Effects of dilazep on cytoplasmic calcium ion concentrations and arachidonic acid metabolism in activated human platelets.

The effects of dilazep (tetrahydro-1H-1,4-diazepine-1,4(5H)-dipropanol bis(3,4,5-trimethoxybenzoate)-di-hydrochloride monohydrate, Comelian), a coronary and cerebral vasodilator and an antiplatelet drug, on the cytoplasmic Ca2+ concentration ([Ca2+]i) and arachidonic acid (AA) metabolism in activated human platelets were investigated. [Ca2+]i (free calcium ion concentration) of aequorin-loaded platelets was estimated by using the platelet ionized calcium aggregometer. AA metabolism was studied by the determination of AA metabolites, hydroxyheptadecatrienoic acid and 12-hydroxyeicosatetraenoic acid, using reversed-phase high performance liquid chromatography. When platelets were preincubated with dilazep (0-0.5 mmol/l), the drug inhibited both platelet aggregation and [Ca2+]i elevation induced by thrombin, AA and collagen in a concentration dependent manner, while only aggregation was inhibited after stimulation with the Ca ionophore A23187 (calcimycin). Both influx and release of Ca2+ into platelet cytoplasm induced by thrombin or AA were inhibited by dilazep, while neither of them was affected when induced by A23187. Oral ingestion of dilazep as a 100-mg capsule significantly depressed the [Ca2+]i elevation induced by thrombin, AA and collagen after 3 h. Dilazep inhibited endogenous AA metabolism by platelets induced by thrombin, although it enhanced exogenous one. Thus, dilazep inhibited platelet aggregation induced by any agonists including A23187, while [Ca2+]i elevation was inhibited by the drug only when the receptor-mediated agonist was used. Furthermore, it is suggested that dilazep inhibited AA liberation from platelet membrane phospholipids, leading to reduced production of all endogenous AA metabolites after platelet activation although metabolites of exogenous AA could be increased.

A novel platelet aggregating factor found in a patient with defective collagen-induced platelet aggregation and autoimmune thrombocytopenia.

We found a novel platelet aggregating factor in a patient with steroid-responsive immune thrombocytopenic purpura that is associated with defective collagen-induced platelet functions. The aggregating factor and platelet functions were analyzed. The patient, a 58-year-old female, had purpura and prolonged bleeding time despite adequate platelet counts (greater than 140,000/microL) after steroid therapy. The patient's platelets responded normally to all agonists except collagen. Platelet adhesion to collagen fibrils was decreased. The patient's plasma induced irreversible aggregation and ATP release in normal platelet-rich plasma (PRP). This platelet aggregating factor was found in F(ab')2 fragments of the patient's IgG, which caused thromboxane B2 synthesis, elevation of cytoplasmic Ca2+ levels, and phosphorylation of 40 kDa protein in normal platelets. Platelet aggregation by the patient's IgG was inhibited by prostacyclin, dibutyryl cAMP, diltiazem, disodium ethylenediaminetetraacetate, and antimycin A plus iodoacetate, but ADP scavengers, cyclo-oxygenase inhibitors, and heparin had little or no effect. The aggregating activity of the patient's IgG absorbed to and eluted from normal platelets. The patient's Fab fragments did not induce platelet aggregation in eight of ten normal PRP but specifically inhibited aggregation induced by collagen and by the patient's IgG. The major component of an immunoprecipitate made with the patient's IgG from radiolabeled membrane proteins of normal platelet extract had a 62 kDa mol wt, while no such precipitate appeared in extracts of the patient's platelets. These results indicated that platelet aggregation by the patient's IgG was induced by the reaction of an antibody with a specific antigen on the normal platelet membrane through stimulus-response coupling. This antigen may be a collagen receptor on the platelet, most likely a polypeptide of 62 kDa under reducing condition. The defect of collagen-induced aggregation of the patient's platelets seemed to be due to alteration of the membrane protein related to this putative collagen receptor.

Requirement of free arachidonic acid for leukotriene B4 biosynthesis by 12-hydroperoxyeicosatetraenoic acid-stimulated neutrophils.

Stimulation of human neutrophils with 12-hydroperoxyeicosatetraenoic acid (12-HPETE) led to formation of 5S, 12S-dihydroxyeicosatetraenoic acid (DiHETE), but leukotriene B4 (LTB4) or 5-hydroxyeicosatetraenoic acid (5-HETE) was not detectable by reversed-phase high-performance liquid chromatography analysis. N-formylmethionylleucylphenylalanine (FMLP) induced the additional synthesis of small amounts of LTB4 in 12-HPETE-stimulated neutrophils. The addition of arachidonic acid greatly increased the synthesis of LTB4 and 5-HETE by neutrophils incubated with 12-HPETE. In experiments using [1-14C]arachidonate-labeled neutrophils, little radioactivity was released by 12-HPETE alone or by 12-HPETE plus FMLP, while several radiolabeled compounds, including LTB4 and 5-HETE, were released by A23187. These findings demonstrate that LTB4 biosynthesis by 12-HPETE-stimulated neutrophils requires free arachidonic acid which may be endogenous or exogenous.

Decreased plasma prostacyclin-regenerating activities in diabetics.

We assessed prostacyclin-regenerating activities (PGI2-RA) in 27 each of diabetic patients and of normal subjects by incubating plasma with "exhausted" rat aortic tissues, and compared these activities with platelet aggregability, vascular complications and the levels of fasting blood sugar (FBS) as an indicator of diabetic control. Patients' plasma showed a significantly decreased PGI2-RA when compared with normal (p less than 0.001). The activities in the patients with enhanced ADP-induced platelet aggregation in plasma or in whole blood were significantly decreased as compared with those with normal platelet functions (p less than 0.01). The patients with diabetic retinopathy showed significantly decreased PGI2-RA compared with those without this complication (p less than 0.02). A significant negative correlation was found between PGI2-RA and FBS levels before or during treatment. This abnormality found in patients' plasma appeared to be reversible and related to the degree of diabetic control. These results suggest that the decreased PGI2-RA could play a pathogenetic role together with platelet hyperaggregability in the development of microvascular and thrombotic complications in some diabetic patients.

Altered arachidonate metabolism by leukocytes and platelets in myeloproliferative disorders.

The arachidonate metabolism by leukocytes and platelets was studied in 14 patients with myeloproliferative disorders including 7 patients with chronic myeloid leukemia (CML), 5 with polycythemia vera (PV) and 2 with essential thrombocythemia (ET). When the leukocytes were incubated with arachidonate and A23187, leukotriene B4 and hydroxyeicosatetraenoic acids (HETEs) were constantly detected using reversed-phase high-performance liquid chromatography in normal subjects, while selective deficiency of 5-lipoxygenase products (leukotriene B4 and 5-HETE) was found in 4 patients with CML. this novel abnormality of the leukocytes seemed to be derived from the possible deficiency of 5-lipoxygenase in these patients' polymorphonuclear neutrophils (PMNNs). The formation of 15-HETE appeared to be almost normal in all the patients. Platelet 12-lipoxygenase deficiency was detected in 2 patients with PV and 2 with CML in whom one was associated with the deficiency of 5-lipoxygenase products. These bicellular abnormalities of the arachidonate metabolism might contribute to understand dysfunctions of PMNNs and platelets in some patients with myeloproliferative disorders.

Effects of etamsylate on platelet functions and arachidonic acid metabolism.

The effects of etamsylate on human platelet aggregation and ATP release as well as on the arachidonate metabolism by the platelet have been studied. Etamsylate enhanced these platelet functions induced by arachidonic acid (AA), thromboxane A2, collagen and calcium ionophore A23187 but not those induced by ADP and epinephrine. In experiments with cyclooxygenase-inhibited platelets, AA-induced platelet aggregation was completely inhibited and it was not enhanced by etamsylate, while A23187-induced aggregation was partially inhibited and this aggregation was enhanced by etamsylate. Platelet AA metabolism including thrombin-induced AA liberation from phospholipids as well as the lipoxygenase and cyclo-oxygenase pathways was not significantly affected by etamsylate. These results suggested that etamsylate enhanced platelet response to thromboxane A2 and calcium ionophore and that this could be included as a mechanism for its potentiating effect on platelet functions.