Association of age-related decrease in platelet membrane fluidity with platelet lipid peroxide

The influence of age on platelet lipid peroxide (LPO), platelet membrane fluidity and the composition of fatty acid was investigated in female Wistar rats widely ranging in age from 14 to 720 days old. LPO levels were significantly higher (p<0.05) in the platelets of upper age groups than in those of lower age groups, showing a significantly positive correlation with age (r=0.84, p<0.0001). Membrane fluidity, assessed by 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence polarization, was significantly reduced with age. The composition of fatty acid demonstrated an age-related elevation (p<0.05) in the unsaturation index. The rises in the LPO levels revealed a significantly positive correlation with DPH-polarization (r=0.73, p<0.0001). Thus our results suggested that the age-related deterioration of platelet membrane fluidity, despite a significant elevation in the unsaturation index, was due to the age-related higher basal levels of LPO in platelets.     

Association of fibronectin with the platelet cytoskeleton

Triton-insoluble cytoskeletons prepared from either normal or thrombasthenic platelets were found to contain approximately 1.3 micrograms of fibronectin/10(9) platelets as measured by a radioimmunoassay. Total endogenous platelet fibronectin was quantitatively retained on the platelet cytoskeleton, whereas 70% of exogenously added fibronectin that bound the surface of thrombin-activated platelets was recovered with the Triton-insoluble cytoskeleton. The exogenously added fibronectin specifically bound platelets and cytoskeletons with the same affinity giving an apparent binding constant of 1.47 X 10(-7) M. The possibility that fibrin associated with the platelet cytoskeleton could serve as the fibronectin receptor was investigated by measuring the binding constant of fibronectin for polymerizing fibrin and by measuring the amount of fibronectin associated with cytoskeletons of thrombasthenic platelets which contain 4-fold less fibrin than controls. The binding constant of fibronectin for polymerizing fibrin was 14-fold lower than that for cytoskeletons and cytoskeletons prepared from thrombasthenic platelets contained approximately the same amount of fibronectin as controls. Therefore, it is unlikely that fibrin is the platelet fibronectin receptor. These results support the hypothesis that platelet fibronectin is released from platelet alpha granules upon thrombin stimulation and becomes bound to the platelet surface and cytoskeleton either directly or through some intermediate protein that spans the membrane and interacts both with fibronectin and the internal cell cytoskeleton.     

Association of fibrin with the platelet cytoskeleton

We have previously postulated that surface membrane proteins become specifically associated with the internal platelet cytoskeleton upon platelet activation (Tuszynski, G.P., Walsh, P.N., Piperno, J., and Koshy, A. (1982) J. Biol. Chem. 257, 4557-4563). Four lines of evidence are in support of this general hypothesis since we now show that platelet surface receptors for fibrin become specifically associated with the platelet Triton-insoluble cytoskeleton. 1) Fibrin was detected immunologically in the washed Triton-insoluble cytoskeletons of thrombin-activated platelets under conditions where fibrin polymerization and resultant precipitation was blocked with Gly-Pro-Arg-Pro, a synthetic peptide that inhibits polymerization of fibrin monomer. 2) Radiolabeled fibrin bound to thrombin-activated platelets and became associated with the cytoskeleton. 3) The amount of radiolabeled fibrin bound to thrombin-activated thrombasthenic platelets and their cytoskeletons amounted to about 20% of the fibrin bound to thrombin-activated control platelets and their cytoskeletons. 4) The association of fibrin with cytoskeletons and with the platelet surface was nearly quantitatively blocked by an antibody prepared against cytoskeletons (anti-C), an antibody against isolated membranes of Pronase-treated platelets (anti-M1), and a monoclonal antibody to the platelet surface glycoprotein complex, GPIIb-GPIII (anti-GPIII). These antibodies blocked ADP and thrombin-induced platelet aggregation as well as thrombin-induced clot retraction. Analysis of the immunoprecipitates obtained with anti-C, anti-M1, and anti-GPIII from detergent extracts of 125I-surface labeled platelets revealed that these antibodies recognized GPIIb-GPIII. These data suggest that thrombin activation of platelets results in the specific association of fibrin with the platelet cytoskeleton, that this association may be mediated by the GPIIb-GPIII complex, and that these mechanisms may play an important role in platelet aggregation and clot retraction induced by thrombin.     

Association of actin with the platelet membrane

Human platelet membrane-actin associations were studied by means of differential extraction of purified membranes and low-shear viscometry of membrane-F-actin mixtures. As indicated by resistance to extraction with 0.6 M potassium iodide, a significant amount of platelet actin appears to be tightly associated with the membrane. When tested by falling-ball viscometry, both whole and KI-extracted membranes increased the low-shear viscosity of preformed rabbit skeletal muscle F-actin at physiologically reasonable pH and ionic conditions. This membrane-associated actin gelation activity was dependent upon low free calcium concentration (10(-8)-10(-7) M). The results are consistent with specific associations between actin and platelet membranes and may be relevant to membrane-cytoskeletal interactions believed to occur in the intact cell.     

Complexes of thrombin with secreted platelet proteins

A labeled 77-kDa complex formed when 125I-thrombin was added to platelet suspensions or to the supernatant solution of ionophore-activated platelets. Prostacyclin inhibited complex formation with whole platelets but not with the supernatant solution of ionophore-activated platelets. This is evidence that the complex formed with a factor secreted from activated platelets. Smaller complexes of 70 and 58 kDa formed between labeled thrombin and lysed platelets. The 77-kDa complex was necessary for the formation of a thrombin-thrombospondin complex.     

Fluorescence of equine platelet tropomyosin labeled with acrylodan

Equine platelet tropomyosin was labeled with the sulfhydryl-specific fluorescent reagent 6-acryloyl-2-dimethylaminonaphthalene (acrylodan). The extent of labeling at 4 degrees C could be regulated between 0.5 and 1.3 acrylodans per tropomyosin chain by varying the reaction time from 1 to 4.5 h. Acrylodan-labeled platelet tropomyosin, AD-P-TM, was highly fluorescent, having an emission maximum near 518 nm on excitation at 365 nm. Steady-state measurements of polarization of the fluorescence of AD-P-TM in both low and high ionic strength solutions gave Perrin plots that exhibited sharp changes in slope near 50 degrees C, indicative of a sharp increase in mobility of the label at that temperature. This correlates with the melting temperature of the platelet tropomyosin coiled coil observed by circular dichroism [G. P. C?té, W. G. Lewis, M. D. Pato, and L. B. Smillie, (1978) FEBS Lett. 91, 237-241]. Perrin plots of carboxypeptidase A-treated platelet tropomyosin that was labeled with acrylodan after digestion resembled more closely those of acrylodan-labeled cardiac tropomyosin rather than those of AD-P-TM, suggesting that the observed emission arose from label at Cys-153 on each truncated platelet tropomyosin chain. In solutions containing 150 mM KCl and 5 mM MgCl2, addition of actin at up to a sixfold molar excess over AD-P-TM caused both the fluorescence emission intensities and fluorescence polarization values of samples to increase. In the presence of actin, the wavelength of maximal emission was shifted to shorter values by about 5 to 7 nm. These changes indicate that actin does bind to AD-P-TM and that the binding affects the environment of the label, both by making it more hydrophobic and by reducing the freedom of the label to tumble in solution.     

Assessment of platelet production with 75Se selenomethionine

Incorporation of intravenously injected ⁷⁵Se selenomethionine into platelets has been found to vary with alterations in the rate of platelet production. It appears to label newly produced platelets during their formation in megakaryocytes and provides a method by which thrombopoiesis may be studied in vivo. This technique may be applicable to clinical studies of disordered platelet production.     

Determinants of platelet activation in hypertensives with microalbuminuria

Microalbuminuria is a predictor of adverse outcome in hypertension. We evaluated in vivo platelet activation, by urinary 11-dehydrothromboxane (TX)B₂ and plasma P-selectin, in hypertensives with or without microalbuminuria, and its possible association with oxidative stress, by urinary 8-iso-prostaglandin (PG)F_2α and endothelial dysfunction. Sixty essential hypertensive patients, with (n = 30) or without (n = 30) microalbuminuria, and 30 controls were studied. Endothelial function was assessed by nitric oxide products, intercellular adhesion molecule (ICAM)-1, and asymmetric dimethylarginine (ADMA) levels. Urinary 11-dehydro-TXB₂ excretion was higher in microalbuminuric (median 805 pg/mg creatinine) compared to nonmicroalbuminuric patients or controls (414 and 291 pg/mg, respectively; P < 0.0001). Plasma P-selectin was significantly higher in patients with microalbuminuria (median 136 ng/ml) as compared to those without microalbuminuria or controls (85 and 65 ng/ml; P < 0.0001). Urinary 8-iso-PGF_2α excretion was also enhanced in microalbuminuric (median 279 pg/mg creatinine) compared to nonmicroalbuminuric patients or controls (157 and 146 pg/mg, respectively; P < 0.0001). A significant impairment in endothelial function was found in microalbuminuric patients, with decreased nitric oxide and increased ICAM-1 and ADMA levels. Multivariate regression analysis showed that urinary 8-iso-PGF_2α excretion (beta = 0.49; P < 0.0001) and microalbuminuria (beta = 0.36; P < 0.001) were independently related to 11-dehydro-TXB₂ in hypertensives. Vitamin E supplementation (900 mg daily for 1 month) in 10 hypertensives with microalbuminuria was associated with normalization in median 11-dehydro-TXB₂ and 8-iso-PGF_2α. We conclude that lipid peroxidation is a major determinant of persistent platelet activation in hypertensive patients with microalbuminuria.     

The interaction of thrombospondin with platelet glycoprotein GPIIb-IIIa

The interaction of human platelet thrombospondin (TSP) with human platelet glycoproteins GPIIb-IIIa was studied using a solid-phase binding assay. Polystyrene test tubes were coated with TSP, and 125I-labeled GPIIb-IIIa was added, allowed to bind, and the bound radioactivity was measured. After 90 min, the binding became time independent, and in most experiments, more than 10% of the exogenously added radioactivity was bound to the tube. Analysis of the bound radioactivity by polyacrylamide gel electrophoresis and autoradiography indicated that it was from labeled GPIIb-IIIa. Several lines of evidence indicate that the binding of GPIIb-IIIa to TSP was specific. (a) TSP immobilized on plastic or Sepharose bound 3-10-fold more GPIIb-IIIa than immobilized bovine serum albumin. (b) Addition of unlabeled excess GPIIb-IIIa reversed the binding of 125I-labeled GPIIb-IIIa to immobilized TSP. (c) Addition of EDTA inhibited the binding of GPIIb-IIIa to TSP by more than 90%, whereas addition of 1 mM CaCl2 and 1 mM MgCl2 potentiated the binding by more than 100%. (d) Monoclonal antibodies against TSP and GPIIb-IIIa inhibited the binding by 30-70% as compared with control and polyclonal anti-fibrinogen anti-serum. (e) A plot of GPIIb-IIIa bound versus GPIIb-IIIa added was best described as a rectangular hyperbola by regression analysis with half-saturation at 60 ng/ml GPIIb-IIIa. Similar results were obtained when labeled TSP was added to tubes coated with GPIIb-IIIa. These results show that TSP and GPIIb-IIIa can specifically interact in vitro and suggest that GPIIb-IIIa may function as a platelet TSP receptor during platelet aggregation.     

Interaction of platelet factor 4 with human platelets

Human washed resting platelets bound 125I-labeled platelet factor 4 in a reaction which was saturable and approached equilibrium within 15-30 min. Scatchard plot analysis of the binding isotherms suggested a single class of specific binding sites. Excess of unlabeled protein and low- and high-affinity heparin competed for platelet factor 4 binding sites on the platelet surface and caused a partial displacement of this molecule. Anti-platelet factor 4 Fab fragments caused inhibition of binding of 125I-platelet factor 4 to platelets. Most of the labeled platelet factor 4 which was bound to intact platelets was recovered in the Triton X-100-insoluble cytoskeletal fraction prepared from the same platelets after their stimulation by thrombin. The association with the cytoskeleton was inhibited by anti-platelet factor 4 Fab fragments and by low-affinity heparin. Anti-platelet factor 4 125I-labeled Fab fragments bound to resting platelets, and this binding was greatly increased following platelet stimulation with thrombin. This suggested that endogenously secreted platelet factor 4 also binds to the platelet surface. No significant binding to platelets of 125I-labeled beta-thromboglobulin and 125I-labeled anti-beta-thromboglobulin Fab fragments was observed. Fab fragments of monospecific anti-human platelet factor 4 antibody raised in rabbits inhibited platelet aggregation and secretion induced by low concentrations of thrombin. Fab fragments of anti-beta-thromboglobulin antibody had no inhibitory effect. We suggest that the binding of alpha-granule-derived platelet factor 4 to the specific sites on the surface of platelets may modulate platelet aggregation and secretion induced by low levels of platelet agonists.     

Biological activity of lipids of pine pollen on platelet aggregation in correlation with the platelet activating factor

Pollen lipids of a pine species were separated by thin layer chromatography systems. The purified neutral and polar lipid classes were examined for their possible platelet aggregation activity and for their effect on Platelet Activating Factor activity. The lipid fraction comigrating on thin layer chromatography with glycerylether standards was shown to have a remarkable inhibition of Platelet Activating Factor activity on washed rabbit platelets in a concentration of 4.5.10(-6) M. At a ten fold higher concentration these lipids also induced platelet aggregation.     

Disturbances of platelet function in patients with multiple myeloma

Coagulation disturbances in patients with multiple myeloma are presented. Platelet disfunction was especially observed, but only in patients with marked hyperproteinemia and a high level of immunoglobulins in plasma, whereas normal adhesion and aggregation were noticed in 3 out of 17 patients without protein disturbances. Chemotherapy as well as plasma exchange caused not only a decrease of the paraprotein level in plasma, but also a normalization of the failure previously observed in platelet function. Our experiments confirm the opinion that protein M in plasma is responsible for the disturbances observed in multiple myeloma.     

Isolation of human platelet plasma membranes with polylysine beads

Human platelet plasma membranes were isolated with polylysine beads according to the technique developed by Jacobson and Branton (1977, Science [Wash. D. C.] 195:302--304). Lactoperoxidase-catalyzed surface iodination revealed that ninefold greater 125I specific activity was associated with the membranes isolated on beads than with whole platelets. Enrichment in the bead membrane preparation of the activities of membrane marker enzymes, bis(p-nitrophenyl)phosphate phosphodiesterase and Na,K-ATPase, was 8.0 and 4.4, respectively. Contamination with enzymes of other organelles, cytochrome oxidase and beta-glucuronidase, was relatively low as compared with membranes isolated by sucrose gradient centrifugation. Analysis by SDS polyacrylamide gel electrophoresis showed that a full complement of surface glycoproteins was present on the membranes isolated with polylysine beads. The polylysine bead technique is a rapid, reproducible and efficient method for the preparation of relatively pure platelet plasma membranes.