The effect of a novel compound, 3-isobutyryl-2-isopropylpyrazolo [1, 5-a] pyridine (ibudilast, KC-404), on human platelet aggregation and its mechanism of action were investigated.
In vitro, KC-404 inhibited human platelet aggregation induced by ADP, collagen, adrenalin, platelet activating factor and arachidonic acid but not by ristocetin. Together, KC-404 and agents which increased cAMP (prostaglandin I₂, prostaglandin E₁ (PGE₁), forskolin) or cGMP (3-morpholinosydnonimine (SIN-1)) produced synergistic inhibitory effects on platelet aggregation.
KC-404 inhibited human platelet cAMP phosphodiesterase (PDE) (IC₅₀: 50μM) and cGMP-PDE (IC₅₀: 5.2μM) activities. cAMP and cGMP concentration of human platelets were not increased by KC-404 itself. PGE₁, an adenylate cyclase stimulator, increased cAMP content; KC-404 enhanced the effect of PGE₁ on cAMP accumulation. SIN-1, which stimulates guanylate cyclase, increased cGMP content; KC-404 enhanced the effect of SIN-1 on cGMP accumulation.
These results suggest that effects of KC-404 on accumulation of cyclic nucleotides and inhibition of platelet aggregation are mediated via inhibition of platelet cyclic nucleotide phosphodiesterase activities.

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Fibronectin (FN) is an extracellular matrix and plasma glycoprotein that binds to heparin. Three different heparin binding sites of fibronectin have been described. These binding sites differ in their affinity to bind to heparin. Their characteristics and functional relationships remain to be discussed. We have developed an assay employing heparin coupled Sepharose CL-6B to show that the binding of ¹²⁵I-FN to heparin was time-dependent, saturable, reversible and independent on divalent ions. Moreover, sodium concentration was crucial for the binding. We produced the polyclonal antibody, designated as FK-2, against human FN. In enzyme linked immunosorbent assay, FK-2 recognized only FN and did not react with fibrinogen or von Willebrand factor (vWf). Using our developed assay for measuring FN binding to heparin, FK-2 inhibited the binding in a dose-dependent manner with IC₅₀ of 29μg/ml. Moreover, a monoclonal anti-vWf antibody (52K-8) also showed the inhibition of fibronectin binding to heparin in a dose-dependent manner with IC₅₀ of 160μg/ml.

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Human blood was incubated in the waterbath at various temperature (0-50°C) for various time (5-90min). Then the effect of temperature on several parameters (APTT, PT, fibrinogen, activities of clotting factors, platelet aggregation) were studied. The results were as follows.
1. APTT and PT didn't change below 40°C, but they were prolonged significantly at 43°C, which was the border temperature for some normal and tumor cells.
2. Prothrombin activity decreased at 50°C, and Factor X did at 45°C. Fibrinogen was clottable between 0°C and 45°C, but became to be unclottable at 50°C. On the other hand, the changes of fibrin clot strand was observed at 43°C and above under scaning electron microscope.
4. Platelets lost their aggregative function at 43°C when collagen was used as inducer, at 40°C using ADP.
From these results, we concluded that 43°C might be the critical temperature in blood coagulation system as well as in the case of hyperthermia.

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In order to assess the hemostatic system activation in infectious disease, plasma levels of thrombin-antithrombin III complex (TAT), plasmin-α₂-plasmin inhibitor complex (PIC) and D-dimer were measured together with antithrombin III, protein C and von Willebrand factor antigen (vWF: Ag) in 65 patients with various infection without a sign of disseminated intravascular coagulation (DIC). Plasma levels of TAT, PIC, D-dimer and vWF: Ag were elevated, and protein C but not antithrombin III decreased. The magnitude of elevation of TAT, PIC and D-dimer were not directly correlated with CRP. The protein C levels tended to correlate negatively with CRP. Although there was a positive correlation between antithrombin III and protein C, protein C seemed to decrease more easily than antithrombin III. Levels of D-dimer changed in parallel with the DIC score and D-dimer may be useful to predict the pre-DIC states in these patients.

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Lupus anticoagulant (LA) positive SLE patients seem to have a thrombotic tendency. We have examined that plasma levels of antigen and protein C (PC) activity were lower in LA positive than in negative SLE patients. To elucidate the mechanism of thrombosis in LA positive SLE patients, we investigate the effects of IgG fraction obtained from LA and antiphospholipid antibody (APA) positive SLE patients on the activation of PC in the reaction of human umbilical vein endothelial cells (HUVEC) and thrombin. The results were as follows:
1. The activation rate of PC was inhibited by the addition of IgG obtained from LA and APA positive SLE patients (inhibition rate: 17.8%, p<0.05).
2. The activation rate of PC was reduced much more by the addition of IgG fraction obtained from the patients who showed high level of APA.
3. Among five antiphospholipid antibodies, anticardiolipin antibody strongly inhibited the activation of PC.
4. IgG fraction obtained from LA and APA positive SLE patients did not injure HUVEC.
In addition to the previous reports which showed the reduced plasma level of PC in LA positive SLE patients, the activation rate of PC on HUVEC was inhibited by their IgG. From these results, we concluded that LA positive SLE patients show higher possibility of thrombosis than LA negative patients.

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Whole blood viscosity, plasma viscosity, whole blood and plasma passage time were all significantly decreased and shortened compared to pre-drug values after intravenously administered Iloprost, a stable analogue of prostacyclin, to patients with peripheral arterial occlusive disease at a flow rate of 1ng/kg/min for 30 minutes once a day for seven consecutive days.
Plasminogen, antithrombin-III (ATIII) and α₂-macroglobulin were significantly decreased along with inhibited platelet aggregation.
When Iloprost was administered once a day for 6 consecutive days, whole blood viscosity at low shear rate and fibrinogen were decreased significantly.
These findings suggest that Iloprost inhibits not only platelet aggregaton, but also stimulates fibrinolytic activity of blood and enhances blood fluidity and ameliorates microcirculation in the ischemic leg of patients with peripheral arterial occlusive disease.

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Six hemophilic patients with asymptomatic human immunodeficiency virus-1 (HIV-1) infection were treated with a natural human interferon-α (IFNα, HLBI-PO, Sumitomo Pharmaceutical, Osaka, Japan). Patients were given 40-200 I. U. orally once a day in powdered atherocollagen held in the mouth to promote mucosal absorption.
The efficacy, safety and usefulness of this therapy have been evaluated for the past one year. Although a remarkable (2.5-3.5 times) but only a transient increase of CD4 cell counts was shown in 2 out of 6 patients, no significant change of peripheral lymphocyte counts, CD4 cell counts and CD 8 cell counts was shown at the end of this study. In 2 cases (Case 2 and 5) value of HIV-1 antigen (p-24) assayed by ELISA turned weakly positive from negative condition and in 2 cases (Case 5 and 6) the virus was isolated from the blood 16 weeks and 46 weeks after the starting of IFNα therapy, respectively.
In a few cases subjective and/or objective symptoms were clearly improved. No adverse reactions were found. During this clinical study, each patient has remained as an asymptomatic carrier of HIV infection (CDC Group II) with relatively decreased cellular immunity.
From these obserevations it would be concluded that in the hemophiliacs with HIV infection the oral IFNα therapy seems to be not so effective from the virological and immunological points of view. In order to totally evaluate the usefulness of oral IFNα further and more investigations are required.

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