMKs and mouse MKs. Among these, GPIbβ was dephosphorylated at serine166 compared with WT, confirmed by immunoblot using a site-specific antibody. GPIbβ protein expression was also decreased in DKO iMKs, coinciding with the loss of serine166 phosphorylation, though causality remained to be determined. Finally, endogenous interaction between the GPIb complex and GRK6 was detected in WT iMKs. In conclusion, thrombocytopenia in DKO mice results from both impaired platelet production and enhanced platelet clearance. These findings identify GRK5 and GRK6 as dual regulators of platelet homeostasis, by (1) ensuring efficient thrombopoiesis, and (2) preventing premature platelet clearance by maintaining platelet quiescence and limiting excessive activation. Building on prior reports highlighting the critical role of GPIbβ in thrombopoiesis, this study reveals a potential regulatory pathway linking GRK5/6 to GPIbβ signaling, with implications for understanding the pathogenesis of thrombocytopenia, particularly in Bernard-Soulier syndrome and related platelet disorders.

A stabilized finite element technique for transport phenomena within and around immersed porous bodies in flow

Physics of Fluids · 2025-10-01

Mass transport in and around porous objects immersed in fluid flow is prevalent in a wide range of industrial and biomedical applications. These include medical devices, drug delivery, membrane-based processes, and pathophysiology of various disease scenarios, such as thrombosis. Numerical modeling using techniques, such as finite element method, is an important avenue for quantitative analysis of such transport processes. However, the presence of large discontinuities in concentrations, driven by discontinuous diffusivity and porosity, can lead to spurious numerical oscillations in finite element solutions. Here, we adopt a numerically consistent jump-stabilized finite element formulation, coupled with immersed non-conforming discretizations of the porous domain, to mitigate such spurious oscillatory behavior. We demonstrate that the resulting stabilized numerical method is robust in the pure advection (hyperbolic) and the pure diffusion (parabolic) limits of the transport equation. The stabilization contribution includes a tunable diffusion contribution to the system, ensuring that the solution does not become over-diffused. Subsequently, we present a series of illustrative simulation case studies, to show that the resulting stabilized algorithm can model transport processes in two- and three-dimensional settings, involving high spatial heterogeneity in porosity and highly arbitrary porous domain geometries that can vary non-trivially in space and time.

From imaging to computational domains for physics-driven molecular biology simulations: Hindered diffusion in platelet masses

PLoS Computational Biology · 2025-07-07

When formed in vivo, murine hemostatic thrombi exhibit a heterogeneous architecture comprised of distinct regions of densely and sparsely packed platelets. In this study, we utilize high-resolution electron microscopy alongside machine learning and physics-based simulations to investigate how such clot microstructure impacts molecular diffusivity. We used Serial Block Face - Scanning Electron Microscopy (SBF-SEM) to image select volumes of hemostatic masses formed in a mouse jugular vein, producing high-resolution 2D images. Images were segmented using machine learning software (Cellpose), whose training was augmented by manually segmented images. The segmented images were then utilized as 2D computational domains for Lattice Kinetic Monte-Carlo (LKMC) simulations. This process constitutes a computational pipeline that combines purely data-derived biological domains with physics-driven simulations to estimate how molecular movement is hindered in a hemostatic platelet mass. Using our pipeline, we estimated that the 2D hindered diffusion rates of a globular protein range from 2% to 40% of the unhindered rate, with denser packing regions lending to lower molecular diffusivity. These data suggest that coagulation reactions rates, thrombin generation and activity, as well as platelet releasate activity may be drastically impacted by the internal geometry of a hemostatic thrombus.

From imaging to computational domains for physics-driven molecular biology simulations: Hindered diffusion in platelet masses

bioRxiv (Cold Spring Harbor Laboratory) · 2025-02-08

Abstract When formed in vivo, murine hemostatic thrombi exhibit a heterogeneous architecture comprised of distinct regions of densely and sparsely packed platelets. In this study, we utilize high-resolution electron microscopy alongside machine learning and physics-based simulations to investigate how such clot microstructure impacts molecular diffusivity. We used Serial Block Face – Scanning Electron Microscopy (SBF-SEM) to image select volumes of hemostatic masses formed in a mouse jugular vein, producing large stacks of high-resolution 2D images. Images were segmented using machine learning software (Cellpose), whose training was augmented by manually segmented images. The segmented images were then utilized as a computational domain for Lattice Kinetic Monte-Carlo (LKMC) simulations. This process constitutes a computational pipeline that combines purely data-derived biological domains with physics-driven simulations to estimate how molecular movement is hindered in a hemostatic platelet mass. Using our pipeline, we estimated that the hindered diffusion rates of a globular protein range from 2% to 40% of the unhindered rate, with denser packing regions lending to lower molecular diffusivity. These data suggest that coagulation reactions rates, thrombin generation and activity, as well as platelet releasate activity may be drastically impacted by the internal geometry of a hemostatic thrombus. Author Summary Hemostasis and coagulation are two exquisitely complex, intertwined, and tightly regulated biological processes. Dysregulation of either process may lead to severe health consequences or death. Coagulation reactions have been extensively studied under static laboratory conditions, which are different from in vivo conditions. It is therefore imperative to understand if and how the chemical reactions underlying coagulation are regulated by the environment where they occur. In vivo experimentation enables us to image hemostasis, but not chemical reactions. Physics-driven molecular simulations of chemical reactions can bridge the gap, provided the physical environment is correctly represented computationally. The present work serves as a much-needed foundation for image-to-computation for physics based molecular simulations in biological domains.

HTRS2025.O6A.6 Reduced Thrombin-Mediated Platelet Activation is a Major Contributor to Impaired Hemostasis in Settings of Coagulation Deficiency

Research and Practice in Thrombosis and Haemostasis · 2025-11-01

Background: Hemostasis requires the dynamic integration of platelet activation and thrombin generation. However, the specific roles of various hemostatic system components in time and space remain to be elucidated. While traditionally considered an amplification mechanism, we hypothesized that the intrinsic tenase is critical for the proper spatial localization of thrombin, and is necessary to maintain the hemostatic response in part via spatially regulated PAR-mediated platelet activation. We further hypothesized that increasing platelet sensitivity to thrombin would ameliorate excess bleeding in contexts of coagulation deficiency.

Platelet Interactions with the Vessel Wall: The Kinetics of Hemostatic Plug Formation

2025-01-01

Plasma growth factors maintain constitutive translation in platelets to regulate reactivity and thrombotic potential

Blood Advances · 2024-01-01 · 18 citations

ABSTRACT: Mechanisms of proteostasis in anucleate circulating platelets are unknown and may regulate platelet function. We investigated the hypothesis that plasma-borne growth factors/hormones (GFHs) maintain constitutive translation in circulating platelets to facilitate reactivity. Bio-orthogonal noncanonical amino acid tagging (BONCAT) coupled with liquid chromatography-tandem mass spectrometry analysis revealed constitutive translation of a broad-spectrum translatome in human platelets dependent upon plasma or GFH exposure, and in murine circulation. Freshly isolated platelets from plasma showed homeostatic activation of translation-initiation signaling pathways: phosphorylation of p38/ERK upstream kinases, essential intermediate MNK1/2, and effectors eIF4E/4E-BP1. Plasma starvation led to loss of pathway phosphorylation, but it was fully restored with 5-minute stimulation by plasma or GFHs. Cycloheximide or puromycin infusion suppressed ex vivo platelet GpIIb/IIIa activation and P-selectin exposure with low thrombin concentrations and low-to-saturating concentrations of adenosine 5'-diphosphate (ADP) or thromboxane analog but not convulxin. ADP-induced thromboxane generation was blunted by translation inhibition, and secondary-wave aggregation was inhibited in a thromboxane-dependent manner. Intravenously administered puromycin reduced injury-induced clot size in cremaster muscle arterioles, and delayed primary hemostasis after tail tip amputation but did not delay neither final hemostasis after subsequent rebleeds, nor final hemostasis after jugular vein puncture. In contrast, these mice were protected from injury-induced arterial thrombosis and thrombin-induced pulmonary thromboembolism (PE), and adoptive transfer of translation-inhibited platelets into untreated mice inhibited arterial thrombosis and PE. Thus, constitutive plasma GFH-driven translation regulates platelet G protein-coupled receptor reactivity to balance hemostasis and thrombotic potential.