ADP And Epinephrine-Induced Release Of Platelet Fibrinogen

1981 ◽  
Author(s):  
K L Kaplan
Keyword(s):  
Adenine Nucleotide ◽  
Human Platelets ◽  
Dense Granule ◽  
Protein Release ◽  
Fibrinogen Concentration ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Fibrinogen Binding ◽  
Induced Aggregation

Added fibrinogen is said to be essential for induction of platelet aggregation and release by ADP and epinephrine. Furthermore, both ADP and epinephrine induce binding of fibrinogen to the platelet surface. In the present study gel-filtered human platelets were examined to determine whether they would aggregate and release platelet fibrinogen in response to ADP or epinephrine without exogenous fibrinogen. Platelets gel-filtered into Tyrode’s buffer containing ImM Mg++, no added Ca++, and 0.35% bovine serum albumin had a fibrinogen concentration in the supernatant of less than 1 nM. After aggregation by ADP or epinephrine the fibrinogen concentration in the supernatant ranged from 11 to 41 nM. ADP and epinephrine each induced biphasic aggregation and release of platelet factor 4 and β-thromboglobulin as well as of fibrinogen. Protein release by epinephrine was coincidental with dense granule adenine nucleotide release. Because Ca++ ions affect fibrinogen binding to platelets, the effect of Ca++ on aggregation and protein release was examined and it was found that both were inhibited by added Ca++ at 1-2 mM. The ability of gel-filtered platelets to undergo ADP and epinephrine induced aggregation and release in the absence of exogenous fibrinogen suggests that released platelet fibrinogen can support these processes. The concentrations of released fibrinogen in these experiments were lower than those reported to be necessary for exogenous fibrinogen for support of aggregation, suggesting that released fibrinogen may interact more efficiently with the platelet membrane. The amount of released fibrinogen in these experiments is similar to the Kd for high-affinity fibrinogen binding reported by Niewiarowski et al. (30nM). Finally, although inhibition of ADP and epinephrine induced aggregation and release by physiologic Ca++ concentrations implies that these processes do not occur in vivo, it is likely that platelet fibrinogen released by collagen or thrombin does function physiologically.

scholarly journals ADP and epinephrine-induced release of platelet fibrinogen

Blood ◽  
1981 ◽  
Vol 58 (4) ◽  
pp. 797-802 ◽  
Author(s):  
KL Kaplan ◽  
MJ Dauzier ◽  
S Rose
Keyword(s):  
Platelet Aggregation ◽  
Bovine Serum Albumin ◽  
Adenine Nucleotides ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Dense Granule ◽  
Fibrinogen Concentration ◽  
Platelet Factor ◽  
Granule Proteins ◽  
Induced Aggregation

Abstract Human platelets gel-filtered into Tyrode's buffer containing 1 mM Mg++ and 0.35% bovine serum albumin were studied to determine whether they would undergo biphasic aggregation and release of alpha-granule proteins in response to adenosine diphosphate (ADP) or epinephrine without addition of exogenous fibrinogen. Fibrinogen concentration in the supernatant of unaggregated gel-filtered platelets was less than 1 pmole/ml. With addition of ADP or epinephrine, biphasic aggregation was seen, with release of platelet fibrinogen, beta-thromboglobulin, and platelet factor 4. Fibrinogen concentration in the supernatant after aggregation ranged from 15 to 70 pmole/ml. Release of the alpha-granule proteins by epinephrine was coincidental with release of the dense granule adenine nucleotides. Aggregation and alpha-granule protein release by both ADP and epinephrine were inhibited by added Ca++ at 1-- 2 mM. The ability of gel-filtered platelets to undergo ADP- and epinephrine-induced aggregation and release in the absence of exogenous fibrinogen suggests that released platelet fibrinogen may be able to fulfill the requirement for fibrinogen in ADP- and epinephrine-induced platelet aggregation and release.

scholarly journals ADP and epinephrine-induced release of platelet fibrinogen

Blood ◽  
1981 ◽  
Vol 58 (4) ◽  
pp. 797-802
Author(s):  
KL Kaplan ◽  
MJ Dauzier ◽  
S Rose
Keyword(s):  
Platelet Aggregation ◽  
Bovine Serum Albumin ◽  
Adenine Nucleotides ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Dense Granule ◽  
Fibrinogen Concentration ◽  
Platelet Factor ◽  
Granule Proteins ◽  
Induced Aggregation

Human platelets gel-filtered into Tyrode's buffer containing 1 mM Mg++ and 0.35% bovine serum albumin were studied to determine whether they would undergo biphasic aggregation and release of alpha-granule proteins in response to adenosine diphosphate (ADP) or epinephrine without addition of exogenous fibrinogen. Fibrinogen concentration in the supernatant of unaggregated gel-filtered platelets was less than 1 pmole/ml. With addition of ADP or epinephrine, biphasic aggregation was seen, with release of platelet fibrinogen, beta-thromboglobulin, and platelet factor 4. Fibrinogen concentration in the supernatant after aggregation ranged from 15 to 70 pmole/ml. Release of the alpha-granule proteins by epinephrine was coincidental with release of the dense granule adenine nucleotides. Aggregation and alpha-granule protein release by both ADP and epinephrine were inhibited by added Ca++ at 1-- 2 mM. The ability of gel-filtered platelets to undergo ADP- and epinephrine-induced aggregation and release in the absence of exogenous fibrinogen suggests that released platelet fibrinogen may be able to fulfill the requirement for fibrinogen in ADP- and epinephrine-induced platelet aggregation and release.

scholarly journals The secretory release reaction initiated by complement proteins C5b-9 occurs without platelet aggregation through glycoprotein IIb-IIIa

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 462-467 ◽  
Author(s):  
B Ando ◽  
T Wiedmer ◽  
PJ Sims
Keyword(s):  
Cellular Protein ◽  
Dense Granule ◽  
Surface Glycoprotein ◽  
Gamma Chain ◽  
Cell Surface Glycoprotein ◽  
Platelet Surface ◽  
Complement Proteins ◽  
Fibrinogen Binding ◽  
Dense Granule Secretion

The secretory and aggregation responses of stirred platelets exposed to complement proteins C5b-9 was investigated. C5b-9 assembly on the platelet surface resulted in the release of dense granule adenosine triphosphate (ATP) accompanied by a decrease in sample turbidity, but no detectable cell lysis. Inhibition of cellular protein kinase C completely blocked the C5b-9 initiated release of ATP, confirming the secretory nature of this response. Addition of fibrinogen (up to 1 mg/mL) had no effect on either the release of ATP or the decreased turbidity observed for C5b-9 cells. Addition of the peptides Arg-Gly- Asp-Ser (RGDS) and fibrinogen gamma-chain carboxyl-terminal (gamma 397- 411) at concentrations sufficient to completely block fibrinogen binding to GP IIb-IIIa had no effect on either C5b-9 induced dense granule secretion or the associated turbidity change. Microscopic examination and electronic particle counting of the stirred platelet suspensions confirmed that no aggregation of C5b-9 platelets occurred even when these cells were stirred in the presence of fibrinogen. The capacity of the C5b-9 proteins to initiate platelet secretion without activation of cell surface glycoprotein (GP) IIb-IIIa suggests a mechanism for intravascular dissemination of activated platelets during complement activation in vivo.

scholarly journals The secretory release reaction initiated by complement proteins C5b-9 occurs without platelet aggregation through glycoprotein IIb-IIIa

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 462-467 ◽  
Author(s):  
B Ando ◽  
T Wiedmer ◽  
PJ Sims
Keyword(s):  
Cellular Protein ◽  
Dense Granule ◽  
Surface Glycoprotein ◽  
Gamma Chain ◽  
Cell Surface Glycoprotein ◽  
Platelet Surface ◽  
Complement Proteins ◽  
Fibrinogen Binding ◽  
Dense Granule Secretion

Abstract The secretory and aggregation responses of stirred platelets exposed to complement proteins C5b-9 was investigated. C5b-9 assembly on the platelet surface resulted in the release of dense granule adenosine triphosphate (ATP) accompanied by a decrease in sample turbidity, but no detectable cell lysis. Inhibition of cellular protein kinase C completely blocked the C5b-9 initiated release of ATP, confirming the secretory nature of this response. Addition of fibrinogen (up to 1 mg/mL) had no effect on either the release of ATP or the decreased turbidity observed for C5b-9 cells. Addition of the peptides Arg-Gly- Asp-Ser (RGDS) and fibrinogen gamma-chain carboxyl-terminal (gamma 397- 411) at concentrations sufficient to completely block fibrinogen binding to GP IIb-IIIa had no effect on either C5b-9 induced dense granule secretion or the associated turbidity change. Microscopic examination and electronic particle counting of the stirred platelet suspensions confirmed that no aggregation of C5b-9 platelets occurred even when these cells were stirred in the presence of fibrinogen. The capacity of the C5b-9 proteins to initiate platelet secretion without activation of cell surface glycoprotein (GP) IIb-IIIa suggests a mechanism for intravascular dissemination of activated platelets during complement activation in vivo.

scholarly journals GSK3β is a negative regulator of platelet function and thrombosis

Blood ◽  
2008 ◽  
Vol 111 (7) ◽  
pp. 3522-3530 ◽  
Author(s):  
Dongjun Li ◽  
Shelley August ◽  
Donna S. Woulfe
Keyword(s):  
Platelet Function ◽  
Glycogen Synthase ◽  
Human Platelets ◽  
Dense Granule ◽  
Negative Regulator ◽  
Wild Type ◽  
Single Allele ◽  
Induced Aggregation

Abstract Glycogen synthase kinase (GSK)3β is a ser-thr kinase that is phosphorylated by the kinase Akt. Although Akt has been shown to regulate platelet function and arterial thrombosis, its effectors in platelets remain unknown. We show here that agonist-dependent phosphorylation of GSK3β in platelets is Akt dependent. To determine whether GSK3β regulates platelet function, platelets from mice lacking a single allele of GSK3β were compared with those of wild-type (WT) controls. GSK3β+/− platelets demonstrated enhanced agonist-dependent aggregation, dense granule secretion, and fibrinogen binding, compared with WT platelets. Treatment of human platelets with GSK3 inhibitors renders them more sensitive to agonist-induced aggregation, suggesting that GSK3 suppresses platelet function in vitro. Finally, the effect of GSK3β on platelet function in vivo was evaluated using 2 thrombosis models in mice. In the first, 80% of GSK3β+/− mice (n = 10) formed stable occlusive thrombi after ferric chloride carotid artery injury, whereas the majority of wild-type mice (67%) formed no thrombi (n = 15). In a disseminated thrombosis model, deletion of a single allele of GSK3β in mice conferred enhanced sensitivity to thrombotic insult. Taken together, these results suggest that GSK3β acts as a negative regulator of platelet function in vitro and in vivo.

Platelet α-Granules: Content and Characteristics of Secretion

1979 ◽  
Author(s):  
Karen Kaplan ◽  
Johan M. Broekman ◽  
Arthur Chernoff ◽  
Barbara Linder ◽  
DeWitt Goodman
Keyword(s):  
Dienoic Acid ◽  
Arterial Disease ◽  
Human Platelets ◽  
Dense Granule ◽  
Distinct Pattern ◽  
Platelet Factor ◽  
Alternative Pathways ◽  
Granule Release

Platelet α-granule contents and secretion were studied using specific radioimmunoas says and cell culture assays. Washed human platelets were disrupted by nitrogen cavitation and subcellular fractions were separated by ultracentrifugation through sucrose density gradients. α-Granules were found to contain platelet factor 4 (PF4), 8-thromboglobulin (8TG) fibrinogen, and the platelet-derived growth factor: their contents were distinct from those of dense granules and of lysos omes. A distinct pattern of release of the four agranule components was ob served. Release of α- and dense-granule components was induced by ADP and epinephrine; α-granule release was, however, more sensitive than dense granule to low levels of thrombin, collagen, and the endoperoxide analogue (15S)-hydroxy-IIα, 9α (epoxymethano)prosta-5Z, l3E-dienoic acid. Studies with cyclooxygenase (CO) inhibitors suggested that α-granule release in vitro can be mediated both by the CO pathway and by other mechanisms. Thus, aspirin and indomethacin inhibited collagen and thrombin induced release, but “the inhibitory effects were progressively overcome with higher levels of these agents. Studies in vivo showed that normal plasma levels of PF4 and 8TG were only slightly reduced by aspirin, whereas the elevated basal levels of these components found in patients with arterial disease were reduced by aspirin to a greater extent. The in viv data thus support the in vitro findings that both CO mediated and alternative pathways are involved in release of α-granule components.

Adenosine Diphosphate-Induced Aggregation of Human Platelets in Flow Through Tubes: III. Shear and Extrinsic Fibrinogen-Dependent Effects

1994 ◽  
Vol 71 (01) ◽  
pp. 078-090 ◽  
Author(s):  
H L Goldsmith ◽  
M M Frojmovic ◽  
Susan Braovac ◽  
Fiona McIntosh ◽  
T Wong
Keyword(s):  
Shear Rate ◽  
Single Cells ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Size Analysis ◽  
Fibrinogen Concentration ◽  
Human Fibrinogen ◽  
Shear Rates ◽  
Rate Dependent ◽  
Induced Aggregation

SummaryThe effect of shear rate and fibrinogen concentration on adenosine diphosphate-induced aggregation of suspensions of washed human platelets in Poiseuille flow at 23°C was studied using a previously described double infusion technique and resistive particle counter size analysis (1). Using suspensions of multiple-centrifuged and -washed cells in Tyrodes-albumin [3 × 105 μl−1; (17)] with [fibrinogen] from 0 to 1.2μM, the, rate and extent of aggregation with 0.7 μM ADP in Tyrodes-albumin were measured over a range of mean transit times from 0.2 to 43 s, and at mean tube shear rates, Ḡ, = 41.9, 335 and 1,335 s−1. As measured by the decrease in singlet concentration, aggregation at 1.2 μM fibrinogen increased with increasing Ḡ up to 1,335 s1, in contrast to that previously reported in citratcd plasma, in which aggregation reached a maximum at Ḡ = 335 s−1. Without added fibrinogen, there was no aggregation at Ḡ = 41.9 s1; at Ḡ = 335 s1, there was significant aggregation but with an initial lag time, aggregation increasing further at Ḡ = 1,335 s−1. Without added fibrinogen, aggregation was abolished at all Ḡ upon incubation with the hexapeptide GRGDSP, but was almost unaffected by addition of an F(ab’)2 fragment of an antibody to human fibrinogen. Aggregation in the absence of added fibrinogen was also observed at 37°C. The activation of the multiple-washed platelets was tested using flow cytometry with the fluorescently labelled monoclonal antibodies FITC-PAC1 and FITC-9F9. It was shown that 57% of single cells in unactivated PRT expressed maximal GPIIb-IIIa fibrinogen receptors (MoAb PAC1) and 54% expressed pre-bound fibrinogen (MoAb 9F9), with further increases on ADP activation. However, incubation with GRGDSP and the F(ab’)2 fragment did not inhibit the prebound fibrinogen. Moreover, relatively unactivated cells (8% expressing receptor, 14% prebound fibrinogen), prepared from acidified cPRP by single centrifugation with 50 nM of the stable prostacyclin derivative, ZK 36 374, and resuspension in Tyrodes-albumin at 5 × 104 μl−1, aggregated with 2 and 5 μM ADP at Ḡ = 335 and 1,335 s−1 in the absence of added fibrinogen. We therefore postulate that a protein such as von Willebrand factor, secreted during platelet isolation or in flow at sufficiently high shear rates, may yield the observed shear-rate dependent aggregation without fibrinogen.

Effects of a Monoclonal Antibody to Glycoprotein IIb/IIIa (P256) and of Enzymically Derived Fragments of P256 on Human Platelets

1991 ◽  
Vol 65 (04) ◽  
pp. 432-437 ◽  
Author(s):  
A W J Stuttle ◽  
M J Powling ◽  
J M Ritter ◽  
R M Hardisty
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Calcium Ions ◽  
Human Platelet ◽  
Human Platelets ◽  
In Vivo Detection ◽  
Fibrinogen Binding ◽  
Specific Antagonist

SummaryThe anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10−9−3 × 10−8 M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10−7 M. P256–induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In–111–labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications

Fibrinogen binding to the integrin αIIbβ3 modulates store-mediated calcium entry in human platelets

Blood ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2648-2656 ◽  
Author(s):  
Juan A. Rosado ◽  
Else M. Y. Meijer ◽  
Karly Hamulyak ◽  
Irena Novakova ◽  
Johan W. M. Heemskerk ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Adenosine Diphosphate ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Integrin Αiibβ3 ◽  
Fibrinogen Binding

Abstract Effects of the occupation of integrin αIIbβ3 by fibrinogen on Ca++signaling in fura-2–loaded human platelets were investigated. Adding fibrinogen to washed platelet suspensions inhibited increases in cytosolic [Ca++] concentrations ([Ca++]i) evoked by adenosine diphosphate (ADP) and thrombin in a concentration-dependent manner in the presence of external Ca++ but not in the absence of external Ca++ or in the presence of the nonselective cation channel blocker SKF96365, indicating selective inhibition of Ca++entry. Fibrinogen also inhibited store-mediated Ca++ entry (SMCE) activated after Ca++ store depletion using thapsigargin. The inhibitory effect of fibrinogen was reversed if fibrinogen binding to αIIbβ3 was blocked using RDGS or abciximab and was absent in platelets from patients homozygous for Glanzmann thrombasthenia. Fibrinogen was without effect on SMCE once activated. Activation of SMCE in platelets occurs through conformational coupling between the intracellular stores and the plasma membrane and requires remodeling of the actin cytoskeleton. Fibrinogen inhibited actin polymerization evoked by ADP or thapsigargin in control cells and in cells loaded with the Ca++ chelator dimethyl BAPTA. It also inhibited the translocation of the tyrosine kinase p60src to the cytoskeleton. These results indicate that the binding of fibrinogen to integrin αIIbβ3 inhibits the activation of SMCE in platelets by a mechanism that may involve modulation of the reorganization of the actin cytoskeleton and the cytoskeletal association of p60src. This action may be important in intrinsic negative feedback to prevent the further activation of platelets subjected to low-level stimuli in vivo.

Partially desulfated heparin modulates the interaction between anti-protamine/heparin antibodies and platelets

2016 ◽  
Vol 115 (02) ◽  
pp. 324-332 ◽  
Author(s):  
Rabie Jouni ◽  
Heike Zöllner ◽  
Ahmad Khadour ◽  
Jan Wesche ◽  
Anne Grotevendt ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Factor ◽  
Standard Drug ◽  
Platelet Surface ◽  
Minimal Impact ◽  
Platelet Survival ◽  
Heparin Complexes ◽  
The Impact

SummaryProtamine (PRT) is the standard drug to neutralise heparin. PRT/heparin complexes induce an immune response similar to that observed in heparin-induced thrombocytopenia (HIT). Partially desulfated heparin (ODSH) was shown to interfere with anti-platelet factor 4/heparin antibodies (Abs), which are responsible for HIT. In this study, we analyse the impact of ODSH on the interaction between anti-PRT/heparin Abs and platelets. The ability of ODSH to prevent anti-PRT/heparin Ab-induced platelet destruction in vivo was investigated using the NOD/ SCID mouse model. ODSH improved platelet survival in the presence of PRT, heparin and anti-PRT/heparin Abs (median platelet survival after 300 minutes (min) with 20 μg/ml ODSH: 75 %, range 70–81 % vs without ODSH: 49%, range 44–59%, p=0.006). Furthermore, when ODSH was applied 60 min after Ab injection platelet survival was improved (median platelet survival after 300 min with ODSH: 83 %, range 77–93 % vs without ODSH: 59 %, range 29–61 %, p=0.02). In in vitro experiments ODSH inhibited platelet activation at concentrations > 16 μg/mL (p< 0.001), as well as PRT/heparin complex binding to platelets (mean fluorescence intensity [MFI] without ODSH: 85 ± 14 vs with ODSH: 15 ± 0.6, p=0.013). ODSH also displaced pre-bound complexes from the platelet surface (MFI without ODSH: 324 ± 43 vs with 32 μg/ml ODSH: 53 ± 9, p< 0.001). While interfering with platelet activation by anti-PRT/heparin Abs, up to a concentration of 16 μg/ml, ODSH had only minimal impact on neutralisation of heparin by PRT. In conclusion, our study shows that ODSH is able to inhibit platelet activation and destruction suggesting a potential clinical use to reduce anti-PRT/heparin Ab-mediated adverse effects.

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