Integrins and other adhesion receptors are essential components for outside-in and inside-out signaling through the cell membrane. The platelet glycoprotein IIb-IIIa (also known as fibrinogen receptor or integrin q&) is activated by platelet agonists, inhibited by cyclic-nucleotide-elevating agents, and is involved in the activation of protein tyrosine kinases including the 125-kDa focal adhesion kinase (~~1 2 5~~~) .However, the molecular details of glycoprotein IIb-IIIa regulation are not well understood. Here we report that in ADP-activated human platelets CAMP-and cGMPdependent protein-kinase-mediated phosphorylation of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) at Ser157 correlates well with glycoprotein IIb-IIIa inhibition. Human platelets contain similar concentrations of glycoprotein IIb-IIIa complexes (fibrinogen binding sites) and VASP. Using gel-filtered platelets, CAMP-elevating agents [e.g. prostaglandin E, and the forskolin analog 6-(3-dimethylaminopropionyl)forskolin (NKH 477)] caused VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa activation up to 70-100%. NO-generating, cGMP-elevating agents [e.g. 3-morpholinosydnonimine hydrochloride (SIN1) and sodium nitroprusside] stimulated VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa activation up to a maximal extent of 30-50%. The effects of CAMP-and cGMP-elevating agents on VASP phosphorylation and fibrinogen binding were reversible and could be mimicked by membrane-permeant selective activators of platelet CAMP-or cGMP-dependent protein kinase, respectively. Using threshold concentrations, the nitrovasodilator SIN 1 potentiated the effects of the forskolin analog NKH 477 with respect to inhibition of platelet aggregation, VASP phosphorylation and glycoprotein IIb-IIIa inhibition. It is proposed that the inhitition of glycoprotein IIb-IIIa induced by cyclic nucleotide involves CAMPand cGMP-dependent protein-kinase-mediated VASP phosphorylation at Ser157.Adhesion, activation and aggregation of platelets and platelet -vessel-wall interactions are tightly regulated under physiological conditions and often impaired in diseases including congenital hemorrhagic diseases, atherosclerosis, hypertension and diabetes 11, 21. Important components of this vascular cell -cell and cell -matrix interaction are integrins and other adhesion receptors [3 -81. The platelet glycoprotein IIb-IIIa ((xllbp3) is one of the best-studied integrins and represents the functional fibrinogen receptor which is absent or impaired in the hereditary disorder Glanzmann's thrombasthenia [l, 5-81. Activated platelets contain at their sur-

Summary. Background and objectives: AVE5026 is a novel, hemisynthetic, ultra-low-molecular-weight heparin (ULMWH), which is in clinical development for prevention of venous thromboembolism. Its unique structural features result from the highly selective depolymerization of heparin by the phosphazene base that protects the antithrombin (AT)-binding site from destruction. In the present paper, we describe the chemical and biological characteristics of AVE5026, as well as its effects on experimental thrombosis as compared to those of the low-molecular-weight heparin (LMWH) enoxaparin after a single subcutaneous (s.c.) administration in certain animal models. Method and results: AVE5026 has a higher antifactor Xa (anti-FXa) activity (160 U mg ) as a result of its structure being strongly enriched in specific ATbinding oligosaccharides. In human plasma, potent inhibition of thrombin generation by AVE5026 was closely related to its anti-FXa activity. In a rat venous thrombosis model, AVE5026 showed a dose-dependent antithrombotic activity comparable to that of enoxaparin (ED50-AVE5026 = 1.6 mg kg ). Interestingly, non-occlusive venous thrombosis in rabbits was inhibited by an ED50 of 0.1 mg kg )1 AVE5026, whereas 0.316 mg kg )1 enoxaparin was not active. In a canine model, similarly to enoxaparin (ED50 = 1.3 mg kg ). At equipotent doses, AVE5026 did not affect bleeding parameters, whereas enoxaparin showed increased hemorrhage in rats, rabbits and dogs. Conclusion: These unique structural attributes distinguish AVE5026 from the LMWH class. Based on these data in wellestablished arterial and venous thrombosis models, AVE5026 could represent a valuable alternative in thrombosis prevention with an improved benefit-risk profile as compared to that of enoxaparin.

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The protozoan parasite Babesia equi, a causative agent of equine piroplasmosis, was continuously cultivated in horse erythrocytes. The parasites were isolated from a carrier horse at a time when no parasite was detected in a thin blood smear. The culture medium consisted of modified medium 199 supplemented with 40% non-heat-inactivated horse serum in a humidified atmosphere containing 5% CO2, 2% O2, and 93% N2 at 37 degrees C. Parasites were detected after 2 days in culture. When the percentage of parasitized erythrocytes (PPE) reached 1%, the cultures were transferred into a humidified atmosphere of 5% CO2 in air. After 7 days the cultures were split at a ratio of 1:2, and after another 5 days they were split at a ratio of 1:4. From them on, cultures were split at a ratio of 1:4 routinely at 2-day intervals. The PPE ranged between 10% and 25%. Supplementation with hypoxanthine was essential for the initiation and propagation of cultures. In established cultures, hypoxanthine could be replaced by equimolar concentrations of adenosine or guanosine. Parasites from cultures could be cryopreserved and resuscitated. Cultures were maintained for more than 300 days.

Antagonists of the platelet fibrinogen receptor (GP IIb/IIIa receptor) are expected to be a promising new class of antithrombotic agents. The binding of fibrinogen to the fibrinogen receptor depends on an Arg-Gly-Asp-Ser (RGDS) tetrapeptide recognition motif. Structural modifications of the RGDS lead have led to the discovery of a non-peptide RGD mimetic GP IIb/IIIa antagonist 44 (S 1197). Compound 44 inhibited, in a dose dependent and reversible manner, human and dog platelet aggregation as well as 125I-fibrinogen binding to ADP-activated human gel filtered platelets and isolated GP IIb/IIIa with K(i) values of 9 nM and 0.17 nM, respectively. A pharmacophore mapping procedure with QXP and a 3D-QSAR analysis applying the GRID/GOLPE methodology yielded a stable, rather predictive model and revealed structural features which are important for binding. Hydrophobic substitutions both at the hydantoin nucleus and at the C-terminus increase the affinity toward the fibrinogen receptor. The crystalline ethyl ester prodrug 48 (HMR 1794) is an orally active antithrombotic agent which is a promising drug candidate for the treatment of thrombotic diseases in humans.

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A series of novel, highly potent P2Y₁₂ antagonists as inhibitors of platelet aggregation based on a phenylpyrazole glutamic acid piperazine backbone is described. Exploration of the structural requirements of the substituents by probing the structure-activity relationship along this backbone led to the discovery of the N-acetyl-(S)-proline cyclobutyl amide moiety as a highly privileged motif. Combining the most favorable substituents led to remarkably potent P2Y₁₂ antagonists displaying not only low nanomolar binding affinity to the P2Y₁₂ receptor but also a low nanomolar inhibition of platelet aggregation in the human platelet rich plasma assay with IC₅₀ values below 50 nM. Using a homology and a three-dimensional quantitative structure-activity relationship model, a binding hypothesis elucidating the impact of several structural features was developed.

The effect of recombinant hirudin (r-hirudin; HBW 023) was studied in four different models of thrombosis as well as on bleeding time. Arteriolar thrombus formation was induced in rats either by argon laser injury or by photochemical reaction. r-Hirudin, given by intravenous infusion, significantly inhibited arteriolar thrombus formation and arterial bleeding time at a dose of 40 μg/kg·min. In rabbit jugular veins and carotid arteries, occluding thrombi were produced by stenosis and endothelial damage. r-Hirudin reduced the incidence of thrombosis dose dependently (ED50 0.7 and 1.0 mg/kg i.v. in arteries and veins, respectively). Caval vein thrombosis was initiated in rats by insertion of a stainless steel coil. This thrombosis was dose dependently reduced by the injection of r-hirudin (ED50 0.16 mg/kg i.v.). The duration of the antithrombotic effect of 0.25 mg/kg of r-hirudin in caval veins was about 60 min, whereas the arterial bleeding time was significantly prolonged in these rats for only 20 min. The plasma level of r-hirudin had dropped to 0.12 μg/ml 60 min after injection. These results suggest that the duration of the effect on bleeding time may be shorter than the antithrombotic action of r-hirudin.