Artificial cerebrospinal fluid restores aspirin-inhibited physiological hemostasis through recovery of platelet aggregation function.

BACKGROUND: Optimal hemostasis provides safety and reliability during neurosurgery which improves surgical outcomes. Previously, artificial cerebrospinal fluid (aCSF) and its component sodium bicarbonate were found to facilitate physiological hemostasis by amplifying platelet aggregation. This study aimed to verify whether aCSF amplifies platelet-dependent hemostasis in the presence of antiplatelet agents. METHODS: We prepared platelet-rich plasma (PRP) or washed platelets using aspirin (acetylsalicylic acid, (ASA)) or normal saline (NS). We evaluated samples treated with a commercially available aCSF solution or NS for amplification of aggregation, activation of integrin αIIbß3, phosphatidylserine (PS) exposure, P-selectin (CD62P) expression, and formation of microparticles (MPs). We assessed the effect of aCSF on in vivo hemostasis in the presence of ASA by measuring the tail bleeding time in ASA-or NS-injected C57BL/6 N mice. RESULTS: Compared with NS, aCSF amplified ASA-inhibited platelet aggregation by recovering platelet activation including PS exposure, MP release, CD62P expression, and integrin αIIbß3 activation. When using washed platelets, aCSF almost completely counteracted the inhibition of platelet aggregation by ASA. Prolonged bleeding time from the amputated tail of ASA-injected mice was significantly shortened by the treatment with aCSF compared to NS. Sodium bicarbonate also directly amplified ASA-inhibited platelet aggregation. CONCLUSIONS: aCSF and sodium bicarbonate facilitate physiological hemostasis through the recovery of inhibited platelet aggregation even in the presence of ASA. The utilization of aCSF in the operative field may be advantageous for facilitating hemostasis in patients with impaired platelet function and contribute to improving outcomes of neurosurgery.

Dominant negative form of alpha4 inhibits the BCR crosslinking-induced phosphorylation of Bcl-xL and apoptosis in an immature B cell line WEHI-231.

We previously demonstrated that c-Jun N-terminal kinase (JNK) phosphorylates serine 62 of Bcl-xL to induce the degradation of Bcl-xL and apoptosis in WEHI-231 cells upon BCR crosslinking. In order to elucidate the regulatory mechanisms underlying the phosphorylation of Bcl-xL, we prepared an assay system in which JNK phosphorylated Bcl-xL in HEK293T cells. Consequently, we found that a signal transduction molecule, alpha4, enhanced the phosphorylation of Bcl-xL by JNK, while the co-expression of C-terminal alpha4 (220-340) diminished the phosphorylation of Bcl-xL induced by JNK. Furthermore, full-length alpha4 associated with both JNK and Bcl-xL, whereas C-terminal alpha4 (220-340) associated only with Bcl-xL, not JNK. In addition, WEHI-231 cells transfected with the cDNA of C-terminal alpha4 (220-340) exhibited decreased phosphorylation of Bcl-xL and stronger resistance to apoptosis induced by BCR crosslinking. These results indicate that alpha4 is an important regulatory molecule of apoptosis induced by BCR crosslinking in WEHI-231 cells and that C-terminal alpha4 (220-340) functions as a dominant negative form.

Protein phosphatase 6 controls BCR-induced apoptosis of WEHI-231 cells by regulating ubiquitination of Bcl-xL.

Crosslinking BCR in the immature B cell line WEHI-231 causes apoptosis. We found that Bcl-xL was degraded by polyubiquitination upon BCR crosslinking and in this study explored the mechanism that controls the degradation of Bcl-xL. Ser(62) of Bcl-xL was phosphorylated by JNK to trigger polyubiquitination, and this was opposed by serine/threonine protein phosphatase 6 (PP6) that physically associated with Bcl-xL. We show BCR crosslinking decreased PP6 activity to allow Ser(62) phosphorylation of Bcl-xL. CD40 crosslinking rescues BCR-induced apoptosis, and we found PP6 associated with CD40 and PP6 activation in response to CD40. Our data suggest that PP6 activity is regulated to control apoptosis by modulating Ser(62) phosphorylation of Bcl-xL, which results in its polyubiquitination and degradation.

CaMKII phosphorylates serine 10 of p27 and confers apoptosis resistance to HeLa cells.

Protein phosphatase (PP) 6 is a serine threonine phosphatase which belongs to the PP2A subfamily of protein phosphatases. PP6 has been implicated in the control of apoptosis. A dominant negative form PP6 (DN-PP6) mutant cDNA was prepared and transfected into HeLa cells to investigate the regulation of apoptosis. HeLa cells expressing DN-PP6 showed increased resistance to apoptosis induced by TNF and cycloheximide. CaMKII phosphorylation and the expression of p27 were increased in DN-PP6 transfectants. Transient expression or activation of CaMKII increased the expression of p27. Furthermore, CaMKII phosphorylated serine 10 of p27, which induces the translocation of p27 from nucleus to cytoplasm and increases the stability of p27. Overexpression of wild type but not the S10A mutant p27 cDNA increased the expression of Bcl-xL and conferred apoptosis resistance to HeLa cells. These results indicated that PP6 and CaMKII regulated apoptosis by controlling the expression level of p27.