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GRK6 regulates the hemostatic response to injury through its rate-limiting effects on GPCR signaling in platelets.

ABSTRACT: G protein-coupled receptors (GPCRs) mediate the majority of platelet activation in response to agonists. However, questions remain regarding the mechanisms that provide negative feedback toward activated GPCRs to limit platelet activation and thrombus formation. Here we provide the first evidence that GPCR kinase 6 (GRK6) serves this role in platelets, using GRK6-/- mice generated by CRISPR-Cas9 genome editing to examine the consequences of GRK6 knockout on GPCR-dependent signaling. Hemostatic thrombi formed in GRK6-/- mice are larger than in wild-type (WT) controls during the early stages of thrombus formation, with a rapid increase in platelet accumulation at the site of injury. GRK6-/- platelets have increased platelet activation, but in an agonist-selective manner. Responses to PAR4 agonist or adenosine 5'-diphosphate stimulation in GRK6-/- platelets are increased compared with WT littermates, whereas the response to thromboxane A2 (TxA2) is normal. Underlying these changes in GRK6-/- platelets is an increase in Ca2+ mobilization, Akt activation, and granule secretion. Furthermore, deletion of GRK6 in human MEG-01 cells causes an increase in Ca2+ response and PAR1 surface expression in response to thrombin. Finally, we show that human platelet activation in response to thrombin causes an increase in binding of GRK6 to PAR1, as well as an increase in the phosphorylation of PAR1. Deletion of GRK6 in MEG-01 cells causes a decrease in PAR1 phosphorylation. Taken together, these data show that GRK6 regulates the hemostatic response to injury through PAR- and P2Y12-mediated effects, helping to limit the rate of platelet activation during thrombus growth and prevent inappropriate platelet activation.

SUBMITTER: Chen X

PROVIDER: S-EPMC6960467 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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GRK6 regulates the hemostatic response to injury through its rate-limiting effects on GPCR signaling in platelets.

Chen Xi X Gupta Shuchi S Cooper Matthew M DeHelian Daniel D Zhao Xuefei X Naik Meghna U MU Wurtzel Jeremy G T JGT Stalker Timothy J TJ Goldfinger Lawrence E LE Benovic Jeffrey J Brass Lawrence F LF McKenzie Steven E SE Naik Ulhas P UP Ma Peisong P

Blood advances 20200101 1

G protein-coupled receptors (GPCRs) mediate the majority of platelet activation in response to agonists. However, questions remain regarding the mechanisms that provide negative feedback toward activated GPCRs to limit platelet activation and thrombus formation. Here we provide the first evidence that GPCR kinase 6 (GRK6) serves this role in platelets, using GRK6-/- mice generated by CRISPR-Cas9 genome editing to examine the consequences of GRK6 knockout on GPCR-dependent signaling. Hemostatic t ...[more]

PMID: 31899801

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Project description:BACKGROUND: The regulation of adult stem cell migration through human hematopoietic tissue involves the chemokine CXCL12 (SDF-1) and its receptor CXCR4 (CD184). In addition, human leukocyte elastase (HLE) plays a key role. When HLE is located on the cell surface (HLE(CS)), it acts not as a proteinase, but as a receptor for ?(1)proteinase inhibitor (?(1)PI, ?(1)antitrypsin, SerpinA1). Binding of ?(1)PI to HLE(CS) forms a motogenic complex. We previously demonstrated that ?(1)PI deficiency attends HIV-1 disease and that ?(1)PI augmentation produces increased numbers of immunocompetent circulating CD4(+) lymphocytes. Herein we investigated the mechanism underlying the ?(1)PI deficiency that attends HIV-1 infection. METHODS AND FINDINGS: Active ?(1)PI in HIV-1 subjects (median 17 µM, n?=?35) was significantly below normal (median 36 µM, p<0.001, n?=?30). In HIV-1 uninfected subjects, CD4(+) lymphocytes were correlated with the combined factors ?(1)PI, HLE(CS) (+) lymphocytes, and CXCR4(+) lymphocytes (r(2)?=?0.91, p<0.001, n?=?30), but not CXCL12. In contrast, in HIV-1 subjects with >220 CD4 cells/µl, CD4(+) lymphocytes were correlated solely with active ?(1)PI (r(2)?=?0.93, p<0.0001, n?=?26). The monoclonal anti-HIV-1 gp120 antibody 3F5 present in HIV-1 patient blood is shown to bind and inactivate human ?(1)PI. Chimpanzee ?(1)PI differs from human ?(1)PI by a single amino acid within the 3F5-binding epitope. Unlike human ?(1)PI, chimpanzee ?(1)PI did not bind 3F5 or become depleted following HIV-1 challenge, consistent with the normal CD4(+) lymphocyte levels and benign syndrome of HIV-1 infected chimpanzees. The presence of IgG-?(1)PI immune complexes correlated with decreased CD4(+) lymphocytes in HIV-1 subjects. CONCLUSIONS: This report identifies an autoimmune component of HIV-1 disease that can be overcome therapeutically. Importantly, results identify an achievable vaccine modification with the novel objective to protect against AIDS as opposed to the current objective to protect against HIV-1 infection.

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GRK6 regulates the hemostatic response to injury through its rate-limiting effects on GPCR signaling in platelets.

Publications

GRK6 regulates the hemostatic response to injury through its rate-limiting effects on GPCR signaling in platelets.

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