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Impaired Glycoprotein VI-Mediated Signaling and Platelet Functional Responses in CD45 Knockout Mice.

ABSTRACT: BACKGROUND AND OBJECTIVE:?CD45 is a receptor protein tyrosine phosphatase present on the surface of all hematopoietic cells except for erythrocytes and platelets. Proteomics studies, however, have demonstrated the presence of a CD45 c-terminal catalytic peptide in platelets. Therefore, we investigated the functional role of this truncated isoform of CD45 in platelets, which contains the c-terminal catalytic domain but lacks the extracellular region. METHODS AND RESULTS:?We used an antibody specific to the c-terminus of CD45 to confirm the presence of a truncated CD45 isoform in platelets. We also examined ex vivo and in vivo platelet function using CD45 knockout (KO) mice. Aggregation and secretion mediated by the glycoprotein VI (GPVI) receptor was impaired in CD45 KO platelets. Consequently, CD45 KO mice had impaired hemostasis indicated by increased tail bleeding times. Also, using a model of pulmonary embolism we showed that CD45 KO mice had defective in vivo thrombus formation. Next, we investigated whether or not the truncated isoform of CD45 had a role in GPVI signaling. The full-length isoform of CD45 is known to regulate Src family kinase (SFK) activation in lymphocytes. We find a similar role for the truncated isoform of CD45 in platelets. SFK activation was impaired downstream of the GPVI receptor in the CD45 KO murine platelets. Consequently, Syk, PLC?2, and pleckstrin phosphorylations were also impaired in CD45 KO murine platelets. CONCLUSION:?We conclude that the truncated CD45 isoform regulates GPVI-mediated signaling and platelet functional responses by regulating SFK activation.

SUBMITTER: Inamdar VV

PROVIDER: S-EPMC7192241 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Impaired Glycoprotein VI-Mediated Signaling and Platelet Functional Responses in CD45 Knockout Mice.

Inamdar Vaishali V VV Kostyak John C JC Badolia Rachit R Dangelmaier Carol A CA Manne Bhanu Kanth BK Patel Akruti A Kim Soochong S Kunapuli Satya P SP

Thrombosis and haemostasis 20190621 8

<h4>Background and objective</h4> CD45 is a receptor protein tyrosine phosphatase present on the surface of all hematopoietic cells except for erythrocytes and platelets. Proteomics studies, however, have demonstrated the presence of a CD45 c-terminal catalytic peptide in platelets. Therefore, we investigated the functional role of this truncated isoform of CD45 in platelets, which contains the c-terminal catalytic domain but lacks the extracellular region.<h4>Methods and results</h4> We used an ...[more]

PMID: 31226719

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Project description:Essentials Collagen and thrombin when used simultaneously generate highly activated platelets. The effect of thrombin stimulation on subsequent glycoprotein VI (GPVI) function was observed. Soluble fibrin, but not protease activated receptor (PAR) activation, prevented GPVI activation. Circulating soluble fibrin in coagulopathic blood may cause an acquired GPVI signaling defect. SUMMARY:Background In coagulopathic blood, circulating thrombin may drive platelet dysfunction. Methods/Results Using calcium dye-loaded platelets, the effect of thrombin exposure and soluble fibrin generation on subsequent platelet GPVI function was investigated. Exposure of apixaban-treated platelet-rich plasma (12% PRP) to thrombin (1-10 nm), but not ADP or thromboxane mimetic U46619 exposure, dramatically blocked subsequent GPVI activation by convulxin, collagen-related peptide or fibrillar collagen. Consistent with soluble fibrin multimerizing and binding GPVI, the onset of convulxin insensitivity required 200-500 s of thrombin exposure, was not mimicked by exposure to PAR-1/4 activating peptides, was not observed with washed platelets, and was blocked by fibrin polymerization inhibitor (GPRP) or factor XIIIa inhibitor (T101). PAR-1 signaling through G?q was not required because vorapaxar blocked thrombin-induced calcium mobilization but had no effect on the ability of thrombin to impair GPVI-signaling. Convulxin insensitivity was unaffected by the metalloprotease inhibitor GM6001 or the ?IIb ?3 antagonist GR144053, indicating negligible roles for GPVI shedding or ?IIb ?3 binding of fibrin. Thrombin treatment of washed platelets resuspended in purified fibrinogen also produced convulxin insensitivity that was prevented by GPRP. Exposure of apixaban/PPACK-treated whole blood to thrombin-treated fibrinogen resulted in > 50% decrease in platelet deposition in a collagen microfluidic assay that required soluble fibrin assembly. Conclusions Conversion of only 1% plasma fibrinogen in coagulopathic blood would generate 90 nm soluble fibrin, far exceeding ~1 nmGPVI in blood. Soluble fibrin, rather than thrombin-induced platelet activation throuh PAR-1 and PAR-4, downregulated GPVI-signaling in response to stimuli, and may lead to subsequent hypofunction of endogenous or transfused platelets.

Project description:Essentials RAS proteins are expressed in platelets but their functions are largely uncharacterized. TC21/RRas2 is required for glycoprotein VI-induced platelet responses and for thrombus stability in vivo. TC21 regulates platelet aggregation by control of αIIb β3 integrin activation, via crosstalk with Rap1b. This is the first indication of functional importance of a proto-oncogenic RAS protein in platelets. Background Many RAS family small GTPases are expressed in platelets, including RAC, RHOA, RAP, and HRAS/NRAS/RRAS1, but most of their signaling and cellular functions remain poorly understood. Like RRAS1, TC21/RRAS2 reverses HRAS-induced suppression of integrin activation in CHO cells. However, a role for TC21 in platelets has not been explored. Objectives To determine TC21 expression in platelets, TC21 activation in response to platelet agonists, and roles of TC21 in platelet function in in vitro and in vivo thrombosis. Results We demonstrate that TC21 is expressed in human and murine platelets, and is activated in response to agonists for the glycoprotein (GP) VI-FcRγ immunoreceptor tyrosine-based activation motif (ITAM)-containing collagen receptor, in an Src-dependent manner. GPVI-induced platelet aggregation, integrin αIIb β3 activation, and α-granule and dense granule secretion, as well as phosphorylation of Syk, phospholipase Cγ2, AKT, and extracellular signal-regulated kinase, were inhibited in TC21-deficient platelets ex vivo. In contrast, these responses were normal in TC21-deficient platelets following stimulation with P2Y, protease-activated receptor 4 and C-type lectin receptor 2 receptor agonists, indicating that the function of TC21 in platelets is GPVI-FcRγ-ITAM-specific. TC21 was required for GPVI-induced activation of Rap1b. TC21-deficient mice did not show a significant delay in injury-induced thrombosis as compared with wild-type controls; however, thrombi were unstable. Hemostatic responses showed similar effects. Conclusions TC21 is essential for GPVI-FcRγ-mediated platelet activation and for thrombus stability in vivo via control of Rap1b and integrins.

Dataset Information

Impaired Glycoprotein VI-Mediated Signaling and Platelet Functional Responses in CD45 Knockout Mice.

Publications

Impaired Glycoprotein VI-Mediated Signaling and Platelet Functional Responses in CD45 Knockout Mice.

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