Kenjiro Tanoue

Monoclonal antibody to glycoprotein Ib inhibits both thrombinand ristocetin-induced platelet aggregations

A monoclonal antibody named TM60, which inhibited both thrombin- and ristocetin-induced platelet aggregations, was obtained by hybridoma technique. TM60 inhibited binding of von Willebrand factor to platelets under the presence of ristocetin. The subclass of TM60 was IgG2a. TM60 did not inhibit ADP-, collagen-A-23187-, arachidonic acid- and PAF-induced platelet aggregations, but inhibited polylysine-, polybrene- and cationized ferritin-induced platelet aggregations. ATP-release from platelets induced by thrombin was also inhibited by TM60. Immunoprecipitation and SDS-PAGE experiments demonstrated that TM60 recognized an epitope on GPIb whose molecular weight was 165,000 under non-reduced and 145,000 under reduced conditions.

PS-liposome and ox-LDL bind to different sites of the immunodominant domain (#155-183) of CD36: a study with GS95, a new anti-CD36 monoclonal antibody.

CD36, a multifunctional adhesive receptor on a variety of cells such as monocytes and platelets, has been implicated in clearance of modified LDL and in the removal of apoptotic or senescent cells. We recently developed a new anti-CD36 monoclonal antibody, GS95. We determined the binding site of phosphatidylserine (PS)-liposome on CD36 by flow cytometric analysis of competitive bindings between phospholipid-liposomes or synthetic CD36 peptides and FITC-labeled anti-CD36 antibodies (GS95, OKM5, and FA6-152). The epitope of GS95 was mapped to the amino acid sequence #162-183 of CD36 that was partially overlapped with, but distinct from, #155-183, which has been reported as the epitopes of two commercially available antibodies, OKM5 and FA6-152. Oxidized-LDL dose-dependently inhibited bindings of both GS95 and OKM5 antibodies to platelet CD36, while PS-liposome inhibited the binding of GS95 but not OKM5 or FA6-152. These results indicate that the binding site of PS-liposome on platelet CD36 is not identical to that of oxidized-LDL and may be located in the amino acid sequence #162-183.

Urinary excretion of the vitronectin receptor (integrin αVβ3) in patients with Fabry disease

Abstract A renal disorder is one of the important manifestations of Fabry disease, but the details of the pathogenesis have not been clarified yet. We examined the possibility that the vitronectin receptor (VNR, integrin α V β 3 ), one of the integrins, is involved in the progression of the renal injury in Fabry disease. We measured the urinary excretion of β 3 originating from VNR in Fabry patients by immunoblotting analysis and enzyme-linked immunosorbent assay (ELISA). Immunofluorescent microscopic analyses for VNR and globotriaosylceramide were performed on urinary sediments from Fabry patients. Furthermore, β 3 and vitronectin in kidney tissues were analyzed immunohistochemically. Immunoblotting analysis and ELISA showed that the urinary excretion of β 3 originating from VNR was significantly increased in the Fabry group compared with both the pathological and healthy control groups. Immunofluorescent microscopy revealed the expression of VNR and accumulation of globotriaosylceramide in urinary sediments from the Fabry patients. Increased expression of β 3 was observed in glomerular epithelial cells, and in Bowmans capsular epithelial layer and tubular cells, and the amount of vitronectin was moderately increased in the kidney tissues from the Fabry patients. The urinary excretion of VNR was increased, and the expression of VNR was observed in Fabry kidney tissues. The expression of VNR may be involved in the progression of the renal injury in this disease.

Activation of platelets in cancer, especially with reference to genesis of disseminated intravascular coagulation.

Seventy-five cancer patients were evaluated on a scale of coagulation abnormalities related to DIC, one point given for each of the following criteria fulfilled and the score (0 to 4) being used. 1. Platelet count less than 150 x 10(3)/mu 1. 2. PT prolonged more than 1 sec over control or APTT prolonged more than 10 sec over control. 3. Fibrinogen less than 250 mg/dl (mean fibrinogen value of the cancer patients minus 1SD). 4. FDP greater than or equal to 20 micrograms/ml. The patients were distributed with 27% for score 0, 38% for 1, 20% for 2, 7% for 3 and 8% for 4. Platelet mode volume in score 4 was smaller than that of the other groups. Platelet aggregation by epinephrine was decreased in score 3 and 4 (P less than 0.01), while it was increased in score 0 (P less than 0.05). ADP-induced aggregation was increased in score 0 and 1 (P less than 0.01 - 0.05). The mean value of beta-thromboglobulin in cancer patients (44 +/- 24 ng/ml) was significantly higher than that of control (22 +/- 13 ng/ml) (P less than 0.01). These results suggest the existence of hyperfunction of platelets in cancer patients and possibility of a triggering mechanism of such activated platelets in the genesis of DIC in cancer.

Effect of cetiedil on platelet aggregation and thromboxane synthesis

Cetiedil was found to inhibit platelet aggregation and thromboxane synthesis induced by thrombin and arachidonic acid. When platelets were activated by thrombin, half maximal inhibition (ED50 effective dose of cetiedil necessary for 50% inhibition) for platelet aggregation was 100 microM while that for thromboxane B2 (TXB2) production was 50 microM. When arachidonic acid was used, the ED50 for platelet aggregation was 100 microM while that for TXB2 production was 150 microM. The presence of calcium ions did not affect on the inhibitory effects of cetiedil. The cAMP level in platelets did not increase after incubation with cetiedil. Cetiedil appears to inhibit the activation of platelets related to thromboxane synthesis.

Appearance of platelet-clumping activity in heparinized plasma after acidification — it's mainly due to aggregation of fibrinogen

Platelet-clumping activity was found after acidifying heparinized rabbit plasma to pH 5.5 in the presence of calcium ions. We tried to isolate the activity from plasma by gel filtration under low salt and high salt condition, and we found that the activity was related to the formation of plasma protein aggregates. Immunological examination and the results of fibrin monomer affinity chromatography indicated that fibrinogen was the main component of the plasma aggregates. This phenomenon was not related to the activation of blood coagulation. The binding of fibrinogen in the presence of heparin or calcium ions at low ionic strength may induce the appearance of platelet-clumping activity.