Tail Bleeding Research Articles

Exposure of Mice to Thirdhand Smoke Modulates In Vitro and In Vivo Platelet Responses.

Smoking is a risk factor for a variety of deleterious conditions, such as cancer, respiratory disease and cardiovascular disease. Thrombosis is an important and common aspect of several cardiovascular disease states, whose risk is known to be increased by both first- and secondhand smoke. More recently, the residual cigarette smoke that persists after someone has smoked (referred to as thirdhand smoke or THS) has been gaining more attention, since it has been shown that it also negatively affects health. Indeed, we have previously shown that 6-month exposure to THS increases the risk of thrombogenesis. However, neither the time-dependence of THS-induced thrombus formation, nor its sex dependence have been investigated. Thus, in the present study, we investigated these issues in the context of a shorter exposure to THS, specifically 3 months, in male and female mice. We show that the platelets from 3-month THS-exposed mice exhibited enhanced activation by agonists. Moreover, we also show that mice of both sexes exposed to THS have decreased tail bleeding as well as decreased thrombus occlusion time. In terms of the role of sex, intersex disparities in thrombus development and hemostasis as well as in platelet aggregation were, interestingly, observed. Together, our findings show that exposing mice to THS for 3 months is sufficient to predispose them to thrombosis; which seems to be driven, at least in part, by an increased activity in platelets, and that it does not manifest equally in both sexes.

Effect of a chromium‐fructooligosaccharide complex on glycemic parameters in obese and obese‐NIDDM rats

The incidence and odds ratio of obesity and Type 2 diabetes, a common sequela of obesity, are increasing in incidence to the point of becoming epidemic in Western society. To determine the potential beneficial effects of a chromium‐fructooligosaccharide complex (FCC) on glycemic responses of male obese and obese NIDDM rats, groups of 3 ‐ 4 month old lean and obese LA/Ntul//‐cp and SHR/Ntul//‐cp rats (NIDDM‐rats, n= 6 rats/group) reared normally on Purina #5012 chow from the time of weaning, were subjected to an oral glucose tolerance (OGT) after a brief 4‐hour fast. A second group of NIDDM rats (n=5) aged one year were also administered the OGT and FCC challenges. NIDDM rats develop IGT by adolescence and renal disease, cataracts, and impaired nerve conduction velocity typical of DM by adulthood. Animals were administered a glucose solution equivalent to 2.5 grams/kg BW via intragastric gavage, or the same glucose plus the FCC (2.5 g/kg BW, containing 200µg Cr and 2.25 mg/serving) or the FCC alone over a brief (1‐2 minute) duration. Measures of blood glucose (BG) were determined at 30‐minute intervals from zero to +120 minutes post gavage via tail bleeding, and the area under the curve (AUC) of each group determined. Final body weights (BW) of obese were greater than lean (p=0.05), and obese‐NIDDM > Obese rats (p=<0.05) and increased in NIDDM rats with older age. OGT of lean rats remained normal throughout, and FCC resulted in minor decreases in AUC and in BG concentrations at all time points (p= n.s.) The OGT of obese rats demonstrated significant increases in BG at +60, +90‐, and +120‐minutes post gavage but remained non‐diabetic, and FCC decreased the glycemic increase and AUC by ~50%, similar to the glycemic response off lean littermates. The OGT of young NIDDM was significantly impaired and FCC resulted in a 32% reduction in the AUC. The OGT and AUC of OLD NIDDM rats was greater than in young rats and was decreased by 25% with FCC. OGT and OGG+FCC were similar at both ages in NIDDM rats. These results are consistent with an FCC‐mediated improvement in glycemic control in congenic animal models for obesity and obesity+NIDDM and suggest that the FCC complex may be a useful nutraceutical adjunct in the dietary treatment of obesity and other glucose intolerant states as they occur in man and animals.

OC-06: Identification of predictive hemostatic biomarkers for cancer diagnosis: results from the HYPERCAN study

ABL1 kinase T315I positive chronic myelogenous leukemia (CML) is treated with the tyrosine kinase inhibitor (TKI) ponatinib. However, its use is associated with a high incidence (29%) of arterial occlusive events.Determine the molecular basis of ponatinib-induced thrombosis.A C57BL/6 mouse model of ponatinib-induced rose bengal thrombosis was created. We characterized ponatinib or combined ponatinib with pioglitazone and measured aortic ROS and apoptosis with antibodies to nitrotyrosine and caspase 3, respectively, using immunohistoperoxidase. The influence of ponatinib influence on collagen-rich peptide (CRP)- and thrombin-induced JON/A and P-selectin expression and ADP-induced fibrinogen binding was examined. Aorta RNAseq studies were performed.Treatment of 20-week old mice with >3 mg/kg oral ponatinib daily for 14 days shortened carotid artery thrombosis times (18.7+3.7 min) compared to control (32.3+4.8 min, p<0.0001) (see Blood Adv. 3 (2019) 2312). Ponatinib-treated mice have normal CBCs, PT, aPTT, and contact- and tissue factor-induced TGT. Aortic adventitia of ponatinib-treated mice express increased nitrotyrosine and caspase 3. Treated mice have shortened tail bleeding times (55+12 min) versus untreated mice (102+9.3 min). CRP- or thrombin-induced JON/A and P-selectin occurred at lower threshold concentrations in ponatinib-treated mice. Concurrent pioglitazone treatment corrected ponatinib-induced thrombosis, aortic ROS and caspase 3 expression, and CRP-induced platelet hyper-reactivity. The mechanism for ponatinib-induced thrombosis and its correction by pioglitazone was sought. Aorta RNAseq data was examined on Reactome’s Hemostasis Enrichment Gene Set. Ponatinib-treated mice have 293/488 up-regulated gene sets (see Fig. 1). The most up-regulated aortic gene is F5. Other up-regulated aortic genes with ponatinib treatment include F8, F2, F10, and F7 and APBB1P, SERPINB2 (PAI-3), and TIMP. Additionally, related integrins (ITGB2, ITGA2B, ITGAL, are ITGAX), receptors (MPL, GP5, GP9), and adhesive proteins (SELL and AMICA1) also are increased. Further, the kinases of the GPVI activation cascade (LCK, LAT, SYK, LYN, FYN) were elevated. Combining pioglitazone with ponatinib treatment reduced the upregulation of 100 gene sets seen with ponatinib alone. Pioglitazone corrected the ponatinib-induced prothrombotic phenotype. F8, F2 and the genes of and Lyn were knocked off the list of top genes expressed.These investigations indicate that ponatinib treatment increases gene expression of aortic prothrombotic and platelet signalling genes that promote GPVI activation. Addition of pioglitazone reduces the prothrombotic characteristics conferred by ponatinib treatment. These data indicate that cancer-associated thrombosis can be managed by non-anticoagulant pharmacologic agents.

Human and mouse PAR4 are functionally distinct receptors: Studies in novel humanized mice

BackgroundHuman and mouse platelets both express protease‐activated receptor (PAR) 4 but sequence alignment reveals differences in several functional domains. These differences may result in functional disparities between the receptors which make it difficult to translate PAR4 studies using mice to human platelet physiology. ObjectivesTo generate transgenic mice that express human, but not mouse, PAR4 and directly compare human and mouse PAR4 function in the same platelet environment. MethodsTransgenic mice were made using a genomic clone of the F2RL3 gene (encoding PAR4) and backcrossed with Par4 KO mice. For certain experiments, mice were bred with GRK6 KO mice. Tail bleeding time and platelet function in response to PAR4‐activating peptide were assessed. ResultsHuman F2RL3 was successfully integrated into the mouse genome, transgenic mice were crossed to the mPar4 KO background (PAR4 tg/KO), and PAR4 was functionally expressed on platelets. Compared to WT, PAR4 tg/KO mice exhibited shortened tail bleeding time and their platelets were more responsive to PAR4‐AP as assessed by α‐granule release and integrin activation. The opposite was observed with thrombin. Knocking out GRK6 had no effect on human PAR4‐expressing platelets, unlike mouse Par4‐expressing platelets. PAR4 tg/KO platelets exhibited greater Ca2+ area under the curve and more robust extracellular vesicle release than WT stimulated with PAR4‐AP. ConclusionThese data suggest that (1) human PAR4‐ and mouse Par4‐mediated signaling are different and (2) the feedback regulation mechanisms of human and mouse PAR4 are different. These functional differences are important to consider when interpreting PAR4 studies done with mice.

Blocking human protein C anticoagulant activity improves clotting defects of hemophilia mice expressing human protein C

AbstractHemophilia A and B are hereditary coagulation defects resulting in unstable blood clotting and recurrent bleeding. Current factor replacement therapies have major limitations such as the short half-life of the factors and development of inhibitors. Alternative approaches to rebalance the hemostasis by inhibiting the anticoagulant pathways have recently gained considerable interest. In this study, we tested the therapeutic potential of a monoclonal antibody, HAPC1573, that selectively blocks the anticoagulant activity of human activated protein C (APC). We generated F8−/− or F9−/− hemophilia mice expressing human protein C by genetically replacing the murine Proc gene with the human PROC. The resulting PROC+/+;F8−/− or PROC+/+;F9−/− mice had bleeding characteristics similar to their corresponding F8−/− or F9−/− mice. Pretreating the PROC+/+;F8−/− mice with HAPC1573 shortened the tail bleeding time. HAPC1573 pretreatment significantly reduced mortality and alleviated joint swelling, similar to those treated with either FVIII or FIX, of either PROC+/+;F8−/− or PROC+/+;F9−/− mice in a needle puncture–induced knee-joint bleeding model. Additionally, we found that HAPC1573 significantly improved the thrombin generation of PROC+/+;F8−/− mice but not F8−/− mice, indicating that HAPC1573 enhanced the coagulant activity of hemophilia mice by modulating human APC in vivo. We further documented that HAPC1573 inhibited the APC anticoagulant activity to improve the clotting time of human plasma deficient of FVIII, FIX, FXI, FVII, VWF, FV, or FX. These results demonstrate that selectively blocking the anticoagulant activity of human APC may be an effective therapeutic and/or prophylactic approach for bleeding disorders lacking FVIII, FIX, or other clotting factors.

Effects of MaiLiuPian on carotid thrombosis in rats and acute pulmonary embolism in mice and its antithrombotic mechanism.

Mailiupian (MLP) is a new patent functional food that consists of Crataegi Fructus, Notoginseng Radix, and Ginkgo Folium, which was reported to be active in improving the microcirculation based on formulation screening. However, whether it is effective in inhibiting thrombus and its mechanism has not been evaluated. Therefore, in the present study, the models of arterial thrombosis induced by FeCl3 and the models of APE by ADP were established to evaluate the antithrombosis effect of MLP. Results showed that MLP markedly reduced the weight and size of wet thrombosis in FeCl3 -induced rats and decreased the recovery time from symptoms of APE mice. MLP was proved to prolong APTT, PT, TT and improve the levels of t-PA and 6-keto-PGF1α significantly, meanwhile, PAI-1 and TXB2 were reduced apparently. By comparing tail bleeding time, MLP showed antithrombotic effects, but without the risk of bleeding, taking aspirin as a control. PRACTICAL APPLICATIONS: Our experiments proved that MLP, a new patent health food, acted on both coagulation and fibrinolytic systems and the platelet aggregation to play antithrombosis roles, providing a theoretical basis for applications of MLP in preventing or curing thrombosis diseases.

Targeting platelet-derived CXCL12 impedes arterial thrombosis

AbstractThe prevention and treatment of arterial thrombosis continue to be clinically challenging, and understanding the relevant molecular mechanisms in detail may facilitate the quest to identify novel targets and therapeutic approaches that improve protection from ischemic and bleeding events. The chemokine CXCL12 augments collagen-induced platelet aggregation by activating its receptor CXCR4. Here we show that inhibition of CXCR4 attenuates platelet aggregation induced by collagen or human plaque homogenate under static and arterial flow conditions by antagonizing the action of platelet-secreted CXCL12. We further show that platelet-specific CXCL12 deficiency in mice limits arterial thrombosis by affecting thrombus growth and stability without increasing tail bleeding time. Accordingly, neointimal lesion formation after carotid artery injury was attenuated in these mice. Mechanistically, CXCL12 activated via CXCR4 a signaling cascade involving Bruton's tyrosine kinase (Btk) that led to integrin αIIbβ3 activation, platelet aggregation, and granule release. The heterodimeric interaction between CXCL12 and CCL5 can inhibit CXCL12-mediated effects as mimicked by CCL5-derived peptides such as [VREY]4. An improved variant of this peptide, i[VREY]4, binds to CXCL12 in a complex with CXCR4 on the surface of activated platelets, thereby inhibiting Btk activation and preventing platelet CXCL12-dependent arterial thrombosis. In contrast to standard antiplatelet therapies such as aspirin or P2Y12 inhibition, i[VREY]4 reduced CXCL12-induced platelet aggregation and yet did not prolong in vitro bleeding time. We provide evidence that platelet-derived CXCL12 is involved in arterial thrombosis and can be specifically targeted by peptides that harbor potential therapeutic value against atherothrombosis.

Sigesbeckia orientalis L. Derived Active Fraction Ameliorates Perioperative Neurocognitive Disorders Through Alleviating Hippocampal Neuroinflammation.

Neuroinflammation is closely related to the pathogenesis of perioperative neurocognitive disorders (PNDs), which is characterized by the activation of microglia, inflammatory pathways and the release of inflammatory mediators. Sigesbeckia orientalis L. (SO) is a traditional Chinese medicine which demonstrates anti-inflammatory activities in different models. In this study, we aim to isolate the active fraction from the extract of SO with higher anti-inflammatory potential and confirm if the selected fraction exerts neuroprotection against the development of PND in an animal model. Moreover, the components in the selected fraction would be determined by UPLC-PDA analysis. Three fractions were prepared by column chromatography packed with three different macroporous resins. Anti-inflammatory activities of prepared fractions were accessed in microglial BV2 cultures by nitric oxide release, gene expression of inflammatory cytokines and activation of inflammatory JNK and NF-kB pathway molecules. Our results demonstrated that the fraction prepared from D101 macroporous resin (D101 fraction) exhibited a more potent anti-neuroinflammatory effect. The neuroprotective effect of D101 fraction was further examined in postoperative mice. Our results showed that surgery-induced cognitive dysfunction was attenuated by the D101 fraction treatment. This fraction also reduced microglial activation, inflammatory cytokines and inhibiting JNK and NF-kB pathway molecules in the hippocampus. In addition, surgery induced dendritic spine loss while D101 fraction ameliorated the spine loss in the hippocampus. For safety concerns, anti-thrombotic effect was examined by tail bleeding assay and no significant change of the bleeding pattern was found. UPLC-PDA analysis indicated that flavonoids (rutin, isochlorogenic acid A, isochlorogenic acid C) and terpenoid (darutoside) were the most important components in the D101 fraction. Our results support a therapeutic, as well as the translational potential for D101 fraction in ameliorating postoperative neuroinflammation and subsequent PND in the clinical setting without increasing bleeding tendencies.

Deletion of Grin1 in mouse megakaryocytes reveals NMDA receptor role in platelet function and proplatelet formation

AbstractThe process of proplatelet formation (PPF) requires coordinated interaction between megakaryocytes (MKs) and the extracellular matrix (ECM), followed by a dynamic reorganization of the actin and microtubule cytoskeleton. Localized fluxes of intracellular calcium ions (Ca2+) facilitate MK-ECM interaction and PPF. Glutamate-gated N-methyl-D-aspartate receptor (NMDAR) is highly permeable to Ca2+. NMDAR antagonists inhibit MK maturation ex vivo; however, there are no in vivo data. Using the Cre-loxP system, we generated a platelet lineage–specific knockout mouse model of reduced NMDAR function in MKs and platelets (Pf4-Grin1−/− mice). Effects of NMDAR deletion were examined using well-established assays of platelet function and production in vivo and ex vivo. We found that Pf4-Grin1−/− mice had defects in megakaryopoiesis, thrombopoiesis, and platelet function, which manifested as reduced platelet counts, lower rates of platelet production in the immune model of thrombocytopenia, and prolonged tail bleeding time. Platelet activation was impaired to a range of agonists associated with reduced Ca2+ responses, including metabotropic like, and defective platelet spreading. MKs showed reduced colony and proplatelet formation. Impaired reorganization of intracellular F-actin and α-tubulin was identified as the main cause of reduced platelet function and production. Pf4-Grin1−/− MKs also had lower levels of transcripts encoding crucial ECM elements and enzymes, suggesting NMDAR signaling is involved in ECM remodeling. In summary, we provide the first genetic evidence that NMDAR plays an active role in platelet function and production. NMDAR regulates PPF through a mechanism that involves MK-ECM interaction and cytoskeletal reorganization. Our results suggest that NMDAR helps guide PPF in vivo.

Dual antiplatelet therapy improves functional recovery and inhibits inflammation after cerebral ischemia/reperfusion injury

BackgroundDual antiplatelet therapy with aspirin and clopidogrel (ASA + CPG) during the first 21 days has been shown to reduce the risk of major ischemic events in patients with transient ischemic attack (TIA) or minor stroke. However, the mechanisms underlying combination treatment with ASA + CPG in experimental ischemic stroke has not been fully elucidated.MethodsMinor cerebral ischemia was induced in mice by transient distal middle cerebral artery occlusion (tdMCAO). Two doses of ASA + CPG (12 and 24 mg/kg/day) or vehicle were administered by gavage daily. Neurological behaviors were assessed using the modified Garcia scores, Rotarod test, Y maze, and open field test. Platelet function was assessed in vitro by flow cytometry and in vivo by bleeding and clotting time. The neutrophil ratio and the levels of inflammatory cytokines were measured by flow cytometry and the Meso Scale Discovery (MSD) electrochemilunimescence, respectively.ResultsSensorimotor function was partially recovered with ASA + CPG treatment after ischemia. Anxiety levels and cognitive functions showed improvement in the ASA + CPG group at 12 mg/kg/day after 21 days. Both tail bleeding time and flow cytometry showed significantly decreased platelet function after ASA + CPG treatment. Notably, ASA + CPG at 12 mg/kg/day prolonged clotting time at 28 days after injury. Furthermore, the ratio of neutrophils, an indicator of inflammation, was reduced with 12 mg/kg/day ASA + CPG treatment in the bone marrow (BM) at 21 days and in the peripheral blood (PB) at 21 and 28 days after tdMCAO. Both doses of ASA + CPG decreased pro-inflammatory cytokine interleukin (IL)-6 expression 21 days after stroke. Taken together, these results demonstrated that combination treatment with ASA + CPG improved long-term neurological function after stroke and may inhibit platelet-neutrophil interaction by decreasing the concentration of pro-inflammatory cytokine, IL-6.ConclusionsThese findings indicate a neuroprotective effect of combination treatment with ASA + CPG for a duration of 21 days in an experimental acute minor stroke model. These findings provide further evidence that dual antiplatelet therapy may be a viable neuroprotective treatment to decrease the recurrence of stroke.

Pennisetum glaucum Protein Extract Protects RBC, Liver, Kidney, Small Intestine from Oxidative Damage and Exhibits Anticoagulant, Antiplatelet Activity

Objective High level of exogenous ROS in the circulation affects RBC membrane integrity which facilitates the generation of endogenous RBC ROS, implicated in series of physiological changes primarily associated with thrombosis and vital tissue damage. Although, Pennisetum glaucum (pearl millet) stores abundance of proteins, their therapeutic potential is least explored. Thus, the purpose of this study is to examine the role of Pennisetum Glaucum Protein Extract (PGE) on oxidative stress induced cell/tissue damage and thrombosis. Method In this investigation, protein characterization was done by using SDS-PAGE, Native-PAGE, PAS-staining and HPLC. In-vitro oxidative stress was induced in RBC using sodium nitrite. While, in-vivo oxidative stress was induced in experimental rats using diclofenac. Stress markers and biochemical parameters were evaluated. Role of PGE on thrombosis was assessed by using, in-vitro plasma recalcification time, activated partial thromboplastin time, prothrombin time, mouse tail bleeding time (In-vivo) and platelet aggregation. Results: PGE revealed varied range of molecular weight proteins on SDS-PAGE. PGE normalized the sodium nitrite induced oxidative damage of RBC and diclofenac induced oxidative damage in liver, kidney and small intestine. PGE exhibited anticoagulant effect by increasing the coagulation time of both PRP and PPP and mouse tail bleeding time. Furthermore, PGE prolonged the clotting time of only APTT but did not affect PT. PGE inhibited agonists ADP and epinephrine induced platelet aggregation. Conclusion Our findings suggest, PGE could be a better contender in the management of oxidative stress and its associated diseases. Abbreviations PGE Pennisetum Glaucum protein Extract APPT Activated Partial Thromboplastin Time PT Prothrombin Time ROS Reactive Oxygen Species PRP Platelet Rich Plasma PPP Platelet Poor Plasma SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis PAS Periodic Acid-schiff Staining OD Optical Density INR International Normalized Ratio PBS Phosphate Buffered Saline SOD Superoxide Dismutase TCA Trichloro Acetatic Acid DTNB Di-Thio-bis-NitroBenzoic acid SGOT Serum Glutamate Oxaloacetate Transaminase SGPT Serum Glutamate Pyruvate Transaminase ALP Alkaline Phosphatase DFC Diclofenac Syl Silymarin MED Minimum Edema Dose MHD Minimum Hemorrhagic Dose

Genetic Deletion of HLJ1 Does Not Affect Blood Coagulation in Mice.

HLJ1 (also called DNAJB4) is a member of the DNAJ/Hsp40 family and plays an important role in regulating protein folding and activity. However, there is little information about the role of HLJ1 in the regulation of physiological function. In this study, we investigated the role of HLJ1 in blood coagulation using wild-type C57BL/6 mice and HLJ1-null (HLJ1-/-) mice. Western blot analysis and immunohistochemistry were used to assess the expression and distribution of HLJ1 protein, respectively. The tail bleeding assay was applied to assess the bleeding time and blood loss. A coagulation test was used for measuring the activity of extrinsic, intrinsic and common coagulation pathways. Thromboelastography was used to measure the coagulation parameters in the progression of blood clot formation. The results showed that HLJ1 was detectable in plasma and bone marrow. The distribution of HLJ1 was co-localized with CD41, the marker of platelets and megakaryocytes. However, genetic deletion of HLJ1 did not alter blood loss and the activity of extrinsic and intrinsic coagulation pathways, as well as blood clot formation, compared to wild-type mice. Collectively, these findings suggest that, although HLJ1 appears in megakaryocytes and platelets, it may not play a role in the function of blood coagulation under normal physiological conditions.

Bacteria-propelled microtubular motors for efficient penetration and targeting delivery of thrombolytic agents

Effective thrombolysis is critical to rapidly rebuild blood flow for thrombosis patients. Drug delivery systems have been developed to address inadequate pharmacokinetics of thrombolytic agents, but challenges still remain in the timely removal of blood clots regarding the dense fibrin networks. Herein, rod-shaped tubular micromotors were developed to achieve efficient penetration and thorough destruction of thrombi. By using electrospun fiber fragments as the template, urokinase (uPA)-loaded polydopamine (PDA) microtubes with surface decorated fucoidan (FuPDAuPA) were prepared at the aspect ratio of around 2. One E. coli Nissle 1917 (EcN) was assembled into one microtube to construct a FuPDAuPA@EcN hybrid micromotor through PDA adhesion and L-aspartate induction. The pharmacokinetic analysis indicates that the encapsulation of uPA into micromotors extends the half-life from 0.4 to 5.6 h and increases the bioavailability over 10 times. EcN-propelled motion elevates adsorption capacities of FuPDAuPA@EcN for more than four times compared with that of FuPDAuPA. The fucoidan-mediated targeting causes 2-fold higher thrombolysis capacity in vitro and over 10-fold higher uPA accumulation in thrombi in vivo. In the treatment of venous thrombi at mouse hindlimbs, intravenous administration of FuPDAuPA@EcN completely removed blood clots with almost full recovery of blood flows and apparently alleviated tail bleeding. It should be noted that FuPDAuPA@EcN treatment at a reduced uPA dose caused no significant difference in the blood flow rate compared with those of FuPDAuPA. The synergistic action of fucoidan-induced targeting and EcN-driven motion provides a prerequisite for promoting thrombolytic efficacy and reducing uPA dose and bleeding side effect. Statement of significanceThe standard treatment to thrombosis patient is intravenous infusion of thrombolytic agents, but the associated bleeding complications and impairment of normal haemostasis greatly offset the therapeutic benefits. Drug delivery systems have been developed to address the limitations of inadequate pharmacokinetics of thrombolytic agents, but challenges still exist in less efficient penetration into dense networks for thorough destruction of thrombi. Up to now only few attempts have been made to construct nano-/micromotors for combating thrombosis and there is no single case that antithrombosis is assisted by bacteria or cells-propelled motors. Herein, bacteria-propelled microtubes were developed to carry urokinase for efficient penetration into blood clots and effective thrombolysis. The synergistic action of bacteria-driven motion and specific ligand-induced targeting holds a promising treatment strategy for life-threatening cardiovascular diseases such as thrombosis and atherosclerosis.

Pharmacological actions of dieckol on modulation of platelet functions and thrombus formation via integrin αIIbβ3 and cAMP signaling

Background and purposeDieckol is a phlorotannin that can be found in seaweeds, particularly in Eisenia bicyclis (brown algae) and is known to have anti-oxidant, anti-inflammatory, and anti-microbial properties. It also possesses anti-thrombotic and pro-fibrinolytic activities; however, the mechanistic aspects of anti-platelet and anti-thrombotic activity are yet to be explored. Study design and methodologyWe investigated the pharmacological effects of dieckol on the modulation of platelet functions using human, rat, and mice models. Inhibitory effects of dieckol on platelet aggregation were assessed using platelet-rich plasma and washed platelets, followed by measurement of dense granule secretions, fibrinogen binding to integrin αIIbβ3, fibronectin adhesion assay, platelet spreading on immobilized fibrinogen, and clot retraction. Cyclic nucleotide signaling events were evaluated, such as cyclic-AMP production followed by vasodilator-stimulated phosphoprotein (VASP) stimulation. The in vivo anti-thrombotic potential was evaluated in mice using an acute pulmonary thromboembolism model and tail bleeding assay. ResultsDieckol markedly inhibited platelet aggregation and granule secretion; furthermore, it down-regulated integrin αIIbβ3–mediated inside-out and outside-in signaling events, including platelet adhesion, spreading, and clot retraction, whereas it upregulated the cAMP–PKA–VASP pathway. Dieckol-treated mice significantly survived the thrombosis than vehicle treated mice, without affecting hemostasis. Histological examinations of lungs revealed minimum occluded vasculature in dieckol-treated mice. ConclusionDieckol possesses strong anti-platelet and anti-thrombotic properties and is a potential therapeutic drug candidate to treat and prevent platelet-related cardiovascular disorders.

Platelet SHARPIN regulates platelet adhesion and inflammatory responses through associations with αIIbβ3 and LUBAC

AbstractPlatelets form hemostatic plugs to prevent blood loss, and they modulate immunity and inflammation in several ways. A key event during hemostasis is activation of integrin αIIbβ3 through direct interactions of the β3 cytoplasmic tail with talin and kindlin-3. Recently, we showed that human platelets express the adapter molecule Shank-associated RH domain interacting protein (SHARPIN), which can associate directly with the αIIb cytoplasmic tail and separately promote NF-κB pathway activation as a member of the Met-1 linear ubiquitination activation complex (LUBAC). Here we investigated the role of SHARPIN in platelets after crossing Sharpin flox/flox (fl/fl) mice with PF4-Cre or GPIbα-Cre mice to selectively delete SHARPIN in platelets. SHARPIN-null platelets adhered to immobilized fibrinogen through αIIbβ3, and they spread more extensively than littermate control platelets in a manner dependent on feedback stimulation by platelet adenosine diphosphate (ADP) (P < .01). SHARPIN-null platelets showed increased colocalization of αIIbβ3 with talin as assessed by super-resolution microscopy and increased binding of soluble fibrinogen in response to submaximal concentrations of ADP (P < .05). However, mice with SHARPIN-null platelets showed compromised thrombus growth on collagen and slightly prolonged tail bleeding times. Platelets lacking SHARPIN also showed reduced NF-κB activation and linear ubiquitination of protein substrates upon challenge with classic platelet agonists. Furthermore, the loss of platelet SHARPIN resulted in significant reduction in inflammation in murine models of colitis and peritonitis (P < .01). Thus, SHARPIN plays differential and context-dependent roles in platelets to regulate important inflammatory and integrin adhesive functions of these anucleate cells.

Tissue dynamics of von Willebrand factor characterized by a novel fluorescent protein–von Willebrand factor chimera

BackgroundTissue dynamics of von Willebrand factor (VWF) that are vital to its biological function have not been fully characterized. ObjectiveTo develop a new fluorescent protein‐‐VWF chimera (FP‐VWF) that has similar hematologic function to wild‐type VWF and use it to monitor the tissue dynamics of VWF distribution. MethodsGenotyping, platelet counting, tail bleeding time assay, agarose gels, western blot, platelet aggregation, proteolytic analysis, and ELISA were applied in characterizing the function of FP‐VWF; fluorescence spectrometer and confocal fluorescence microscope were used to monitor the plasma and tissue distribution of FP‐VWF. ResultsThe transgenic mice that carry the FP‐VWF retain hematologic activity of VWF with plasma levels of FP‐VWF reduced by 50% and there are reduced high molecular weight FP‐VWF multimers compared to the wild‐type mice. The GPIb‐binding and ADAMTS‐13 (A Disintegrin and Metalloprotease with ThrombSpondin type 1 motif, member 13) proteolytic efficiency of FP‐VWF are similar to wild‐type VWF. The tissue distribution of FP‐VWF was probed directly through its intrinsic fluorescence at normal or stimulated status, which indicated that the medicine‐stimulated endogenous FP‐VWF seems primarily released from the aorta and cleared in the spleen. Similar results were observed in non‐fluorescent mice through a standard immunofluorescence approach. The fluorescence signals of FP‐VWF were also similar to the standard dye‐based approach in detecting the FeCl3‐induced blood clotting in vivo. ConclusionsTogether, these results suggest that this novel FP‐VWF chimera is valuable in probing the tissue dynamics of VWF in quite a few biological and pharmaceutical applications.

Sorghum Protein Extract Protects RBC from Sodium Nitrite-Induced Oxidative Stress and Exhibits Anticoagulant and Antiplatelet Activity

Oxidative stress plays a critical role in the progression of diabetes, arthritis, cancer, eryptosis, cardiovascular disease, and thrombosis. Currently, antioxidants from natural sources are in high demand due to their beneficial role in the management of said diseases. The purpose of the study was to evaluate the protective effect of sorghum protein buffer extract (SBE) on sodium nitrite-induced oxidative stress and thrombosis. Protein characterization of SBE was done using SDS-PAGE. Oxidative stress in RBC was induced using sodium nitrite (NaNO2) and the key stress markers such as lipid peroxidation (LPO), protein carbonyl content (PCC), and the level of antioxidant enzymes (SOD and CAT) were measured. The anticoagulant effect of SBE was identified by employing in-vitro plasma recalcification time, activated partial thromboplastin time (APTT), prothrombin time (PT), and in-vivo mouse tail bleeding time. SBE antiplatelet activity was examined using agonist adenosine diphosphate (ADP) and epinephrine-induced platelet aggregation. Non-toxic property of SBE was identified using in-vitro direct haemolytic, haemorrhagic, and edema forming activities using experimental mice. SBE revealed similar protein banding pattern under both reduced and non-reduced conditions on SDS-PAGE. Interestingly, SBE normalized the level of LPO, PCC, SOD, and CAT in stress-inducedRBCs. Furthermore, SBE showed anticoagulant effect in platelet rich plasma by enhancing the clotting time from the control 250 s to 610 s and bleeding time from the control 200 s to more than 500 s (p<0.01) in a dose dependent manner. In addition, SBE prolonged the clot formation process of only APTT but not PT. SBE inhibited the agonists ADP and epinephrine induced platelet aggregation. SBE did not hydrolyze RBC cells, devoid of edema and haemorrhage properties. This study demonstrates for the first time the anticoagulant, antiplatelet, and antioxidant properties of SBE. Thus, the observed results validate consumption of sorghum as good for health and well-being.

Hypofibrinogenemia with preserved hemostasis and protection from thrombosis in mice with an Fga truncation mutation

AbstractGenetic variants within the fibrinogen Aα chain encoding the αC-region commonly result in hypodysfibrinogenemia in patients. However, the (patho)physiological consequences and underlying mechanisms of such mutations remain undefined. Here, we generated Fga270 mice carrying a premature termination codon within the Fga gene at residue 271. The Fga270 mutation was compatible with Mendelian inheritance for offspring of heterozygous crosses. Adult Fga270/270 mice were hypofibrinogenemic with ∼10% plasma fibrinogen levels relative to FgaWT/WT mice, linked to 90% reduction in hepatic Fga messenger RNA (mRNA) because of nonsense-mediated decay of the mutant mRNA. Fga270/270 mice had preserved hemostatic potential in vitro and in vivo in models of tail bleeding and laser-induced saphenous vein injury, whereas Fga−/− mice had continuous bleeding. Platelets from FgaWT/WT and Fga270/270 mice displayed comparable initial aggregation following adenosine 5′-diphosphate stimulation, but Fga270/270 platelets quickly disaggregated. Despite ∼10% plasma fibrinogen, the fibrinogen level in Fga270/270 platelets was ∼30% of FgaWT/WT platelets with a compensatory increase in fibronectin. Notably, Fga270/270 mice showed complete protection from thrombosis in the inferior vena cava stasis model. In a model of Staphylococcus aureus peritonitis, Fga270/270 mice supported local, fibrinogen-mediated bacterial clearance and host survival comparable to FgaWT/WT, unlike Fga−/− mice. Decreasing the normal fibrinogen levels to ∼10% with small interfering RNA in mice also provided significant protection from venous thrombosis without compromising hemostatic potential and antimicrobial function. These findings both reveal novel molecular mechanisms underpinning fibrinogen αC-region truncation mutations and highlight the concept that selective fibrinogen reduction may be efficacious for limiting thrombosis while preserving hemostatic and immune protective functions.

Ultrasound-Propelled Janus Rod-Shaped Micromotors for Site-Specific Sonodynamic Thrombolysis.

Antithrombosis therapy is confronted with short half-lives of thrombolytic agents, limited therapeutic effects, and bleeding complications. Drug delivery systems of thrombolytic agents face challenges in effective penetration into thrombi, which are characterized by well-organized fibrin filled with abundant activated platelets. Herein, Janus rod (JR)-shaped micromotors are constructed by side-by-side electrospinning and cryosection, possessing advantages in controlling the Janus structure and aspect ratio of microrods. Silicon phthalocyanine (Pc) and CaO2 nanoparticles (NPs) are loaded into the separate sides of JRs, and Arg-Gly-Asp (RGD) peptides are grafted on the surface to obtain Pc/Ca@r-JRs for the sonodynamic therapy (SDT) of thrombosis without using any thrombolytic agents. Decomposition of CaO2 NPs ejects O2 bubbles from one side of JRs, and ultrasonication of O2 bubbles produces the cavitation effect, both generating mechanical force to drive the thrombus penetration. The integration of ultrasonication-propelled motion and RGD mediation effectively increases the targeting capabilities of r-JRs to activated platelets. In addition to mechanical thrombolysis, ultrasonication of the released Pc produces 1O2 to destruct fibrin networks of clots. In vitro thrombolysis of whole blood clots shows that ultrasonication of Pc/Ca@r-JRs has a significantly higher thrombolysis rate (73.6%) than those without propelled motion or RGD-mediated clot targeting. In a lower limb thrombosis model, intravenous administration of Pc/Ca@r-JRs indicates 3.4-fold higher accumulations at the clot site than those of JRs, and ultrasonication-propelled motion further increases thrombus retention 2.1 times. Treatment with Pc/Ca@r-JRs and ultrasonication fully removes thrombi and significantly prolongs tail bleeding time. Thus, this study has achieved precise and prompt thrombolysis through selective targeting to clots, efficient penetration into dense networks of thrombi, and SDT-executed thrombolysis.

DMAG, a novel countermeasure for the treatment of thrombocytopenia

BackgroundThrombocytopenia is one of the most common hematological disease that can be life-threatening caused by bleeding complications. However, the treatment options for thrombocytopenia remain limited.MethodsIn this study, giemsa staining, phalloidin staining, immunofluorescence and flow cytometry were used to identify the effects of 3,3ʹ-di-O-methylellagic acid 4ʹ-glucoside (DMAG), a natural ellagic acid derived from Sanguisorba officinalis L. (SOL) on megakaryocyte differentiation in HEL cells. Then, thrombocytopenia mice model was constructed by X-ray irradiation to evaluate the therapeutic action of DMAG on thrombocytopenia. Furthermore, the effects of DMAG on platelet function were evaluated by tail bleeding time, platelet aggregation and platelet adhesion assays. Next, network pharmacology approaches were carried out to identify the targets of DMAG. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to elucidate the underling mechanism of DMAG against thrombocytopenia. Finally, molecular docking simulation, molecular dynamics simulation and western blot analysis were used to explore the relationship between DAMG with its targets.ResultsDMAG significantly promoted megakaryocyte differentiation of HEL cells. DMAG administration accelerated platelet recovery and megakaryopoiesis, shortened tail bleeding time, strengthened platelet aggregation and adhesion in thrombocytopenia mice. Network pharmacology revealed that ITGA2B, ITGB3, VWF, PLEK, TLR2, BCL2, BCL2L1 and TNF were the core targets of DMAG. GO and KEGG pathway enrichment analyses suggested that the core targets of DMAG were enriched in PI3K–Akt signaling pathway, hematopoietic cell lineage, ECM-receptor interaction and platelet activation. Molecular docking simulation and molecular dynamics simulation further indicated that ITGA2B, ITGB3, PLEK and TLR2 displayed strong binding ability with DMAG. Finally, western blot analysis evidenced that DMAG up-regulated the expression of ITGA2B, ITGB3, VWF, p-Akt and PLEK.ConclusionDMAG plays a critical role in promoting megakaryocytes differentiation and platelets production and might be a promising medicine for the treatment of thrombocytopenia.Graphical