Aggregation of platelets by muscle actin. A multivalent interaction model of platelet aggregation by ADP

Filamentous muscle actin (F-actin) aggregated blood platelets while G-actin was ineffective. This aggregation could be blocked by ATP suggesting a possible role of actin-bound ADP in this process. Actin-bound ADP caused platelet aggregation at concentrations significantly lower than equivalent concentrations of free ADP. Thus, actin potentiates the aggregating action of ADP. An actin antibody or DNase I inhibited this aggregation showing the requirement of actin in this process. Like other physiological agents, Ca⁺⁺ was necessary for platelet aggregation by actin. Platelets fixed in formaldehyde were not aggregated by actin showing the need for viable platelets. Since F-actin contains 1 mole of bound ADP/mole protein, it is postulated that actin potentiates ADP-induced aggregation by providing multiple interaction sites for platelets.     

Alkaline extraction and reverse-phase high-performance liquid chromatography of adenine and pyridine nucleotides in human platelets

The levels of adenine (ATP, ADP, AMP) and pyridine (NAD, NADH) nucleotides in human platelets have been measured by a simple and reproducible method. A rapid alkaline extraction allows a complete recovery of the compounds concerned. The metabolic ATP and ADP in the cytosolic fraction, the amount released upon thrombin stimulation, and the ADP bound to F-actin have also been evaluated. Analysis was performed by reverse-phase, isocratic high-performance liquid chromatography on a 5-microns Lichrosorb RP-18 column with uv detection at 254 nm.     

Evidence that F-actin can hydrolyze ATP independent of monomer-polymer end interactions

The rate of ATP hydrolysis in solutions of F-actin at steady state in 50 mM KC1, 0.1 mM CaC12 was inhibited by AMP and ADP. The inhibition was competitive with ATP (Km of about 600 microM) with Ki values of 9 microM for AMP and 44 microM for ADP. ATP hydrolysis was inhibited greater than 95% by 1 mM AMP. AMP had no effect on the time course of actin polymerization, ATP hydrolysis during polymerization, or the critical actin concentration. Simultaneous measurements of G-actin/F-actin subunit exchange and nucleotide exchange showed that nucleotide exchange occurred much more rapidly than subunit exchange; during the experiment over 50% of the F-actin-bound nucleotide was replaced when less than 1% of the F-actin subunits had exchanged. When AMP was present it was incorporated into the polymer, preventing incorporation of ADP from ATP in solution. F-actin with bound Mg2+ was much less sensitive to AMP than F-actin with bound Ca2+. These data provide evidence for an ATP hydrolysis cycle associated with direct exchange of F-actin-bound ADP for ATP free in solution independent of monomer-polymer end interactions. This exchange and hydrolysis of nucleotide may be enhanced when Ca2+ is bound to the F-actin protomers.     

Methodological problems of direct bioluminescent ADP assay in platelets and erythrocytes

We have developed a method for ADP bioluminescent measurement in platelets and erythrocytes which complements our previous method for ATP assay. When the different parameters of the system under investigation are taken into account, a linea range between 10(-9) and 10(-7) g/ml can be obtained without incubation or troublesome extraction. This makes the method easy and useful for identifying any disease-induced alterations in ATP and/or ADP levels in these blood cells. The data obtained correlate well with those of a bioluminescent method requiring extraction with ethanol/EDTA and incubation, giving the reference intervals of 3.5-5.5 mumol/10(11) PLT for ATP determination and 1.9-3.7 mumol/10(11) PLT for ADP determination in platelets, and 3.2-3.8 mumol/g Hgb for ATP determination and 0.56-0.73 mumol/g Hgb for ADP in erythrocytes. This assay was applied to quality control on blood bags in transfusion centers and proved to be a rapid and reliable method for testing the viability of stored blood cells.     

Determination of the ADP concentration available to participate in energy metabolism in an actin-rich cell, the platelet.

Almost all cells contain actin, which in its polymerized form, F-actin, binds 1 molecule of ADP/monomer. Little is known about the availability to metabolism of this bound ADP. A comparison was therefore made between perchloric acid and EDTA/ethanol extracts of human blood platelets. When the cells were extracted under conditions where the ATPase activity was negligible, the ethanol extracts had a 75% higher ATP/ADP ratio and a higher adenylate energy charge than perchloric acid extracts. The methods differed in that a considerable portion of protein-bound ADP was not extracted by ethanol. This bound ADP behaved as though it were unavailable to energy metabolism and should thus be considered as a compartment separate from the bulk metabolic pool of extragranular platelet adenine nucleotides. These results suggest that the level of ADP obtained with the common acid extraction overestimates the level available to participation in metabolism.     

Rapid exchange of actin-bound nucleotide in perfused rat heart

In the rat heart the actin-bound nucleotide contained both ATP and ADP. The ratio of bound ATP to bound ADP depended on the functional state of the heart; it was higher in hearts stopped reversibly in diastole (low Ca(2+), high Mg(2+), or high K(+)), than in stimulated (inotropic agents or pacing) hearts. Immunoblotting and gel electrophoresis showed the existence of G-actin (30% of total actin) in the cytoplasm of the heart. Pure actin was isolated from rat hearts: in G-actin the bound nucleotide readily exchanged with ATP or ADP, and in F-actin the bound nucleotide did not exchange with ATP or ADP. The free and bound nucleotides were separated in the intact heart by extraction with 75% methanol at -15 degrees C. In rat hearts perfused with (32)P-labeled orthophosphate the actin-bound nucleotide rapidly exchanged with the cytoplasmic ATP. The full exchange of the bound ATP was immediate, whereas the full exchange of the bound ADP was slower. The full exchange of the bound ATP was independent of the heartbeat frequency, whereas the full exchange of the bound ADP was frequency dependent. The data suggest that the transformation of actin monomer-ATP to actin polymer-ADP is a part of the normal contraction-relaxation cycle of the rat heart.     

Isolation and characterization of covalently cross-linked actin dimer

Covalently cross-linked actin dimer was isolated from rabbit skeletal muscle F-actin reacted with phenylenebismaleimide (Knight, P., and Offer, G. (1978) Biochem. J. 175, 1023-1032). The UV spectrum of the purified cross-linked actin dimer, in a nonpolymerizing buffer, was very similar to that of native F-actin and not to the spectrum of G-actin. Cross-linked actin dimer polymerized to filaments that were indistinguishable in the electron microscope from F-actin made from native G-actin and that were similar to native F-actin in their ability to activate the Mg2+-ATPase of myosin subfragment-1. The critical concentrations of polymerization of cross-linked actin dimer in 0.5 mM and 2.0 mM MgCl2, 2 to 4 microM, and 1 to 2 microM, respectively, were similar to the values for native G-actin. Cross-linked actin dimer contained 2 mol of bound nucleotide/mol of dimer. One bound nucleotide exchanged with ATP in solution with a t 1/2 of 55 min and with ADP with a t 1/2 of 5 h. The second bound nucleotide exchanged much more slowly. The more rapidly exchangeable site contained 10 to 15% bound ADP.Pi and 85 to 90% bound ATP while the second site contained much less, if any, bound ADP.Pi. Cross-linked actin dimer had an ATPase activity in 0.5 mM MgCl2 that was 7 times greater than the ATPase activity of native G-actin and that was also stimulated by cytochalasin D. These data are discussed in relation to the possible role of ATP in actin polymerization and function with the speculation that the cross-linked actin dimer may serve simultaneously as a useful model for each of the two different ends of native F-actin.     

The bound nucleotide of actin

The extent of actin polymerization has been studied for samples in which the bound nucleotide of the actin was ATP, ADP, or an analog of ATP that was not split (AMPPNP). The equilibrium constants for the addition of a monomer to a polymer end were determined from the concentration of monomer coexisting with the polymer. An analysis of these results concludes that the bound ATP on G-actin provides little energy to promote the polymerization of the actin. AMPPNP was incorporated into F-actin and the interaction of F-actin · AMPPNP with myosin was studied. F-actin · AMPPNP activated the ATPase of myosin to the same extent as did F-actin · ADP. However, the rate of superprecipitation was slower in the case of F-actin · AMPPNP than in the control.     

Influence of the bound nucleotide on the molecular dynamics of actin

Rotational dynamics of actin spin-labelled with maleimide probes at the reactive thiol Cys-374 were studied. Replacement of the bound nucleotide by Br8ATP in G-actin and Br8ADP in F-actin causes significant increase of the rotational correlation time of the spin probe, indicating reduced motion in both G and F-actin. The orientation dependence of the electron paramagnetic resonance spectra in oriented F-actin filaments revealed an altered molecular order of the probe when the nucleotide was a Br-substituted one. The bound nucleotide affects the myosin S1 ATPase activation by actin; both Vmax and K(actin) decreased significantly when the bound nucleotide of actin was Br8ADP.     

The 110,000-dalton actin- and calmodulin-binding protein from intestinal brush border is a myosin-like ATPase

A 110-kDa protein present in chicken intestinal brush-border microvilli is believed to laterally link the actin filament bundle that forms the structural core of the microvilli with the microvillar plasma membrane. We have purified a 110-kDa protein to greater than 95% homogeneity by extraction of brush borders with solution containing 0.6 M KCl and 5 mM ATP, followed by gel filtration chromatography, sedimentation as a complex with exogenous actin, and hydroxylapatite chromatography. The 110-kDa protein-calmodulin complex bound F-actin in the absence but not the presence of ATP and had K+,EDTA-ATPase (0.2 mumol/min/mg) and Ca2+-ATPase (0.2 mumol/min/mg) activities and Mg2+-ATPase activity (0.03 mumol/min/mg) that was not activated by F-actin. The actin-binding and ATPase activities of the complex were similar to those of purified brush-border myosin. However, immunoblot analysis showed no reactivity between the 110-kDa protein and polyclonal antibody against purified chicken brush-border myosin. Also, peptide maps of 110-kDa protein and myosin obtained by limited proteolysis with chymotrypsin and Staphylococcus aureus V8 protease had few, if any, peptides in common. Immunoblot analysis also showed that myosin heavy chain was stable under the conditions of the preparation.     

Cytochalasins induce actin polymerization in human leukocytes.

We studied the effect of cytochalasins (B, D, and E) on the F-actin content in human neutrophils and lymphocytes using NBD-phallacidin labeling followed by flow cytometry. All three cytochalasins induced a concentration- and time-dependent increase in the F-actin content in both cell types. The order of potency was cytochalasin D greater than E greater than B. The increase in F-actin content was accompanied by a decrease in the G-actin content as measured by DNase I inhibition assay. These observations suggest that in intact cells cytochalasins may function differently compared to purified and semipurified systems, and their effects may be modified through other actin-binding or sequestering proteins. 2-deoxyglucose (20 mM) caused a decrease in the basal F-actin content and significantly reduced the change induced by the cytochalasins. These results suggest that the state of actin in intact cells is regulated by cytosolic ATP levels, primarily by the integrity of the glycolytic pathway. Based on these observations, we conclude that the mechanism of action of cytochalasins in intact cells is more complex than current models suggest.     

Actin deoxyroboncuclease I interaction. Depolymerization and nucleotide exchange

Deoxyribonuclease I (DNase I) forms a 1:1 complex with globular actin (G-actin) and also will depolymerize filamentous actin (F-actin) to form a 1:1 complex. The effect of DNase I on the exchange of the actin nucleotide has been investigated. When DNase I is added to G-actin, the rate of nucleotide exchange is decreased from 1.16 +/- 0.25 X 10(-4) s-1 to 0.28 +/- 0.09 X 10(-4) s-1 (0 degrees C). The presence of ATP or ADP in the actin has little effect on the rate of exchange of the nucleotide for ATP. This suggests that the weaker affinity of ADP than ATP for actin is due to a slower association rate of ADP. The rate of the nucleotide exchange in the actinDNase I complex is increased by the addition of NaCl or MgCl2. When DNase I is added to F-actin, the rate of nucleotide exchange (6.2 +/- 1.6 X 10(-4) x-1, 0 degrees C) is similar to the rate of depolymerization as measured by loss of viscosity. The actinDNase I complex formed by depolymerization of F-actin exchanges nucleotide at a 4-fold faster rate than the G-actinDNase I complex in the same ionic conditions. This and other experiments suggest that DNase I binds first to F-actin before dissociating the monomer from the filament. These results are discussed in terms of possible mechanisms of action depolymerization.     

Evidence for an ATP cap at the ends of actin filaments and its regulation of the F-actin steady state

The correlation between the time courses of actin polymerization under continuous sonication and the associated ATP hydrolysis has been studied. ATP hydrolysis was not mechanistically coupled to polymerization, i.e. not necessary for polymerization, but occurred on F-actin in a subsequent monomolecular reaction. Under sonication, polymerization was complete in 10 s while hydrolysis of ATP on the polymer required 200 s. A value of 0.023 s-1 was found for the first order rate constant of ATP hydrolysis on the polymer at 25 degrees C, pH 7.8, in the presence of 0.2 mM ATP, 0.1 mM CaCl2, and 1 mM MgCl2, independent of the F-actin concentration. The conversion of ATP X F-actin to ADP X F-actin was accompanied by an increase in fluorescence of a pyrenyl probe covalently attached to actin, consistent with a 2-fold greater fluorescence for ADP X F-actin than for ATP X F-actin, with a rate constant of 0.022 s-1. In contrast, the fluorescence of F-actin labeled with 7-chloro-4-nitrobenzeno-2-oxa-1,3-diazole did not change significantly when ATP or ADP was bound. The direct consequence of the uncoupling between polymerization and ATP hydrolysis is the formation of an ATP cap at the ends of the filaments, which maintains the stability of the polymer, while most of the filament contains bound ADP. The heterogeneity of the filament with respect to ATP and ADP results in a nonlinear relationship between the rate of elongation and the concentration of G-actin with a discontinuity at the critical concentration, where the rate of growth is zero. In this respect, F-actin in ATP behaves similarly to microtubules in GTP.     

High microfilament concentration results in barbed-end ADP caps.

Current theory and experiments describing actin polymerization suggest that site-specific cleavage of bound nucleotide following F-actin filament formation causes the barbed ends of microfilaments to be capped first with ATP subunits, then with ADP bound to inorganic phosphate (ADP.Pi) at steady-state. The barbed ends of depolymerizing filaments consist of ADP subunits. The decrease in stability of the barbed-end cap accompanying the transition from ADP.Pi to ADP allows nucleotide hydrolysis and subsequent loss of Pi to regulate F-actin filament dynamics. We describe a novel computational model of nucleotide capping that simulates both the spatial and temporal properties of actin polymerization. This model has been used to test the effects of high filament concentration on the behavior of the ATP hydrolysis cycle observed during polymerization. The model predicts that under conditions of high microfilament concentration an ADP cap can appear during steady-state at the barbed ends of filaments. We show that the presence of the cap can be accounted for by a kinetic model and predict the relationship between the nucleotide concentration ratio [ATP]/[ADP], the F-actin filament concentration, and the steady-state distribution of barbed-end ADP cap lengths. The possible consequences of this previously unreported phenomenon as a regulator of cytoskeletal behavior are discussed.     

Comparative studies on the energetics of platelet responses induced by different agonists.

The correlation between energy consumption and platelet responses induced by collagen, A23187 and ADP was investigated and compared with the energetics of thrombin-stimulated platelets established in earlier work. Aggregation, measured as single-platelet disappearance, and secretion correlated quantitatively with the increment but not with the total consumption of energy, suggesting that the former reflects the energy cost of these responses. The cost of complete aggregation was 2-3 mumol of ATP equivalents/10(11) platelets with collagen, ADP and thrombin as the stimulus. The cost of complete dense-granule secretion was 0.5-0.8 mumol of ATP equivalents/10(11) platelets with all agonists tested. The cost of combined secretion of alpha-granule and acid hydrolase granule contents was 5-7 mumol of ATP equivalents/10(11) platelets with thrombin and collagen. However, in the presence of A23187 much more energy was consumed during aggregation and secretion. Also ADP triggered more energy consumption during secretion than was seen with the other inducers. The effect of inhibitors of aggregation and secretion was investigated in thrombin-stimulated platelets. Raising the cellular cyclic AMP content sharply decreased the increment in energy consumption as well as aggregation and secretion. The cytoskeleton-disrupting agents cytochalasin B and colchicine left the increment in energy consumption intact, but decreased the basal consumption seen in unstimulated platelets. This was accompanied by normal (cytochalasin B) or diminished (colchicine) aggregation and secretion. Apart from the latter exception, all inhibitors decreased secretion and incremental energy consumption in parallel, thereby preserving the energy-versus-secretion relationship established in earlier work. In contrast, aggregation and energy consumption varied independently, suggesting that the coupling with energy consumption is much weaker for this response.     

Secretion, subcellular localization and metabolic status of inorganic pyrophosphate in human platelets. A major constituent of the amine-storing granules.

The platelet content of PPi is 1.90 +/- mumol/10(11) platelets (S.E.M., n = 19) or about 10.5 nmol/mg of protein, several hundred times that found for rat liver. Some 80% of this PPi is secreted by platelets treated with thrombin with a time course and dose-response relationship similar to secretion of ATP, ADP and 5-hydroxytryptamine (serotonin) from the platelet dense granules. During platelet aggregation induced by ADP and adrenaline, substantial amounts of PPi were secreted, but no release of acid hydrolases was observed. Subcellular-fractionation studies showed that the PPi is highly enriched in the same fraction that contains the storage organelles which store ATP, ADP, Ca2+ and 5-hydroxytryptamine. Inorganic pyrophosphatase was present mainly in the soluble fraction and in the mitochondria. Secretion studies done with platelets prelabelled with [32P]Pi showed that the sequestered PPi was relatively metabolically inactive, as is the ATP and ADP in the storage organelles. The possible participation of PPi in the formation of a bivalent-cation-nucleotide complex associated with amine storage is discussed.     

Fluorescence study of the high pressure-induced denaturation of skeletal muscle actin.

Ikkai & Ooi [Ikkai, T. & Ooi, T. (1966) Biochemistry 5, 1551-1560] made a thorough study of the effect of pressure on G- and F-actins. However, all of the measurements in their study were made after the release of pressure. In the present experiment in situ observations were attempted by using epsilon ATP to obtain further detailed kinetic and thermodynamic information about the behaviour of actin under pressure. The dissociation rate constants of nucleotides from actin molecules (the decay curve of the intensity of fluorescence of epsilon ATP-G-actin or epsilon ADP-F-actin) followed first-order kinetics. The volume changes for the denaturation of G-actin and F-actin were estimated to be -72 mL x mol(-1) and -67 mL x mol(-1) in the presence of ATP, respectively. Changes in the intensity of fluorescence of F-actin whilst under pressure suggested that epsilon ADP-F-actin was initially depolymerized to epsilon ADP-G-actin; subsequently there was quick exchange of the epsilon ADP for free epsilon ATP, and then polymerization occurred again with the liberation of phosphate from epsilon ATP bound to G-actin in the presence of excess ATP. In the higher pressure range (> 250 MPa), the partial collapse of the three-dimensional structure of actin, which had been depolymerized under pressure, proceeded immediately after release of the nucleotide, so that it lost the ability to exchange bound ADP with external free ATP and so was denatured irreversibly. An experiment monitoring epsilon ATP fluorescence also demonstrated that, in the absence of Mg(2+)-ATP, the dissociation of actin-heavy meromyosin (HMM) complex into actin and HMM did not occur under high pressure.     

Interaction of F-actin-AMPPNP with myosin subfragment-1 and troponin-tropomyosin: influence of an extra phosphate at the nucleotide binding site in F-actin on its function

An unsplitable analogue of ATP (adenylyl imidodiphosphate; AMPPNP) was incorporated into F-actin [Cooke, R. (1975) Biochemistry 14, 3250-3256]. The resulting polymers (F-actin-AMPPNP) activated the ATPase activity of myosin subfragment-1 (S1) as efficiently as normal F-actin; neither the maximum velocity at infinite actin concentration (Vmax) nor the affinity of actin to S1 in the presence of ATP (1/KATPase) changed, which indicates that the terminal phosphate of the bound nucleotide at the cleft region between the two domains of the actin molecule [Kabsch, W., Mannherz, H.G., & Suck, D. (1985) EMBO J. 4, 2113-2118] is not directly involved in a myosin binding site. However, the interaction of F-actin with troponin-tropomyosin was strongly modulated by the replacement of ADP with AMPPNP. The troponin-tropomyosin complex strongly enhanced the activation of S1-ATPase activity by F-actin-AMPPNP in the presence of Ca2+, although it has no effect on the activation by normal F-actin-ADP. KATPase was enhanced about threefold by troponin-tropomyosin in the presence of Ca2+, while Vmax was not markedly changed. F-actin-AMPPNP is highly potentiated by troponin-tropomyosin even with low S1 to actin ratios and at high ATP conditions. In the absence of Ca2+, the activation by F-actin-AMPPNP was inhibited normally by troponin-tropomyosin. The results suggest that the terminal beta-phosphate of the bound nucleotide in F-actin is located in a region which is important for regulation of the interaction with myosin.     

Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein

Exposure of cryptic actin filament fast growing ends (barbed ends) initiates actin polymerization in stimulated human and mouse platelets. Gelsolin amplifies platelet actin assembly by severing F-actin and increasing the number of barbed ends. Actin filaments in stimulated platelets from transgenic gelsolin-null mice elongate their actin without severing. F-actin barbed end capping activity persists in human platelet extracts, depleted of gelsolin, and the heterodimeric capping protein (CP) accounts for this residual activity. 35% of the approximately 5 microM CP is associated with the insoluble actin cytoskeleton of the resting platelet. Since resting platelets have an F- actin barbed end concentration of approximately 0.5 microM, sufficient CP is bound to cap these ends. CP is released from OG-permeabilized platelets by treatment with phosphatidylinositol 4,5-bisphosphate or through activation of the thrombin receptor. However, the fraction of CP bound to the actin cytoskeleton of thrombin-stimulated mouse and human platelets increases rapidly to approximately 60% within 30 s. In resting platelets from transgenic mice lacking gelsolin, which have 33% more F-actin than gelsolin-positive cells, there is a corresponding increase in the amount of CP associated with the resting cytoskeleton but no change with stimulation. These findings demonstrate an interaction between the two major F-actin barbed end capping proteins of the platelet: gelsolin-dependent severing produces barbed ends that are capped by CP. Phosphatidylinositol 4,5-bisphosphate release of gelsolin and CP from platelet cytoskeleton provides a mechanism for mediating barbed end exposure. After actin assembly, CP reassociates with the new actin cytoskeleton.     

Characteristics of EYFP-actin and visualization of actin dynamics during ATP depletion and repletion

Disruption of theactin cytoskeleton in proximal tubule cells is a key pathophysiologicalfactor in acute renal failure. To investigate dynamic alterations ofthe actin cytoskeleton in live proximal tubule cells,LLC-PK₁₀ cells were transfected with an enhanced yellowfluorescence protein (EYFP)-actin construct, and a clone with stableEYFP-actin expression was established. Confluent live cells werestudied by confocal microscopy under physiological conditions or duringATP depletion of up to 60 min. Immunoblots of stabletransfected LLC-PK₁₀ cells confirmed the presence of EYFP-actin, accounting for 5% of total actin. EYFP-actin predominantly incorporated in stress fibers, i.e., cortical and microvillar actin as shown by excellent colocalization with Texas red phalloidin. Homogenous cytosolic distribution of EYFP-actin indicatedcolocalization with G-actin as well. Beyond previous findings, weobserved differential subcellular disassembly of F-actin structures:stress fibers tagged with EYFP-actin underwent rapid and completedisruption, whereas cortical and microvillar actin disassembled atslower rates. In parallel, ATP depletion induced the formation ofperinuclear EYFP-actin aggregates that colocalized with F-actin. DuringATP depletion the G-actin fraction of EYFP-actin substantiallydecreased while endogenous and EYFP-F-actin increased. Duringintracellular ATP repletion, after 30 min of ATP depletion, there was ahigh degree of agreement between F-actin formation from EYFP-actin andendogenous actin. Our data indicate that EYFP-actin did not alter thecharacteristics of the endogenous actin cytoskeleton or the morphologyof LLC-PK₁₀ cells. Furthermore, EYFP-actin is a suitableprobe to study the spatial and temporal dynamics of actin cytoskeletonalterations in live proximal tubule cells during ATP depletion and ATP repletion.