Photochemically-induced Thrombosis Research Articles

Interaction between JAK2-Mutated Neutrophils and Platelets Initiates Thrombosis Via Neutrophil Extracellular Traps

Thrombosis is one of the most common complications of myeloproliferative neoplasms with JAK2-V617F mutation. Recently, individuals with clonal hematopoiesis (CH) harboring JAK2-V617F mutation were also shown to have 12-fold higher risk of thrombosis compared to JAK2 non-mutated individuals (N Engl J Med. 2017;377:111-121). The finding that individuals with JAK2-V617F CH do not have altered blood counts suggests the mechanisms other than increases in the hematopoietic cells which induce thrombosis associated with JAK2-V617F hematopoiesis. JAK2-V617F neutrophils are primed to neutrophil extracellular traps (NETs), which can promote thrombosis (Sci Transl Med. 2018;10: eaan8292). NETs were initially reported as an immunological reaction against infection. However, NETs also play various roles in non-infectious (sterile) settings, and the mechanism how sterile NETs are initiated in vivo remains largely unknown. Here, we studied the mechanisms how NETs are initiated and promote thrombosis in the presence of JAK2-V617F hematopoiesis. First, we isolated neutrophils and platelets from Jak2-V617F transgenic ( Jak2VF) and wild-type (WT) mice, and co-cultured in each cell/genotype combination (see Figure1) at a 1:50 ratio. Of note, Jak2VF neutrophils formed NETs with Jak2VF platelets, but not with WT platelets. Also, Jak2VF platelets slightly increased formation of NETs by WT neutrophils. However, culture supernatant of Jak2VF platelets did not induce formation of NETs by Jak2VF neutrophils. These findings indicated that direct interaction, between Jak2VF neutrophils and Jak2VF platelets, initiates sterile NETs in the presence of JAK2-V617F hematopoiesis. Surprisingly, platelet activation of Jak2VF platelets measured by P-selectin release was inferior to WT platelets. Thus, it was unlikely that overactivation of Jak2VF platelets fully initiate NETs. Subsequently, we sought an initiator on Jak2VF platelets to form NETs in the presence of JAK2-V617F hematopoiesis. Unbiased proteomics analysis of WT and Jak2VF platelets identified several proteins significantly enriched in Jak2VF platelets, including a membrane protein PODXL, which is expressed on podocytes in kidney as well as platelets and other hematopoietic cells. Interestingly, PODXL on platelet is associated with aggregation with other hematopoietic cells depending on CD41, SLC44A2 and P-selectin. Therefore, we hypothesized that PODXL enhances aggregation of Jak2VF platelets with neutrophils and initiates formation of NETs (Figure 2). We confirmed overexpression of PODXL in Jak2VF platelets and in platelets of another murine model, hematopoietic cell-specific Jak2V617F/+ mice by FACS and immunofluorescence imaging. The formations of NETs measured by intensity of citrullinated histone H3 (citH3) correlated with aggregation of PODXL-expressing Jak2VF platelets on cell surface. Presently, we are conducting to confirm the findings utilizing patients' samples with JAK2-V617F. To test if a JAK inhibitor prevents PODXL-associated formation of NETs, we collected platelets from ruxolitinib-treated Jak2VF mice. Platelets from ruxolitinib-treated mice similarly expressed PODXL and induced formation of NETs with neutrophils from untreated Jak2VF mice, compared to platelets from untreated Jak2VF mice, suggesting that ruxolitinib does not alter platelets' ability to initiate formation of NETs. Next, we tested if these mechanisms induce venous and arterial thrombosis in vivo, utilizing models of half ligation of inferior vena cava (IVC) and photochemically induced thrombosis (PIT) in femoral artery. In both models, Jak2VF or Jak2V617F/+ mice formed thrombosis more quickly than WT controls. Jak2-mutated mice formed white thrombus presenting citH3-positive neutrophils aggregated with PODXL-expressing platelets at the core of the thrombus, while WT mice mainly formed red thrombus without any nucleated cells in these models. Therefore, we are generating platelet-specific knockout mice of PODXL to directly clarify if PODXL on Jak2VF-platelet is the initiator of NETs and thrombosis. In conclusion, PODXL on JAK2-V617F platelets may be a key player in the pathogenesis of thrombosis associated with formation of NETs with JAK2-V617F neutrophils in myeloproliferative neoplasms.

Driver gene KRAS aggravates cancer-associated stroke outcomes

The incidence of cancer-associated stroke has increased with the prolonged survival times of cancer patients. Recent genetic studies have led to progress in cancer therapeutics, but relationships between oncogenic mutations and stroke remain elusive. Here, we focused on the driver gene KRAS, which is the predominant RAS isoform mutated in multiple cancer types, in cancer associated stroke study. KRASG13D/− and parental human colorectal carcinoma HCT116 cells were inoculated into mice that were then subjected to a photochemically-induced thrombosis model to establish ischemic stroke. We found that cancer inoculation exacerbated neurological deficits after stroke. Moreover, mice inoculated with KRASG13D/− cells showed worse neurological deficits after stroke compared with mice inoculated with parental cells. Stroke promoted tumor growth, and the KRASG13D/− allele enhanced this growth. Brain RNA sequencing analysis and serum ELISA showed that chemokines and cytokines mediating pro-inflammatory responses were upregulated in mice inoculated with KRASG13D/− cells compared with those inoculated with parental cells. STAT3 phosphorylation was promoted following ischemic stroke in the KRASG13D/− group compared with in the parental group, and STAT3 inhibition significantly ameliorated stroke outcomes by mitigating microglia/macrophage polarization. Finally, we compared the prognosis and mortality of colorectal cancer patients with or without stroke onset between 1 January 2007 and 31 December 2020 using a hospital-based cancer registry and found that colorectal cancer patients with stroke onset within 3 months after cancer diagnosis had a worse prognosis. Our work suggests an interplay between KRAS and ischemic stroke that may offer insight into future treatments for cancer-associated stroke.

Change in the central control of the bladder function of rats with focal cerebral infarction induced by photochemically-induced thrombosis.

The photochemically-induced thrombosis (photothrombosis) method can create focal cerebral infarcts anywhere in the relatively superficial layers of the cerebrum; it is easy to implement and minimally invasive. Taking advantage of this versatility, we aimed to establish a new rat model of urinary frequency with focal cerebral infarction, which was characterized by its simplicity, nonlethal nature, and high reproducibility. The prefrontal cortex and the anterior cingulate cortex, which are involved in lower urinary tract control, were targeted for focal cerebral infarction, and urinary parameters were measured by cystometrogram. Cystometric analysis indicated that micturition intervals significantly shortened in photothrombosis-treated rats compared with those in the sham operative group on Days 1 and 7 (P < 0.01), but prolonged after 14 days, with no difference between the two groups. Immunopathological evaluation showed an accumulation of activated microglia, followed by an increase in reactive astrocytes at the peri-infarct zone after photothrombotic stroke. Throughout this study, all postphotothrombosis rats showed cerebral infarction in the prefrontal cortex and anterior cingulate cortex; there were no cases of rats with fatal cerebral infarction. This model corresponded to the clinical presentation, in that the micturition status changed after stroke. In conclusion, this novel model combining nonlethality and high reproducibility may be a suitable model of urinary frequency after focal cerebral infarction.

MiR-214 Alleviates Ischemic Stroke-Induced Neuronal Death by Targeting DAPK1 in Mice.

BackgroundIschemic stroke induces neuronal cell death and causes brain dysfunction. Preventing neuronal cell death after stroke is key to protecting the brain from stroke damage. Nevertheless, preventative measures and treatment strategies for stroke damage are scarce. Emerging evidence suggests that microRNAs (miRNAs) play critical roles in the pathogenesis of central nervous system (CNS) disorders and may serve as potential therapeutic targets.MethodsA photochemically induced thrombosis (PIT) mouse model was used as an ischemic stroke model. qRT-PCR was employed to assess changes in miRNAs in ischemic lesions of PIT-stroke mice and primary cultured neurons subjected to oxygen-glucose deprivation (OGD). 2,3,5-triphenyltetrazolium chloride (TTC) staining was performed to evaluate brain infarction tissues in vivo. TUNEL staining was employed to assess neuronal death in vitro. Neurological scores and motor coordination were investigated to evaluate stroke damage, including neurological deficits and motor function.ResultsIn vivo and in vitro results demonstrated that levels of miR-124 were significantly decreased following stroke, whereas changes in death-associated protein kinase 1 (DAPK1) levels exhibited the converse pattern. DAPK1 was identified as a direct target of miR-124. N-methyl-D-aspartate (NMDA) and OGD-induced neuronal death was rescued by miR-124 overexpression. Upregulation of miR-124 levels significantly improved PIT-stroke damage, including the overall neurological function in mice.ConclusionWe demonstrate the involvement of the miR-124/DAPK1 pathway in ischemic neuronal death. Our results highlight the therapeutic potential of targeting this pathway for ischemic stroke.

Protective Effects of PGC-1α Activators on Ischemic Stroke in a Rat Model of Photochemically Induced Thrombosis.

The pharmacological induction and activation of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), a key regulator of ischemic brain tolerance, is a promising direction in neuroprotective therapy. Pharmacological agents with known abilities to modulate cerebral PGC-1α are scarce. This study focused on the potential PGC-1α-modulating activity of Mexidol (2-ethyl-6-methyl-3-hydroxypyridine succinate) and Semax (ACTH(4–7) analog) in a rat model of photochemical-induced thrombosis (PT) in the prefrontal cortex. Mexidol (100 mg/kg) was administered intraperitoneally, and Semax (25 μg/kg) was administered intranasally, for 7 days each. The expression of PGC-1α and PGC-1α-dependent protein markers of mitochondriogenesis, angiogenesis, and synaptogenesis was measured in the penumbra via immunoblotting at Days 1, 3, 7, and 21 after PT. The nuclear content of PGC-1α was measured immunohistochemically. The suppression of PGC-1α expression was observed in the penumbra from 24 h to 21 days following PT and reflected decreases in both the number of neurons and PGC-1α expression in individual neurons. Administration of Mexidol or Semax was associated with preservation of the neuron number and neuronal expression of PGC-1α, stimulation of the nuclear translocation of PGC-1α, and increased contents of protein markers for PGC-1α activation. This study opens new prospects for the pharmacological modulation of PGC-1α in the ischemic brain.

Different exercises can modulate the differentiation/maturation of neural stem/progenitor cells after photochemically induced focal cerebral infarction.

IntroductionExercise therapies during rehabilitation significantly promote recovery from various deficits after cerebral infarction, which is mediated by neuronal plasticity with distinct inputs. Although adult neurogenesis can also be modulated by neuronal activity before synaptogenesis, how distinct exercises contribute to the neurological reorganization of the injured cerebral cortex remains unclear. In the present study, we aimed to elucidate the effects of different exercise therapies on motor recovery and neuronal reorganization after photochemically induced focal cerebral infarction.MethodsHere, we examined the effects of three different exercises—(a) forced lower‐intensity and (b) higher‐intensity treadmill exercises, and (c) voluntary exercise with wheel running—on motor recovery and adult neurogenesis in a rat model of focal cerebral infarction. Photochemically induced thrombosis (PIT) was used to generate focal infarction in rats that was mostly confined to their motor cortices.ResultsBeam walking tests showed that recovery after PIT‐induced cortical infarction differed in acute and chronic stages and was influenced by the type of exercise. Furthermore, forced low‐intensity training had more positive effects on functional recovery than other exercises or control. To evaluate the production of newly generated cells including de novo neurogenesis, we performed lineage analysis with BrdU labeling and immunofluorescence experiments. Lower‐intensity treadmill exercise increased the number of BrdU/NeuN colabeled cells, but not total BrdU‐retaining or BrdU/Sox2‐colabeled cells, in the peri‐infarct region of the ipsilateral cortex. In contrast, high‐intensity treadmill or voluntary exercises had the opposite effects.ConclusionsThese results suggest that neuronal maturation can be differently modulated by distinct exercises and that low‐intensity treadmill exercise could result in more potent generation of mature neurons. This also suggests the possibility that the generation of neural stem/progenitor cells and differentiation might be modulated by rehabilitation‐mediated neural plasticity.

Beneficial actions of prothymosin alpha-mimetic hexapeptide on central post-stroke pain, reduced social activity, learning-deficit and depression following cerebral ischemia in mice

Prothymosin alpha (ProTα)-mimetic hexapeptide (amino acid: NEVDQE, P6Q) inhibits cerebral or retinal ischemia-induced behavioral, electrophysiological and histological damage. P6Q also abolishes cerebral hemorrhage induced by ischemia with tissue plasminogen activator (tPA). In the present study we examined the beneficial effects of P6Q on other post-stroke prognostic psychology-related symptoms, which obstruct the motivation toward physical therapy. Intravenous (i.v.) administration with tPA (10 mg/kg) at 6 h after photochemically induced thrombosis (PIT) in mice resulted in bilateral central post-stroke pain in thermal and mechanical nociception tests and loss of social activity in the nest building test, both of which were significantly blocked by P6Q (30 mg/kg, i.v.) given at 5 h after PIT. P6Q (30 mg/kg, i.v.) also improved the memory-learning deficit in the step-through test and depression-like behavior in the tail suspension test when it was given 1 day after bilateral common carotid arteries occlusion (BCCAO) in mice. Thus, these studies suggest that P6Q could be a promising candidate to prevent negative prognostic psychological symptoms following focal and global ischemia.

Lysophosphatidic acid LPA1 and LPA3 receptors play roles in the maintenance of late tissue plasminogen activator-induced central poststroke pain in mice

We developed a mouse model for central post-stroke pain (CPSP), a centrally-originated neuropathic pain (NeuP). In this mode, mice were first injected with Rose Bengal, followed by photo-irradiation of left middle cerebral artery (MCA) to generate thrombosis. Although the MCA thrombosis was soon dissolved, the reduced blood flow remained for more than 24 h due to subsequent occlusion of microvessels. This photochemically induced thrombosis (PIT) model showed a hypersensitivity to the electrical stimulation of both sides of paw, but did not show any abnormal pain in popular thermal or mechanical nociception tests. When tissue-type plasminogen activator (tPA) was injected 6 h after the PIT stress, tPA-dependent hypersensitivity to the electrical paw stimulation and stable thermal and mechanical hyperalgesia on both sides for more than 17 or 18 days after the PIT treatment. These hyperalgesic effects were abolished in lysophosphatidic acid receptor 1 (LPA1)- and lysophosphatidic acid receptor 3 (LPA3)-deficient mice. When Ki-16425, an LPA1 and LPA3 antagonist was treated twice daily for 6 days consecutively, the thermal and mechanical hyperalgesia at day 17 and 18 were significantly reversed. The liquid chromatography–mass spectrometry (LC–MS/MS) analysis revealed that there is a significant increase in several species of LPA molecules in somatosensory S-I and medial dorsal thalamus (MD), but not in striatum or ventroposterior thalamus. All these results suggest that LPA1 and LPA3 signaling play key roles in the development and maintenance of CPSP.

N1-Nonyl-1,4-diaminobutane ameliorates brain infarction size in photochemically induced thrombosis model mice

Inhibitors for polyamine oxidizing enzymes, spermine oxidase (SMOX) and N1-acetylpolyamine oxidase (PAOX), were designed and evaluated for their effectiveness in a photochemically induced thrombosis (PIT) mouse model. N1-Nonyl-1,4-diaminobutane (C9-4) and N1-tridecyl-1,4-diaminobutane (C13-4) competitively inhibited the activity of PAOX and SMOX in a manner comparable to N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL72527), an irreversible inhibitor of both enzymes. The two compounds were then tested for their effects in the PIT model. Both intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) administration of C9-4 decreased infarct volumes significantly. By contrast, C13-4 reduced the volume of brain infarction by i.c.v. administration, but no reduction was observed after i.p. administration. C9-4 administered by i.p. injection reduced the volume of brain infarction significantly at doses of more than 3 mg/kg, and the dosage of 5 mg/kg or 10 mg/kg demonstrated the most potent effect and were more effective than equivalent doses of the other inhibitors such as MDL72527 and N-benzylhydroxylamine. I.P. injection of 5 mg/kg of C9-4 provided a therapeutic time window of longer than 12 h. This report demonstrates that C9-4 is a potent inhibitor of the polyamine oxidizing enzymes and is useful lead compound for candidate drugs with a long therapeutic time window, to be used in the treatment of ischemic stroke.

Specific Combination of Salvianolic Acids As Core Active Ingredients of Danhong Injection for Treatment of Arterial Thrombosis and Its Derived Dry Gangrene.

Although single-targeting anti-platelet agents are used extensively in clinics, their limitations in resistance and bleeding have started a trend of combination therapy. Danhong injection (DHI) is a widely prescribed injection medicine for cardiovascular and cerebrovascular diseases in China. However, its precise clinical efficacy and functional components remain unexplored. In this study, we investigated the anti-thrombotic role and its chemical basis of DHI. In a photochemically-induced thrombosis model, DHI effectively dissolved thrombus and ameliorated its derived dry gangrene. DHI inhibited multiple GPCR agonists-induced platelet adhesion, aggregation and downstream Ca2+ and cAMP signaling pathways. A functional screen of DHI library identified its major active components as a cluster of seven salvianolic acids. A combination of salvianolic acid A and C synergistically inhibited platelet aggregation in vitro while salvianolic acid B antagonized this effect. Our study revealed the anti-thrombotic activity of DHI. The multi-targeting mechanism of DHI proves the effectiveness of a natural anti-thrombotic combination therapy. The identification of salvianolic acids as a core anti-thrombotic activity of DHI and the discovery that their different combinations could either synergistically or antagonistically provide a better guidance for safer clinical application and paves the way for further development of DHI.

Effects of task-specific rehabilitation training on tau modification in rat with photothrombotic cortical ischemic damage

Although stroke elicits progressive cognitive decline and is a leading cause of dementia, molecular interplay between stroke and Alzheimer's disease (AD) pathology has not been fully elucidated. Furthermore, studies on the effects of post-stroke rehabilitation on AD pathology are limited. We evaluated the acute effect of stroke on tau modification, and the molecular effects of task-specific training (TST) on tau modification using a model of photochemically-induced thrombosis (PIT)-induced cortical infarction. Following PIT in the dominant side of sensorimotor cortex, the rehabilitation group received 4-weeks of TST rehabilitation once daily by single pellet reaching training, whereas the sedentary control group did not received any type of training. Cortical expression levels of proteins related to tau modification were evaluated on post-stroke day 1 (PSD1) and 28; functional tests were also evaluated performed every week. The expression levels of acetyl-tau, phosphorylated-tau (p-tau), cyclooxygenase-2 and Akt-mTORC1-p70S6K pathway in infarcted cortices on PSD1 were significantly greater, whereas the expression levels of p-AMPK were significantly lower than in the paired contralateral sides. TST rehabilitation for 4 weeks greatly improved functional motor performance but not memory, which concurred with the down-regulations of ipsilateral p-AMPK, cyclooxygenase-2, Akt-mTORC1-p70S6K pathway, and p-tau in rehabilitation group. PIT-induced cortical infarction was found to induce cortical tau modification through the Akt-mTORC1-p70S6K activation, and to suppress the expression of AMPK-related proteins. TST rehabilitation greatly improved motor function, but not memory, and suppressed p-tau expression and neuroinflammation. Nevertheless, the role of TST-mediated regulation of tau hyperphosphorylation required further clarification.

Abstract WMP79: Characterization of Acute Stoke Induced by Photochemically-induced Thrombosis (PIT) in Nonhuman Primate (NHP)

Introduction: Tissue plasminogen activator (tPA) is the standard treatment for acute stroke but its widespread use is constrained by its narrow therapeutic window: better treatments are needed. Clinical stroke is heterogeneous in presentation and responsiveness to therapeutics. There are several methods of inducing acute cerebral ischemia in NHP and each model induces a differential pathology following occlusion. The goal of the current study was to characterize the PIT model of acute stroke in nonhuman primates. Methods: First, the time course of brain infarction between PIT and permanent middle cerebral artery (pMCA) occlusion was compared. PIT and pMCA occlusion were performed in cynomolgus macaques (n = 3/procedure). Total brain infarction volume and the volume of viable tissue surrounding the ischemic core that eventually becomes necrotic (“penumbra”) over time was measured using FLAIR MR imaging, 3, 6, 24 hours, 3, 5 and 7 days post-occlusion. In addition, functionality (Neurologic Deficit Score; NDS) was assessed 24 hours, 3, 5 and 7 days post-occlusion. Second, tPA’s (n = 7) therapeutic time window was determined. tPA (0.9 mg/kg) was i.v. infused 1, 3 and 4.5 hours post-occlusion. Control animals (n = 7) received a saline infusion 3 hours post-occlusion. Total brain infarction volume and presence of intracerebral hemorrhage 24 hours post-occlusion were measured. NDS was also determined. Results: First, significant brain infarction was observed 6 hours post-occlusion with FLAIR MR imaging in both models. Significant penumbral tissue was observed up to 6 hours post-occlusion. The brain infarction volume plateaued 5-7 days post-occlusion. In the second stage, tPA showed robust efficacy when it was infused 1 hour after occlusion. However, when tPA was infused either 3 or 4.5 hours post-occlusion, infarct volume and NDS were not different compared to saline treatment. Furthermore, significant intracranial hemorrhaging was observed when tPA was infused 3 or 4.5 hours post-occlusion. Conclusion: The current study suggests that the PIT model mimics human thrombotic stroke. In addition, since significant risks associated with tPA can also observed, the PIT model can be used to compare novel treatments with tPA in terms of intracranial hemorrhaging.

Aggravation of brain infarction through an increase in acrolein production and a decrease in glutathione with aging

We previously reported that tissue damage during brain infarction was mainly caused by inactivation of proteins by acrolein. This time, it was tested why brain infarction increases in parallel with aging. A mouse model of photochemically induced thrombosis (PIT) was studied using 2, 6, and 12 month-old female C57BL/6 mice. The size of brain infarction in the mouse PIT model increased with aging. The volume of brain infarction in 12 month-old mice was approximately 2-fold larger than that in 2 month-old mice. The larger brain infarction in 12 month-old mice was due to an increase in acrolein based on an increase in the activity of spermine oxidase, together with a decrease in glutathione (GSH), a major acrolein-detoxifying compound in cells, based on the decrease in one of the subunits of glutathione biosynthesizing enzymes, γ-glutamylcysteine ligase modifier subunit, with aging. The results indicate that aggravation of brain infarction with aging was mainly due to the increase in acrolein production and the decrease in GSH in brain.

Toxic acrolein production due to Ca2+ influx by the NMDA receptor during stroke

Background and purposeN-Methyl-d-aspartate (NMDA) receptors have a high permeability to Ca2+, contributing to neuronal cell death after stroke. We recently found that acrolein produced from polyamines is a major toxic compound during stroke. Thus, it was determined whether over-accumulation of Ca2+ increases the production of acrolein from polyamines in a photochemically-induced thrombosis mouse model of stroke and in cell culture systems. MethodsA unilateral infarction was induced in mouse brain by photoinduction after injection of Rose Bengal. The volume of the infarction was analyzed using the public domain National Institutes of Health image program. Protein-conjugated acrolein levels at the locus of infarction and in cells were measured by Western blotting. Levels of polyamines were measured by high-performance liquid chromatography. ResultsWhen the size of brain infarction was decreased by N1, N4, N8-tribenzylspermidine, a channel blocker of the NMDA receptors, levels of Ca2+ and protein-conjugated acrolein (PC-Acro) were reduced, while levels of polyamines were increased at the locus of infarction. When cell growth of mouse mammary carcinoma FM3A cells and neuroblastoma Neuro2a cells was inhibited by Ca2+, the level of polyamines decreased, while that of PC-Acro increased. It was also shown that Ca2+ toxicity was decreased in an acrolein toxicity decreasing FM3A mutant cells recently isolated. In addition, 20–40 μM Ca2+ caused the release of polyamines from ribosomes. The results indicate that acrolein is produced from polyamines released from ribosomes through Ca2+ increase. ConclusionThe results indicate that toxicity of Ca2+ during brain infarction is correlated with the increase of acrolein.

Betaine (N,N,N-trimethylglycine) averts photochemically-induced thrombosis in pial microvessels invivo and platelet aggregation invitro.

Betaine (N,N,N-trimethylglycine) is an important food component with established health benefits through its homocysteine-lowering effects, and is used to lower total homocysteine concentration in plasma of patients with homocystinuria. It is well established that hyperhomocysteinemia is an established risk factor for cardiovascular disease and stroke. However, the possible protective effect of betaine on coagulation events invivo and invitro has thus far not been studied. Betaine was given to mice at oral doses of either 10 mg/kg (n = 6) or 40 mg/kg (n = 6) for seven consecutive days, and control mice (n = 6) received water only. The thrombotic occlusion time in photochemically induced thrombosis in pial arterioles was significantly delayed in mice pretreated with betaine at doses of 10 mg/kg (P < 0.001) and 40 mg/kg (P < 0.01). Similar effects were observed in pial venules with 10 mg/kg (P < 0.05) and 40 mg/kg (P < 0.05) betaine. Invitro, in whole blood samples collected from untreated mice (n = 3-5), betaine (0.01-1 mg/mL) significantly reversed platelet aggregation induced by adenosine diphosphate (5 µM). The number of circulating platelets and plasma concentration of fibrinogen invivo were not significantly affected by betaine pretreament compared with the control group. Lipid peroxidation (LPO) in mice pretreated with betaine was significantly reduced compared with the control group. Moreover, betaine (0.01-1 mg/mL) caused a dose-dependent and significant prolongation of PT (n = 5) and aPTT (n = 4-6). In conclusion, our data show that betaine protected against coagulation events invivo and invitro and decreased LPO in plasma.

Detection of ischemic neuronal damage with [18F]BMS‐747158‐02, a mitochondrial complex‐1 positron emission tomography ligand: Small animal PET study in rat brain

The acute and subacute ischemic neuronal damage in rat brain caused by photochemically induced thrombosis (PIT) was imaged using [¹⁸F]BMS-747158-02 ([¹⁸F]BMS) for mitochondrial complex-1 (MC-1) and [¹¹C](R)-PK11195 ([¹¹C](R)-PK) for peripheral benzodiazepine receptor [PBR; translocator protein] at preischemic "Normal," 1 (day 1), and 7 days (day 7) after ischemic insult. When [¹⁸F]BMS was intravenously injected into "Normal" rat, it was rapidly taken up into the brain, in which it showed a homogeneous distribution, and the uptake was suppressed by rotenone, a specific MC-1 inhibitor. The specificity of [¹⁸F]BMS binding to MC-1 was also confirmed by living brain slice imaging. At day 1, [¹⁸F]BMS uptake was low in infarct and peri-infarct regions where neuronal damage was detected by 2,3,5-triphenyltetrazolium chloride (TTC) staining. At day 7, the damaged areas determined using [¹⁸F]BMS revealed some discrepancy from those detected by TTC staining, suggesting that TTC stained not only surviving cells but also activated microglial cells in the peri-infarct region. This was also confirmed by [¹¹C](R)-PK imaging and immunohistochemical assessment with Iba1 antibody. In contrast, the uptake pattern of [¹⁸F]BMS was consistent with immunohistochemical assessment with NeuN antibody at both days 1 and 7. These results demonstrated that [¹⁸F]BMS could be a promising positron emission tomography ligand to assess the neuronal damage induced by ischemic insult in both acute and subacute phases.

Abstract 414: Combined Tissue Factor/Factor VIIa Inhibitor and Acetylsalicylic Acid Show Increased Antithrombotic Potency Without Affecting Bleeding Time

Since increased tissue factor (TF) in human atherosclerotic plaques is believed to trigger thrombotic events, TF/factor VIIa (FVIIa) inhibition might have prophylactic potential for atherothrombosis such as acute coronary syndrome (ACS). Although recent clinical observation by rivaroxaban provides a scientific rationale for the addition of anticoagulant to current antiplatelet treatment, these might have potential to increase bleeding. It is therefore important that emerging anticoagulants demonstrate additive antithrombotic effects with no concomitant increase in bleeding for ACS indication. We recently discovered ER-410660, a potent TF/FVIIa inhibitor. It suppressed venous thrombosis without affecting bleeding time and suggested that ER-410660 could act as a novel anticoagulant with a low bleeding risk. In the photochemically-induced thrombosis (PIT) model, the thrombus after vessel-wall injury has been developed by the deposition of TF, platelets, and fibrin. Therefore, we hypothesized that ER-410660 would be effective in the PIT model and could show enough margin against bleeding. The antithrombotic effects of ER-410660 and acetylsalicylic acid (ASA), both alone and in combination, was assessed using a rat PIT model. We also measured the bleeding time of ER-410660 in combination with ASA using a rat tail-transection model. Intravenous (iv) pretreatment with ER-410660 prolonged the time to occlusion (TTO) of the artery. The TTO was doubled at 0.99 mg/kg (95% CI, 0.51-1.9). Orally administered ASA also showed antithrombotic effect, and the TTO was doubled at approximately 12 mg/kg. Combined administration of 1 mg/kg ER-410660 (iv) and 10 mg/kg ASA (orally) showed an additive antithrombotic effect, with no cessation of blood flow during the 30-min observation period. Intravenous administration of ER-410660 (0.6, 2, and 6 mg/kg) to rats treated with ASA (10 mg/kg) did not significantly increased when compared with ASA alone, even at the highest dose. In conclusion, a TF/FVIIa inhibitor suppressed arterial thrombosis without affecting the bleeding time associated with ASA. Owing to these favorable characteristics, anticoagulation via TF/FVIIa inhibition may be a superior approach to the prophylaxis of atherothrombotic diseases.

The novel and orally active thrombin receptor antagonist E5555 (Atopaxar) inhibits arterial thrombosis without affecting bleeding time in guinea pigs

Thrombin is a powerful agonist for platelets, the action of which is mediated by the thrombin receptor protease-activated receptor-1 (PAR-1). Recently, we discovered that E5555 (1-(3- tert-butyl-4-methoxy-5-morpholinophenyl)-2-(5,6-diethoxy-7-fluoro-1-imino-1,3-dihydro-2 H-isoindol-2-yl) ethanone hydrobromide) is a potent thrombin receptor antagonist. We evaluated the anti-platelet and anti-thrombotic effects of E5555. E5555 inhibited the binding of a high-affinity thrombin receptor-activating peptide ([ 3H]haTRAP) to PAR-1 with a half maximal inhibitory concentration (IC 50) value of 0.019 μM. E5555 showed potent inhibitory effects on human platelet aggregation induced by thrombin and TRAP with IC 50 values of 0.064 and 0.031 μM, respectively, but had no effect on platelet aggregation induced by either ADP or collagen. Similarly, E5555 showed potent and selective inhibitory effects on guinea pig platelet aggregation induced by thrombin and TRAP with IC 50 values of 0.13 and 0.097 μM, respectively. The antithrombotic activity of E5555 in vivo was evaluated in a photochemically-induced thrombosis (PIT) model using guinea pigs. Oral administration of E5555 at 30 and 100 mg/kg prolonged the time to occlusion by 1.8-fold and 2.4-fold, respectively, compared with controls. Furthermore, E5555 did not prolong bleeding time in guinea pigs at the highest tested dosage of 1000 mg/kg. The drug interactions between E5555 and tissue plasminogen activator (tPA) were evaluated. Intravenous administration of 1 mg/kg tPA significantly prolonged bleeding time, and its effects were not altered by the oral co-administration of 300 mg/kg E5555. These results suggest that E5555 could be a therapeutic option for atherothrombotic disease.

Multiparametric assessment of acute and subacute ischemic neuronal damage: A small animal positron emission tomography study with rat photochemically induced thrombosis model

We evaluated sequential changes in rat brain function up to 14 days after focal ischemic insult with a small animal positron emission tomography (PET). Unilateral focal ischemic cerebral damage was induced by left middle cerebral artery occlusion with a photochemically induced thrombosis (PIT) method. PET scans were conducted with [(11)C](R)-PK11195 ([(11)C](R)-PK) for peripheral benzodiazepine receptor (PBR), [(11)C]flumazenil ([(11)C]FMZ) for central benzodiazepine receptor (CBR), and [(18)F]fluoro-2-deoxy-D-glucose ([(18)F]FDG) for glucose metabolism at before (as "Normal") and after PIT. At 1 and 3 days after PIT, [(18)F]FDG indicated lower uptake in the infarct area. Interestingly, unexpectedly high-[(18)F]FDG uptake was observed in the peri-infarct area surrounding the infarct area at day 7. The high-[(18)F]FDG uptake region completely overlapped with the high-[(11)C](R)-PK uptake region at day 7, which resulted in the underestimation of neuronal damage. Immunohistochemical data also suggested that the high-[(18)F]FDG uptake peak at day 7 was caused by inflammation including microglial cell activation. In contrast, imaging with [(11)C]FMZ indicated cortical neuronal damage on days 7 and 14 without any disturbance by microglial formation. These results indicated that [(18)F]FDG might not be a suitable ligand for ischemic neuronal damage detection from acute to subacute phases.

Effectiveness of Bisphosphonates for the Reduction of Clinical Fractures – An Observational Study

Inhibitors for polyamine oxidizing enzymes, spermine oxidase (SMOX) and N1-acetylpolyamine oxidase (PAOX), were designed and evaluated for their effectiveness in a photochemically induced thrombosis (PIT) mouse model. N1-Nonyl-1,4-diaminobutane (C9-4) and N1-tridecyl-1,4-diaminobutane (C13-4) competitively inhibited the activity of PAOX and SMOX in a manner comparable to N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL72527), an irreversible inhibitor of both enzymes. The two compounds were then tested for their effects in the PIT model. Both intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) administration of C9-4 decreased infarct volumes significantly. By contrast, C13-4 reduced the volume of brain infarction by i.c.v. administration, but no reduction was observed after i.p. administration. C9-4 administered by i.p. injection reduced the volume of brain infarction significantly at doses of more than 3 mg/kg, and the dosage of 5 mg/kg or 10 mg/kg demonstrated the most potent effect and were more effective than equivalent doses of the other inhibitors such as MDL72527 and N-benzylhydroxylamine. I.P. injection of 5 mg/kg of C9-4 provided a therapeutic time window of longer than 12 h. This report demonstrates that C9-4 is a potent inhibitor of the polyamine oxidizing enzymes and is useful lead compound for candidate drugs with a long therapeutic time window, to be used in the treatment of ischemic stroke.