| Co-Investigator(Kenkyū-buntansha) |
AKAMATSU Noriko Tokyo Metropolitan Institute of Medical Scienced Dept.of Cardiovas Res., Researc, 循環器病研究部門, 研究員 (30124431)
YAMAGUCHI Atsumi Tokyo Metropolitan Institute of Medical Scienced Dept.of Cardiovas Res., Researc, 循環器病研究部門, 研究員 (70124500)
SUZUKI Hidenori Tokyo Metropolitan Institute of Medical Scienced Dept.of Cardiovas Res., Researc, 循環器病研究部門, 研究員 (30158977)
AOKI Kazumasa Tokyo Metropolitan Institute of Medical Scienced Dept.of Cardiovas Res., Researc, 循環器病研究部門, 研究員 (10184029)
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| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1998: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1997: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
We studied mechanisms of platelet adhesion to surface consisting of FXIII-cross-linked fibrin. Human' fibronectin-depleted fibrinogen (Fbgen) (f. c. 2.0mg/ml) , thrombin (lU/ml) and CaCl_2 (2 mM) in a final 100 mu1 were mixed in wells of 96-immunoplates. Fibrin surface was then blocked with 0.3% BSA and PPACK, a thrombin-inhibitory peptide. For comparison, immobilized Fbgen surface (imFbgen) was also prepared by coating Fbgen(30 mug/ml) on the wells. Human platelets were loaded with BCECF-AM, an fluorescent dye, washed and finally suspended to a concentration of 50 x 10^3/ml. 100mu1 of the BCECF-loaded platelets were placed on the surfaces of fibrin and imFbgen. After intervals, non-adhesive platelets were removed and adhering platelets were measured with Fluoloskan. Platelet adhesion to fibrin and imFbgen were 15.7*2.3 % (M*SD, n=11) and 12.8*2.3 % (n=9), respectively, after 1 hr incubation at 37ﾟC.T74, an aggregation-inhibiting monoclonal anti-beta3 antibody, inhibited platelet adhes
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ion to imFbgen by 90%, but, in a sharp contrast, adhesion to fibrin was inhibited only by 20%. Adhesion to imFbgen and fibrin of the platelets previously treated with 4mM EDTA at pH 7.4 at 37ﾟC for 1 hr were 28 and 52 %, respectively, of control platelets treated with EDTA at 22ﾟC.RGD-peptides dose-dependently inhibited platelet adhesion to both imFbgen and fibrin. Dodecapeptide of C-terminus of fibrinogen gamma chain inhibited platelet adhesion to imFbgen but not to fibrin. Adhesion of Type II thrombasthenic platelets to imFbgen and fibrin were 26.7 and 75.4%, respectively, of normal platelets. Scannning electron microscopic studies revealed that thrombasthenic platelets, as likely as EDTA-treated platelets, as a round shape, adhered to but did not spread on fibrin. Some anti-beta1 antibodies such as JBS5 and LM534 inhibited platelet adhesion to fibrin but not to imFbgen. In addition, C32TG cells, a melanoma-derived cell line, and previously reported to retract fibrin clot, adhered to fibrin which was inhibited by beta1 as well as beta3 antibodies. These results indicate that platelet adhesion to fibrin is mediated by not only beta3 but also beta1 integrins. They also suggest that conversion of fibrinogen to fibrin is associated with exposure of a new adhesive domain and loss of functional C-terminal dodecapeptide of fibrinogen gamma chain. Less
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