Sheehan J P, Lan H C
University of Texas Health Science Center at San Antonio, Department of Medicine/Hematology, San Antonio, TX, USA.
Blood. 1998 Sep 1;92(5):1617-25.
Systemic administration of ISIS 2302, a 20-mer antisense phosphorothioate oligonucleotide targeting human intercellular adhesion molecule-1 mRNA, causes prolongation of plasma clotting times in both monkey and human studies. The anticoagulant effects of ISIS 2302 were investigated with both in vitro coagulation assays in human plasma and purified enzyme systems. At high oligonucleotide plasma concentrations (>100 microgram/mL), prolongation of the prothrombin and thrombin times was observed. In a thrombin time assay using purified components, high concentrations of ISIS 2302 inhibited thrombin clotting activity both by stimulating inhibition by heparin cofactor II and directly competing with fibrinogen for binding to anion binding exosite I. In contrast, low concentrations of ISIS 2302 (<100 microgram/mL) showed a selective, linear prolongation of the activated partial thromboplastin time (PTT). The rate limiting effect of 50 microgram/mL ISIS 2302, which prolonged the PTT to 1.5 times control, was identified by sequential modification of the clotting assay. Delaying addition of oligonucleotide until after contact activation failed to correct prolongation of the PTT. The calcium-dependent steps of the intrinsic pathway were individually assessed by adding sufficient activated coagulation factor to correct the PTT in plasma deficient in that specific factor. Addition of factor XIa, IXa, VIIIa, or Va failed to correct the PTT in the presence of ISIS 2302. In contrast, 0.2 nmol/L factor Xa corrected prolongation of the PTT in factor X-deficient plasma with or without oligonucleotide present. ISIS 2302 (50 microgram/mL) did not prolong a modified Russel viper venom time, suggesting no significant inhibition of prothrombinase. Thus, 50 microgram/mL ISIS 2302 prolonged the PTT by selectively inhibiting intrinsic tenase activity. ISIS 2302 showed partial inhibition of intrinsic tenase activity (to approximately 35% of control) at clinically relevant oligonucleotide concentrations in a chromogenic assay. This activity was oligonucleotide sequence-independent but required the phosphorothioate backbone, suggesting that inhibition of intrinsic tenase is a general property of this class of oligonucleotides. These results are relevant to both the therapeutic use of phosphorothioate oligonucleotides and the potential design of inhibitors of the intrinsic tenase complex, a novel target for anticoagulation.
ISIS 2302是一种针对人类细胞间黏附分子-1 mRNA的20聚体反义硫代磷酸酯寡核苷酸，在猴子和人类研究中，全身给药都会导致血浆凝血时间延长。通过在人血浆和纯化酶系统中进行体外凝血试验，对ISIS 2302的抗凝作用进行了研究。在高寡核苷酸血浆浓度（>100微克/毫升）时，观察到凝血酶原时间和凝血酶时间延长。在使用纯化成分的凝血酶时间测定中，高浓度的ISIS 2302通过刺激肝素辅因子II的抑制作用以及直接与纤维蛋白原竞争结合阴离子结合外位点I来抑制凝血酶的凝血活性。相比之下，低浓度的ISIS 2302（<100微克/毫升）显示活化部分凝血活酶时间（PTT）有选择性的线性延长。通过对凝血试验进行顺序改良，确定了50微克/毫升ISIS 2302的限速作用，它将PTT延长至对照的1.5倍。将寡核苷酸的添加延迟至接触激活后未能纠正PTT的延长。通过添加足够的活化凝血因子来纠正缺乏该特定因子的血浆中的PTT，分别评估了内源性途径中钙依赖性步骤。在存在ISIS 2302的情况下，添加因子XIa、IXa、VIIIa或Va未能纠正PTT。相比之下，0.2纳摩尔/升因子Xa在存在或不存在寡核苷酸的情况下都能纠正因子X缺乏血浆中PTT的延长。ISIS 2302（50微克/毫升）并未延长改良的罗素蝰蛇毒时间，表明对凝血酶原酶没有显著抑制作用。因此，50微克/毫升ISIS 2302通过选择性抑制内源性凝血酶原酶活性来延长PTT。在显色试验中，ISIS 2302在临床相关的寡核苷酸浓度下对内源性凝血酶原酶活性有部分抑制作用（降至对照的约35%）。这种活性与寡核苷酸序列无关，但需要硫代磷酸酯主链，这表明抑制内源性凝血酶原酶是这类寡核苷酸的普遍特性。这些结果对于硫代磷酸酯寡核苷酸的治疗应用以及内源性凝血酶原酶复合物抑制剂的潜在设计都具有重要意义，内源性凝血酶原酶复合物是抗凝的一个新靶点。
