Megakaryocytes (MKs) and platelet-like particles (PLPs) have generally been obtained by culturing embryonic stem (ES) cells over feeder cells. However, using feeder cells need many labor-consuming processes and the MK and PLP fractions obtained are often contaminated by such cells and their fragments. Here we describe our new culture system for differentiating mouse ES cells to MKs and PLPs without using feeder cells. ES cells are differentiated to cells with MK-like morphology and properties, including proplatelet formation, high ploidy (>8N), and CD41 expression. The culture medium contained PLPs expressing platelet glycoproteins, CD41 and GPIb. Integrin αIIbβ3 of PLPs can be activated by thrombin. Addition of the metalloproteinase inhibitor TAPI-2 to the culture increased the surface expression of GPIbα and augmented the adhesion of PLPs to immobilized von Willebrand factor through decreasing the shedding of GPIbα. Thus our mouse ES cells culture system is a suitable and efficient method for obtaining MKs and functional PLPs that obviates the need for feeder cells.

10.2739/kurumemedj.56.61

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Potentiation by collagen or epinephrine of platelet responsiveness to aggregation. The possible role of arachidonic acid released from platelet membranes

Blood & Vessel (1977)

N Yoshida Masaaki Moroi Michio Matsuda Nobuo Aoki

When human platelets in plasma were exposed to a small amount (nonaggregating concentration) of collagen, epinephrine or arachidonic acid, their responsiveness to aggregating agents were potentiated and they were aggregated by a subsequent addition of nonaggregating concentration of the stimulants. Otherwise the nonaggregating concentrations of the stimulants were uncapable to induce platelet aggregation. The potentiation of platelet responsiveness to aggregating agents was also caused by collagen- or epinephrine-treated platelet menbranes, but not by arachidonic acid-treated membranes. Furthermore, the soluble fraction of collagen- or epinephrine-treated membranes contained some material responsible for platelet potentiation, indicating that the responsible material was released from platelet membranes by collagen or epinephrine. It is suggested that the material may be arachidonic acid since the soluble fraction of collage- or epinephrine-treated membranes contained a larger amount of the precursor of prostaglandin F2α than the untreated membranes and arachidonic acid is the precursor of Prostaglandin F2α. The potentiation of platelet aggregability by the small amounts of the stimulants was increased in nephrotic syndrome whose albumin concentration in plasma is low, and the increased potentiation was abolished by the addition of albumin. The abolishment of the potentiation of platelets by albumin could be explained by postulating that some part of arachidonic acid released from membranes immediately binds to albumin and becomes unavailable to platelets.

10.2491/jjsth1970.8.254

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Characterization of a patient with glycoprotein (GP) VI deficiency possessing neither anti‐GPVI autoantibody nor genetic aberration

Journal of Thrombosis and Haemostasis (2006)

H. Kojima Masaaki Moroi Stéphanie Jung Shinya Goto Noriko Tamura

GPVI

10.1111/j.1538-7836.2006.02173.x

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Isolation and characterization of α2-plasmin inhibitor brom human plasma

Blood & Vessel (1978)

Masaaki Moroi Kazuo TATSUYA Michio Matsuda N Yoshida Nobuo Aoki

A procedure is presented for purifying a novel proteinase inhibitor in human plasma, whose apparent unique biological property is to inhibit efficiently the lysis of fibrin clot induced by plasminogen activator. The final product is homogeneous as judged by disc gel electrophoresis, sodium dodecyl sulfate gel electrophoresis, and immunoelectrophoresis. ts molecular weight estimated by sodium dodecyl sulfate gel electrophoresis or sedimentation equilibrium was 67, 000 and 63, 000, respectively. The inhibitor is a glycoprotein consisting of a single polypeptide chain containing 11.7% cabohydrate. It migrates in α2-globulin region in immunoelectrophoresis. The inhibitor is chemically and immunologically different from all the other known inhibitors in plasma.Inhibition of plasmin by the inhibitor is almost instantaneous even at 0°, in contrast to the slow inhibition of urokinase (plasminogen activator in urine). Plasminogen activation by urokinase or urokinase-induced clot lysis is inhibited by the inhibitor mainly through a mechanism of instantaneous inhibition of plasmin formed and not through the inhibition of urokinase. Consequently, this newly identified inhibitor is suggested to be named α2plasmin inhibitor or α2-proteinase inhibitor. A specific antibody directed against the inhibitor neutralizes virtually all inhibitory activity of plasma to activator-induced clot lysis. Immunochemical quantitation of the inhibitor with specific antiserum to the inhibitor and the purified inhibitor as a standard indicates that the concentration of the inhibitor in serum of healthy man is in or near the range of 5 to 7mg per 100ml, which is the lowest concentration among the concentrations of the proteinase inhibitors in plasma.The inhibitor and plasmin or urokinase form a complex which cannot be dissociated with denaturing and reducing agents. The formation of the enzyme-inhibitor complex occurs on a 1:1 molar basis. In the complex formation between the inhibitor and plasmin, the inhibitor is cross-linked with the light chain which contains the active site of plasmin.

Sodium dodecyl sulfate

Immunoelectrophoresis

10.2491/jjsth1970.9.60

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On the interaction of inhibitor and proteases Evidence for the formation of a covalent crosslinkage and non-covalent weak bondings between the inhibitor and proteases

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics (1977)

Masaaki Moroi Nobuo Aoki

Trypsin inhibitor

Sodium dodecyl sulfate

Protease inhibitor (pharmacology)

10.1016/0005-2744(77)90255-8