Thrombosthenin

Actin and myosin molecules, and thrombosthenin, are contractile proteins that enable platelets to contract. 

Thrombosthenin, the Contractile Protein from Blood Platelets and Its Relation to Other Contractile Proteins M. Bettex-Galland and E. F. LOscher... 

THROMBOSTHENIN, THE CONTRACTILE PROTEIN FROM BLOOD PLATELETS AND ITS RELATION TO OTHER CONTRACTILE PROTEINS... 

F. Thrombosthenin, the Contractile Protein from Blood Platelets... 

This review will deal primarily with this contractile protein from human blood platelets, which we have named thrombosthenin in view of its origin and function. A brief review on blood platelets in general and their role in hemostatis will be included. The role of thrombosthenin for platelet function will be discussed and its properties compared with those of other contractile proteins. 

In 1959 Bettex-Galland and Liischer succeeded in extracting from human blood platelets such a (iontractile protein, which was subsecpiently named thrombosthenin. Its solubility properties, as well as its dependence for activity on the presence of ATP and metal ions, soon led to its classification as a member of the actomyosin group. Work on thrombosthenin has since continued, both with respect to its properties as a complex protein with enzymatic activity and to its biological significance. 

Thrombosthenin is extracted from a concentrated suspension of washed blood platelets obtained by any of the described methods of isolation (Maupin, 1954b). Bettex-Galland and Liischer (1961), starting with 50 liters of freshly (collected citrated human blood isolated by differential centrifugation in the (told, from the buffy layers, 20 to 30 ml of a highly concentrated suspension of washed platelets. Since the isolation of thrombosthenin is based on its solubility properties, special ce,re must be taken to eliminate the leucocytes their content of deoxyribonuclcoproteids... 

Thrombosthenin from Pig Platelets. The method described by Grette (1962) for the extraction of the contractile protein from pig platelets uses butanol for the lysis of platelets, and the precipitation is effected at an ionic strength of 0.2 y in the presence of Mg++ ions. 

It is noteworthy that, in spite of the differences in origin and in experimental procedure, the yield and the (Karacteristic properties of the active material from pig platelets are the same as for thrombosthenin of human origin. 

The presence of ATP leads to an increased solubility at lower ionic strength. Grette (1962) reports that solutions of porcine thrombosthenin tend toward precipitation at an ionic strength of only 0.08 g provided 5 X 10 M ATP is present. Perhaps it is not correct to speak of a solubilizing effect of ATP on thrombosthenin more likely this seemingly increased... 

Lowering the pH also leads to a decreased solubility of thrombosthenin. Grette (1962) obtained precipitation of porcine thrombosthenin at pH 6.5. The addition of small amounts of Mg++ ions seems to enhance the precipitating effect. Care must be taken, however, because even at pH 6 signs of progressive denaturation become discernible. It seems preferable for preparative purposes to work at a neutral pH. 

At an ionic strength of about 0.1 g with Mg++ ions and ATP present, thrombosthenin forms a flocculant precipitate which contracts rapidly to a smaller volume. Since the contracting units in the precipitate are randomly arranged, the magnitude of contraction is the same in every direction. The result is a small contracted pellet, still showing the con-... 

This phenomenon, termed superprecipitation by Szent-Gyorgyi (1951), is typical for the contractile proteins of the actomyosin group. It becomes even more spectacular if concentrated solutions of thrombosthenin are spun out to small fibers and allowed to shrink (Bettex-Galland and Luscher, 1961). Precipitates obtained from dilute solutions are not stable enough to show macroscopically visible contraction. In this case a fine granular... 

Fig. 4. Supcrijrecipitation of thrombosthenin at 20°C. Upper series shows the contraction of precipitated thrombosthenin under the presence of 10 M ATP. Lower series sedimentation of precipitate without added ATP. From Bettex-Galland and Luscher (1961).

The speed of the contraction of a superprecipitate is greatly dependent upon temperature. For thrombosthenin at room temperature (about 20°C) 15 to 20 min are required for complete contraction. At 37°C this process requires only 1 to 2 min. At 0°C superprecipitation does not take place. Accurate and comparable measurements of the contraction rates... 

Fig. 5, Enzymatic cleavage of ATP by thrombosthenin. Left half of picture shows chromatogram of nucleotides after incubation of ATP with thrombosthenin. ADP has been formed at the expense of ATP. AMP ha.s remained unchanged. Right half of pictures shows chromatogram obtained prior to incubation. Numbers on to ) of curves give KHCOa concentrations used in elution of nucleotides from column. From Bettex-Galland and Liischer (1961).

At low ionic strength, Mg ions up to a concentration of 10 M increasingly activate the thrombosthenin ATPase. Still higher concentra-... 

Fig. 7. Influence of magnesium ion concentration on ATPase activity of thrombosthenin at low (left) and high ionic strength (right). ATP, 10 Af temperature, 20°C. From Beltex-Galland and Luscher (1961).

This difference in the effects of the two metal ions explains why the ATP loss suffered by the blood platelets in model systems containing only Mg++ ions is not as pronounced as the disappearance of the nucleotide during normal blood coagulation (Born, 1958). It has been mentioned before that ADP production during VM of the platelets was of biological importance. This production most likely is due mainly to the activation of the thrombosthenin ATPase by the influx of plasmatic Ca++ ions their pronounced activator role therefore appears of particular significance. 

Tephorine and Diparcol, both synthetic antihistamines known to inhibit clot retraction (Bounameaux, 1957 Hugues, 1959), only partially inhibit ATPase activity and superprecipitation of thrombosthenin. Finally, monoiodoacetate, a powerful inhibitor of clot retraction, inhibits neither phenomenon. 

The findings reported above may be summarized as follows Inhibitors of the ATPase activity of thrombosthenin always are inhibitors of superprecipitation, whereas they do not necessarily affect manifestations of the contractile activity of the whole platelet, such as clot retraction, to the same extent. 

At low ionic strengths, ATP causes the superprecipitation of thrombosthenin. Applied at higher ionic strength, i.e., under conditions where the protein is in solution, it exerts an entirely different effect—it causes the dissociation of the thrombosthenin complex into its two components. This dissociation is connected with a considerable decrease in viscosity that is easily determined quantitatively. 

With Ztj values between 0.1 and 0.2, solutions of thrombosthenin appear less viscous than comparable preparations of actomyosin obtained from striated muscle. As shown in Fig. 8, the sudden decrease in viscosity which accompanies the addition of ATP is followed by a slow rise owing to the enzymatic breakdown of the nucleotide, until the original level is almost reached again. On repeated addition of ATP the same sequence of events is observed. 

Based on such quantitative determinations of the viscosity changes induced by adding ATP to soluble thrombosthenin, the so-called ATP sensitivity of thrombosthenin has been calculated according to the method... 

Fw. 8. Influence of ATP on viscosity of thrombosthenin. Extract reprecipitated once protein concentration, 033% ionic strength, 0.6/t pH 7 temperature, 25°C. ATP is added as a 10" M solution in 0.6 M KCl. All added solutions represent 2 % of volume of extract. From Bettex-Galland and Luscher (1961). 

The changes in viscosity observed in solutions of muscle actomyosin on addition of ATP are explained by a dissociation of the actomyosin complex into its component parts, actin and myosin. Since thrombosthenin behaves similarly, it must be assumed that it too has dissociated into the corresponding components, which have been termed thrombosthenin A and thrombosthenin M, (A for actin-like, M for myosin-like) (Bettex-Galland et al., 1962, 1963a). 

Thrombosthenin M was isolated from thrombosthenin by the use of polyethenesulfonate. This compound belonging to the group of interaction inhibitors (Bdrdny and Jaisle, 1960) potentiates the dissociation at such a low ionic strength that thrombosthenin M precipitates, whereas... 

An alternative way of preparing thrombosthenin A uses the supernatant from the isolation of thrombosthenin M as a starting material. This solution is first concentrated and low molecular components are removed by gel filtration. This method has not yet been used to any great extent for preparative purposes (Bettex-Galland et al., 1963a). 

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