Characterization of in vitro and in vivo platelet responses to thrombin and thrombin receptor-activating peptides in guinea pigs.
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.
Chiu
P J
PJ
Department of Pharmacology (2600), Schering-Plough Research Institute, Kenilworth, NJ 07033-0530, USA.
Tetzloff
G G
GG
Foster
C
C
Chintala
M
M
Sybertz
E J
EJ
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000991:Antithrombins / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects*; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007205:Indium Radioisotopes; D008168:Lung / Q000166:cytology; D008297:Male; D010446:Peptide Fragments / Q000494:pharmacology*; D010455:Peptides / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects; D011869:Radioligand Assay; D018179:Receptors, Thrombin / Q000819:agonists / Q000187:drug effects*; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000494:pharmacology*
D000590:Amino Acid Chloromethyl Ketones; D000991:Antithrombins; D007205:Indium Radioisotopes; D010446:Peptide Fragments; D010455:Peptides; D018179:Receptors, Thrombin; C072032:thrombin receptor peptide (42-55); D013917:Thrombin; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone