Reinhold D S, Gershman H
Thromb Res. 1985 Feb 15;37(4):513-27. doi: 10.1016/0049-3848(85)90097-0.
It has been previously proposed that platelet stimulation may involve two platelet-thrombin complexes: an initial platelet-thrombin complex (P-T) which is converted to an activated platelet-thrombin complex (P*-T). By using the release of radioactive serotonin as a measure of thrombin stimulation, we have demonstrated that under appropriate conditions, a hirudin sensitive and a hirudin insensitive complex can be differentiated. At short platelet-thrombin preincubation times (0-2 minutes) at 4 degrees C, in a buffer containing 18.7 mM phosphate, added hirudin almost completely inhibited the release of radioactive serotonin obtained upon subsequent warming to 37 degrees C (only the hirudin sensitive complex exists). If platelets were preincubated with thrombin for longer periods of time (30 minutes), hirudin became less effective in inhibiting the release obtained upon subsequent warming to 37 degrees C. The same results were obtained whether or not the platelets were washed after incubation at 4 degrees C and before warming to 37 degrees C. We postulate that this change in hirudin sensitivity may reflect a slow conversion of the first platelet-thrombin complex (P-T) to an activated platelet-thrombin complex (P*-T) which can undergo release upon warming. This transition appears to be much faster in acetatetris buffer since at short platelet-thrombin incubation times at 4 degrees C, added hirudin had little or no effect on the release obtained upon warming to 37 degrees C. The difference in the ability of hirudin to inhibit thrombin-induced release in the two buffers was shown to depend upon the presence of phosphate and on variations in ionic strength, and not due to a change in the inhibition constant (Ki) for hirudin.
此前有人提出，血小板刺激可能涉及两种血小板 - 凝血酶复合物：初始血小板 - 凝血酶复合物（P - T），它会转化为活化血小板 - 凝血酶复合物（P* - T）。通过使用放射性血清素的释放作为凝血酶刺激的指标，我们已经证明，在适当条件下，可以区分出血清素敏感复合物和血清素不敏感复合物。在4℃下血小板 - 凝血酶预孵育时间较短（0 - 2分钟）时，在含有18.7 mM磷酸盐的缓冲液中，加入水蛭素几乎完全抑制了随后升温至37℃时获得的放射性血清素的释放（仅存在血清素敏感复合物）。如果血小板与凝血酶预孵育更长时间（30分钟），水蛭素在抑制随后升温至37℃时获得的释放方面效果就会变差。无论血小板在4℃孵育后且在升温至37℃之前是否洗涤，都得到了相同的结果。我们推测，水蛭素敏感性的这种变化可能反映了第一种血小板 - 凝血酶复合物（P - T）缓慢转化为活化血小板 - 凝血酶复合物（P* - T），后者在升温时会发生释放。在醋酸 - 三缓冲液中这种转变似乎要快得多，因为在4℃下血小板 - 凝血酶孵育时间较短时，加入的水蛭素对升温至37℃时获得的释放几乎没有影响。结果表明，水蛭素在两种缓冲液中抑制凝血酶诱导释放能力的差异取决于磷酸盐的存在以及离子强度的变化，而不是由于水蛭素的抑制常数（Ki）发生了改变。
