The purification, complete amino acid sequence, and biological activity are described for several homologous snake venom proteins that are platelet glycoprotein (GP) IIb-Ila antagonists and potent inhibitors of platelet aggregation. The primary structures of kistrin (from Agkistrodon rhodostoma), bitan (from BiWs arietans), three isoforms of trigramin (from Trimeresusus gramineus), and an isoform of echistatin (from Echis carinatus) were determined by automated sequence analysis and fast atom bombardment mass spectrometry analysis. Each of the proteins in this family, which range from 47 to 83 residues, contains an Arg-Gly-Asp amino acid sequence found in protein ligands that binds to GPIIb-Ila, a high (17 ± 1%) cysteine content conserved in the primary sequence, and a homologous N-terminal region absent only in the echistatin isoforms. Each protein directly inhibits the interaction of purified platelet GPIIb-Hla to immobilized fibrinogen about 100 times more effectively than does the pentapeptide Gly-Arg-Gly-Asp-Ser; IC50 values range from 1.1 to 3.0 nM. The IC50 value for the inhibition of platelet aggregation, using human platelet-rich plasma stimulated with ADP, ranges from 110 to 550 nM for the various proteins, about 1000-fold more potent than Gly-Arg-Gly-Asp-Ser. Kistrin binds reversibly to both resting and ADP-activated human platelets with high affinity (Kd = 10.8 nM and 1.7 nM, respectively) and to purified GPIIb-IIIa with a lower affinity (Kd = x100 nM). Finally, kistrin injected at 1.0 mg/kg into rabbits reversibly inhibits platelet aggregation ex vivo over 30 min without induction of thrombocytopenia. We propose that these proteins are members of a general class of proteins found in the venom of pit vipers that inhibit platelet aggregation by antagonism of the GPIIb-Ila-fibrinogen interaction and as such serve as potential antithrombotic agents.

Stimulation of platelets activates GPIIbIIIa, the heterodimeric integrin receptor, to bind fibrinogen (Fg), which results in platelet aggregation. GPIIbIIIa/Fg binding inhibitors are potentially suitable for acute use during and after thrombolytic therapy as antithrombotic agents. Incorporation of the tripeptide sequence Arg-Gly-Asp (RGD), a common structural element of many integrin ligands, into cyclic peptides produced a series of peptides of the general structure BrAc-(AA1)-RGD-Cys-OH, which were prepared by solid-phase peptide synthesis. Cyclization was accomplished by reaction of the N-terminal bromoacetyl group with the cysteine sulfhydryl at pH 8 at high dilution, resulting in thioether-bridged cyclic peptides [cyclo-S-Ac-(AA1)-RGD-Cys-OH]. Use of alpha-substituted bromoacetyl groups gave rise to an analogous series of acetyl-substituted thioether-bridged cyclic peptides. Oxidation of the thioethers produced separable diastereomeric sulfoxide-bridged cyclic peptides. After thorough evaluation in a GPIIbIIIa ELISA assay and a platelet aggregation assay, G-4120 (70A; AA1 = D-Tyr; sulfoxide bridge) was selected for further investigation as an antithrombotic agent. G-4120 was equipotent in the platelet aggregation assay to kistrin, a highly potent inhibitor of fibrinogen-mediated platelet aggregation isolated from snake venom (IC50 = 0.15 microM).

Background and Purpose-SolCD39 is a soluble form of recombinant human ecto-ATP/ADPase (NTPDase1) and represents a new class of antithrombotic agents. SolCD39 blocks and reverses platelet activation, preventing recruitment of additional platelets into a growing thrombus. The purpose of this study was to examine the effect of solCD39 on neurological deficit, infarct size, and extent of edema after transient middle cerebral artery occlusion (MCAO) in rats. Methods-Physiologically controlled Sprague-Dawley rats underwent 2-hour MCAO by retrograde insertion of an intraluminal suture coated with poly-L-lysine. The agent (solCD39) was administered intravenously before MCAO or at 1-hour or 3-hour recirculation. Other groups received vehicle (Tris-buffered saline) or human albumin (as a "positive" neuroprotective control; 25%, 0.5% of body weight) at 1-hour recirculation. Neurological status was evaluated during occlusion (at 60 minutes) and daily for 3 days after MCAO. Brains were perfusion-fixed at 72 hours, and infarct volumes and brain swelling were determined. Results-Pretreatment with solCD39 significantly improved the neurological score at 72 hours compared with the vehicle group (4.4Ϯ0.6 versus 7.6Ϯ0.6, respectively; Pϭ0.008). Cortical infarct areas were significantly reduced at multiple levels by pretreatment with solCD39. Total striatal infarct area was also significantly reduced compared with vehicle by both solCD39 pretreatment (48% mean reduction) and solCD39 treatment at 3-hour recirculation (51% mean reduction). Treatment with SolCD39 significantly reduced total infarct volume (corrected for brain swelling) by an average of 71% to 72% when administered either before ischemia or at 3 hours of recirculation compared with vehicle. Treatment with albumin significantly reduced neurological score and total, cortical, and subcortical infarction at multiple levels, as expected. Conclusions-Treatment

The role of protein kinase C (PKC) in interleukin‐1β‐ (II‐ 1β)‐, tumor necrosis factor‐α‐ (TNF‐α)‐, and lipopolysaccharide‐(LPS)‐induced vascular cell adhesion molecule‐1 (VCAM‐1) expression on human umbilical vein endothelial cells (HUVEC) was studied. PKC inhibition or downregulation diminished VCAM‐1 mRNA accumulation and protein expression. Interleukin‐1β, TNF‐α, and LPS induce nuclear factor (NF)‐kB‐like binding activity, which precedes VCAM‐1 transcription. PKC inhibition did not prevent NF‐xB‐like binding activity, indicating that this is PKC‐independent, and NF‐kB‐like binding activity is insufficient for transcription of VCAM‐1.

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