Different Abilities of Thrombin Receptor Activating Peptide and Thrombin to Induce Platelet Calcium Rise and Full Release Reaction

1995 ◽  
Vol 74 (05) ◽  
pp. 1323-1328 ◽  
Author(s):  
Dominique Lasne ◽  
José Donato ◽  
Hervé Falet ◽  
Francine Rendu
Keyword(s):  
Essential Role ◽  
Calcium Influx ◽  
Dense Granule ◽  
Receptor Interaction ◽  
Thrombin Receptor ◽  
Release Reaction ◽  
External Calcium ◽  
Maximum Rise ◽  
Granule Release

SummarySynthetic peptides (TRAP or Thrombin Receptor Activating Peptide) corresponding to at least the first five aminoacids of the new N-terminal tail generated after thrombin proteolysis of its receptor are effective to mimic thrombin. We have studied two different TRAPs (SFLLR, and SFLLRN) in their effectiveness to induce the different platelet responses in comparison with thrombin. Using Indo-1/AM- labelled platelets, the maximum rise in cytoplasmic ionized calcium was lower with TRAPs than with thrombin. At threshold concentrations allowing maximal aggregation (50 μM SFLLR, 5 μM SFLLRN and 1 nM thrombin) the TRAPs-induced release reaction was about the same level as with thrombin, except when external calcium was removed by addition of 1 mM EDTA. In these conditions, the dense granule release induced by TRAPs was reduced by over 60%, that of lysosome release by 75%, compared to only 15% of reduction in the presence of thrombin. Thus calcium influx was more important for TRAPs-induced release than for thrombin-induced release. At strong concentrations giving maximal aggregation and release in the absence of secondary mediators (by pretreatment with ADP scavengers plus aspirin), SFLLRN mobilized less calcium, with a fast return towards the basal level and induced smaller lysosome release than did thrombin. The results further demonstrate the essential role of external calcium in triggering sustained and full platelet responses, and emphasize the major difference between TRAP and thrombin in mobilizing [Ca2+]j. Thus, apart from the proteolysis of the seven transmembrane receptor, another thrombin binding site or thrombin receptor interaction is required to obtain full and complete responses.

Endobrevin/VAMP-8-Mediated Dense Granule Release Is Required for Efficient Thrombus Formation in Vivo.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1836-1836
Author(s):  
Price S. Blair ◽  
Qiansheng Ren ◽  
Gwenda J. Graham ◽  
James R. Dilks ◽  
Sidney W. Whiteheart ◽  
...  
Keyword(s):  
Vascular Injury ◽  
Thrombus Formation ◽  
Dense Granule ◽  
Wild Type ◽  
Platelet Accumulation ◽  
Induced Aggregation ◽  
Kinetics Of ◽  
Granule Release

Abstract Individuals whose platelets lack dense core or alpha-granules suffer varying degrees of abnormal bleeding, implying that granule cargo contributes to hemostasis. Despite these clinical observations, little is known regarding the effects of impaired platelet granule secretion on thrombus formation in vivo. The release of cargo from platelet granules requires a group of membrane proteins called SNAREs (Soluble NSF Attachment Protein Receptors) that mediate fusion of granule membranes to the plasma membrane and open canalicular system. Endobrevin/VAMP-8 is the primary vesicular-SNARE (v-SNARE) responsible for efficient release of dense core and a-granule contents. To evaluate the importance of VAMP-8-mediated secretion on the kinetics of thrombus formation in vivo, we measured platelet accumulation following laser-induced vascular injury in VAMP-8−/− mice. Three different phases of thrombus formation - initiation, maximal accumulation, and stabilized platelet accumulation - were tested. Analysis of initial thrombus formation from wild-type and VAMP-8−/− mice showed that average platelet accumulation in VAMP- 8−/− mice was 23% of accumulation in wild-type mice (P=0.009) at 30 sec following injury. There was a trend towards smaller maximal thrombus size in VAMP-8−/− mice, but the difference was not statistically significant (P=0.1). Average stabilized platelet accumulation at 180 sec in VAMP-8−/− mice was 40% of wild-type mice (P=0.05). Thus, thrombus formation is delayed and decreased in VAMP-8−/− mice, but not absent. Dense granule release occurs more rapidly than alpha-granule release, which does not occur for 2–3 min following laser-induced vascular injury. Agonist-induced dense granule release from VAMP-8−/− platelets is defective. To directly evaluate the role of dense granule release on the kinetics of thrombus formation, we assessed thrombus formation in the mouse model of Hermansky-Pudlak syndrome, ruby-eye, which lack dense granules. Thrombus formation following laser-induced vascular injury was nearly abolished in ruby-eye mice such that maximal platelet accumulation was 15% that of wild-type mice. In vitro, the thrombin doses required to induce irreversible aggregation in wild-type, VAMP-8−/−, and ruby-eye platelets were 25 mU, 50 mU, and 150 mU, respectively. Incubation with apyrase had little effect on thrombin-induced aggregation of VAMP-8−/− or ruby-eye platelets. In contrast, incubation of wild-type platelets with apyrase reduced their thrombin sensitivity compared to that of ruby-eye platelets. Supplementation with a substimulatory ADP concentration reversed the thrombin-induced aggregation defect in VAMP-8−/− and ruby-eye mice. Thus, defective ADP release is the primary abnormality leading to impaired aggregation in VAMP-8−/− and ruby-eye mice. Tail bleeding times were assessed in VAMP- 8−/− mice to evaluate the role of VAMP-8 in hemostasis. In contrast to ruby-eye mice, which have a markedly prolonged bleeding time, tail bleeding times in VAMP-8−/− mice were not significantly prolonged compared to those in wild-type mice. These results demonstrate the importance of VAMP-8 and dense granule release in the initial phases of thrombus formation and validate the distal platelet secretory machinery as a potential target for anti-platelet therapies.

THE ROLE OF GPIIb-IIIa IN MODULATION OF ADHESION REACTIONS

1987 ◽  
Author(s):  
J C Mattson ◽  
D W Estry ◽  
D Peterson ◽  
R LaFevre ◽  
J Chirco
Keyword(s):  
Shape Change ◽  
Atp Release ◽  
Dense Granule ◽  
Clot Retraction ◽  
Virtual Absence ◽  
Fibrinogen Binding ◽  
Related Defect ◽  
Rice University ◽  
Granule Release

We have previously reported that patients with Glanzmann’s Thrombasthenia (GT) fail to adhere to a carbon-formvar surface and undergo contact-induced shape change in a non-flow system. The ability of ADP to reverse this adhesion defect suggested that it may be secondary to defective dense granule release rather that a direct requirement for GPIIb-IIIa. To further assess the role of GPIIb-IIIa in adhesion, we examined the effect of two mouse monoclonal antibodies to the GPIIb-IIIa complex, AP2 (IgG, kappa) from T. Kunicki, Milwaukee Blood Center and MAb36 (IgM, lambda) from D. Peterson, Rice University. AP2 (1:50 dil) and MAb36 (1:200 dil) both completely abolished aggregation by ADP, collagen and epinephrine and prevented clot retraction. In a transmission EM (TEM) whole mount assay of adhesion and contact-induced shape change, both antibodies inhibited platelet attachment to the substrate and impaired spreading in those few platelets that did attach. This antibody-induced adhesion defect was reversed by the addition of 2×10−6 m ADP just prior to exposure of platelets to the activating surface. In parallel studies, antibody treated platelets demonstrated a dose-related defect in ATP release as measured in a Lumiaggregometer with total absence of release at antibody dilutions that abolished aggregation. Using a colloidal gold-fibrinogen probe, virtual absence of binding of exogenous fibrinogen was demonstrated in antibody treated platelets induced to. spread by ADP stimulation. These studies suggest that while GPIIb-IIIa may play a role in adhesion in non-flow systems, as suggested by the altered adhesion seen in GT platelets, adhesion and adhesion-induced shape change can be supported by ADP stimulation in the absence of fibrinogen binding to GPIIb-IIIa.

Role of arachidonic acid in depolarization-induced modulation of ion currents in Aplysia giant neurons

1990 ◽  
Vol 64 (2) ◽  
pp. 341-350 ◽  
Author(s):  
R. O. Carlson ◽  
I. B. Levitan
Keyword(s):  
Arachidonic Acid ◽  
Calcium Influx ◽  
Reversal Potential ◽  
Inward Current ◽  
Inward Currents ◽  
Pharmacologic Agents ◽  
Inwardly Rectifying ◽  
Aplysia Neuron ◽  
External Calcium

1. The effects of membrane depolarization on inward currents subsequently elicited by hyperpolarization were studied with the use of two-electrode, voltage-clamp techniques in the giant neurons LP1 and R2 of Aplysia. 2. Several successive sets of brief depolarizing pulses, or bursts, were used to depolarize the giant neurons. Two distinct inward currents elicited by hyperpolarization were found to be altered after these sets of depolarizing pulses. These currents were distinguished by their voltage dependence, reversal potential, and sensitivity to 1 mM BaCl2. One of the inward currents was increased after depolarization. It was outwardly rectifying, reversed at -50 mV, and not blocked by Ba2+, suggesting it was a chloride current (ICl). The other inward current, which was decreased after depolarization, was inwardly rectifying, reversed at -70 mV, and completely inhibited by Ba2+. These are characteristics of the inwardly rectifying potassium current (IR), a current previously described to be inhibited after depolarization in Aplysia neuron R 15. Depolarization typically increased the putative ICl and decreased IR for minutes, with the decrease in IR consistently outlasting the increase in an initial brief net increase in inward current followed by a long-lasting decrease. 3. Several criteria suggest arachidonic acid (AA) may mediate depolarization-induced modulation of IR. Previously, free AA has been shown to constitutively inhibit IR in the resting state. Also, depolarization has been reported to stimulate liberation of AA from storage in Aplysia ganglia. Consistent with previous results in neuron R 15, depolarization-induced modulation of IR in giant neurons was dependent on external calcium. Indomethacin and 4-bromophenacylbromide (BPB), pharmacologic agents that activate IR through inhibition of AA turnover, altered the effect of depolarization on IR. In contrast serotonin (5HT), which activates IR through adenosine 3',5'-cyclic monophosphate (cAMP), did not alter the effect of depolarization. Also, extended perfusion with bovine serum albumin (BSA), which strips AA from lipid storage in neurons, decreased the depolarization-induced modulation of IR. We conclude that the calcium influx accompanying depolarization activates the phospholipase responsible for liberation of AA from phospholipid, and the liberated AA then acts to inhibit IR. The molecular mechanism of this AA-mediated inhibition remains to be determined. 4. Depolarization-induced modulation of ICl was also dependent on external calcium but was not affected by BPB and only slightly decreased with indomethacin. This suggested AA was probably not involved in this modulation. However, 5HT opposed the modulation of IC1 induced by previous depolarization, suggesting cAMP may be involved in this effect of depolarization.

Lyn and PKCδ order SHIP1 embargo

Blood ◽  
2009 ◽  
Vol 114 (14) ◽  
pp. 2856-2857 ◽  
Author(s):  
Ulhas P. Naik
Keyword(s):  
Dense Granule ◽  
Regulatory Role ◽  
Negative Regulatory Role ◽  
Granule Release

In this issue of Blood, Chari and colleagues provide a novel mechanism for the unique negative regulatory role of PKCδ in platelet dense granule release downstream of collagen signaling.

scholarly journals Selective inhibition of protein kinase C. Effect on platelet-activating-factor-induced platelet functional responses

1992 ◽  
Vol 283 (1) ◽  
pp. 159-164 ◽  
Author(s):  
C T Murphy ◽  
J Westwick
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Platelet Activating Factor ◽  
Selective Inhibition ◽  
Dense Granule ◽  
High Dose ◽  
Functional Responses ◽  
Kinase C ◽  
Granule Release

The role of protein kinase C (PKC) in platelet-activating-factor (PAF)-induced platelet activation was examined by using two selective inhibitors of PKC, namely Ro 31-7549/001 and Ro 31-8220/002. Both inhibitors dose-dependently inhibited PAF-induced phosphorylation of the major 40-47 kDa protein substrate of PKC, with 50% inhibition at 4.5 microM-Ro 31-7549/001 and 0.7 microM-Ro 31-8220/002. Inhibition of PKC had no effect on maximal elevation of intracellular Ca2+ [Ca2+]i produced by either a high or a low dose of PAF, but significantly increased the duration of the Ca2+ signal and the thromboxane B2 (TxB2) generation in high-dose PAF-stimulated platelets. The inhibitors also abrogated the effect of the PKC activator phorbol 12-myristate 13-acetate on PAF-induced [Ca2+]i elevation. Sub-maximal PAF-induced dense-granule release and platelet aggregation were dose-dependently inhibited by Ro 31-7549/001 and Ro 31-8220/002. The findings suggest that endogenously activated PKC holds a bifurcating role in PAF-activated platelets, negatively affecting duration of both [Ca2+]i and TxB2 generation, and positively influencing dense-granule release and aggregation.

scholarly journals P2 Receptors and Platelet Activation

2002 ◽  
Vol 2 ◽  
pp. 424-433 ◽  
Author(s):  
Satva P. Kunapuli
Keyword(s):  
Shape Change ◽  
Adenosine Diphosphate ◽  
Receptor Activation ◽  
Dense Granule ◽  
Receptor Subtypes ◽  
P2 Receptor ◽  
Fibrinogen Receptor ◽  
Granule Release ◽  
Platelet Shape

Adenosine diphosphate (ADP) plays a crucial role in hemostasis and thrombosis by activating platelets. In platelets, the classical P2T receptor is now resolved into three P2 receptor subtypes: the P2Y1, the P2Y12, and the P2X1 receptors. Both pharmacological and molecular biological approaches have confirmed the role of the P2Y1 and P2Y12 receptors in the ADP-induced platelet fibrinogen receptor activation. The P2Y1 and the P2X1 receptors independently contribute to platelet shape change. Whereas the P2Y12 receptor mediates the potentiation of dense granule release reaction, both the P2Y1 and P2Y12 receptors play an important role in the ADP-induced phospholipase A2 activation. The signaling events downstream of these receptors leading to the physiological effects remain elusive, and they are yet to be delineated.

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