ON THE NATURE OF FORCES OPERATING IN BLOOD CLOTTING : I. THE PARTICIPATION OF ELECTROSTATIC ATTRACTION

The results of the study of the inhibiting effect of neutral salts upon the clotting tendency of fibrinogen by thrombin may be summarised as follows: Salts like NaCl and KCl inhibit only weakly. Salts of the same cation (K•) with monovalent anions of different ionic radius are the more active the larger the anion (Cl',Br',I'). Salts of the same cation with anions of different valency are the more active the higher the charge of the anion (1–1 <1–2 <1–3 <1–4). Salts with the same anion with cations of different valency show stronger inhibition in the case of cations of higher charge (K•,Na• < Mg••, Ca••, Sr••, Ba••). Salts with the same anion and cations of the same charge, but of different radius, are the more active the larger the cation (but with an inversion between Mg•• and Ca•• in the series of the alkali earths, which is not infrequent in biocolloids). These results show that the clotting of fibrinogen with thrombin is, at least partly, caused by a coacervation process, due to electrostatic attraction between positive and negative groups. Its nature and localisation will be dealt with in the next paper of this series

THE REACTION BETWEEN ACTOMYOSIN AND ADENOSINE TRIPHOSPHATE

1. A study is made of the effect of adenosine triphosphate (ATP) upon the viscosity of solutions of actomyosin in 0.5 M KCl. 2. The observed effects are discussed in terms of an initial drop of the viscosity (viscosity response) and its subsequent slow reversal (recovery effect). The latter is ascribed to a decrease in the ATP concentration through enzymatic hydrolysis. 3. The recovery effect is inhibited by Mg, activated by Ca, in accordance with the effect of these ions on the activity of myosin-ATPase. 4. The viscosity response is not inhibited, probably promoted by Mg. It is not promoted, probably inhibited by Ca. 5. The viscosity response is induced not only by ATP, but to a certain extent also by inosinetriphosphate, inorganic triphosphate, and inorganic pyrophosphate, not by adenosine diphosphate or monophosphate. 6. The viscosity response could be obtained with enzymatically inactive myosin. 7. It is concluded that the effect of ATP upon myosin does not depend on its enzymatic hydrolysis

ON THE NATURE OF FORCES OPERATING IN BLOOD CLOTTING : II. THE CLOTTING OF FIBRINOGEN AS A TWO-STEP REACTION

It is found that clotting of fibrinogen by thrombin does not occur on the acid side of the isoelectric point of the fibrinogen. At such pH values, however, a primary reaction between thrombin and fibrinogen takes place, leading to the formation of profibrin, a compound of thrombin and fibrinogen. At pH values at which clotting is possible, fibrinogen is negatively, thrombin positively charged, whereas profibrin has a pattern of positive and negative charges. The primary reaction, the formation of profibrin by combination of thrombin and fibrinogen, is inhibited by urea but not by neutral salts. The combination of thrombin with fibrinogen most probably takes place by hydrogen bonds. The second reaction, the polymerisation of profibrin to fibrin, is inhibited by neutral salts in the same way as complex or autocomplex coacervates. It is caused therefore by electrostatic attraction between the positive and the negative charges of the profibrin

The Role of Calcium Ions in the Acceleration of Resting Muscle Glycolysis by Extracellular Potassium

The activation of the glycolysis of resting muscle by increased extracellular potassium is dependent upon the simultaneous presence of calcium, but not of sodium ions. This regulation of metabolism by a membrane characteristic seems to act upon an early link in the glycolytic enzyme chain

A STUDY OF THE ADENOSINE TRIPHOSPHATASE ACTIVITY OF MYOSIN AND ACTOMYOSIN

1. An experimental study was made on the adenosine triphosphatase action of crystalline myosin and actomyosin preparations under different conditions. 2. No enzymatic activity was found in the absence of salts. Activation was given by KCl and CaCl2, whereas MgCl2 in the presence of other ions inhibited. 3. The effect of pH is complex. In stabilizing buffers or at low temperature, there are two optima (pH 6.2 to 6.5 and pH 9.2) provided Ca is present. Without Ca only the acid optimum is found. The highest activities are reached in glycine buffer at pH 9.2 in the presence of Ca. 4. The study of the Mg-Ca antagonism revealed that the inhibition due to Mg is fully developed with Mg:Ca ratios less than 1, the inhibition usually exceeding 90 per cent. 5. It is shown that in the muscle the myosin-ATPase is most probably also subjected to the inhibitory action of the Mg ions. 6. From data in the literature it is calculated that the liberation of inorganic phosphate during muscular activity takes place at a rate of at least 0.200 mg. P per mg. myosin per minute. 7. From the results of the present study it is found that the myosin in the muscle can liberate inorganic phosphate from ATP at a rate of at most 0.003 mg. P per mg. myosin per minute. 8. It is concluded therefore that myosin-ATPase cannot be responsible for the liberation of the main part of the phosphate in contracting muscle, and therefore cannot have the rôle in muscular metabolism ascribed to it in recent hypotheses and discussions

THE EFFECT UPON ACTOMYOSIN OF STOICHIOMETRIC AMOUNTS OF ADENOSINETRIPHOSPHATE REGENERATED IN A COUPLED ENZYME SYSTEM

Pyruvate kinase and phosphoenolpyruvate, added to actomyosin, cause a maintenance of the response of the actomyosin to stoichiometric amounts of ATP. This steady state maintenance depends on the presence of Mg ions

The Accumulation of Calcium Ions by Sarcotubular Vesicles

The accumulation of Ca++ by microsomal (sarcotubular) preparations of rabbit skeletal muscle in the presence of oxalate, and the concurrent splitting of nucleoside triphosphate, displayed moderate nucleotide specificity in the sequence ATP > GTP, CTP, ITP > UTP > (ADP) > ATetraP for the former, ATP > (ADP) > ITP > GTP > CTP > UTP > ATetraP for the latter process. The "calcium pump" was weakly inhibited by caffeine, and was inhibited together with the ATPase by pyridoxalphosphate. Carnosine had no effect as such nor in the presence of pyridoxalphosphate except at high concentration; thiourea and p-chloromercuribenzoate were inhibiting while iodoacetate was inactive. Ca++ accumulation and ATPase were inhibited by atabrine (not tested on ATPase), dinitrophenol, and amytal. High concentrations of oligomycin and rutamycin inhibited Ca++ uptake while slightly stimulating ATPase. Antimycin A stimulated the Ca++ uptake. These results are discussed in the light of their possible relation to partial reactions in oxidative phosphorylation. The Ca++ uptake and relaxing factor activities did not behave identically throughout. This is in part ascribed to changes in reactivity of actomyosin in the relaxation test, in part to the participation of relaxing substances other than the calcium pump

A STUDY OF INOTROPIC MECHANISMS IN THE PAPILLARY MUSCLE PREPARATION

The length-tension diagram, the force-velocity relation, the characteristics of the series elasticity, and the duration of the active state have been studied on the papillary muscle preparation of the cat heart, and on other examples of cardiac muscle. Positive inotropic changes such as the staircase phenomenon and post-extrasystolic potentiation occur without lengthening, but frequently with shortening, of the duration of the active state. They are accompanied by an increased velocity of contraction, and may be caused either by an intensification of the active state or by an alteration of the force-velocity characteristics of the contractile component. The changes in the force-velocity relation point to an adaptation of the velocity-efficiency relation in dependence on the frequency of contraction

Energetics of Shortening Muscles in Twitches and Tetanic Contractions : II. Force-Determined Shortening Heat

The extra heat liberation accompanying muscular shortening, the force-determined shortening heat, is defined as the difference between the heat produced when shortening occurs and that produced in an isometric contraction developing the same amount of force and performing the same amount of internal work. Based on this definition, the initial energy production in twitches and tetanic contractions (E) is given by E = A + f (P, t) + αFx + W, where A is the activation heat, f(P, t), the tension-related heat (a heat production associated with the development and maintenance of tension), αFx, the force-determined shortening heat, and W, the external work. It is demonstrated that this equation accurately accounts for the time-course of heat evolution and the total initial energy production in both twitches and tetani at 0°C. The force-determined shortening heat is liberated, during shortening, in direct proportion to (a) the distance shortened, and (b) the force against which shortening occurs. The normalized value of the force-determined shortening heat coefficient, αF/Po, is the same in both the twitch and the tetanus. Finally, this formulation of the muscle's energy production also accounts for the total energy production in afterload isotonic twitches at 20°C, where a Fenn effect is not demonstrable

Inotropic Effects of Trains of Impulses Applied during the Contraction of Cardiac Muscle

The application of a train of supramaximal stimuli during the absolute refractory period of a cardiac muscle preparation has two effects: a depression of the contraction during which it is applied, and a large potentiation of subsequent contractions. The former is ascribed to a direct effect upon the cell membrane, and is an indication of the continued control of the contractile event by this membrane. The latter is explained as a sudden liberation of norepinephrine by a stimulation of embedded nerve elements, which norepinephrine then distributes itself through the tissue and finally diffuses away