Grunkemeier J, Wan C, Horbett T
Department of Chemical Engineering, Seattle, WA 98195, USA.
J Biomater Sci Polym Ed. 1996;8(3):189-209. doi: 10.1163/156856296x00246.
Previously, we found that when fibrinogen-coated polyurethanes resided in a buffer for a period of time (the 'residence time') platelet adhesion to these materials decreased. Other changes in adsorbed fibrinogen such as decreases in polyclonal antibody binding and SDS elutability supported the conclusion that fibrinogen undergoes postadsorptive conformational changes. Subsequently we measured the binding of monoclonal antibodies to the three putative platelet binding sites on fibrinogen, using a single mid-range concentration of antibody. We found that binding of a monoclonal antibody to the platelet binding site at the C-terminus of the gamma chain of fibrinogen changed little with residence time, while binding of monoclonal antibodies to the other two putative binding sites on fibrinogen either increased with residence time (RGDF at A alpha 95-98), or first increased and then decreased with residence time (RGDS at A alpha 572-575). In the current study, we measured antibody binding affinity, Ka, by measuring antibody binding at a series of antibody concentrations. This is a more sensitive method for detecting changes in adsorbed fibrinogen than measuring antibody binding from a single antibody concentration. The Ka was determined for two antibodies, M1 (4A5), which binds to a platelet binding domain of fibrinogen (gamma 402-411) and R1 (155 B 1616), which binds to residues 87-100 of the A alpha chain (containing an RGDF site). A summary of the results for the M1 antibody are as follows. The Ka was higher for M1 binding to fibrinogen adsorbed to Immulon I than to Biomer, Biospan or poly(ethylene terephthalate), suggesting that fibrinogen adsorbed to Immulon I is more platelet adhesive than fibrinogen adsorbed to the other polymers. On Biospan, the Ka decreased from 2.8 x 10(9) to 1.0 x 10(9) M-1 after a 24 h 37 degrees C residence time, which correlated with the decrease in platelet adhesiveness of adsorbed fibrinogen observed previously under these conditions. The change in Ka was greater when adsorbed fibrinogen was kept under denaturing conditions. For example, the Ka decreased from 2.8 x 10(9) to 0.8 x 10(9) M-1 after a 1 h 70 degrees C residence time whereas it remained approximately the same, 2.9 x 10(9) M-1, after a 24 h 0 degree C residence time.
此前，我们发现当纤维蛋白原包被的聚氨酯在缓冲液中放置一段时间（“停留时间”）后，血小板对这些材料的黏附性会降低。吸附的纤维蛋白原的其他变化，如多克隆抗体结合减少和SDS洗脱性降低，支持了纤维蛋白原在吸附后发生构象变化的结论。随后，我们使用单一中等浓度的抗体，测量了单克隆抗体与纤维蛋白原上三个假定的血小板结合位点的结合情况。我们发现，单克隆抗体与纤维蛋白原γ链C末端的血小板结合位点的结合随停留时间变化不大，而单克隆抗体与纤维蛋白原上其他两个假定结合位点的结合要么随停留时间增加（Aα链95 - 98位的RGDF），要么先增加后随停留时间减少（Aα链572 - 575位的RGDS）。在当前研究中，我们通过测量一系列抗体浓度下的抗体结合情况来测定抗体结合亲和力Ka。这是一种比从单一抗体浓度测量抗体结合更灵敏的检测吸附纤维蛋白原变化的方法。测定了两种抗体的Ka，M1（4A5）可结合纤维蛋白原的血小板结合域（γ402 - 411），R1（155 B 1616）可结合Aα链的87 - 100位残基（包含一个RGDF位点）。M1抗体的结果总结如下。M1与吸附在Immulon I上的纤维蛋白原结合时的Ka高于与Biomer、Biospan或聚对苯二甲酸乙二酯结合时的Ka，这表明吸附在Immulon I上的纤维蛋白原比吸附在其他聚合物上的纤维蛋白原更具血小板黏附性。在Biospan上，37℃停留24小时后，Ka从2.8×10⁹降至1.0×10⁹ M⁻¹，这与之前在这些条件下观察到的吸附纤维蛋白原的血小板黏附性降低相关。当吸附的纤维蛋白原处于变性条件下时，Ka的变化更大。例如，70℃停留1小时后，Ka从2.8×10⁹降至0.8×10⁹ M⁻¹，而在0℃停留24小时后，Ka保持大致相同，为2.9×10⁹ M⁻¹。
