thrombin-receptor-peptide-(42-55) and phenylalanyl-prolyl-arginine-chloromethyl-ketone

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.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antithrombins; Blood Platelets; Guinea Pigs; In Vitro Techniques; Indium Radioisotopes; Lung; Male; Peptide Fragments; Peptides; Platelet Aggregation; Radioligand Assay; Receptors, Thrombin; Thrombin

The aims were (1) to determine whether thrombin, which is increased in the presence of coronary thrombosis, can directly stimulate the production of lysophosphatidylcholine, which has arrhythmogenic properties, in ventricular myocytes; (2) whether the effect is dependent upon extracellular [Ca2+]; and (3) whether it is mediated directly through stimulation of the thrombin receptor.. Lipids were extracted from isolated adult rabbit ventricular myocytes and lysophosphatidylcholine was isolated by HPLC and quantified using a recently developed radiometric assay employing 3H-acetic anhydride.. Thrombin (0.05 U.ml-1) stimulation of ventricular myocytes resulted in a nearly sixfold increase in lysophosphatidylcholine levels [0.26(SEM 0.03) to 1.61(0.42) nmol.mg-1 protein] within 1 min. The increase in myocytic lysophosphatidylcholine content was prevented by preincubation of thrombin with the proteolytic site inhibitors phenyly-prolyl-arginyl-chloromethyl ketone (PPACK) and dansylarginine N-(3-ethyl-1,5-pentanediyl)amide. The increase in lysophosphatidylcholine content in response to thrombin was not present at an extracellular calcium concentration ([Ca2+)]o) = 500 microM, but was marked at a physiological level of [Ca2+]o = 1.8 mM. Stimulation of myocytes with the thrombin receptor activating peptide SFLLRNPNDKYEPF (100 microM for 1 min) resulted in a similar increase in lysophosphatidylcholine content [1.61(0.27) nmol.mg-1 protein].. The marked increase in lysophosphatidylcholine content in cardiac myocytes in response to thrombin has important implications as an arrhythmogenic mechanism during early myocardial ischaemia.

Topics: Amino Acid Chloromethyl Ketones; Animals; Arginine; Calcium; Cells, Cultured; Dansyl Compounds; Female; Lysophosphatidylcholines; Male; Myocardium; Peptide Fragments; Rabbits; Receptors, Cell Surface; Receptors, Thrombin; Stimulation, Chemical; Thrombin

We previously reported that thrombin produces endothelium-dependent relaxation and endothelium-independent constrictions in canine coronary arteries. To determine whether these opposing vascular effects of thrombin are mediated by the same receptor mechanism, but at different cell types, we investigated the effects of thrombin receptor agonist peptide (TRAP) on isolated canine coronary arteries with and without intact endothelium. In coronary arteries with intact endothelium, addition of 0.01-3.0 microM TRAP, a 14-amino acid residue peptide (SFLLRNPNDKYEPF) homologous to the newly exposed N-terminus after cleavage of the cloned human thrombin receptor, produced rapid, dose-dependent relaxation (Emax = -89.6 +/- 2.3%, n = 26). Threshold concentration was 0.03 microM, and IC50 value was 0.3 microM. Mechanical disruption of the endothelium completely abolished the TRAP-induced relaxation; instead a dose-dependent contraction was observed. Expressed as a percentage of the maximum 70 mM KCl-induced contraction, the maximum contraction observed with 3 microM TRAP was 62.0 +/- 4.1% (n = 32). Pretreatment of endothelium-intact coronary arteries with either 3 microM hemoglobin or 0.25 mM NG-monomethyl-L-arginine (L-NMMA), specific inhibitors of endothelium-derived relaxing factor or nitric oxide (EDRF/NO), also inhibited the relaxation and unmasked the constrictor effect. The pharmacokinetic characteristics of the opposing coronary vascular effects of TRAP are similar to those observed with thrombin, but specific thrombin inhibitors, such as hirudin and D-phenylalanyl-prolyl-L-arginine chloromethyl ketone (PPACK), which inhibit both thrombin relaxant and constrictor effects, had no effect on TRAP-induced responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Chloromethyl Ketones; Amino Acid Oxidoreductases; Amino Acid Sequence; Animals; Arginine; Arteries; Coronary Vessels; Dogs; Dose-Response Relationship, Drug; Endothelium, Vascular; Hemoglobins; In Vitro Techniques; Male; Molecular Sequence Data; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide Synthase; omega-N-Methylarginine; Peptide Fragments; Receptors, Thrombin; Thrombin