Fernandez F A, Buchanan M R, Hirsh J, Fenton J W, Ofosu F A
Canadian Red Cross Society, Blood Transfusion Service, Hamilton, Ontario, Canada.
Thromb Haemost. 1987 Jun 3;57(3):286-93.
Previous studies have demonstrated that standard anticoagulant tests are poor indices of the antithrombotic potential of glycosaminoglycans which are weak catalysts of the thrombin-antithrombin III reaction. In this study we investigated whether the catalysis of thrombin inhibition by plasma could serve as a reliable index for assessing the antithrombotic effectiveness of glycosaminoglycans. Equal volumes of 125I-thrombin and control or test plasma were incubated for up to 10 min at 37 degrees C. Inactivation of thrombin was then determined after 7.9% SDS-polyacrylamide gel electrophoresis and subsequent autoradiography. Increasing concentrations of heparin (greater than 0.066 micrograms/mL or 0.01 USP units/mL) and dermatan sulfate (greater than 0.1 micrograms/mL) could be readily demonstrated in undiluted plasma by enhanced formation of complexes of thrombin with antithrombin III and heparin cofactor II respectively. However, the detection of any catalytic effect of the two glycosaminoglycans decreased significantly with increasing plasma dilutions. When ex vivo plasmas obtained from rabbits that had been injected with the minimum dose of any one of seven glycosaminoglycans required to achieve their optimal antithrombotic effect were assessed for their ability to catalyse thrombin inhibition, there was approximately a 2-fold increase in the amount of thrombin inactivated 30 s after the thrombin had been added to the plasma. The enhanced inhibition of thrombin was achieved by catalysis of antithrombin III and/or heparin cofactor II activities. These results suggest that measurement of the catalysis of thrombin inactivation in undiluted plasma is a sensitive and reliable index for estimating the antithrombotic potential of glycosaminoglycans in rabbits.
先前的研究表明，标准抗凝试验不能很好地反映糖胺聚糖的抗血栓形成潜力，因为糖胺聚糖是凝血酶 - 抗凝血酶III反应的弱催化剂。在本研究中，我们调查了血浆对凝血酶抑制的催化作用是否可作为评估糖胺聚糖抗血栓形成有效性的可靠指标。将等量的125I - 凝血酶与对照血浆或测试血浆在37℃下孵育长达10分钟。然后在7.9％SDS - 聚丙烯酰胺凝胶电泳及随后的放射自显影后测定凝血酶的失活情况。通过分别增强凝血酶与抗凝血酶III和肝素辅因子II的复合物形成，可在未稀释的血浆中轻松检测到浓度不断增加的肝素（大于0.066微克/毫升或0.01美国药典单位/毫升）和硫酸皮肤素（大于0.1微克/毫升）。然而，随着血浆稀释度的增加，两种糖胺聚糖的任何催化作用的检测都显著降低。当对从注射了七种糖胺聚糖中任何一种以达到其最佳抗血栓形成效果所需的最小剂量的兔子获得的离体血浆进行评估，以检测其催化凝血酶抑制的能力时，在将凝血酶加入血浆后30秒，凝血酶失活量增加了约2倍。通过催化抗凝血酶III和/或肝素辅因子II的活性实现了对凝血酶的增强抑制。这些结果表明，测量未稀释血浆中凝血酶失活的催化作用是估计兔子体内糖胺聚糖抗血栓形成潜力的敏感且可靠的指标。
