scholarly journals Affinity labelling of the Ca2+-activated neutral proteinase (calpain) in intact human platelets

1993 ◽  
Vol 289 (1) ◽  
pp. 93-99 ◽  
Author(s):  
J Anagli ◽  
J Hagmann ◽  
E Shaw
Keyword(s):  
Human Platelets ◽  
Actin Binding ◽  
Subsequent Degradation ◽  
Affinity Labelling ◽  
Intracellular Labelling ◽  
Diazomethyl Ketones ◽  
Fluoromethyl Ketone ◽  
Calpain Inhibitors ◽  
Major Target

Two irreversible calpain inhibitors, benzyloxycarbonyl (Cbz)-Leu-Leu-Tyr-Ch2F and Cbz-Leu-Leu-Tyr-CHN2, were shown earlier [Anagli, Hagmann and Shaw (1991) Biochem. J. 274, 497-502] to penetrate intact platelets and to inactivate calpain. This permitted an evaluation of certain functions attributed to this proteinase. For example, in platelets pretreated with these inhibitors, talin and actin-binding protein were protected from subsequent degradation when the Ca2+ level was raised. On the other hand, additional properties of stimulated platelets attributed to calpain remained unaffected by this treatment, and such hypotheses may be dismissed. Radioiodinated inhibitors permitted confirmation of the labelling of calpain by the procedures used. Although Cbz-Leu-Leu-Tyr-CHN2 is more effective in vitro than the corresponding fluoromethyl ketone, we now show that the latter penetrates more readily. These two inhibitors, and two additional ones, t-butyloxycarbonyl-Val-Lys(Cbz)-Leu-Tyr- CHN2 and Cbz-Leu-Tyr-CH2F, have been radioiodinated to permit a comparison of their intracellular labelling patterns in activated platelets. Calpain is the major target of all four inhibitors. Although they are closely related peptide structures, variations with respect to the labelling of additional proteins were observed. These were minor in the case of the peptidyl diazomethyl ketones, but were major in the case of the fluoromethyl ketones. However, in contrast to calpain, this labelling was neither time-dependent nor Ca(2+)-dependent. Radiolabelling and cellular fractionation studies were used to localize active calpain during platelet activation. Calpain appears to be activated in the cytosol and translocated to the membrane or cytoskeletal sites.

scholarly journals Regulation of F-Actin Binding to Platelet Moesin In Vitro by Both Phosphorylation of Threonine 558 and Polyphosphatidylinositides

1999 ◽  
Vol 10 (8) ◽  
pp. 2669-2685 ◽  
Author(s):  
Fumihiko Nakamura ◽  
Laiqiang Huang ◽  
Kersi Pestonjamasp ◽  
Elizabeth J. Luna ◽  
Heinz Furthmayr
Keyword(s):  
Kinase Inhibitor ◽  
Human Platelets ◽  
Actin Binding ◽  
Calyculin A ◽  
High K ◽  
Triton X ◽  
Cellular Control ◽  
Actin Binding Site ◽  
Nonionic Detergents

Activation of human platelets with thrombin transiently increases phosphorylation at558threonine of moesin as determined with phosphorylation state-specific antibodies. This specific modification is completely inhibited by the kinase inhibitor staurosporine and maximally promoted by the phosphatase inhibitor calyculin A, making it possible to purify the two forms of moesin to homogeneity. Blot overlay assays with F-actin probes labeled with either [32P]ATP or125I show that only phosphorylated moesin interacts with F-actin in total platelet lysates, in moesin antibody immunoprecipitates, and when purified. In the absence of detergents, both forms of the isolated protein are aggregated. Phosphorylated, purified moesin co-sediments with α- or β/γ-actin filaments in cationic, but not in anionic, nonionic, or amphoteric detergents. The interaction affinity is high (Kd, ∼1.5 nM), and the maximal moesin:actin stoichiometry is 1:1. This interaction is also observed in platelets extracted with cationic but not with nonionic detergents. In 0.1% Triton X-100, F-actin interacts with phosphorylated moesin only in the presence of polyphosphatidylinositides. Thus, both polyphosphatidylinositides and phosphorylation can activate moesin’s high-affinity F-actin binding site in vitro. Dual regulation by both mechanisms may be important for proper cellular control of moesin-mediated linkages between the actin cytoskeleton and the plasma membrane.

scholarly journals Focal adhesion kinase (pp125FAK) cleavage and regulation by calpain

1996 ◽  
Vol 318 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Prasad COORAY ◽  
Yuping YUAN ◽  
Simone M SCHOENWAELDER ◽  
Christina A MITCHELL ◽  
Hatem H SALEM ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Receptor Tyrosine Kinase ◽  
Human Platelets ◽  
Calpain Inhibitor ◽  
Ionophore A23187 ◽  
Dramatic Reduction ◽  
Calpain Inhibitors ◽  
Autokinase Activity

Focal adhesion kinase (125 kDa form; pp125FAK) is a widely expressed non-receptor tyrosine kinase that is implicated in integrin-mediated signal transduction. We have identified a novel means of pp125FAK regulation in human platelets, in which this kinase undergoes sequential proteolytic modification from the native 125 kDa form to 90, 45 and 40 kDa fragments in thrombin-, collagen- and ionophore A23187-stimulated platelets. The proteolysis of pp125FAK was prevented by pretreating platelets with the calpain inhibitors calpeptin or calpain inhibitor-1, and was reproduced in vitro by incubating immunoprecipitated pp125FAK with purified calpain. Proteolysis of pp125FAK resulted in a dramatic reduction in its autokinase activity and led to its dissociation from the cytoskeletal fraction of platelets. These studies define a novel signal-terminating role for calpain, wherein proteolytic modification of pp125FAK attenuates its autokinase activity and induces its subcellular relocation within the cell.

scholarly journals Investigation of the role of calpain as a stimulus-response mediator in human platelets using new synthetic inhibitors

1991 ◽  
Vol 274 (2) ◽  
pp. 497-502 ◽  
Author(s):  
J Anagli ◽  
J Hagmann ◽  
E Shaw
Keyword(s):  
Cell Shape ◽  
Human Platelets ◽  
Model System ◽  
Actin Binding ◽  
Platelet Response ◽  
Calpain Activity ◽  
Stimulus Response ◽  
Affinity Labelling

A series of peptidyl diazomethanes and monofluoromethane with structures specific for calpain have been synthesized and tested for their ability to inhibit calpain activity in vivo, using human platelets as a model system. Calpain activity in vivo was determined by observing proteolysis of actin-binding protein and talin, two known substrates of calpain. Very potent inhibitors, which emerged from this study, were used to investigate the role of calpain in some platelet response processes. Our results show that calpain-mediated proteolysis in platelets is not an obligatory event leading to change of cell shape, adhesion to glass and spreading, aggregation and 5-hydroxytryptamine release. Two of the inhibitors were iodinated with 125I and used to radiolabel the enzyme in vivo. To our knowledge, this work also represents the first report describing the affinity labelling of calpain in human platelets using irreversible radioactive inhibitors.

Increased Protein Synthesis by Human Platelets during Phagocytosis of Latex Particles in Vitro

1976 ◽  
Vol 35 (02) ◽  
pp. 350-357 ◽  
Author(s):  
Hana Bessler ◽  
Galila Agam ◽  
Meir Djaldetti
Keyword(s):  
Protein Synthesis ◽  
Fold Increase ◽  
Human Platelets ◽  
Polystyrene Latex ◽  
Latex Particles ◽  
The Third ◽  
Platelet Protein ◽  
Dose Dependent ◽  
Phagocytotic Activity

SummaryA three-fold increase of protein synthesis by human platelets during in vitro phagocytosis of polystyrene latex particles was detected. During the first two hours of incubation, the percentage of phagocytizing platelets and the number of latex particles per platelet increased; by the end of the third hour, the first parameter remained stable, while the number of latex particles per cell had decreased.Vincristine (20 μg/ml of cell suspension) inhibited platelet protein synthesis. This effect was both time- and dose-dependent. The drug also caused a decrease in the number of phagocytizing cells, as well as in their phagocytotic activity.

The Interrelationship between Thromboxane Biosynthesis, Aggregation and 5-Hydroxytryptamine Secretion in Human Platelets in Vitro

1980 ◽  
Vol 43 (01) ◽  
pp. 038-040 ◽  
Author(s):  
L C Best ◽  
T K Holland ◽  
P B B Jones ◽  
R G G Russell
Keyword(s):  
Human Platelet ◽  
Human Platelets ◽  
Dense Granule ◽  
Thromboxane B2 ◽  
Essential Requirement ◽  
Direct Mechanism ◽  
Thromboxane Synthetase ◽  
Thromboxane Production ◽  
Higher Doses

SummaryPlatelet aggregation, secretion of 5-hydroxy tryptamine and production of thromboxane B2 were monitored simultaneously in human platelet suspensions in the absence and presence of cyclooxygenase or thromboxane synthetase inhibitors. Aggregation, secretion and thromboxane B2 formation in response to either sodium arachidonate or epinephrine were blocked by aspirin or by 1-N-butyl imidazole suggesting that thromboxane biosynthesis was an essential requirement for platelet activation by these agents. In contrast, thrombin and collagen could apparently induce aggregation and secretion via two pathways: at low doses involving thromboxane production, but at higher doses by a direct mechanism independent of thromboxane biosynthesis. In the case of ADP, inhibition of thromboxane production blocked secretion but had little effect on aggregation, indicating that secretion was probably dependent on thromboxane biosynthesis which probably occurred as a result of aggregation. Thus it appears that although the processes of thromboxane production, release of dense granule constituents and aggregation may often be intimately linked, each process can occur independently of the other, depending upon the stimulus used.

The Effects of Brinolase (Fibrinolytic Enzyme from Aspergillus Oryzae) on Platelet Aggregation of Dog and Man

1972 ◽  
Vol 28 (01) ◽  
pp. 031-048 ◽  
Author(s):  
W. H. E Roschlau ◽  
R Gage
Keyword(s):  
Platelet Aggregation ◽  
Aspergillus Oryzae ◽  
Degradation Products ◽  
Blood Platelet ◽  
Human Platelets ◽  
Fibrinolytic Enzyme ◽  
Inhibitory Effects ◽  
Blood Platelet Aggregation

SummaryInhibition of blood platelet aggregation by brinolase (fibrinolytic enzyme from Aspergillus oryzae) has been demonstrated with human platelets in vitro and with dog platelets in vivo and in vitro, using both ADP and collagen as aggregating stimuli. It is suggested that the optimal inhibitory effects of brinolase occur indirectly through the generation of plasma fibrinogen degradation products, without compromising platelet viability, rather than by direct proteolysis of platelet structures.

Effects of Bupranolol, a New β-Blocker, on Platelet Functions of Rabbit and Human in Vitro

1976 ◽  
Vol 36 (02) ◽  
pp. 376-387 ◽  
Author(s):  
Teruhiko Umetsu ◽  
Kazuko Sanai ◽  
Tadakatsu Kato
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Human Platelets ◽  
Rabbit Platelet ◽  
Rabbit Platelets ◽  
Β Blocker ◽  
Platelet Aggregates ◽  
Induced Aggregation ◽  
Platelet Functions

SummaryThe effects of bupranolol, a new β-blocker, on platelet functions were investigated in vitro in rabbits and humans as compared with propranolol, a well-known β-blocker. At first, the effect of adrenaline on ADP-induced rabbit platelet aggregation was studied because adrenaline alone induces little or no aggregation of rabbit platelets. Enhancement of ADP-induced rabbit platelet aggregation by adrenaline was confirmed, as previously reported by Sinakos and Caen (1967). In addition the degree of the enhancement was proved to be markedly affected by the concentration of ADP and to increase with decreasing concentration of ADP, although the maximum aggregation (percent) was decreased.Bupranolol and propranolol inhibited the (adrenaline-ADP-)induced aggregation of rabbit platelets, bupranolol being approximately 2.4–3.2 times as effective as propranolol. Bupranolol stimulated the disaggregation of platelet aggregates induced by a combination of adrenaline and ADP, but propranolol did not. Platelet adhesion in rabbit was also inhibited by the β-blockers and bupranolol was more active than propranolol. With human platelets, aggregation induced by adrenaline was inhibited by bupranolol about 2.8–3.3 times as effectively as propranolol.From these findings. We would suggest that bupranolol might be useful for prevention or treatment of thrombosis.

Reaction of Acetaldehyde with Human Platelets

1992 ◽  
Vol 67 (01) ◽  
pp. 126-130 ◽  
Author(s):  
Olivier Spertini ◽  
Jacques Hauert ◽  
Fedor Bachmann
Keyword(s):  
Platelet Aggregation ◽  
Inhibitory Action ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Reaction Medium ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Neutral Ph

SummaryPlatelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37° C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100° C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (<0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (≥0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 pM AcH.AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation.SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.This in vitro study shows that AcH has a major inhibitory action on platelet aggregation and may account for the prolonged ex vivo inhibition of aggregation observed in chronic alcoholics even in the absence of alcoholemia.

Thrombin Stimulated Platelet Accumulation on Protein Coated Glass Capillaries: Role of Adhesive Platelet α-Granule Proteins

1989 ◽  
Vol 62 (03) ◽  
pp. 989-995 ◽  
Author(s):  
Juliette N Mulvihill ◽  
J Andrew Davies ◽  
Florence Toti ◽  
Jean-Marie Freyssinet ◽  
Jean-Pierre Cazenave
Keyword(s):  
Human Platelets ◽  
Capillary Perfusion ◽  
Coated Glass ◽  
Bovine Collagen ◽  
Glass Capillaries ◽  
Human Thrombin ◽  
Platelet Accumulation ◽  
Granule Proteins ◽  
Von Willebrand

SummaryThe generation of trace amounts of thrombin at artificial surfaces in contact with blood is likely to be a contributing factor in thrombosis on biomaterials. Using an in vitro capillary perfusion system, platelet accumulation on glass surfaces, uncoated or precoated with purified bovine collagen or human plasma proteins, was determined in the presence or absence of preadsorbed purified human thrombin. Static adsorption for 15 min at 22° C from solutions of thrombin 100 NIH units (33 μg)/ml gave surface concentrations in the range 0.019-0.101 μg/cm2. Protein coated capillaries, thrombin treated or untreated, were perfused for 2 min at 37° C with suspensions of washed 111In-labeled human platelets in Tyrode's-albumin buffer containing 40% washed red blood cells, under conditions of controlled, non pulsatile laminar flow (50 s−1 or 2,000 s−1). Platelet accumulation was increased in the presence of surface adsorbed thrombin on uncoated and albumin or fibrinogen coated glass but little affected on fibronectin or collagen coated glass. On von Willebrand factor (vWF) coated glass, thrombin enhancement was observed only at high shear forces. In experiments using antibodies against human platelet α-granule proteins, thrombin stimulated platelet deposition in uncoated glass capillaries was inhibited at 2,000 s−1 by anti-vWF and to a lesser extent by anti-fibrinogen but not by antithrombospondin antibodies.

Binding of Human Platelet Glycoprotein lb and Actin to Fragments of Actin-Binding Protein

1992 ◽  
Vol 67 (02) ◽  
pp. 252-257 ◽  
Author(s):  
Anne M Aakhus ◽  
J Michael Wilkinson ◽  
Nils Olav Solum
Keyword(s):  
Actin Filaments ◽  
High Speed ◽  
Binding Protein ◽  
Protein A ◽  
Actin Binding ◽  
Platelet Glycoprotein ◽  
Actin Binding Protein ◽  
Crossed Immunoelectrophoresis ◽  
Triton X ◽  
Calpain Inhibitors

SummaryActin-binding protein (ABP) is degraded into fragments of 190 and 90 kDa by calpain. A monoclonal antibody (MAb TI10) against the 90 kDa fragment of ABP coprecipitated with the glycoprotein lb (GP lb) peak observed on crossed immunoelectrophoresis of Triton X-100 extracts of platelets prepared without calpain inhibitors. MAb PM6/317 against the 190 kDa fragment was not coprecipitated with the GP lb peak under such conditions. The 90 kDa fragment was adsorbed on protein A agarose from extracts that had been preincubated with antibodies to GP lb. This supports the idea that the GP Ib-ABP interaction resides in the 90 kDa region of ABP. GP lb was sedimented with the Triton-insoluble actin filaments in trace amounts only, and only after high speed centrifugation (100,000 × g, 3 h). Both the 190 kDa and the 90 kDa fragments of ABP were sedimented with the Triton-insoluble actin filaments.

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