Masanori Tsujimoto

Cilostazol ameliorates collagenase-induced cerebral hemorrhage by protecting the blood-brain barrier.

Intracranial hemorrhage remains a devastating disease. Among antiplatelet drugs, cilostazol, a phosphodiesterase 3 inhibitor, was recently reported to prevent secondary hemorrhagic stroke in patients in a clinical trial. The aim of this study was to evaluate whether pre-treatment with cilostazol could decrease the intracranial hemorrhage volume and examine the protective mechanisms of cilostazol. We evaluated the pre-treatment effects of the antiplatelet drug cilostazol on the collagenase-induced intracranial hemorrhage volume and neurological outcomes in mice. To estimate the mechanism of collagenase injury, we evaluated various vascular components in vitro, including endothelial cells, vascular smooth muscle cells, pericytes, and a blood–brain barrier model. Cilostazol pre-treatment reduced the intracranial hemorrhage volume with sufficient inhibition of platelet aggregation, and motor function was improved by cilostazol treatment. Blood–brain barrier permeability was increased by collagenase-induced intracranial hemorrhage, and cilostazol attenuated blood–brain barrier leakage. Terminal deoxynucleotidyl transferase dUTP nick-end labeling and western blot analysis showed that cilostazol prevented pericyte cell death by inducing cyclic adenosine monophosphate-responsive element-binding protein phosphorylation. Cilostazol also prevented endothelial cell death and protected collagen type 4, laminin, and vascular endothelial- and N-cadherins from collagenase injury. In conclusion, cilostazol reduced collagenase-induced intracranial hemorrhage volume by protecting the blood–brain barrier.

Release of Phosphorylated HSP27 (HSPB1) from Platelets Is Accompanied with the Acceleration of Aggregation in Diabetic Patients

We investigated the relationship between HSP27 phosphorylation and collagen-stimulated activation of platelets in patients with diabetes mellitus (DM). Platelet-rich plasma was prepared from blood of type 2 DM patients. The platelet aggregation was analyzed in size of aggregates by an aggregometer using a laser scattering method. The protein phosphorylation was analyzed by Western blotting. Phosphorylated-HSP27 and PDGF-AB released from platelets were measured by ELISA. The phosphorylated-HSP27 levels at Ser-78 and Ser-82 induced by collagen were directly proportional to the platelet aggregation. Total HSP27 levels in platelets were decreased concomitantly with the phosphorylation. The released HSP27 levels were significantly correlated with the phosphorylated levels of HSP27 in the platelets stimulated by 0.3 μg/ml collagen. The low dose collagen-stimulated release of HSP27 was detected but relatively small in healthy donors. The released levels of PDGF-AB were in parallel with the levels of released HSP27. Area under the curve (AUC) of small aggregation (9-25 μm) induced by 0.3 μg/ml collagen was inversely proportional to the levels of released HSP27. AUC of large aggregation (50-70 μm) was directly proportional to the levels of released HSP27. Exogenous recombinant phosphorylated- HSP27 hardly affected the aggregation or the released levels of PDGF-AB induced by collagen. These results strongly suggest that HSP27 is released from human platelets accompanied with its phosphorylation induced by collagen, which is correlated with the acceleration of platelet aggregation in type 2 DM patients.

Long-term Magnetic Resonance Angiography Follow-up for Recanalized Vessels after Mechanical Thrombectomy

BACKGROUND Mechanical thrombectomy is an effective revascularization therapy for acute intracranial large vessel occlusion. We retrospectively evaluated magnetic resonance angiography (MRA) follow-up data to assess the long-term patency of recanalized vessels after mechanical thrombectomy. METHODS We retrospectively reviewed medical records of consecutive patients who had undergone mechanical thrombectomy for intravenous tissue plasminogen activator-failed/ineligible acute intracranial major vessel occlusion between October 2010 and April 2013 at our institution. MRA follow-up was performed at baseline and at 24 ± 6 hours and 3 months after mechanical thrombectomy. RESULTS Forty-nine patients underwent mechanical thrombectomy for acute intracranial major vessel occlusion. Mean age was 69.7 ± 11.5 years, and baseline median National Institute of Health Stroke Scale score was 15 (range, 8-24). Occlusion was found in the internal carotid artery in 18 patients (36.7%), middle cerebral artery in 26 patients (53%), and vertebral-basilar arteries in 5 patients (10.2%). Successful recanalization, as defined by a thrombolysis in cerebral infarction flow grade of 2b or 3, was achieved in 40 patients (81.6%). MRA follow-up at 24 hours after the treatment revealed that reocclusion of recanalized vessels was observed in 3 of 38 patients (7.9%). Long-term MRA follow-up showed that 2 of 27 patients (8.3%) developed diffuse severe stenosis of treated vessels. Both the patients had undergone treatment for middle cerebral artery occlusion with the Merci retriever and had been administered only anticoagulants, but not any antiplatelets. CONCLUSIONS Reocclusion or late stenosis of successfully recanalized vessels was observed in 16.2% of patients. Long-term MRA follow-up of recanalized vessels will be useful, in particular, for the patient with middle cerebral artery occlusion who undergoes mechanical thrombectomy.

Sphingosine 1-phosphate (S1P) suppresses the collagen-induced activation of human platelets via S1P4 receptor

Sphingosine 1-phosphate (S1P) is as an extracellular factor that acts as a potent lipid mediator by binding to specific receptors, S1P receptors (S1PRs). However, the precise role of S1P in human platelets that express S1PRs has not yet been fully clarified. We previously reported that heat shock protein 27 (HSP27) is released from human platelets accompanied by its phosphorylation stimulated by collagen. In the present study, we investigated the effect of S1P on the collagen-induced platelet activation. S1P pretreatment markedly attenuated the collagen-induced aggregation. Co-stimulation with S1P and collagen suppressed collagen-induced platelet activation, but the effect was weaker than that of S1P-pretreatment. The collagen-stimulated secretion of platelet-derived growth factor (PDGF)-AB and the soluble CD40 ligand (sCD40L) release were significantly reduced by S1P. In addition, S1P suppressed the collagen-induced release of HSP27 as well as the phosphorylation of HSP27. S1P significantly suppressed the collagen-induced phosphorylation of p38 mitogen-activated protein kinase. S1P increased the levels of GTP-bound Gαi and GTP-bound Gα13 coupled to S1PPR1 and/or S1PR4. CYM50260, a selective S1PR4 agonist, but not SEW2871, a selective S1PR1 agonist, suppressed the collagen-stimulated platelet aggregation, PDGF-AB secretion and sCD40L release. In addition, CYM50260 reduced the release of phosphorylated-HSP27 by collagen as well as the phosphorylation of HSP27. The selective S1PR4 antagonist CYM50358, which failed to affect collagen-induced HSP27 phosphorylation, reversed the S1P-induced attenuation of HSP27 phosphorylation by collagen. These results strongly suggest that S1P inhibits the collagen-induced human platelet activation through S1PR4 but not S1PR1.

Superficial temporal artery-middle cerebral artery bypass using local anesthesia and a sedative without endotracheal general anesthesia.

OBJECT Superficial temporal artery (STA)-middle cerebral artery (MCA) bypasses have continually evolved, and new strategies have been advocated for reducing anesthetic or surgical morbidity and mortality. Further simplifying, and decreasing the invasiveness of, STA-MCA bypass by performing this operation without endotracheal general anesthesia was believed to be feasible in certain subsets of patients. METHODS The authors performed STA-MCA bypass using local anesthesia with a sedative in 10 patients with hemodynamically compromised occlusive cerebrovascular disease, as well as multiple comorbidities, between February 2010 and September 2011. The technique is based on the preoperative identification of the point at which the donor and recipient vessels are in closest proximity. Preoperative use of CT angiography allowed the authors to identify the target point precisely and use a minimally invasive procedure. All patients received dexmedetomidine as the sole sedative agent, together with scalp-blocking local anesthesia, with an unsecured airway. RESULTS Successful STA-MCA bypass surgeries were achieved via a preselected minimally invasive approach in all cases. There was good hemodynamic stability throughout surgery. No airway or ventilation complications occurred, and no patients were converted to general anesthesia. Subjectively, patients tolerated the technique well with a high rate of satisfaction. There were no perioperative morbidities or deaths. Postoperative MR angiography confirmed a patent bypass in all patients. All patients remained symptom free and returned to normal daily life following the operation. CONCLUSIONS This initial experience confirms the feasibility of performing STA-MCA bypass without endotracheal general anesthesia. This novel technique produced a high degree of patient satisfaction.

Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase.

Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase.

Thrombin Receptor-Activating Protein (TRAP)-Activated Akt Is Involved in the Release of Phosphorylated-HSP27 (HSPB1) from Platelets in DM Patients

It is generally known that heat shock protein 27 (HSP27) is phosphorylated through p38 mitogen-activated protein (MAP) kinase. We have previously reported that HSP27 is released from human platelets associated with collagen-induced phosphorylation. In the present study, we conducted an investigation into the effect of thrombin receptor-activating protein (TRAP) on the release of HSP27 in platelets in type 2 diabetes mellitus (DM) patients. The phosphorylated-HSP27 levels induced by TRAP were directly proportional to the aggregation of platelets. The levels of phosphorylated-HSP27 (Ser-78) were correlated with the levels of phosphorylated-p38 MAP kinase and phosphorylated-Akt in the platelets stimulated by 10 µM TRAP but not with those of phosphorylated-p44/p42 MAP kinase. The levels of HSP27 released from the TRAP (10 µM)-stimulated platelets were correlated with the levels of phosphorylated-HSP27 in the platelets. The released platelet-derived growth factor-AB (PDGF-AB) levels were in parallel with the HSP27 levels released from the platelets stimulated by 10 µM TRAP. Although the area under the curve (AUC) of small aggregates (9–25 µm) induced by 10 µM TRAP showed no significant correlation with the released HSP27 levels, AUC of medium aggregates (25–50 µm), large aggregates (50–70 µm) and light transmittance were significantly correlated with the released HSP27 levels. TRAP-induced phosphorylation of HSP27 was truly suppressed by deguelin, an inhibitor of Akt, in the platelets from a healthy subject. These results strongly suggest that TRAP-induced activation of Akt in addition to p38 MAP kinase positively regulates the release of phosphorylated-HSP27 from human platelets, which is closely related to the platelet hyper-aggregation in type 2 DM patients.

Delayed Stenosis in the Intracranial Vessels following Endovascular Treatment for Acute Stroke

PURPOSE To evaluate delayed stenosis of the vessels after endovascular thrombectomy using magnetic resonance (MR) angiography. MATERIALS AND METHODS Of 82 consecutive patients who underwent successful endovascular treatment for acute intracranial large vessel occlusion between October 2010 and October 2014 at a single institution, 57 patients for whom 3-month radiologic follow-up examinations using MR angiography were available were included in the analysis. MR angiography images were assessed to detect delayed stenosis, which was defined as a decrease in the diameter of treated vessels > 50% compared with MR angiography images obtained 24 hours after endovascular treatment. RESULTS MR angiography images obtained 3 months after endovascular treatment revealed delayed stenosis of treated vessels in five (8.8%) of 57 patients. All cases of delayed stenosis were asymptomatic and occurred in the middle cerebral artery (MCA). Further serial radiologic follow-up showed gradual improvement of all delayed stenosis over 12 months. CONCLUSIONS Endovascular treatment poses a risk of delayed stenosis of treated vessels, especially in the MCA. MR angiography is a useful modality in long-term follow-up to evaluate delayed stenosis after endovascular treatment.

Diabetes mellitus and carotid artery plaques exhibiting high-intensity signals on MR angiography are related to increased platelet reactivity after carotid artery stenting

Background Increased platelet reactivity after carotid artery stenting (CAS) may cause thromboembolic complications. Objective This study aimed to investigate the incidence of increased platelet reactivity after CAS and to determine the factors related to it. Methods Patients who underwent CAS were recruited prospectively. They received pre-procedural antiplatelet therapy comprising some combination of aspirin (100 mg/day), clopidogrel (75 mg/day), and/or cilostazol (200 mg/day) for a minimum of 7 days. ADP- and collagen-induced platelet aggregation were measured before and 4 days after CAS. Changes in platelet reactivity were reported as changes in the categorized platelet reactivity grade based on the effective dose 50%. Clinical characteristics of patients with and without increased platelet reactivity were compared. Results Among 38 consecutive patients who underwent CAS, 18 (47%) exhibited increased platelet reactivity. Diabetes mellitus (OR 15.0; 95% CI 2.1 to 106.5; p=0.007) and carotid artery plaques exhibiting high-intensity signals (HIS) on time-of-flight MR angiography (TOF-MRA) (OR 25.2; 95% CI 2.0 to 316.2; p=0.013) were independently associated with increased platelet reactivity in a multivariate analysis. Conclusions Increased platelet reactivity occurred in nearly half of the studied patients subjected to CAS and was independently associated with diabetes mellitus and carotid artery plaques exhibiting HIS on TOF-MRA.

Rho-kinase regulates human platelet activation induced by thromboxane A2 independently of p38 MAP kinase

We have previously demonstrated that ristocetin, an activator of GPIb/IX/V, induces the release of soluble CD40 ligand (sCD40L) via thromboxane A2 production in human platelets. It has been shown that thromboxane A2 induces the activation of Rho-kinase, a downstream effector of Rho, in human platelets. In the present study, we investigated the exact roles of Rho-kinase in thromboxane A2-induced platelet activation. We found that U46619, a thromboxane receptor (TP) agonist, induced the phosphorylation of cofilin, a target of Rho-kinase signaling, and that the cofilin phosphorylation by U46619 was suppressed by Y27632 or fasudil, specific inhibitors of Rho-kinase. Y27632 and fasudil markedly decreased large platelet aggregate formation by U46619. The release of sCD40L and secretion of platelet-derived growth factor (PDGF)-AB stimulated by U46619 were inhibited by Y27632 and fasudil. SB203580, a specific inhibitor of p38 mitogen-activated protein (MAP) kinase, reduced the sCD40L release and PDGF-AB secretion. Y27632 and fasudil failed to affect the phosphorylation of p38 MAP kinase whereas SB203580 had little effect on the phosphorylation of cofilin induced by U46619. In conclusion, our results strongly suggest that Rho-kinase regulates thromboxane A2-induced human platelet activation independently of p38 MAP kinase.