Relaxing Factor in Extracts of Blood Platelets and its Function in the Cells

Grette, K.

doi:10.1038/198488a0

Cited by 35 publications

(7 citation statements)

References 7 publications

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“…The following day the platelets were centrifuged in a graduated conical centrifuge tube in a GLC centrifuge at 1400 g for 30 min. The pellets were resuspended in 3 vol of homogenizing medium (30 mM KCI, 5 mM MgC12, 10 mM potassium oxalate, and 20 mM Tris-HCI, pH 7.0) (Grette, 1963). 1 ml portions were homogenized in a smooth-walled glass tube with a motor-driven teflon pestle (Arthur H. Thomas Co., Philadelphia, Pa., Cat.…”

Section: Preparation Of Platelet Fractionsmentioning

confidence: 99%

Calcium Uptake and Associated Adenosine Triphosphatase Activity of Isolated Platelet Membranes

Robblee

Shepro

Belamarich

1973

The Journal of General Physiology

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A platelet subcellular fraction, sedimenting between 14,000 and 40,000 g and consisting primarily of membrane vesicles, accumulates up to 200-400 nmoles calcium/mg protein in the presence of ATP and oxalate. Steady-state levels of calcium accumulation are attained in 40-60 min. Calcium uptake requires adenosine triphosphate (ATP), is enhanced by oxalate, and is accompanied by the release of inorganic phosphate. Calcium accumulation and phosphate release require magnesium and are inhibited by Salyrgan (10 /UM) and adenosine diphosphate (ADP) (1 mM), but not by ouabain (0.1 mM). The ATPase activity is stimulated by low concentrations of calcium (5-10 JUM) and is inhibited by 2 mM EGTA. Electron microscopic histochemistry using lead nitrate to precipitate released phosphate results in lead precipitates localized primarily at the inner surface of membrane vesicles. These results provide evidence for a membrane ATPase that is stimulated by low concentrations of calcium and may be involved in the transport of calcium across the membrane. It is postulated that the observed calcium uptake activity is an in vitro manifestation of a calcium extrusion pump in the intact platelet.

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Section: Preparation Of Platelet Fractionsmentioning

confidence: 99%

Calcium Uptake and Associated Adenosine Triphosphatase Activity of Isolated Platelet Membranes

Robblee

Shepro

Belamarich

1973

The Journal of General Physiology

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“…DISCUSSION Although contractile properties have been attributed to many vertebrate cells, apart from muscle, only platelets have so far been shown to contain an actomyosin-like protein. Moreover, it has been claimed on the basis of physicochemical studies that both actin and myosin can be distinguished in platelets (13) and that even a relaxing principle may play a role (30). With improvement in fixation, embedding, and other electron microscopic techniques thin fibrils resembling those in smooth FIGURE 2 Details of glycerinated platelets which have been reacted with HMM.…”

Section: Methodsmentioning

confidence: 99%

The Actin and Myosin Filaments of Human and Bovine Blood Platelets

Zucker‐Franklin¹,

Grusky²

1972

J. Clin. Invest.

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A B S T R A C T The contractility of platelets has been attributed to an actomyosin-like protein which has been well defined on a physicochemical basis. Moreover, platelets contain ±80 A filaments which resemble actin filaments in smooth muscle. Studies were undertaken on human and bovine platelets to better define the morphologic structures which may subserve this contractile function. In order to identify actin, the ability of the filaments to react with heavy meromyosin (HMM) was tested. Accordingly, platelets were glycerinated and treated with HMM. In addition, platelet actin was extracted, reacted with HMM, and examined by negative staining. In both instances typical arrowhead structures with clearly defined polarity and a periodicity of ±360 A formed. As is the case with purified muscle actin, the complexes were dissociable with Mg-ATP. The formation of myosin-like filaments was observed when osmotically shocked platelets were incubated with MgCla and excess ATP. These "thick" filaments measured 250-300 A in width, tapered at both ends and often occurred in clumps. They resembled aggregates of thick filaments described in contracted smooth muscle. Extraction of platelets by methods suitable for the demonstration of myosin showed filaments with an average length of 0.3 /, a smooth shaft, and frayed or bulbous ends. These appeared identical to those seen in synthetically prepared myosin of striated muscle. It is suggested that the filaments described here represent the actin and myosin of platelets.

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“…These changes include pseudopod formation, movement of granules, increased metabolism, and the specific release of certain platelet constituents such as adenine nucleotides, calcium, 5-hydroxytryptamine, and certain enzymes (Holmsen et at., 1969). Release of calcium (Murer, 1969) is of particular interest since comparisons with other secretory cells (Douglas, 1968; Stormorken, 1969) and limited experimental evidence (Grette, 1962;Sneddon, 1972) suggest that Ca2+ may be the agent that mediates other changes.The most potent physiological stimulator of platelets is thrombin, the enzyme that catalyzes conversion of fibrinogen to fibrin as well as other reactions in the overall process of blood coagulation. Thrombin is one of a class of proteolytic enzymes that are distinguishable by an active serine believed to function as a nucleophilic catalyst in the hydrolysis of peptides (for review, see Magnussion, 1971).…”

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Kinetics of the thrombin-induced release of calcium(II) by platelets

Detwiler¹,

Feinman²

1973

Biochemistry

168

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The kinetics of the thrombin-induced release of Ca2+ from human blood platelets have been followed using the calcium-complexing dye murexide with a dual-wavelength spectrophotometer. The observed reaction shows three phases, a lag followed by an exponential phase and a final slow zero-order change. From the dependence of the kinetic parameters on concentration, at least four steps must be proposed for the reaction: (i) a rapid formation of a thrombinplatelet complex, (ii) a first-order transformation of the complex, (iii) a slower first-order release of Ca2+, and (iv) slow turnover of thrombin leading to release of additional Ca2+. P JL latelets are anucleate blood cells that play crucial roles in hemostasis and thrombosis by forming compact, adhesive aggregates (for reviews, see Johnson, 1971, Marcus, 1969, and Mustard and Packham, 1970. A variety of physiological agents, such as thrombin, collagen, and ADP, induce the profound morphological and biochemical changes in platelets that lead ultimately to aggregation and contraction of the aggregate. These changes include pseudopod formation, movement of granules, increased metabolism, and the specific release of certain platelet constituents such as adenine nucleotides, calcium, 5-hydroxytryptamine, and certain enzymes (Holmsen et at., 1969). Release of calcium (Murer, 1969) is of particular interest since comparisons with other secretory cells (Douglas, 1968; Stormorken, 1969) and limited experimental evidence (Grette, 1962;Sneddon, 1972) suggest that Ca2+ may be the agent that mediates other changes.The most potent physiological stimulator of platelets is thrombin, the enzyme that catalyzes conversion of fibrinogen to fibrin as well as other reactions in the overall process of blood coagulation. Thrombin is one of a class of proteolytic enzymes that are distinguishable by an active serine believed to function as a nucleophilic catalyst in the hydrolysis of peptides (for review, see Magnussion, 1971). It is similar to trypsin, with specificity for arginyl and lysyl peptide and ester bonds. However, thrombin has a much higher degree of specificity than trypsin. For example, thrombin will hydrolyze only 4 out of 100 or so trypsin-sensitive bonds in fibrinogen. Like other serine proteases, thrombin is inhibited by DFP, a reagent that forms a covalent, inactive diisopropylphosphoryl derivative (DIP-enzyme)* 1 by reacting with the active serine.The mechanism by which thrombin modifies platelets is not known. The fact that the reaction is observed with trypsin but not with DIP-thrombin (Davey and Luscher, 1967

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Relaxing Factor in Extracts of Blood Platelets and its Function in the Cells

Cited by 35 publications

References 7 publications

Calcium Uptake and Associated Adenosine Triphosphatase Activity of Isolated Platelet Membranes

Calcium Uptake and Associated Adenosine Triphosphatase Activity of Isolated Platelet Membranes

The Actin and Myosin Filaments of Human and Bovine Blood Platelets

Kinetics of the thrombin-induced release of calcium(II) by platelets

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