Movement of Calcium Ions and their Role in the Activation of Platelets

1978 ◽  
Vol 40 (02) ◽  
pp. 212-218 ◽  
Author(s):  
P Massini ◽  
R Käser-Glanzmann ◽  
E F Lüscher
Keyword(s):  
Plasma Membrane ◽  
Platelet Activation ◽  
Calcium Ions ◽  
Binding Sites ◽  
Shape Change ◽  
Release Reaction ◽  
Dense Bodies ◽  
Activated Platelets ◽  
Different Types ◽  
Ca Ions

SummaryThe increase of the cytoplasmic Ca-concentration plays a central role in the initiation of platelet activation. Four kinds of movements of Ca-ions are presumed to occur during this process: a) Ca-ions liberated from membranes induce the rapid shape change, b) Vesicular organelles release Ca-ions into the cytoplasm which initiate the release reaction, c) The storage organelles called dense bodies, secrete their contents including Ca-ions to the outside during the release reaction, d) At the same time a rearrangement of the plasma membrane occurs, resulting in an increase in its permeability for Ca-ions as well as in an increase in the number of Ca-binding sites.Since most processes occurring during platelet activation are reversible, the platelet must be equipped with a mechanism which removes Ca-ions from the cytoplasm. A vesicular fraction obtained from homogenized platelets indeed accumulates Ca actively. This Ca- pump is stimulated by cyclic AMP and protein kinase; it may be involved in the recovery of platelets after activation.It becomes increasingly clear that the various manifestations of platelet activation are triggered by a rise in the cytoplasmic Ca2+-concentration. The evidence for this and possible mechanisms involved are discussed in some detail in the contributions by Detwiler et al. and by Gerrard and White to this symposium. In this article we shall discuss four different types of mobilization of Ca-ions which occur in the course of the activation of platelets. In addition, at least one transport step involved in the removal of Ca2+ must occur during relaxation of activated platelets.

scholarly journals Movements of Calcium Ions and Their Role in the Activation of Platelets

1977 ◽  
Author(s):  
P. Massini ◽  
R. Käser-Glanzmann ◽  
E.F. Lüscher
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Platelet Activation ◽  
Calcium Ions ◽  
Binding Sites ◽  
Shape Change ◽  
Release Reaction ◽  
Dense Bodies ◽  
Ca Ions

The increase of the cytoplasmic Ca-concentration plays a central role in the initiation of platelet activation. Four kinds of movements of Ca-ions are presumed to occur during this process: (a) Ca-ions liberated from membranes induce the rapid shape change. (b) Vesicular organelles release Ca-ions into the cytoplasm which initiate the release reaction, (c) The storage organelles, called dense bodies, secrete their contents including Ca-ions to the outside during the release reaction, (d) At the same time a rearrangement of the plasma membrane occurs, resulting in an increase in its permeability for Ca-ions as well as in an increase in the number of Ca-binding sites.Since most processes occurring during platelet activation are reversible the platelet must be equipped with a mechanism which removes Ca-ions from the cytoplasm. A vesicular fraction of platelet homogenate indeed accumulates Ca actively. This Ca-pump is stimulated by cyclic AMP and protein kinase; it might be involved in the recovery of platelets after activation.

scholarly journals The effect of aggregation and release on platelet prothrombin- converting activity

Blood ◽  
1979 ◽  
Vol 54 (3) ◽  
pp. 659-672 ◽  
Author(s):  
AC Cox ◽  
P Inyangetor ◽  
CT Esmon ◽  
BN White
Keyword(s):  
Platelet Aggregation ◽  
Prostaglandin E1 ◽  
Dense Body ◽  
Shape Change ◽  
Factor Xa ◽  
Dense Bodies ◽  
Activated Platelets ◽  
Induced Aggregation ◽  
Body Secretion ◽  
Platelet Shape

Abstract Platelets provide a procoagulant activity for the conversion of prothrombin to thrombin during normal hemostatis. This activity designated as platelet prothrombin-converting activity (PPCA) was monitored as rate of thrombin production in a two-stage assay using gel- filtered bovine platelets, factor Xa, and prothrombin. Expression of PPCA was not associated with ADP-induced release or platelet shape change but was associated with aggregation. Release of the contents of dense bodies, measured by release of 14C-5-hydroxytryptamine, was not required for expression of PPCA during platelet aggregation. During the PPCA assay, 5-hydroxytrypamine was released, but only after onset of thrombin production. Furthermore, the release of 5-hydroxytryptamine was retarded during the assay by the addition of 2 mM theophylline and 100 nM prostaglandin E1 without a comparable reduction in PPCA. In addition, 125I-factor-Xa was bound in greater amounts to platelets (aspirin-treated) after ADP-induced aggregation (without detectable release) than to unactivated control platelets. Finally, the PPCA of the ADP-activated platelets was saturated with respect to factors Xa and Va at less than 1 nM concentrations, indicating that the aggregation induced by ADP leads to the exposure of specific procoagulant sites by some process other than dense body secretion.

Changes in G-Actin after Platelet Activation in Platelet Rich Plasma

1992 ◽  
Vol 68 (06) ◽  
pp. 727-730 ◽  
Author(s):  
S Heptinstall ◽  
J Glenn ◽  
P Spangenberg
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Actin Polymerization ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Inhibition Assay ◽  
Activated Platelets ◽  
Lysis Buffer ◽  
Two Phases ◽  
Rapid Conversion

SummaryWe have used the DNase I inhibition assay to study changes in G-actin after platelet activation in platelet-rich plasma (PRP) induced by ADP. Because of problems associated with depolymerization of F-actin after lysis of ADP-activated platelets in the presence of plasma, G-actin was measured using a lysis buffer that contained formaldehyde to prevent any depolymerization of F-actin.Different patterns of response were seen depending on the concentration of ADP used, and these were modified by avoiding aggregation by either not stirring the sample or by adding EDTA. The results show rapid conversion of G-actin to F-actin in association with shape change, and there is a further decrease in G-actin associated with irreversible platelet aggregation. Thus evidence is presented that actin polymerization occurs in two phases after ADP stimulation.

CD63 Associates with the αIIbβ3 Integrin-CD9 Complex on the Surface of Activated Platelets

2001 ◽  
Vol 85 (01) ◽  
pp. 134-141 ◽  
Author(s):  
Eileen McMillan-Ward ◽  
Jeffrey Easton ◽  
Catherine Robertson ◽  
Archibald McNicol ◽  
Sara Israels
Keyword(s):  
Extracellular Matrix ◽  
Plasma Membrane ◽  
Platelet Activation ◽  
Molecular Complexes ◽  
Dense Granule ◽  
Integral Membrane Proteins ◽  
Matrix Proteins ◽  
Activated Platelets ◽  
Αiibβ3 Integrin ◽  
Dependent Interaction

SummaryThe tetraspanins are integral membrane proteins expressed on cell surface and granular membranes of hematopoietic cells and have been identified in multi-molecular complexes with specific integrins. In resting platelets, CD63, a member of the tetraspanin superfamily, is present in dense granule and lysosomal membranes and, following platelet activation, translocates to the plasma membrane. In the present study, platelet activation by thrombin leads to incorporation of CD63 into the Triton-insoluble actin cytoskeletal fraction. This incorporation was inhibited by preincubation of platelets with RGDS or EGTA and did not occur in platelets from a patient with Glanzmann’s thrombasthenia, suggesting that it was dependent upon αIIbβ3. In activated platelets, the anti-CD63 MoAb, D545, co-immunoprecipitated CD63 with other surface-labeled proteins, including αIIbβ3 and another tetraspanin, CD9. The association of CD63 with CD9 and αIIbβ3 was not inhibited by preincubation of platelets with RGDS or EGtA. D545 did not inhibit the adhesion of activated platelets to purified extracellular matrix proteins, but significantly decreased adhesion of thrombin-activated platelets to neutrophils in a rosetting assay. D545 also caused disaggregation of platelets stimulated by ADP, but had no effect on aggregation induced by other agonists. These results are consistent with the proposal that CD63 becomes part of an αIIbβ3-CD9-CD63 integrintetraspanin complex in activated platelets – an association that may modulate the function of αIIbβ3-dependent interaction with other cells such as neutrophils.

scholarly journals The effect of aggregation and release on platelet prothrombin- converting activity

Blood ◽  
1979 ◽  
Vol 54 (3) ◽  
pp. 659-672
Author(s):  
AC Cox ◽  
P Inyangetor ◽  
CT Esmon ◽  
BN White
Keyword(s):  
Platelet Aggregation ◽  
Prostaglandin E1 ◽  
Dense Body ◽  
Shape Change ◽  
Factor Xa ◽  
Dense Bodies ◽  
Activated Platelets ◽  
Induced Aggregation ◽  
Body Secretion ◽  
Platelet Shape

Platelets provide a procoagulant activity for the conversion of prothrombin to thrombin during normal hemostatis. This activity designated as platelet prothrombin-converting activity (PPCA) was monitored as rate of thrombin production in a two-stage assay using gel- filtered bovine platelets, factor Xa, and prothrombin. Expression of PPCA was not associated with ADP-induced release or platelet shape change but was associated with aggregation. Release of the contents of dense bodies, measured by release of 14C-5-hydroxytryptamine, was not required for expression of PPCA during platelet aggregation. During the PPCA assay, 5-hydroxytrypamine was released, but only after onset of thrombin production. Furthermore, the release of 5-hydroxytryptamine was retarded during the assay by the addition of 2 mM theophylline and 100 nM prostaglandin E1 without a comparable reduction in PPCA. In addition, 125I-factor-Xa was bound in greater amounts to platelets (aspirin-treated) after ADP-induced aggregation (without detectable release) than to unactivated control platelets. Finally, the PPCA of the ADP-activated platelets was saturated with respect to factors Xa and Va at less than 1 nM concentrations, indicating that the aggregation induced by ADP leads to the exposure of specific procoagulant sites by some process other than dense body secretion.

scholarly journals Identification of a 33-Kd protein associated with the alpha-granule membrane (GMP-33) that is expressed on the surface of activated platelets

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 372-379
Author(s):  
MJ Metzelaar ◽  
HF Heijnen ◽  
JJ Sixma ◽  
HK Nieuwenhuis
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Binding Sites ◽  
Major Protein ◽  
Activated Platelets ◽  
Granule Membrane ◽  
Microscopic Studies ◽  
Number Of Binding Sites ◽  
Thrombin Activation ◽  
A Minor

To identify antigens on the platelet plasma membrane that are exposed after activation, we developed a monoclonal antibody (MoAb) designated RUU-SP 1.77. The RUU-SP 1.77 antigen is present on the membrane of resting platelets at a basal level and is strongly expressed on the plasma membrane after thrombin activation. Freshly fixed platelets bound 4,150 +/- 1,935 (mean +/- SD) RUU-SP 1.77 molecules per platelet; on fixed thrombin-stimulated platelets the number of binding sites was upregulated to 19,050 +/- 5,120 (kd 4.5 +/- 0.8 nmol/L). MoAb RUU-SP 1.77 recognized a major protein of 33 Kd and a minor 28-Kd protein, both under nonreduced and reduced conditions. Immunoelectron microscopic studies showed the presence of the protein associated with the membrane of alpha-granules. Due to the localization associated with the alpha-granule membrane, we have designated it GMP-33 (granule membrane protein with a molecular weight of 33 Kd). Based on structural properties, we conclude that GMP-33 is a protein associated with the alpha-granule membrane that has not been described before.

Activated Platelets Release Two Types of Membrane Vesicles: Microvesicles by Surface Shedding and Exosomes Derived From Exocytosis of Multivesicular Bodies and -Granules

Blood ◽  
1999 ◽  
Vol 94 (11) ◽  
pp. 3791-3799 ◽  
Author(s):  
Harry F.G. Heijnen ◽  
Anja E. Schiel ◽  
Rob Fijnheer ◽  
Hans J. Geuze ◽  
Jan J. Sixma
Keyword(s):  
Platelet Activation ◽  
Annexin V ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Multivesicular Bodies ◽  
Factor X ◽  
Activated Platelets ◽  
Immuno Electron Microscopy ◽  
Different Types ◽  
Platelet Aggregates

Platelet activation leads to secretion of granule contents and to the formation of microvesicles by shedding of membranes from the cell surface. Recently, we have described small internal vesicles in multivesicular bodies (MVBs) and -granules, and suggested that these vesicles are secreted during platelet activation, analogous to the secretion of vesicles termed exosomes by other cell types. In the present study we report that two different types of membrane vesicles are released after stimulation of platelets with thrombin receptor agonist peptide SFLLRN (TRAP) or -thrombin: microvesicles of 100 nm to 1 μm, and exosomes measuring 40 to 100 nm in diameter, similar in size as the internal vesicles in MVBs and -granules. Microvesicles could be detected by flow cytometry but not the exosomes, probably because of the small size of the latter. Western blot analysis showed that isolated exosomes were selectively enriched in the tetraspan protein CD63. Whole-mount immuno-electron microscopy (IEM) confirmed this observation. Membrane proteins such as the integrin chains IIb-β3 and β1, GPIb, and P-selectin were predominantly present on the microvesicles. IEM of platelet aggregates showed CD63+ internal vesicles in fusion profiles of MVBs, and in the extracellular space between platelet extensions. Annexin-V binding was mainly restricted to the microvesicles and to a low extent to exosomes. Binding of factor X and prothrombin was observed to the microvesicles but not to exosomes. These observations and the selective presence of CD63 suggest that released platelet exosomes may have an extracellular function other than the procoagulant activity, attributed to platelet microvesicles.

scholarly journals Studies with a monoclonal antibody against activated platelets: evidence that a secreted 53,000-molecular weight lysosome-like granule protein is exposed on the surface of activated platelets in the circulation

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 838-845 ◽  
Author(s):  
HK Nieuwenhuis ◽  
JJ van Oosterhout ◽  
E Rozemuller ◽  
F van Iwaarden ◽  
JJ Sixma
Keyword(s):  
Cardiopulmonary Bypass ◽  
Platelet Activation ◽  
Binding Sites ◽  
Double Labeling ◽  
Flow Cytofluorometry ◽  
Activated Platelets ◽  
Normal Individuals

Abstract To define the role of activated platelets we have attempted to prepare monoclonal antibodies specific for activated platelets. The IgG2b antibody of one of the clones, designated 2.28, was studied in more detail. Native platelets from normal individuals bound 650 125I-2.28 molecules/platelet, whereas thrombin-activated platelets bound 12,600 molecules/platelet with high affinity (4.6 nmol/L). Immunoelectrophoretic analysis revealed that 2.28 reacted with a 53,000- mol wt protein. Immunocytochemistry showed that the antigen is located in a special subclass of platelet granules in unstimulated platelets and is exposed on the surface of thrombin-activated platelets. Double- labeling studies with immunogold labels disclosed simultaneous localization of 2.28 binding sites and cathepsin D in the same granules both in megakaryocytes and endothelial cells, thereby indicating that the antigen may be localized in lysosomes. By using flow cytofluorometry, in vivo platelet activation was studied in patients undergoing cardiac surgery with cardiopulmonary bypass. Increased numbers of platelets that expressed the 2.28 antigen on their surface were observed after extracorporeal perfusion. The percentage of 2.28- positive platelets in the circulation was 3.9% +/- 2.7% (SD) in controls (n = 20), 5.5% +/- 3.0% in patients (n = 10) before cardiopulmonary bypass surgery, 24.6% +/- 13.5% after the bypass, and 8.5% in two patients with acute deep venous thrombosis. These data indicate that 2.28 may serve as a useful probe of in vitro and in vivo platelet activation.

scholarly journals Studies with a monoclonal antibody against activated platelets: evidence that a secreted 53,000-molecular weight lysosome-like granule protein is exposed on the surface of activated platelets in the circulation

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 838-845 ◽  
Author(s):  
HK Nieuwenhuis ◽  
JJ van Oosterhout ◽  
E Rozemuller ◽  
F van Iwaarden ◽  
JJ Sixma
Keyword(s):  
Cardiopulmonary Bypass ◽  
Platelet Activation ◽  
Binding Sites ◽  
Double Labeling ◽  
Flow Cytofluorometry ◽  
Activated Platelets ◽  
Normal Individuals

To define the role of activated platelets we have attempted to prepare monoclonal antibodies specific for activated platelets. The IgG2b antibody of one of the clones, designated 2.28, was studied in more detail. Native platelets from normal individuals bound 650 125I-2.28 molecules/platelet, whereas thrombin-activated platelets bound 12,600 molecules/platelet with high affinity (4.6 nmol/L). Immunoelectrophoretic analysis revealed that 2.28 reacted with a 53,000- mol wt protein. Immunocytochemistry showed that the antigen is located in a special subclass of platelet granules in unstimulated platelets and is exposed on the surface of thrombin-activated platelets. Double- labeling studies with immunogold labels disclosed simultaneous localization of 2.28 binding sites and cathepsin D in the same granules both in megakaryocytes and endothelial cells, thereby indicating that the antigen may be localized in lysosomes. By using flow cytofluorometry, in vivo platelet activation was studied in patients undergoing cardiac surgery with cardiopulmonary bypass. Increased numbers of platelets that expressed the 2.28 antigen on their surface were observed after extracorporeal perfusion. The percentage of 2.28- positive platelets in the circulation was 3.9% +/- 2.7% (SD) in controls (n = 20), 5.5% +/- 3.0% in patients (n = 10) before cardiopulmonary bypass surgery, 24.6% +/- 13.5% after the bypass, and 8.5% in two patients with acute deep venous thrombosis. These data indicate that 2.28 may serve as a useful probe of in vitro and in vivo platelet activation.

scholarly journals The role of protein phosphorylation and cytoskeletal reorganization in microparticle formation from the platelet plasma membrane

1994 ◽  
Vol 299 (1) ◽  
pp. 303-308 ◽  
Author(s):  
Y Yano ◽  
J Kambayashi ◽  
E Shiba ◽  
M Sakon ◽  
E Oiki ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Protein Phosphorylation ◽  
Shape Change ◽  
Cytochalasin D ◽  
Cytoskeletal Reorganization ◽  
Phosphatase Inhibitors ◽  
Activated Platelets ◽  
Protein Phosphatase Inhibitors ◽  
Microparticle Formation

Platelets activated by various agonists produce vesicles (microparticles; MPs) from the plasma membrane. However, the mechanism of this MP formation remains to be elucidated. To investigate the possible involvement of protein phosphorylation and cytoskeletal reorganization in MP formation, the effects of various inhibitors on MP formation were investigated. Flow cytometry was employed to detect the amount of MP formation by using monoclonal antibodies against glycoprotein (GP) IIb-IIIa (NNKY 1-32) or GPIIb (Tab). The relationship between changes in cytoskeletal architecture and MP formation in the platelets activated by thrombin plus collagen was observed by scanning electron microscopy (SEM). MPs were observed in the vicinity of the terminals of pseudopods, suggesting that MPs may be related by budding of the pseudopods. Cytochalasin D (10 microM) inhibited MP formation from the activated platelets almost completely. Moreover, SEM of the cytochalasin D-treated platelets revealed the absence of shape change, pseudopod formation and MPs. These findings suggest that cytoskeletal reorganization is necessary for MP formation. Since cytoskeletal reorganization is considered to be regulated by a dynamic phosphorylation-dephosphorylation process, we investigated the effects of the protein phosphatase inhibitors, calyculin A (CLA) and okadaic acid (OA), on MP formation. Flow cytometry showed that these two inhibitors doubled MP formation in activated platelets. SEM of the platelets treated with CLA or OA demonstrated more prominent shape change and pseudopod formation in these platelets than in those without inhibitor. From these results, we conclude that cytoskeletal reorganization, which is controlled by phosphorylation, is involved in MP formation.

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