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.

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It is firmly established that smoking is a risk factor of cardiovascular disease, stroke and peripheral vascular disease. Although smoking alters the hemostatic process, the influence of smoking on human platelet activation remains controversial. For patients undergoing surgery, cessation of smoking prior to the procedure is recommended as it increases the risk of postoperative morbidity or mortality. The presented study investigated the effects of smoking cessation on human platelet activation induced via collagen (n=19 patients). Blood samples were taken on four occasions: Before smoking cessation, and at 4, 8 and 12 weeks after smoking cessation. Platelet aggregation using citrated platelet-rich plasma (PRP) was monitored using a PA-200 aggregometer, which determined the size of platelet aggregates using laser scattering methods. A low dose of collagen (1 µg/ml) accelerated platelet aggregation at 4 or 8 weeks after smoking cessation when compared with results before cessation. After 12 weeks, levels of platelet aggregation induced by collagen were almost equal to those recorded prior to smoking cessation. The secretion levels of collagen-induced platelet-derived growth factor (PDGF)-AB at 4 or 8 weeks after smoking cessation were significantly higher than those before smoking was stopped. Furthermore, smoking cessation markedly strengthened the collagen-induced phosphorylation of p38 mitogen-activated protein (MAP) kinase after 4 weeks. The results of the current study indicated that smoking cessation causes temporary short-term human platelet hyper-activation. The further suggest that the incidence of complications due to human platelet hyper-reactivity may be lowered by considering the period of smoking abstinence.

ABSTRACT. A novel sandwich enzyme-linked immunosorbent assay (ELISA) was established to determine the serum insulin concentrations in domestic cats. By using a solid-phase mouse anti-bovine insulin monoclonal antibody and a peroxidase-conjugated guinea pig antirat insulin polyclonal antibody, feline serum insulin concentrations in the range of 0.1 to 3.6 ng/ml could be measured. The intraassay CV and interassay CV were less than 6% and less than 10%, respectively. The present insulin assay will strongly help studies on feline diabetes mellitus. Diabetes mellitus is one of the most common endocrine diseases in cats, with peak occurence in middle aged and older cats. Determination of serum insulin is very helpful for the diagnosis and classification of diabetes mellitus. Previous reports showed that several radioimmunoassay (RIA) kits designed to measure human insulin concentrations could be applied to feline samples [2,4]. However, only relative values of serum insulin were provided since those RIA kits were calibrated with human insulin but not with feline insulin. There has been no commercially available non-radiometric assay designed for and calibrated with feline insulin. In the present study, we established a novel sandwich enzyme-linked immunosorbent assay (ELISA) to determine the serum insulin concentrations in cats.To establish the ELISA system, we newly produced a monoclonal antibody against bovine insulin (BAN25433). Balb/c mice were immunized with purified bovine insulin (Sigma, St. Louis, MO, U.S.A.). Splenocytes from the immunized mice were fused to P3X53 mouse myeloma cells. Antigen-producing hybridoma clones were selected by the reactivity to bovine insulin. Purified BAN 25433 antibody was used as a solid phase antibody. Plastic ELISA plates (Sumilon MS-8896F, Tokyo) were coated with 50 l/ well of BAN25433 antibody (8 g/ml) in bicarbonate buffer (pH 9.6) at 4C overnight and then blocked with 200 ml/ well of ApplieDuo (1:10, Seikagaku Co., Tokyo) at room temperature (RT) for 1 hr.To prepare the standard feline insulin, an approximately 8 g of frozen feline pancreatic tissue was kindly gifted from Dr. Tsujimoto, Laboratory of Veterinary Internal Medicine, The University of Tokyo. Feline insulin in the pancreatic tissue was extracted by ethanol-HCl [1], partially purified with an affinity column (HiTrap, Amersham Pharmacia Biotech, Uppsala, Sweden) with BAN25433 antibody, and then thoroughly purified by a reverse-phase high-performance liquid chromatography as previously described [1]. The protein concentration was determined (Protein Assay Kit, Bio-Rad, Hercules, CA, U.S.A.) and the feline insulin standard solution was prepared. The other ELISA reagents used in this study including peroxidase-conjugated anti-rat insulin antibody, antibody diluting solution, washing buffer, substrate solution containing 3,3',5,5'-tetramethylbenzidine (TMB) and stop solution (1N sulfuric acid) were the parts of a commercial ELISA kit for rat insulin (Morinaga Institute of Biological Science, Inc., Yokohama).The ELIS...

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.