Leon Salganicoff

Subcellular localization of human platelet antiheparin proteins

Abstract The distribution of two platelet antiheparin proteins, platelet factor 4 (PF4) and low affinity platelet factor 4 (LA-PF4) measured by radioimmunoassays and of antiheparin activity measured by a chromogenic assay, was determined in human platelet granule fractions separated by sucrose density centrifugation. Subcellular fractions were characterized by the levels of serotonin (dense granule marker) and β-N-acetylglucosaminidase (lysosomal enzyme marker). PF4 and LA-PF4 antigens and antiheparin activity showed an identical distribution pattern with the highest specific activities occurring in a fraction lighter than that containing serotonin. The fraction with the highest specific activity of β-N-acetylglucosaminidase was observed at a lighter sucrose density than the fraction most enriched in platelet antiheparin proteins. These results indicate that PF4, LA-PF4 and heparin-neutralizing activity are located in the same granule fraction (α-granules) which is different from both the serotonin and the β-N-acetylglucosaminidase-containing fractions. The PF4 and LA-PF4 amounted to 13% of the total proteins of α-granules as determined by radioimmunoassay and SDS polyacrylamide gel densitometric scanning.

Energy metabolism of blood platelets: I. Isolation and properties of platelet mitochondria☆

A method for the isolation of a coupled mitochondrial preparation from pig platelets by disruption with a French pressure cell modified for use at pressures of approximately 800 psi is described. Electron micrographs showed this preparation to contain intact mitochondria and α-granules. This preparation was well-coupled; respiratory control ratios of 3–7 were obtained with α-ketoglutarate and 2–4 with succinate. The most rapidly oxidized substrate was α-glycerophosphate at rates as high as 140 natoms of oxygen/min/mg of protein. None of the citric acid cycle intermediates were oxidized at rates greater than 20 natoms/min/mg; rates of respiration with pyruvate plus malate were only 4–7 natoms/min/mg. The ADP:O ratios with various substrates and the response of the respiratory chain to site-specific inhibitors were the same as in other mammalian mitochondria. Room temperature difference spectra of dithionite-treated vs aerobic mitochondria showed the α-bands of the cytochromes of the electron transport chain to be at 603, 560, and 550 nm for (a + a3), b, and (c1 + c) at relative concentrations of 1 to 0.54 to 1.06, respectively.

Subcellular fractionation of pig platelets

Subcellular components were obtained from pig platelets, disrupted by means of a French press and separated into 4 primary fractions. The granule fraction (10 000 g) was subjected to a sucrose gradient fractionation. Primary fractions and the granule subfractions were studied electron microscopically and biochemically by following the distribution of markers of membranes, lysosomes or alpha-granules, mitochondria and dense granules. With this technique of platelet homogenization, 80% of the serotonin and 93% of the beta-N-acetylglucosaminidase were found to be particulate. In the gradient, mitochondria were sharply banded in a fraction (density 1.16--1.17) having a specific activity 10--100 times higher than the other fractions of the gradient. Serotonin-containing granules were found in a pellet of density greater than 1.27 and contained 60% of the serotonin and adenine nucleotides of the granule fraction. The lysosome markers that were monitored, acid phosphatase and beta-N-acetylglucosaminidase, exhibited different distribution patterns. Acid phosphatase showed the highest specific activity in the microsomal fraction with only 2.8% in the granule fraction, and this latter amount also appeared to be associated with membranes upon further fractionation. Beta-N-acetylglucosaminidase was present in both the granule fraction and in the microsomal fraction with nearly the same specific activity. However, that present in the granule fraction was clearly associated with granules that distributed over a wide range of densities on a sucrose gradient. The calcium distribution was followed to attempt to determine its subcellular location; 19% was found in the same subfraction as the serotonin-containing granules, but at least 50% of the particulate calcium was associated with granules distinctly separate from the storage granules.

Adenine nucleotide metabolism of blood platelets. IX. Time course of secretion and changes in energy metabolism in thrombin-treated platelets.

Changes in the energy metabolism of washed human platelets were compared with the kinetics of secretion induced by thrombin (5 units/ml). A 50% decrease in the level of metabolic ATP (3H-labelled), which was essentially complete in 30s, was matched in rate by adenine nucleotide secretion from storage in dense granules. Incubation of platelets with antimycin before thrombin addition increased the rate of fall in metabolic ATP, but did not affect the rate of adenine nucleotide secretion. beta-N-Acetylglucosaminidase secretion, which was slower than adenine nucleotide secretion in control platelets, was noticeably inhibited by antimycin, confirming previous reports that different regulatory mechanisms exist for dense and alpha-granule secretion. The rates of rephosphorylation of metabolic ADP to ATP via glycolysis and oxidative phosphorylation were estimated by measuring lactate production and O2 consumption in resting and thrombin-stimulated platelets and compared to the level of metabolic ATP (9-10 nmol/mg of platelet protein in the resting state). The rate of ATP production was stimulated at least two fold from 12 nmol to 24 nmol/min/mg within seconds of thrombin addition. This increased rate was maintained over the observed period of 5 min although the level of metabolic ATP had decreased to 4-5 nmol/mg within 30 s; the turnover of the remaining metabolic ATP thus increased four fold over the resting state although the actual stimulation of energy production was only two fold.

Mitochondrial activity in pompe’s disease

Mitochondrial oxidative metabolism was examined in two infants with Pompes disease. The clinical diagnosis was confirmed by the demonstration of intralysosomal glycogen accumulation and a deficiency of acid alpha-D-glucosidase in muscle biopsies. Light and electron microscopy studies demonstrated a normal number of mitochondria with normal ultrastructure. Spectrophotometric measurements revealed that the specific activities of citrate synthase and the partial reactions of electron transport were markedly elevated in the skeletal muscle homogenates prepared from both infants with Pompes disease when calculated as micromoles per minute per gram wet weight of tissue. However, when respiratory chain enzyme activities were expressed relative to citrate synthase as a marker mitochondrial enzyme, a different pattern emerged, in which all Pompe muscle respiratory enzymes, except complex IV, were decreased relative to control subjects. These observations demonstrate that caution should be exercised when analyzing and interpreting data obtained from tissue homogenates in general and, in particular, in those prepared from tissues in which the wet weight of tissue may be altered, for example, by pathologic accumulation of carbohydrate or lipid.

Polyamine effects on succinate-linked and αketoglutarate-linked rat liver mitochondrial respiration

Summary At 0.6–1.17 mM Mg ++ , physiological spermine levels strikingly enhance respiratory control ratios of rat liver mitochondria with αketoglutarate by suppressing respiration which occurs after added ADP is converted to ATP. Respiration with added ADP is enhanced at 0.6 but not at 1.17 mM Mg ++ . Conversely, with succinate, spermine at high concentrations depresses respiratory control ratios but only slightly affects respiration. Spermidine with αketoglutarate increases respiratory control ratios but suppresses respiration both during and after added ADP-ATP conversion. However, suppression is greater after this conversion. With succinate, spermidine also increases respiratory control but its effects on respiration during or after added ADP-ATP conversion vary with Mg ++ concentration. Thus, polyamines seem to affect mitochondrial metabolism.

An hypothesis on the consolidation and PGE1-induced deconsolidation of a platelet plug.

Consolidation is the final stage in haemostasis in which a platelet plug blocking a bleeding area of a vessel: (a) becomes impermeable to circulating plasma proteins and (b) contracts to resist blood pressure. Hypothesis: The impermeabilization step of consolidation is accomplished through fluid uptake by the platelets from a hydrated intercellular glue formed during thrombin activation. Dehydration occurs through inhibition of the Na+,K+-ATPase of platelets with sodium and water uptake. However, and uniquely, due to the high cellular density of the platelet plug, access of peripheral plasma fluids to the plug is limited forcing the platelets to take up preferentially the fluid of the interplatelet space. The increased adhesion properties of the dehydrated glue simultaneously furthers a decreased hydraulic permeability and an improved coupling of the contractile forces among platelets. In ‘Deconsolidation’, the fluid uptake process can be reversed and amplified by agents that increase cAMP, reactivating the Na+,K+-ATPase and expressing CFTR or equivalent Cl− secretory channels that force the extrusion of fluid from the platelets, with rehydration of the intercellular polymer and a large increase in the interplatelet space.

Use of Zn—pyrophosphatase in the high-performance liquid chromatographic analysis of cell extracts containing 32P-labelled inositol phosphates

A simple method is described for eliminating the interference of pyrophosphate and pyrophosphorylated nucleosides in the high-performance liquid chromatographic determination of inositol 1,3,4-triphosphate and inositol 1,4,5-triphosphate of 32P-labelled extracts of cells. Treatment of the extract with pyrophosphatase, but substituting Zn2+ for Mg2+ as the cofactor, converts all nucleoside triphosphates and pyrophosphate to their di- and monoesters. Such change shifts their position in the elution profile, allowing a clear identification and quantification of the inositol phosphates. Typical overall recoveries near 80% or higher of added markers.

A method to prepare degranulated human platelets: Use for studies of platelet aggregation and ca2+ mobilization

A method for the preparation of a suspension of thrombin-degranulated human platelets is described. Two peptides (RGDS and GPRP) are used to prevent fibrinogen binding and consequent aggregation, and to prevent fibrin polymerization during thrombin activation. A mixture of creatine phosphokinase and creatine phosphate is used to remove ADP. Hirudin and TAMe are used to neutralize thrombin after the platelets have been activated. [(14)C] Serotonin and PF4 release and electron microscopy demonstrate that the preparation is completely degranulated. After all inhibitors are removed and fibrinogen added, the preparation aggregates rapidly to a mixture of agonists composed of ADP, epinephrine and the synthetic analog of prostaglandin H(2)/thromboxane A(2), U46619. ADP and epinephrine when added individually are both able to induce a clearly detectable aggregation, while U46619 induces only a shape change. The preparation is also suitable for intracellular Ca(2+) studies and we find that the mixture of agonists produces an increase in the intracellular calcium concentration to about 1 µM.

45Ca Efflux and Agonist Induced Changes in Force in a Model of Thrombin Activated Platelets

(45)Ca(2+) and (3)H sorbitol were loaded by incubation into a model of thrombin activated, irreversibly aggregated platelets. Total Ca, measured by atomic absorption, was approximately 4.0 nmoles/mg wet weight. 55% of the total Ca(2+) was exchangeable with (45)Ca(2+), 14% was extracellular and 42% cellular, either surface or intracellular. Changes in the efflux of the marker into a buffer containing Mg-EGTA were correlated with the contractile responses of the preparation after addition of agonists. For the contracting agonists tested individually (ADP, epinephrine, and the endoperoxide analogue, U46619) the fractional efflux rate increased in phases, the descending component stabilizing at a rate higher than the basal. When agonists of different classes were added sequentially in supramaximal amounts, the increases in the stable component of the efflux were also additive and correlated well with the increases in the force of contraction. Washout of the agonist returned the efflux to the baseline. Agents increasing cyclic AMP, like prostaglandin E(1), produced a small decrease in the basal level of the efflux of (45)Ca. When contracting agonists were added to the pretreated preparation, a simultaneous decrease in efflux and force generated were found. The inhibition was dose dependent on the relaxing agonist.