Red and White Thrombus Characteristics in Patients Undergoing Carotid Endarterectomy.

OBJECTIVE: The study aimed to compare the characteristics of red and white thrombi in patients undergoing carotid endarterectomy. MATERIAL AND METHODS: The study was conducted in 81 patients with ischemic stroke who underwent carotid endarterectomy for carotid artery stenosis. Carotid plaques were graded by two pathologists. Thrombus materials were divided into two groups: white and red. The parameters of assessment were plaque rupture, lipid core, fibrous cap thickness, inflammation, intraplaque hemorrhage, calcification, necrotic core, and neovascularization. Normally distributed data were evaluated using Mann-Whitney U and Chi-squared tests. RESULTS: The ratio of white and red thrombus was 19.8% and 80.2%, respectively. Lipid core, plaque rupture, necrotic core, neovascularization, intraplaque hemorrhage, obstruction, and inflammation were observed more in red thrombus, which were statistically significant. Calcification and fibrous cap thickness were not statistically significant in the two groups. Moreover, intimal smooth muscle cells were present in all thrombus types. CONCLUSION: In our study, we found that red thrombi had more unstable characteristics than white thrombi. Thus, the risk for ischemic cerebrovascular events is more in red thrombi. However, this finding cannot be generalized due to the small number of patients in this study. Therefore, studies involving more patients are needed.

Left Carotid Artery Thrombosis Due to Thromboangiitis Obliterans.

ABSTRACT: Thromboangiitis obliterans (TAO, Buerger disease) is a segmental, non-atherosclerotic vasculitis that causes occlusion of the small and medium sized vessels of the distal extremities. In rare cases, it can affect vessels in the gastrointestinal, cerebrovascular, coronary, and renal systems. The etiology of thromboangiitis obliterans is unknown, but there is a strong association with smoking in the development and the progression of the disease. We present the case of a 42-year-old homeless female smoker, who was found dead outdoors. Although originally suspected to be a possible trauma-related death, autopsy revealed a thrombus in her left carotid artery, which caused an acute cerebral infarction. It was concluded that thromboangiitis obliterans, likely precipitated by smoking, was the cause of the thrombosis and subsequent death.

Nix-mediated mitophagy regulates platelet activation and life span.

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 FeCl3-induced carotid arterial thrombosis without affecting the expression of platelet glycoproteins (GPs) such as GPIb, GPVI, and αIIbß3 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.

Intraluminal Thrombi in the Cervico-Cephalic Arteries.

Background and Purpose- Intraluminal thrombus (ILT) is an uncommon finding among patients with ischemic stroke. We report clinical-imaging manifestations, treatment offered, and outcome among patients with ischemic stroke/transient ischemic attack and ILT in their cervico-cephalic arteries. Methods- Sixty-one of 3750 consecutive patients with acute ischemic stroke/transient ischemic attack (within 24 hours of onset) and ILT on initial arch-to-vertex computed tomography angiography from April 2015 through September 2017 constituted the prospective study cohort. Functional outcome was assessed using the modified Rankin Scale score with functional independence at discharge defined as modified Rankin Scale score ≤2. Results- Prevalence of ILT on computed tomography angiography was 1.6% (95% CI, 1.2%-2.1%). Median age was 67 years (interquartile range, 56-73), and 40 subjects (65%) were male. The initial clinical presentation included transient ischemic attack in 12 (20%) and stroke in 49 patients (80%); most strokes (76%) were mild (National Institutes of Health Stroke Scale ≤5). The most common ILT location was cervical carotid or vertebral artery (n=48 [79%]) followed by intracranial (n=11 [18%]) and tandem lesions (n=2 [3%]). The most common initial treatment strategy was combination antithrombotics (heparin with single antiplatelet agent) among 57 patients (93%). Follow-up computed tomography angiography (n=59), after a median 6 days (interquartile range 4-10 days), revealed thrombus resolution in 44 patients (75% [completely in 27%]). Twenty four of 30 patients (80%) with >50% residual carotid stenosis underwent carotid revascularization (endarterectomy in 15 and stenting in 9 patients) without peri-procedural complications a median of 9 days after symptom onset. In-hospital stroke recurrence occurred in 4 patients (6.6%). Functional independence was achieved in 46 patients (75%) at discharge. Conclusions- Patients presenting with acute stroke/transient ischemic attack with ILT on baseline imaging have a favorable clinical course in hospital with low stroke recurrence, high rate of thrombus resolution, and good functional outcome when treated with combination antithrombotic therapy.

Adamts18 deficiency increases arterial thrombus formation associated with vascular defects in mice.

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 FeCl3-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.

PEGylation of Lumbrokinase improves pharmacokinetic profile and enhances anti­thrombotic effect in a rat carotid artery thrombosis model.

The present study aimed to prepare injectable Lumbrokinase (LK) with long circulation time in addition to enhanced anti-thrombotic efficacy. Following preparation, the pharmacokinetic and anti­thrombotic effect of the drug in a rat carotid artery thrombosis model was evaluated. The drug was prepared by conjugation of LK with mPEG­SC20000 as previously reported. The pharmacokinetics of the mPEG­SC20000­LK were then examined and the anti­thrombotic activity in an artery­vein bypass thrombosis rat model was evaluated. Finally, the parameters of fibrinolysis including thromboxane B2, prostaglandin F1α, tissue plasminogen activator and plasminogen activator inhibitor­1 were compared between native LK and mPEG­SC20000­LK in a FeCl3­induced carotid artery thrombosis rat model. mPEG­SC20000­LK was successfully prepared by PEGylation of LK with mPEG20000­SC in optimal conditions. Pharmacokinetic analysis demonstrated that the biological half­life of the mPEG20000­SC­LK increased by 2.2­fold compared with native LK. In vivo anti­thrombotic analysis indicated that mPEG20000­SC­LK exhibited a greater anti­thrombotic effect in artery­vein bypass thrombosis and FeCl3-induced carotid artery thrombosis models compared with native LK. In conclusion, injectable PEGylated LK with prolonged half-life and enhanced anti­thrombotic effect is a potential anti­thrombotic agent for long­acting treatment of the thrombus diseases.

Apixaban Inhibits Cerebral Microembolic Signals Derived from Carotid Arterial Thrombosis in Rabbits.

Cerebral microembolic signal (MES) is an independent predictor of stroke risk and prognosis. The objective of this study is to assess the effects of apixaban, as a representative of the novel oral anticoagulant class, on a rabbit model of cerebral MES. A clinical transcranial Doppler ultrasound instrument was used to assess MESs in the middle cerebral artery in a 30% FeCl3-induced carotid arterial thrombosis model in male New Zealand White rabbits. Ascending doses of apixaban were evaluated as monotherapy and in combination with aspirin on both arterial thrombosis and MES. Pharmacokinetic and pharmacodynamic responses were also evaluated. The effective dose for 50% inhibition (ED50) of thrombus formation for monotherapy was 0.04 mg/kg per hour apixaban, i.v. (0.03 µM plasma exposure) for the integrated blood flow, 0.13 mg/kg per hour apixaban (0.10 µM plasma exposure) for thrombus weight, and 0.03 mg/kg per hour apixaban (0.02 µM plasma exposure) for MES. Dual treatment with aspirin (5 mg/kg, PO) and apixaban (0.015 mg/kg per hour, i.v.) resulted in a significant reduction in cerebral MES (P < 0.05) compared with monotherapy with either agent. Pharmacokinetic analysis of apixaban and pharmacodynamic assays using activated partial thromboplastin time (aPTT) and prothrombin time (PT) for apixaban- and arachidonic acid-induced platelet aggregation for aspirin were used to confirm the exposure-response relationships. In summary, our study demonstrates that apixaban in a concentration-dependent manner inhibits both arterial thrombosis and MES, suggesting a potential association between factor Xa (FXa) blockade and the reduction in MES in patients at risk of ischemic stroke.

Successful endovascular stroke therapy in a 103-year-old woman.

People older than 80 years of age constitute the most rapidly growing age group in the world. Several trials confirming superior efficacy of endovascular therapy did not have an upper age limit and showed favorable treatment effects, regardless of age. Current American Heart Association/American Stroke Association guidelines do not restrict treatment based on age as long as other eligibility criteria are met. A 103-year-old woman presented 2 h after stroke onset secondary to a left internal carotid artery terminus (ICA-T) occlusion. Admission National Institutes of Health Stoke Scale (NIHSS) score was 38, with no early ischemic changes on imaging, pre-stroke modified Rankin Scale score was 0, and she lived independently with minimal help. After initiation of intravenous thrombolysis, the patient underwent successful mechanical thrombectomy with Thombosis in Cerebral Infaction-3 recanalization. She showed remarkable recovery (NIHSS score of 1 at 48 h). Stroke onset to recanalization was 3 h 40 min. Our objective in documenting the oldest patient to successfully undergo stroke intervention is to corroborate that with the current evidence, appropriate patients undergoing rapid treatment may allow us to advance the limits of endovascular therapy.

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel.

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

Effects of febuxostat on platelet-derived microparticles and adiponectin in patients with hyperuricemia.

Platelet-derived microparticles (PDMPs) and adiponectin play an important role in the development of atherothrombosis. We investigated the effect of febuxostat on circulating PDMP levels and adiponectin in hyperuricemic patients. Levels of PDMP and biomarkers were measured using an ELISA at baseline and after 2 and 6 months of treatment. Plasma levels of PDMPs and biomarkers were higher, while those of adiponectin were lower in hyperuricemic patients than in normouricemic controls. Uric acid and interleukin (IL)-6 levels decreased significantly in hyperuricemic patients after 2 months of febuxostat treatment. However, PDMP and biomarkers decreased significantly in hyperuricemic patients after only 6 months of febuxostat treatment and adiponectin increased significantly. These results suggest that the effects of febuxostat for PDMPs seen may be the effect on xanthine oxidase but not the decrease of uric acid, and febuxostat may be beneficial for primary prevention of atherothrombosis in hyperuricemic patients.

Murine arterial thrombus induction mechanism influences subsequent thrombodynamics.

BACKGROUND: The mechanism of thrombotic induction in experimental models can vary greatly, as can the applied evaluative measures, making comparisons among models difficult. OBJECTIVES: This study comparatively evaluated the arterial thrombodynamic response among injury mechanisms. METHODS: Thrombotic responses were induced in mouse carotid arteries, with subsequent intravital imaging using rhodamine-6G-labeled platelets to quantitate platelet accumulation over 30minutes. Nine induction methods were evaluated: brief pinch, temporary hard ligation, cautery/heat, needle puncture, intralumenal wire (scratch), intralumenal adventitia/collagen (2 different models), and brief exposures to either iron-based surface electrolytic injury or ferric chloride. RESULTS: The accumulation of platelets was variable among induction methods, with a greater response to more severe injury mechanisms, free radical injury, and exposed collagen. Temporal profiles were generated by normalizing data to peak platelet accumulation for each run; rapid platelet development and subsequent detachment were found for mechanical injuries that maintained vessel integrity (pinch and ligation injuries), with more sustained growth for more severe mechanical (wire) injury or breach of the vessel (needle puncture or intralumenal collagen). A delayed but extended temporal response was seen with free radical injury (both electrolytic and ferric chloride). CONCLUSIONS: These findings demonstrate a dependence of platelet thrombodynamics on the method of induction, with collagen exposure causing greater, more prolonged activity, while free-radical injury effected a delayed but sustained platelet thrombus formation with slower resolution. A better understanding of how these various injury models relate to clinical causes of arterial thrombosis is needed for optimal translational interpretation of murine models of thrombosis.

Inflammatory cytokine TSLP stimulates platelet secretion and potentiates platelet aggregation via a TSLPR-dependent PI3K/Akt signaling pathway.

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 αIIbß3 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 αIIbß3 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.

Reduced thrombosis in Klkb1-/- mice is mediated by increased Mas receptor, prostacyclin, Sirt1, and KLF4 and decreased tissue factor.

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.

Intraluminal thrombus, intraplaque hemorrhage, plaque thickness, and current smoking optimally predict carotid stroke.

BACKGROUND AND PURPOSE: Intraplaque hemorrhage (IPH) is associated with acute and future stroke. IPH is also associated with lumen markers of stroke risk including stenosis, plaque thickness, and ulceration. Whether IPH adds further predictive value to these other variables is unknown. The purpose of this study was to determine whether IPH improves carotid-source stroke prediction. METHODS: In this retrospective cross-sectional study, patients undergoing stroke workup were imaged with MRI and IPH detection. Seven hundred twenty-six carotid-brain image pairs were analyzed after excluding vessels with noncarotid plaque stroke sources (420) and occlusions (7) or near-occlusions (3). Carotid imaging characteristics were recorded, including percent diameter and mm stenosis, plaque thickness, ulceration, intraluminal thrombus, and IPH. Clinical confounders were recorded, and a multivariable logistic regression model was fitted. Backward elimination was used to determine essential carotid-source stroke predictors with a threshold 2-sided P<0.10. Receiver operating characteristic analysis was performed to determine discriminatory value. RESULTS: Significant predictors of carotid-source stroke included intraluminal thrombus (odds ratio=103.6; P<0.001), IPH (odds ratio=25.2; P<0.001), current smoking (odds ratio=2.78; P=0.004), and thickness (odds ratio=1.24; P=0.020). The final model discriminatory value was excellent (area under the curve=0.862). This was significantly higher than the final model without IPH (area under the curve=0.814), or models using only stenosis as a continuous variable (area under the curve=0.770) or cutoffs of 50% and 70% (area under the curve=0.669), P<0.001. CONCLUSIONS: After excluding patients with noncarotid plaque stroke sources, optimal discrimination of carotid-source stroke was obtained with intraluminal thrombus, IPH, plaque thickness, and smoking history but not ulceration and stenosis.