J Artif Organs (2006) 9:251–258 © The Japanese Society for Artificial Organs 2006 DOI 10.1007/s10047-006-0345-0 ORIGINAL ARTICLE Yosuke Okamura, PhD · Makoto Handa, MD, PhD Hidenori Suzuki, PhD · Yasuo Ikeda, MD, PhD Shinji Takeoka, PhD New strategy of platelet substitutes for enhancing platelet aggregation at high shear rates: cooperative effects of a mixed system of fibrinogen γ-chain dodecapeptide- or glycoprotein Iba-conjugated latex beads under flow conditions Abstract To construct platelet substitutes that have hemostatic properties over a wide range of shear rates, we used fibrinogen γ-chain carboxy-terminal sequence HHLGGAKQAGDV (H12), which recognizes activated platelets at low shear rates, and a recombinant water- soluble moiety of the platelet glycoprotein (rGPIbα), which recognizes von Willebrand factor at high shear rates. Three kinds of samples were prepared for this purpose: H12- conjugated latex beads (H12-latex beads), rGPIbα-latex beads, and H12/rGPIbα-latex beads. These samples were evaluated in thrombocytopenia-imitation blood at various flow conditions. Based on ADP-induced platelet aggrega- tion studies, the H12-latex beads significantly enhanced platelet aggregation via H12 binding with GPIIb/IIIa acti- vated on the surface of activated platelets, whereas the rGPIbα-latex beads did not support platelet aggregation. In the case of the H12/rGPIbα-latex beads, the function of H12 was suppressed by steric hindrance from the larger rGPIbα bound to the latex bead. A mixture of the H12- latex beads and the rGPIbα-latex beads adhered to a col- lagen surface over a wide range of shear rates. In particular, at high shear rates, a cooperative effect was observed in the enhancement of platelet thrombus formation compared with H12-latex beads or rGPIbα-latex beads alone. We pro- Received: October 27, 2005 / Accepted: June 12, 2006 Y. Okamura · S. Takeoka (*) Graduate School of Science and Engineering, Waseda University, Tokyo 169-8555, Japan Tel. +81-3-5286-3217; Fax +81-3-5286-3217 e-mail: takeoka@waseda.jp Y. Okamura · M. Handa Department of Transfusion Medicine and Cell Therapy, School of Medicine, Keio University, Tokyo, Japan H. Suzuki Center for Electron Microscopy, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Y. Ikeda Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan pose that a mixed system of H12- and rGPIbα-conjugated nanoparticles is a more effective platelet substitute than each of the beads used alone and has enhanced platelet aggregation properties. Key words Platelet substitute · Fibrinogen γ-chain dodecapeptide · Platelet glycoprotein (GP) Ibα · Thrombocytopenia-imitation blood · Shear rate Introduction Platelet transfusion plays an important role in the support- ive therapy of thrombocytopenia caused by cancer or hematologic malignancies, or in the perioperative period. However, a shortage of platelet concentrates has always been a serious issue because of the short storage period (3 days in Japan), insufficient donations, and the imbalance of demand and supply. Furthermore, there is the issue of the risk of viral and bacterial infections from transfusion. For these reasons, a number of trials have been conducted to develop platelet substitutes such as infusible platelet mem- branes (IPMs), 1 solubilized platelet membrane protein- conjugated liposomes (plateletsomes), 2 fibrinogen-bonded red blood cells, 3 fibrinogen-coated albumin microcapsules (synthocytes), 4 and arginine-glycine-asparaginic acid (RGD) peptide-bound red blood cells (thromboerythro- cytes). 5 However, these platelet substitutes consist of materials derived from blood components. We also conjugated fibrinogen 6 to biocompatible carriers such as polymerized albumin particles (polyAlb) 7–9 and phospholipid vesicles (liposomes). 10–13 Fibrinogen conju- gates facilitated platelet aggregation on an activated platelet-immobilized surface in vitro by the recruitment of flowing platelets in the aggregates after their attach- ment. 6 However, fibrinogen isolated from human blood tends to precipitate at 4°C within a few hours. 6 Recently, we have focused on fibrinogen dodecapeptide (HHLGGAKQAGDV; H12) 14–18 and confirmed that the H12-conjugates showed minimal interaction with