Abstract

We have studied a prototype of platelet substitutes focusing on a dodecapeptide, HHLGGAKQAGDV (H12)11), which is specific for fibrinogen γ-chain carboxy-terminal sequence (γ 400–411). In this study, we conjugated H12 to the surface of polymerized albumin particles (polyAlb) as biocompatible and biodegradable carriers to produce particles having hemostatic ability, and evaluated their in vitro and in vivo effects.

H12 (H12; 9.6 x 103 /particles) containing cysteine to the N-terminal was conjugated to polyAlb (260 ± 60 nm), and the effect of H12-polyAlb on platelet thrombus formation was evaluated in vitro with thrombocytopenic whole blood ([platelet] = 2.0 x 104 /μL, anticoagulated with PPACK) under flow conditions (shear rate; 150 s−1). Thrombocytopenic rats were made by busulphan injection at a dose of 20 mg/kg, and a 2.5 mm length x 1.0 mm depth template-guided incision (QuikheelTM, Becton-Dickinson, San Jose, CA) was made 1 cm from the tip of tail. The tail was immersed in a 50 mL cylinder of saline and the time taken to stop bleeding was measured.

When thrombocytopenic blood in the presence of H12-polyAlb ([rHSA]=0.14 mg/mL) was flowed on collagen-plate, the surface coverage of DiOC6-labeled platelets evaluated by fluorescence microscopy was increased to 3.9 ± 1.1 % (n=3) from 2.1 ± 0.4 % (n=3) obtained in the absence of H12-polyAlb. In the same experiments, rhodamine-labeled H12-polyAlb was found to be involved in platelet-platelet interactions by binding to activated platelet surface, thus enhancing platelet thrombus formation. Next, in vivo hemostatic effect was tested by measuring tail bleeding time of thrombocytopenic rats 5 minutes after the intravenous administration of H12-polyAlb. The bleeding times of normal ([platelet] = 8.1 ±0.9 x 105 /μL) and thrombocytopenic rats ([platelet] = 2.0 ±0.3 x 105 /μL) were 187 ± 51 s and 609 ± 153 s (n=6), respectively. H12-polyAlb administration at a dose of 4 mg/kg significantly shortened the bleeding time to 342 ± 73 s (n=10), whereas polyAlb was without effects (553 ± 104 s, n=6). These results indicate that H12-polyAlb can be a suitable candidate for an alternative to human platelet concentrates infused into thrombocytopenic patients.

1)
Takeoka, S. et al.
Biochem. Biophys. Res. Commun.
,
312
,
773
–779 (
2003
).

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