Balasubramanian Viji, Vele Oana, Nemerson Yale
Thrombosis Research, Mount Sinai School of Medicine, Box 1269, Annenberg 24-92, 1 Gustave L. Levy Place, NY, NY 10029, USA.
Thromb Haemost. 2002 Nov;88(5):822-6.
The presence of thrombogenic blood-borne or circulating tissue factor (cTF) has recently been demonstrated. These observations have implicated cTF to be a key determinant of thrombus propagation by depositing on platelets in nascent thrombi. Previously, we detected cTF by detergent solubilization and addition of phospholipids. We now report the direct demonstration of TF activity in ex-vivo thrombi. Collagen-coated substrates were exposed to native blood at shear rates of 0, 650, and 2,000 s(-1) for 10 min in a modified rotating Teflon cone and plate viscometer. Substrates were then gently rinsed to remove 'loose' (unadherent) components of blood. cTF activity was measured by adding a solution containing 10 nM FVIIa, 100 nM FX, and 5 mM CaCl(2) to the substrates exposed to blood. Samples of this mixture were obtained at intervals for 30 min and the amount of Xa generated was quantified by adding a chromogenic substrate, Spectrozyme Xa, and measuring the increase in OD at 405 nm. Our studies show that a minimal amount of generated Xa (approximately 1nM) can be measured from ex-vivo thrombi. Static and shear samples generated the same amount of Xa, with the exception of blood subjected to 650 s(-1) shear. At 650 s(-1) shear rate, the amount of Xa generated reached a maximum of 4 nM at 5 min and then decreased to approximately 1 nM. Immunohistological stains and fluorescent images demonstrate the presence of cTF antigen at 650 s(-1) wall shear rate.
最近已证实存在促血栓形成的血源性或循环组织因子（cTF）。这些观察结果表明，cTF通过沉积在新生血栓中的血小板上，是血栓形成的关键决定因素。此前，我们通过去污剂溶解和添加磷脂来检测cTF。我们现在报告体外血栓中TF活性的直接证明。在改良的旋转聚四氟乙烯锥板粘度计中，将胶原包被的底物在0、650和2000 s(-1)的剪切速率下暴露于天然血液中10分钟。然后轻轻冲洗底物以去除血液中的“松散”（未粘附）成分。通过向暴露于血液的底物中添加含有10 nM FVIIa、100 nM FX和5 mM CaCl(2)的溶液来测量cTF活性。每隔一段时间从该混合物中取样30分钟，并通过添加显色底物Spectrozyme Xa并测量405 nm处OD的增加来定量生成的Xa量。我们的研究表明，从体外血栓中可以测量到极少量生成的Xa（约1 nM）。静态和剪切样本生成的Xa量相同，但650 s(-1)剪切的血液除外。在650 s(-1)的剪切速率下，生成的Xa量在5分钟时达到最大值4 nM，然后降至约1 nM。免疫组织化学染色和荧光图像显示在650 s(-1)壁剪切速率下存在cTF抗原。
