Tail Bleeding Assay Research Articles

The humanized platelet glycoprotein VI Fab inhibitor EMA601 protects from arterial thrombosis and ischaemic stroke in mice.

Glycoprotein VI (GPVI) is a platelet collagen/fibrin(ogen) receptor and an emerging pharmacological target for the treatment of thrombotic and thrombo-inflammatory diseases, notably ischaemic stroke. A first anti-human GPVI (hGPVI) antibody Fab-fragment (ACT017/glenzocimab, KD: 4.1 nM) recently passed a clinical phase 1b/2a study in patients with acute ischaemic stroke and was found to be well tolerated, safe, and potentially beneficial. In this study, a novel humanized anti-GPVI antibody Fab-fragment (EMA601; KD: 0.195 nM) was developed that inhibits hGPVI function with very high potency in vitro and in vivo. Fab-fragments of the mouse anti-hGPVI IgG Emf6.1 were tested for functional GPVI inhibition in human platelets and in hGPVI expressing (hGP6tg/tg) mouse platelets. The in vivo effect of Emf6.1Fab was assessed in a tail bleeding assay, an arterial thrombosis model and the transient middle cerebral artery occlusion (tMCAO) model of ischaemic stroke. Using complementary-determining region grafting, a humanized version of Emf6.1Fab (EMA601) was generated. Emf6.1Fab/EMA601 interaction with hGPVI was mapped in array format and kinetics and quantified by bio-layer interferometry. Emf6.1Fab (KD: 0.427 nM) blocked GPVI function in human and hGP6tg/tg mouse platelets in multiple assays in vitro at concentrations ≥5 µg/mL. Emf6.1Fab (4 mg/kg)-treated hGP6tg/tg mice showed potent hGPVI inhibition ex vivo and were profoundly protected from arterial thrombosis as well as from cerebral infarct growth after tMCAO, whereas tail-bleeding times remained unaffected. Emf6.1Fab binds to a so far undescribed membrane proximal epitope in GPVI. The humanized variant EMA601 displayed further increased affinity for hGPVI (KD: 0.195 nM) and fully inhibited the receptor at 0.5 µg/mL, corresponding to a >50-fold potency compared with ACT017. EMA601 is a conceptually novel and promising anti-platelet agent to efficiently prevent or treat arterial thrombosis and thrombo-inflammatory pathologies in humans at risk.

Abstract Tu040: A Novel Anti-Thrombotic and Anti-AF Drug without an Adverse Bleeding Problem

Introduction: Aging is an independent risk factor of atrial fibrillation (AF), which leads to a high mortality due to thromboembolic stroke. But available antithrombotic therapies are limited for the aged AF patients due to aging-associated bleeding risk. We recently revealed critical roles of activated cardiac stress kinase JNK2 in both enhanced AF risk and thrombogenesis in aging through a mechanism of heart-platelet crosstalk. Here, we assessed anti-thrombotic potentials of JNK2 inhibition. Methods: Young (Yg) and aged (2 vs 24 mo) wildtype (WT) mice with and without a JNK2-specific inhibitor (JNK2I, 5 mg/kg i.p.; every other day/4 doses) or meloxicam (0.5 mg/kg/day, i.p.; five days) treatment were used for in vivo FeCl 3 -induced carotid artery occlusion (thrombus formation) and tail bleeding assays. The effects of cardiac JNK2-specific inhibition in thrombogenesis and bleeding were assessed in our unique cardiac-specific mice of MKK7D (inducible overexpression of active MKK7D, a JNK upstream activator) and MKK7D-JNK2dn (overexpressed inactive dominant negative (dn) JNK2 to competitively inhibit endogenous JNK2 in MKK7D mice). Results: Aged mice had a shortened artery occlusion time compared to Yg (5.8±0.2 min vs. 7.1±0.2 min, p<0.001, n=12,7). JNK2I reversed the occlusion time in aged mice to that seen in Yg (p=NS, n=8,7). Yg MKK7D mice also showed a shortened occlusion time compared to WT-controls (p<0.001, n=9,8) that was rescued by cardiac-specific JNK2 inhibition in MKK-JNK2dn mice (p=NS, n=5,8), suggesting a key role of cardiac JNK2 in thrombus formation. Next, we found that 40% of aged WT mice had continuous bleeding compared to its absence in Yg WT. However, meloxicam (an anti-platelet NSAID drug) increased continuous bleeding risk in both aged (83% had it) and Yg (20%) and prolonged the initial clotting time in aged mice (385±56s vs 84±18s in aged controls; p<0.001, n=8,6). In contrast, JNK2I eliminated this bleeding issue, evidenced from the absence of continuous bleeding and shortened initial clotting time compared to aged controls (p=NS, n=8,8). Conclusion: Heart JNK2 enhances thrombotic risk in the blood. JNK2 inhibition could be a promising anti-thrombotic and anti-AF therapeutic approach without adverse bleeding for aged AF patients.

Abstract 154: Hierarchical Redundancy And Contextual Roles Of Vesicle-associated Membrane Proteins (VAMPs) In Platelet Function

Background: Platelet granule secretion, driven by the SNARE complex involving Vesicle-Associated Membrane Proteins (VAMPs) and t-SNAREs, is essential for vascular integrity. We dissected individual and combinatorial contributions of all four platelet VAMPs to granule secretion, hemostasis, and intracellular cargo trafficking. Research Questions: 1. Are specific VAMPs critical for granule fusion, or does total VAMP quantity (mass action) dictate secretion? 2. How do differential VAMP deficiencies impact hemostasis in arterial and venous microenvironments? 3. Do VAMPs influence platelet cargo loading and gene expression? Goals: Investigate individual and combinatorial roles of VAMPs in platelet biology and their context dependent contributions to hemostasis. Approach: We generated V7 -/- 8 -/- and V2 Δ 3 Δ 7 -/- 8 -/- mice, assessing granule secretion (dense, alpha, lysosomal) via ATP, PF4/P-selectin, and β-hexosaminidase/LAMP1 release assays, respectively. Hemostasis in arterial and venous models was evaluated with tail bleeding, FeCl3, and venous puncture injury assays. Cargo levels were analyzed using proteomic arrays, and platelet RNA sequencing identified transcriptional changes. Results: Global V7/V8 deletion modestly reduced secretion compared to isolated V8 deficiency. Surprisingly, while platelet-specific V2 Δ 3 Δ 7 -/- 8 -/- exhibited the most dramatic decrease, complete abolition was absent (p<0.01). Alpha and lysosomal secretion were more affected than densegranule release. Both knockout models bled profusely and failed to form stable arterial thrombi (p<0.01). Notably, only V2 Δ 3 Δ 7 -/- 8 -/- mice displayed significantly prolonged venous bleeding (p<0.01). RNA sequencing revealed >800 upregulated and >500 downregulated transcripts in V7 - /- 8 -/- and V2 Δ 3 Δ 8 -/- platelets, suggesting shared transcriptional control by V7 and secondary VAMPs (V2 and V3). Conclusions: VAMP functions exhibit hierarchical redundancy, with V8 presence minimally impacting dense granule secretion and arterial hemostasis. Alpha granule release is more sensitive to VAMP loss. The requirements for platelet granule secretion for hemostasis are context dependent. The deletion of VAMPs disrupts platelet cargo trafficking and transcription.

The Prediction of Milk Whey Extract (MWE) Bioactive Compounds Based on Proximate Analysis and its Effects on Blood Coagulation: A New Approach

The present work demonstrates the milk whey extraction, proximate analysis, and biochemical characterization of bioactive compounds in MWE. Effects of MWE on plasma coagulation and platelet aggregation. Proximate analysis of MWE was done according to AOAC. Whey protein banding was confirmed in 12%, 15%, and 18% SDS-PAGEs. A quantitative analysis of bioactive compounds was done. The anticoagulant effects of MWE were tested using plasma recalcification time in both PRP and PPP, further confirmed by PT and APTT assays and in tail bleeding assays at concentrations of 0-100 µg. The non-toxic property of MWE was screened by edema, hemorrhage, and direct hemolytic activities. MWE proximate analysis showed the presence of both micro- and macronutrients. Qualitative analysis confirmed the presence of proteins and carbohydrates. A similar protein-banding pattern was observed in 12%, 15%, and 18% SDS-PAGE’s in both reduced and non-reduced conditions. MWE prolonged the clotting time of human citrated plasma, both PRP and PPP, against the control 210 sec to 770 sec, suggesting its anticoagulant property. MWE delayed the clot progress of only the APTT ratio (8.99 ± 0.09), and PT was not altered, suggesting its role in an intrinsic pathway of blood coagulation. MWE exhibited antiplatelet activity in PRP against ADP and epinephrine-induced platelet aggregation. The percentage of inhibition was 74% and 100% for ADP and epinephrine, respectively. Interestingly, MWE exhibits nontoxic properties, it does not cause hemolysis, hemorrhage, or edema. Milk whey extraction and studies confirm bioactive compounds, and these exhibit antithrombotic, antiplatelet, and non-toxic properties. Hence, MWE not only acts as a good nutritive source but also may prove tobe a therapeutic bioactive compound in the management of cardiovascular disease.

A low bleeding risk thrombolytic agent: citPA5.

Alteplase is a cornerstone thrombolytic agent in clinical practice but presents a potential bleeding risk. Stroke patients need pre-screening to exclude haemorrhagic stroke before using alteplase. In this study, we develop a new thrombolytic agent citPA5, characterized by an enhanced safety profile and minimal bleeding tendency. A clot lysis agent, named citPA5, is developed based on rtPA with point mutations to completely suppress its proteolytic activity in the absence of fibrin. In the presence of fibrin, citPA5 exhibited significantly higher fibrinolytic activity (a 15.8-fold increase of kcat/Km). Furthermore, citPA5 showed resistance to endogenous fibrinolysis inhibitor, PAI-1, resulting in enhanced potency. In a series of safety evaluation experiments, including thrombelastography assay, mice tail bleeding assay, and a murine intracerebral haemorrhage (ICH) model, citPA5 did not cause systemic bleeding or worsen ICH compared with alteplase. This highlights the low risk of bleeding associated with citPA5. Finally, we found that citPA5 effectively improved cerebral blood flow and reduced infarct volume in a carotid embolism-induced stroke model. This clot lysis agent, citPA5, not only exhibits a low risk of bleeding but also demonstrates highly effective thrombolysis capabilities. As a result, citPA5 shows great potential for administration prior to the classification of stroke types, making it possible for use in ambulances at the onset of stroke when symptoms are identified. The findings presented in this study also suggest that this strategy could be applied to develop a new generation of fibrinolytic drugs that offer greater safety and specificity in targeting fibrin.

A mouse model of the protease-activated receptor 4 Pro310Leu variant has reduced platelet reactivity

BackgroundProtease-activated receptor 4 (PAR4) mediates thrombin signaling on platelets and other cells. Our recent structural studies demonstrated that a single nucleotide polymorphism in extracellular loop 3 and PAR4-P310L (rs2227376) leads to a hyporeactive receptor. ObjectivesThe goal of this study was to determine how the hyporeactive PAR4 variant in extracellular loop 3 impacts platelet function in vivo using a novel knock-in mouse model (PAR4-322L). MethodsA point mutation was introduced into the PAR4 gene F2rl3 via CRISPR/Cas9 to create PAR4-P322L, the mouse homolog to human PAR4-P310L. Platelet response to PAR4 activation peptide (AYPGKF), thrombin, ADP, and convulxin was monitored by αIIbβ3 integrin activation and P-selectin translocation using flow cytometry or platelet aggregation. In vivo responses were determined by the tail bleeding assay and the ferric chloride–induced carotid artery injury model. ResultsPAR4-P/L and PAR4-L/L platelets had a reduced response to AYPGKF and thrombin measured by P-selectin translocation or αIIbβ3 activation. The response to ADP and convulxin was unchanged among genotypes. In addition, both PAR4-P/L and PAR4-L/L platelets showed a reduced response to thrombin in aggregation studies. There was an increase in the tail bleeding time for PAR4-L/L mice. The PAR4-P/L and PAR4-L/L mice both showed an extended time to arterial thrombosis. ConclusionPAR4-322L significantly reduced platelet responsiveness to AYPGKF and thrombin, which is in agreement with our previous structural and cell signaling studies. In addition, PAR4-322L had prolonged arterial thrombosis time. Our mouse model provides a foundation to further evaluate the role of PAR4 in other pathophysiological contexts.

Carbonization of quercetin into nanogels: a leap in anticoagulant development.

Quercetin, a flavonoid abundantly found in onions, fruits, and vegetables, is recognized for its pharmacological potential, especially for its anticoagulant properties that work by inhibiting thrombin and coagulation factor Xa. However, its clinical application is limited due to poor water solubility and bioavailability. To address these limitations, we engineered carbonized nanogels derived from quercetin (CNGsQur) using controlled pyrolysis and polymerization techniques. This led to substantial improvements in its anticoagulation efficacy, water solubility, and biocompatibility. We generated a range of CNGsQur by subjecting quercetin to varying pyrolytic temperatures and then assessed their anticoagulation capacities both in vitro and in vivo. Coagulation metrics, including thrombin clotting time (TCT), activated partial thromboplastin time (aPTT), and prothrombin time (PT), along with a rat tail bleeding assay, were utilized to gauge the efficacy. CNGsQur showed a pronounced extension of coagulation time compared to uncarbonized quercetin. Specifically, CNGsQur synthesized at 270 °C (CNGsQur270) exhibited the most significant enhancement in TCT, with a binding affinity to thrombin exceeding 400 times that of quercetin. Moreover, variants synthesized at 310 °C (CNGsQur310) and 290 °C (CNGsQur290) showed the most substantial delays in PT and aPTT, respectively. Our findings indicate that the degree of carbonization significantly influences the transformation of quercetin into various CNGsQur forms, each affecting distinct coagulation pathways. Additionally, both intravenous and oral administrations of CNGsQur were found to extend rat tail bleeding times by up to fivefold. Our studies also demonstrate that CNGsQur270 effectively delays and even prevents FeCl3-induced vascular occlusion in a dose-dependent manner in mice. Thus, controlled pyrolysis offers an innovative approach for generating quercetin-derived CNGs with enhanced anticoagulation properties and water solubility, revealing the potential for synthesizing self-functional carbonized nanomaterials from other flavonoids for diverse biomedical applications.

Apoptosis Signal-Regulating Kinase 1 (Ask1) Inhibition Prevents In Vivo Thrombosis without Compromising Hemostasis

Cardiovascular diseases are currently among the leading causes of death in mankind. Although significant progress has been made in prevention and treatment, the currently available pharmacological interventions have a significant rate of severe bleeding side effects due to their effect on hemostasis. Recently, we have shown that apoptosis signal-regulating kinase 1 (ASK1) plays an important role in platelet activation and thrombus formation. Here, we show that two chemically distinct ASK1 inhibitors, dose-dependently inhibited thrombin-induced phosphorylation of ASK1 and its downstream effectors MKK3/4/6, p38 MAPK, and cPLA2 in human platelets, as detected by western blotting using phospho-specific antibodies. ASK1 inhibitors also dose-dependently inhibited platelet a granule release and α IIbβ 3 activation which are the readouts for inside-out signaling events as analyzed by flow cytometry. Moreover, pretreatment of human platelets with ASK1 inhibitors completely impaired clot retraction and platelet spreading on immobilized fibrinogen two events regulated by outside-in signaling. To investigate the in-vivo effect of Ask1 inhibitor on thrombus formation, we intraperitoneally injected C57BL/6 mice with Ask1 inhibitor (1mg/kg), and the blood was withdrawn at 1-, 4-, and 24-hour time points and analyzed for in vitro thrombus formation on collagen matrix under arterial flow. Ask1 inhibitor significantly decreased thrombus growth ( P<0.01) which was sustained for at least 24 hours. Platelet adhesion to collagen was not affected as determined by surface coverage.In a 10% FeCl 3 -induced carotid artery injury model of thrombosis, we found that Ask1 inhibitor (100μg/kg)showed a significantly extended time of vessel occlusion ( P<0.01) . Moreover, Ask1 inhibitor (100μg/kg) treated C57BL/6 mice were protected from collagen/epinephrine-induced pulmonary thromboembolism ( P<0.001). Furthermore, in a well-established ischemic stroke model of transient middle cerebral artery occlusion, C57BL/6 mice treated with Ask1 inhibitor (1mg/kg) showed increased survival and improved neurological deficits than vehicle treated mice. The extent of the brain damage was assessed by staining the brain sections with 2% 2,3,5-triphenyltetrazoliun chloride, which showed significant reduction in infarct size and edema ( P<0.0001). Interestingly, mice treated with Ask1 inhibitor (1mg/kg), a 10-fold more than the amount needed to inhibit thrombosis, had no effect on hemostasis as observed by both the tail bleeding assay and laser-induced cremaster injury model. Whereas clopidogrel (5-30mg/kg) showed significantly increased ( P<0.0001) tail bleeding time. Next, we performed cerebral bleeding assay in which brain injury was induced by inserting a 26-gauge needle through the skull in the cerebrum 2mm laterally to the bregma and 4mm deep into the cerebrum and rotated 360 degrees. After 20 minutes the extent of bleeding in the brain was assessed by imaging. Treatment of C57BL/6 mice with clopidogrel (30mg/kg) showed significantly enhanced bleeding ( P<0.05) compared to vehicle treated mice. However, Ask1 inhibitor treated mice had same extent of bleeding as that of vehicle treated mice. Additionally, a liver bleeding assay was performed by excising a calibrated piece (~4-5mg) of inferior edge of the right lobe of the liver of the mouse under anesthesia. After 20 minutes the bleeding was assessed by counting red blood cells in the peritoneal lavage. As observed in brain bleeding assay, treatment of mice with clopidogrel (30mg/kg), showed significantly increased liver bleeding ( P<0.05) compared to vehicle treated mice whereas Ask1 inhibitor treated mice had same extent of liver bleeding as that of vehicle treated mice. Taken together, our results provide evidence that ASK1 inhibitors are novel therapeutic agents to combat thrombotic disorders without any side effects of bleeding, which is inherent to current anti-thrombotic drugs.

Guianensin, a Simulium guianense salivary protein, has broad anti-hemostatic and anti-inflammatory properties.

Salivary glands from blood-feeding arthropods secrete several molecules that inhibit mammalian hemostasis and facilitate blood feeding and pathogen transmission. The salivary functions from Simulium guianense, the main vector of Onchocerciasis in South America, remain largely understudied. Here, we have characterized a salivary protease inhibitor (Guianensin) from the blackfly Simulium guianense. A combination of bioinformatic and biophysical analyses, recombinant protein production, in vitro and in vivo experiments were utilized to characterize the molecula mechanism of action of Guianensin. Kinetics of Guianensin interaction with proteases involved in vertebrate inflammation and coagulation were carried out by surface plasmon resonance and isothermal titration calorimetry. Plasma recalcification and coagulometry and tail bleeding assays were performed to understand the role of Guianensin in coagulation. Guianensin was identified in the sialotranscriptome of adult S. guianense flies and belongs to the Kunitz domain of protease inhibitors. It targets various serine proteases involved in hemostasis and inflammation. Binding to these enzymes is highly specific to the catalytic site and is not detectable for their zymogens, the catalytic site-blocked human coagulation factor Xa (FXa), or thrombin. Accordingly, Guianensin significantly increased both PT (Prothrombin time) and aPTT (Activated partial thromboplastin time) in human plasma and consequently increased blood clotting time ex vivo. Guianensin also inhibited prothrombinase activity on endothelial cells. We show that Guianensin acts as a potent anti-inflammatory molecule on FXa-induced paw edema formation in mice. The information generated by this work highlights the biological functionality of Guianensin as an antithrombotic and anti-inflammatory protein that may play significant roles in blood feeding and pathogen transmission.

GPIIb/IIIa Receptor Targeted Rutin Loaded Liposomes for Site-Specific Antithrombotic Effect.

Rutin (RUT) is a flavonoid obtained from a natural source and is reported for antithrombotic potential, but its delivery remains challenging because of its poor solubility and bioavailability. In this research, we have fabricated novel rutin loaded liposomes (RUT-LIPO, nontargeted), liposomes conjugated with RGD peptide (RGD-RUT-LIPO, targeted), and abciximab (ABX-RUT-LIPO, targeted) by ethanol injection method. The particle size, ζ potential, and morphology of prepared liposomes were analyzed by using DLS, SEM, and TEM techniques. The conjugation of targeting moiety on the surface of targeted liposomes was confirmed by XPS analysis and Bradford assay. In vitro assessment such as blood clot assay, aPTT assay, PT assay, and platelet aggregation analysis was performed using human blood which showed the superior antithrombotic potential of ABX-RUT-LIPO and RGD-RUT-LIPO liposomes. The clot targeting efficiency was evaluated by in vitro imaging and confocal laser scanning microscopy. A significant (P < 0.05) rise in the affinity of targeted liposomes toward activated platelets was demonstrated that revealed their remarkable potential in inhibiting thrombus formation. Furthermore, an in vivo study executed on Sprague Dawley rats (FeCl3 model) demonstrated improved antithrombotic activity of RGD-RUT-LIPO and ABX-RUT-LIPO compared with pure drug. The pharmacokinetic study performed on rats demonstrates the increase in bioavailability when administered as liposomal formulation as compared to RUT. Moreover, the tail bleeding assay and clotting time study (Swiss Albino mice) indicated a better antithrombotic efficacy of targeted liposomes than control preparations. Additionally, biocompatibility of liposomal formulations was determined by an in vitro hemolysis study and cytotoxicity assay, which showed that they were hemocompatible and safe for human use. A histopathology study on rats suggested no severe toxicity of prepared liposomal formulations. Thus, RUT encapsulated nontargeted and targeted liposomes exhibited superior antithrombotic potential over RUT and could be used as a promising carrier for future use.

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.

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.

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.

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.

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.

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.

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.

Influence of Vincristine, Clinically Used in Cancer Therapy and Immune Thrombocytopenia, on the Function of Human Platelets.

Vincristine is a clinically used antimicrotubule drug for treating patients with lymphoma. Due to its property of increasing platelet counts, vincristine is also used to treat patients with immune thrombocytopenia. Moreover, antiplatelet agents were reported to be beneficial in thrombotic thrombocytopenic purpura (TTP). Therefore, we investigated the detailed mechanisms underlying the antiplatelet effect of vincristine. Our results revealed that vincristine inhibited platelet aggregation induced by collagen, but not by thrombin, arachidonic acid, and the thromboxane A2 analog U46619, suggesting that vincristine exerts higher inhibitory effects on collagen-mediated platelet aggregation. Vincristine also reduced collagen-mediated platelet granule release and calcium mobilization. In addition, vincristine inhibited glycoprotein VI (GPVI) signaling, including Syk, phospholipase Cγ2, protein kinase C, Akt, and mitogen-activated protein kinases. In addition, the in vitro PFA-100 assay revealed that vincristine did not prolong the closure time, and the in vivo study tail bleeding assay showed that vincristine did not prolong the tail bleeding time; both findings suggested that vincristine may not affect normal hemostasis. In conclusion, we demonstrated that vincristine exerts antiplatelet effects at least in part through the suppression of GPVI signaling. Moreover, this property of antiplatelet activity of vincristine may provide additional benefits in the treatment of TTP.

Pyruvate Dehydrogenase Kinase Modulates Platelet Function and Thrombosis

Background and objective: Pyruvate dehydrogenase kinases (PDK 1-4) are mitochondrial enzymes that regulate the activity of pyruvate dehydrogenase (PDH) complex, which converts pyruvate (end product of glycolysis in cytosol) to acetyl coenzyme A that is further oxidized to produce energy (oxidative phosphorylation; OXPHOS). Like most normal cells, resting platelets rely primarily on OXPHOS to generate ATP, whereas activated platelets exhibit a high level of aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen, a phenomenon referred to as the Warburg effect in tumor cells). It seems paradoxical that why platelets prefer aerobic glycolysis instead of OXPHOS. The rate of ATP generation is faster in aerobic glycolysis compared to OXPHOS, which is well suited for high-energy demand of activated platelets to form blood clot. We tested the hypothesis that manipulating aerobic glycolysis (metabolic reprogramming), by down regulating PDK activity in the mitochondria of platelets, will inhibit platelet function and thereby thrombosis.Methods: Global knock out or floxed PDK mice does not exists, therefore we used dichloroacetic acid (DCA), a potent inhibitor of PDKs, known to divert metabolism back from aerobic glycolysis to OXPHOS. DCA has been successfully used in clinical trials to treat several type of cancer. In human and murine platelets, using standardized platelet in vitro assays, we determined the mechanistic role of PDK in regulating platelet function. Rate of glycolysis and mitochondrial respiration was measured using the Seahorse XF24 extracellular flux analyzer. Using intravital microscopy, thrombus growth was evaluated in vitro (microfluidics flow chamber) and in vivo (FeCl3-induced carotid artery thrombosis) models.Results: Using, extracellular flux analysis, we first confirmed that activated platelets have higher aerobic glycolysis. With both human and mice platelets, we found that DCA (10 and 25 mM) significantly inhibited in vitro platelet aggregation to collagen and convulxin-induced signaling, but not thrombin or ADP-induced signaling in a dose-dependent manner (P<0.05 vs. vehicle treated). DCA also inhibited static adhesion of washed platelets to collagen in dose-dependent manner. Furthermore, we found that DCA significantly inhibited tyrosine phosphorylation of Syk and PLCγ2 in the GPVI signaling pathway with both mice and human platelets (P<0.05 vs. vehicle treated). Metabolic profiling, using extracellular flux analysis, showed that DCA significantly inhibited extracellular acidification rate in convulxin-stimulated platelets in dose-dependent manner. Using microfluidics flow chamber, we found that both mouse and human whole blood treated with DCA formed small thrombi when perfused over collagen for 10 min at a arterial shear rate of 1500s-1 (P<0.05 vs. vehicle control), suggesting that inhibiting PDK with DCA limits thrombus growth. Finally, wild-type mice pre-treated with DCA (600 mg/Kg body weight) were less susceptible to thrombosis in FeCl3-induced arterial thrombosis model (P<0.05 vs. control, N=10 mice/group), without altered hemostasis (tail-bleeding assay). The precise mechanism how aerobic glycolysis modulates GPVI signaling pathway is unclear and is currently under investigation.Conclusion: Our results suggest that PDK present in mitochondria regulates platelet function and thrombus growth, most likely via GPVI signaling pathway. DisclosuresNo relevant conflicts of interest to declare.

Phosphorylation of Y311 on Pkcδ Regulates Protease Activated Receptor 4-Mediated Signaling in Platelets

Background: Protein kinase C (PKC) is known to regulate various aspects of platelet functions. In platelets, PKCδ is known to both positively and negatively regulate dense granule secretion in response to PAR and GPVI agonists, respectively. Hence, we speculate that the differential regulation of PKCδ in platelets is due to differences in tyrosine phosphorylation of PKCδ.Results: We investigated several phosphorylation sites on PKCδ and found that Y311 is phosphorylated downstream of PAR agonists to a greater extent than GPVI agonists in human and murine platelets when feedback is blocked. Hence, the focus of this study is to characterize the function of PKCδ tyrosine Y311 in platelets. Therefore, we generated PKCδY311F knock-in mice to study the contribution of Y311 on PKCδ to PAR-4-mediated platelet activation. PKCδY311F mice are viable and have no gross abnormalities. Washed platelets from PKCδY311F mice have reduced aggregation and dense granule secretion after stimulation with AYPGKF, a PAR-4 agonist (Fig. 1) indicating its importance in PAR-4 mediated signaling. In an in vivo tail-bleeding assay, PKCδY311 mice have significantly enhanced tail-bleeding times compared to wild-type (WT) littermate controls (Fig. 2). Finally, PKCδY311F mice exhibit longer time to occlusion compared to WT control using a ferric chloride in vivo thrombosis model. This indicates that the phosphorylation of PKCδ Y311 is pro-thrombotic. At the molecular level, PKCδY311 was phosphorylated in Fyn-/- null platelets but not in Lyn-/- platelets by AYPGKF, indicating that Lyn phosphorylates this tyrosine residue.Conclusions: Phosphorylation of PKCδ on Y311 is important for regulation of platelet functional responses initiated by thrombin. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.