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Background: QiShen YiQi (QSYQ) dropping pill, a component-based Chinese medicine consisting of benefiting Qi (YQ) and activating blood (HX) components, has been reported to exert a beneficial effect on cerebral ischemia-induced stroke. However, its efficacy and pharmacological mechanism on acute thromboembolic stroke is not clear., Purpose: This study is to explore the preventative effect and pharmacological mechanism of QSYQ and its YQ/HX components on the formation of platelet-leukocyte aggregation (PLA) in acute thromboembolic stroke., Study Design and Methods: In vivo thromboembolic stroke model and FeCl 3 -induced carotid arterial occlusion models were used. Immunohistochemistry, Western blot, RT-qPCR, and flow cytometry experiments were performed to reveal the pharmacological mechanisms of QSYQ and its YQ/HX components., Results: In thromboembolic stroke rats, QSYQ significantly attenuated infarct area, improved neurological recovery, reduced PLA formation, and inhibited P-selection (CD62P)/ P-selectin glycoprotein ligand-1 (PSGL-1) expressions. The YQ component preferentially down-regulated PSGL-1 expression in leukocyte, while the HX component preferentially down-regulated CD62P expression in platelet. In carotid arterial thrombosis mice, QSYQ and its YQ/HX components inhibited thrombus formation, prolonged vessel occlusion time, reduced circulating leukocytes and P-selectin expression. PLA formation and platelet/leukocyte adhesion to endothelial cell were also inhibited by QSYQ and its YQ/HX components in vitro., Conclusion: QSYQ and YQ/HX components attenuated thromboembolic stroke and carotid thrombosis by decreasing PLA formation via inhibiting CD62P/PSGL-1 expressions. This study shed a new light on the prevention of thromboembolic stroke., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Abstract: Desialylation, governed by sialidases or neuraminidases, is strongly implicated in a wide range of human disorders, and accumulative data show that inhibition of neuraminidases, such as neuraminidases 1 sialidase, may be useful for managing atherosclerosis. Several studies have reported promising effects of oseltamivir phosphate, a widely used anti-influenza sialidase inhibitor, on human cancer cells, inflammation, and insulin resistance. In this study, we evaluated the effects of oseltamivir phosphate on atherosclerosis and thrombosis and potential liver toxicity in LDLR-/- mice fed with high-fat diet. Our results showed that oseltamivir phosphate significantly decreased plasma levels of LDL cholesterol and elastin fragmentation in aorta. However, no effect was observed on both atherosclerotic plaque size in aortic roots and chemically induced thrombosis in carotid arteries. Importantly, oseltamivir phosphate administration had adverse effects on the liver of mice and significantly increased messenger RNA expression levels of F4/80, interleukin-1β, transforming growth factor-β1, matrix metalloproteinase-12, and collagen. Taken together, our findings suggest that oseltamivir phosphate has limited benefits on atherosclerosis and carotid thrombosis and may lead to adverse side effects on the liver with increased inflammation and fibrosis., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

In spite of current treatment strategies, myocardial infarction and stroke are still major causes of death worldwide. These events are triggered by damage of an atherosclerotic plaque, resulting in occlusive thrombus formation. Mouse studies have significantly contributed to our understanding of the mechanisms of atherogenesis and of thrombosis following plaque injury, but the extent to which the mouse serves as an accurate model of human disease is open to discussion. In this review, we provide a detailed overview and comparison of the described mouse models for atherothrombosis including their (dis)advantages. Herein guidance is provided on how to select a suitable atherothrombosis model for research questions primarily relevant to the field of thrombosis.

The ferric chloride models of arterial thrombosis are useful tools with which to investigate the cellular and molecular mechanisms that contribute to arterial thrombosis. Recent insights have, however, revealed the complex and multifaceted mechanism by which ferric chloride induces thrombus formation. Here, we discuss the strengths and weaknesses of the ferric chloride models of arterial thrombosis. Particular focus is given to the phenotypes of different knockout mice in the ferric chloride models and how these compare to other models with independent modes of initiation. Further, we discuss the relevance of the ferric chloride models to the human pathology of atherothrombotic disease.

Background: The role of neutrophil extracellular traps (NETs) in procoagulant activity (PCA) in stroke patients caused by thromboembolic occlusion of the internal carotid artery (ICA) remains unclear. Our objectives were to evaluate the critical role of NETs in the induction of hypercoagulability in stroke and to identify the functional significance of NETs during atherothrombosis., Methods: The levels of NETs, activated platelets (PLTs), and PLT-derived microparticles (PMPs) were detected in the plasma of 55 stroke patients and 35 healthy controls. NET formation and thrombi were analysed using immunofluorescence. Exposed phosphatidylserine (PS) was evaluated with flow cytometry and confocal microscopy. PCA was analysed using purified coagulation complex, thrombin, and fibrin formation assays., Findings: The plasma levels of NETs, activated PLTs, and PMP markers in the carotid lesion site (CLS) were significantly higher than those in the aortic blood. NETs were decorated with PS in thrombi and the CLS plasma of ICA occlusion patients. Notably, the complementary roles of CLS plasma and thrombin-activated PLTs were required for NET formation and subsequent PS exposure. PS-bearing NETs provided functional platforms for PMPs and coagulation factor deposition and thus increased thrombin and fibrin formation. DNase I and lactadherin markedly inhibited these effects. In addition, NETs were cytotoxic to endothelial cells, converting these cells to a procoagulant phenotype. Sivelestat, anti-MMP9 antibody, and activated protein C (APC) blocked this cytotoxicity by 25%, 39%, or 52%, respectively., Interpretation: NETs played a pivotal role in the hypercoagulability of stroke patients. Strategies that prevent NET formation may offer a potential therapeutic strategy for thromboembolism interventions., Funding: This study was supported by grants from the National Natural Science Foundation of China (61575058, 81873433 and 81670128) and Graduate Innovation Fund of Harbin Medical University (YJSKYCX2018-58HYD)., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Platelet activation requires fully functional mitochondria, which provide a vital energy source and control the life span of platelets. Previous reports have shown that both general autophagy and selective mitophagy are critical for platelet function. However, the underlying mechanisms remain incompletely understood. Here, we show that Nix, a previously characterized mitophagy receptor that plays a role in red blood cell maturation, also mediates mitophagy in platelets. Genetic ablation of Nix impairs mitochondrial quality, platelet activation, and FeCl 3 -induced carotid arterial thrombosis without affecting the expression of platelet glycoproteins (GPs) such as GPIb, GPVI, and α IIb β 3 Metabolic analysis revealed decreased mitochondrial membrane potential, enhanced mitochondrial reactive oxygen species level, diminished oxygen consumption rate, and compromised adenosine triphosphate production in Nix -/- platelets. Transplantation of wild-type (WT) bone marrow cells or transfusion of WT platelets into Nix-deficient mice rescued defects in platelet function and thrombosis, suggesting a platelet-autonomous role (acting on platelets, but not other cells) of Nix in platelet activation. Interestingly, loss of Nix increases the life span of platelets in vivo, likely through preventing autophagic degradation of the mitochondrial protein Bcl-xL. Collectively, our findings reveal a novel mechanistic link between Nix-mediated mitophagy, platelet life span, and platelet physiopathology. Our work suggests that targeting platelet mitophagy Nix might provide new antithrombotic strategies., (© 2019 by The American Society of Hematology.)

Dysfunction of vascular endothelial cells causes the risk of thrombosis. Aim of this study is to evaluate the antithrombotic effect of Okinawa mozuku (brown seaweed, Cladosiphon okamuranus) extract by using cultured vascular endothelial cells and rat carotid arterial thrombosis model induced by ferric chloride (FeCl 3 ). The cell line (TKM-33) established from human umbilical vein endothelial cells were cultured with or without Okinawa mozuku extract. After incubation for 24 h, the conditioned medium was collected to evaluate urokinase-type plasminogen activator (u-PA) activity. Next, rats were fed with water or water containing 5% of Okinawa mozuku extract for 8 wk. After 8 wk of treatments, the rats were provided for the carotid arterial thrombosis model, and fibrinolytic factor and coagulation factor in blood were measured. Okinawa mozuku extract significantly augmented u-PA activity in the conditioned medium. The decrease of carotid artery blood flow induced by 40% FeCl 3 injury in rats fed with Okinawa mozuku extract was less than that in control rats. Thus, oral administration of Okinawa mozuku extract prevented thrombus formation in this model. Oral administration of Okinawa mozuku extract significantly increased u-PA activity in euglobulin fraction, compared with control group. On the other hand, platelet aggregation activity, activated partial thromboplastin time, and active PAI-1 level in plasma exhibited no significant differences between control and Okinawa mozuku groups. These results indicate that oral administration of Okinawa mozuku enhances fibrinolytic activity in plasma and prevents thrombus formation which is induced by injury of vascular endothelial cells.

Lumbrokinase (LK) has strong fibrinolytic and thrombolytic activities, but it has a short half-life, can be easily inactivated, and may cause hemorrhage as a side effect. This study develops a potential thrombolytic therapy by fabricating N,N,N-Trimethyl Chitosan (TMC) nanoparticles modified with the cyclic Arg-Gly-Asp-Phe-Lys peptide (c-RGD) and loaded with LK (i.e. c-RGD-LK-NPs). The binding of c-RGD to platelet membrane GPIIb/IIIa receptors is expected to enable targeted delivery of the c-RGD-conjugated TMC to the thrombus. The synthesized c-RGD-LK-NPs had a mean particle size of 232.0 nm, zeta potential of 19.8 mV, entrapment efficiency of 52.7% ± 2.5%, and loading efficiency of 17.4% ± 0.65%. Transmission electron microscopy showed that they were generally spherical. The c-RGD-LK-NPs gave a cumulative in vitro LK release of 80.6% over 8 h, and the activity of LK was close to 80%, indicating that the nanoparticles protected the activity of LK. In vitro blood clot lysis assays were carried out and in vivo thrombolysis effect was tested in Sprague-Dawley rats carotid artery thrombus model. In all cases, the c-RGD-LK-NPs showed superior performance compared with the free LK and the unmodified TMC nanoparticles loaded with LK. The c-RGD-LK-NPs reagent is expected to be potentially useful in treating thromboembolic diseases.

Salvia deserta Schang (SDS) belongs to the same family as Salvia miltiorrhiza bunge, one of the antithrombotic Chinese herbal medicines. In our study, EtOAc root extracts were analyzed for their effects on adenosine diphosphate (ADP)-induced platelet aggregation in rabbits and FeCl 3 -induced rat common carotid artery thrombosis as well as on rat blood plasma concentrations of thromboxane B2 (TXB 2 ), 6-keto-prostaglandin F1 alpha (6-keto-PGF 1α ), antithrombin-III (AT-III), protein C (PC), plasminogen (PLG), plasminogen activator inhibitor (PAI-1), von Willebrand factor (vWF) and tissue-type plasminogen activator (t-PA). EtOAc extracts from SDS roots had significant inhibitory effects on ADP-induced maximum platelet aggregation rate (10.2 ± 2.6 vs control 35.7 ± 5.2; P < 0.05), reduced the FeCl 3 -induced rat common carotid artery thrombus weight and thrombus area ratio (P < 0.05), significantly decreased plasma TXB 2 , vWF and PAI-1 levels and increased 6-keto-PGF 1α and t-PA levels in a dose dependent manner (all P < 0.05). Thus, the ratio of TXB 2 /6-keto-PGF 1α was significantly decreased (P < 0.05), while the ratio of t-PA/PAI-1 was significantly increased (P < 0.05). In addition, enhanced AT-III and PC activities indicated coagulation inactivation effects of EtOAc SDS root extracts. EtOAc extraction from SDS showed antithrombotic effects, which are likely due to platelet adhesion and aggregation inhibition as well as anticoagulant activities.

ADAMTS18 is a member of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) that are known for their crucial role in development, angiogenesis, inflammation and coagulation. It was previously reported that ADAMTS18 cleaved by thrombin induced platelet fragmentation, through which thrombus were dissolved. However, it remains unclear whether this represents a dominant physiologic mechanism controlling thrombus growth in vivo. Here, we used an established Adamts18 knockout (KO) mouse model to determine its function in thrombus formation. ADAMTS18 deficiency accelerated FeCl 3 -induced carotid artery thrombosis and aggravated postischemic cerebral infarction in mice. However, this accelerated thrombus phenotype in Adamts18 KO mice was not due to the lack of ADAMTS18-mediated-platelet fragmentation. Moreover, Adamts18 deficiency exerted little effects on mouse platelet functions. The underlying molecular mechanisms could be attributed in part to the abnormal vascular remodeling, including deficiency of carotid body (glomus) and aberrant carotid basal lamina. These results indicate a novel function of ADAMTS18 in vascular remodeling and associated thrombus formation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Introduction: Kunitz proteinase inhibitor (KPI) domain-containing forms of the amyloid β-protein precursor (AβPP) inhibit cerebral thrombosis. KPI domain-lacking forms of AβPP are abundant in brain. Regions of AβPP other than the KPI domain may also be involved with regulating cerebral thrombosis. To determine the contribution of the KPI domain to the overall function of AβPP in regulating cerebral thrombosis we generated a reactive center mutant that was devoid of anti-thrombotic activity and studied its anti-thrombotic function in vitro and in vivo., Methods: To determine the extent of KPI function of AβPP in regulating cerebral thrombosis we generated a recombinant reactive center KPI R13I mutant devoid of anti-thrombotic activity. The anti-proteolytic and anti-coagulant properties of wild-type and R13I mutant KPI were investigated in vitro. Cerebral thrombosis of wild-type, AβPP knock out and AβPP/KPI R13I mutant mice was evaluated in experimental models of carotid artery thrombosis and intracerebral hemorrhage., Results: Recombinant mutant KPI R13I domain was ineffective in the inhibition of pro-thrombotic proteinases and did not inhibit the clotting of plasma in vitro. AβPP/KPI R13I mutant mice were similarly deficient as AβPP knock out mice in regulating cerebral thrombosis in experimental models of carotid artery thrombosis and intracerebral hemorrhage., Conclusions: We demonstrate that the anti-thrombotic function of AβPP primarily resides in the KPI activity of the protein., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A thrombus (blood clot) is formed in injured vessels to maintain the integrity of vasculature. However, obstruction of blood vessels by thrombosis slows blood flow, leading to death of tissues fed by the artery and is the main culprit of various life-threatening cardiovascular diseases. Herein, we report a rationally designed nanomedicine that could specifically image obstructed vessels and inhibit thrombus formation. On the basis of the physicochemical and biological characteristics of thrombi such as an abundance of fibrin and an elevated level of hydrogen peroxide (H 2 O 2 ), we developed a fibrin-targeted imaging and antithrombotic nanomedicine, termed FTIAN, as a theranostic system for obstructive thrombosis. FTIAN inhibited the generation of H 2 O 2 and suppressed the expression of tumor necrosis factor-alpha (TNF-α) and soluble CD40 ligand (sCD40L) in activated platelets, demonstrating its intrinsic antioxidant, anti-inflammatory, and antiplatelet activity. In a mouse model of ferric chloride (FeCl 3 )-induced carotid thrombosis, FTIAN specifically targeted the obstructive thrombus and significantly enhanced the fluorescence/photoacoustic signal. When loaded with the antiplatelet drug tirofiban, FTIAN remarkably suppressed thrombus formation. Given its thrombus-specific imaging along with excellent therapeutic activities, FTIAN offers tremendous translational potential as a nanotheranostic agent for obstructive thrombosis.

Arterial thrombosis and its related diseases are major healthcare problems worldwide. Blebbistatin is an inhibitor of myosin II, which plays an important role in thrombosis. The aim of our study is to explore the effect and potential mechanism of blebbistatin on arterial thrombosis. A ferric chloride (FeCl 3 ) solution at a concentration of 5% was used to induce carotid artery thrombosis in mice. Immunohistochemistry and immunofluorescence staining were used to detect the expression or activation of non-muscle myosin heavy chain IIA (NMMHC IIA), tissue factor (TF), GSK3β and NF-κB. Blebbistatin (1 mg/kg, i.p.) significantly reduced carotid artery thrombosis induced by FeCl 3 solution in mice, inhibited NMMHC IIA expression and impeded TF expression via the GSK3β-NF-κB signalling pathway in mouse arterial vascular tissues. The present study demonstrates that blebbistatin may impede TF expression partly via the Akt/GSK3β-NF-κB signalling pathways in the endothelium in a FeCl 3 model, shedding new insights into the pathogenesis of arterial thrombosis and providing new clues for the development of antithrombotic drugs., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

The precise mechanism for reduced thrombosis in prekallikrein null mice (Klkb1(-/-)) is unknown. Klkb1(-/-) mice have delayed carotid artery occlusion times on the rose bengal and ferric chloride thrombosis models. Klkb1(-/-) plasmas have long-activated partial thromboplastin times and defective contact activation-induced thrombin generation that partially corrects upon prolonged incubation. However, in contact activation-induced pulmonary thromboembolism by collagen/epinephrine or long-chain polyphosphate, Klkb1(-/-) mice, unlike F12(-/-) mice, do not have survival advantage. Klkb1(-/-) mice have reduced plasma BK levels and renal B2R mRNA. They also have increased expression of the renal receptor Mas and plasma prostacyclin. Increased prostacyclin is associated with elevated aortic vasculoprotective transcription factors Sirt1 and KLF4. Treatment of Klkb1(-/-) mice with the Mas antagonist A-779, COX-2 inhibitor nimesulide, or Sirt1 inhibitor splitomicin lowers plasma prostacyclin and normalizes arterial thrombosis times. Treatment of normal mice with the Mas agonist AVE0991 reduces thrombosis. Klkb1(-/-) mice have reduced aortic tissue factor (TF) mRNA, antigen, and activity. In sum, Klkb1(-/-) mice have a novel mechanism for thrombosis protection in addition to reduced contact activation. This pathway arises when bradykinin delivery to vasculature is compromised and mediated by increased receptor Mas, prostacyclin, Sirt1, and KLF4, leading to reduced vascular TF., (© 2015 by The American Society of Hematology.)

Aims: Thymic stromal lymphopoietin (TSLP) plays an important role in inflammatory diseases and is over-expressed in human atherosclerotic artery specimens. The present study investigated the role of TSLP in platelet activation and thrombosis models in vitro and in vivo, as well as the underlying mechanism and signaling pathway., Methods and Results: Western blotting and flow cytometry demonstrated that the TSLP receptor was expressed on murine platelets. According to flow cytometry, platelet stimulation with TSLP induced platelet degranulation and integrin αIIbβ3 activation. A TSLPR deficiency caused defective platelet aggregation, defective platelet secretion and markedly blunted thrombus growth in perfusion chambers at both low and high shear rates. TSLPR KO mice exhibited defective carotid artery thrombus formation after exposure to FeCl3. TSLP increased Akt phosphorylation, an effect that was abrogated by the PI3K inhibitors wortmannin and LY294002. The PI3K inhibitors further diminished TSLP-induced platelet activation. TSLP-mediated platelet degranulation, integrin αIIbβ3 activation and Akt phosphorylation were blunted in platelets that lacked the TSLP receptor., Conclusion: This study demonstrated that the functional TSLPR was surface-expressed on murine platelets. The inflammatory cytokine TSLP triggered platelet activation and thrombus formation via TSLP-dependent PI3K/Akt signaling, which suggests an important role for TSLP in linking vascular inflammation and thrombo-occlusive diseases., (© 2015 S. Karger AG, Basel.)

Background: Fibrin is a major component of arterial and venous thrombi and represents an ideal candidate for molecular imaging of thrombosis. Here, we describe imaging properties and target uptake of a new fibrin-specific positron emission tomographic probe for thrombus detection and therapy monitoring in 2 rat thrombosis models., Methods and Results: The fibrin-binding probe FBP7 was synthesized by conjugation of a known short cyclic peptide to a cross-bridged chelator (CB-TE2A), followed by labeling with copper-64. Adult male Wistar rats (n=26) underwent either carotid crush injury (mural thrombosis model) or embolic stroke (occlusive thrombosis model) followed by recombinant tissue-type plasminogen activator treatment (10 mg/kg, IV). FBP7 detected thrombus location in both animal models with a high positron emission tomographic target-to-background ratio that increased over time (>5-fold at 30-90 minutes, >15-fold at 240-285 minutes). In the carotid crush injury animals, biodistribution analysis confirmed high probe uptake in the thrombotic artery (≈0.5%ID/g; >5-fold greater than blood and other tissues of the head and thorax). Similar results were obtained from ex vivo autoradiography of the ipsilateral versus contralateral carotid arteries. In embolic stroke animals, positron emission tomographic-computed tomographic imaging localized the clot in the internal carotid/middle cerebral artery segment of all rats. Time-dependent reduction of activity at the level of the thrombus was detected in recombinant tissue-type plasminogen activator-treated rats but not in vehicle-injected animals. Brain autoradiography confirmed clot dissolution in recombinant tissue-type plasminogen activator-treated animals, but enduring high thrombus activity in control rats., Conclusions: We demonstrated that FBP7 is suitable for molecular imaging of thrombosis and thrombolysis in vivo and represents a promising candidate for bench-to-bedside translation., (© 2014 American Heart Association, Inc.)

There is considerable interest in defining new agents or targets for antithrombotic purposes. The 5-HT2A receptor is a G-protein coupled receptor (GPCR) expressed on many cell types, and a known therapeutic target for many disease states. This serotonin receptor is also known to regulate platelet function. Thus, in our FDA-approved drug repurposing efforts, we investigated the antiplatelet activity of cyproheptadine and pizotifen, two antidepressant 5-HT2A Receptor antagonists. Our results revealed that cyproheptadine and pizotifen reversed serotonin-enhanced ADP-induced platelet aggregation in vitro and ex vivo. And the inhibitory effects of these two agents were found to be similar to that of EMD 281014, a 5-HT2A Receptor antagonist under development. In separate experiments, our studies revealed that these 5-HT2A receptor antagonists have the capacity to reduce serotonin-enhanced ADP-induced elevation in intracellular calcium levels and tyrosine phosphorylation. Using flow cytometry, we also observed that cyproheptadine, pizotifen, and EMD 281014 inhibited serotonin-enhanced ADP-induced phosphatidylserine (PS) exposure, P-selectin expression, and glycoprotein IIb-IIIa activation. Furthermore, using a carotid artery thrombosis model, these agents prolonged the time for thrombotic occlusion in mice in vivo. Finally, the tail-bleeding time was investigated to assess the effect of cyproheptadine and pizotifen on hemostasis. Our findings indicated prolonged bleeding time in both cyproheptadine- and pizotifen-treated mice. Notably, the increases in occlusion and bleeding times associated with these two agents were comparable to that of EMD 281014, and to clopidogrel, a commonly used antiplatelet drug, again, in a fashion comparable to clopidogrel and EMD 281014. Collectively, our data indicate that the antidepressant 5-HT2A antagonists, cyproheptadine and pizotifen do exert antiplatelet and thromboprotective effects, but similar to clopidogrel and EMD 281014, their use may interfere with normal hemostasis.

Factor XI (fXI) is a homodimeric zymogen that is converted to a protease with 1 (1/2-fXIa) or 2 (fXIa) active subunits by factor XIIa (fXIIa) or thrombin. It has been proposed that the dimeric structure is required for normal fXI activation. Consistent with this premise, fXI monomers do not reconstitute fXI-deficient mice in a fXIIa-dependent thrombosis model. FXI activation by fXIIa or thrombin is a slow reaction that can be accelerated by polyanions. Phosphate polymers released from platelets (poly-P) can enhance fXI activation by thrombin and promote fXI autoactivation. Poly-P increased initial rates of fXI activation 30- and 3000-fold for fXIIa and thrombin, respectively. FXI monomers were activated more slowly than dimers by fXIIa in the presence of poly-P. However, this defect was not observed when thrombin was the activating protease, nor during fXI autoactivation. The data suggest that fXIIa and thrombin activate fXI by different mechanisms. FXIIa may activate fXI through a trans-activation mechanism in which the protease binds to 1 subunit of the dimer, while activating the other subunit. For activation by thrombin, or during autoactivation, the data support a cis-activation mechanism in which the activating protease binds to and activates the same fXI subunit.

Noninvasive detection of fibrin in vivo using diagnostic imaging modalities may improve clinical decision-making on possible therapeutic options in atherosclerosis, cancer and thrombus-related pathologies such as pulmonary embolism and deep venous thrombosis. The aim of this study was to assess the potential of a novel (111)In-labeled fibrin-binding peptide (FibPep) to visualize thrombi in mice noninvasively using single-photon emission computed tomography (SPECT). FibPep and a negative control peptide (NCFibPep) were synthesized and their fibrin-binding properties were assessed in vitro. FibPep showed enhanced binding compared with NCFibPep to both fibrin and blood clots. FibPep bound to fibrin with a dissociation constant (K(d)) of 0.8 μ m, whereas NCFibPep displayed at least a 100-fold lower affinity towards fibrin. A FeCl3 -injury carotid artery thrombosis mouse model was used to evaluate the peptides in vivo. FibPep and NCFibPep displayed rapid blood clearance and were eliminated via the renal pathway. In vivo SPECT imaging using FibPep allowed clear visualization of thrombi. Ex vivo biodistribution showed significantly increased uptake of FibPep in the thrombus-containing carotid in comparison to the noninjured carotid (5.7 ± 0.7 and 0.6 ± 0.4% injected dose per gram (%ID g(-1)), respectively; p < 0.01; n = 4), whereas nonspecific NCFibPep did not (0.4 ± 0.2 and 0.3 ± 0.0%ID g(-1), respectively; n = 4). In conclusion, FibPep displayed high affinity towards fibrin in vitro and rapid blood clearance in vivo, and allowed sensitive detection of thrombi using SPECT imaging. Therefore, this particular imaging approach may provide a new tool to diagnose and monitor diseases such as atherosclerosis and cancer., (Copyright © 2013 John Wiley & Sons, Ltd.)

Introduction: The thrombin mutant W215A/E217A (WE thrombin) has greatly reduced procoagulant activity, but it activates protein C in the presence of thrombomodulin and inhibits binding of platelet glycoprotein Ib to von Willebrand factor and collagen under flow conditions. Both thrombomodulin-dependent protein C activation and inhibition of platelet adhesion could contribute to the antithrombotic activity of WE thrombin., Materials and Methods: To assess the role of thrombomodulin, we administered WE thrombin to thrombomodulin-deficient (TM(Pro/Pro)) mice and measured the time to occlusive thrombus formation in the carotid artery after photochemical injury of the endothelium., Results and Conclusions: Doses of WE thrombin ≥10μg/kg prolonged the thrombosis time of wild-type mice (>1.6-fold), while doses ≥100μg/kg only slightly prolonged the thrombosis time of TM(Pro/Pro) mice. We conclude that thrombomodulin plays a predominate role in mediating the antithrombotic effect of WE thrombin in the arterial circulation of mice after endothelial injury. Thrombomodulin-independent effects may occur only when high doses of WE thrombin are administered., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Selective inhibitors of cyclooxygenase (COX)-2 increase the risk of myocardial infarction and thrombotic events, but the responsible mechanisms are not fully understood., Methods and Results: We found that ferric chloride-induced arterial thrombus formation was significantly greater in COX-2 knockout compared with wild-type mice. Cross-transfusion experiments excluded the likelihood that COX-2 knockout platelets, despite enhanced aggregation responses to collagen and thrombin, are responsible for increased arterial thrombus formation in COX-2 knockout mice. Importantly, we observed that COX-2 deletion decreased prostacyclin synthase and production and peroxisome proliferator-activated receptor- and sirtuin-1 (SIRT1) expression, with consequent increased upregulation of tissue factor (TF), the primary initiator of blood coagulation. Treatment of wild-type mice with a prostacyclin receptor antagonist or a peroxisome proliferator-activated receptor-δ antagonist, which predisposes to arterial thrombosis, decreased SIRT1 expression and increased TF activity. Conversely, exogenous prostacyclin or peroxisome proliferator-activated receptor-δ agonist completely reversed the thrombotic phenotype in COX-2 knockout mice, restoring normal SIRT1 levels and reducing TF activity. Furthermore, inhibition of SIRT1 increased TF expression and activity and promoted generation of occlusive thrombi in wild-type mice, whereas SIRT1 activation was sufficient to decrease abnormal TF activity and prothrombotic status in COX-2 knockout mice., Conclusions: Modulation of SIRT1 and hence TF by prostacyclin/peroxisome proliferator-activated receptor-δ pathways not only represents a new mechanism in controlling arterial thrombus formation but also might be a useful target for therapeutic intervention in the atherothrombotic complications associated with COX-2 inhibitors.

The clinical use of niacin to treat dyslipidemic conditions is limited by noxious side effects, most commonly facial flushing. In mice, niacin-induced flushing results from COX-1-dependent formation of PGD₂ and PGE₂ followed by COX-2-dependent production of PGE₂. Consistent with this, niacin-induced flushing in humans is attenuated when niacin is combined with an antagonist of the PGD₂ receptor DP1. NSAID-mediated suppression of COX-2-derived PGI₂ has negative cardiovascular consequences, yet little is known about the cardiovascular biology of PGD₂. Here, we show that PGD₂ biosynthesis is augmented during platelet activation in humans and, although vascular expression of DP1 is conserved between humans and mice, platelet DP1 is not present in mice. Despite this, DP1 deletion in mice augmented aneurysm formation and the hypertensive response to Ang II and accelerated atherogenesis and thrombogenesis. Furthermore, COX inhibitors in humans, as well as platelet depletion, COX-1 knockdown, and COX-2 deletion in mice, revealed that niacin evoked platelet COX-1-derived PGD₂ biosynthesis. Finally, ADP-induced spreading on fibrinogen was augmented by niacin in washed human platelets, coincident with increased thromboxane (Tx) formation. However, in platelet-rich plasma, where formation of both Tx and PGD₂ was increased, spreading was not as pronounced and was inhibited by DP1 activation. Thus, PGD₂, like PGI₂, may function as a homeostatic response to thrombogenic and hypertensive stimuli and may have particular relevance as a constraint on platelets during niacin therapy.

The present study was aimed to study the effect of RGD peptide conjugation on the bio-distribution behaviour of long circulatory liposomes in the thrombosed rat model. Further, thrombolysis study was also performed to evaluate the therapeutic activity of the prepared liposomes. Liposomes were prepared by film hydration method and peptide was subsequently conjugated on the preformed liposomes using carbodiimide chemistry. Prepared liposomes were characterized for size and size distribution, entrapment efficiency and in vitro drug release. In vitro targeting ability of the liposomes was determined by platelets binding assay. In vivo studies were performed in the rat model containing human blood clot inoculated in the carotid artery. Results of the study showed that RGD peptide conjugated liposomes significantly accumulated to the site of blood clot and higher thrombolytic activity was observed with peptide modified liposomes as compared to plain streptokinase solution and long circulatory liposomes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objective: Plant-derived α-linolenic acid (ALA) may constitute an attractive cardioprotective alternative to fish-derived n-3 fatty acids. However, the effect of dietary ALA on arterial thrombus formation remains unknown., Methods and Results: Male C57Bl/6 mice were fed a high-ALA or low-ALA diet for 2 weeks. Arterial thrombus formation was delayed in mice fed a high-ALA diet compared with those on a low-ALA diet (n=7; P<0.005). Dietary ALA impaired platelet aggregation to collagen and thrombin (n=5; P<0.005) and decreased p38 mitogen-activated protein kinase activation in platelets. Dietary ALA impaired arterial tissue factor (TF) expression, TF activity, and nuclear factor-κB activity (n=7; P<0.05); plasma clotting times and plasma thrombin generation did not differ (n=5; P=not significant). In cultured human vascular smooth muscle and endothelial cells, ALA inhibited TF expression and activity (n=4; P<0.01). Inhibition of TF expression occurred at the transcriptional level via the mitogen-activated protein kinase p38 in smooth muscle cells and p38, extracellular signal-regulated kinases 1 and 2, and c-Jun N-terminal kinases 1 and 2 in endothelial cells., Conclusions: ALA impairs arterial thrombus formation, TF expression, and platelet activation and thereby represents an attractive nutritional intervention with direct dual antithrombotic effects.

Platelet hyperactivity associated with hyperlipidemia contributes to development of a pro-thrombotic state. We previously showed that oxidized LDL (oxLDL) formed in the setting of hyperlipidemia and atherosclerosis initiated a CD36-mediated signaling cascade leading to platelet hyperactivity. We now show that the guanine nucleotide exchange factors Vav1 and Vav3 were tyrosine phosphorylated in platelets exposed to oxLDL. Pharmacologic inhibition of src family kinases abolished Vav1 phosphorylation by oxLDL in vitro. Coimmunoprecipitations revealed the tyrosine phosphorylated form of src kinase Fyn was associated with Vav1 in platelets exposed to oxLDL. Using a platelet aggregation assay, we demonstrated that Vav1 deficiency, Fyn deficiency, or Vav1/Vav3 deficiency protected mice from diet-induced platelet hyperactivity. Furthermore, flow cytometric analysis revealed that Vav1/Vav3 deficiency significantly inhibited oxLDL-mediated integrin αIIbβIII activation of platelets costimulated with ADP. Finally, we showed with an in vivo carotid artery thrombosis model that genetic deletion of Vav1 and Vav3 together may prevent the development of occlusive thrombi in mice fed a high-fat diet. These findings implicate Vav proteins in oxLDL-mediated platelet activation and suggest that Vav family member(s) may act as critical modulators linking a prothrombotic state and hyperlipidemia.

Homocysteine is an endogenous sulphydryl aminoacid irreversibly catabolized by transsulfuration to cysteine or remethylated to methionine. Increased plasma levels of homocysteine are an independent risk factor for atherosclerosis and cardiovascular disease. Accurate and reliable quantification of this amino acid in plasma samples is essential in clinical practice to explore the presence of a hyperhomocysteinemia, for instance after an ischemic event, or to control a possible adjunctive risk factor in patients at higher risk. In this review, LC-ESI-MS/MS methods are discussed and compared with other analytical methods for plasma homocysteine. LC-ESI-MS/MS is a technique combining the physicochemical separation of liquid chromatography with the analysis of mass spectrometry. It is based on stable-isotope dilution and possesses inherent accuracy and precision. Quantitative analysis is achieved by using commercially available homocystine-d(8) as an internal standard. Taking advantage of the high sensitivity and specificity, approaches involving LC-ESI-MS/MS require less laborious sample preparation, no derivatization and produce reliable results.

Objectives: Thrombomodulatory factors have been implicated in plaque instability. The aim was to examine the relationship between thrombomodulatory gene expression, timing of clinical events and plaque histology., Design of Study: Plaques were obtained from 40 consecutive patients undergoing carotid endarterectomy and divided into three groups (group 1, early symptomatic, within 1 month; group 2, late symptomatic, 1-6 months and group 3, asymptomatic). Total RNA was isolated to determine the expression of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), plasminogen activator inhibitor-1 (PAI-1), tissue factor (TF), tissue factor pathway inhibitor (TFPI), thrombomodulin (TM), CD68 and vascular endothelial-cadherin (VE-Cadherin)., Results: Expression of t-PA, PAI-1, TF, TFPI, TM, CD68 and VE-cadherin were significantly increased in the early symptomatic group (p=0.019, 0.028, 0.018, 0.025, 0.038, 0.016 and 0.027 respectively), but the level of gene expression in the late symptomatic group was indistinguishable from the asymptomatic group. The incidence of plaque rupture and intraplaque haemorrhage was significantly increased in the early symptomatic groups (58% versus 18%/18% group 2/3, and 55% versus 6%/9% respectively, p<0.05 for both)., Conclusions: Expression of thrombomodulatory genes is increased in unstable plaques, though levels after 1 month are comparable to asymptomatic plaques. This transient rise may influence plaque instability, and rapid resolution mirrors the clinical reduction in risk of further thrombo-embolic events.

Tissue factor (TF) initiates coagulation, regulates hemostasis, and plays a critical role in mediating arterial thrombosis. TF is up-regulated in vascular smooth muscle cells (VSMCs) in atherosclerosis and arterial injury. To examine the biologic role of VSMC-derived TF, we crossed TF(flox/flox) mice with SM22alphaCre(+/-) mice. TF mRNA and activity were decreased in the aortic media of TF-deficient mice by 96% and 94.8%, respectively. There were no differences in TF activity measured in plasma or concentrated microparticles. TF-deficient mice were generated with the expected frequency, showed no evidence of bleeding or increased mortality, and had similar activated partial thromboplastin and tail vein bleeding times. Thrombus-mediated flow reduction in response to ferric chloride injury of the carotid arteries was significantly attenuated in VSMC-specific TF-deficient. Stable occlusion was seen in 11 of 12 wild-type mice, but in only 6 of 16 VSMC-specific TF-deficient mice (P = .001). These data suggest that VSMC-derived TF is critical in a macrovascular model of arterial thrombosis. This mouse model should be valuable in determining the contribution of VSMC-derived TF in other TF-mediated phenomena, such as restenosis.

Background: In patients with coronary artery disease and reduced ejection fraction, amiodarone reduces mortality by decreasing sudden death. Because the latter may be triggered by coronary artery thrombosis as much as ventricular arrhythmias, amiodarone might interfere with tissue factor (TF) expression and thrombus formation., Methods and Results: Clinically relevant plasma concentrations of amiodarone reduced TF activity and impaired carotid artery thrombus formation in a mouse photochemical injury model in vivo. PTT, aPTT, and tail bleeding time were not affected; platelet number was slightly decreased. In human endothelial and vascular smooth muscle cells, amiodarone inhibited tumor necrosis factor (TNF)-alpha and thrombin-induced TF expression as well as surface activity. Amiodarone lacking iodine and the main metabolite of amiodarone, N-monodesethylamiodarone, inhibited TF expression. Amiodarone did not affect mitogen-activated protein kinase activation, TF mRNA expression, and TF protein degradation. Metabolic labeling confirmed that amiodarone inhibited TF protein translation., Conclusions: Amiodarone impairs thrombus formation in vivo; in line with this, it inhibits TF protein expression and surface activity in human vascular cells. These pleiotropic actions occur within the range of amiodarone concentrations measured in patients, and thus may account at least in part for its beneficial effects in patients with coronary artery disease.

Objectives: Although remarkable therapeutic advances in the treatment of acute coronary syndromes (ACS) have been made with anti-platelet therapy, the therapeutic options may be limited by considerable side effects. Pigment epithelium-derived factor (PEDF) has anti-oxidative properties and may play a protective role against atherosclerosis. In this study, we investigated whether PEDF prevented occlusive thrombus formation in rats., Methods and Results: Occlusive thrombus formation was induced by treating rats with ligation and cuff placement at the left common carotid artery. Intravenous injection of PEDF dose-dependently inhibited thrombus formation and blocked the increase in immunoreactivity of P-selectin, a marker of platelet activation, NADPH oxidase activity and superoxide generation in thrombi. In vitro, PEDF significantly decreased collagen-induced reactive oxygen species generation in platelets and subsequently suppressed the platelet activation and aggregation. Plasma and intraplatelet levels of PEDF in the coronary circulation in patients with ACS were significantly lower than those in age- and gender-matched controls without coronary artery disease., Conclusions: These results demonstrated that PEDF administration could inhibit occlusive thrombus formation by blocking the platelet activation and aggregation through its anti-oxidative properties. Our present study suggests that pharmacological up-regulation or substitution of PEDF may offer a promising strategy for the treatment of arterial thrombosis.

The recombinant protein SAK-RGD-K2-Hir is characterized by its fibrin-specific properties of plasminogen activation combined with antithrombin and antiplatelet activities. It was previously shown in our in-vitro studies to be a more potent and faster-acting thrombolytic agent compared with standard r-SAK. In order to document the effects of the thrombolytic potential of SAK-RGD-K2-Hir we examined this protein in an electrically induced carotid artery thrombosis model and stasis-induced venous model in rats. In the arterial thrombosis model, a bolus injection of SAK-RGD-K2-Hir was less effective than rt-PA and r-SAK. However, the most effective in the improvement and maintenance of carotid patency and in arterial thrombus mass reduction was SAK-RGD-K2. In contrast, all r-SAK derivatives reduced venous thrombus weight significantly in comparison to r-SAK and r-Hir. However, the most observable decrease in thrombus weight was obtained after application of recombinant proteins containing the r-Hir. The bleeding time was significantly prolonged in the animals treated with proteins containing r-Hir at a dose of 1.0 mg/kg. There were no observable changes in plasma fibrinogen concentration. In conclusion, our findings show thrombolytic activity in intravenous bolus injection of the novel thrombolytic agent SAK-RGD-K2-Hir in rats. Although this protein compares favourably with r-SAK in rat venous thrombolysis, we were unable to confirm the beneficial effects of SAK-RGD-K2-Hir over r-SAK and rt-PA in the carotid artery thrombolysis model. Furthermore, our results also suggest that SAK-RGD-K2-Hir bears a risk of bleeding, but this may be true for higher doses.

In vivo markers that allow for detection of ferritin within atheromatous plaque may be useful for identifying iron-catalyzed hydroxyl-radical formation and subsequent lipid peroxidation. Recently, a positive contrast MR technique--GRadient echo Acquisition for Superparamagnetic particles/suscePtibility (GRASP)--was used to identify the presence of magnetic entities in phantom models. The aim of the current study was to determine the feasibility of using GRASP in conjunction with conventional T(2) (*)-weighted (T(2) (*)W) gradient-echo (GRE) sequences for identifying ferritin/hemosiderin deposition using in vitro and in vivo models of thrombus. In vitro thrombi were prepared by incubating blood with ferritin. MRI was performed using conventional GRE sequences and GRASP. The results indicate that GRASP was able to verify ferritin deposition in in vitro thrombi. In vivo thrombi were created using a crush injury model in rabbits. The signal enhancement obtained using conventional GRE sequences and GRASP was compared with the location of iron deposition by histology. In all of the animals the GRASP signal correlated with signal loss by conventional GRE, and ferritin/hemosiderin deposition by histology. GRASP sequences in combination with conventional GRE sequences may be used to detect the presence of ferritin deposition in in vitro thrombi and in vivo crush-injured rabbit carotid arteries., ((c) 2006 Wiley-Liss, Inc.)

Although there are some in vitro evidence that angiotensin II (Ang II) may promote thrombosis, there is still no data concerning effect of Ang II on arterial thrombus formation. In the present study we have investigated the influence of Ang II on electrically induced arterial thrombosis in a common carotid artery of renovascular hypertensive rats. Furthermore, we examined if Ang II effect is mediated via AT1 receptor. We measured some coagulation and fibrinolytic parameters at the same time. Since platelets play crucial role in the initiation of arterial thrombosis their contribution in the mode of Ang II action was also determined. Intravenous infusion of Ang II caused significant increase in arterial thrombus weight, which was reversed by losartan, selective AT1 receptor antagonist. The prothrombotic effect of Ang II was accompanied by increase in haemostatic and decrease in fibrinolytic potential of rat plasma. While number of data has clearly demonstrated that Ang II can augment human platelets aggregation, at least in rats, platelets were not involved in the mechanism of Ang II action. Our study shows that Ang II via AT1 receptor accelerates arterial thrombosis in renovascular hypertensive rat, therefore may be considered as a risk factor of myocardial infarction or stroke.

Aim: To study the effect of honokiol on arterial thrombosis and endothelial cells., Methods: Rabbit platelet aggregation was performed with Borns turbid method. Thrombosis was produced by the endothelial injury stimulated with electric current. Rat aortic endothelial cells (RAEC) were cultured and cell viability was assessed using the MTT assay. Nitric oxide (NO) concentrations in serum-free media of RAEC were determined using the kinetic cadmium-reduction method. The stable metabolite prostacyclin was measured in serum-free media of RAEC by radioimmunoassay., Results: Honokiol (37.6-376 micromol/L) decreased rabbit platelet aggregation in vitro in a concentration-dependent manner, while intravenously injection of honokiol (0.12-12 microg/kg) significantly inhibited rabbit platelet aggregation induced by collagen ex vivo. In the electrical current-stimulated carotid thrombosis model in rats, honokiol (5-50 microg/kg, iv) prolonged the thrombus occlusion time in a does-dependent manner. In vitro honokiol (0.376-37.6 micromol/L) effectively protected cultured RAEC against oxidized low density lipoprotein (ox-LDL) injury, and significantly increased 6-keto-PGF1alpha (the stable metabolite of prostacyclin) in serum-free media of RAEC. Honokiol also increased NO level in RAEC serum-free medium at a lower concentration range (0.0376-0.376 micromol/L), but honokiol 3.76 micromol/L decreased NO level., Conclusion: Honokiol is a potent arterial thrombosis inhibitor. Endothelial cell protection and the stimulation of prostacyclin release may be its main anti-thrombosis mechanism. Stimulation of NO release in endothelial cells may play a role, but it is not a key factor.

Clinic-experimental complex investigation and ELISA determination of protein HLDF as also primary antibodies (Abs) to HLDF values in blood and cerebro-spinal fluid (CSF) of patients with hypertensive crises and acute aterotrombotic ischemic stroke were performed. Statistically improved difference in serum HLDF and Abs content in examined patients in comparison with age-matched controls as also significant intergroup's differences in protein and Abs dynamic content were determined. Correlation between content and dynamics of investigated factors in CSF and blood serum with determined clinical and instrumental parameters was revealed. Pathogenetic and prognostic significance of revealed changes in molecular factors was viewed.

Objective: This study was undertaken to investigate the origin and pathophysiological importance of plasminogen activator inhibitor (PAI-1) in atherosclerosis., Methods and Results: We used the ferric chloride model to induce carotid artery injury in apolipoprotein E knockout (apoE-/-) and wild-type (WT) mice. ApoE-/- mice fed high-fat diet for 4 months developed severe hypercholesterolemia and had significantly elevated plasma PAI-1 levels (2.3+/-0.3 versus 0.6+/-0.1 ng/mL in WT mice; P<0.05). These mice exhibited a prothrombotic phenotype with shortened times to thrombotic arterial occlusion (8.6 versus 11.5 minutes; P<0.001) and reduced recanalization rates (12% versus 51%; P<0.0001) compared with WT mice. In situ hybridization, reverse transcriptase-polymerase chain reaction, and immunohistochemistry showed a significantly upregulated PAI-1 expression in P-selectin-positive (activated) endothelial cells lining normal-appearing arterial segments and within the advanced atherosclerotic lesions of apoE-/- mice. No significant upregulation of PAI-1 expression was found in the other organs studied, and only trace amounts of PAI-1 mRNA were detected in murine platelets. Importantly, deletion of the PAI-1 gene reversed the prothrombotic tendency and reduced neointimal growth after injury in apoE-/- mice despite the persistence of excessive hypercholesterolemia., Conclusions: These results suggest that increased vascular expression of PAI-1 may contribute to the elevated circulating levels of the inhibitor and be responsible, at least in part, for the prothrombotic phenotype in apoE-/- mice.

Objective: Tissue factor (TF) is normally expressed at low levels in the media of blood vessels, but it is readily induced after vessel injury. It is not known whether vascular damage per se or thrombus formation is responsible for this phenomenon., Methods and Results: Cyclic flow variations (CFVs), attributable to recurrent thrombus formation, were induced in stenotic rabbit carotid arteries with endothelial injury. CFVs were observed for 30 minutes and 2, 4, and 8 hours in different groups of animals. Another group of rabbits pretreated with hirudin before inducing arterial damage to inhibit thrombus formation was observed for 8 hours. Arterial sections were immunostained for TF. Undamaged arteries served as controls. In additional rabbits, in situ hybridization experiments were performed. No TF expression was observed in the media of control vessels, whereas a progressive increase in TF mRNA and protein expression was observed in carotid arteries as CFVs progressed. No increase in TF expression was observed in animals pretreated with hirudin. In vitro experiments demonstrated that TF mRNA is induced in smooth muscle cells stimulated with activated platelets as well as with some platelet-derived mediators., Conclusions: This phenomenon may contribute to sustain intravascular thrombus formation after the initial thrombogenic stimulus.

Objective: We sought to fabricate a compliant engineered vascular graft (inner diameter of approximately 4.5 mm and length of 6 cm) lined with endothelial progenitor cells derived from circulating peripheral canine blood and to verify its nonthrombogenicity potential in vivo., Methods: Autologous circulating endothelial progenitor cells derived from the peripheral veins of 6 adult mongrel dogs were isolated by using a density gradient method. The cells were proliferated in vitro in EGM-2 culture medium, prelined on the luminal surface of in situ-formed collagen type I meshes as an extracellular matrix, and wrapped with a segmented polyurethane thin film with multiple micropores as a compliant scaffold. After canine carotid arteries were bilaterally implanted with these grafts for 1 and 3 months, microscopic observation, histologic staining, and immunochemical staining were performed to evaluate morphogenesis., Results: After 33.3 +/- 10.5 days of culture in vitro, 4.2 +/- 1.2 x 10(6) endothelial progenitor cells were obtained. Eleven of the 12 engineered vascular grafts were patent. The grafts possessed smooth, glistening, and ivory-colored luminal surfaces at the predetermined observation period up to 3 months. The intimal layer was covered with confluent, cobblestone-like monolayered cells that were positively stained with factor VIIIB-related antigen. The thickness of the neoarterial walls was approximately 300 microm at 3 months after implantation. A few smooth muscle cells were observed in the medial tissue, and fibroblasts dominated the adventitial tissue., Conclusion: Circulating endothelial progenitor cells could be a substitute source of endothelial cells for endothelialization on small-diameter-vessel prostheses to ensure nonthrombogenicity.

The anti-thrombotic effects of LB30057, a direct thrombin inhibitor, were evaluated with in vivo rat and dog thrombosis models. In rats, 1 mg/kg of LB30057 inhibited half of the clot formations in the inferior vena cava at 5 minutes after intravenous application. When measured at 2 hours after oral application, 100 mg/kg prevented approximately half of the clot formations in the inferior vena cava and 50 mg/kg prolonged the mean occlusion time from 15.6 +/- 1.3 minutes to 47.2 +/- 8.3 minutes in the carotid artery. In dogs, the formation of thrombus in the jugular vein was reduced to half at a dose range of 20-30 mg/kg at 6 hours after oral application. In addition, the LB30057 dosage required to reduce venous clot formation by approximately 80-90% in dogs was only about 10% of that required for the same reduction in rats. This is probably due to the variation in its time-dependent blood concentration profiles in each species; for example, the plasma half-life of LB71350 in dogs was longer than that in rats (153.0 +/- 3.0 vs. 129.7 +/- 12.7 min at 30 mg/kg, i.v., respectively). AUC, T(max), C(max), and BA in dogs were 59, 8.9, 9.17, and 13.3 times higher than those in rats at oral 30 mg/kg, respectively. Taken together, these results suggest that LB30057 administered orally is effective in the prevention of arterial and venous thrombosis in rats and dogs. It therefore represents a good lead compound for investigations to discover a new, orally available, therapeutic agent for treating thrombotic diseases.

Alterations in factors involved in the regeneration of the neuronal network in the hippocampus of rats with microsphere embolism (ME) were examined. Nine hundred microspheres (48 microm in diameter) were injected into the right hemisphere, and immunochemical and immunohistochemical studies on the hippocampus were performed on the seventh day thereafter. Hematoxylin-eosin staining showed progressive and severe degeneration of the hippocampus after ME. The protein levels of brain-derived neurotrophic factor (BDNF), 43-kDa growth-associated protein (GAP-43), and adhesion protein L1 (L1) in the ipsilateral hippocampus of the ME animal, determined by Western blot analysis or enzyme immunoassay, were increased, unaltered, and decreased, respectively. In contrast, the immunohistochemical study showed increases in a marker of axonal sprouting GAP-43, and a neurotrophic factor BDNF, and a decrease in an adhesion molecule L1 in some areas of the hippocampal ischemic penumbra of such animals. These results suggest that some factors for regeneration of the neuronal network in the ischemic penumbra responded to sustained cerebral ischemia for a certain period, although functional network of the nerve cells in the microsphere-injected hemisphere would be unlikely established after ME.

Investigation into the influence of specific genes and gene products upon the pathophysiology of cerebral ischaemia has been greatly enhanced by the use of genetically modified mice. A simple model of global cerebral ischaemia in mouse is bilateral common carotid artery occlusion (BCCAo) and the neuropathological impact of BCCAo has been investigated in several mouse strains. Bilateral carotid occlusion produces extensive neuronal damage in C57Bl/6J strain mice and this damage is linked to posterior communicating artery (PcomA) hypoplasticity in the circle of Willis. In the present study, we investigated the effect of BCCAo in MF1 strain mice and compared them with C57Bl/6J mice. The neuropathological consequences of BCCAo were assessed using standard histochemical staining and heat shock protein 70 (HSP70) immunohistochemical staining (to demarcate cells that had been ischaemically stressed). The effect of BCCAo on mean arterial blood pressure (MABP) was also measured. The plasticity of the circle of Willis was recorded using carbon black perfusion. MF1 mice displayed significantly less ischaemic neuronal damage and HSP70 immunoreactivity compared to C57Bl/6J mice following 10-20 min BCCAo. Moreover, ischaemic neuronal damage and HSP70 immunoreactivity in MF1 mice subjected to extended BCCAo (25-45 min) was never as extensive or widespread as that observed in C57Bl/6J mice after 20 min BCCAo. MABP in MF1 mice (102+/-5 mmHg) was significantly higher than in C57Bl/6J mice (87+/-5) during 20 min BCCAo. MABP in MF1 mice during 20 and 40 min (103+/-12 mmHg) BCCAo remained above pre-occlusion values for the entire occlusion period. MF1 mice had significantly greater circle of Willis plasticity (more PcomAs) than C57Bl/6J mice did. These data indicate that MF1 mice are less susceptible to BCCAo than C57Bl/6J mice and that this could be due to maintained increases in MABP during BCCAo and the lower prevalence of abnormalities of the circle of Willis in MF1 mice.

Objectives: The aim of the present study was to test the hypothesis that retrovirus-mediated in vivo tissue factor pathway inhibitor (TFPI) gene transfer to the arterial wall would efficiently inhibit thrombosis without causing significant changes in systemic hemostatic variables., Background: Acute coronary syndromes (unstable angina and acute myocardial infarction) are usually caused by atherosclerotic plaque rupture, with consequent activation of the coagulation cascade and circulating platelets. Tissue factor (TF) exposure represents an early event in this pathophysiologic sequence, leading to activation of the extrinsic coagulation pathway and thrombin formation. Tissue factor pathway inhibitor is a naturally occurring inhibitor of the extrinsic pathway., Methods: In the present study, the gene coding for rabbit TFPI was inserted in a retroviral vector under control of a tetracycline-inducible promoter. Replication-defective, infectious, recombinant retroviruses were used to transfect rabbit carotid arteries with either TFPI or a reporter gene--green fluorescent protein (GFP)., Results: Retroviral-mediated arterial gene transfer of TFPI resulted in potent inhibition of intravascular thrombus formation in stenotic and injured rabbit carotid arteries, whereas transfection of the contralateral carotid artery with GFP had no effect on thrombosis. No significant changes in systemic hemostatic variables (prothrombin time and partial thromboplastin time) were observed when thrombosis was inhibited., Conclusions: These data suggest that retroviral-mediated transfection of the arterial wall with TFPI might represent an attractive approach for the treatment of thrombotic disorders.

Objective: To assess whether patients with carotid artery occlusion (CAO) who have clinical features suggesting a haemodynamic origin have a poor haemodynamic or metabolic state of the brain., Methods: In 117 patients with ischaemic symptoms of the eye or brain that were transient or at most moderately disabling and associated with a CAO, we compared CO(2) reactivity, quantitative flow measurement by magnetic resonance (MR) angiography, metabolic ratios measured by (1)H-MR spectroscopy, collateral blood flow patterns and the presence of infarcts of the borderzone type between patients grouped by the following clinical features: (1) presence or absence of at least one of the 'classical' haemodynamic symptoms: limb shaking, retinal claudication, precipitation of symptoms by exercise, by rising from a sitting or lying position, by transition from a cold to a warm environment, or by documented hypotension, and (2) symptoms having occurred after demonstration of the CAO or only before the occlusion was documented., Results: Patients with (n = 16) and without (n = 101) one of the 'classical' haemodynamic symptoms did not differ in any of the measured indices. Patients with recurrent symptoms after documentation of the CAO (n = 56) had lower CO(2) reactivity (difference 8.3%, 95% confidence interval 0.1-16.5) than those with symptoms only before documentation of the occlusion (n = 61), whereas no significant differences were found in any of the other measured indices. The difference in CO(2) reactivity was no longer significant after adjustment for the interval between the patients' last symptoms and the CO(2) reactivity measurement., Conclusion: In patients with CAO we could not find an association between symptoms that have been associated with hypoperfusion and a poor haemodynamic or metabolic state of the brain.

Tissue factor (TF) is a low-molecular-weight glycoprotein that initiates the extrinsic clotting cascade and is considered a major regulator of arterial thrombogenicity. TF pathway inhibitor (TFPI) is a major physiological inhibitor of TF-initiated coagulation. The aim of this study was to define the complex interplay between TF and TFPI and the regulation of vascular thrombogenicity in a model of vascular remodeling. To determine the levels and pattern of vascular expression of TF and TFPI associated with vascular remodeling, a murine model of flow cessation was studied. TF activity of the arteries increased after ligation (P<0.05). Quantitative analysis of homogenates of remodeled carotid arteries revealed increased TF expression but unchanged TFPI expression compared with normal carotid arteries, resulting in enhanced TF activity. To determine the potential therapeutic role of TFPI in this thrombogenic state, mice were treated with intravascular adenoviral delivery of either murine TFPI (Ad-mTFPImyc) or a control adenovirus (Ad-DeltaE1). Overexpression of TFPI decreased vascular TF activity compared with viral control (P<0.01). Overexpression of TFPI inhibited neointimal formation (P=0.038), resulting in enhanced luminal area (P=0.001) 4 weeks after flow cessation. In this murine model of vascular remodeling, an imbalance between TF and TFPI expression is generated, resulting in increased TF activity. Overexpression of TFPI in this model inhibits vascular TF activity and results in attenuation of vascular remodeling associated with flow interruption.

The importance of low-density lipoprotein (LDL) in the etiology of atherosclerosis is well recognized. We have established a reproducible stenosis model in hypercholesterolemic hamsters, and the process of arterial stenosis by thrombus or neointima was studied and compared with that in normal hamsters. The level of plasma LDL was 4.6 times higher in hamsters fed a high-cholesterol diet than in hamsters fed normal food. Endothelial injury in right common carotid arteries was induced using a modified catheter. Arterial blood flow was monitored continuously using a Doppler flow probe. Arterial patency after the initiation of injury in high-cholesterol hamsters was significantly changed as compared with that of normal hamsters. Neointima was observed 2 wk after the vascular injury. The neointimal area of high-cholesterol hamsters was significantly larger than that of normal hamsters. To characterize the stenosis in hypercholesterolemic hamsters, we measured platelet aggregation, thrombin time, activated partial thromboplastin time, and proliferating smooth muscle cells (SMC) in vitro and in vivo. The half-maximal inhibitory concentration value for platelet aggregation induced by thrombin or collagen, the DNA synthesis stimulated by platelet-derived growth factor (PDGF)-BB, and 5-bromo-2-deoxy-uridine labeling indices (proliferating index of SMC in vivo) in high-cholesterol hamsters were each significantly higher than the comparable value from normal hamsters. However, specific binding of PDGF-BB in SMC was not different between the two types of hamsters. Furthermore, we investigated the inhibitory effects of probucol or losartan on neointima formation using this model. Probucol, but not losartan, significantly reduced the neointimal area in hypercholesterolemic hamsters. These findings indicated that high levels of plasma LDL strongly contributed to the development of thrombus and neointima formation via both up-regulation of platelet aggregation and the enhancement of SMC proliferation. This stenosis model may be useful for the investigation of hypercholesterolemia-associated cardiovascular diseases.

Brief ischemia induces tolerance to subsequent more severe insults, and induction of the 70 kDa heat shock/stress protein, hsp72, has been suggested to play a role. This study tested the requirement for hsp72 expression in a gerbil tolerance model in which postischemic temperature was varied to modulate the level of hsp72 induction. Gerbils were subjected to 2 min bilateral common carotid artery occlusion and kept under halothane anesthesia for 90 min, during which rectal temperature was either maintained at 37 degrees C (normothermic, NT) or elevated to 39.5 degrees C (hyperthermic, HT) during 15-60 min recirculation. Hsp72 mRNA expression was determined by in situ hybridization with a (35)S-labeled oligonucleotide probe at 3, 24 and 48 h. Separate groups were subjected to a test challenge of 5 min ischemia 48 h after the priming insult, and CA1 neuron counts were obtained at 1 week. Significant protection was observed in both NT and HT groups. However, while 90% of hippocampi from NT animals showed detectable protection of CA1 neurons, less than half showed detectable hsp72 mRNA induction. These results indicate that, within the limits of experimental detection, hsp72 expression is not required for induction of ischemic tolerance.

The assumption that fibrin and crosslinked fibrin impart irreversibility to arterial thrombi is explored with procedure developed for measuring changes in platelet function, morphology and fibrinogen metabolism in aging occlusive thrombi, in which the condition of stasis is imposed uniformly. Arterial thrombi containing autologous (111)In labeled platelets were generated in vivo by bilateral mechanical injury of porcine carotid arteries. Vessels containing the platelet-rich thrombi were harvested and incubated intact (37 degrees C) for intervals ranging from 30 min to 12 h. The isolated vessels were then bisected and agitated in culture medium containing tick anticoagulant and hirudin for 60 min. Disaggregated platelets were evaluated for yield (from (111)In radioactivity) viability (dense body ATP secretion) and morphology (electron microscopy). Western analysis of fibrin(ogen) in thrombus extracts was performed using anti-fibrinogen Bbeta- and gamma-chain monoclonal antibodies for thrombi at each time point. A stable recovery of nearly 50% of platelets was observed during 12 h of thrombus aging. As thrombi aged, viability of disaggregated platelets gradually decreased with platelet necrosis the predominant feature beyond 6 h. By western analysis of thrombus extracts, nearly 50% of fibrinogen was cleaved to fibrin and extensively crosslinked within 30 min of injury with no evidence of fibrinolysis. With the exception of a declining proportion of gamma-monomer, these features remain relatively constant during 12 h of thrombus maturation. It is concluded that neither fibrin nor crosslinked fibrin are dominant factors imparting cohesion within platelet thrombi. Furthermore, under conditions of complete arterial occlusion imposed by this experimental design, there is no evidence of endogenous fibrinolysis.

Recent studies have shown a crucial role of intercellular adhesion molecule 1 (ICAM-1) in expansion of infarction after focal cerebral ischemia. The purpose of the present study was to assess whether ICAM-1 is involved in selective neuronal vulnerability and reactive gliosis after transient forebrain ischemia. ICAM-1 knockout mice and wild-type mice were subjected to transient forebrain ischemia for 5, 10 or 15 min, and the hippocampus and caudoputamen were examined 7 days later with conventional histological and immunohistochemical methods. Bilateral common carotid artery occlusion with less than 10% of baseline cortical microperfusion for 10 or 15 min resulted in ischemic neuronal damage in the hippocampus and caudoputamen. The frequency and the severity of neuronal damage were similar in wild-type and knockout mice. Proliferation of reactive astrocytes in the hippocampus was also similar in both types of mice. Therefore, it is highly unlikely that ICAM-1 plays a key role in delayed neuronal death after transient global ischemia or in astroglial responses after ischemic neuronal injury.