Duncan H. Haynes

Platelet microparticles and calcium homeostasis in acute coronary ischemias

Elevation of free cytoplasmic calcium is the common pathway of platelet activation, leading to shape change, shedding of platelet microparticles (PMP), aggregation, and secretion of internal granules, including expression of CD62p on the surface. Platelet activation is well documented in unstable angina (UA) and acute myocardial infarction (MI). We investigated the following markers of platelet activation in 55 patients undergoing coronary angiography for suspected CAD: free cytoplasmic calcium, [Ca2+]cyt, PMP, CD62p expression, and platelet/leukocyte (P/L) interaction. [CA2+]cyt was measured by Fluo‐3 and the other measurements were by flow cytometry. Patients were classified into three groups: unstable angina (UA, n = 11), recent myocardial infarction (MI, n = 11), and patient controls (CTL, n = 33). Blood was drawn before infusion of heparin through femoral lines at the time of catheterization for assays. Results: (1) PMP values were significantly higher in both UA and MI than in CTL, P < 0.05. There was no difference between UA and MI. (2) P/L interaction was significantly elevated only in UA, P < 0.05. (3) CD62p expression on free platelets did not differ significantly between any of the three groups. (4) The resting [Ca2+]cyt, thrombin‐induced CA2+ influx, and release of CA2+ from internal stores were all significantly higher in platelets from the combined patient group (UA + MI) than in the patient group, P < 0.001 Conclusions: Results on calcium hemostasis and PMP were significantly different in patients with acute coronary syndromes than those with stable angina or no coronay ischemia; this may reflect underlying pathophysiology of acute coronary ischemia. P/L interaction was higher only in the UA group, suggesting a role of leukocytes in the UA. Am. J. Hematol. 54:95–101, 1997

1-Anilino-8-naphthalenesulfonate: a fluorescent probe of ion and ionophore transport kinetics and trans-membrane asymmetry.

SummaryThe kinetics of the transport of the 1-anilino-8-naphthalenesulfonate (ANS−, an anionic fluorescent probe of the membrane surface) across phospholipid vesicle membranes have been studied using a stopped-flow rapid kinetic technique. The method has been used to gain detailed information about the mechanism of transport of this probe and to study ionophore-mediated cation transport across the membrane. The technique has also been exploited to study differences between the inside and outside surfaces of vesicles containing phosphatidyl choline (PC).The following is a summary of the major conclusions of this study. (a) Binding of ANS− on the outside surface occurs within times shorter than 100 μsec while permeation occurs in the time range 5–100 sec. (b) Net transport of ANS− occurs with cotransport of alkali cations. (c) The transport rate is maximal in the region of the crystalline to liquidcrystalline phase transition, and the increase correlates with changes in the degree of aggregation of the vesicles. (d) Incorporation of phosphatidic acid (PA), phosphatidyl ethanolamine (PE) or cholesterol into PC membranes decreases the rate of ANS− transport. (e) Neutral ionophores (I) of the valinomycin type increase ANS− permeability in the presence of alkali cations (M+) by a mechanism involving the transport of a ternaryI−M+-ANS− complex. The equilibrium constants for formation of these complexes and their rate constants for their permeation are presented. The maximal turnover number for ANS− transport by valinomycin in dimyristoyl PC vesicles at 35°C was 46 per sec. (f) The partitioning of the ionophore between the aqueous and membrane phases and the rate of transfer of an ionophore from one membrane have been determined in kinetic experiments. (g) A method is described for the detection ofI−M+ complexes on the membrane surface by their enhancement effects on ANS− fluorescence at temperature below the phase transition temperature on “monolayer” vesicles. The apparent stability constants for severalI−M+ complexes are given. (h) Analysis of the effect of ionic strength on the ANS− binding to the inside outside surfaces indicates that the electrostatic surface potential (at fixed ionic strength and surface change) is larger for the inside surface than for the outside surface. (i) Analysis of the dependence of the maximal ANS− binding for the inside and outside surfaces of vesicles made from PC and a variable mole fraction of PA, PE or cholesterol indicate that the latter three are located preferentially on the inside surface.

Fluorescence study of the divalent cation-transport mechanism of ionophore A23187 in phospholipid membranes

The mechanism for transport of divalent cations across phospholipid bilayers by the ionophore A23187 was investigated. The intrinsic fluorescence of the ionophore was used in equilibrium and rapid-mixing experiments as an indicator of ionophore environment and complexation with divalent cations. The neutral (protonated) form of the ionophore binds strongly to the membrane, with a high quantum yield relative to that in the aqueous phase. The negatively charged form of the ionophore binds somewhat less strongly, with a lower quantum yield, and does not move across the membrane. Complexation of the negatively charged form with divalent cations was measured by the decrease in fluorescence. An apparent rate constant (kapp) for transport of the ionophore across the membrane was determined from the rate of fluorescence changes observed in stopped-flow rapid kinetic experiments. The variation of kapp was studied as a function of pH, temperature, ionophore concentration, membrane lipid composition, and divalent cation concentration and type. Analysis and comparison with equilibrium constants for protonation and complexation show that A23187 and its metal:ionophore complexes bind near the membrane-water interface in the lipid polar-head region. The interfacial reactions occur rapidly, compared with the transmembrane reactions, and are thus in equilibrium during transport. The transport cycle can be described as follows: a 1:1 complex is formed between the membrane bound A23187-(Am-) and the aqueous divalent cation with dissociation constant K1 approximately 4.6 x 10(-4) M. This is in equilibrium with a 1:2 (metal:ionophore) complex (K2 approximately 3.0 x 10(-4) [ionophore/lipid]) that is responsible for transporting the divalent cations across the membrane. The rate constant for translocation of the 1:2 complex is 0.1-0.3 s-1. Dissociation of the complex of the trans side and protonation occur rapidly. The rate constant for translocation of H+ . A23187- is 28 s-1. A theory is presented that is capable of reproducing the kinetic data at any calcium concentration. The cation specificity for ionophore complex transport (kapp), determined at low ionophore concentration for a series of divalent cations, was found to be proportional to the equilibrium constant for 1:1 complexation. The order of ion specificity for these processes was found to be Ca2+ greater than Mg2+ greater Sr2+ greater than Ba2+. Interactions with Na+ were not observed. Maximal values of kapp were observed for vesicles prepared from pure dimyristoyl phosphatidylcholine. Inclusion of phosphatidyl ethanolamine, phosphatidic acid, or dipalmatoyl phosphatidylcholine resulted in lower values of kapp. Calcium transport by A23187 is compared with that of X537A, and it is shown that the former is 67-fold faster. The difference in rates is due to differences in the ability of each ionophore to form a 1:2 complex from a 1:1 complex.

Cyclic AMP stimulates Ca2+-ATPase-mediated Ca2+ extrusion from human platelets

The effect of cAMP on active Ca2+ extrusion across the plasma membrane of intact human platelets was studied using quin2, a fluorimetric indicator of free Ca2+ in the cytoplasmic compartment ([Ca2+]cyt). Elevations of cAMP were achieved by incubation with dibutyryl-cAMP or by forskolin, which was found to selectively elevate cAMP without affecting cGMP levels. Progress curves of Ca2+ extrusion from quin2-overloaded platelets were measured. The rate vs. [Ca2+]cyt characteristic was calculated as previously described (Johansson, J.S. and Haynes, D.H. (1988) J. Membr. Biol. 104, 147-163). Forskolin, at a maximally effective concentration of 10 microM, was shown to stimulate Ca2+ extrusion by increasing by a factor of 1.6 +/- 0.5 the Vm of a saturable component, previously identified with a Ca(2+)-Mg(2+)-ATPase located in the plasma membrane. Neither the Km (80 nM) or Hill coefficient (1.7 +/- 0.3) of the Ca(2+)-ATPase was affected. Forskolin had no effect on the linear, non-saturable component of extrusion (previously identified with a Na+/Ca2+ exchanger) over the [Ca2+]cyt range examined (50-1500 nM). Dibutyryl-cAMP (Bt2-cAMP, 1 mM) stimulated the Ca(2+)-Mg(2+)-ATPase component of Ca2+ extrusion by a factor of 2.0 +/- 0.6. Separate experiments showed that 10 microM forskolin reduces the resting [Ca2+]cyt from 112 nM to 96 nM. Mathematical analysis showed that this can be accounted for by the above-mentioned increase in Vm of the pump, countered by a 37-74% increase in the rate constant for passive Ca2+ leakage across the plasma membrane. The results suggest two mechanisms by which prostacyclin-induced elevation of cAMP inhibits platelet aggregation: (a) lowering of resting [Ca2+]cyt and (b) increasing the rate of Ca2+ extrusion after the initial influx or triggered release event.

Evidence for a role of phosphatidyl ethanolamine as a modulator of membrane-membrane contact.

SummaryPhosphatidyl ethanolamine (PE) is shown to be effective in producing membrane aggregation. The aggregation of PE and PE/PC (phosphatidyl choline) mixed vesicles was studied as a function of pH and cation composition of the medium. The kinetics and equilibria were studied in stopped-flow rapid mixing experiments, in which PE vesicles prepared at pH 9.2 were “jumped” to pH 7.H+ ions protonate PE− and promote vesicle aggregation in a cooperative fashion. Vesicles containing PC have a decreased tendency to aggregate compared to pure PE vesicles. The apparent rate constant for aggregation was about two orders of magnitude below that for diffusion controlled aggregation and was virtually the same for PE and PE/PC mixed vesicles.A theoretical description of equilibrium for vesicle aggregation is developed in terms of three parameters: the equilibrium constant for the protonation of PE (KA), the equilibrium constant for aggregation (Keq) and the number of PE molecules in an effective area that the two vesicles must interact in order to aggregate (Neff). These parameters are compared with values and trends expected for electrostatic calculations based on dipolar repulsion and short-range binding, to which hydrogen bonding may contribute. The results are interpreted in a self-consistent fashion to indicate: (i) that PE and PC mix randomly, (ii) that head-to-tail binding occurs between PE(PC) molecules on apposing vesicles, (iii) that electrostatic screening accounts for the decrease inKA as a function of the molar fraction of PC per vesicle, (iv) that the PE must be 90% protonated before aggregation can occur, and (v) that for all the lipid systems we considered, the point at which the extent of dimerization is half maximal is close to the physiological pH, indicating that PE may have a regulatory effect in the aggregation of biological systems.

Thrombin-induced calcium movements in platelet activation.

The thrombin-induced Ca2+ fluxes and their coupling to platelet aggregation of the human platelet were studied using quin2 as a measure of the cytoplasmic Ca2+ concentration [( Ca2+]cyt) and chlorotetracycline (CTC) as a measure of internally sequestered Ca2+. Evidence is given that the CTC fluorescence change is proportional to the free internal Ca2+ concentration in the dense tubular lumen. The intracellular quin2 concentration was 1 mM and analysis showed that it did not perturb the processes reported herein. The value of [Ca2+]cyt at rest and during thrombin activation was analyzed in terms of Ca2+ influx, Ca2+ release, Ca2+ sequestration, and Ca2+ extrusion. Influx was distinguished from internal release by removing extracellular Ca2+ 1 min before thrombin activation. In the presence of 2 mM external Ca2+, the thrombin-induced Ca2+ influx accounts for most of the increase in [Ca2+]cyt (over 80%). Thrombin-induced Ca2+ influx and release have somewhat different EC50 values (0.17 U/ml vs. 0.35 U/ml). The contribution of influx can be inhibited by verapamil, bepridil and Cd2+ (IC50 values of 19 microM, 2 microM and 50 microM). The influx results were analyzed in terms of a thrombin-activated channel. Indomethacin pretreatment experiments suggest that activation of the arachidonic pathway accounts for approx. 50% of the influx-related [Ca2+]cyt elevation. Elevation of [Ca2+]cyt by intracellular release is not inhibited by verapamil or Cd2+ but is inhibited by bepridil with a high IC50 (25 microM). It is only 15-20% inhibited by indomethacin and is thus not dependent on thromboxane A2 formation. The release reaction does not require Ca2+ influx. The rate of thrombin-activated platelet aggregation is shown to have an approximately fourth-power dependence on [Ca2+]cyt with an apparent Km of 0.4 microM. Comparisons of aggregation rates of the partially thrombin-activated vs. fully thrombin-activated, partially verapamil-inhibited conditions suggest that this dependence on [Ca2+]cyt is the major determinant of the aggregation behavior. Analysis shows that calcium influx is the major pathway for elevating [Ca2+]cyt by thrombin when physiological concentrations of external Ca2+ are present.

Intracellular calcium storage and release in the human platelet. Chlorotetracycline as a continuous monitor.

the calcium-sensitive fluorescent probe chlorotetracycline was used to monitor calcium movement in human platelets. The chlorotetracycline fluorescence signal is a linear measure of the level of free calcium in the dense tubules and in the mitochondria, with probe sensitivity in the millimolar range. Experiments perturbing the system with the calcium ionophore A23187 shows that the level of free internal calcium in the organelle depends upon the cytoplasmic level, which, in turn, depends upon the passive permeability of the plasma membrane. Chlorotetracycline in the cytoplasmic compartment does not respond to changes in the cytoplasmic calcium concentration, which is held in the micromolar to submicromolar range by an extrusion system. The calcium concentration in the cytoplasmic compartment can be directly manipulated by the calcium ionophore A23187 and is measured in parallel experiments with Quin 2, a high-affinity indicator. The calcium transport systems of the organelles are shown to be less susceptible to short circuit by A23187. Analysis shows that mitochondrial uptake is slow (ti/2 = 20 minutes), produces a large increase in chlorotetracycline fluorescence, and is inhibited by sodium azide plus oligomycin. Uptake by the dense tubules is more rapid (t1/2 = 2 minutes), produces a smaller increase in chlorotetracycline fluorescence, is inhibited by trifluoperazine, and is less sensitive to A23187. The Km is estimated as 1 /*m or lower. Studies show that the chlorotetracycline technique is useful for the monitoring of calcium uptake and release by the platelet organelles, and suggests that the Quin-2/chlorotetracycline technique will be useful as a diagnostic of both physiological and pathological activation mechanisms.

Stimulation of dense tubular Ca2+ uptake in human platelets by cAMP

Elevation of intracellular cAMP is shown to increase the rate (V) and maximal extent of Ca2+ uptake by the dense tubules in intact human platelets. Elevation of [cAMP] was accomplished by preincubation with the adenylate cyclase activator forskolin or with dibutyryl-cAMP (Bt2-cAMP). The free concentration of Ca2+ in the dense tubular lumen ([Ca2+]dt) was monitored using the fluorescence of chlorotetracycline (CTC) according to protocols developed in this laboratory. The free cytoplasmic Ca2+ concentration ([Ca2+]cyt) was monitored in parallel experiments with quin2. Both [Ca2+]cyt and [Ca2+]dt were analyzed in terms of competition between pump and leak mechanisms in the plasma membrane (PM) and dense tubular membrane (DT). When platelets are incubated in media with approx. 1 microM external Ca2+, [Ca2+]cyt is approx. 50 nM and [Ca2+]dt is very low. When 2 mM external Ca2+ is added, [Ca2+]cyt rises to approx. 100 nM and the process of dense tubular Ca2+ uptake can be resolved. Forskolin (10 microM) and Bt2-cAMP increase the rate of dense tubular Ca2+ uptake (V) to 2.1 +/- 0.60 and 1.70 +/- 40 times control values (respectively). The agents also increase the final [Ca2+]dt to 1.70 +/- 0.21 and 1.72 +/- 0.60 times control values (respectively). Titrations with ionomycin (Iono) showed that the increase was due to an increase in the Vm of the dense tubular Ca2+ pump. With [Iono] = 500 nM, [Ca2+]cyt was raised to greater than or equal to 1.0 microM and Vm of the dense tubular pump was elicited. (At [Iono] = 1.0 microM, the final [Ca2+]dt values were degraded 15% due to shunting of Ca2+ uptake.) Analysis showed that forskolin (10 microM) and Bt2-cAMP (1 mM) increase the Vm by a factors of 1.56 +/- 40 and 1.56 +/- 40, respectively. Analysis showed that neither agent changed the Km of the pump significantly from its control value of 180 nM. Neither agent changed the rate constant for passive leakage of Ca2+ across the DT membrane (1.7 min-1).

Deliberate quin2 overload as a method for in situ characterization of active calcium extrusion systems and cytoplasmic calcium binding: application to the human platelet

SummaryThe objectives of the title were accomplished by a four-step experimental procedure followed by a simple graphical and mathematical analysis. Platelets are (i) overloaded with the indicator quin2 to cytoplasmic concentrations of 2.9mm and (ii) are exposed to 2mm external Ca2+ and 1.0 μm ionomycin to rapidly achieve cytoplasmic Ca2+ ([Ca2+]cyt) of ca. 1.5 μm. (iii) The external Ca2+ is removed by EGTA addition, and (iv) the active Ca2+ extrusion process is then monitored as a function of time. Control experiments show that the ionophore shunts dense tubular uptake and does not contribute to the Ca2+ efflux process during phases iii–iv and that the extrusion process is sensitive to metabolic inhibitors.The progress curves for the decline of quin2 fluorescence (resulting from active Ca2+ extrusion) were analyzed as a function of [Ca2+]cyt using a mathematical model involving the probability that an exported Ca2+ was removed from a quin2 complex (vs. a cytoplasmic binding element). The observed rates of decline of quin2 fluorescence at a particular [Ca2+]cyt are dependent upon (i) the absolute rate of the extrusion system (a function of itsKm, Vm and Hill coefficient (n)), (ii) the intrinsic Ca2+ buffer capacity of the cytoplasm (a function of the total site concentration ([B]T) and itsKd) and (iii) the buffer capacity of the intracytoplasmic quin2 (a function of its concentration andKd). The contribution of (iii) was known and varied and was used to determine (ii) and (i) as a function of [Ca2+]cyt.The Ca2+ binding data were verified by45Ca2+ experimentation. The data fit a single binding site ([B]T=730±200 μm) with an averageKd of 140±10nm. This can be accounted for by platelet-associated calmodulin. The rate of the Ca2+ extrusionvs. [Ca2+]cyt curve can be described by two components: A saturable one withVm=2.3±0.3 nmol min−1 mg-membrane−1,Km=80±10 andn=1.7±0.3 (probably identified with a Ca2+-ATPase pump) and a linear one (probably identified with a Na+/Ca2+ exchanger).

Rapid kinetic studies of active Ca2+ transport in sarcoplasmic reticulum

SummaryA method is reported for the rapid and continuous monitoring of active Ca2+ transport events occurring in isolated skeletal sarcoplasmic reticulum (SR). The method is based on the quantitative evaluation of increases in the fluorescence of 1-anilino-8-naphthalenesulfonate (ANS−), resulting from active transport. The method, which has a time resolution of 20 msec, was applied to the kinetics of Ca2+ transport by a Ca2+-ATPase-rich SR fraction and the effects and loci of action of Mg2+ and monovalent cations (M+) were investigated. The turnover number of the enzyme and its ability to establish gradients were investigated in the absence of the complicating effects of precipitating anions. The results are explicable in terms of the model of Kanazawa et al. (Kanazawa, T., Yamada, S., Yamamoto, T., Tonomura, Y., 1971,J. Biochem. (Tokyo)70:95) and are difficult to reconcile with models in which the enzyme is considered to be electrogenic. The major observations of the study are as follows:1)Active uptake of 29 μM free Ca2+ in the presence of 5mm KCl, initiated by the addition of 10−4m Mg2+ and 2×10−4m ATP, occurs with at1/2 of ca. 9 sec. The process results in an internal free Ca2+ concentration of 13mm.2)Preincubation with 50mm KCl and 5mm MgCl2, followed by initiation of active uptake by the addition of ATP to give a final concentration of ca. 2.5mm Mg2+ and ca. 2.5mm MgATP, gave faster and larger uptakes. Thet1/2 for the reaction was ca. 600 msec and the internal free Ca2+ concentration was 70±20mm. The turnover number of 7.1±0.8 sec−1 was calculated for the enzyme at mid-reaction under the assumption of a stoichiometry of 2 Ca2+ per cycle.3)The accelerative effects of Mg2+ andM+ on the rate of transport were investigated. Experiments in which the cations were added to or omitted from the incubation medium showed that the presence of both classes of activator in the internal aqueous space was necessary for maximal activation of the transport system. The concentration dependencies of these effects were investigated. Analysis shows that the monovalent cation effect is probably based on the countertransport according to the model of Kanazawa et al. (1971) while the Mg2+ effect referable to the inside surface is primarily catalytic. No Mg2+ counter-transport could be demonstrated under conditions in which the internal monovalent cation concentration was adequate.4)Under conditions in which the Mg2+ concentration is adequate for stimulation but theM+ concentration is not (and vice versa), the active uptake can be resolved into two phases. The rapid phase is complete within the first 50 msec and corresponds to 0.26–2.06 Ca2+ released to the internal phase per Ca2+-ATPase. These results correspond closely to those of published studies measuring the rate at which Ca2+ becomes inaccessible to the external solution. The comparison shows that Ca2+ is released to the internal aqueous phase almost as rapidly as it becomes inaccessible to the outside phase. Analysis of the concentration dependencies shows that K+/Ca2+ or Mg2+/Ca2+ competition for occupation of the inwardly-oriented translocator (of the phosphorylated enzymes) is involved in the fast phase of Ca2+ release. When the internal concentrations of both K+ and Mg2+ are adequate, the slow phase is speeded up to such an extent that the first partial turnover can no longer be kinetically isolated from the subsequent turnovers. Under these conditions, the rate of enzyme dephosphorylation, the binding of K+ to the translocator, and its return to an outward orientation are no longer rate limiting. The rate constant for the outward-to-inward reorientation of this translocator is ca. 13.8 sec−1. The average turnover number for the first several turnovers, obtained under conditions of maximal stimulation, is ca. 7.1. The latter value was somewhat influenced by trans-inhibition by internal Ca2+. It is concluded that this outward-to-inward transition of the Ca2+-laden translocator is rate-limiting to the first turnover and that the rate of the inward-to-outward transition of the K+-laden translocator becomes limiting in the final phases of the transport process.5)Two major lines of evidence against electrogenic models of pump function were the stimulatory effect of internal K+ on the transport reaction and the lack of a stimulatory effect as an inwardly-directed Cl− gradient. Also the mechanism of the reponse of the KCl impermeable vesicles to valinomycin was investigated. Those findings also run counter to the expectations of electrogenic pump mechanisms.