Kazuki Niwa

Sulfation of silk sericin and anticoagulant activity of sulfated sericin

—Silk sericin extracted from a cocoon was sulfated by chlorosulfonic acid. Sulfation of sericin was confirmed by FT-IR and the reaction efficiency was calculated as 44.9%. 1H-NMR suggested that sulfation mainly occurred at serine residues in the sericin molecule. The sulfated sericin was separated in three fractions by gel-filtration chromatography using Sephacryl S-200. The sulfate group content and amino-acid composition of each fraction were almost identical, while the anticoagulant activity differed for each fraction of sulfated sericin. Higher anticoagulant activity was observed for the higher-molecular-mass fraction. The anticoagulant activity of sulfated sericin was estimated at 1/10 to 1/20 of heparin.

Fibroblasts Spread on Immobilized Fibrin Monomer by Mobilizing a β1-class Integrin, Together with a Vitronectin Receptor αvβ3 on Their Surface

Human and murine fibroblasts were found to spread far more avidly on fibrin monomer monolayers than on immobilized fibrinogen, indicating that removal of fibrinopeptides by thrombin is a prerequisite for the fibrin-mediated augmentation of cell spreading. In fact, cell spreading was not efficiently augmented on monolayers of a thrombin-treated dysfibrinogen lacking the release of fibrinopeptide A due to an Aα Arg-16 → Cys substitution. Since a synthetic Arg-Gly-Asp (RGD)-containing peptide inhibited the fibrin-mediated cell spreading, subsequent dissociation of the carboxyl-terminal globular domain of the Aα-chains appears to render the RGD segments accessible to the cell-surface integrins. In support of this, fibrin-augmented cell spreading was inhibited by an antibody recognizing a 12-kDa peptide segment with γ Met-89 at its amino terminus, which is located in close association with the RGD segment at Aα 95-97 in the helical coiled-coil interdomainal connector. The fibrin-mediated augmentation of cell spreading was inhibited not only by an antibody against human vitronectin receptor (LM 609) but also by an antibody against the β1 subunit of integrin (mAb13), suggesting that the β1-class integrin together with a vitronectin receptor, αvβ3, is mobilized onto the surface of fibroblasts upon contact with the fibrin monomer monolayer.

Sulfated Sericin is a Novel Anticoagulant Influencing the Blood Coagulation Cascade

Sulfated sericins influence on factors in the blood coagulation cascade was investigated to elucidate its anticoagulant mechanism. Prolongation of the prothrombin time (PT) and activated partial thromboplastin time (APTT) were observed in the presence of sulfated sericin. Fluorogenic peptide substrates on thrombin (factor IIa) and factor Xa were used to study the influence of sulfate sericin on their respective activities. Sulfated sericin inhibited neither thrombin nor factor Xa in the presence of antithrombin III (AT III). Gel electrophoresis was used to examine fibrinogen–fibrin conversion by thrombin in the presence of sulfated sericin. FPA and FPB release from fibrinogen by thrombin proceeded in the presence of sulfated sericin. The behavior of polymerization of fibrin monomer (FM) was affected by the presence of sulfated sericin. No initial lag time in the polymerization process was observed by addition of sulfated sericin to FM. This result means that sulfated sericin will interfere in the build-up of normal double-strand fibrils of FM during formation of fibrin fiber. Scanning electron microscopy (SEM) observations supported this inference. The anticoagulant mechanism of sulfated sericin is inferred to interfere with the initial polymerization process of FM.

Immediate Mechanism of the Osteoconductivity of the Polarized Hydroxyapatite

We investigated the immediate response in the vicinity of the implanted polarized hydroxyapatite (HA). One of the important immediate events was found to be the fibrin adsorption on the implanted HA. The activation of the fibrin adsorption on the polarized HA was confirmed by the observation by SEM observation and immunohistochemical detection. After the implantation, the coagulation cascade induced the wound healing. The adsorbed fibrin was formed network structure and worked for later cell responses as the scaffolds. The acceleration of fibrin scaffolds formation in the vicinity of the implanted polarized HA contributed to the stimulation and activation of plate ets and osseous cells. The platelets were activated from the results of the signal transduction and cytoskeleton changes and released growth factors. The growth factors released from platelets led to bone regeneration by stimulating migration and proliferation of the osseous cells. In the vicinity of the implanted HA samples, the coagulation cascade reaction to repair wound healing led to the new bone formation by mediation of the platelets and osseous cells. The mechanism of osteoconduction at earlier stage had close relationships to the electrostatic properties of the implant.