Chiu P J, Tetzloff G G, Foster C, Chintala M, Sybertz E J
Department of Pharmacology (2600), Schering-Plough Research Institute, Kenilworth, NJ 07033-0530, USA.
Eur J Pharmacol. 1997 Feb 19;321(1):129-35. doi: 10.1016/s0014-2999(96)00931-4.
Guinea pig platelets are similar to human platelets in their responsiveness to thrombin receptor-activating peptides and other agonists. Therefore, guinea pigs anesthetized with Inactin (90 mg/kg i.p.) were used to assess in vivo activities of thrombin and thrombin receptor-activating peptides (TRAPs) using 111 In-labeled platelets and a microcomputer-based system. The aggregatory responses are expressed as percent change for a 20 min period over basal radioactivity (AUC). Reversible accumulation of platelets occurred in the pulmonary microcirculation in response to stimuli. Human thrombin (50 and 100 U/kg i.v.) caused a dose-related platelet accumulation. Responses of similar magnitude were induced by SFLLRN (TRAP-(1-6)) and Ala-Phe(p-F)-Arg-Cha-HArg-Tyr-NH2 (high-affinity thrombin receptor-activating peptide, 0.03, 0.1 and 0.3 mg/kg i.v.). High-affinity thrombin receptor-activating peptide, a new synthetic oligopeptide agonist, is about 3-fold more potent than TRAP-(1-6), a wild-type sequence. Similarly, high-affinity thrombin receptor-activating peptide is about 4 times more potent than TRAP-(1-6) in the radioligand binding study using platelet membrane. By comparison, high-affinity thrombin receptor-activating peptide manifested an aggregatory activity (EC60 = 1.2 microM) about 15 times more potent than that of TRAP-(1-6)(EC60 = 18.6 microM) in washed guinea pig platelets. The intrapulmonary platelet aggregation in response to thrombin, TRAP-(1-6) and high-affinity thrombin receptor-activating peptide was characterized by long duration (approximately 30 min); a reduction in response (18-54%) tended to occur with repeated challenges, presumably due to desensitization and consumption. The response to thrombin (100 U/kg) was greatly inhibited by (D)-Phe-Pro-Arg-chloromethyl ketone (PPACK), a potent thrombin inhibitor (250 micrograms/kg + 6 micrograms/kg per min i.v. x 30): AUC, 150 +/- 552 vs. 7171 +/- 1052 in the control period (n = 8, P < 0.05). The response to high-affinity thrombin receptor-activating peptide (0.03 mg/kg), which acts on thrombin receptor directly, was not affected by PPACK. It is concluded that guinea pigs are an appropriate preparation for evaluation of in vivo activity of thrombin inhibitors as well as thrombin receptor agonists and antagonists.
豚鼠血小板对凝血酶受体激活肽和其他激动剂的反应性与人类血小板相似。因此，使用以Inactin（90mg/kg腹腔注射）麻醉的豚鼠，采用111In标记的血小板和基于微型计算机的系统来评估凝血酶和凝血酶受体激活肽（TRAPs）的体内活性。聚集反应以相对于基础放射性（AUC）在20分钟内的变化百分比表示。血小板在肺部微循环中因刺激而发生可逆性聚集。人凝血酶（50和100U/kg静脉注射）引起剂量相关的血小板聚集。SFLLRN（TRAP-(1-6)）和Ala-Phe(p-F)-Arg-Cha-HArg-Tyr-NH2（高亲和力凝血酶受体激活肽，0.03、0.1和0.3mg/kg静脉注射）诱导出相似程度的反应。高亲和力凝血酶受体激活肽，一种新的合成寡肽激动剂，其效力比野生型序列TRAP-(1-6)高约3倍。同样，在使用血小板膜的放射性配体结合研究中，高亲和力凝血酶受体激活肽的效力比TRAP-(1-6)高约4倍。相比之下，在洗涤过的豚鼠血小板中，高亲和力凝血酶受体激活肽表现出的聚集活性（EC60 = 1.2μM）比TRAP-(1-6)（EC60 = 18.6μM）高约15倍。对凝血酶、TRAP-(1-6)和高亲和力凝血酶受体激活肽的肺内血小板聚集的特点是持续时间长（约30分钟）；重复刺激时反应往往会降低（18 - 54%），可能是由于脱敏和消耗。对凝血酶（100U/kg）的反应被强效凝血酶抑制剂（D）-Phe-Pro-Arg-氯甲基酮（PPACK，250μg/kg + 6μg/kg每分钟静脉注射×30）大大抑制：AUC，对照期为7171±1052，处理后为150±552（n = 8，P < 0.05）。对直接作用于凝血酶受体的高亲和力凝血酶受体激活肽（0.03mg/kg）的反应不受PPACK影响。结论是，豚鼠是评估凝血酶抑制剂以及凝血酶受体激动剂和拮抗剂体内活性的合适实验对象。
