Regulation of Syk Function by Ubiquitination in Human Platelets.

Abstract Platelets have an integral function in maintaining hemostasis. Signaling pathways are strictly regulated to ensure the cessation of bleeding without precipitating a thrombotic episode. An important initial physiological step in this process is the interaction of platelets with freshly exposed subendothelial collagen after vascular injury. A major platelet receptor involved is the GPVI/FcRγ-chain complex. Upon activation of this receptor, the immunoreceptor tyrosine-based activation motif (ITAM) present in the FcRγ-chain is phosphorylated by Src family kinases. This causes the tyrosine kinase Syk to bind to the ITAM where it is autophosphorylated, initializing a signaling cascade that activates a number of proteins including PLCγ2, PI-3 kinase and small G proteins. Syk appears to play an early pivotal role in GPVI/FcRγ-chain signal transduction and its regulation is crucial. Recent studies have shown that platelet aggregation, in response to collagen-related peptide (CRP), is potentiated in c-Cbl knockout mice. c-Cbl, a protein containing an E3 ligase responsible for substrate recognition for ubiquitin, appears to regulate numerous cytoplasmic kinases in other cell systems. This protein modification has emerged as one of the most common regulatory processes in all eukaryotes, second only to possibly phosphorylation. It appears to target proteins for proteosomal degradation although recently other mechanisms have been described, ranging from protein kinase activation to translation control. In the work presented here, we demonstrate that Syk associates with c-Cbl after GPVI/FcRγ-chain activation and therefore explored the possibility that Syk is ubiquitinated during GPVI/FcRγ-chain stimulation in human platelets. We have found that Syk is rapidly ubiquitinated upon activation by collagen, CRP and the snake venom protein convulxin, but not thrombin. PP2 and SU6656, two Src kinase inhibitors, prevented Syk phosphorylation and its ubiquitination, indicating that the process is downstream of Src kinases and probably requires Syk phosphorylation. The ubiquitination of Syk did not cause any apparent degradation of the protein as evidenced by the lack of effect of proteosomal and lysosomal inhibitors. We have been able to separate ubiquitinated Syk from its non-ubiquitinated counterpart. We have used an in vitro kinase assay to compare the activity of ubiquitinated Syk to non-ubiquitinated Syk. Surprisingly, when we compared the specific activity of the two Syk fractions, we found that the ubiquitinated Syk appeared to be about five-fold more active. Using a phosphospecific antibody to Syk (Tyr525/Tyr526) that measures activated Syk, we found that the majority (75%) of the active Syk is in the ubiquitinated fraction. In addition we found that Syk is not ubiquitinated in c-Cbl deficient mice. We therefore propose that c-Cbl plays a regulatory role in GPVI/FcRγ-chain stimulation through ubiquitination of Syk. The fact that 75% of active Syk is ubiquitinated suggests that ubiquitination plays an essential role in the regulation of its function. A possible function of ubiquitination may be targeting Syk to appropriate signaling molecules.

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Rapid ubiquitination of Syk following GPVI activation in platelets

Blood ◽

10.1182/blood-2004-09-3689 ◽

2005 ◽

Vol 105 (10) ◽

pp. 3918-3924 ◽

Cited By ~ 50

Author(s):

Carol A. Dangelmaier ◽

Patricia G. Quinter ◽

Jianguo Jin ◽

Alexander Y. Tsygankov ◽

Satya P. Kunapuli ◽

...

Keyword(s):

Platelet Activation ◽

Kinase Inhibitors ◽

Specific Activity ◽

High Specific Activity ◽

Kinase Assay ◽

Intermediate Step ◽

Glycoprotein Vi ◽

In Vitro Kinase Assay ◽

Deficient Mice

AbstractSpleen tyrosine kinase (Syk) activation is a key intermediate step in the activation of platelets by the physiologic agonist collagen. We have found that Syk is rapidly ubiquitinated upon activation of platelets by collagen, collagen-related peptide (CRP), and convulxin. The Src family kinase inhibitors prevented Syk phosphorylation and its ubiquitination, indicating that the process is downstream of Src kinases. The ubiquitination of Syk did not cause degradation of the protein as evidenced by the lack of effect of proteasomal and lysosomal inhibitors. We separated ubiquitinated Syk from its nonubiquitinated counterpart and used an in vitro kinase assay to compare their activities. We found that the ubiquitinated Syk appeared to be about 5-fold more active. Using a phosphospecific antibody to Syk (Tyr525/Tyr526) that measures activated Syk, we found that most (60%-75%) of the active Syk is in the ubiquitinated fraction. This result explains the apparent high specific activity of ubiquitinated Syk. In c-Cbl–deficient mice, Syk is not ubiquitinated, implicating c-Cbl as the E3 ligase involved in Syk ubiquitination. Furthermore, Syk is not dephosphorylated in these mice. We propose that c-Cbl plays a regulatory role in glycoprotein VI (GPVI)/Fc receptor γ (FcRγ)-chain–dependent platelet activation through its interaction with Syk.

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AMPK α2 subunit is involved in platelet signaling, clot retraction, and thrombus stability

Blood ◽

10.1182/blood-2010-04-279612 ◽

2010 ◽

Vol 116 (12) ◽

pp. 2134-2140 ◽

Cited By ~ 30

Author(s):

Voahanginirina Randriamboavonjy ◽

Johann Isaak ◽

Timo Frömel ◽

Benoit Viollet ◽

Beate Fisslthaler ◽

...

Keyword(s):

Kinase Inhibitors ◽

Adenosine Monophosphate ◽

Human Platelets ◽

Whole Body ◽

Initial Increase ◽

Clot Retraction ◽

Compound C ◽

Platelet Signaling ◽

Upstream Kinase

Abstract The adenosine monophosphate (AMP)–activated protein kinase (AMPK) is a regulator of energy balance at the cellular and whole-body levels, but little is known about the role of AMPK in platelet activation. We report that both the α1 and α2 AMPK isoforms are expressed by human and murine platelets and that thrombin elicits the phosphorylation of AMPKα as well as the upstream kinase, liver kinase B1 (LKB1). In human platelets, the kinase inhibitors iodotubercidin and compound C significantly inhibited thrombin-induced platelet aggregation and clot retraction without affecting the initial increase in [Ca2+]i. Clot retraction was also impaired in platelets from AMPKα2−/− mice but not from wild-type littermates or AMPKα1−/− mice. Moreover, rebleeding was more frequent in AMPKα2−/− mice, and the FeCl3-induced thrombi formed in AMPKα2−/− mice were unstable. Mechanistically, AMPKα2 was found to phosphorylate in vitro the Src-family kinase, Fyn, and isoform deletion resulted in the attenuated threonine phosphorylation of Fyn as well as the subsequent tyrosine phosphorylation of its substrate, β3 integrin. These data indicate that AMPKα2—by affecting Fyn phosphorylation and activity—plays a key role in platelet αIIbβ3 integrin signaling, leading to clot retraction and thrombus stability.

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Kinase Inhibitors of Novel Pyridopyrimidinone Candidates: Synthesis and In Vitro Anticancer Properties

Journal of Chemistry ◽

10.1155/2019/2635219 ◽

2019 ◽

Vol 2019 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Nagy M. Khalifa ◽

Mohamed A. Al-Omar ◽

Hamad M. Alkahtani ◽

Ahmed H. Bakheit

Keyword(s):

Kinase Inhibitors ◽

Human Cancer ◽

Ethyl Cyanoacetate ◽

Diethyl Malonate ◽

Aromatic Aldehydes ◽

Docking Studies ◽

Braf V600e ◽

Kinase Assay ◽

Human Cancer Cell Lines

A new class of pyridopyrimidinone compounds containing different nitrogenous heterocycles were synthesized starting from the key precursor 2-hydrazinyl-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one via condensation with series of aromatic aldehydes and cyclization using different reagents as ethyl acetoacetate, ethyl cyanoacetate, diethyl malonate, and ammonium isothiocyanate. The bioassay results showed compound 6 to be highly effective towards three human cancer cell lines (HepG2, PC-3, and HCT-116) in vitro with promising activity values (IC50: 0.5 μM) relative to the standard doxorubicin (IC50: 0.6 μM). Kinase inhibitory evaluation of compound 6 displays hopeful inhibitory action against BRAF V600E, EGFR, and PDGFRβ at100 μM. The molecular docking studies supported the initial kinase assay.

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Membrane Glycoproteins of Human and Rabbit Platelets: Studies In Vitro and after Circulation in Vivo

10.1055/s-0039-1689638 ◽

1975 ◽

Author(s):

J. N. George ◽

P. C. Lewis ◽

D. A. Sears

Keyword(s):

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Age Distribution ◽

Human Platelets ◽

Membrane Glycoproteins ◽

Rabbit Platelets ◽

Trypsin Hydrolysis ◽

Initial Recovery

The initial events of hemostasis and thrombosis involve platelet contact interactions and may be mediated by surface glycoproteins. Human and rabbit platelets were labeled with 125I-diazotized diiodosulfanilic acid (I), which reacts covalently with proteins, and proteins were separated by SDS-polyacrylamide gel electrophoresis. Only exposed membrane proteins were labeled because: 1) protein specific activity of membranes was 4-7 times that of whole platelets, 2) different proteins were labeled when I was reacted with isolated membranes, and 3) trypsin-hydrolysis of labeled intact platelets altered the radioactive peaks. Like Phillips (Biochem. 11, 4582, 72) and Nachman et al. (JBC 248, 2928, 73) we found that lactoperoxidase iodinated the 93,000 dalton glycoprotein (GP) of human platelets. In contrast, I labeled both the 93,000 and 118,000 dalton membrane GP of human platelets, and all 3 membrane GP of rabbit platelets.Rabbit platelets labeled simultaneously with I and 51Cr had identical density and therefore age distribution of the 2 labels. After infusion into rabbits, initial recovery of I was 23% of the Cr recovery. After 3 hrs, I disappearance was exponential and more rapid (T/2 = 17 hrs) than the linear Cr disappearance (T/2 = 30 hrs, p < .01). This was due to in vivo removal of I from circulating platelets since 1 did not elute more rapidly from platelets harvested after 3 hrs circulation and incubated in plasma at 37° (T/2 of I elution = 43 hrs, Cr = 33 hrs). Platelets harvested after 14-20 hrs circulation had the same distribution of I on the membrane GP as before circulation. We postulate that this symmetrical label loss indicates uniform loss of membrane GP, suggesting that platelets lose pieces of their plasma membrane during circulation. This could occur during contact interaction in the process of hemostasis.

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Structure-Based Drug Design of c-Kit Inhibitors for Use in the Treatment of Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v108.11.1906.1906 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1906-1906

Author(s):

David S. Maxwell ◽

Ashutosh Pal ◽

Zhenghong Peng ◽

Alexandr Shavrin ◽

Stefan Faderl ◽

...

Keyword(s):

Crystal Structure ◽

Acute Myeloid Leukemia ◽

Drug Design ◽

Cell Lines ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Kinase Assay ◽

Structure Based Drug Design ◽

Acute Myeloid

Abstract Inhibitors of c-Kit kinase have shown clinical relevance in various myeloid disorders, including acute myeloid leukemia (AML). Research in our lab has been oriented towards structure-based drug design of c-Kit inhibitors based on the available crystal structure. We describe the design, synthesis, and preliminary results from the in-vitro testing of several c-Kit kinase inhibitors in both enzymatic and cell-based assays. The design resulted from in-silico screening of several targeted libraries via docking to the crystal structure of c-Kit, followed by aggressive post-filtering by several criteria to significantly bias synthesis efforts towards candidate compounds with best chance for success. This led to 128 structures built from 8 common structural cores, from which 2 cores were initially selected based on the synthetic feasibility. Five compounds were initially synthesized, and were immediately followed by 60 compounds with variations to probe local structure-activity relationships. The initial set of compounds, designated APCKxxx, was tested in a c-Kit kinase assay; two compounds were found to have an IC50 in the high nM to low uM range. These compounds have been tested in a MTT-based assay using OCIM2 and OCI/AML3 cell lines. In the c-Kit expressing OCI/AML3 cell line, all five compounds possessed an EC50 < 500 nM and two had and EC50 ~100 nM. Our most recent results show that these compounds also show efficacy in some imatinib-resistant cell lines. We will discuss these results and our strategies for the second generation of compounds that are optimized for better activity, selectivity, and ADME properties.

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Identification of Inhibitors for a Virally Encoded Protein Kinase by 2 Different Screening Systems: In Vitro Kinase Assay and In-Cell Activity Assay

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057104270269 ◽

2005 ◽

Vol 10 (1) ◽

pp. 36-45 ◽

Cited By ~ 9

Author(s):

Helmut Mett ◽

Kerstin Hölscher ◽

Heidrun Degen ◽

Christina Esdar ◽

Birgit Felden De Neumann ◽

...

Keyword(s):

Protein Kinase ◽

Kinase Activity ◽

Kinase Inhibitors ◽

Cell Activity ◽

Kinase Assay ◽

Activity Assay ◽

Drug Candidates ◽

Cellular Context ◽

In Vitro Kinase Assay

The human cytomegalovirus (HCMV) protein kinase pUL97 represents an important determinant for viral replication and thus is a promising target for the treatment of HCMV. The authors screened a compound library of nearly 5000 entities based on known kinase inhibitors in 2 distinct ways. A radioactive in vitro kinase assay was performed with recombinant pUL97, purified from baculovirus-infected insect cells, on myelin basic protein-coated FlashPlates. About 20% of all compounds tested inhibited pUL97 kinase activity by more than 50% at a concentration of 10 μM. These hits belonged to various structural classes. To elucidate their potential to inhibit pUL97 in a cellular context, all compounds of the library were also tested in a cell-based activity assay. For this reason, a HEK293 cell line was established that ectopically expressed pUL97. When these cells were incubated with ganciclovir (GCV), pUL97 phosphorylated GCV to its monophosphate, which subsequently became phosphorylated to cytotoxic metabolites by cellular enzymes. Thereby, pUL97 converted cells into a GCV-sensitive phenotype. Inhibition of the pUL97 kinase activity resulted in protection of the cells against the cytotoxic effects of GCV. In total, 199 compounds of the library were cellular active at nontoxic concentrations, and 93 of them inhibited pUL97 in the in vitro kinase assay. Among these, promising inhibitors of HCMV replication were identified. The 2-fold screening system described here should facilitate the development of pUL97 inhibitors into potent drug candidates. ( Journal of Biomolecular Screening 2005:36-45)

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Potassium uptake and release by human blood platelets

Blood ◽

10.1182/blood.v48.2.185.bloodjournal482185 ◽

1976 ◽

Vol 48 (2) ◽

pp. 185-197

Author(s):

JS Wiley ◽

J Kuchibhotla ◽

CC Shaller ◽

RW Colman

Keyword(s):

Time Course ◽

Specific Activity ◽

Blood Platelets ◽

Gel Filtration ◽

Lactic Dehydrogenase ◽

Human Platelets ◽

First Order Kinetics ◽

A Minor ◽

Human Blood Platelets

Thrombin is known to reduce the K+ content of human platelets, but the subcellular origin of the lost K+ is not known. The effect of aggregating agents on K+ release was studied in platelets labeled in plasma by preincubation with 42KCI. Platelets were separated from plasma by gel filtration through Sepharose 2B equilibrated with K+ - free Tyrode's buffer. Platelet K+ was 116nEq/10(8) platelets, of which 23% was found to be extracellular immediately after gel filtration. K+ influx was 65 nEq/10(8) platelets/hr at pH 7.5 and was more rapid at pH 7.9. About 70% of cell K+ exchanged with plasma in 4 hr with first- order kinetics, while a minor fraction of about 30% exchanged with a slower time course. This slowly exchanging fraction of platelet K+ was thought to arise from heterogeneity in the platelet population. Epinephrine and ADP aggregated gel-filtered platelets and released serotonin, but with loss of only 5%-10% of cell K+ and no beta- glucuronidase. In contrast, thrombin released up to 30% of platelet K+, whether aggregation occurred or was prevented by not stirring the cells. The specific activity of K+ released by all aggregating agents was identical to the specific activity of total platelet K+. Thrombin (0.01–0.2 NIH U/ml) released serotonin and also beta-glucuronidase (an enzyme of the alpha-granule), and there was a linear relation between release of K+ and this enzyme (r = 0.88). No lysis of platelets occurred, since lactic dehydrogenase was not detected. Pretreatment of platelets with aspirin in vitro inhibited thrombin-induced release of serotonin but had no effect on the loss of K+ or beta-glucuronidase. In contrast, the ingestion of aspirin by mouth inhibited the release of serotonin, beta-glucuronidase, and K+ by thrombin. The data suggested that the K+ loss induced by thrombin was primarily derived from release of alpha-granules and that these organelles contained about 20% of the total platelet K+ in a freely exchangeable and nonsequestered state.

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Potassium uptake and release by human blood platelets

Blood ◽

10.1182/blood.v48.2.185.185 ◽

1976 ◽

Vol 48 (2) ◽

pp. 185-197 ◽

Cited By ~ 13

Author(s):

JS Wiley ◽

J Kuchibhotla ◽

CC Shaller ◽

RW Colman

Keyword(s):

Time Course ◽

Specific Activity ◽

Blood Platelets ◽

Gel Filtration ◽

Lactic Dehydrogenase ◽

Human Platelets ◽

First Order Kinetics ◽

A Minor ◽

Human Blood Platelets

Abstract Thrombin is known to reduce the K+ content of human platelets, but the subcellular origin of the lost K+ is not known. The effect of aggregating agents on K+ release was studied in platelets labeled in plasma by preincubation with 42KCI. Platelets were separated from plasma by gel filtration through Sepharose 2B equilibrated with K+ - free Tyrode's buffer. Platelet K+ was 116nEq/10(8) platelets, of which 23% was found to be extracellular immediately after gel filtration. K+ influx was 65 nEq/10(8) platelets/hr at pH 7.5 and was more rapid at pH 7.9. About 70% of cell K+ exchanged with plasma in 4 hr with first- order kinetics, while a minor fraction of about 30% exchanged with a slower time course. This slowly exchanging fraction of platelet K+ was thought to arise from heterogeneity in the platelet population. Epinephrine and ADP aggregated gel-filtered platelets and released serotonin, but with loss of only 5%-10% of cell K+ and no beta- glucuronidase. In contrast, thrombin released up to 30% of platelet K+, whether aggregation occurred or was prevented by not stirring the cells. The specific activity of K+ released by all aggregating agents was identical to the specific activity of total platelet K+. Thrombin (0.01–0.2 NIH U/ml) released serotonin and also beta-glucuronidase (an enzyme of the alpha-granule), and there was a linear relation between release of K+ and this enzyme (r = 0.88). No lysis of platelets occurred, since lactic dehydrogenase was not detected. Pretreatment of platelets with aspirin in vitro inhibited thrombin-induced release of serotonin but had no effect on the loss of K+ or beta-glucuronidase. In contrast, the ingestion of aspirin by mouth inhibited the release of serotonin, beta-glucuronidase, and K+ by thrombin. The data suggested that the K+ loss induced by thrombin was primarily derived from release of alpha-granules and that these organelles contained about 20% of the total platelet K+ in a freely exchangeable and nonsequestered state.

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Substrate Recognition by Osteoclast Precursors Induces C-src/Microtubule Association

The Journal of Cell Biology ◽

10.1083/jcb.137.1.247 ◽

1997 ◽

Vol 137 (1) ◽

pp. 247-258 ◽

Cited By ~ 68

Author(s):

Yousef Abu-Amer ◽

F. Patrick Ross ◽

Paul Schlesinger ◽

M. Mehrdad Tondravi ◽

Steven L. Teitelbaum

Keyword(s):

Specific Activity ◽

Substrate Recognition ◽

Kinase Assay ◽

Osteoclast Precursors ◽

Cell Substrate ◽

Exogenous Substrate ◽

In Vitro Kinase Assay ◽

Intracellular Vesicles

The osteoclast is distinguished from other macrophage polykaryons by its polarization, a feature induced by substrate recognition. The most striking component of the polarized osteoclast is its ruffled membrane, probably reflecting insertion of intracellular vesicles into the bone apposed plasmalemma. The failure of osteoclasts in c-src−/− osteopetrotic mice to form ruffled membranes indicates pp60c-src (c-src) is essential to osteoclast polarization. Interestingly, c-src itself is a vesicular protein that targets the ruffled membrane. This being the case, we hypothesized that matrix recognition by osteoclasts, and their precursors, induces c-src to associate with microtubules that traffic proteins to the cell surface. We find abundant c-src associates with tubulin immunoprecipitated from avian marrow macrophages (osteoclast precursors) maintained in the adherent, but not nonadherent, state. Since the two proteins colocalize only within adherent avian osteoclast-like cells examined by double antibody immunoconfocal microscopy, c-src/tubulin association reflects an authentic intracellular event. C-src/tubulin association is evident within 90 min of cell-substrate recognition, and the event does not reflect increased expression of either protein. In vitro kinase assay demonstrates tubulin-associated c-src is enzymatically active, phosphorylating itself as well as exogenous substrate. The increase in microtubule-associated kinase activity attending adhesion mirrors tubulin-bound c-src and does not reflect enhanced specific activity. The fact that microtubule-dissociating drugs, as well as cold, prevent adherence-induced c-src/tubulin association indicates the protooncogene complexes primarily, if not exclusively, with polymerized tubulin. Association of the two proteins does not depend upon protein tyrosine phosphorylation and is substrate specific, as it is induced by vitronectin and fibronectin but not type 1 collagen. Finally, consistent with cotransport of c-src and the osteoclast vacuolar proton pump to the polarized plasmalemma, the H+-ATPase decorates microtubules in a manner similar to the protooncogene, specifically coimmunoprecipitates with c-src from the osteoclast light Golgi membrane fraction, and is present, with c-src, in preparations enriched with acidifying vesicles reconstituted from the osteoclast ruffled membrane.

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A Barbourin-albumin Fusion Protein that Is Slowly Cleared In Vivo Retains the Ability to Inhibit Platelet Aggregation In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0037-1616148 ◽

2001 ◽

Vol 86 (09) ◽

pp. 902-908 ◽

Cited By ~ 21

Author(s):

Janey Marques ◽

Jacob George ◽

Ian Smith ◽

Varsha Bhakta ◽

William Sheffield

Keyword(s):

Platelet Aggregation ◽

Fusion Protein ◽

Human Platelets ◽

Rabbit Serum ◽

Inhibit Platelet Aggregation ◽

Venom Protein ◽

Nickel Chelate ◽

Pichia Pastoris Yeast

SummaryBarbourin is a 73 amino acid venom protein that inhibits platelet aggregation. Recombinant barbourin (BARH6), rabbit serum albumin (RSAH6), and a barbourin-RSA fusion protein (barbourin-linker-albumin; BLAH6) were secreted from Pichia pastoris yeast, and purified by nickel-chelate affinity chromatography via their C-terminal hexahisti-dine (H6) tags. BARH6 and BLAH6 did not differ in their IC50s for inhibition of platelet aggregation using either human platelets stimulated with thrombin or ADP, or rabbit platelets stimulated with ADP. BARH6 and BLAH6 were also effective in inhibiting platelet aggregation in whole blood, and formed complexes with platelet integrin αIIbβ3. The terminal catabolic half-life of BLAH6 approached that of RSAH6[3.4 ± 0.2 versus 4.0 ± 0.1 days (n = 4 ± SD)], but was substantially increased relative to that of BARH6 [0.15 ± 0.03 days (n = 3 ± SD)]. Our results suggest that fusion to albumin slows the clearance of barbourin in vivo, while preserving its ability to inhibit platelet aggregation.

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