Amyloid ß protein negatively regulates human platelet activation induced by thrombin receptor-activating protein.

Amyloid ß protein deposition in cerebral vessels, a characteristic of Alzheimer's disease, is a risk factor for intracerebral hemorrhage. Amyloid ß protein directly modulates human platelet function; however, the exact mechanism of action is unclear. Therefore, we investigated the effects of amyloid ß protein on human platelet activation using an aggregometer with laser scattering. Amyloid ß protein decreased platelet aggregation induced by thrombin receptor-activating protein, but not by collagen and ADP. Amyloid ß protein also suppressed platelet aggregation induced by SCP0237 and A3227. Platelet-derived growth factor-AB secretion and phosphorylated-heat shock protein 27 release by thrombin receptor-activating protein were inhibited by amyloid ß protein. Additionally, thrombin receptor-activating protein-induced phosphorylation of JNK and p38 MAP kinase was reduced by amyloid ß protein. Collectively, our results strongly suggest that amyloid ß protein negatively regulates protease-activated receptor-elicited human platelet activation. These findings may indicate a cause of intracerebral hemorrhage due to amyloid ß protein.

A case of segmental arterial mediolysis with subarachnoid hemorrhage due to anterior cerebral artery dissection followed by internal carotid artery dissection.

BACKGROUND: Segmental arterial mediolysis (SAM) causes subarachnoid hemorrhage (SAH) due to intracranial aneurysm rupture and arterial dissection. We encountered a case of SAM-related SAH due to ruptured dissection of the A1 segment of the anterior cerebral artery concomitant with internal carotid artery (ICA) dissection. CASE DESCRIPTION: A 53-year-old man presented with SAH due to a ruptured right A1 dissecting aneurysm. The aneurysm was trapped; however, 7 days after the onset of SAH, he experienced right hemiparesis and aphasia. Angiography showed left ICA dissection; urgent carotid artery stenting was performed, leading to symptom improvement. Abdominal computed tomography angiography showed aneurysms of the celiac and superior mesenteric arteries. He was diagnosed with SAM based on clinical, imaging, and laboratory findings. CONCLUSION: In the acute phase of SAM-related SAH, cerebral ischemia could occur due to both cerebral vasospasm and intracranial or cervical artery dissection.

Synergistic effect of collagen and CXCL12 in the low doses on human platelet activation.

CXCL12, also known as stromal cell-derived factor-1, is a chemokine classified into CXC families, which exerts its function by binding to specific receptors called CXCR4 and CXCR7. Human platelets express CXCR4 and CXCR7 on the plasma membrane. It has been reported that CXCL12 potentiates to induce platelet aggregation in cooperation with agonists including collagen. However, the precise roles and mechanisms of CXCL12 in human platelet activation are not fully elucidated. In the present study, we investigated the effect of simultaneous stimulation with low doses of collagen and CXCL12 on the activation of human platelets. The simultaneous stimulation with collagen and CXCL12 induced the secretion of platelet-derived growth factor (PDGF)-AB and the release of soluble CD40 ligand (sCD40L) from human platelets in addition to their aggregation, despite the fact that the simultaneous stimulation with thrombin receptor-activating peptide (TRAP) or adenosine diphosphate (ADP), and CXCL12 had little effects on the platelet aggregation. The agonist of Glycoprotein (GP) Ⅵ convulxin and CXCL12 also induced platelet aggregation synergistically. The monoclonal antibody against CXCR4 but not CXCR7 suppressed the platelet aggregation induced by simultaneous stimulation with collagen and CXCL12. The phosphorylation of p38 mitogen-activated protein kinase (MAPK), but not p44/p42 MAPK, was induced by the simultaneous stimulation. In addition, the simultaneous stimulation with collagen and CXCL12 induced the phosphorylation of HSP27 and the subsequent release of phosphorylated-HSP27 from human platelets. SB203580, a specific inhibitor of p38 MAPK, attenuated the platelet aggregation, the phosphorylation of p38 MAPK and HSP27, the PDGF-AB secretion, the sCD40L release and the phosphorylated-HSP27 release induced by the simultaneous stimulation with collagen and CXCL12. These results strongly suggest that collagen and CXCL12 in low doses synergistically act to induce PDGF-AB secretion, sCD40L release and phosphorylated-HSP27 release from activated human platelets via p38 MAPK activation.

Correlation Between the VerifyNow P2Y12 Assay and the Newly Developed APAL System in Neuroendovascular Patients.

OBJECTIVE: Light transmission aggregometry (LTA) is the gold standard method for assessing platelet function. Recently, a new parameter called adenosine diphosphate (ADP)-induced platelet aggregation level (APAL) was developed to aid interpretation of LTA results. APAL is a score calculated based on platelet aggregation patterns upon exposure to 1 µM and 10 µM ADP and is determined using an automated coagulation analyzer. We compared APAL and VerifyNow P2Y12 assay for neuroendovascular patients. METHODS: 42 patients who have received antiplatelet therapy were studied. Platelet function tests were performed on CS-2400 for APAL and VerifyNow P2Y12 assay was used for P2Y12 reaction unit (PRU) and % inhibition. RESULTS: Moderate correlations were observed between APAL and PRU (r=0.64, p<0.001) and between APAL and % inhibition (r=-0.74, p<0.001). The optimal threshold for APAL was 8.2 for PRU (threshold=240) and 8.1 for % inhibition (threshold=26%). The percentage of agreement between the above thresholds was 90.9% between PRU and APAL and 77.3% between % inhibition and APAL. CONCLUSIONS: The APAL system exhibits moderate correlation with PRU and % inhibition. APAL testing is a good choice for a clinical laboratory already in possession of Sysmex CS series analyzers. In this setting, APAL testing can significantly decrease the cost of platelet function testing for patients on antiplatelet therapy.

Cigarette Smoking Cessation Temporarily Enhances the Release of Phosphorylated-HSP27 from Human Platelets.

Objective Cigarette smoking is a risk factor for arteriopathy, including acute coronary syndrome, stroke and peripheral vascular disease. Thus, cessation is strongly recommended in order to reduce these risks. We recently demonstrated that smoking cessation causes temporary hyper-aggregability of human platelets. We previously showed that heat shock protein 27 (HSP27) is released from human platelets stimulated by collagen, accompanied by its phosphorylation. Accumulating evidence indicates potent roles of extracellular HSP27 as a modulator of inflammation. In the present study, using the stored samples obtained in the previous study, we investigated the effect of cigarette smoking cessation on the release of phosphorylated-HSP27 from collagen-activated human platelets (n=15 patients). Methods We enrolled patients who visited smoking cessation outpatient services between January 2012 and November 2014. Platelet-rich plasma, chronologically obtained before and after the cessation, was stimulated by collagen using a PA-200 aggregometer in the previous study. The levels of phosphorylated-HSP27 released from platelets were determined by an enzyme-linked immunosorbent assay. The phosphorylation of HSP27 in platelets was evaluated by a Western blot analysis. Results Cessation of cigarette smoking significantly upregulated the levels of collagen-stimulated release of phosphorylated-HSP27 at four and eight weeks after quitting smoking compared to before cessation. However, there was no significant difference between the levels before cessation and those at 12 weeks after cessation. The levels of phosphorylated-HSP27 stimulated by collagen in the platelets at four weeks after smoking cessation were remarkably enhanced compared to before cessation. Conclusion Cigarette smoking cessation temporarily enhances the collagen-stimulated release of phosphorylated-HSP27 from human platelets in the short term.

Olive polyphenol reduces the collagen-elicited release of phosphorylated HSP27 from human platelets.

Hydroxytyrosol (HT) and oleuropein (OLE) are natural polyphenols found in extra virgin olive oil. Accumulating evidence indicates that ingestion of olive oil contributes to reduce the risk of cardiovascular diseases and stroke. It has been reported that HT and OLE inhibit human platelet aggregation. We have shown that collagen induces the phosphorylation of heat shock protein 27 (HSP27) in human platelets, resulting in the release of HSP27, an extracellular pro-inflammatory agent. In this study, we investigated the effects of HT and OLE on the collagen-stimulated human platelet activation. The PDGF-AB secretion and the soluble CD40 ligand (sCD40L) release by collagen were reduced by HT or OLE. HT and OLE significantly suppressed the phosphorylation of HSP27 and the release of phosphorylated-HSP27. These findings suggest that olive polyphenol reduces the collagen-stimulated phosphorylation of HSP27 in human platelets and the release. Our results may provide a novel anti- inflammatory effect of olive polyphenol.

Evaluation of the Newly Developed Adenosine Diphosphate-Induced Platelet Aggregation Level System in Aggregometer on Automated Coagulation Analyzer.

BACKGROUND: Light transmission aggregometry (LTA) is the gold standard for platelet function assessment. The automated coagulation analyzer from Sysmex that performs LTA offers the advantage of being a walk-away technology. Recently, a new parameter "ADP-induced platelet aggregation level (APAL)" was developed to support the interpretation of results. APAL is calculated as a score from 0.0 to 10.0 based on platelet aggregation patterns with 1 and 10 µM adenosine diphosphate (ADP). Here, the basic performance of the newly developed APAL system and comparison with the maximum aggregation rate of ADP (ADP-MA) was evaluated. METHODS: The within-run precision was calculated by conducting five replicate analyses of the platelet-rich plasma (PRP) from healthy volunteers and 0.05 µM of cangrelor-spiked PRP. Cangrelor is a P2Y12 inhibitor that does not require liver CYP activation. The reference interval was calculated from the results of 67 healthy volunteers. The effect of the antiplatelet P2Y12 agent was evaluated using several concentrations of cangrelor. A comparative study was performed using 103 PRP samples with different levels of aggregation. Each test was analyzed with both APAL and ADP-MA. RESULTS: The percentage coefficient of variation in within-run precision was within 7% for APAL and 10 µM ADP-MA. Reference interval of APAL and 10 µM ADP-MA was 7.1 - 10.0 and 80.0 - 99.2%, respectively. APAL signifi-cantly decreased with the addition of 0.02 µM cangrelor, while 10 µM ADP-MA was barely affected. A significant correlation was observed between APAL and 10 µM ADP-MA (r = 0.94; p < 0.0001). CONCLUSIONS: The newly developed APAL system exhibited an acceptable performance. APAL score showed a good correlation with ADP-MA and was adequate to detect the weak effect of P2Y12 inhibitors. APAL is a new platelet aggregation scoring system with the potential to monitor the effects of P2Y12 inhibitor over a wide range.

Rac Regulates the TRAP-Induced Release of Phosphorylated-HSP27 from Human Platelets via p38 MAP Kinase but Not JNK.

BACKGROUND/AIMS: Thrombin induces the activation of human platelets through protease-activated receptor (PAR) 1 and PAR4, and Rac, a member of the Rho family of small GTPases, is implicated in PAR activation. We previously reported that phosphorylated-heat shock protein 27 (HSP27) is released from the thrombin receptor-activating peptide (TRAP)-stimulated platelets of diabetic patients. In the present study, we investigated the role of Rac in the TRAP-elicited release of phosphorylated-HSP27 from human platelets. METHODS: Platelet aggregation was measured using an aggregometer with laser scattering. Protein phosphorylation was analyzed by Western blotting. The levels of phosphorylated-HSP27 and platelet-derived growth factor-AB (PDGF-AB) were measured by enzyme-linked immunosorbent assays. RESULTS: NSC23766, an inhibitor of Rac-guanine nucleotide exchange factor interaction, suppressed the TRAP-elicited release of phosphorylated-HSP27 as well as platelet aggregation. The TRAP-induced phosphorylation of HSP27, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) was attenuated by NSC23766. SB203580, a p38 MAPK inhibitor, but not SP600125, a JNK inhibitor, suppressed the release of phosphorylated-HSP27 in addition to HSP27 phosphorylation. On the other hand, both SB203580 and SP600125 reduced the TRAP-stimulated secretion of PDGF-AB. CONCLUSION: Our results strongly suggest that Rac acts as a positive regulator of the PAR-elicited release of phosphorylated-HSP27 from human platelets via p38 MAPK but not JNK.

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.

Duration of the horizontal decubitus position for prevention of cerebrospinal fluid leakage following transection of a tight filum terminale.

OBJECT: The untethering of a tethered spinal cord in patients with a tight filum terminale is a relatively simple procedure that can prevent or improve neurological symptoms. Postoperatively, patients are usually kept in the horizontal decubitus position to prevent a CSF leak. However, the optimal period for keeping patients flat has not been determined yet. The authors compared 2 cohorts with different periods of horizontal decubitus; one with 72 hours and the other with 8 days. METHODS: The authors retrospectively analyzed surgical results in 2 cohorts of pediatric patients who had tethered spinal cord with a tight filum terminale. One cohort was maintained flat for 8 days and the other cohort for 72 hours postoperatively. The patients' charts were reviewed for demographic data, clinical presentation, surgical therapy, and clinical course. RESULTS: Three hundred fifty-four patients underwent sectioning of a tight filum terminale. Of those, 238 were kept lying flat for 8 days postoperatively, and 116 were maintained flat for 72 hours. Magnetic resonance imaging was performed 1 to 2 weeks after the surgery. None of the patients in either cohort developed a CSF leak. Pseudomeningocele, which was confirmed by MRI, developed in 1 patient who had been kept flat for 8 days. The occurrence rates of a CSF leak and pseudomeningocele were not significantly different in either cohort. CONCLUSIONS: Keeping patients flat for longer than 72 hours did not change the rate of postoperative CSF leakage or pseudomeningocele. Seventy-two hours or less would be an appropriate period for maintaining patients flat after transection of a tight filum terminale.

[Effect of an oral anti-estrogen agent (mepitiostane) on the regression of intracranial meningiomas in the elderly].

We report here the regression of meningioma following treatment with the anti-estrogen agent mepitiostane in a series of cases. The first case was that of a 72-year-old woman who presented with coma status due to non-communicating hydrocephalus. A large presumed meningioma within the cerebello-pontine angle was detected on gadolinium-enhanced magnetic resonance imaging (MRI). The patient recovered from the neurological deficit following endoscopic third ventriculostomy treatment, and was administered mepitiostane (10mg/day) orally. Gadolinium-enhanced MRI showed a marked regression (85%) of the meningioma following 60 months of oral medication. The second case was that of a 79-year-old woman with no neurological deficit; however, a presumed meningioma located in the frontal skull base was detected on gadolinium-enhanced MRI. Mepitiostane (10mg/day) was administered orally. Again, a marked regression (88%) of the meningioma was demonstrated after 115 months of oral medication. The third case was that of a 71-year-old woman who presented with right visual disturbance and a visual field defect. Gadolinium-enhanced MRI demonstrated a presumed meningioma located in the left sphenoidal bone. Mepitiostane (20mg/day) was administered orally. An 79% regression of the meningioma was observed after 21 months of oral medication. In these three cases, the marked reduction in meningioma following anti-estrogen agent (mepitiostane) administration suggested that this oral medication could be an effective therapeutic option in elderly patients.

Obliteration of the choroid plexus after endoscopic coagulation.

OBJECT: Endoscopic choroid plexus coagulation (CPC) with or without endoscopic third ventriculostomy (ETV) has been shown to be effective for selected patients with hydrocephalus. However, whether the effect of the coagulation is temporary and the choroid plexus regenerates or can be obliterated has remained largely unknown. The authors evaluate the effectiveness of CPC and report 3 cases of obliteration demonstrated by direct endoscopic observation. METHODS: The authors retrospectively analyzed the surgical results of patients with hydrocephalus primarily treated by CPC with or without ETV. Charts were reviewed for demographic data, clinical presentations, surgical therapies, and clinical outcomes. RESULTS: Eighteen patients with hydrocephalus were surgically treated using endoscopic CPC between July 2002 and July 2012. In 12 patients, ETV was concurrently performed. The etiology of hydrocephalus was posthemorrhagic in 5 patients, myelomeningocele in 3, postmeningitis in 2, congenital aqueductal stenosis in 1, hydranencephaly in 1, porencephaly in 1, and idiopathic in 5. The mean age at surgery was 8 months (range 0.3-24 months). The mean follow-up was 64 months. In 9 cases (50%), control of hydrocephalus was successful and the patients did not require further surgeries. In 9 patients (50%), treatment failed. Of these, 3 patients underwent repeat ETV 2, 3, and 38 months after the initial surgery. Endoscopic observation of the previous coagulation site revealed no regeneration of the choroid plexus in 2 patients, who underwent repeat ETV 2 and 3 months after CPC. In 1 patient who underwent repeat ETV 38 months after CPC, no regeneration of the choroid plexus, except for that in the proximity of the foramen of Monro, was observed. CONCLUSIONS: Endoscopic CPC with or without ETV can be a safe and effective treatment alternative to shunt placement in infantile hydrocephalus. Obliteration of the choroid plexus can persist in the relatively long term following CPC, which may contribute to the long-term control of hydrocephalus in successful cases.

Alteration in radiological subtype of spinal lipoma: case report.

We experienced a rare case of lipomyelomeningocele diagnosed at birth by magnetic resonance imaging which transformed the radiological appearance after 2 months into the transitional-type spinal lipoma with rapid increase in size of lipoma. Intraoperative findings revealed the presence of the dural sac extended dorsally outside the vertebral canal, which was characteristic of lipomyelomeningocele. Although there have been several reports showing that the size of spinal lipoma changed during development, there have been no reports which demonstrated alteration of radiological subtype with rapid increase of lipoma. Herein, we describe the first case of lumbosacral lipoma which changed radiological subtype from lipomyelomeningocele into transitional-type spinal lipoma.