Thrombosthenin M

Cohen, Isaac; Bohak, Zvi; VriesA, De; Katchalski, Ephraim

doi:10.1111/j.1432-1033.1969.tb00702.x

Cited by 80 publications

(54 citation statements)

References 27 publications

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“…Cohen et al (2) suggested that thrombin may digest thrombosthenin M when it is still bound to the platelet membrane and Baenziger et al (16) described a thrombin-sensitive protein in platelet membranes with a subunit molecular weight of 190,000. Further careful work will be necessary before deciding if thrombin can digest platelet myosin, either on or off the membrane, and what the physiological significance of such a reaction might be.…”

Section: Resultsmentioning

confidence: 99%

Isolation and Characterization of Myosin and Two Myosin Fragments from Human Blood Platelets

Adelstein

Pollard

Kuehl

1971

Proc. Natl. Acad. Sci. U.S.A.

120

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Platelet m'4osin (thlrombosthenin Nl) and two additional proteins corresponding to the head and rod portion of the myosin molecule have been prepared from human blood platelets. Characterization of these proteins by SDS-polyacrylamide gel electrophoresis, actin binding studies, assay of enzymic ATPase activity, and electron microscopy has shown that the platelet contractile proteins closely resemble the corresponding muscle proteins. Platelet myosin and platelet myosin-head bind to both muscle and platelet actin and have an EDTA + Kstimulated ATPase activity, which is suppressed by Mg2+ in high salt concentration, whereas platelet rod does not possess either of these properties; platelet myosin and platelet myosin rod aggregate to form thick filaments at low ionic strength. Both intact platelet myosin and myosin head form typical arrowhead-shaped complexes with either platelet or muscle F-actin.Clot retraction, which is important for hemostasis in higher organisms, is mediated by the contraction of blood platelets. This contraction of platelets is thought to be caused by thrombosthenin, a complex of contractile proteins that shares many imlortalit properties with muscle actomyosin (1).Thromobosthenin. has been separated into two fractions that have the properties of actin (thrombosthenin A) and myosin (thromobosthenin AI), aind that form functional hybrids with muscle actin or myosin (1). Several attempts have been made to purify the myosin-like component of thrombostenin, but the resulting enzyme has had extremely low specific activity compared with muscle myosin and has not been tested for the functional properties that characterize rnyosin: ability to bind to tactin and to interact enzymically with actin (2, 3).We have developed methods for fractionating human thrombosthenin, and have identified and partially characterized intact platelet myosin, platelet actin, and two additional proteins, which correspond to the head and rod portions of the platelet myosin. Characterization of these proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, ATPase assay (ATP phosphohydrolase EC 3.6.1.3), actin binding studies, and electron microscopy has shown. that the platelet contractile proteins closely resemble the corresponding muscle proteins in many of their properties. MATERIALS AND METHODSPlatelets. Human platelet concentrates 1-to 5-days-old were obtained from the NIH Blood Bank, pooled, washed three times in 10 volumes of 0.9%o NaCl-0.3%O sodium citrate, 3 mMI dithiothreitol (DTT), and 1 mMi EDTA, and sedimented each time at 27,000 X g. After the third centrifugation, the pellets of platelets were stored at -15'C for up to 6 weeks before use. In one case, the platelets were stored in 50% glycerol at -15'C, without any significant difference in the results obtained. The washed platelets were essentially free from leucocyte and erythrocyte contamination. Purification proceduresAll steps were performed at 2-40C; deionized water was used throughout the investigation.

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Section: Resultsmentioning

confidence: 99%

Isolation and Characterization of Myosin and Two Myosin Fragments from Human Blood Platelets

Adelstein

Pollard

Kuehl

1971

Proc. Natl. Acad. Sci. U.S.A.

120

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“…4. This extract also possesses calcium-activated ATPase activity, which in platelets has been ascribed (10) Fig. 2. intact platelets, nor did the thrombin-sensitive band in the pyrophosphate extract disappear after incubation up to 1 hr and thrombin levels to 3 U/ml.…”

Section: Extraction Of Thrombin-sensitive Proteinmentioning

confidence: 95%

“…Sialic acid was assayed according to Warren (9). Membranes were extracted with pyrophosphate buffer by the method of Cohen et al (10).…”

mentioning

confidence: 99%

A Thrombin-Sensitive Protein of Human Platelet Membranes

Baenziger

Brodie

Majerus

1971

Proc. Natl. Acad. Sci. U.S.A.

323

201

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The action of thrombin on intact human platelets has been studied with the aid of polyacrylamide gel electrophoresis in sodium dodecylsulfate. A single major membrane protein band with a molecular weight of 190,000 disappears after thrombin treatment, while a new membrane protein with a molecular weight of 107,000 appears. This may represent hydrolysis of the thrombinsensitive protein. When platelets are disrupted or when the thrombin-sensitive protein is solubilized from membranes prior to thrombin treatment, no hydrolysis occurs. The effect of thrombin on the platelet membrane protein is complete within 2 min which suggests that hydrolysis of this membrane protein may trigger the physiological effects of thrombin on platelets.Platelets are anucleate circulating particles, with a diameter of 2-4 Mum in man, which function in hemostasis. These "cells" are metabolically active and catalyze the formation of fatty acids, phospholipids, glycogen, and even protein (1). Both glycolytic and Krebs cycle oxidative pathways are active in these cells.Blood platelets are rapidly converted from free floating cells to an aggregated mass of intact platelets during the process of hemostasis or of thrombus formation. This obvious change in the platelet surface is accompanied by a variety of morphological and biochemical changes which have been termed "viscous metamorphosis" (1). The most potent agent known to induce this process is the proteolytic enzyme thrombin, which is generated from reactions of the coagulation pathway. Characterization of the role of thrombin in producing viscous metamorphosis is integral to understanding thrombus formation and may also provide some insight into the membrane specification of cell-cell interactions.Previous work done in our laboratory (2) has shown that thrombin causes a 7-fold increase in the rate of incorporation of glycerol into phosphatidyl serine in the platelet membrane. This burst in the rate of lipid synthesis is maximal within 2 min of the addition of thrombin and such synthesis returns to the pre-thrombin rate by 20 min. Studies of the mechanism of this thrombin effect indicate that proteolysis is involved, since the effect can be mimicked by trypsin and inhibited by prior incubation of trypsin with soybean trypsin inhibitor. Human platelets, collected and isolated as described previously (2), were washed twice in a pH 6.5 isotonic buffer containing 0.113 M NaCl, 0.0043 M K2HPO4, 0.0043 M\I Na2HPO4, 0.0244 M NaH2PO4, and 1 mg/ml glucose. After washing, l)latlets were resuspended in either 0.154 M NaCl-0.154 A Tris HCl (pH 7.4) 9:1 with 1 mg/ml glucose, or in a pH 7.5 buffer containing 0.109 M NaCl, 0.043 M K2HP04, 0.0106 M Na2HPO4, 0.0083 MI NaH2PO4, and 1 mg/ml glucose.Platelets were incubated at 370C at 1-2 X 109 cells/ml in resuspension buffer with or without 1 U/ml thrombin and 2.5 mM CaCl2. The cells were disrupted by no-clearance-pestle homogenization (5), glycerol loading, and hypotonic lysis (6) Samples were solubilized in 1-3%o SDS-1%o 0-mercaptoethanol -0.1 AM so...

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“…The labeling pattern of solubilized 14C-epinephrine labelled membranes, showed that a protein located in the platelet "myosin region" (1,12) and the Schiff-positive, 155,000 glycoprotein I in the area of the glycoprotein I "complex," have the selective ability to firmly bind epinephrine.…”

Section: Resultsmentioning

confidence: 99%

<SUP>14</SUP>C-Epinephrine Interaction with Platelet Membrane Binding Sites

Vainer

1977

Cell Struct. Funct.

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Thrombosthenin M

Cited by 80 publications

References 27 publications

Isolation and Characterization of Myosin and Two Myosin Fragments from Human Blood Platelets

Isolation and Characterization of Myosin and Two Myosin Fragments from Human Blood Platelets

A Thrombin-Sensitive Protein of Human Platelet Membranes

<SUP>14</SUP>C-Epinephrine Interaction with Platelet Membrane Binding Sites

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