Tail Bleeding Research Articles

Mechanosensing Via a Gp2b3a-Src-Axis Regulates Platelet Migration in Vitro and In Vivo

Platelets are vascular first responders in thrombosis and hemostasis, but also central players in inflammation. Compared to platelets recruited to thrombi, immune responsive platelets use distinct effector functions, including Arp2/3 dependent migration along substrate gradients (haptotaxis), which prevents inflammatory bleeding and contributes to host defense. How platelet migration in this context is regulated on a cell biological level is incompletely understood. Here, we use in vitro assays to show that in contrast to retractile function, polarized actin polymerization at the leading edge in combination with Myosin II dependent retraction at the rear is necessary for initiation and maintenance of migratory behavior. In migrating platelets, myosin II function is regulated via ROCK and MLCK, but blocking these pathways does not abrogate platelet polarization preceding movement. Rather, Arp2/3 dependent actin nucleation is necessary to initiate polarization. Using morphodynamic analyses integrating shape as well as indicators of migration/retraction behavior, we performed a drug screen to identify relevant upstream regulators of actin nucleation. These experiments revealed coordinated integrin GPIIbIIIa outside-in signaling via Galpha13, that triggers tyrosinkinase c-Src dependent lamellipodium formation. This cascade functions independent of soluble agonists ADP and Thromboxane or chemotactic signals. Inhibitors of this signaling cascade interfere with the migratory ability of platelets. In particular, clinically employed Src inhibitor Dasatinib abrogates migration at nanomolar concentrations, notably below its threshold to impede other platelet functions like procoagulant activation or adhesion. In murine inflammation models, low dose Dasatinib treatment translated to reduced migration of platelets visualized by 4D intravital microscopy, as well as increased pulmonary hemorrhage in acute lung injury, with no effect on tail bleeding times and vascular occlusion in a Ferric Chloride induced carotid thrombosis assay. In summary, we define a distinct signaling pathway essential for platelet migration in inflammation and uncover a novel mechanism explaining Dasatinib-related platelet dysfunction and bleeding phenotype. This underscores the differential molecular regulation of platelets in inflammatory/mucosal hemostasis and could explain the occurrence of mucosa-associated bleeding in up to 40% of patients treated with Dasatinib.

Platelet ERO1α Is Crucial for Ca2+ Mobilization and Platelet Activation in Atherothrombosis

Endoplasmic reticulum oxidoreductase 1α (ERO1α) is a key oxidase of protein disulfide isomerase (PDI) during protein folding in the ER. Platelet ERO1α was reported to interact with PDI and αIIbβ3 integrin and regulate platelet function. However, the regulatory mechanism and pathophysiological role remain unknown. In the present study, we demonstrate that platelet ERO1α orchestrates intracellular Ca2+ signaling during platelet activation and contributes to thrombogenesis. Intravital microscopy using megakaryocyte-specific Ero1α conditional knockout (CKO) and KO mice reveals that platelet ERO1α is crucial for platelet thrombus formation in mouse models of laser-induced cremaster arteriolar thrombosis and FeCl3-induced carotid arterial thrombosis. Deletion of Ero1α does not affect bleeding times and blood loss at the site of tail amputation, implying that ERO1α is not required for hemostasis. Studies using Ero1α-null platelets, novel function-blocking antibodies, and small-molecule inhibitors reveal that intracellular, but not extracellular, ERO1α promotes P-selectin exposure, αIIbβ3 integrin activation, and platelet aggregation induced by multiple agonists, such as thrombin and collagen-related peptide. Importantly, the regulatory function of ERO1α is independent of PDI activity. Mass spectrometry and immunoprecipitation assays identify stromal interaction molecule 1 (STIM1) and sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2) as ERO1α binding partners. We have found that ERO1α-STIM1 binding is enhanced during platelet activation, whereas ERO1α-SERCA2 binding is reduced. Intriguingly, platelet ERO1α interacts with STIM1 through Cys49 and Cys56 residues and promotes its function by oxidizing the Cys49-Cys56 disulfide bond, contributing to Ca2+ store content and enhancing Ca2+ mobilization during platelet activation. Using a high throughput screen, we identify a small-molecule ERO1α inhibitor, B20, which inhibits ERO1α activity with an IC50 value of 10.0 μM but does not affect the activities of H2O2 and PDI. Treatment of platelets with B20 recapitulates the functional defects as seen in Ero1α-null platelets. Intravenous injection of B20 and eptifibatide, an αIIbβ3 integrin antagonist, into WT mice significantly inhibits platelet thrombus formation following laser- or FeCl3-induced vascular injury. While treatment of mice with eptifibatide markedly prolongs tail bleeding times and causes blood loss, B20 does not impair hemostatic function. Furthermore, treatment with B20 reduces the infarct volume in ischemic stroke. These results indicate that platelet ERO1α is a novel regulator of Ca2+ signaling and promotes platelet activation and aggregation in a PDI-independent manner, contributing to the pathogenesis of arterial thrombosis and thromboinflammation. Targeting ERO1α may be an attractive therapeutic strategy for treating patients with thrombotic disease.

Novel Snake Venom Derived Hemocoagulase Reverses Anticoagulant Effect of Factor Xa Inhibitors, Restores Hemostatic Clot Formation, and Limits Bleeding in Vitro and In Vivo

Background: Direct oral anticoagulants (DOACs) such as rivaroxaban and apixaban are factor Xa (FXa) inhibitors widely used to treat thromboembolism and prevent stroke. Despite improvements in patient outcomes compared to vitamin K antagonists, major bleeding still occurs and there is an urgent unmet need for reversal agents in the event of major bleeding or when emergency surgery is needed. Slounase is a snake venom derived batroxobin (a thrombin-like enzyme) containing FXa that converts fibrinogen to fibrin in a manner distinctly different from thrombin. We recently reported slounase enhances platelet-fibrin clot formation in vivo in heparin-anticoagulated mice suggesting slounase may bypass coagulation to restore hemostasis and prevent bleeding. Here, we investigate if slounase reverses the anticoagulant effect of FXa inhibitors and restores hemostatic clot formation as a novel bypassing agent. Methods: In this study we used in vitro thromboelastography (TEG) to test the effect of slounase on whole blood from healthy human donors spiked with a range of apixaban or rivaroxaban doses. Clot formation kinetics were recorded for 60 minutes and the parameters reaction time and maximum amplitude (MA) were analyzed. The effect of rivaroxaban and apixaban on hemostasis in vivo was determined in wild type (WT) mice pretreated with apixaban (30mg/kg) or rivaroxaban (10mg/kg) via oral gavage using intravital microscopy laser-induced cremaster arteriole thrombosis model. WT mice pretreated with apixaban or rivaroxaban were further treated with slounase (1U/kg) and reversal of anticoagulant effect of FXa inhibition was determined in vivo under intravital microscopy. The dynamics of platelet recruitment and fibrin formation within the growing clot were analyzed for change in fluorescent intensity over the course of thrombus formation in response to vascular injury. Additionally, mice were pretreated with FXa inhibitors via oral gavage and then intravenously dosed slounase (1U/kg) 10 minutes prior to tail bleeding assay to test the effect on bleeding time and blood loss. Results: A high dose of apixaban and rivaroxaban spiked in human whole blood caused full inhibition of the TEG parameters and an inability to form a stable thrombus in vitro. However, slounase treatment dose dependently restored the parameters of clot formation into the range of control. The in vivo laser-induced cremaster arteriole model was used to test slounase as an intervention to apixaban and rivaroxaban pretreatment. The severe hemostatic defect was determined by limited platelet accumulation and fibrin formation at the site of vascular injury in both groups. Additionally, platelet adherence was transient and fibrin formation was unstable in mice treated with rivaroxaban and apixaban. Intravenous intervention with slounase (1U/kg) increased platelet accumulation and fibrin formation at the site of vascular injury. Slounase treatment reversed the anticoagulant effect of FXa inhibitors in vivo and restored the hemostatic clot formation in response to vascular injury in mice treated with FXa inhibitors. In the in vivo tail bleeding assay WT mice pretreated with FXa inhibitors bled continually until intervention via cauterization. Slounase pretreatment significantly decreased blood loss and bleeding time into the range of WT controls. Conclusions: Slounase is a potential bypassing agent for reversal of anticoagulation by DOACs rivaroxaban and apixaban. Using TEG, our data demonstrated the anticoagulant effect of FXa inhibitors in vitro and the reversal of anticoagulation by slounase. Slounase dose dependently restored clot formation in human whole blood pretreated with an inhibitory dose of FXa inhibitors. Slounase intervention improved hemostasis in vivo by increasing platelet accumulation and fibrin formation at the site of vascular injury. Additionally, slounase treatment decreased bleeding and blood loss in an in vivo bleeding assay demonstrating its potential to quickly reverse effects of DOACs and prevent bleeding episodes. This study further demonstrates slounase is a novel bypassing agent that promotes platelet procoagulant activity and converts fibrinogen to fibrin independent of thrombin activity.

The protective effect of rivaroxaban with or without aspirin on inflammation, oxidative stress, and platelet reactivity in isoproterenol-induced cardiac injury in rats.

Coronary artery diseases are principal sources of mortality and disability in global human population. Progressively, rivaroxaban is being evaluated for the prevention of atherosclerotic thrombi, particularly with anti-platelet agents. Hence, the current report aimed to investigate the cardioprotective effect of rivaroxaban on isoproterenol (ISO)-induced cardiac injury model in rats and the possible synergistic effect when combined with aspirin. Male Wistar rats were randomly assigned into five different groups. Cardiac injury was induced by subcutaneous injection of ISO (85mg/kg) for 2 consecutive days. Rat tail bleeding time was performed prior to sacrifice. Cardiac enzymes, platelet activity, inflammatory, and oxidative stress biomarkers levels were measured using enzyme-linked immunoassay (ELISA). Pre-administration of rivaroxaban alone and on combination with aspirin prevented ISO-induced increase in cardiac thiobarbituric acid reactive substances (TBARS), interleukin 6 (IL-6), and thromboxane B2 (TXB2) levels. Moreover, a significant prolongation of bleeding time was demonstrated among aspirin, rivaroxaban, and aspirin plus rivaroxaban treated groups. On the other hand, the combination treatment of aspirin plus rivaroxaban showed no marked difference in these biomarkers and bleeding time relative to either drug administered separately. However, a prominent decrease of cardiac 6-keto prostaglandin F1α (6-Keto-PGF1α) level was displayed in the combination treatment when compared with ISO and rivaroxaban-treated groups, whereas no significant improvement was seen in cardiac glycoprotein V (GPV) levels except in aspirin-treated group. The study results demonstrated that rivaroxaban decreases cardiac oxidative stress, inflammation, and platelets reactivity. However, the addition of rivaroxaban to aspirin did not seem to show synergistic antioxidant, anti-inflammatory, or antiplatelet effect.

A calcium and zinc composite alginate hydrogel for pre-hospital hemostasis and wound care

We developed, characterized, and examined the hemostatic potential of sodium alginate-based Ca2+ and Zn2+ composite hydrogel (SA-CZ). SA-CZ hydrogel showed substantial in-vitro efficacy, as observed by the significant reduction in coagulation time with better blood coagulation index (BCI) and no evident hemolysis in human blood. SA-CZ significantly reduced bleeding time (≈60 %) and mean blood loss (≈65 %) in the tail bleeding and liver incision in the mice hemorrhage model (p ≤ 0.001). SA-CZ also showed enhanced cellular migration (1.58-fold) in-vitro and improved wound closure (≈70 %) as compared with betadine (≈38 %) and saline (≈34 %) at the 7th-day post-wound creation in-vivo (p < 0.005). Subcutaneous implantation and intra-venous gamma-scintigraphy of hydrogel revealed ample body clearance and non-considerable accumulation in any vital organ, proving its non-thromboembolic nature. Overall, SA-CZ showed good biocompatibility along with efficient hemostasis and wound healing qualities, making it suitable as a safe and effective aid for bleeding wounds.

D121 Located within the DRY Motif of P2Y12 Is Essential for P2Y12-Mediated Platelet Function.

Platelets are anucleate cells that mediate hemostasis. This occurs via a primary signal that is reinforced by secreted products such as ADP that bind purinergic receptors (P2Y1 and P2Y12) on the platelet surface. We recently identified a human subject, whom we termed platelet defect subject 25 (PDS25) with a platelet functional disorder associated with the P2Y12 receptor. PDS25 has normal blood cell counts and no history of bleeding diathesis. However, platelets from PDS25 have virtually no response to 2-MeSADP (a stable analogue of ADP). Genetic analysis of P2Y12 from PDS25 revealed a heterozygous mutation of D121N within the DRY motif. Rap1b activity was reduced in platelets from PDS25, while VASP phosphorylation was enhanced, suggesting that signaling from the P2Y12 receptor was interrupted by the heterozygous mutation. To explore this further, we produced knock-in mice that mimic our subject. Bleeding failed to cease in homozygous KI mice during tail bleeding assays, while tail bleeding times did not differ between WT and heterozygous KI mice. Furthermore, occlusions failed to form in most homozygous KI mice following carotid artery injury via FeCl3. These data indicate that the aspartic acid residue found in the DRY motif of P2Y12 is essential for P2Y12 function.

Effect of warfarin on tail bleeding time of male wistar rats model type 2 diabetes mellitus

Diabetes mellitus (DM) is a chronic metabolic disease that characterized by elevated blood glucose levels. Warfarin is an anticoagulant drug that is often used to treat and prevent microvascular and macrovascular complications. This study wanted to find out the effect of warfarin on the tail bleeding time of male Wistar rats with type 2 diabetes mellitus model. This study used quasi-experimental with posttest research design with an in vivo control group with male Wistar rats model DMT2 induced by nicotinamide and streptozotocin, the experimental group was given warfarin at a dose of 2 mg/Kg of body weight. The number of samples as many as 24 tails. The samples were divided into 4 groups, namely DM + Warfarin, DM + Aquades, Normal + Warfarin, Normal + Aquades. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests. The results of the average duration of tail bleeding time for each group is DM + Warfarin for 1200 ± 0.00 seconds, DM + Aquades for 913 ± 194.84 seconds, Normal group + Warfarin for 1173.3 ± 65.31 seconds, Normal + Aquades for 619.3 ± 319.64 seconds. The Kruskal-Wallis test showed that there was an effect of warfarin on tail bleeding time.

(1R,3S)-THCCA-Asn: To show the discovery of selective inhibitor of thrombin by successfully combining virtual screening and biological assay

Thrombin is the most potent platelet aggregator. To discover the selective inhibitor of thrombin that is important to curing platelet aggregation-related diseases, docking experiments were performed to dock (1R,3S)-2,3,4,9-tetrahydro-β-carboline-3- carboxylic acid, [(1R,3S)-THCCA], and (1S,3S)-2,3,4,9-tetrahydro-β-carboline-3- carboxylic acid, [(1S,3S)-THCCA], into the p pocket of bovine thrombin. The ideal match supported that (1R,3S)-THCCA could be used as a potential lead compound. In this case 20 natural amino acids were theoretically introduced into the 3-carboxyl of (1R,3S)-THCCA and 20 derivatives, (1R,3S)-THCCA-amino acids, were docked into p pocket of bovine thrombin to perform virtual screening. The screening revealed that comparing to (1R,3S)-THCCA itself the DockScores of 16 derivatives were higher, and (1R,3S)-THCCA-Asn (4j) got the highest DockScore. Thus, 16 derivatives were synthesized for experimental study. The in vitro anti-platelet aggregation assay showed that at 100 μM of concentration the 16 derivatives failed to inhibit the platelet aggregation induced by both adenosine diphosphate and arachidonic acid. On the other hand, however, the IC50 value of the 16 derivatives inhibiting the platelet aggregation induced by platelet activating factor and thrombin ranged from 9.44 μM to 194.64 μM and from 0.07 μM to 9.56 μM, respectively. The in vitro anti-platelet aggregation assay suggested that the 16 derivatives selectively inhibited the platelet aggregation induced by thrombin. In particular, the IC50 of (1R,3S)-THCCA-Asn (4j) had the lowest value. On rat model at 1 nmol/kg of dosage the 16 derivatives effectively prevented thrombus formation. It is worth pointing out that even at 0.01 nmol/kg of dosage, 4j still effectively prevented thrombus formation. 4j hardly has effects on the proliferation of mammalian cells and rat tail bleeding time. In conclusion, the combination of virtual screening and biological assays successfully lead to the discovery of 4j as a promising candidate of selective inhibitor of thrombin.

A Novel Antibody Targeting the Second Extracellular Loop of the Serotonin 5-HT2A Receptor Inhibits Platelet Function

Serotonin (5-hydroxytriptamine or 5-HT) is known to be a weak platelet agonist, and is involved in thrombus formation. While 5-HT cannot induce platelet aggregation on its own, when secreted from the alpha granules, it binds to its G-protein Coupled Receptor (GPCR; i.e., 5HT2AR), thereby acting to amplify platelet functional responses (e.g., aggregation). Thus, 5HT2AR-mediated responses are more involved in the secondary amplification of platelet aggregation in the growing thrombus. Therefore, even though 5-HT can be seen as a weak inducer of platelet activation, it is an important amplifier of aggregation triggered by agonists such as ADP, collagen, and epinephrine, thereby enhancing thrombogenesis. The 5HT2AR/5HT2A signaling pathway is of clinical interest to the scientific and medical communities as it has been implicated in the genesis of several forms of cardiovascular disorders. However, efforts to develop antagonists for 5HT2AR as therapeutic agents in cardiovascular diseases have thus far failed due to these reagents having deleterious side-effects, and/or to lack of selectivity, amongst other reasons. In light of research efforts that identified that the 5HT2AR ligand binding domain resides in the second extracellular loop (EL2; amino acids P209-N233), we developed an antibody, i.e., referred to as 5HT2ARAb, against the EL2 region, and characterized its pharmacological activity in the context of platelets. Thus, we utilized platelets from healthy human donors, as well as C57BL/6J mice (10–12 weeks old) to analyze the inhibitory effects of the 5HT2ARAb on platelet activation in vitro, ex vivo, and on thrombogenesis in vivo as well as on 5HT2AR ligand binding. Our results indicate that the 5HT2ARAb inhibits 5-HT-enhanced platelet activation in vitro and ex vivo, but has no apparent effects on that which is agonist-induced. The 5HT2ARAb was also found to prolong the thrombus occlusion time, and it did so without modulating the tail bleeding time, in mice unlike the P2Y12 antagonist clopidogrel and the 5HT2AR antagonist ketanserin. Moreover, it was found that the 5HT2ARAb does so by directly antagonizing the platelet 5HT2AR. Our findings document that the custom-made 5HT2ARAb exhibits platelet function blocking activity and protects against thrombogenesis without impairing normal hemostasis.

Cav3.2 T‐type calcium channel regulates mouse platelet activation and arterial thrombosis

BackgroundCav3.2 is a T‐type calcium channel that causes low‐threshold exocytosis. T‐type calcium channel blockers reduce platelet granule exocytosis and aggregation. However, studies of the T‐type calcium channel in platelets are lacking. ObjectiveTo examine the expression and role of Cav3.2 in platelet function. MethodsGlobal Cav3.2−/− and platelet‐specific Cav3.2−/− mice and littermate controls were used for this study. Western blot analysis was used to detect the presence of Cav3.2 and activation of the calcium‐responsive protein extracellular signal‐regulated kinase (ERK). Fura‐2 dye was used to assess platelet calcium. Flow cytometry and light transmission aggregometry were used to evaluate platelet activation markers and aggregation, respectively. FeCl3‐induced thrombosis and a microfluidic flow device were used to assess in vivo and ex vivo thrombosis, respectively. ResultsCav3.2 was expressed in mouse platelets. As compared with wild‐type controls, Cav3.2−/− mouse platelets showed reduced calcium influx. Similarly, treatment with the T‐type calcium channel inhibitor Ni2+ decreased the calcium influx in wild‐type platelets. As compared with controls, both Cav3.2−/− and Ni2+‐treated wild‐type platelets showed reduced activation of ERK. ATP release, P‐selectin exposure, and αIIbβ3 activation were reduced in Cav3.2−/− and Ni2+‐treated wild‐type platelets, as was platelet aggregation. On in vivo and ex vivo thrombosis assay, Cav3.2 deletion caused delayed thrombus formation. However, tail bleeding assay showed intact hemostasis. ConclusionThese results suggest that Cav3.2 is required for the optimal activation of platelets.

Inhibition of Hypoxia-Inducible Factor Prolyl-Hydroxylase Modulates Platelet Function.

Hypoxia-inducible factor-1α (HIF-1α) involves in redox reactions. Considering the role of reactive oxygen species (ROS) in platelet function, whether it regulates platelet function remains unclear. Using an inhibitor of HIF prolyl-hydroxylase, IOX-2, we intend to investigate its effect on platelet function. Human platelets were treated with IOX-2 (0, 10, 25, and 50 μM) followed by analysis of platelet aggregation, granule secretion, receptor expression, platelet spreading, or clot retraction. Additionally, IOX-2 (10 mg/kg) was injected intraperitoneally into mice to measure tail bleeding time and arterial thrombosis. IOX-2 significantly inhibited collagen-related peptide (CRP; 0.25 μg/mL) or thrombin (0.03 U/mL)-induced platelet aggregation and ATP release dose dependently without affecting P-selectin expression and the surface levels of glycoprotein (GP)Ibα, GPVI, or αIIbβ3. In addition, IOX-2-treated platelets presented significantly decreased spreading on fibrinogen or collagen and clot retraction. Moreover, IOX-2 administration into mice significantly impaired the in vivo hemostatic function of platelets and arterial thrombus formation without affecting the number of circulating platelets and coagulation factors (FVIII and FIX). Further, IOX-2 significantly upregulated HIF-1α in platelets, decreased ROS generation, and downregulated NOX1 expression. Finally, IOX-2 increased the phosphorylation level of VASP (Ser157/239), and inhibited the phosphorylation of p38 (Thr180/Tyr182), ERK1/2 (Thr202/Tyr204), AKT (Thr308/Ser473), and PKCδ (Thr505) in CRP- or thrombin-stimulated platelets. In conclusion, inhibition of HIF prolyl-hydroxylase modulates platelet function and arterial thrombus formation, possibly through upregulation of HIF-1α expression and subsequent inhibition of ROS generation, indicating that HIF-1α might be a novel target for the treatment of thrombotic disorders.

A Factor XIa Inhibitor Engineered from Banded Krait Venom Toxin: Efficacy and Safety in Rodent Models of Arterial and Venous Thrombosis.

Activated factor XI (FXIa) is an important antithrombotic drug target. Clinical and pre-clinical data have demonstrated that its inhibition attenuates thrombosis with minimal risk of excessive bleeding. We isolated Fasxiator from the venom of banded krait Bungarus fasciatus and subsequently engineered FasxiatorN17R,L19E, with improved affinity (Ki = 0.9 nM) and selectivity towards FXIa. Here, we assess the in vivo efficacy and bleeding risk of rFasxiatorN17R, L19E in pre-clinical animal models. Rats injected intravenously (i.v.) with bolus rFasxiatorN17R, L19E showed the specific in vivo attenuation of the intrinsic coagulation pathway, lasting for at least 60 min. We performed the in vivo dose-ranging experiments for rFasxiatorN17R, L19E as follows: FeCl3-induced carotid artery occlusion in rats (arterial thrombosis); inferior vena cava ligation in mice (venous thrombosis); tail bleeding time in both rats and mice (bleeding risk). Head-to-head comparisons were made using therapeutic dosages of unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) for arterial and venous thrombosis, respectively. In the arterial thrombosis model, 2 mg/kg i.v. rFasxiatorN17R,L19E achieved a similar antithrombotic efficacy to that of UFH, with >3-fold lower bleeding time. In the venous thrombosis model, the 10 mg/kg subcutaneous (s.c.) injection of rFasxiatorN17R,L19E achieved similar efficacy and bleeding levels to those of LMWH enoxaparin. Overall, rFasxiatorN17R,L19E represents a promising molecule for the development of FXIa-targeting anticoagulants.

Phosphorylation on Syk Y342 is important for both ITAM and hemITAM signaling in platelets

Immune cells express receptors bearing an immune tyrosine activation motif (ITAM) containing two YXXL motifs or hemITAMs containing only one YXXL motif. Phosphorylation of the ITAM/hemITAM is mediated by Src family kinases allowing for the binding and activation of spleen tyrosine kinase (Syk). It is believed that Syk must be phosphorylated on tyrosine residues for activation, and Tyr342, а conserved tyrosine in the interdomain B region, has been shown to be critical for regulating Syk in FcεR1-activated mast cells. Syk is a key mediator of signaling pathways downstream of several platelet pathways including the ITAM bearing glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor and the hemITAM containing C-type lectin-like receptor-2 (CLEC-2). Since platelet activation is a crucial step in both hemostasis and thrombosis, we evaluated the importance of Syk Y342 in these processes by producing an Syk Y342F knock-in mouse. When using a CLEC-2 antibody as an agonist, reduced aggregation and secretion were observed in Syk Y342F mouse platelets when compared with control mouse platelets. Platelet reactivity was also reduced in response to the GPVI agonist collagen-related peptide. Signaling initiated by either GPVI or CLEC-2 was also greatly inhibited, including Syk Y519/520 phosphorylation. Hemostasis, as measured by tail bleeding time, was not altered in Syk Y342F mice, but thrombus formation in response to FeCl3 injury was prolonged in Syk Y342F mice. These data demonstrate that phosphorylation of Y342 on Syk following stimulation of either GPVI or CLEC-2 receptors is important for the ability of Syk to transduce a signal.

Discovery of 7, 4′-dimethoxy-3-hydroxyflavone as a protease-activated receptor 4 antagonist with antithrombotic activity and less bleeding tendency in mice

There is growing evidence of the importance of protease-activated receptor 4 (PAR4), one of thrombin receptors, as a therapeutic target in thrombotic cardiovascular diseases. In the present study, we utilized ligand-based virtual screening, bioassay, and structure–activity relationship study to discover PAR4 antagonists with new chemical scaffolds from natural origin, and examined their application as antiplatelet agents. By using these approaches, we have identified a flavonoid, 7, 4′-dimethoxy-3-hydroxyflavone, that exhibits anti-PAR4 activity. 7, 4′-Dimethoxy-3-hydroxyflavone inhibited PAR4-mediated human platelet aggregation, GPIIb/IIIa activation, and P-selectin secretion. Also, it inhibited PAR4 downstream signaling pathways, including Ca2+/protein kinase C, Akt, and MAP kinases ERK and p38, in human platelets, and suppressed PAR4-mediated β-arrestin recruitment in CHO-K1 cells exogenously expressed human PAR4. In a microfluidic system, 7, 4′-dimethoxy-3-hydroxyflavone reduced thrombus formation on collagen-coated chambers at an arterial shear rate in recalcified whole blood. Furthermore, mice treated with 7, 4′-dimethoxy-3-hydroxyflavone were significantly protected from FeCl3-induced carotid arterial occlusions, without significantly affecting tail bleeding time. In conclusion, 7, 4′-dimethoxy-3-hydroxyflavone represents a new class of nature-based PAR4 antagonist, it shows effective in vivo antithrombotic properties with less bleeding tendency, and could be a potential candidate for developing new antiplatelet agents.

GRK2 regulates ADP signaling in platelets via P2Y1 and P2Y12

AbstractThe critical role of G protein–coupled receptor kinase 2 (GRK2) in regulating cardiac function has been well documented for >3 decades. Targeting GRK2 has therefore been extensively studied as a novel approach to treating cardiovascular disease. However, little is known about its role in hemostasis and thrombosis. We provide here the first evidence that GRK2 limits platelet activation and regulates the hemostatic response to injury. Deletion of GRK2 in mouse platelets causes increased platelet accumulation after laser-induced injury in the cremaster muscle arterioles, shortens tail bleeding time, and enhances thrombosis in adenosine 5′-diphosphate (ADP)-induced pulmonary thromboembolism and in FeCl3-induced carotid injury. GRK2−/− platelets have increased integrin activation, P-selectin exposure, and platelet aggregation in response to ADP stimulation. Furthermore, GRK2−/− platelets retain the ability to aggregate in response to ADP restimulation, indicating that GRK2 contributes to ADP receptor desensitization. Underlying these changes in GRK2−/− platelets is an increase in Ca2+ mobilization, RAS-related protein 1 activation, and Akt phosphorylation stimulated by ADP, as well as an attenuated rise of cyclic adenosine monophosphate levels in response to ADP in the presence of prostaglandin I2. P2Y12 antagonist treatment eliminates the phenotypic difference in platelet accumulation between wild-type and GRK2−/− mice at the site of injury. Pharmacologic inhibition of GRK2 activity in human platelets increases platelet activation in response to ADP. Finally, we show that GRK2 binds to endogenous Gβγ subunits during platelet activation. Collectively, these results show that GRK2 regulates ADP signaling via P2Y1 and P2Y12, interacts with Gβγ, and functions as a signaling hub in platelets for modulating the hemostatic response to injury.

Danshensu prevents thrombosis by inhibiting platelet activation via SIRT1/ROS/mtDNA pathways without increasing bleeding risk

BackgroundCoronary thrombosis and its correlated disorders are main healthcare problems globally. The therapeutic effects of current treatments involving antiplatelet drugs are not fully satisfactory. Danshensu (DSS) is an important monomer obtained from Salvia miltiorrhiza roots that have been widely employed for vascular diseases in medicinal practices. Nonetheless, the underlying mechanisms of DSS are not fully unraveled. PurposeThe objective of this study was to penetrate the antithrombotic and antiplatelet mechanisms of DSS. MethodsNetwork pharmacology assay was used to forecast the cellular mechanisms of DSS for treating thrombosis. The work focused the impacts of DSS on platelet activation by analyzing aggregation and adhesion in vitro. Flow cytometry, western blotting, CM-H2DCFDA staining and mitochondrial function assays were performed to reveal the molecular mechanisms. The model of common carotid artery thrombus induced by ferric chloride was established. The wet weight of thrombus was measured, and the thrombosis was observed by hematoxylin and eosin (H&E) staining, in order to support the inhibitory effect of DSS on thrombosis. ResultsData mining found the antithrombotic effect of DSS is related to platelet activation and the core target is silent information regulator 1 (SIRT1). We confirmed that DSS dose-dependently inhibited platelet activation in vitro. DSS was further demonstrated to induce the expression of SIRT1 and decreased reactive oxygen species (ROS) burden and thereby prevented mitochondrial dysfunction. Mitochondrial function tests further indicated that DSS prevented mitochondrial DNA (mtDNA) release, which induced activation of platelet in a dendritic cell specific intercellular-adhesion-molecule-3 grabbing non-integrin (DC-SIGN)-dependent manner. In carotid artery injury model induced by ferric chloride, DSS inhibited the development of carotid arterial thrombosis. More encouragingly, in tail bleeding time assay, DSS did not augment bleeding risk. ConclusionThese findings indicated that DSS effectively inhibited platelet activation by depressing the collection of ROS and the release of platelet mtDNA without arousing hemorrhage risk. DSS might represent a promising candidate drug for thrombosis and cardiovascular disease therapeutics.

Orally delivered rutin in lipid-based nano-formulation exerts strong antithrombotic effects by protein disulfide isomerase inhibition

Thrombosis occurs in both macrovasculature and microvasculature, causing various cardio-cerebral vascular diseases. The lack of effective and safe antithrombotic drugs leads to a public health crisis. Mounting evidence suggests that protein disulfide isomerase (PDI) plays a critical role in the initial stage of thrombus formation, motivating the research of the feasibility of PDI inhibitors as novel anti-thrombotics. Rutin, one of the most potent PDI inhibitors, was reported to suppress platelet aggregation and thrombosis in animal models, but further studies and clinical translation were restricted due to its low aqueous solubility and oral bioavailability. In this work, we fabricated rutin-loaded lipid-based nano-formulation (NanoR) and characterized their physical–chemical properties, release profiles, pharmacokinetic process, and pharmacodynamic function against thrombosis in macrovessels and microvessels. NanoR provided increased solubility and dissolution of rutin to achieve earlier T max and higher C max than the sodium salt of rutin (NaR) after oral gavage. Ex vivo studies demonstrated that NanoR significantly inhibited thrombin generation and clot formation in the plasma of mice. Importantly, such effect was reversed by exogenous recombinant PDI, demonstrating the specificity of the NanoR. In direct current-induced arterial thrombosis model and ferric chloride-induced microvascular thrombosis model, NanoR exhibited greatly enhanced antithrombotic activity compared with NaR. NanoR also showed good safety performance according to tail bleeding assay, global coagulation tests, and histological analysis. Overall, our current results indicated that NanoR offers a promising antithrombotic treatment with potential for clinical translation.

Angiopoietin-2 Inhibition of Thrombomodulin-Mediated Anticoagulation-A Novel Mechanism That May Contribute to Hypercoagulation in Critically Ill COVID-19 Patients.

Hypercoagulation and endothelial dysfunction play central roles in severe forms of COVID-19 infections, but the molecular mechanisms involved are unclear. Increased plasma levels of the inflammatory cytokine and TIE2 receptor antagonist Angiopoietin-2 were reported in severely ill COVID-19 patients. In vitro experiments suggest that Angiopoietin-2 bind and inhibits thrombomodulin. Thrombomodulin is expressed on the luminal surface of endothelial cells where it is an important member of the intrinsic anticoagulant pathway through activation of protein C. Using clinical data, mouse models, and in vitro assays, we tested if Angiopoietin-2 plays a causal role in COVID-19-associated hypercoagulation through direct inhibition of thrombin/thrombomodulin-mediated physiological anticoagulation. Angiopoietin-2 was measured in 61 patients at admission, and after 10 days in the 40 patients remaining in the ICU. We found that Angiopoietin-2 levels were increased in COVID-19 patients in correlation with disease severity, hypercoagulation, and mortality. In support of a direct effect of Angiopoietin-2 on coagulation, we found that injected Angiopoietin-2 in mice associated to thrombomodulin and resulted in a shortened tail bleeding time, decreased circulating levels of activated protein C, and increased plasma thrombin/antithrombin complexes. Conversely, bleeding time was increased in endothelial-specific Angiopoietin-2 knockout mice, while knockout of Tie2 had no effect on tail bleeding. Using in vitro assays, we found that Angiopoietin-2 inhibited thrombomodulin-mediated anticoagulation and protein C activation in human donor plasma. Our data suggest a novel in vivo mechanism for Angiopoietin-2 in COVID-19-associated hypercoagulation, implicating that Angiopoietin-2 inhibitors may be effective in the treatment of hypercoagulation in severe COVID-19 infection.

Chickpea Protein Hydrolysate Ameliorates the Impact of Feeding a High-Fat-Diet-Induced Obesity in Mice by Modulating Inflammation

ObjectivesTo evaluate the efficacy of a chickpea (Cicer arietinum) protein hydrolysate (CPH) on ameliorating markers of obesity in a mouse model of metabolic dysfunction when fed a high fat diet. MethodsCPH was produced using an Alcalase® and Flavourzyme® dual-system. Fifty-two male C57BL/6J mice (6 weeks of age) were randomized into four groups (n = 13/group) and fed daily, for a period of 16 weeks, as follows: standard diet (SD), high-fat diet (HFD), HFD + 400 mg CPH/kg body weight (BW) (400 CPH), and HFD + 800 mg CPH/kg BW (800 CPH). BW was measured weekly and blood glucose biweekly via tail bleeding. Feces were collected during week 16. Tissue samples were collected at the end of the study. Protein expression was measured in blood plasma, and metabolites relative concentrations in feces. Gene expression analyses were performed on liver, adipose, and pancreas tissues. ResultsWeight gain for the 800 CPH group was significantly lower (19%, P < 0.01) than the weight gain in the HFD group. Both, adipose and liver tissue weights were significantly lower (12% and 26%, P < 0.05, respectively) in 800 CPH than in HFD. Group 800 CPH had significantly reduced (P < 0.05) plasma protein levels of interleukin (IL)-1 α (14%), IL-4 (35%), IL-9 (32%), lymphotactin (37%), MCP-1 (48%), MIP-1 γ (40%), and TNF- α (40%) as compared to the HFD group. In liver, the 400 CPH group had upregulation (fold change [FC] >2) of genes related to lipid metabolism (Acsl4) and adipokine signaling (Adipor2) and downregulation (FC 0.3–0.5) of genes related to insulin signaling (Pik3r1, Ikbkb, Map2k1) when compared to HFD. In adipose tissue, the 800 CPH group modulated (FC > 2) genes related to inflammation (IL1B and IL18R1), and carbohydrate metabolism (Hk2) compared to the HFD group. The 400 CPH group had significant (FC > 2) upregulation of Tlr4 in pancreas tissue as compared to HFD. More than two hundred metabolites were identified in feces; branched-chain amino acids leucine and isoleucine decreased their relative concentrations in 800 CPH in comparison to HFD. ConclusionsFindings indicated that mice fed CPH had lower BW, reduced levels of inflammatory proteins in plasma, downregulated genes related to insulin signaling, and upregulated genes related to lipid metabolism, and adipokine signaling. Funding SourcesUSDA Pulses Division.

The FVB mouse strain as a spontaneous model of degenerative mitral valve disease

Mitral valve disease is a complex entity with diverse origins, degeneration occurring following an extrinsic aggression (infection, autoimmune attack, drug), genetic malformation or being due to a hemodynamic abnormality (dilatation of the left ventricle, myocardial infarction). The aim is to present the main cardiovascular characteristics of the FVB mouse strain with a particular focus on spontaneous mitral valve degeneration and the serotonergic system in comparison with the C57BL/6 J, the usual background strain of many cardiovascular studies. Body weight, systolic blood pressure, heart rate, urinary 5-hydroxyindoleacetic acid (5-HIAA), whole blood and plasma serotonin, tail bleeding time, blood cell count, plasma TGF-β1 and plasma natriuretic peptide concentrations were measured in FVB/NJ and FVB/NRj mice strains (males and females) obtained from Janvier and Charles River Labs in comparison to the usual C57BL/6 background at different ages (12, 16, 20 and 24-week-old). To know if the mitral valve was remodeled, cardiac anatomy and function were assessed by echocardiography and mitral valves were finally characterized by histology. The expression of 5-HT2A and 5-HT2B receptors was measured by RTqPCR in the anterior leaflet of the valve. No significant difference in C57BL/6 J compared to FVB mice strains regarding body weight increase, arterial blood pressure and heart rate was observed. A progressive augmentation of plasma pro-ANP was observed in FVB mice. However, no cardiac hypertrophy or left-ventricular fibrosis were observed. Conversely, FVB mice demonstrated a high prevalence of fibromyxoid highly cellularized and enriched in glycosaminoglycans lesions, inducing major mitral leaflets thickening without increase in length (100% of 24-week-old FVB/NRj males). The increased thickness was correlated with urinary 5-HIAA and blood platelet count. Whole blood serotonin concentration was similar in the two strains but, in FVB, a reduction of plasma serotonin was observed together with an increase of the bleeding time. No difference regarding serotonergic receptors expression was observed. Finally, echocardiography identified left atrial and left ventricular remodeling associated with thickening of both mitral leaflets and mitral insufficient in 30% of FVB mice but no systolic protrusion of mitral leaflets towards the atrium meaning the absence of mitral valve prolapse. Spontaneous mitral myxomatous degeneration is highly prevalent in FVB mice. A contribution of the serotonergic system is suggested