Laser-induced Thrombosis Research Articles

Imaging Hypoxia to Predict Primary Neuronal Cell Damage in Branch Retinal Artery Occlusion.

To develop a reliable method to generate a mouse model of branch retinal artery occlusion (BRAO) using laser-induced thrombosis of a major artery in the mouse retina. Also, to develop a reliable method to detect retinal hypoxia as predictive biomarker for the risk of neuronal cell damage in BRAO. A reliable and reproducible model of laser-induced BRAO was developed in mouse retina using Rose Bengal. To characterize retinal hypoxia in BRAO, pimonidazole immunostaining and HYPOX-4 molecular imaging methods were used. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) was used to characterize neuronal cell damage in the BRAO retina. Expression of mRNA in retinal tissues from BRAO and age-matched control retinas were analyzed using qRT-PCR. Occlusion of a branch retinal artery near the optic nerve head (ONH) caused a pattern of retinal tissue hypoxia covering about 12.5% of the entire retina. TUNEL-positive cells were localized in all layers in BRAO retinal tissue cross sections. In addition, qRT-PCR data analysis suggests that BRAO is associated with both inflammation and hypoxia. This study provides a reliable method for BRAO in mouse retina and demonstrates the utility of molecular imaging method to detect retinal hypoxia as predictive biomarker for the risk of neuronal cell damage in BRAO. In addition, our data suggest that BRAO retinas are associated with inflammation and also associated with hypoxia-related neuronal cell damage. Imaging areas of retinal hypoxia may provide accurate diagnosis, evaluating retinal tissue injury from BRAO.

Thioredoxin-related transmembrane protein 1 negatively regulates coagulation and phosphatidylserine exposure

BackgroundFive secreted platelet protein disulfide isomerases (PDIs) and 1 transmembrane PDI regulate platelet function and thrombosis. Thioredoxin-related transmembrane protein 1 (TMX1) was the first member of the PDI family found to negatively regulate platelet aggregation and platelet accumulation in vivo. The effect of TMX1 on coagulation is unknown. ObjectivesTo determine the effect of TMX1 on coagulation. MethodsTMX1-/- mice were used to study platelet accumulation and fibrin deposition in vivo in the laser-induced thrombosis injury model. Annexin V deposition at the site of vascular injury was studied using conditional TMX1 knockout mice. Annexin V binding to platelets was studied using human platelets, anti-TMX1 antibodies, and TMX1-deficient platelets. ResultsTMX1-/- mice had increased fibrin deposition that was reversed with infusion of recombinant TMX1. Infusion of recombinant TMX1 inhibited platelet accumulation and fibrin deposition in wild-type mice and inhibited fibrin deposition in β3-null mice. Platelet accumulation is absent in β3-null mice, suggesting that TMX1 inhibits coagulation independently of platelets. Annexin V binding was increased in activated human platelets incubated with an anti-TMX1 antibody and mouse platelets lacking TMX1. Addition of recombinant TMX1 decreased annexin V binding to platelets. Annexin V binding was increased at the site of vascular injury in Tie2-Cre/TMX1fl/fl mice deficient in endothelial cell TMX1. ConclusionTMX1 decreases coagulation at the site of vascular injury and negatively regulates phosphatidylserine exposure on endothelial cells and platelets.

HDAC Inhibitor CS014 Attenuates Thrombosis Alone and in Combination with Rivaroxaban without Increased Risk of Bleeding

Introduction: Targeting the human platelet for prevention of occlusive thrombotic events remains an active area of research due to the high incidence of myocardial infarction and stroke due to platelet-rich thrombotic clots. While, many patients taking FXa anticoagulant therapy require further inhibition of thrombosis, dual therapy including anticoagulant plus antiplatelet drugs result in an increased risk for bleeding. The current study sought to determine if a histone deacetylase (HDAC) inhibitor, either alone or in combination with the FXa inhibitor rivaroxaban, is able to effectively inhibit platelet and fibrin formation at the site of vascular injury without causing a significant increase in the bleeding risk in mice. Methods and Results: Thrombosis and bleeding models in the mouse were assessed in the presence of the HDAC inhibitors valproic acid (VPA) or the newly developed HDAC inhibitor, CS014. Both CS014 and VPA significantly, dose-dependently inhibit platelet accumulation and fibrin formation at the site of a vascular injury using the laser-induced cremaster thrombosis assay model. Further, to determine if CS014 has the potential to be used in conditions where an anticoagulant is already on board, mice were dosed for 5 days with rivaroxaban alone or in combination with CS014 and were assessed for prevention of laser-induced thrombosis in the cremaster arteriole. Both rivaroxaban and CS014 alone cause significant attenuation of clot formation. To determine if the combination results in an increased risk for bleeding, blood from mice treated with CS014 or rivaroxaban alone, or in combination was assessed for coagulation activity and clot strength using thromboelestography (TEG). Rivaroxaban was found to significantly delay onset of clotting in TEG whereas CS014 did not show any delay in coagulation or clotting with TEG. In combination CS014 was not observed to cause any additional delay in clotting time or decrease in maximal clot strength compared to rivaroxaban alone. Finally, a tail vein bleeding assay was conducted to determine if CS014 alone or in combination with rivaroxaban result in an increased risk for bleeding. Neither CS014 alone, rivaroxaban alone, or the combination of CS014 plus rivaroxaban resulted in increased bleeding time in the resected tail experiment. Conclusions: The data presented here suggests that while CS014 and rivaroxaban are each effective antithrombotic agents, they work well together to minimize clot formation without altering coagulation or clotting compared to rivaroxaban alone. Hence, CS014 has the potential to enrich the toolbox of anticoagulant therapies in patients with a high risk of thrombotic events.

Utility of Platelet Endothelial Cell Adhesion Molecule 1 in the Platelet Activity Assessment in Mouse and Human Blood.

In our previous study, we introduced the platelet endothelial cell adhesion molecule 1 (PECAM-1)/thrombus ratio, which is a parameter indicating the proportion of PECAM-1 in laser-induced thrombi in mice. Because PECAM-1 is an antithrombotic molecule, the higher the PECAM-1/thrombus ratio, the less activated the platelets. In this study, we used an extracorporeal model of thrombosis (flow chamber model) to verify its usefulness in the assessment of the PECAM-1/thrombus ratio in animal and human studies. Using the lipopolysaccharide (LPS)-induced inflammation model, we also evaluated whether the PECAM-1/thrombus ratio determined in the flow chamber (without endothelium) differed from that calculated in laser-induced thrombosis (with endothelium). We observed that acetylsalicylic acid (ASA) decreased the area of the thrombus while increasing the PECAM-1/thrombus ratio in healthy mice and humans in a dose-dependent manner. In LPS-treated mice, the PECAM-1/thrombus ratio decreased as the dose of ASA increased in both thrombosis models, but the direction of change in the thrombus area was inconsistent. Our study demonstrates that the PECAM-1/thrombus ratio can more accurately describe the platelet activation status than commonly used parameters such as the thrombus area, and, hence, it can be used in both human and animal studies.

Multidirectional Effects of Tormentil Extract on Hemostasis in Experimental Diabetes.

In our previous study, we showed that ellagitannin- and procyanidin-rich tormentil extract (TE) decreased experimental arterial thrombosis in normoglycemic rats through platelet inhibition. TE also slightly increased coagulation and attenuated fibrinolysis; however, these effects did not nullify the antithrombotic effect of TE. The present study aimed to assess whether TE exerts antithrombotic activity in streptozotocin (STZ)-induced diabetes, which is characterized by pre-existing increased coagulation and impaired fibrinolysis, in vivo and ex vivo thrombosis assays. TE (100, 200, or 400 mg/kg, p. o.) was administered for 14 days to STZ-induced diabetic rats and mice. TE at 100 mg/kg dose decreased the thrombus area in the mice model of laser-induced thrombosis through its potent antiplatelet effect. However, TE at 200 mg/kg dose increased thrombus weight in electrically induced arterial thrombosis in rats. The prothrombotic effect could be due to increased coagulation and attenuated fibrinolysis. TE at 400 mg/kg dose also improved vascular functions, which was mainly reflected as an increase in the arterial blood flow, bleeding time prolongation, and thickening of the arterial wall. However, TE at 400 mg/kg dose did not exert antithrombotic effect. Summarizing, the present results show that TE may exert multidirectional effects on hemostasis in STZ-induced diabetic rats and mice. TE inhibited platelet activity and improved endothelial functions, but it also showed unfavorable effects by increasing the activity of the coagulation system and by inhibiting fibrinolysis. These contrasting effects could be the reason for model-specific influence of TE on the thrombotic process in STZ-induced diabetes.

Changes in the Relative Nitric Oxide Content in the Cortex of a Rat Brain in the Acute Ischemia Model

This study uses the electron paramagnetic resonance (EPR) method to study changes in the relative nitric oxygen content in the cortex of a rat brain in conditions of acute ischemia. Laser-induced vascular thrombosis is used as the model of ischemia. Rose bengal is administered into the bloodstream beforehand. In response to laser beaming, it generates reactive oxygen species, which induces protein coagulation, vascular wall damage, and, as a consequence, the formation of thrombus. It is shown histologically that the acute ischemic process ends a day after the laser surgery, and active remission, with the corresponding morphological changes in the area of the lesion, starts within three days. The relative nitric oxide content is determined 1 and 3 days after the brain vascular photothrombosis using the NO–Fe–DETC paramagnetic complex as a spin trap. The nitric oxide content is estimated by the extreme lower field of the EPR spectrum of this complex. It is shown that a significant increase in the relative nitric oxide (NO) content occurs on the third day after laser surgery and coincides with the beginning of remission after the acute ischemia. A possible role of nitric oxide in the remission is discussed in the study.

Selatogrel, the reversible and specific P2Y12 receptor antagonist, does not interfere with haemostasis

Abstract Background Combination of a P2Y12 receptor (P2Y12R) antagonist (clopidogrel, prasugrel, ticagrelor) with aspirin is the recommended standard of care for patients with acute coronary syndrome. Selatogrel is a reversible and potent antagonist of P2Y12R. Interestingly, in an experimental thrombosis model in rat, at equivalent antithrombotic effect, blood loss was lower in the presence of selatogrel, compared with clopidogrel or ticagrelor. Purpose To characterise the lower risk of bleeding previously observed with selatogrel Methods Mechanistic studies were performed to profile laser-induced thrombosis in wild-type and P2Y12 deficient mice with real-time intravital microscopy. Ticagrelor and clopidogrel were used as selatogrel comparators. Results Selatogrel, ticagrelor and clopidogrel dose-dependently inhibited laser-induced platelet thrombus formation. At maximal antithrombotic effect, only small mural platelets aggregates, corresponding to the haemostatic seals, were present. The phenotype of these haemostatic seals depended on the P2Y12R antagonist used. In the presence of clopidogrel or ticagrelor, the stability of haemostatic seals was reduced. In contrast, in the presence of selatogrel, the apparent stability was not disturbed. Moreover, equivalent antithrombotic dosing regimens of ticagrelor and clopidogrel interfered with laser-induced calcium mobilisation in the endothelium, restricted subsequent neutrophil adhesion and thus reduced fibrin-mediated stabilisation of the haemostatic seals in wild type mice. The effects of ticagrelor were also observed in P2Y12R-deficient mice, indicating that the effects are P2Y12R independent and off-target. In contrast, an equivalent antithrombotic dosing regimen of selatogrel did not interfere with the process of haemostasis in wild-type or P2Y12R-deficient mice. The degree of interference with the stability of the haemostatic seals correlated with the blood loss profile. The dosing regimens of clopidogrel and ticagrelor, corresponding to the equivalent antithrombotic effects, induced a more pronounced increase in blood loss than that observed with selatogrel. Conclusion Our data offer a novel mechanistic explanation for the differences in bleeding risk of clopidogrel, ticagrelor and selatogrel. Clopidogrel and ticagrelor were found to interfere with haemostasis due to off-target activities. In contrast, selatogrel did not interfere with haemostasis in wild-type and P2Y12-deficient mice, inferring that the process of haemostasis, as defined by formation of haemostatic seals, is independent of P2Y12R. In addition, our data emphasize that the absence of interference with haemostasis is paramount to preserve the advantage of P2Y12R antagonism. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Idorsia Pharmaceuticals Ltd. Allschwil, Switzerland

Injury Length and Arteriole Constriction Shape Clot Growth and Blood-Flow Acceleration in a Mouse Model of Thrombosis.

Quantitative relationships between the extent of injury and thrombus formation in vivo are not well understood. Moreover, it has not been investigated how increased injury severity translates to blood-flow modulation. Here, we investigated interconnections between injury length, clot growth, and blood flow in a mouse model of laser-induced thrombosis. Approach and Results: Using intravital microscopy, we analyzed 59 clotting events collected from the cremaster arteriole of 14 adult mice. We regarded injury length as a measure of injury severity. The injury caused transient constriction upstream and downstream of the injury site resulting in a 50% reduction in arteriole diameter. The amount of platelet accumulation and fibrin formation did not depend on arteriole diameter or deformation but displayed an exponentially increasing dependence on injury length. The height of the platelet clot depended linearly on injury length and the arteriole diameter. Upstream arteriolar constriction correlated with delayed upstream velocity increase, which, in turn, determined downstream velocity. Before clot formation, flow velocity positively correlated with the arteriole diameter. After the onset of thrombus growth, flow velocity at the injury site negatively correlated with the arteriole diameter and with the size of the above-clot lumen. Injury severity increased platelet accumulation and fibrin formation in a persistently steep fashion and, together with arteriole diameter, defined clot height. Arterial constriction and clot formation were characterized by a dynamic change in the blood flow, associated with increased flow velocity.

New approaches for the assessment of platelet activation status in thrombus under flow condition using confocal microscopy.

The goal of the study was the assessment of heterogeneous platelet activation status in thrombus. In a ferric(III) chloride (FeCl3) thrombosis (intravital) model of C57BL/6J mice, the area of irreversibly activated (phosphatidylserine (PS)-positive) platelets was assessed after 1-s exposure of a vessel to FeCl3. In a laser-induced thrombosis (intravital) model of GFP mice, the area of the thrombus composed of PS-negative platelets was evaluated. The ratio of the area of PECAM-1 to the area of the thrombus was used as a marker to assess the activity of PS-negative platelets. In the in vitro flow chamber model, the thrombus area (PS-negative and PS-positive platelets) and the platelet activation index (ratio of the area of PS-positive platelets to the area of thrombus) were determined. To assess platelet activation status with these models, acetylsalicylic acid (ASA) and iloprost (Ilo) were used. In the FeCl3 thrombosis, ASA (10mg/kg, 100mg/kg) decreased the area of PS-positive platelets. In the laser thrombosis, ASA (10mg/kg) decreased the thrombus area, but the decrease in platelet activity was evident even at 3mg/kg by an increased PECAM-1/thrombus ratio. In the flow chamber, ASA (0.02mg/ml, 0.2mg/ml) equally decreased the platelet activation index, whereas only at 0.2mg/ml, it decreased the thrombus area. Ilo (3.6ng/ml, 36ng/ml) decreased the thrombus area but at 36ng/ml increased the platelet activation index. We showed that intravital models and flow chamber provide a detailed assessment of platelet activation status and the mechanism of drug action.

The selective sphingosine 1-phosphate receptor 1 modulator RP101075 improves microvascular circulation after cerebrovascular thrombosis.

Sphingosine-1-phosphate receptor (S1PR) modulators provide protection in preclinical and clinical studies for ischemic stroke, but the influences of S1PR modulation on microvascular thrombosis remain poorly understood. This study investigates the impact of a selective S1PR1 modulator RP101075 on microvascular circulation in a mouse model of laser-induced thrombosis. The flow velocity of cortical arterioles in mice was measured in vivo under 2-photon laser scanning microscopy. Thrombosis was induced in cortical arterioles by laser irritation. At 30 min after laser-induced thrombosis, mice were treated with either RP101075 or vehicle. RP101075 did not alter the flow velocity of cortical arterioles under physiologic conditions. Laser-induced thrombosis led to a pronounced reduction of flow velocity in cortical arterioles that persisted for ≥90 min. The reduction of flow velocity in cortical arterioles following thrombosis was significantly attenuated following RP101075 treatment. RP101075 did not significantly affect coagulation time, bleeding time, heart rate, and blood pressure. In addition, RP101075 treatment reduced thrombus volume, which was accompanied by a reduction of leukocyte content in the thrombus. Our findings demonstrate that the selective S1PR1 modulator RP101075 improves microvascular circulation after thrombosis, implying a component of improved microvascular circulation to the benefit of S1PR modulation in cerebral ischemia.-Li, H., Zhou, X., Li, Y., Ma, X., Gonzales, R. J., Qiu, S., Shi, F.-D., Liu, Q. The selective sphingosine 1-phosphate receptor 1 modulator RP101075 improves microvascular circulation after cerebrovascular thrombosis.

Potent Anti-Platelet Metabolite, 12-HETrE, Inhibits Platelet Activation and Thrombosis In Vivo Via Activation of the IP Receptor

Platelet activation is a critical component of clot formation. Under pathophysiological conditions platelet-mediated clot formation can lead to an occlusive thrombotic event resulting in myocardial infarction and stroke. Regulation of platelet reactivity relies on an understanding of the complex intra-platelet signaling that takes place following activation of various receptors on the platelet surface. Our lab recently identified a novel oxidized metabolite known as 12-HETrE that is formed from 12-lipoxygenase (12-LOX) oxidation of the ω-6 fatty acid DGLA in the platelet. Since DGLA constitutes one of the predominant fatty acids contained in the phospholipids on the platelet membrane and is also a rich source of dietary fatty acid intake through a variety of plant oils, we had originally sought to determine if this metabolite contained inherent regulatory properties on the human platelet. We successfully showed not only that 12-HETrE potently regulates hemostasis and thrombosis in vivo, but additionally identified that the metabolite signals inhibition of platelet activity through a GαS-linked GPCR resulting in formation of cAMP and activation of PKA and VASP. In the current study, we identified for the first time the receptor activated by 12-HETrE in the human platelet. To identify which GPCR is activated by 12-HETrE, we utilized both pharmacologic as well as genetic approaches. Our ex vivo pharmacological data suggested 12-HETrE predominantly signals through the IP receptor in the human platelet. This finding was confirmed using an IP receptor knockout mouse as well as human cell lines expressing or absent IP receptor on their surface. Finally, these findings were confirmed in vivo with wild type or IP knockout mice and assessing the ability of 12-HETrE to inhibit laser-induced thrombosis. Hence, these findings show for the first time that 12-HETrE does in fact regulate inhibition of platelets through the IP receptor. Since 12-HETrE has already been shown to inhibit thrombosis without a significant increase in bleeding diathesis, the current study has elucidated a new target for IP receptor activation and an inhibition of platelet activity and occlusive thrombus formation and should be evaluated for its potential as a new class of anti-thrombotic therapeutics. DisclosuresNo relevant conflicts of interest to declare.

Short-term treatment with nitrate is not sufficient to induce in vivo antithrombotic effects in rats and mice

In humans, short-term supplementation with nitrate is hypotensive and inhibits platelet aggregation via an nitric oxide (NO)-dependent mechanism. In the present work, we analyzed whether short-term treatment with nitrate induces antithrombotic effects in rats and mice. Arterial thrombosis was evoked electrically in a rat model in which renovascular hypertension was induced by partial ligation of the left renal artery. In mice expressing green fluorescent protein, laser-induced thrombosis was analyzed intravitally by using confocal microscope. Sodium nitrate (NaNO3) or sodium nitrite (NaNO2) was administered orally at a dose of 0.17 mmol/kg, twice per day for 3 days. Short-term nitrate treatment did not modify thrombus formation in either rats or mice, while nitrite administration led to pronounced antithrombotic activity. In hypertensive rats, nitrite treatment resulted in a significant decrease in thrombus weight (0.50 ± 0.08 mg vs. VEH 0.96 ± 0.09 mg; p < 0.01). In addition, nitrite inhibited ex vivo platelet aggregation and thromboxane B2 (TxB2) generation and prolonged prothrombin time. These effects were accompanied by significant increases in blood NOHb concentration and plasma nitrite concentration. In contrast, nitrate did not affect ex vivo platelet aggregation or prothrombin time and led to only slightly elevated nitrite plasma concentration. In mice, nitrate was also ineffective, while nitrite led to decreased platelet accumulation in the area of laser-induced endothelial injury. In conclusion, although nitrite induced profound NO-dependent antithrombotic effects in vivo, conversion of nitrates to nitrite in rats and mice over short-term 3-day treatment was not sufficient to elicit NO-dependent antiplatelet or antithrombotic effects.

Reevaluation of antithrombotic fruits and vegetables: great variation between varieties.

In the quest for prevention of atherothrombotic diseases, an antithrombotic diet may offer a promising approach. The major stumbling block in finding an effective diet is the lack of pathophysiological relevant techniques to detect potential antithrombotic effects of various diet components. Platelet function and coagulation/fibrinolysis tests currently in use do not allow assessment of global thrombotic status and their value in screening diet-components for antithrombotic effects. Recently, we combined the point-of-care shear-induced ex vivo thrombosis test (Global Thrombosis Test-GTT) with the Flow-mediated Vasodilation (FMV) in vivo test and found that the combination improved the assessment of thrombotic status in humans and could be used for screening diet-components for antithrombotic effects. In the present experiments, a combination of GTT, hemostatometry, laser-induced thrombosis tests and FMV were employed for screening. The results show that the overall antithrombotic effect is determined by the effect on thrombus formation and endogenous thrombolytic activities. This study showed a great variation in the observed antithrombotic effect between the tested varieties. Antithrombotic activities were independent from polyphenolic content or antioxidant activities. The presented experimental techniques seem to be suitable for establishing an antithrombotic diet, which may be effective in the prevention of atherothrombotic cardiovascular diseases in humans.

Abstract 19967: Differential Roles of the NADPH-Oxidase 1 and 2 in Platelet Activation and Thrombosis

Objective: Reactive oxygen species (ROS) generated from activated platelets is known to regulate platelet activation. However, it remains unclear whether and how different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs) play roles in different platelet activation pathways. Here we investigated the role of NOX1 and NOX2 in different platelet activation pathways using NOX1 and NOX2 knockout mice. Approach and Results: NOX1-/- platelets showed selective defects in G protein coupled receptor (GPCR)-mediated platelet activation induced by thrombin, protease-activated receptor 4 agonist peptide (PAR4AP) and thromboxane A2 analog U46619, but was not affected in platelet activation induced by collagen-related peptide (CRP), a glycoprotein VI (GPVI) agonist. In contrast, NOX2-/- platelets showed potent inhibition of CRP-induced platelet activation, and also showed partial inhibition of thrombin-induced platelet aggregation and secretion. Consistently, production of reactive oxygen species (ROS) was inhibited in NOX1-/- platelets stimulated with thrombin, but not CRP, whereas NOX2-/- platelets showed reduced ROS generation induced by CRP or thrombin. Interestingly, laser-induced arterial thrombosis was impaired in NOX2-/- mice, and in thrombocytopenic mice transfused with NOX2-/- platelets, suggesting an important role for NOX2-dependent platelet ROS production in the laser-induced injury model of thrombosis. Conclusions: NOX1 and NOX2 play differential roles in different platelet activation pathways: NOX1 mediates GPCR-mediated ROS production and platelet activation, whereas NOX2 plays a general role in GPVI- and GPCR-induced ROS production and platelet activation in vitro , and in laser-induced thrombosis in vivo .

Evaluation of antithrombotic effect: Importance of testing components and methodologies.

The beneficial antithrombotic effect of some dietary components may offer the most promising approach of prevention of cardiovascular diseases and arterial thrombosis. The major stumbling block in finding effective dietary components is the lack of physiologically relevant techniques which can detect potential antithrombotic effect in humans. The presently used platelet function and coagulation tests do not allow the assessment of global thrombotic status and their value in screening dietary components for antithrombotic effect is questionable. Most of these in vitro tests ignore the effect of flow and shear stress, thrombin generation and vascular endothelium, the major contributors to arterial thrombogenesis in humans. As a gold standard, we employed the helium-neon (He-Ne) laser-induced thrombosis test in murine carotid artery and mesenteric microvessels, as the pathomechanism of this test closely reflects arterial thrombogenesis in humans. Results obtained with laser thrombosis test were compared with various shear-induced in vitro platelet function tests which use native blood (Haemostatometry, Thrombotic Status Analyser, Global Thrombosis Test-GTT). Contribution of vascular endothelium to thrombogenesis was assessed by measuring flow-mediated vasodilation (FMV) in vivo. The combination of the two shear-induced ex vivo thrombosis tests (Haemostatometry and GTT) with FMV correlated most closely with the laser-thrombosis test. Our findings suggest that combining the commercially available point-of-care GTT with the FMV test could provide a better assessment of the overall thrombotic status than either of the two tests alone.

Animal Models of Arterial and Venous Thrombosis

The intersection of the availability of genetically altered mice, new technologies for intravital microscopy and high speed computing systems has led to the use of animal models to build on the concepts that have emerged from in vitro studies of the molecular and cellular biology of hemostasis and thrombosis. In an effort to improve the understanding of the etiology and pathogenesis of thrombosis, thrombus formation has been initiated in experimental systems via mechanical disruption, laser-induced, photochemical-induced and ferric chloride-induced injury to the vessel wall, among others. None of these methods are physiologic, and as such, remain models that require extrapolation from a living animal – a mouse – to human biology. We have focused on laser-induced injury of the arteriole vessel wall in the cremasteric muscle of the mouse. Using high speed digital imaging of fluorescently labeled components and real time intravital microscopy, our group has been able to demonstrate that platelet accumulation and fibrin generation during thrombus formation occur simultaneously, that tissue factor and collagen are independent initiators of platelet activation, and that monocyte-derived microparticles deliver tissue factor to the site of thrombus development. Perhaps the most important and unanticipated observation has been that thiol isomerases, thought only to be involved in protein biosynthesis via the formation of disulfide bonds in the endoplasmic reticulum, play a critical role in thrombus formation. Protein disulfide isomerase (PDI), ERp5 and ERp57 are among the vascular thiol isomerases that are known to be important for the initiation of thrombus formation. Laser-induced thrombosis in mice is associated with PDI, ERp5 and ERp57 secretion by platelets and endothelium. Inhibition of these thiol isomerases blocks platelet thrombus formation and fibrin generation. The integrins αIIbβ3 and αVβ3 play a key role in this process, binding directly with the secreted thiol isomerases and capturing them in the vicinity of vessel wall injury. These enzymes are required for the initiation of platelet thrombus formation and fibrin generation, but the mechanism by which they function remains to be elucidated. At present, it would appear that there is an electron transfer pathway involving these enzymes that regulates the initiation of thrombus formation. The mechanism by which PDI participates in thrombus generation is being evaluated by using trapping mutant forms to identify substrates of PDI that participate in the network pathways linking thiol isomerases, platelet receptor activation and fibrin generation. Several proteins, including vitronectin, thrombospondin and Factor V, have been identified as forming covalent disulfide intermediates with PDI. We are currently exploring PDI as an antithrombotic target using isoquercetin and quercetin 3-rutinoside, inhibitors of PDI identified by high throughput screening. Tail bleeding times are equivalent for mice treated with quercetin-3-rutinoside and isoquercetin compared to untreated mice. In an in vivo mouse pulmonary embolism model, PDI inhibitors rescue a high percentage of mice from death. The b-domain of PDI binds to quercetin-3-rutinoside, and infusion of the isolated b’ domain into a mouse treated with quercetin-3-rutinoside restores thrombus formation. This suggests a method for reversal of bleeding if these PDI inhibitors are found to be complicated by bleeding. The antithrombotic properties of quercetin and isoquercetin in humans have been tested. A pharmacokinetic study with quercetin and isoquercetin determined optimal oral delivery with isoquercetin. The effectiveness of these PDI inhibitors in human studies is being evaluated in a clinical trial evaluating prophylaxis of thromboembolic events in patients with cancer-associated thrombosis. PDI is a novel target for antithrombotic therapy and is unique in that its inhibition simultaneously blocks platelet thrombus formation and fibrin generation. Disclosures Zwicker: Quercegen Pharma: Research Funding.

Apolipoprotein Α-IV Is a Novel Ligand of Platelet αIIbβ3 Integrin and an Endogenous Thrombosis Inhibitor: Measurement of Single-Molecular Interactions By Biomembrane Force Probe

Platelet adhesion and aggregation at sites of vascular injury are key events in thrombosis and hemostasis. Platelet β3 integrins and their ligands are essential in mediating these processes. Therefore the understanding of β3 integrin-ligand interactions is crucial in elucidating mechanisms of thrombosis and hemostasis. In an effort to identify unknown ligands for β3 integrin, we used immobilized human platelet β3 integrin to capture proteins from human plasma. The isolated proteins were further analysed by 2D electrophoresis and mass spectrometry, and apolipoprotein A-IV (apoA-IV) was identified. ApoA-IV is an abundant plasma lipid binding protein secreted by the small intestine during dietary lipid absorption. Several studies in different ethnic populations have suggested that the level of apoA-IV is inversely correlated with cardiovascular diseases. However, the roles of apoA-IV in platelets and thrombosis are completely unknown.A single-molecule technique, biomembrane force probe (BFP), was employed to detect direct interactions between apoA-IV and platelet αIIbβ3 integrin. The BFP adhesion frequency assay demonstrated that apoA-IV bound to αIIbβ3 integrin on ADP treated platelets. ApoA-IV also bound to purified activated αIIbβ3 integrin or the integrin expressed on Chinese hamster ovary (CHO) cells. In comparison, apoA-IV did not significantly bind to αIIbβ3 integrin on resting platelets, GPIb-complex expressed on CHO cells, αMβ2 integrin expressed on K562 cells, nor purified α5β1 and αvβ3 integrins. Importantly, apoA-IV-αIIbβ3 interactions in these experiments could be completely inhibited by a blocking monoclonal antibody (M1) against β3 integrin. These data clearly demonstrated the specificity of apoA-IV for αIIbβ3 integrin. Furthermore, 2D kinetics measurements revealed that the effective 2D affinity of apoA-IV-αIIbβ3 is 43% of that between fibrinogen and αIIbβ3. The BFP competition assay showed that apoA-IV competitively inhibited fibrinogen-αIIbβ3 interactions at its physiological concentration.Platelet functional studies in vitro showed that recombinant apoA-IV significantly inhibited both mouse and human platelet aggregation following stimulation with various agonists. Consistently, platelet aggregation in platelet rich plasma of apoA-IV deficient mice (apoA-IV-/-) was enhanced. Depletion of human plasma apoA-IV also enhanced ADP-induced human platelet aggregation. In ex vivo perfusion chambers, recombinant apoA-IV inhibited human and mouse thrombus growth and dissolved pre-formed thrombi, while absence of apoA-IV in blood enhanced ex vivo thrombus growth under both low and high shear stresses. Using two in vivo intravital microscopy thrombosis models and a carotid artery thrombosis model, we demonstrated that FeCl3- and laser-induced thrombosis were enhanced in apoA-IV-/-mice, while transfusion of recombinant apoA-IV markedly attenuated this process. In addition, we found recombinant apoA-IV significantly decreased platelet P-selectin expression, and consistently more P-selectin expression was observed on ADP treated platelets from apoA-IV-/- mice, suggesting that apoA-IV occupancy may inhibit fibrinogen or other prothrombotic ligands mediated αIIbβ3 outside-in signaling.We further found that the N-terminus of apoA-IV plays a key role in its inhibitory function and the exposure of N-terminus is negatively regulated by its C-terminus. Furthermore, mutation of the two aspartic acid (D) residues at apoA-IV N-terminal 5 and 13 abolished its binding for αIIbβ3 integrin as demonstrated by BFP adhesion frequency assay, resulting in the loss of these inhibitory effects.These findings suggest that D5 and D13 of apoA-IV are the potential binding sites for αIIbβ3 integrin.Thus, apoA-IV is identified as a novel endogenous inhibitor of thrombosis and represents a new link between lipoprotein metabolism and platelet function, both of which play critical roles in cardiovascular diseases. These findings may also contribute to hemostasis, P-selectin mediated postprandial platelet activation and inflammation. DisclosuresNo relevant conflicts of interest to declare.

The disulfide isomerase ERp57 is required for fibrin deposition in vivo

ERp57 is required for platelet function; however, whether ERp57 contributes to fibrin generation is unknown. Using an inhibitory anti-ERp57 antibody (mAb1), Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice, and mutants of ERp57, we analyzed the function of ERp57 in laser-induced thrombosis. Fibrin deposition was decreased in Pf4-Cre/ERp57(fl/fl) mice, consistent with a role for platelet ERp57 in fibrin generation. Fibrin deposition was further decreased with infusion of mAb1 and in Tie2-Cre/ERp57(fl/fl) mice, consistent with endothelial cells also contributing to fibrin deposition. Infusion of eptibifatide inhibited platelet and fibrin deposition, confirming a role for platelets in fibrin deposition. Infusion of recombinant ERp57 corrected the defect in fibrin deposition but not platelet accumulation, suggesting a direct effect of ERp57 on coagulation. mAb1 inhibited thrombin generation in vitro, consistent with a requirement for ERp57 in coagulation. Platelet accumulation was decreased to similar extents in Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice and normal mice infused with mAb1. Infusion of completely inactivated ERp57 or ERp57 with a non-functional second active site inhibited fibrin deposition and platelet accumulation, indicating that the isomerase activity of the second active site is required for these processes. ERp57 regulates thrombosis via multiple targets.

Platelets are required for enhanced activation of the endothelium and fibrinogen in a mouse thrombosis model of APS

AbstractAntiphospholipid syndrome (APS) is defined by thrombosis, fetal loss, and the presence of antiphospholipid antibodies, including anti–β2-glycoprotein-1 autoantibodies (anti-β2GP1) that have a direct role in the pathogenesis of thrombosis in vivo. The cellular targets of the anti-β2GP1 autoantibody/β2GP1 complex in vivo were studied using a laser-induced thrombosis model of APS in a live mouse and human anti-β2GP1 autoantibodies affinity-purified from APS patients. Cell binding of fluorescently labeled β2GP1 and anti-β2GP1 autoantibodies revealed their colocalization on the platelet thrombus but not the endothelium. Anti-β2GP1 autoantibodies enhanced platelet activation, monitored by calcium mobilization, and endothelial activation, monitored by intercellular adhesion molecule-1 expression. When eptifibatide was infused to block platelet thrombus formation, enhanced fibrin generation and endothelial cell activation were eliminated. Thus, the anti-β2GP1 autoantibody/β2GP1 complex binds to the thrombus, enhancing platelet activation, and platelet secretion leads to enhanced endothelium activation and fibrin generation. These results lead to a paradigm shift away from the concept that binding of the anti-β2GP1 autoantibody/β2GP1 complex activates both endothelial cells and platelets toward one in which activation of platelets in response to anti-β2GP1 autoantibody/β2GP1 complex binding leads to subsequent enhanced endothelium activation and fibrin generation.

Abstract 225: Apolipoprotein A-IV Is a ß3 Integrin Ligand and an Endogenous Inhibitor of Platelets: Novel Mechanisms of Prevention and Treatment for Atherothrombosis

Apolipoprotein A-IV (apoA-IV) is a lipid binding protein secreted by the intestine during dietary lipid absorption. Several clinical studies in different ethnic populations have demonstrated that apoA-IV levels inversely correlate with cardiovascular diseases. However, the roles of apoA-IV in platelet aggregation and atherothrombosis are completely unknown. Here we isolated apoA-IV from plasma with beads coated with human platelet β3 integrin. Using a biomembrane force probe that detects single-molecule interactions, we clearly demonstrated that apoA-IV specifically binds to purified αIIbβ3 on beads and native αIIbβ3 on platelets or Chinese hamster ovary cells. ApoA-IV and αIIbβ3 interaction could be blocked by a monoclonal antibody against β 3 integrin, M1. Importantly, recombinant apoA-IV can competitively block fibrinogen-αIIbβ3 interaction, and specifically inhibited human and mouse platelet aggregation. Consistently, platelet aggregation was enhanced in mice lacking apoA-IV following stimulation with various agonists. In ex vivo perfusion chambers, apoA-IV inhibited human and mouse thrombus growth and dissolved pre-formed thrombi, while absence of apoA-IV enhanced ex vivo thrombosis under both low and high shear stresses. Furthermore, in vivo intravital microscopy models revealed that FeCl 3 - and laser-induced thrombosis were enhanced in mice lacking apoA-IV, while transfusion of human or mouse recombinant apoA-IV significantly attenuated this process. To further identify the potential binding sites of apoA-IV for platelet αIIbβ3 integrin, we deleted apoA-IV N-terminal 38 amino acids and/or the C-terminal 41 amino acids. We observed that deletion of the N-terminus abrogated platelet-inhibitory function. Interestingly, we found that mutation of either of two highly conserved aspartic acid (D) residues at positions 5 and 13 abolished or reduced the inhibitory function of apoA-IV, suggesting that D5 and/or D13 participate in a direct protein-protein interaction between apoA-IV and αIIbβ3 integrin. Thus, apoA-IV is identified as a novel endogenous inhibitor of thrombosis and represents a novel link between lipoprotein metabolism and platelet function, both of which play critical roles in cardiovascular diseases.