The effect of 2-methyl-3-(1, 4, 5, 6-tetrahydronicotinoyl) pyrazolo [1, 5-a] pyridine (KC-764) on human platelet aggregation and its mechanism of action were investigated. The inhibitory activity of KC-764 on arachidonic acid-induced platelet aggregation was most potent with IC₅₀ of 4.2×10^-8 M and was about 100 times more potent than that of acetylsalicylic acid (ASA). KC-764 weakly inhibited prostaglandin G₂-and prostaglandin H₂-induced platelet aggregations. Neither KC-764 nor ASA had effect on platelet aggregation induced with STA₂ (thromboxane A₂ analog). KC-764 inhibited strongly human platelet cyclooxygenase activity. The effect of KC-764 on platelet aggregation, differently from ASA, was reversible. The inhibitory effect of KC-764 on cyclic nucleotide phosphodiesterases was weak. KC-764 had no effect on the concentration of cAMP and cGMP.
These results suggest that the reversible inhibitory effects of KC-764 on platelet aggregations are mediated mainly via inhibition of platelet cyclooxygenase.

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Cationic antibacterial proteins (CAP), intracellular LPS-binding proteins, were prepared from rabbit peritoneal granulocytes using an assay the agglutination of erythrocytes coated with Re-LPS. Molecular weight of CAP estimated by SDS-PAGE was 7kDa (CAP-7) and 18kDa (CAP-18), and CAP-7 had high contents of cationic residues. LPS activates murine macrophages and human blood monocytes to generate tissue factor (tissue thromboplastin). After 18 hours incubation of LPS preparations with CAP (heparin-sepharose fraction), S-LPS, Re-LPS and lipid A-induced tissue factor activities were inhibited. S-LPS was inactivated more rapidly than Re-LPS and lipid A. Within 1 hour incubation, purified CAP-18 inhibited about 75% of the activity of S-LPS. CAP-7 also inhihited LPS-induced tissue factor generation by human blood monocytes. LPS-binding, and LPS-neutralizing activates of CAP-7 were highter than those of CAP-18. Synthetic peptied #197-1 (indentical structure to CAP-7) also showed LPS-binding and LPS-neutralizing activities.
DIC manifestation in endotoxemia, due to gram-negative bacterial infections, are controlled by CAP released from granulocytes. In addition, CAP-7 and CAP-18 may have therapeutic potential for septic and endotoxin shock.

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We report the successful treatment of a subepiglottic hematoma accompanied by progressive dyspnea and vocal difficulty in a 17-year-old hemophilia A patient with inhibitor. The inhibitor titer to human factor VIII was 16 Bethesda Units (B. U.)/ml, whereas that to porcine factor VIII was 5 B. U./ml (cross-reactivity: 31.3%). Then, he was treated by 19, 885u (310.7/kg) (15, 000u for inhibitor neutralizing dose plus about 5, 000u for factor VIII activity (FVIII: C) incremental dose up to 100%) of porcine factor VIII concentrate (MTI-9002; Hyate: C) in a single infusion. The vocal difficulty and respiratry obstruction started disappearing 2 hours later and completely disappeared 2 days later. The recovery of the FVIII: C at 30min and 60min after infusion was 130 and 110%, respectively. Although shivering and a transient and mild decrease in the platelet counts occurred at 30min after the end of the infusion, the symptom disappeared by using hydrocortisone infusion 30min later and the thrombocytopenia recovered within 8 hours. Anamnestic response appeared 7 days after infusion. The peak anti-human and anti-porcine factor VIII titers were 448 and 160 B. U./ml at week 4, respectively. Inhibitor titers decreased to 220 and 84 B. U./ml at week 8. We concluded that only a single but a large infusion more than the calculated neutralizing dose of porcine factor VIII concentrates is effective for lifethreatening hemorrhages in hemophillia A patients with low cross-reactivity.

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We investigated the cell surface-focused urokinase-type plasminogen activator (uPA)/plasmin system regulating plasmin prodution. Plasmin cleaves single-chain uPA (pro-uPA) synthesized from cells at amino acid position Lys158-Ile159 bond and the Lys135-Lys136 bond. uPA receptors are occupied by Amino-terminal fragment (ATF) after cleavage at position Lys135-Lys136 bond. Extensive treatment of pro-uPA/uPA with plasmin results in the cleavages of the Lys23-Tyr24 bond and of the Lys35-Lys36 bond in the Growth Factor-like domain (GFD) before binding to the uPA receptor. These polypeptides with the cleavage in the GFD can not bind to the uPA receptors on U937 cells any more. Plasmin, however, does not cleave amino acid sequences within GFD of receptor-bound pro-uPA/uPA on U937 cells, indicating that receptor-bound ATF is resistant to the plasmin action. These provide a feedback mechanism of pro-uPA/uPA binding to the uPA receptor and subsequent plasmin production.

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Phase I and II clinical studies were performed to evaluate the safety, efficacy and pharmacokinetics of recombinant factor VIII (BL-160) in hemophilia A patients. Three and seven patients were enrolled in the phase I and phase II study respectively. The half life and recovery observed with recombinant factor VIII were comparable to those observed with plasma-derived factor VIII (Hemofil M). Forty-four bleeding episodes were treated with recombinant factor VIII during the phase II study, hemostatic effects were excellent in 39 and good in 5 bleeding episodes. There is no evidence of neoantigenicity resulting from potential contaminating proteins used in production steps. No adverse reactions were reported. These data indicate that recombinant factor VIII is safe and efficacious for the treatment of bleeding episodes in hemophilia A.

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