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Biomedical subjects

T Kokawa

Publications and source records attributed to T Kokawa.

At least 19 recordsLinked to original sources
PubMed1996

Measurement of the free form of TFPI antigen in hyperlipidemia. Relationship between free and endothelial cell-associated forms of TFPI.

Tissue factor pathway inhibitor (TFPI), a protease with three tandem Kunitz-type (K1, K2, and K3) domains, inhibits the initial reaction of the TF-mediated coagulation pathway. TFPI occurs in a free and a lipoprotein-associated form in plasma as well as an endothelial cell-associated form on vascular walls. In a previous study we had demonstrated that free-form TFPI activity was lower in hyperlipidemic patients. In the present study we established a new enzyme immunoassay method for measuring free-form TFPI antigen; this new method uses a monoclonal antibody that recognizes the K3 domain of free-form TFPI but not lipoprotein-associated TFPI. Free-form TFPI antigen was significantly lower in hyperlipidemic patients compared with those in normolipidemic individuals. We applied this new method to measure the amount of endothelial cell-associated TFPI, which can be released by heparin injection, as "free-form TFPI." We found that free-form TFPI antigen in plasma was positively correlated with the endothelial cell-associated form. These results indicate that both of these forms of TFPI are in equilibrium in vivo and that our new method can be used for assessing changes in the levels of endothelial cell-associated TFPI antigen and, hence, for assessing thrombotic tendencies in various disease states.

Adolescent↗
PubMed1995

An anti-tissue factor pathway inhibitor (TFPI) monoclonal antibody recognized the third Kunitz domain (K3) of free-form TFPI but not lipoprotein-associated forms in plasma.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor with three tandem inhibitory domains, which inhibits the initial reactions of the extrinsic blood coagulation pathway through the first and second Kunitz domains. We prepared a monoclonal antibody against recombinant human TFPI (rTFPI) and determined the epitope as the third Kunitz domain, using fragments derived from rTFPI (K1-K2 fragment and K3 fragment) and synthetic peptides. We then developed an enzyme immunoassay (EIA) method using a combination of the monoclonal antibody and a polyclonal antibody. Although TFPI activity is distributed among LDL/VLDL-associated, HDL-associated, and free forms of TFPI after gel-filtration of human plasma, only the free form was detected by the EIA method. After incubation with LDL, the antigenicity of rTFPI was reduced, but that of K3 fragment was not. Gel-filtration analysis of the mixture of radiolabeled rTFPI or K3 with LDL demonstrated that rTFPI, but not K3, bound LDL. From these results, we concluded that the monoclonal antibody against TFPI recognized only a free form of TFPI in plasma, since the epitope of lipoprotein-associated TFPI had been masked by the interaction with lipoproteins.

Amino Acid Sequence↗
PubMed1995

Tissue factor pathway inhibitor activity in human plasma. Measurement of lipoprotein-associated and free forms in hyperlipidemia.

Tissue factor pathway inhibitor (TFPI), a protease inhibitor that is present in free and lipoprotein-associated forms in plasma and that also occurs as an endothelial cell-associated form, can inhibit the initial reactions of the tissue factor-mediated coagulation pathway. Although a positive correlation between plasma TFPI activity and cholesterol concentration in human plasma has been demonstrated, levels of the various forms of TFPI, ie, the LDL/VLDL-associated form, the HDL-associated form, and the free form, have not yet been completely determined in hyperlipidemia. We therefore established a method for the measurement of each of these forms of TFPI in plasma by gel filtration of plasma in buffer containing 1 mol/L NaCl. The recovery of TFPI activity in the free form was markedly greater as assessed by the new method than the recovery reported when other methods have been used. We employed the new method to analyze TFPI activity in 19 hyperlipidemic patients and compared the results with those for normal control subjects. The level of LDL/VLDL-associated TFPI in hyperlipidemic patients was significantly increased compared with control subjects' levels (0.383 +/- 0.112 versus 0.237 +/- 0.077 U/mL), whereas the level of the free form of TFPI in hyperlipidemic patients was significantly decreased (0.381 +/- 0.132 versus 0.495 +/- 0.106 U/mL), the former being positively correlated with cholesterol level, while the latter was negatively correlated.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1995

Decreased plasma tissue factor pathway inhibitor activity in ischemic stroke patients.

Although tissue factor pathway inhibitor (TFPI) plays an essential role in the regulation of blood coagulation, the quantitative changes in its levels in thrombotic disease are still undefined. We compared TFPI activity in ischemic stroke patients and control subjects matched for age and cholesterol level to determine whether TFPI activity is changed in the disease. TFPI activity was significantly lower in the stroke patients (1.01 +/- 0.24 U/ml) than in the control subjects (1.10 +/- 0.16 U/ml). In relation to clinical subtypes of stroke, TFPI activity in atherothrombotic infarction (0.93 +/- 0.19 U/ml) and lacunar infarction (0.99 +/- 0.23 U/ml) was significantly lower than in the control subjects, whereas the level in cardioembolic infarction (1.16 +/- 0.31 U/ml) was not. No relationship could be established between TFPI activity and other haemostatic parameters reflecting the production of thrombin/fibrin and the activation of fibrinolysis. These results may suggest that the moderately lower TFPI activity in stroke patients could be due to atherosclerotic changes rather than to consumptive coagulopathy.

Age Factors↗
PubMed1994

Effect of three Japanese kampo medicines on platelet activation by monoclonal anti-platelet membrane glycoprotein antibodies.

We studied the effect of three Japanese kampo medicines on platelet activation by an anti-CD9 monoclonal antibody (NNKY1-19) and an anti-human Fc gamma receptor II monoclonal antibody (NNKY3-2). Sho-saiko-to (TJ-9) and Sairei-to (TJ-114) partially suppressed platelet aggregation induced by NNKY1-19, while Juzen-taiho-to (TJ-48) suppressed aggregation induced by NNKY3-2. TJ-9 and TJ-114 also suppressed collagen-induced aggregation, but TJ-48 did not. Flow cytometry showed that the three medicines did not affect antibody binding to the platelets. Thus, all three kampo medicines suppressed platelet activation by anti-platelet glycoprotein antibodies without inhibiting antibody binding.

Antibodies, Monoclonal↗
PubMed1993

Flow cytometric analysis of changes in cytoskeletal proteins during platelet destruction and activation using a monoclonal antibody against platelet myosin.

We developed a new monoclonal antibody directed against platelet myosin (NNKY6-19). Using this antibody, we analyzed platelet cytoskeletal changes related to stimulation with thrombin and to long-term storage. Immunoelectron microscopy showed increased binding of NNKY6-19 to pseudopods and the open canalicular system during treatment with thrombin (0.1 U/ml) and during storage for 7 days. Flow cytometry also showed increased binding to platelets by NNKY6-19 and an antiactin monoclonal antibody during storage. The binding of NNKY6-19 showed an increase greater than that with the antiactin antibody after storage of platelets for 7 days and after thrombin treatment. These findings indicated that the increased binding of NNKY6-19 had some relationship to changes in intracellular myosin and platelet morphology. Thus use of NNKY6-19 allowed analysis of subtle changes related to platelet activation, which differed from those detected by antibodies against platelet glycoproteins or by the antiactin antibody. This antibody appears to provide a simple method for studying changes in platelet cytoskeletal and surface proteins.

Actins↗
PubMed1993

Platelet activation induced by an antiplatelet autoantibody against CD9 antigen and its inhibition by another autoantibody in immune thrombocytopenic purpura.

In a patient with immune thrombocytopenic purpura (ITP), we found a novel platelet-activating IgG (act-IgG) and an inhibitory IgG (inhi-IgG) that prevented activation induced by both CD9 monoclonal antibody (mAb) and the act-IgG. Purified IgG from the patient plasma caused a rise in [Ca2+]i and the aggregation of normal platelets, and bound to a 24 kD membrane protein. This aggregation was inhibited by aspirin, staurosporine, an inhibitor of protein kinase C, and F(ab')2 fragments of MALL13, a CD9 mAb. When the platelet count of this patient rose to normal range, the act-IgG disappeared. About 2 weeks later, the relapse of thrombocytopenia was observed. The purified IgG obtained in this period did not activate platelets but inhibited both the rise in [Ca2+]i and platelet aggregation stimulated by NNKY 1-19, a CD9 mAb, as well as the act-IgG, and bound to a 40 kD membrane protein. The inhi-IgG prevented the binding of IV-3, a mAb against Fc gamma receptor II (Fc gamma RII), but did not prevent the binding of NNKY 1-19 to its antigen. We suggest that the activating autoantibody recognized CD9 antigen and activated both the thromboxane- and phospholipase C-dependent pathways, while the inhibitory autoantibody recognized the Fc gamma RII and inhibited CD9 antibody-induced platelet activation mediated via this receptor.

Adult↗
PubMed1993

Detection of platelet antigen for antiplatelet antibodies in idiopathic thrombocytopenic purpura by flow cytometry, antigen-capture ELISA, and immunoblotting: a comparative study.

We compared three methods of detecting platelet antigens for antiplatelet antibodies in patients with idiopathic thrombocytopenic purpura (ITP), i.e., a microtiter well antigen-capture enzyme-linked immunosorbent assay (AC-ELISA), a platelet suspension immunofluorescence test using flow cytometry (PSIFT-FCM), and Western blotting. Using PSIFT-FCM, the reactivity of NNKY1-32, an anti-glycoprotein (GP) IIb/IIIa antibody, and of NNKY5-5 (anti-GPIb) to platelets from 60 ITP patients were examined. By PSIFT-FCM, both the peak channel and the relative fluorescence value were below the mean-2SD for healthy control platelets in 15 patients when NNKY1-32 was used and in 2 patients when NNKY5-5 was used. Western blotting gave an apparent molecular weight for GPIb of 160,000, while GPIIb was 135,000 and GPIIIa was 88,000. By the AC-ELISA, 12 patients were positive for NNKY1-32 and 4 for NNKY5-5. Although NNKY1-32 binding was detected by PSIFT-FCM in 15 of the ITP patients using platelets, only 3 were positive using plasma. By AC-ELISA and Western blotting of plasma, 12 and 10 of the patients were positive for NNKY1-32 and NNKY5-5, respectively. Our results suggest that none of the three methods is good enough to stand alone and that they should be used together in the analysis of platelet antigens for antiplatelet antibodies in ITP.

Autoantibodies↗
PubMed1993

Effects of kami-kihi-to (jia-wei-gui-pi-tang) on autoantibodies in patients with chronic immune thrombocytopenic purpura.

We studied the effect of Kami-kihi-to (Jia-Wei-Gui-Pi-Tang) on the production of autoantibodies in ten patients with chronic immune thrombocytopenic purpura. After administration of Kami-kihi-to, platelet count was increased in seven of the ten patients (p < 0.05). Using Western blotting, we demonstrated the disappearance of autoantibody reaction with antigen in one patient. However, platelet-associated IgG was decreased in eight of ten patients (p < 0.05). Kami-kihi-to appears to promote the suppression of autoantibodies in patients with chronic immune thrombocytopenic purpura. No side effects were observed in any patient. Thus, Kami-kihi-to may be a useful and safe drug in the management of chronic immune thrombocytopenia purpura.

Administration, Oral↗
PubMed1993

Periodic production of antiplatelet autoantibody directed against GPIIIa in cyclic thrombocytopenia.

We report here a female patient with cyclic thrombocytopenia associated with antiplatelet autoantibodies. There was an inverse relationship between the level of platelet-associated IgG and platelet count. Bone marrow megakaryocytes were normal in number even during the thrombocytopenia. The binding of monoclonal antibodies (mAbs) against glycoprotein (GP) IIb/IIIa to patient platelets was significantly inhibited in the thrombocytopenic phase, while these mAbs normally bound to patient platelets obtained during the normal platelet count. Western blotting and mAb-specific immobilization of platelet antigens showed that both plasma autoantibody and the eluted IgG from the patient platelets bound to GPIIIa. These results suggest that the periodic production of antiplatelet autoantibody against GPIIIa caused cyclic destruction of platelets in this patient.

Adult↗
PubMed1993

Flow cytometric analysis of surface membrane proteins on activated platelets and platelet-derived microparticles from healthy and thrombasthenic individuals.

We used flow cytometry to investigate surface membrane protein expression by platelets and platelet-derived microparticles from normal individuals and a patient with Glanzmann's thrombasthenia. Microparticles were detected by both forward scatter and side scatter using FACScan. The binding of coagulation factors on microparticles was investigated by using monoclonal anti-Factor IX (IXa) and anti-Factor X (Xa) antibodies. Furthermore, the procoagulant activity of microparticles was measured with a chromogenic substrate (S-2222) using a microtiter enzyme-linked immunosorbent assay. Both types of platelets showed similar release of microparticles. Microparticles released from platelets after activation with the calcium ionophore A23187 did not bind factors IXa and Xa, but when purified factors Va and Xa were added to the incubation buffer, factor Xa binding increased markedly in both normal and thrombasthenic platelets. Both normal and thrombasthenic platelets showed a similar time-dependent release of microparticles when activated with A23187. However, the binding of an antibody to granule membrane protein-140 also increased time-dependently in normal microparticles, but was little increased in thrombasthenic microparticles. These findings suggest that glycoprotein IIb/IIIa does not participate in the expression of prothrombinase activity on the surface of activated platelets and microparticles, whereas this glycoprotein appears to have an important role in the movement of granule membrane protein-140 from platelets to microparticles.

Adult↗