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MALT1-ubiquitination triggers non-genomic NF-κB/IKK signaling upon platelet activation.

Karim ZA, Vemana HP, Khasawneh FT - PLoS ONE (2015)

Bottom Line: In this connection, it is well known that MALT1, whose activity is modulated by proteasome, plays an important role in the regulation of IKK complex.It was also found to regulate thrombogenesis and physiologic hemostasis.Finally, we observed that the proteasome inhibitor blocks CBM complex formation and the interaction of IKKγ and MALT1; abrogates SNARE formation, and the association of MALT1 with TAK1 and TAB2, which are upstream of the CBM complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA, 91766, United States of America.

ABSTRACT
We have recently shown that IKK complex plays an important non-genomic role in platelet function, i.e., regulates SNARE machinery-dependent membrane fusion. In this connection, it is well known that MALT1, whose activity is modulated by proteasome, plays an important role in the regulation of IKK complex. Therefore, the present studies investigated the mechanism by which IKK signaling is regulated in the context of the platelet proteasome. It was found that platelets express a functional proteasome, and form CARMA/MALT1/Bcl10 (CBM) complex when activated. Using a pharmacological inhibitor, the proteasome was found to regulate platelet function (aggregation, integrin activation, secretion, phosphatidylserine exposure and changes in intracellular calcium). It was also found to regulate thrombogenesis and physiologic hemostasis. We also observed, upon platelet activation, that MALT1 is ubiquitinated, and this coincides with the activation of the IKK/NF-κB-signaling pathway. Finally, we observed that the proteasome inhibitor blocks CBM complex formation and the interaction of IKKγ and MALT1; abrogates SNARE formation, and the association of MALT1 with TAK1 and TAB2, which are upstream of the CBM complex. Thus, our data demonstrate that MALT1 ubiquitination is critical for the engagement of CBM and IKK complexes, thereby directing platelet signals to the NF-κB pathway.

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Proteasome inhibitor inhibits fibrin Clot retraction, thrombosis and hemostasis.(A) Platelets were washed and resuspended at 1 × 108/mL in buffer (see “Experimental Procedures”) in the presence of 500 μg/mL human fibrinogen, and fibrin clot was initiated by 0.025 U/mL thrombin at 37°C. Figure represents time frames of a retracting clot at the indicated time points. (B) Clot retraction kinetics curves in the absence or the presence of proteasome inhibitor (MG132). Clot surface areas were assessed by digital processing and plotted as percentage of maximal retraction (i.e. volume of platelet suspension); **P < 0.01, Mann-Whitney test. (C) Mice were injected either 3% Tween 80 (5 mL/kg, vehicle), 6 mg/kg MG132 (**P < 0.01, Mann-Whitney test) or 2.5 mg/kg bortezomib (***P < 0.001, Mann-Whitney test) in 3% Tween 80 by tail vain injection. (D) One hour post-dosing, FeCl3 induced thrombosis measurements were conducted as described in the “Methods” section; **P < 0.01, Mann-Whitney test (for MG132) and ***P < 0.001, Mann-Whitney test (for bortezomib). Each point represents the bleeding time/occlusion time of a single animal (n = 5).
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pone.0119363.g003: Proteasome inhibitor inhibits fibrin Clot retraction, thrombosis and hemostasis.(A) Platelets were washed and resuspended at 1 × 108/mL in buffer (see “Experimental Procedures”) in the presence of 500 μg/mL human fibrinogen, and fibrin clot was initiated by 0.025 U/mL thrombin at 37°C. Figure represents time frames of a retracting clot at the indicated time points. (B) Clot retraction kinetics curves in the absence or the presence of proteasome inhibitor (MG132). Clot surface areas were assessed by digital processing and plotted as percentage of maximal retraction (i.e. volume of platelet suspension); **P < 0.01, Mann-Whitney test. (C) Mice were injected either 3% Tween 80 (5 mL/kg, vehicle), 6 mg/kg MG132 (**P < 0.01, Mann-Whitney test) or 2.5 mg/kg bortezomib (***P < 0.001, Mann-Whitney test) in 3% Tween 80 by tail vain injection. (D) One hour post-dosing, FeCl3 induced thrombosis measurements were conducted as described in the “Methods” section; **P < 0.01, Mann-Whitney test (for MG132) and ***P < 0.001, Mann-Whitney test (for bortezomib). Each point represents the bleeding time/occlusion time of a single animal (n = 5).

Mentions: It is notable that many cytoskeletal proteins, other than β-actin, are involved in generating contractile forces in different cell types[31–33]. To investigate the possibility that the proteasome may regulate the ability of platelets to generate contractile forces, we analyzed the effect of MG132 on clot retraction. Remarkably, MG132 (10 μM) significantly delayed clot retraction when compared with the control (Fig. 3A). The extent of retraction was assessed from quantitation of clot surface and is expressed as percentage of total clot curve (Fig. 3B). Given that clot retraction is driven by myosin, a motor protein that interacts with actin, we hypothesized that the observed clot retraction was inhibited in proteasome inhibitor treated platelets due to decreased myosin-mediated force applied to the existing actin cables[34]. Since actin-dependent myosin contractility is thought to contribute to thrombus stability in the arterial system[34], we determined whether proteasome inhibition has any effects on thrombogenesis. Thus, we intravenously injected MG132 (6 mg/kg) or bortezomib (2.5 mg/kg) into C57BL6 mice, and initiated occlusive thrombosis 5 minutes later in a surgically exposed external carotid artery by a brief ectopic application of 7.5% FeCl3. This oxidative slur to the vascular wall resulted in the deposition of platelets along the damaged vessel wall that increased over several minutes (Fig. 3C). Typically, complete occlusion of the vessel was found to occur by 2.5 minutes after FeCl3 treatment; however, in animals injected with MG132 or bortezomib, occlusion was significantly delayed to more than 28 minutes (Fig. 3C). We next investigated whether the MG132 would exert negative consequences on hemostasis by measuring the tail bleeding time. It was found that mice injected with 6 mg/kg MG132 or bortezomib (2.5 mg/kg) had a significantly prolonged tail bleeding time when compared with control animals (Fig. 3D). Taken together, the above data provide evidence that the MG132 impairs clot retraction, and both MG132 and bortezomib exhibit anti-thrombotic activity, but they do so along with increasing the risk of bleeding.


MALT1-ubiquitination triggers non-genomic NF-κB/IKK signaling upon platelet activation.

Karim ZA, Vemana HP, Khasawneh FT - PLoS ONE (2015)

Proteasome inhibitor inhibits fibrin Clot retraction, thrombosis and hemostasis.(A) Platelets were washed and resuspended at 1 × 108/mL in buffer (see “Experimental Procedures”) in the presence of 500 μg/mL human fibrinogen, and fibrin clot was initiated by 0.025 U/mL thrombin at 37°C. Figure represents time frames of a retracting clot at the indicated time points. (B) Clot retraction kinetics curves in the absence or the presence of proteasome inhibitor (MG132). Clot surface areas were assessed by digital processing and plotted as percentage of maximal retraction (i.e. volume of platelet suspension); **P < 0.01, Mann-Whitney test. (C) Mice were injected either 3% Tween 80 (5 mL/kg, vehicle), 6 mg/kg MG132 (**P < 0.01, Mann-Whitney test) or 2.5 mg/kg bortezomib (***P < 0.001, Mann-Whitney test) in 3% Tween 80 by tail vain injection. (D) One hour post-dosing, FeCl3 induced thrombosis measurements were conducted as described in the “Methods” section; **P < 0.01, Mann-Whitney test (for MG132) and ***P < 0.001, Mann-Whitney test (for bortezomib). Each point represents the bleeding time/occlusion time of a single animal (n = 5).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352082&req=5

pone.0119363.g003: Proteasome inhibitor inhibits fibrin Clot retraction, thrombosis and hemostasis.(A) Platelets were washed and resuspended at 1 × 108/mL in buffer (see “Experimental Procedures”) in the presence of 500 μg/mL human fibrinogen, and fibrin clot was initiated by 0.025 U/mL thrombin at 37°C. Figure represents time frames of a retracting clot at the indicated time points. (B) Clot retraction kinetics curves in the absence or the presence of proteasome inhibitor (MG132). Clot surface areas were assessed by digital processing and plotted as percentage of maximal retraction (i.e. volume of platelet suspension); **P < 0.01, Mann-Whitney test. (C) Mice were injected either 3% Tween 80 (5 mL/kg, vehicle), 6 mg/kg MG132 (**P < 0.01, Mann-Whitney test) or 2.5 mg/kg bortezomib (***P < 0.001, Mann-Whitney test) in 3% Tween 80 by tail vain injection. (D) One hour post-dosing, FeCl3 induced thrombosis measurements were conducted as described in the “Methods” section; **P < 0.01, Mann-Whitney test (for MG132) and ***P < 0.001, Mann-Whitney test (for bortezomib). Each point represents the bleeding time/occlusion time of a single animal (n = 5).
Mentions: It is notable that many cytoskeletal proteins, other than β-actin, are involved in generating contractile forces in different cell types[31–33]. To investigate the possibility that the proteasome may regulate the ability of platelets to generate contractile forces, we analyzed the effect of MG132 on clot retraction. Remarkably, MG132 (10 μM) significantly delayed clot retraction when compared with the control (Fig. 3A). The extent of retraction was assessed from quantitation of clot surface and is expressed as percentage of total clot curve (Fig. 3B). Given that clot retraction is driven by myosin, a motor protein that interacts with actin, we hypothesized that the observed clot retraction was inhibited in proteasome inhibitor treated platelets due to decreased myosin-mediated force applied to the existing actin cables[34]. Since actin-dependent myosin contractility is thought to contribute to thrombus stability in the arterial system[34], we determined whether proteasome inhibition has any effects on thrombogenesis. Thus, we intravenously injected MG132 (6 mg/kg) or bortezomib (2.5 mg/kg) into C57BL6 mice, and initiated occlusive thrombosis 5 minutes later in a surgically exposed external carotid artery by a brief ectopic application of 7.5% FeCl3. This oxidative slur to the vascular wall resulted in the deposition of platelets along the damaged vessel wall that increased over several minutes (Fig. 3C). Typically, complete occlusion of the vessel was found to occur by 2.5 minutes after FeCl3 treatment; however, in animals injected with MG132 or bortezomib, occlusion was significantly delayed to more than 28 minutes (Fig. 3C). We next investigated whether the MG132 would exert negative consequences on hemostasis by measuring the tail bleeding time. It was found that mice injected with 6 mg/kg MG132 or bortezomib (2.5 mg/kg) had a significantly prolonged tail bleeding time when compared with control animals (Fig. 3D). Taken together, the above data provide evidence that the MG132 impairs clot retraction, and both MG132 and bortezomib exhibit anti-thrombotic activity, but they do so along with increasing the risk of bleeding.

Bottom Line: In this connection, it is well known that MALT1, whose activity is modulated by proteasome, plays an important role in the regulation of IKK complex.It was also found to regulate thrombogenesis and physiologic hemostasis.Finally, we observed that the proteasome inhibitor blocks CBM complex formation and the interaction of IKKγ and MALT1; abrogates SNARE formation, and the association of MALT1 with TAK1 and TAB2, which are upstream of the CBM complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA, 91766, United States of America.

ABSTRACT
We have recently shown that IKK complex plays an important non-genomic role in platelet function, i.e., regulates SNARE machinery-dependent membrane fusion. In this connection, it is well known that MALT1, whose activity is modulated by proteasome, plays an important role in the regulation of IKK complex. Therefore, the present studies investigated the mechanism by which IKK signaling is regulated in the context of the platelet proteasome. It was found that platelets express a functional proteasome, and form CARMA/MALT1/Bcl10 (CBM) complex when activated. Using a pharmacological inhibitor, the proteasome was found to regulate platelet function (aggregation, integrin activation, secretion, phosphatidylserine exposure and changes in intracellular calcium). It was also found to regulate thrombogenesis and physiologic hemostasis. We also observed, upon platelet activation, that MALT1 is ubiquitinated, and this coincides with the activation of the IKK/NF-κB-signaling pathway. Finally, we observed that the proteasome inhibitor blocks CBM complex formation and the interaction of IKKγ and MALT1; abrogates SNARE formation, and the association of MALT1 with TAK1 and TAB2, which are upstream of the CBM complex. Thus, our data demonstrate that MALT1 ubiquitination is critical for the engagement of CBM and IKK complexes, thereby directing platelet signals to the NF-κB pathway.

Show MeSH
Related in: MedlinePlus