Platelet activation is energy-dependent and associated with a hyperglycolytic phenotype, yet the role of key glycolytic enzymes remains unclear. We investigated the role of platelet hexokinases (HK1 and HK2) in regulating thrombin- and convulxin-stimulated bioenergetics, functions, and hemostasis using pharmacological inhibitors 2-deoxyglucose (2DG; pan-HK) and 3-bromopyruvate (3BP; HK2-preferential). Platelet activation increased HK activity, with convulxin producing a stronger response than thrombin. Using 2DG and 3BP, we found that activation-induced glucose uptake, glycolytic ATP production, and glycolysis were dependent on both HK isoforms. Platelet aggregation was reduced but not abolished by HK inhibition, indicating that platelet function is underpinned by metabolic flexibility. Integrin activation and pro-coagulant platelet formation were suppressed by both inhibitors. In contrast, α-granule secretion markers (CD62P, CD40L), platelet spreading, and convulxin-induced reactive oxygen species (ROS) generation were more sensitive to 3BP, suggesting a prominent role for HK2 in secretion, outside-in signaling, and ROS regulation. In vitro thrombus formation over collagen was significantly reduced by both inhibitors. In addition, HKs play a pivotal role in the regulation of in vivo hemostasis with increased bleeding time in mice treated with HK inhibitor. These findings identify HK as a central regulator of platelet metabolism and function, with distinct contributions of HK isoforms to specific platelet responses.

BACKGROUND:Glycoprotein (GP)VI is involved in platelet activation, procoagulant phenotype development and reactive oxygen species (ROS) production. Patients with diabetes have altered platelet reactivity and abnormal clot structure, contributing to elevated thrombosis risk. The role of GPVI in clot formation in hyperglycemia is under-investigated. OBJECTIVES:To compare the effect of GPVI-deficiency on ROS production and ex vivo and in vivo clot formation in normo- vs hyperglycemic mice. METHODS:Wild-type (WT) and GPVI-deficient (GPVI-/-) mice had access to hyperglycemic (30% sucrose) or normoglycaemic (standard) diet for 8-weeks. ROS and mitochondrial activity (CS) were measured in isolated platelets. Clot contraction was performed with whole blood. Thrombus formation in vivo was assessed by inferior vena cava (IVC) ligation. RESULTS:Hyperglycemia increased ROS and CS activity in WT but not GPVI-/- mice. Clot weight was decreased in GPVI-/- vs WT mice in normoglycaemia, but these effects were lost in hyperglycemia. Clot weight after IVC ligation was reduced in GPVI-/- vs WT mice in normoglycaemia, with the inverse observed in hyperglycemia. CONCLUSIONS:Our data suggest that clot formation is affected differentially by absence of GPVI in normo- vs hyperglycemia, supporting a role for GPVI in thrombosis under normoglycaemic conditions only.

Hypofibrinolysis is a documented abnormality in conditions with high risk of vascular occlusion. A key inhibitor of fibrinolysis is α2-antiplasmin (α2AP) and we hypothesise that the Affimer technology, comprising small conformational proteins with two nine amino acid variable regions, can be used to modulate α2AP activity and facilitate fibrinolysis. Using a phage display system, a library of Affimers was screened against α2AP. A total of 28 α2AP-specific Affimers were isolated of which one, termed Affimer A11, inhibited protein function and enhanced fibrinolysis. Affimer A11 displayed a monomeric form and consistently reduced lysis time of clots made from plasma samples of individuals with type 2 diabetes mellitus (n=15; from 150.8±100.9 to 109.8±104.8 mins) and those with cardiovascular disease (n=15; 117.6±40.6 to 79.7±33.3 mins); p<0.01 for both groups. The effects of A11 on fibrinolysis were maintained when clots were made from whole blood samples. Mechanistic studies demonstrated that A11 did not affect clot structure or interfere with incorporation of α2AP into fibrin networks but significantly enhanced plasmin activity and accelerated plasmin generation. Affimer A11 reduced α2AP binding to plasmin(ogen), while molecular modelling demonstrated interactions with α2AP in an area responsible for binding to plasminogen, explaining the effects on both plasmin activity and generation. Affimer A11, at 0.15-0.60 mg/ml, had the ability to bind 70-90% of plasma α2AP. In conclusion, we demonstrate that Affimers are viable tools for inhibiting α2AP function and facilitating fibrinolysis, making them potential future therapeutic agents to reduce thrombosis risk.

OBJECTIVES:Venous thromboembolism (VTE), following Janus kinase inhibitors treatment (JAKi), is poorly understood in rheumatoid arthritis (RA). We investigated whether JAKi augmented immune cell-driven clotting or immunothrombosis in RA. METHODS:Peripheral blood leukocytes (PBLs) isolated from patients with RA and healthy controls were treated with various JAKi classes, before stimulation with Toll-like receptor (TLR)-4 (lipopolysaccharide [LPS]) or TLR3 (polyinosinic-polycytidylic acid-poly (I:C)) agonists. Conditioned supernatants were used in plasma turbidity assays to evaluate clot formation and lysis dynamics, while bulk RNA sequencing, enzyme-linked immunosorbent assay, and bead-based immunoassays were used to explore immunothrombosis mechanisms. RESULTS:Turbidity analyses showed that conditioned media from PBLs treated with LPS and tofacitinib significantly accelerated clot formation when compared to LPS alone, and this effect was tissue factor pathway dependent and accompanied by elevations in immunothrombotic cytokines, including tumour necrosis factor α, interleukin (IL)-1β, and IL-6. PBLs from patients with active RA exhibited significantly greater immunothrombotic potential compared to those with low disease activity, despite comparable baseline cytokine levels. RNA sequencing analysis revealed significant pathway enrichment in tofacitinib/LPS-treated PBLs, including activation of Nuclear Factor (NF)-κB pathways, increased tissue factor expression, and reduced levels of anticoagulant factors such as protein S. Pharmacological inhibition assays with 5 JAK therapies suggested that JAK1/tyrosine kinase 2-dependent effect underscored increased thrombosis but selective JAK3 inhibition did not reproduce the prothrombotic effects. Finally, patients with RA with JAK-associated pulmonary embolism showed interstitial changes compatible with immunothrombosis in 4/6 (67%). CONCLUSIONS:Immunothrombosis offers a novel explanation for JAKi-associated VTE in RA.

It has been proposed that low power, high frequency ultrasound can augment the ability of thrombolytic agents to dissolve clot in patients with venous thromboembolism. We created a bench model to examine what role and mechanism ultrasound may have in this process. Fibrin polymerization was analyzed through modified light-scattering experiments with the inclusion of catheter-mediated ultrasound application. We studied fibrin fiber diameters through scanning electron microscopy of ultrasound treated fibrin clots. Clot porosity was investigated using permeation tests, while fibrinolysis was analyzed through light-scattering experiments, and by changes in porosity of lysing clots under flow. Whilst application of ultrasound did not change initial fibrin polymerization, it did induce a reversible change in maximal turbidity of already formed fibrin clots. This change in turbidity was caused by a reduction in fibrin fiber diameter and was associated with an increase in clot porosity. These reversible structural changes were associated with a linear increase in fibrinolysis rates under static conditions, while an exponential increase in rates was observed under flow. The use of ultrasound augmentation of thrombolysis enhances clot dissolution through greater and more rapid fibrin degradation. This is due to conformational change created by the ultrasound in clot structure, a reversible phenomenon that may increase binding sites for lytic agent, and could potentially allow the use of lower doses and shorter infusion times of ultrasound-assisted thrombolytic to treat venous thromboembolism in vivo.

Beyond haemostasis and thrombosis, platelets are increasingly recognized for playing a crucial role in modulating immunoinflammation. Toll-like receptors (TLRs) constitute the first line of defence against infection and injury, with their engagement stimulating thrombotic and immune responses in platelets. Hence, anti-platelet drugs have been used to treat patients with infections and inflammation. However, due to the increased risk of bleeding with current anti-platelet drugs, alternative therapeutic targets need to be identified to ameliorate the consequences of inflammation-driven platelet hyperactivation. Previously, we demonstrated that resting platelets exhibit a metabolic plasticity that facilitates fuel selection flexibility, while in contrast, thrombin-stimulated platelets become highly glycolytic. Since multiple aspects of platelet activation require energy in terms of ATP, we investigated metabolic alterations in TLR1/TLR2-activated platelets. In this study, we have demonstrated that TLR1/TLR2-induced platelet activation reprogrammed platelets to upregulate glycolysis via CD36-linked mechanisms. In addition, we showed that this glycolytic flux is controlled by hexokinase (HK), which plays a crucial role in TLR1/TLR2-induced platelet aggregation. Targeting platelet metabolism plasticity may offer a novel strategy to inhibit platelet function in TLR-initiated diseases.

Blood platelets are anucleate cells that play a vital role in haemostasis, innate immunity, angiogenesis, and wound healing. However, the inappropriate activation of platelets also contributes to vascular inflammation, atherogenesis, and thrombosis. Platelet activation is a highly complex receptor-mediated process that involves a multitude of signalling intermediates in which Reactive Oxygen Species (ROS) are proposed to play an important role. However, like for many cells, changes in the balance of ROS generation and/or scavenging in disease states may lead to the adoption of maladaptive platelet phenotypes. Here, we review the diverse roles of ROS in platelet function and how ROS are linked to specific platelet activation pathways. We also examine how changes in disease, particularly the plasma oxidised low-density lipoprotein (oxLDL), affect platelet ROS generation and platelet function.

Background: Thromboinflammation of adipose tissue involves accumulation of pro-fibrinogenic factors to adipose tissue in obesity, which promotes immune cell infiltration, affects weight gain and can lead to metabolic dysfunction. The role of neutrophil genes in inflammation are frequently investigated using MRP8-Cre mice to generate neutrophil-specific knockouts. Recent study demonstrated that MRP8-Cre mice have off-target deletions in Serpine1 and Ap1s1 genes. Serpine1, encoding plasminogen activator inhibitor-1, is a key anti-fibrinolytic factor linked to thromboinflammation and metabolic dysfunctions in obesity. In this study, we provide evidence suggesting a critical limitation in using MRP8-Cre model to study adipose tissue, weight-related dysfunctions and/or metabolic disorders. Methods: MRP8-Cre and F13a1-/-MRP8 mice were placed on either control diet or high-fat diet for 16 weeks, with body weight monitored weekly. The expression of Serpine 1, Ap1s1 and Adgre1 (macrophage marker) genes in inguinal and epididymal adipose tissues were analyzed using qRT-PCR and compared to the wild-type mice. Results: MRP8-Cre shows no Serpine1 or Ap1s1 expression in inguinal and epididymal adipose tissues. MRP8-Cre mouse is resistant to weight gain on obesogenic diet and does not show macrophage marker in adipose tissue compared to control obesity model. The resistance to weight gain translates to a neutrophil knockout model, F13a1-/-MRP8 that was not expected to show resistance to weight gain as its global knockout does not exhibit this phenotype. Conclusion: Our work suggests that MRP8-Cre model may not be suitable to investigate metabolic outcomes of neutrophil genes, or pathologies that have underlying etiology in thomboinflammation. ### Competing Interest Statement The authors have declared no competing interest.

Background: Platelet activation is constrained by endothelial-derived prostacyclin (PGI2) through cyclic adenosine-3 ',5 '-monophosphate (cAMP) signaling involving multiple isoforms of adenylyl cyclase (AC). The roles of specific AC isoforms in controlling hemostasis remain unclear and require clarification. Objectives: To understand the specific contribution of AC6 in platelet hemostatic and thrombotic function. Methods: A platelet-specific AC6 knockout mouse was generated. Biochemical approaches were used to determine intracellular signaling, with flow cytometry, tail bleeding time assays, and in vivo thrombosis by ferric chloride were used to measure the hemostatic and thrombotic importance of platelet AC6. Results: Loss of AC6 resulted in diminished accumulation of platelet cAMP in response to PGI2, while basal cAMP was unaffected. We found no differences in phosphodiesterase 3A activity, suggesting the defect was in generation rather than hydrolysis of cAMP. Consistent with this, phosphorylation of protein kinase A substrates, vasodilator-stimulated phosphoprotein, and glycogen synthase kinase were diminished but not ablated. Functional studies demonstrated that the inhibition of thrombin-induced fibrinogen binding and P-selectin expression by PGI2 was severely compromised, while inhibition of glycoprotein VI-mediated platelet activation was largely unaffected. Under conditions of flow formed stable thrombi, but in the absence of AC6, thrombi were insensitive to PGI2. Diminished in vivo sensitivity to PGI2 manifested as significantly reduced tail bleeding and accelerated occlusive arterial thrombus formation in response to vascular injury that were highly unstable and prone to embolization in AC6 knockout mice. Conclusion: These data demonstrate that AC6 is linked directly to PGI2-mediated platelet inhibition and regulation of hemostasis and thrombosis in vivo.

Background The microscopic characteristics of the fibrin clot structure, such as fibrin fiber length and diameter, network fiber density, and pore size, can be correlated with mechanical properties, permeability, susceptibility to lysis, and diseases. Several of these properties are commonly assessed using scanning electron microscopy (SEM); however, there are currently no standardized protocols on clot preparation for fiber diameter determination using SEM. This has led to a large discrepancy in values reported for healthy individuals and precludes interlaboratory comparisons. Objectives To develop a standardized protocol for the preparation of blood plasma clots for SEM analysis and fibrin fiber diameter determination. Methods A standardized sample preparation protocol using widely available materials and matching fibrinogen concentration and molality of normal human blood was established. This protocol, in addition to in-house protocols, was reproduced and tested by 8 laboratories across the world. Testing also included evaluations of sample air drying with hexamethyldisilazane versus critical point drying, fiber diameter measurements on the surface and inside a fractured clot, and manual versus automated measurement of fiber diameter. Results Reported fiber diameter values were in good agreement across laboratories, with an average median diameter of 110 ± 13 nm, coefficient of variation = 12% (hexamethyldisilazane drying) and 99 ± 7 nm, coefficient of variation = 7% (critical point drying). There were only small, practically insignificant differences between the diameters of surface fibers and internal fibers. Manual and automated measurements showed reasonable agreement. Conclusion The standardized protocol is recommended as a reference point for preparing plasma clots from the blood of healthy individuals for SEM.

BACKGROUND:Coronary artery disease plaque rupture leads to occlusive thrombosis, causing acute myocardial infarction requiring immediate life-saving treatment. The blood clot is supported by a mesh of fibrin fibers that are generated through the polymerization of fibrin. We recently showed that fibrin also produces a film on the surface of blood clots. OBJECTIVES:This study aimed to examine whether fibrin film occurs intravascularly on thrombi from patients with myocardial infarction. METHODS:In this observational study, we recruited 42 patients with acute ST-elevation myocardial infarction and obtained intracoronary thrombi using catheter-guided aspiration thrombectomy. We used a protocol that avoids air contact and immediately fixes the sample upon thrombectomy. Thrombi were analyzed by tiled scanning electron micrographs to analyze percentage coverage by fibrin film. RESULTS:We found that all thrombi showed surface areas covered with fibrin film. Some film coverage was discontinuous. Total film coverage was on average 24.1% ± 17.0% (range, 4.6%-77.2%). Percentage of film coverage correlated positively with the time from onset of symptoms to thrombectomy (ischemia duration) and negatively with in vitro clot formation time. Discontinuous film did not correlate with plasmin generation. CONCLUSION:These findings show that fibrin film is an integral structural feature that covers around one-quarter of the surface of all coronary thrombi and that older thrombi contain more film. This new structure on thrombi may have important implications for clot contraction, resistance to thrombolysis, and mechanical clot extraction.

Background: Blood clot formation, triggered by vascular injury, is crucial for hemostasis and thrombosis. Blood clots are composed mainly of fibrin fibers, platelets, and red blood cells (RBCs). Recent studies show that clot surfaces also develop a fibrin film, which provides protection against wound infection and retains components such as RBCs within the clot. However, the role of fibrin films in thrombi remains poorly understood. Objectives: To explore the relationship between fibrin films and inflammation, RBC concentration, platelets, and fibrinolysis activity. Methods: We used laser scanning confocal and scanning electron microscopy, enzymelinked immunosorbent assay, and turbidity and fibrinolysis assays to investigate the interactions between fibrin film and inflamed endothelium, RBCs, platelets, and fibrinolysis. Results: We found that plasma clots forming on top of inflamed endothelial cells show less fibrin film coverage and are characterized by higher fiber density and shorter lag time compared with control cells. Blood clots formed under conditions of high hematocrit showed significantly more fibrin film coverage than low hematocrit clots. We found that platelet adhesion was significantly reduced on clots with film compared with clots without film even when platelets were preactivated. Fibrinolysis was faster in clots without film than in clots with film, partly due to reductions in plasmin generation. Conclusion: Our findings indicate that reductions in fibrin film formation under thromboinflammatory conditions support continued clot growth through effects on increased platelet adhesion and activation. On the other hand, increased fibrin film impairs fibrinolysis. These data show a multifaceted role of the fibrin film in clot growth and stability.

Glycoprotein VI (GPVI) plays a key role in collagen-induced platelet aggregation. Affimers are engineered binding protein alternatives to antibodies. We screened and characterized GPVIbinding Affimers as novel tools to probe GPVI function. Among the positive clones, M17, D22, and D18 bound GPVI with the highest affinities (dissociation constant (KD) D ) in the nanomolar range). These Affimers inhibited GPVI-collagen-related peptide (CRP)-XL/collagen interactions, CRP-XL/collagen-induced platelet aggregation, and D22 also inhibited in vitro thrombus formation on a collagen surface under flow. D18 bound GPVI dimer but not monomer. GPVI binding was increased for D18 but not M17/D22 upon platelet activation by CRP-XL and adenosine 5 '-diphosphate. '-diphosphate. D22 but not M17/D18 displaced nanobody 2 (Nb2) binding to GPVI, indicating similar epitopes for D22 with Nb2 but not for M17/D18. Mapping of binding sites revealed that D22 binds a site that overlaps with Nb2 on the D1 domain, whereas M17 targets a site on the D2 domain, overlapping in part with the glenzocimab binding site, a humanized GPVI antibody fragment antigen-binding fragment. D18 targets a new region on the D2 domain. We found that D18 is a stable noncovalent dimer and forms a stable complex with dimeric GPVI with 1:1 stoichiometry. Taken together, our data demonstrate that Affimers modulate GPVI-ligand interactions and bind different sites on GPVI D1/D2 domains. D18 is dimer-specific and could be used as a tool to detect GPVI dimerization or clustering in platelets. A dimeric epitope regulating ligand binding was identified on the GPVI D2 domain, which could be used for the development of novel bivalent antithrombotic agents selectively targeting GPVI dimer on platelets.

Results: The cell-lysis activity after static incubation showed that no lysis was observed in general bacteria, but No.1-3 bacteria showed particularly strong activity among the 12 species of Bacillus natto.From the shaking culture of No.1, the yield of cell-lysis activity was equivalent to 1.5g of lysozyme per liter.It was found that the bacteria could utilize ammonium sulfate as a nitrogen source, and that the activity was transient and very strong at 37°C for 24 to 48 hours of incubation, after which it gradually decreased.The optimal pH of this enzyme was about 6.0, and pI was about 9.5 by isoelectric point electrophoresis (LKB column method).There was a fairly large difference in cell-lysis activity among commercial natto (10 varieties), but three products (No.1-3) had cell-lysis activities of 1.70, 0.83, and 0.53μg/ml, in order from strongest to weakest.Conclusion(s): Although the cell-lysis activity in natto probably extends to all microorganisms or even cancer, the relationship between this bacteriolysis activity and antibacterial activity and nattokinase activity is still unknown and remains a future challenge.