scholarly journals Platelet SHARPIN regulates platelet adhesion and inflammatory responses through associations with αIIbβ3 and LUBAC

2022 ◽  
Author(s):  
Ana Kasirer-Friede ◽  
Emilia Peuhu ◽  
Johanna Ivaska ◽  
Sanford J. Shattil
Keyword(s):  
Inflammatory Responses ◽  
Super Resolution ◽  
Cytoplasmic Tail ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Littermate Control ◽  
Protein Substrates ◽  
Pathway Activation ◽  
Super Resolution Microscopy

Platelets form hemostatic plugs to prevent blood loss and they modulate immunity and inflammation in several ways. A key event during hemostasis is activation of integrin αIIbβ3 through direct interactions of the β3 cytoplasmic tail with talin and kindlin-3. Recently, we showed that human platelets express the adapter molecule, SHARPIN, that can associate directly with the αIIb cytoplasmic tail and can separately promote NF-κB pathway activation as a member of the Met-1 linear ubiquitination activation complex (LUBAC). Here we investigated the role of SHARPIN in platelets after crossing Sharpin flox/flox (fl/fl) mice with PF4-Cre or GPIbα-Cre mice to selectively delete SHARPIN in platelets. SHARPIN-null platelets adhered to immobilized fibrinogen through αIIbβ3, and they spread more extensively than littermate control platelets in a manner dependent on feedback stimulation by platelet adenosine diphosphate (ADP) (P < 0.01). SHARPIN-null platelets showed increased colocalization of αIIbβ3 with talin as assessed by super-resolution microscopy and increased binding of soluble fibrinogen in response to sub-maximal concentrations of ADP (P < 0.05). However, mice with SHARPIN-null platelets showed compromised thrombus growth on collagen and slightly prolonged tail bleeding times. Platelets lacking SHARPIN also showed reduced NF-κB activation and linear ubiquitination of protein substrates upon challenge with classical platelet agonists. Furthermore, the loss of platelet SHARPIN resulted in significant reduction in inflammation in murine models of colitis and peritonitis (P < 0.01). Thus, SHARPIN plays differential and context-dependent roles in platelets to regulate important inflammatory and integrin adhesive functions of these anucleate cells.

Collagen-Induced Platelet Aggregation: -Evidence Against the Essential Role of Platelet Adenosine Diphosphate

1979 ◽  
Vol 42 (04) ◽  
pp. 1193-1206 ◽  
Author(s):  
Barbara Nunn
Keyword(s):  
Platelet Aggregation ◽  
Time Course ◽  
Essential Role ◽  
Atp Release ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
High Doses ◽  
Induced Aggregation

SummaryThe hypothesis that platelet ADP is responsible for collagen-induced aggregation has been re-examined. It was found that the concentration of ADP obtaining in human PRP at the onset of aggregation was not sufficient to account for that aggregation. Furthermore, the time-course of collagen-induced release in human PRP was the same as that in sheep PRP where ADP does not cause release. These findings are not consistent with claims that ADP alone perpetuates a collagen-initiated release-aggregation-release sequence. The effects of high doses of collagen, which released 4-5 μM ADP, were not inhibited by 500 pM adenosine, a concentration that greatly reduced the effect of 300 μM ADP. Collagen caused aggregation in ADP-refractory PRP and in platelet suspensions unresponsive to 1 mM ADP. Thus human platelets can aggregate in response to collagen under circumstances in which they cannot respond to ADP. Apyrase inhibited aggregation and ATP release in platelet suspensions but not in human PRP. Evidence is presented that the means currently used to examine the role of ADP in aggregation require investigation.

Mechanism of platelet plug formation and role of adenosine diphosphate

1964 ◽  
Vol 206 (6) ◽  
pp. 1267-1274 ◽  
Author(s):  
Theodore H. Spaet ◽  
Marjorie B. Zucker
Keyword(s):  
Connective Tissue ◽  
Divalent Cation ◽  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Suitable Model ◽  
Plug Formation ◽  
Rat Platelets

Traumatized rat omentum was used to demonstrate the development of "platelet plugs" following agitation in platelet-rich plasma. In the absence of divalent cation there was only platelet adhesion to connective tissue fibers; in the presence of divalent cation masses of platelets formed (cohesion) even in plasma adequately anticoagulated with heparin. Exposure of these platelet masses to thrombin produced greater compactness and stability. Human and rat platelets behaved alike with the traumatized rat omentum; platelets from two patients with von Willebrand's disease gave normal reactions whereas platelets from a patient with thrombasthenia showed adhesion only. Exposure of human platelets to washed connective-tissue fragments or to thrombin elicited clumping accompanied by release of serotonin and of adenine nucleotides (AN) of which about one-third was adenosine diphosphate. Intermediate concentrations of connective tissue and thrombin also caused clumping but no liberation of AN or serotonin. ADP caused intense clumping but failed to liberate serotonin or additional ADP. It is suggested that cohesion reaction is mediated by release of ADP. The traumatized omentum appears to be a suitable model for studying the hemostatic process.

scholarly journals Functional Characterization of Membrane Sialyltransferase Activity of Human Platelets

1977 ◽  
Author(s):  
K. K. Wu ◽  
C. Ku ◽  
C. Smith
Keyword(s):  
Enzyme Activity ◽  
Functional Characterization ◽  
Adenosine Diphosphate ◽  
Normal Subjects ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Transferase Activity ◽  
Platelet Surface

To evaluate the role of membrane sialyltransferase in the initiation of platelet aggregation, we studied the stimulatory effect of epinephrine and adenosine diphosphate and the inhibitory effect of aspirin on the platelet surface sialyl transferase activity. The enzyme activity was assayed under optimal conditions as determined previously. The assay mixture consisted of intact washed human platelets, CMP-14C-sialic acid, desialated fetuin, Mn2+ and buffer to a final volume of 1 ml. The enzyme activity was enhanced to 172% of control by 1μH, 152% by 5μM and 146% by 10μM epinephrine. Adenosine diphosphate enhanced the enzyme activity to a lesser extent:103% at 1μM and 113% at 5μM. In contrast, aspirin inhibited the enzyme activity to 46% of control when 10μg/ml of aspirin was used. Higher concentrations of aspirin failed to cause further inhibition. In the in-vivo experiment, 600 mg aspirin was given to normal subjects and the surface enzyme activity was determined 12 hours later. The enzyme activity reduced to 43% following aspirin administration. Furthermore, we studied the enzyme activity in a patient with “aspirin-like” release disorder. While the mean surface enzyme activity of 10 normal subjects was 1.56 + 0.21 (S. D.) pmole-hr-1 per 108 platelets, the enzyme activity of the patient was only 0.91 pmole.hr-l. The results strongly suggest that the membrane sialyltransferase plays an important part in the initiation of platelet release reaction.

A critical role of lipid rafts in the organization of a key FcγRIIa-mediated signaling pathway in human platelets

2003 ◽  
Vol 89 (02) ◽  
pp. 318-330 ◽  
Author(s):  
Stéphane Bodin ◽  
Cécile Viala ◽  
Ashraf Ragab ◽  
Bernard Payrastre
Keyword(s):  
Lipid Rafts ◽  
Critical Role ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Temporal Organization ◽  
Membrane Microdomains ◽  
Cross Linking ◽  
Dependent Manner ◽  
Signaling Complex

SummaryThe involvement of platelet FcγRIIa in heparin-associated thrombocytopenia (HIT) is now well established. However, the precise sequence of molecular events initiated by FcγRIIa cross-linking in platelets remains partly characterized. We investigated here the role of lipid rafts in the spatio-temporal organization of the FcγRIIa-dependent signaling events. Upon cross-linking, FcγRIIa relocated in rafts where the kinase Lyn and the adapter LAT were among the major phosphotyrosyl proteins. Upon stimulation by HIT sera, the second messenger phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) accumulated in rafts in a P2Y12 adenosine diphosphate (ADP) recep- tor-dependent manner. PtdIns(3,4,5)P3 was then essential to specifically recruit phospholipase Cγ2 (PLCγ2) to these membrane microdomains. Controlled disruption of rafts by methyl γ-cyclodextrin reversibly abolished PtdIns(3,4,5)P3 production, PLC activation and platelet responses induced by FcγRIIa cross-linking without affecting the tyrosine phosphorylation events. This work demonstrates that platelet rafts are essential for the integration of a key signaling complex leading to the rapid production of PtdIns(3,4,5)P3 and in turn PLCγ2 activation during HIT.

scholarly journals Detyrosinated microtubules spatially constrain lysosomes facilitating lysosome–autophagosome fusion

2018 ◽  
Vol 218 (2) ◽  
pp. 632-643 ◽  
Author(s):  
Nitin Mohan ◽  
Elena M. Sorokina ◽  
Ione Vilanova Verdeny ◽  
Angel Sandoval Alvarez ◽  
Melike Lakadamyali
Keyword(s):  
Epithelial Cells ◽  
Functional Diversity ◽  
Super Resolution ◽  
Live Cell ◽  
Small Subset ◽  
Post Translational Modifications ◽  
Super Resolution Microscopy ◽  
Small Subpopulation ◽  
Dependent Mechanism

Microtubule post-translational modifications impart functional diversity to microtubules by affecting their dynamics, organization, and interaction with proteins. Using super-resolution microscopy, we show that only a small subpopulation of microtubules are detyrosinated in epithelial cells, while acetylated and tyrosinated microtubules comprise the majority of all microtubules. Surprisingly, lysosomes are enriched by approximately threefold on detyrosinated microtubules. Further, their motility on detyrosinated microtubules is impaired, showing shorter runs and more frequent and longer pauses. Lysosome enrichment is mediated through a kinesin-1–dependent mechanism, since knocking down this motor abolishes enrichment. Finally, correlative live-cell and super-resolution microscopy showed that lysosomes interact with autophagosomes on detyrosinated microtubules. Removal of detyrosinated microtubules or knockdown of kinesin-1 leads to a decrease in the percentage of autolysosomes, a fusion intermediate of autophagosomes and lysosomes. Taken together, our data reveal a new role of detyrosinated microtubules as hubs that spatially concentrate lysosomes on a small subset of microtubules and facilitate their interaction and fusion with autophagosomes to initiate autophagy.

scholarly journals Role of Purinergic Receptor Expression and Function for Reduced Responsiveness to Adenosine Diphosphate in Washed Human Platelets

PLoS ONE ◽  
2016 ◽  
Vol 11 (1) ◽  
pp. e0147370 ◽  
Author(s):  
Juergen Koessler ◽  
Stephanie Hermann ◽  
Katja Weber ◽  
Angela Koessler ◽  
Sabine Kuhn ◽  
...  
Keyword(s):  
Purinergic Receptor ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Receptor Expression ◽  
And Function

scholarly journals Binding of the RNA Chaperone Hfq on Target mRNAs Promotes the Small RNA RyhB-Induced Degradation in Escherichia coli

2021 ◽  
Vol 7 (4) ◽  
pp. 64
Author(s):  
David Lalaouna ◽  
Karine Prévost ◽  
Seongjin Park ◽  
Thierry Chénard ◽  
Marie-Pier Bouchard ◽  
...  
Keyword(s):  
Complex Formation ◽  
Super Resolution ◽  
Rnase E ◽  
Rna Chaperone ◽  
Target Mrnas ◽  
Mrna Targets ◽  
Super Resolution Microscopy

Many RNA-RNA interactions depend on molecular chaperones to form and remain stable in living cells. A prime example is the RNA chaperone Hfq, which is a critical effector involved in regulatory interactions between small RNAs (sRNAs) and cognate target mRNAs in Enterobacteriaceae. While there is a great deal of in vitro biochemical evidence supporting the model that Hfq enhances rates or affinities of sRNA:mRNA interactions, there is little corroborating in vivo evidence. Here we used in vivo tools including reporter genes, co-purification assays, and super-resolution microscopy to analyze the role of Hfq in RyhB-mediated regulation, and we found that Hfq is often unnecessary for efficient RyhB:mRNA complex formation in vivo. Remarkably, our data suggest that a primary function of Hfq is to promote RyhB-induced cleavage of mRNA targets by RNase E. Moreover, our work indicates that Hfq plays a more limited role in dictating regulatory outcomes following sRNAs RybB and DsrA complex formation with specific target mRNAs. Our investigation helps evaluate the roles played by Hfq in some RNA-mediated regulation.

scholarly journals Hyperphosphorylated tau self-assembles into amorphous aggregates eliciting TLR4-dependent inflammatory responses

2021 ◽  
Author(s):  
David Klenerman ◽  
Jonathan Meng ◽  
Yu Zhang ◽  
Dominik Saman ◽  
Suman De ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Mechanistic Model ◽  
Super Resolution ◽  
Hyperphosphorylated Tau ◽  
Toll Like Receptor ◽  
Wild Type ◽  
Toll Like Receptor 4 ◽  
Soluble Aggregates ◽  
Super Resolution Microscopy

Abstract Soluble aggregates of the microtubule-associated protein tau have been challenging to assemble and characterize, despite their important role in the development of tauopathies. We found that sequential hyperphosphorylation by PKA in conjugation with either GSK3-β or SAPK4 enabled recombinant wild-type (WT) tau of isoform 0N4R to spontaneously polymerize into small amorphous aggregates in vitro. We employed tandem mass spectrometry to determine the phosphorylation sites and the degree of phosphorylation, and super-resolution microscopy and electron microscopy to characterize the morphology of aggregates formed. Functionally, in comparison with the unmodified aggregates, which require heparin induction to assemble, these self-assembled hyperphosphorylated tau aggregates more efficiently disrupt membrane bilayers and induce Toll-like receptor 4 (TLR4)-dependent inflammatory responses. Together, our results demonstrate that tau hyperphosphorylation is potentially damaging to cells, providing a mechanistic model of how hyperphosphorylation of tau aggregates drives neuroinflammation in tauopathies.

The Various Procoagulant Effects of PolyP Require Different Minimal Polymer Lengths.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1761-1761
Author(s):  
Stephanie A. Smith ◽  
Jillian Huyck ◽  
Sharon H. Choi ◽  
James H. Morrissey
Keyword(s):  
Tissue Factor ◽  
Inflammatory Responses ◽  
Maximal Activity ◽  
Human Platelets ◽  
Minimum Length ◽  
Factor V ◽  
Pathway Activation ◽  
Clotting Cascade ◽  
Contact Pathway ◽  
Anticoagulant Function

Abstract Introduction: Polyphosphates (polyP) are negatively charged, linear phosphate polymers found throughout biology. PolyP in prokaryotes and unicellular eukaryotes is typically hundreds of phosphate units long, while polyP in dense granules of human platelets is ∼75 phosphates long. PolyP is secreted upon platelet activation, and we recently reported that it is a potent hemostatic regulator, acting at multiple points in the clotting cascade: PolyP triggers the contact pathway, accelerates factor V activation, and abrogates the anticoagulant function of tissue factor pathway inhibitor (TFPI). Our previous experiments utilized relatively heterodisperse polyP preparations. We have now isolated polyP fractions of defined polymer lengths and here report the size dependence of the various procoagulant effects of polyP. Methods: PolyP preparations were carefully size-fractionated by gel electrophoresis (Clark and Wood, Anal. Biochem. 1987, 161:280–290). Clotting assays were performed using pooled normal plasma with or without added recombinant TFPI or 40–50 μM polyP. Clotting was initiated by tissue factor (TF), factor Xa (FXa), or CaCl2 alone (contact pathway). Results: Of the polyP procoagulant activities tested, triggering the contact pathway required the longest polymers: We observed weak triggering of the contact pathway with ∼75mers and increasing procoagulant activity as polymer lengths increased, with maximal contact pathway activation requiring 450mers or longer. On the other hand, the downstream procoagulant effects of polyP required significantly shorter polyP polymers. (When clotting is triggered by either TF or FXa, further shortening of clotting times by polyP is attributed to accelerated factor V activation.) Minimum polymer lengths required to shorten TF or FXa clot times were ∼37mers, with maximal activity requiring 75mers or longer. Finally, the ability of polyP to abrogate TFPI anticoagulant function required the shortest minimum polymer lengths: Detectable anti-TFPI activity was observed with 11mers, with maximal activity requiring 49mers or longer. Conclusions: PolyP exhibited different minimum length requirements for its various procoagulant effects, consistent with the idea that polyP interacts with different proteins at various points in the clotting cascade. Contact pathway activation required very long polymers that were similar in size to those in infectious microorganisms. Therefore, triggering of the contact pathway by polyP may contribute to procoagulant/inflammatory responses to infection. In contrast, acceleration of factor V activation by polyP was maximal with polymers of the size released from human platelets. Even shorter polyP polymers abrogated TFPI function, suggesting that at least some of polyP’s anti-TFPI effect may be mediated by mechanisms other than (or in addition to) its ability to accelerate factor V activation.

Role of Secreted Adenosine Diphosphate in the Synergistic Effects of Cathepsin G on Human Platelets

1999 ◽  
Vol 95 (6) ◽  
pp. 341-346
Author(s):  
R.L Kinlough-Rathbone ◽  
D.W Perry ◽  
M.L Rand ◽  
M.A Packham
Keyword(s):  
Synergistic Effects ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Cathepsin G
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