Fibrinogen-Conjugated Albumin Polymers and Their Interaction with Platelets under Flow Conditions Shinji Takeoka, † Yuji Teramura, † Yosuke Okamura, † Makoto Handa, ‡ Yasuo Ikeda, ‡ and Eishun Tsuchida* ,† Department of Polymer Chemistry, Advanced Research Institute for Science and Engineering, Waseda University, Tokyo 169-8555, Japan; and Department of Internal Medicine and Blood Center, School of Medicine, Keio University, Tokyo, 160-8582, Japan Received May 1, 2001; Revised Manuscript Received August 9, 2001 Albumin polymers, having an average diameter of 1020 ( 250 nm, were prepared by the disulfide polymerization of recombinant human serum albumin (rHSA) by controlling of the pH and temperature. Fibrinogen could be conjugated on the surface of an albumin polymer using N-succinimidyl 3-(2- pyridyldithio)propionate (SPDP). Under flow conditions, the fibrinogen-conjugated albumin polymers (fibrinogen-albumin polymers) were irreversibly attached to the platelet-immobilized surface in the reconstituted blood at a low platelet concentration ([platelet] ) 5.0 × 10 4 /µL, a 5-fold diluted platelet concentration), and the attachment was suppressed by the addition of anti-GPIIb/IIIa monoclonal antibodies. It was confirmed that fibrinogen-albumin polymers specifically interacted with GPIIb/IIIa expressed on the surface of the activated platelets. Although platelets with a low platelet concentration were hardly attached to the platelet-immobilized surface under the flow conditions, the addition of fibrinogen-albumin polymers enhanced the attachment of the remaining platelets to the surface, indicating that the fibrinogen-albumin polymers would help the hemostatic ability of platelets at the site of vascular injury of patients in thrombocytopenia. Introduction Platelet transfusion plays an important role as a supportive therapy for the treatment of cancer or hematologic malignan- cies or during surgical procedures. Recently, the number of applications of platelet concentrates is increasing even though the shortage of platelets has continued due to the short-term storage of platelet concentrates (3 days in Japan). Therefore, it is difficult for stored platelets to be available for use in emergency care such as natural disasters. Another problem is the risk of viral and bacterial infections during transfusions. The development of platelet substitutes is necessary in order to solve these issues. Platelet substitutes, which have so far been developed, utilize the recombinant fragment of the platelet membrane such as glycoprotein (GP) IbR 1,2 and the GPIa/IIa complex 3 or fibrinogen, 4,5 RGDS, 6 etc., as the recognition sites. Recently, Levi et al succeeded in reducing the bleeding time with fibrinogen-coated albumin microcap- sules. 5 Albumin-based particles such as albumin microcapsules, nanospheres, or microspheres have been used as intrave- nously injectable particles since the 1960s because of their high biocompatibility and biodegradability. 7-9 Since the preparation methods of albumin-based particles include spray-drying, coacervation, and emusification using organic solvents, detergents, or cross-linkers as stabilizers, it was difficult to completely remove these additives and control the particle diameter. 8 We have already succeeded in the preparation of albumin polymers, which were polymerized with intermolecular disulfide bonds by changing the pH and temperature in an aqueous system and found that the particle diameter could be controlled from 50 to 300 nm. 10 It was confirmed that the addition of ristocetin to the rGPIbR- conjugated albumin polymers (rGPIbR-albumin polymers) having a 240 nm diameter in the presence of the von Willebrand factor (vWf) caused specific aggregation involv- ing the remaining platelets, and the rGPIbR-albumin poly- mers also became specifically attached to the vWf- immobilized surface under flow conditions. 11 In this study, for the purpose of constructing the particle system which recognizes the platelets attached to the site of vascular injury, we selected fibrinogen, which specifically recognizes GPIIb/IIIa expressed on the activated platelet surface. Fibrinogen is a 340 kDa fibrious glycoprotein having two identical disulfide-linked subunits composed of three nonidentical polypeptide chains: AR,B, and γ present at 0.3 g/dL in plasma, which plays an important role in a blood coagulation system. During bleeding, this protein plays a critical role in secondary hemostasis because of being involved in platelet aggregation. 12,13 As mentioned above, Levi et al. used fibrinogen-coated albumin microcapsules as platelet substitutes, of which the surface fibrinogen was physically adsorbed. On the other hand, we covalently conjugated fibrinogen to the surface of the albumin polymers with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). 14 * Corresponding author. E-mail: eishun@mn.waseda.ac.jp. Telephone: +81-5-3286-3120. Fax: +81-3-3205-4740. † Waseda University. ‡ Keio University. 1192 Biomacromolecules 2001, 2, 1192-1197 10.1021/bm015554o CCC: $20.00 © 2001 American Chemical Society Published on Web 11/02/2001