The development of potent antithrombotic agents from the fibrinogen platelet receptor binding sequences Fg-alpha 572-575 -Arg-Gly-Asp-Ser- and Fg-gamma 400-411 -HHLGGAKQAGDV, believed to be a cryptic RGD-type sequence, is described. The tetrapeptide Ac-RGDS-NH2 itself is capable of inhibiting platelet aggregation in vitro at high concentrations, IC50 91.3 +/- 0.1 microM [in vitro antiaggregatory activity employing dog platelet rich plasma (PRP)/ADP], due to low platelet fibrinogen receptor affinity, Ki 2.9 +/- 1.9 microM (purified, reconstituted human platelet GPIIb/IIIa), relative to fibrinogen, Ki 38.0 +/- 6.0 nM. The peptide is also unstable to plasma, suffering total loss of in vitro activity upon incubation in PRP for 3 h (T1/2 90 min). Only modest improvements in potency were achieved with linear analogues of Ac-RGDS-NH2, while dramatic results were achieved with cyclic analogues, culminating in the cyclic disulfide Ac-cyclo-S,S-[Cys-(N alpha-Me)Arg-Gly-Asp-Pen]-NH2 (SK&F 106760) with improved plasma stability (100% activity after 3 h), affinity (Ki 58 +/- 20 nM purified human receptor), and potency (IC50 0.36 +/- 0.4 microM dog PRP/ADP). The affinity of this peptide is 2 orders of magnitude greater than that of Ac-RGDS-NH2. The affinity of the analogue is also comparable to fibrinogen. This peptide constitutes a first potent small peptide entry into the class of novel antithrombotic agents called fibrinogen receptor antagonists.

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Summary: Endothelin-1, a peptide exhibiting extremely potent cerebral vasoactive properties, is elevated in the cerebrospinal fluid after hemorrhagic stroke and impli cated in cerebral vasospasm. The purpose of this study was to determine changes in endothelin in ischemic rat brain by assaying endothelin tissue and extracellular lev els. Immunoreactive endothelin levels in ischemic brain tissue following permanent or transient focal ischemia produced by middle cerebral artery occlusion was deter mined. In addition, endothelin levels were assayed in stri atal extracellular fluid collected by microdialysis before, during, and after global ischemia produced by two-vessel occlusion combined with hypotension. Twenty-four hours after the onset of permanent middle cerebral artery occlusion, the ischemic cortex level (0.58 ± 0.27 fmoll mg protein) of immunoreactive endothelin was signifi cantly (p < 0.05) increased, by 100%, over that in the nonischemic cortex (0.29 ± 0.13 fmollmg protein). TranEndothelin (ET) is a 21-amino acid peptide orig inally isolated from porcine endothelial cells (Yan agisawa et aI., 1988). Three isoforms of endothelin (i.e., ET-l, -2, and -3) have been identified in geno mic DNA (Inoue et aI., 1989). Both ET-l and ET-3 are present in the brain (Matsumoto et aI., 1989; Shinmi et aI., 1989; Takahashi et aI., 1990) and spe cific binding sites have been identified (Koseki et

The observation that levels of the mitogenic peptide endothelin-1 are elevated in the human coronary sinus after percutaneous transluminal coronary angioplasty (PTCA) has implicated endothelin-1 in the etiology of vascular restenosis. The present study examined this hypothesis in both an in vitro and an in vivo rat model of neointimal formation by using the novel nonpeptide endothelin receptor antagonist SB 209670. In vitro, endothelin-1 (1 nmol/L) induced a ninefold increase in rat aortic vascular smooth muscle [3Hlthymidine incorporation. This endothelin A receptor-mediated effect was completely inhibited by SB 209670 (IC50, 6.2±2.2 nmol/L). In vivo, acute intra-arterial administration of exogenous endothelin-1 (5 to 500 pmol/kg over a 30-minute period immediately after angioplasty) dose-dependently augmented the degree of neointimal formation (by up to 150% when assessed 14 days after surgery). This response was evident as early as 7 days after angioplasty. Hemodynamic studies indicated that this action was unrelated to a systemic pressor action of the peptide. Administration of SB 209670 (2.5 mg/kg IP, twice a day for 3 days before and for 2 weeks after surgery) reduced neointimal formation by '50% relative to control animals. Thus, the data indicate for the first time that (1) endothelin-1 promotes neointimal formation in vivo and (2) tensin II and norepinephrine, endothelin-1 is now recognized as playing an intrinsic role in the endogenous control of systemic hemodynamics.3 Since levels of endothelin-1 are elevated in the human coronary sinus after PTCA, it has been suggested that endothelin-1 is involved in the pathogenesis of vascular restenosis.4In addition to its actions on vascular smooth muscle tonus, endothelin-1 is also a potent mitogen in several cultured cell lines of both cardiovascular and noncarReceived February 1, 1994; accepted March 21, 1994.

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Oxyhemoglobin and endothelin have both been linked to the development of the severe and sustained cerebral vasospasm associated with subarachnoid hemorrhage. The effects of oxyhemoglobin on endothelin biosynthesis in cultured endothelial cells were evaluated. Oxyhemoglobin (0.01 to 100 microM) produced concentration-dependent increases in immunoreactive endothelin levels in bovine pulmonary artery endothelial cell-conditioned medium. The median effective concentration for oxyhemoglobin-induced increases in immunoreactive endothelin levels was approximately 0.5 microM, and the maximum stimulation of immunoreactive endothelin levels was approximately 5.5-fold over basal conditions. In addition to directly stimulating basal production of immunoreactive endothelin, oxyhemoglobin significantly augmented immunoreactive endothelin production following platelet-mediated stimulation of endothelin production. An l-arginine analog inhibitor of nitric oxide synthase, L-NG-monomethyl arginine (L-NMMA, 200 microM), did not significantly affect basal immunoreactive endothelin levels. However, L-NMMA significantly augmented platelet-induced immunoreactive endothelin production. Methylene blue (10 microM), an inhibitor of soluble guanylate cyclase, did not significantly affect basal immunoreactive endothelin levels, nor did it significantly affect the platelet-mediated stimulation of immunoreactive endothelin production in cultured endothelial cells. The present results reveal that oxyhemoglobin can directly stimulate endothelin biosynthesis in cultured endothelial cells. This newly identified property of oxyhemoglobin suggests a potential mechanism for the sustained and severe cerebral vasospasm associated with subarachnoid hemorrhage.

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