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Biomedical subjects

M B Zucker

Publications and source records attributed to M B Zucker.

At least 37 records · Page 2Linked to original sources
PubMed1986

Protease-induced immunoregulatory activity of platelet factor 4.

Intravenous injection of human or mouse serum or platelet material secreted from appropriately stimulated platelets ("releasate") together with antigen alleviates the immunosuppression in SJL/J mice induced by injection of irradiated lymphoma cells or in (CB6)F1 mice induced by injection of concanavalin A. We now report that injection of releasate from 10(6) human platelets restores plaque-forming cells to the unsuppressed number; greater amounts increase responses further. Immunoregulatory activity is released from platelets exposed to thrombin in parallel with other alpha-granule components. Heparin-agarose absorbs activity. Purified platelet factor 4 (PF4) has activity; beta-thromboglobulin and platelet-derived growth factor have little or none. Activity in serum is neutralized by goat anti-human PF4. An enzymatic step is necessary for production of immunoregulatory activity. Releasates boiled immediately after platelet aggregation with 250 nM A23187 or those produced by adding A23187 in the presence of 100 microM serine protease inhibitor (p-amidinophenyl)methanesulfonyl fluoride (APMSF) are ineffective, whereas releasates boiled or mixed with APMSF after incubation for 60 min are active. Activity is generated by incubating a mixture of heparin-absorbed releasate (as enzyme source) and heparin-agarose eluate of releasate made in the presence of APMSF (as substrate source). The enzymatic step does not alter the heparin-neutralizing activity of PF4. Apparently a secreted platelet protease converts PF4 to a form with immunoregulatory activity.

Animals↗
PubMed1986

Binding of thrombin-activated human factor VIII to platelets.

To study association of platelets with factor VIII, the purified protein was 125I-labelled with Bolton-Hunter reagent to a specific activity of 243,000-360,000 cpm/U. Autoradiographs of SDS polyacrylamide gels revealed polypeptides of VIII at Mr about 240 kDa, 90 kDa and intermediate values, as well as some radioactive contaminants, but the light chain (Mr 78/76) seen with silver stain was not labelled. After 2.5-5-fold activation with thrombin, the higher radioactive Mr band disappeared, the band at 90 kDa became more intense, and a band appeared at about 45 kDa. The radioactivity associated with platelets, studied in the presence of haemophilic BaSO4-treated plasma, was maximal after 6-8 min and increased 3-15-fold on activation with thrombin. With activated VIII, autoradiographs of platelet pellets showed only VIIIa but results are expressed as units of unactivated VIII bound. At 0.3-0-0.7 U/ml, 10(8) platelets bound 0.0008-0.004 U VIIIa. The amount bound was not affected by the ratio of unlabelled VIII to VIII labelled in the presence of a 50-fold molar excess of unlabelled Bolton-Hunter reagent. Binding increased to 1.5 U VIIIa/10(8) platelets (about 13,600 molecules per platelet) at 140 U/ml, with no evidence of saturation. Binding was not affected by monoclonal antibodies to platelet glycoproteins IIb/IIIa or Ib, quenching the thrombin before adding platelets, or aggregating the platelets with A23187 in the presence of thrombin. Qualitatively, binding of labelled VIIIa and factor Va studied by others are similar. Binding of 125I-VIIIa to aggregated and unaggregated platelets was normal in patient M.S. whose platelets were shown by others to be deficient in their ability to bind radiolabelled factor Xa and generate coagulant activity. This difference. and the fact that platelet coagulant activity is increased by platelet activation and/or aggregation, suggest that binding of Va or VIIIa alone does not determine the assembly of active proteolytic complexes on the platelet surface.

Autoradiography↗
PubMed1985

A platelet-derived immunoregulatory serum factor with T cell affinity.

A factor in mouse and human serum which enhances immune responses to SRBC and TNP-Ficoll in mice has been described. This factor completely reverses immunosuppression induced by syngeneic lymphoma (RCS) or allogeneic lymph node cells. T lymphocytes bind this factor, since the activity can be removed from serum by absorption with T cell containing spleen cells or cloned cytotoxic T lymphocytes. Furthermore, the factor is active only in the presence of mature T cells. Platelet alpha-granules seem to be the source of this serum factor because 1) the activity is lacking in plasma, in serum prepared in the absence of platelets, and in serum prepared from a patient lacking platelet alpha-granules and 2) isolated mouse or human platelets release an immunostimulatory factor with very similar properties upon incubation with thrombin.

Animals↗
PubMed1985

Platelet cytoskeleton alpha-actinin in normal and thrombasthenic platelets: distribution and immunologic characterization.

The subcellular localization of alpha-actinin (Mr 100,000) in human skeletal muscle is restricted to the Z line, in which it is believed to anchor actin filaments. Recently, this protein was identified in normal and thrombasthenic human platelets by its antigenic cross-reaction with antibodies to chicken gizzard alpha-actinin. In our study, the biochemical interaction between purified platelet alpha-actinin and striated muscle F-actin was examined by electron microscopy of negatively stained preparations. Like its muscle counterpart, platelet alpha-actinin promotes the cross-linking and bundling of actin filaments. Antibodies prepared to human platelet alpha-actinin cross-reacted with chicken gizzard alpha-actinin as shown by immunoelectrophoresis and the western blotting technique. Immunoblots prepared with normal and thrombasthenic platelets with antibodies to human platelet alpha-actinin revealed that this protein is susceptible to proteolysis. Extracts of freshly drawn platelets showed a protein band of 100 K. When the platelet extracts were incubated at 37 degrees C for various times, the immunoblots showed protein bands of 100 and 80 K. The proportion of the 80 K protein band increased with incubation time. This proteolysis can be prevented by chelating agents such as EDTA or the protease inhibitor leupeptin. Indirect immunofluorescent studies of human skin fibroblasts with antibodies to chicken gizzard actin and human skeletal muscle, chicken gizzard, and platelet alpha-actinin revealed the staining pattern characteristic of each protein. The distribution of alpha-actinin in normal and thrombasthenic platelets was assessed by ferritin-labeled immunoelectron microscopy. Ferritin particles were found in the cytoplasm immediately below the membrane and in some granules. There was no labeling associated with the mitochondria.

Actinin↗
PubMed1984

Evidence that von Willebrand factor is not required for the clotting of plasma in the presence of platelets and kaolin (Hardisty-Hutton test).

Antihemophilic factor (AHF) essentially free of von Willebrand factor (vWF) was used to determine whether vWF affected the coagulant activity of AHF in a kaolin-activated system using platelet-rich plasma. The AHF was prepared by sequential chromatography on solid-phase polyelectrolyte (PE E-5), and on Sepharose CL-4B in the presence of a high concentration of CaCl2. Residual traces of vWF antigen were removed by affinity chromatography with immobilized anti-vWF IgG. The essentially vWF-free AHF corrected the clotting defect of plasma from three patients with severe von Willebrand's disease (vWD) as measured by the kaolin-activated clotting time in the presence of normal or vWD platelets ( Hardisty - Hutton test). Addition of hemophilic plasma as a source of vWF did not cause additional improvement, nor did a potent antibody to vWF raised in rabbits inhibit the ability of AHF to shorten the clotting time of vWD plasma. Thus we found no evidence that vWF is necessary for AHF to function in the coagulation of recalcified kaolin-activated vWF-deficient platelet-rich plasma.

Blood Coagulation↗
PubMed1984

Platelet aggregation caused by dithiothreitol.

MacIntyre et al. showed that over 1 mM dithiothreitol (DTT) aggregates blood platelets in the presence of fibrinogen; aggregation is not inhibited by prostaglandin E1. We confirmed their data and found that 70 mM 2-mercaptoethanol was also active. DDT-induced aggregation was not associated with platelet shape change or secretion of dense granule contents, was not inhibited by tetracaine or metabolic inhibitors, was prevented at pH 6.5, and prevented, reversed, or arrested by EDTA, depending on when the EDTA was added. DTT did not cause aggregation of thrombasthenic, EDTA-treated, or cold (0 degree C) platelets, which also failed to aggregate with ADP. Platelets stimulated with DTT bound 125I-labeled fibrinogen. Thus DTT appears to "expose" the fibrinogen receptors. SDS gel electrophoresis of platelet fractions prepared by use of Triton X-114 showed that aggregating concentrations of DTT reduced proteins of apparent Mr 69,000 and 52,000 (probably platelet albumin) and, to a variable extent, glycoproteins Ib, IIb and III. Exposure of unlabeled or 125I-labeled platelets to ADP had no discernible effect on the electrophoretic patterns.

Adenosine Diphosphate↗
PubMed1983

The combining ability of glycoproteins IIb, IIIa and Ca2+ in EDTA-treated nonaggregable platelets.

Platelets deprived of calcium and incubated at 37 degrees C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated nonaggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X-114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37 degrees C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37 degrees C.

Blood Platelets↗
PubMed1983

Identification of fibrinogen derivatives in the Triton-insoluble residue of human blood platelets.

Several proteins (eg. actin, myosin, and actin-binding protein) in the Triton-insoluble residue of thrombin-stimulated platelets are important in the formation of cytoskeletal structures. Electrophoretic analyses have shown that unidentified protein bands of 68,000, 55,000, and 48-50,000 daltons are also present in larger amounts after thrombin stimulation. Since these molecular weights correspond roughly to those of the alpha, beta, and gamma chains of fibrin, and since fibrinogen is found in platelet alpha-granules, these bands were compared to those obtained when purified fibrinogen was treated with thrombin, exposed to 1% Triton X-100-5 mM EGTA, and the resultant Triton-insoluble residue sedimented. Identification of the 68,000-, 55,000-, and 48-50,000-dalton bands as fibrinogen derivatives was confirmed by identifying them in comigration studies and in autoradiographs of Triton-insoluble residues of platelets that were electrophoretically transferred to nitrocellulose paper and treated with antifibrinogen antibody and 125I-protein A. Furthermore, if the platelet suspension was treated with thrombin in the presence of calcium ions, protein bands characteristic of the action of Factor XIII on fibrin were observed, active platelet Factor XIII apparently having been made available by lysis of platelets during preparations. Making use of the electrophoretic properties of tubulin recently described by Best et al [1981], comigration studies using hog brain tubulin indicated that tubulin is not present in significant amounts in the Triton-insoluble residue of platelets as previously suggested. The identification of these proteins as fibrinogen derivatives does not demonstrate a physiological interaction between fibrin and the platelet cytoskeleton, since fibrin is Triton-insoluble and can be pelleted even in the absence of platelet cytoskeletons.

Blood Platelets↗
PubMed1982

Non-decalcified barium sulfate-adsorbed plasma. A potentially useful reagent for studying blood clotting, platelets or complement.

Plasma was prepared by rapid centrifugation of native blood followed by adsorption of vitamin K-dependent clotting factors with BaSO4. Compared to fresh citrated plasma, BaSO4-treated plasma contained about 80% of factor V and factor VII and the same concentration of fibrinopeptide A. Assays were also carried out after overnight incubation of citrated plasma and BaSO4-adsorbed plasma with and without added citrate and compared to assays of fresh citrated plasma. Factor V decreased to about 25% in both citrated samples, factor VIII decreased to 45% in both samples of BaSO4-treated plasma, and fibrinopeptide A did not change. Thus loss of factor V activity depended on reduction in divalent cation concentration whereas loss of factor VIII activity may have resulted from effects of early traces of thrombin.

Adsorption↗
PubMed1980

Relationship of ADP-induced fibrinogen binding to platelet shape change and aggregation elucidated by use of colchicine and cytochalasin B.

ADP causes human, aspirin-treated, gel-filtered platelets to change from their native discoid shape to spiny spheres with pseudopods, bind 125I-labeled fibrinogen, and aggregate if shaken with sufficient fibrinogen. After destruction of the added ADP with the enzyme apyrase, the platelets revert to a disc shape and lose much of their bound fibrinogen. Colchicine (208 muM or 83 microgram/ml) added to ADP-treated platelets before apyrase prevented restoration of the discoid shape but not the loss of bound fibrinogen. It did not inhibit ADP-induced shape change, aggregation, or fibrinogen binding. Cytochalasin B (0.02--0.2 muM or 0.01--0.10 microgram/ml) prevented ADP-induced shape change but not ADP-induced fibrinogen binding or aggregation. Thus, these findings support earlier studies with thrombasthenic and EDTA-treated platelets and with normal platelets at low pH, or in the presence of EDTA to indicate that fibrinogen binding is associated with aggregability but not with platelet shape.

Adenosine Diphosphate↗
PubMed1980

Decreased association of 45calcium with platelets unable to aggregate due to thrombasthenia or prolonged calcium deprivation.

45Calcium uptake was studied with aspirin-treated platelets that were gel-filtered through a column of Sepharose 2B equilibrated with divalent cation-free modified Tyrode's solution to remove readily exchangeable surface-associated calcium. These platelets aggregated almost immediately when exposed to ADP, fibrinogen and at least 30 microM CaCl2. At this calcium ion concentration, 10(8) platelets took up 36.6 +/- SEM 2.7 pmol of 45calcium within 1-2 min. The presence of ADP and fibrinogen did not affect the amount of calcium bound. Over 90% of this platelet-associated calcium was removed by EDTA in 5 min suggesting that it was surface-bound. Calcium uptake increased rapidly for 10 min, then more slowly for up to 2 h. At 60 min, maximal uptake was approached at CaCl2 concentrations between 250 and 300 microM when an average of 276 +/- SEM 18 pmol of calcium was associated with 10(8) platelets. Only 50-60% of this calcium could be removed by EDTA in 5 min, and about 70% in 20 min, suggesting that some of it had been internalized. Platelets from two patients with thrombasthenia that were unable to aggregate took up 50% less calcium than platelets from normal volunteers. Similarly, platelets that had been incubated with EDTA at 37 degrees C, pH 7.8 for 8 min lost the ability to aggregate despite recalcification and took up 40-60% less calcium than CaEDTA-treated controls. Platelets from a patient with the Bernard-Soulier syndrome aggregated and bound calcium normally. Thus the platelets' ability to take up calcium after removal of surface-associated calcium correlates with their ability to aggregate. Since thrombasthenic platelets and platelets rendered incapable of aggregating after prolonged calcium deprivation with EDTA do not bind fibrinogen, we postulate that some of the surface-associated calcium normally binds to the fibrinogen receptors.

Adenosine Diphosphate↗
PubMed1980

Correlation between fibrinogen binding to human platelets and platelet aggregability.

Fibrinogen is essential for aggregating platelets with adenosine diphosphate (ADP) and was recently shown to bind to platelets stimulated with ADP. The present work confirms the specific and saturable nature of the platelet-fibrinogen interaction. Binding of 125iodine-labeled fibrinogen to human gel-filtered platelts was maximal at 1 min, and the receptors were saturated when the fibrinogen concentration in the suspending medium approached 0.8 mg/ml. Assuming that one fibrinogen molecule interacts with a single receptor, experiments with 9 normal donors revealed the presence of 12,896 +/- 2456 receptors per platelet. Much of the bound material dissociated from platelets after incubation with apyrase or EDTA. Binding was markedly inhibited at pH 6.5, in the presence of EDTA, and with platelets from 3 thrombasthenic patients but not with those from a patient with the Bernard-Soulier syndrome. Fibrinogen binding was also virtually absent with platelets that had been incubated with EDTA for 8 min at 37 degrees C and pH 7.8. These platelets could not aggregate when mixed with ADP and adequate CaCl2 and fibrinogen, although they could still change their shape. Thus, ADP-induced binding of fibrinogen correlates with platelet aggregability.

Adenosine Diphosphate↗