Identification Of a Novel Small Molecule Inhibitor Of ASK1 As a Potent Anti-Platelet Agent

Abstract Apoptosis signal-regulating kinase (ASK1) is a serine/threonine kinase, belonging to the MAP kinase-kinase-kinase family, which is activated in response to stress. However, its presence and role in platelets are not known. We found that ASK1 is expressed in platelets and is rapidly activated during platelet stimulation by various agonists in a dose-dependent manner. In addition, we found that TRAF2/6, known endogenous activators of ASK1, are expressed in platelets and associate with ASK1 upon platelet activation with agonists. Furthermore, genetic ablation of Ask1 significantly delayed tail-bleeding time (P=0.2x10-9). While WT mice showed an average bleeding time of 100 s, the Ask1 null mice had an average bleeding time of 576 s. A carotid artery injury induced by 10% FeCl3 showed a significantly increased (P=0.0003) time of occlusion and unstable thrombus formation in Ask1 null mice. Furthermore, we found that loss of Ask1 renders significant protection to the mice from pulmonary thromboembolism induced by a mixture of collagen and epinephrine as determined by increased survival and lack of large occlusive thrombi in the lung. We also found that ADP- and AYPGKF (PAR4 receptor peptide) -induced platelet aggregation was diminished in Ask1 null mice compared to WT mice. Furthermore, PAR4 peptide-induced alpha- and dense-granular secretion was also reduced in Ask1 null platelets compared to WT. Interestingly, we also found that Ask1 null platelets bind less FITC-fibrinogen compared to the WT upon activation by PAR4 peptide. Furthermore, thrombin failed to activate MKK6 and p38 in Ask1 knockout platelets, showing that Ask1 is indispensable for p38 activation by thrombin. These results indicated that ASK1 regulates platelet function by augmenting platelet secretion as well as fibrinogen receptor activation, making it an important target for combating thrombosis. We therefore synthesized a novel and highly specific ASK1 inhibitor, N-(6-(1H-imidazol-1-yl)imidazo[1,2-a]pyridin-2-yl)-4-(tert-butyl)benzamide (IPTB) based on the published report. IPTB has been found to be a very potent inhibitor that inhibits ASK1 activity at nM concentrations. IPTB is also highly specific to ASK1 and does not affect activities of related protein kinases such as ASK2, MEKK1, TAK1, and ERK1. We found that in human platelets, IPTB dose-dependently inhibits p38 activation induced by a variety of platelet agonists. Furthermore, IPTB dose-dependently inhibited ADP and PAR4 peptide-induced platelet aggregation. Interestingly, IPTB also dose-dependently inhibited platelet spreading on immobilized fibrinogen. Our results strongly suggest that the dose of IPTB could be adjusted so that it attenuates thrombosis without affecting hemostasis. This development would make IPTB a novel potential therapeutic agent to be used to combat thrombotic disorders. Disclosures: No relevant conflicts of interest to declare.

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An Antibody Targeted To The Anionic Region Of Human Protease Activated Receptor 4 Inhibits Thrombosis Without Influencing Bleeding

Blood ◽

10.1182/blood.v122.21.2376.2376 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2376-2376

Author(s):

Michele M. Mumaw ◽

Maria de la Fuente ◽

Amal Arachiche ◽

Daniel N. Nobel ◽

Marvin T. Nieman

Keyword(s):

Platelet Aggregation ◽

Bleeding Time ◽

Conflicts Of Interest ◽

Preliminary Evidence ◽

Human Platelets ◽

Control Treatment ◽

Dependent Manner ◽

Protease Activated Receptors

Abstract Protease activated receptors (PARs) are G-protein coupled receptors which are activated by cleavage of their N-terminus by thrombin. This generates a tethered ligand which is then able to activate the corresponding receptor. Human platelets express PAR1 and PAR4, which both have crucial roles in mediating the response of platelets to injury. Our hypothesis is that PAR4 is an ideal target for new anti-platelet therapies because it is required for stable clot formation and has limited tissue distribution. We have previously determined a region on PAR4 that is required for efficient activation by thrombin. A polyclonal antibody (CAN12) targeted to this region of the PAR4 exodomain does not cross react to PAR1. Initial studies determined that CAN12 is able to block thrombin-induced human platelet aggregation with an IC50 of 10 ng/ml. Control IgG does not inhibit aggregation at 2 mg/ml. In mouse platelets, CAN12 inhibits P-selectin expression and integrin activation. In the Rose-Bengal mouse model of carotid artery thrombosis, CAN12 (1 mg/kg) administered 10 minutes prior to injury was able to completely inhibit the formation of a thrombus in a dose dependent manner. The antibody delayed thrombosis to greater than 90 min; the experiment was terminated at 90 minutes. In contrast, control treatment (2 mg/kg IgG or saline) resulted in complete occlusion at ∼40 minutes. Further, the minimal dose of CAN12 required for complete inhibition of thrombosis (0.5 mg/kg) administered fifteen minutes after injury also delayed thrombosis from ∼50 minutes to ∼80 minutes. This indicates that CAN12 is able to disrupt a thrombus after it has been initiated. Preliminary evidence indicates that CAN12 is able to delay the cleavage of PAR4. Importantly, CAN12 (2 mg/kg) treatment does not increase bleeding time or blood loss in the tail clip assay compared to control IgG (2 mg/kg) treatment. There was also no significant increase in bleeding in the saphenous vein assay. The mice treated with CAN12 (2 mg/kg) had an average bleeding time of 102 seconds for 12 clot formations in 20 minutes compared to the control mice (IgG 2 mg/kg) which had an average bleeding time of 143 seconds for 11 clot formations. These data demonstrate that we are able to inhibit platelet aggregation in vitro and thrombosis in vivo without influencing bleeding time. Overall, these studies provide insight towards the development of new anti-platelet therapies and, specifically, PAR4 as an antiplatelet therapy target. Disclosures: No relevant conflicts of interest to declare.

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Evaluation of Two Structurally Distinct Novel Inhibitors of Apoptosis Signal-Regulating Kinase 1 (MAP3K5), As Potent Anti-Platelet Agents

Blood ◽

10.1182/blood.v128.22.3833.3833 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3833-3833

Author(s):

Meghna U. Naik ◽

Xi Chen ◽

Brendan Bachman ◽

Ganesha Rai ◽

David Maloney ◽

...

Keyword(s):

Platelet Aggregation ◽

Carotid Artery ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Critical Role ◽

Receptor Activation ◽

New Drugs ◽

Severe Bleeding ◽

Fibrinogen Receptor

Abstract Platelets play a critical role in both hemostasis and thrombosis. Anti-platelet drugs currently available apart from aspirin are directed against platelet agonist receptors or fibrinogen receptor integrin aIIbb3. These antagonists, although having potent anti-thrombotic activities, cause severe bleeding due to their effect on hemostasis. It is therefore of utmost important to develop new drugs that will protect from thrombosis with minimal effect on hemostasis. Apoptosis signal-regulating kinase (ASK1) is a redox sensitive serine/threonine kinase, belonging to the MAP kinase-kinase-kinase family, which is activated in response to stress. However, its role in platelets is not known. We found that ASK1 is rapidly activated downstream of all platelet agonists. Ablation of Ask1 gene results in impaired platelet functions such as granule secretion, thromboxane A2 generation, as well as fibrinogen receptor activation, which translates into attenuated platelet aggregation compared to WT littermates. We also found that thrombin failed to activate p38 in Ask1 null platelets, showing that Ask1 is indispensable for p38 activation by thrombin. FeCl3-induced carotid artery injury model of thrombosis showed a significantly increased (P=0.0003) time of occlusion and unstable thrombus formation in Ask1 null mice. These results indicated that ASK1 plays a central role in regulating platelet function, making it a potential target for combating thrombosis. We therefore synthesized two novel and highly specific ASK1 inhibitors based on the published reports, N-(6-(1H-imidazol-1-yl)imidazo[1,2-a]pyridin-2-yl)-4-(tert-butyl)benzamide (IPTB) and GS-4997. We found that GS-4997 (500nM) and IPTB (5mM) inhibit agonist-induced ASK1 activation in human platelets. They do not affect activities of related protein kinases such as ASK2, MEKK1, TAK1, and ERK1/2. We also found that IPTB and GS-4997 dose-dependently inhibited activation of p38, a downstream effector kinase, induced by a variety of platelet agonists. Furthermore, these compounds dose-dependently inhibited ADP, collagen, convulxin and PAR4 activating peptide AYPGKF-induced platelet aggregation as well as platelet spreading on immobilized fibrinogen. In-vivo carotid artery thrombosis assay revealed that WT mice injected intraperitoneally with either IPTB (100mg/kg) of GS-4997 (100mg/kg), showed a significantly increased time of occlusion (P=0.028 and P=0.005 respectively) and thrombus formed were unstable as compared to control WT mice treated with saline alone. Furthermore, injection of either IPTB or GS-4997 protected mice against collagen/epinephrine-induced pulmonary thromboembolism. Out of 14 saline-treated mice only two survived whereas, 10 out of 11 mice treated with GS-4997 (100mg/kg) survived (P=0.0002). In case of IPTB 1 out of 12 control mice survived as compared to 9 out of 12 treated mice (P=0.0028). Interestingly, tail-bleeding studies revealed that WT mice treated with either IPTB (1mg/kg) of GS-4997 (1mg/kg), did not affect the average bleeding time (100s) seen in the WT mice treated with saline alone, suggesting that both inhibitors had no effect on in-vivo hemostasis. Moreover, pretreatment of the whole blood with these inhibitors significantly reduced thrombus formation under arterial flow (800s-1) without affecting platelet adhesion to collagen as assessed using a microfluidic device. Our results strongly suggest that both IPTB and GS-4997 protect the mice from thrombosis without affecting hemostasis. Further development of these inhibitors as a potential therapeutic agent to combat thrombotic disorders is highly warranted. Disclosures No relevant conflicts of interest to declare.

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Antithrombotic Effect of Crotalin, a Platelet Membrane Glycoprotein Ib Antagonist From Venom of Crotalus atrox

Blood ◽

10.1182/blood.v91.5.1582.1582_1582_1589 ◽

1998 ◽

Vol 91 (5) ◽

pp. 1582-1589

Author(s):

Mei-Chi Chang ◽

Hui-Kuan Lin ◽

Hui-Chin Peng ◽

Tur-Fu Huang

Keyword(s):

Platelet Aggregation ◽

Latent Period ◽

Bleeding Time ◽

Thrombus Formation ◽

Platelet Membrane ◽

Dependent Manner ◽

Glycoprotein Ib ◽

Platelet Surface ◽

Dose Dependent

A potent platelet glycoprotein Ib (GPIb) antagonist, crotalin, with a molecular weight of 30 kD was purified from the snake venom ofCrotalus atrox. Crotalin specifically and dose dependently inhibited aggregation of human washed platelets induced by ristocetin with IC50 of 2.4 μg/mL (83 nmol/L). It was also active in inhibiting ristocetin-induced platelet aggregation of platelet-rich plasma (IC50, 6.3 μg/mL). 125I-crotalin bound to human platelets in a saturable and dose-dependent manner with a kd value of 3.2 ± 0.1 × 10−7 mol/L, and its binding site was estimated to be 58,632 ± 3,152 per platelet. Its binding was specifically inhibited by a monoclonal antibody, AP1 raised against platelet GPIb. Crotalin significantly prolonged the latent period in triggering platelet aggregation caused by low concentration of thrombin (0.03 U/mL), and inhibited thromboxane B2formation of platelets stimulated either by ristocetin plus von Willebrand factor (vWF), or by thrombin (0.03 U/mL). When crotalin was intravenously (IV) administered to mice at 100 to 300 μg/kg, a dose-dependent prolongation on tail bleeding time was observed. The duration of crotalin in prolonging tail bleeding time lasted for 4 hours as crotalin was given at 300 μg/kg. In addition, its in vivo antithrombotic activity was evidenced by prolonging the latent period in inducing platelet-rich thrombus formation by irradiating the mesenteric venules of the fluorescein sodium-treated mice. When administered IV at 100 to 300 μg/kg, crotalin dose dependently prolonged the time lapse in inducing platelet-rich thrombus formation. In conclusion, crotalin specifically inhibited vWF-induced platelet agglutination in the presence of ristocetin because crotalin selectively bound to platelet surface receptor-glycoprotein Ib, resulting in the blockade of the interaction of vWF with platelet membrane GPIb. In addition, crotalin is a potent antithrombotic agent because it pronouncedly blocked platelet plug formation in vivo.

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Antithrombotic Effect of Crotalin, a Platelet Membrane Glycoprotein Ib Antagonist From Venom of Crotalus atrox

Blood ◽

10.1182/blood.v91.5.1582 ◽

1998 ◽

Vol 91 (5) ◽

pp. 1582-1589 ◽

Cited By ~ 49

Author(s):

Mei-Chi Chang ◽

Hui-Kuan Lin ◽

Hui-Chin Peng ◽

Tur-Fu Huang

Keyword(s):

Platelet Aggregation ◽

Latent Period ◽

Bleeding Time ◽

Thrombus Formation ◽

Platelet Membrane ◽

Dependent Manner ◽

Glycoprotein Ib ◽

Platelet Surface ◽

Dose Dependent

AbstractA potent platelet glycoprotein Ib (GPIb) antagonist, crotalin, with a molecular weight of 30 kD was purified from the snake venom ofCrotalus atrox. Crotalin specifically and dose dependently inhibited aggregation of human washed platelets induced by ristocetin with IC50 of 2.4 μg/mL (83 nmol/L). It was also active in inhibiting ristocetin-induced platelet aggregation of platelet-rich plasma (IC50, 6.3 μg/mL). 125I-crotalin bound to human platelets in a saturable and dose-dependent manner with a kd value of 3.2 ± 0.1 × 10−7 mol/L, and its binding site was estimated to be 58,632 ± 3,152 per platelet. Its binding was specifically inhibited by a monoclonal antibody, AP1 raised against platelet GPIb. Crotalin significantly prolonged the latent period in triggering platelet aggregation caused by low concentration of thrombin (0.03 U/mL), and inhibited thromboxane B2formation of platelets stimulated either by ristocetin plus von Willebrand factor (vWF), or by thrombin (0.03 U/mL). When crotalin was intravenously (IV) administered to mice at 100 to 300 μg/kg, a dose-dependent prolongation on tail bleeding time was observed. The duration of crotalin in prolonging tail bleeding time lasted for 4 hours as crotalin was given at 300 μg/kg. In addition, its in vivo antithrombotic activity was evidenced by prolonging the latent period in inducing platelet-rich thrombus formation by irradiating the mesenteric venules of the fluorescein sodium-treated mice. When administered IV at 100 to 300 μg/kg, crotalin dose dependently prolonged the time lapse in inducing platelet-rich thrombus formation. In conclusion, crotalin specifically inhibited vWF-induced platelet agglutination in the presence of ristocetin because crotalin selectively bound to platelet surface receptor-glycoprotein Ib, resulting in the blockade of the interaction of vWF with platelet membrane GPIb. In addition, crotalin is a potent antithrombotic agent because it pronouncedly blocked platelet plug formation in vivo.

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STAT3 Regulates Collagen-Induced Platelet Aggregation Independent of Its Transcription Factor Activity

Blood ◽

10.1182/blood.v116.21.157.157 ◽

2010 ◽

Vol 116 (21) ◽

pp. 157-157

Author(s):

Zhou Zhou ◽

Francisca C. Gushiken ◽

Angela Bergeron ◽

Vinod K. Vijayan ◽

Rolando Rumbaut ◽

...

Keyword(s):

Shear Stress ◽

Transcription Factor ◽

Platelet Aggregation ◽

Tyrosine Phosphorylation ◽

Thrombus Formation ◽

Human Platelets ◽

Dependent Manner ◽

Transcription Factor Activity ◽

Factor Activity ◽

Dose Dependent

Abstract Abstract 157 Signal Transducer and Activator of Transcription 3 (STAT3) serves as a transcription factor activated by cytokine-induced intracellular signals, which are critical in megakaryopoiesis. This signaling pathway may also be active in anucleated platelets that are primed by proinflammatory cytokines, suggesting that STAT3 plays a role in platelet hyperactivity associated with inflammation. We have recently found that three different classes of STAT3 inhibitors each selectively inhibited collagen-induced aggregation of human platelets by ∼50%. They also blocked thrombus formation (∼80%) on immobilized collagen under an arterial shear stress of 62.5 dyn/cm2. These STAT3 inhibitors also blocked platelet aggregation induced by collagen-related peptide, suggesting that they acted on GP VI-mediated intracellular signaling in platelets. These in vitro results were further verified in two sets of experiments in mouse models. First, an oligonucleotide G-quartet STAT3 inhibitor (1 mg/ml) or a scrambled control oligonucleotide were infused into C57/BJ6 mice daily for three days. Collagen-induced platelet aggregation was then induced and found to be reduced by up to 60% in mice infused with the STAT3 inhibitor, but not with the control oligonucleotide. Photochemical injury-induced thrombosis in the cremaster arterioles was also significantly delayed in the inhibitor-infused mice as compared to control mice. Second, infusing STAT3 inhibitor could result in platelet inhibitor indirectly by acting endothelial cells. To address this concern, we have generated platelet-specific STAT3 null mice that have developed normally and have normal platelet counts. The collagen-, but not TRAP-induced platelet aggregation in the platelet STAT3 KO mice was reduced as compared to their littermates. Platelets from the platelet-specific STAT3 KO mice were also significantly defective in thrombus formation on immobilized collagen under 62.5 dyn/cm2 of fluid shear stress that was generated in a parallel-plate flow chamber system. Consistent with results from these functional assays, collagen induced rapid (peaked at 5 min after stimulation) and dose-dependent tyrosine phosphorylation of STAT3, but not of STAT1 or STAT5 in washed human platelets. The phosphorylation was blocked dose-dependently by two STAT3 inhibitors. Syk inhibitors also blocked collagen-induced STAT3 phosphorylation in a dose-dependent manner, but STAT3 inhibitors had no effect on Syk phosphorylation, suggesting that Syk acts upstream of STAT3. Furthermore, STAT3 inhibitors also dose-dependently reduced collagen-induced tyrosine phosphorylation of PLCγ2, which is a known substrate of Syk. Consistent with this temporal interaction among STAT3, Syk and PLCγ2, activated STAT3 co-immunoprecipitated phosphorylated Syk and PLCγ2 in collagen-activated human platelets. The tri-molecular complex was also immunoprecipitated by an antibody to PLCγ2. Taken together, these data suggest that STAT3 regulates collagen-induced platelet aggregation, independent of its transcription factor activity. The regulation is potentially achieved by STAT3 serving as a protein scaffold linking the kinase Syk with its substrate PLCγ2 to enhance the signal relay in collagen-activated platelets. This cross-talk between collagen and cytokine signaling pathways provides a mechanism for how proinflammatory mediators could prime platelets for activation by hemostatic ligands. Disclosures: No relevant conflicts of interest to declare.

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The Disulfide Isomerase Erp57 Mediates Platelet Aggregation and Hemostasis

Blood ◽

10.1182/blood.v118.21.1129.1129 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1129-1129

Author(s):

Yi Wu ◽

Syed S. Ahmad ◽

Junsong Zhou ◽

Lu Wang ◽

Matthew P Cully ◽

...

Keyword(s):

Platelet Aggregation ◽

Disulfide Bonds ◽

Bleeding Time ◽

Conflicts Of Interest ◽

Cross Reactivity ◽

Functional Assay ◽

Inhibitory Antibody ◽

Platelet Surface ◽

Disulfide Isomerase ◽

Fibrinogen Receptor

Abstract Abstract 1129 A close homologue to protein disulfide isomerase (PDI) called ERp57 forms disulfide bonds in glycoproteins in the endoplasmic reticulum and is expressed on the platelet surface. We generated two rabbit antibodies to ERp57. One antibody strongly inhibited ERp57 in a functional assay and strongly inhibited platelet aggregation. There was minimal cross reactivity of this antibody with PDI by Western blot or in the functional assay. Using flow cytometry this antibody substantially inhibited activation of the alphaIIbbeta3 fibrinogen receptor. Furthermore, adding ERp57 to platelets potentiated aggregation. In contrast, adding a catalytically inactive ERp57 inhibited platelet aggregation. When the inactive ERp57 was infused into mice the tail bleeding time was prolonged. We generated two IgG2a monoclonal antibodies that reacted with ERp57 by immunoblot. One of these antibodies inhibited both ERp57 activity and platelet aggregation. The other antibody did not inhibit ERp57 activity or platelet aggregation. When the antibodies were infused into mice the tail bleeding time was prolonged by the inhibitory antibody, but not by the non-inhibitory antibody. Finally, we found that a commonly used monoclonal antibody to PDI also inhibited ERp57 activity. We conclude that a glycoprotein specific member of the PDI family, ERp57, is required for platelet aggregation and hemostasis. Disclosures: No relevant conflicts of interest to declare.

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Targeting The PI3K/AKT Pathway To Inhibit Platelet Activation and Thrombus Formation

Blood ◽

10.1182/blood.v122.21.3513.3513 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3513-3513

Author(s):

Wenxiu Yi ◽

Wei Li ◽

Lijie Ren ◽

Xinliang Mao ◽

Li Zhu

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Function ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Venous Blood ◽

Pi3k Pathway ◽

Dependent Manner ◽

Clot Retraction ◽

Occlusion Time

Abstract The phosphatidylinositol 3' –kinase (PI3K)-Akt signaling pathway has been shown to be critical in modulating platelet function and increasing number of studies have been focusing on the development of PI3K inhibitors to modulate platelet function. We recently identified a novel small molecule compound S14161, namely 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene, displaying potent antileukemia and antimyeloma activity via inhibition of the PI3K pathway (Mao et al, Blood, 2011, 117:1986). In the present study, we evaluated the effect of S14161 on platelet activation and the underlying mechanisms. Gel-filtered human platelets were isolated from venous blood of healthy adults and the effect of S14161 on platelet aggregation in response to agonists was determined. Results showed that S14161 inhibited platelet aggregation induced by collagen, convulxin, thrombin, PAR1 agonist peptide SFLLRN, and U46619 in a dose dependent manner (2.5-10μM) with the most striking inhibition for collagen by 89.8% (P<0.001, n=3) and for U46619 by 94.3% (P<0.001, n=3), respectively compared to vehicle-treated samples when 10μM S14161 was used. Flow cytometry studies showed that S14161 inhibits convulxin- or thrombin-induced P-selectin expression and fibrinogen binding of single platelet. S14161 also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in signaling. Using a microfluidic chamber we demonstrated that incubation of S14161 decreases platelet adhesion on collagen-coated surface by about 80% at various time points of blood flow in the chambers. Western blot showed that similar to LY294002, the classic PI3K inhibitor, S14161 inhibited phosphorylation of Akt Ser473 and Akt Thr308 in response to collagen, thrombin, or U46619, implying the involvement of PI3K pathway. Additionally, S14161 inhibited MAPK/ERK1/2 phosphorylation. Finally, the effects of S14161 on thrombus formation in vivo were measured using a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of S14161 (2mg/kg) to male C57BL6/J mice significantly extended the first occlusion time (5.05±0.99 min, N=9) compared to the vehicle controls (3.72±0.95 min, N=8) (P<0.05), but did not increase the bleeding time (P>0.05). Taken together, our data showed that S14161 inhibits platelet activation and thrombus formation, and may be developed as a novel therapeutic agent for the prevention of thrombotic disorders. (This study was supported by National Natural Science Foundation of China 81170132 to Li Zhu) Disclosures: No relevant conflicts of interest to declare.

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JAM-A protects from thrombosis by suppressing integrin αIIbβ3-dependent outside-in signaling in platelets

Blood ◽

10.1182/blood-2011-12-397398 ◽

2012 ◽

Vol 119 (14) ◽

pp. 3352-3360 ◽

Cited By ~ 48

Author(s):

Meghna U. Naik ◽

Timothy J. Stalker ◽

Lawrence F. Brass ◽

Ulhas P. Naik

Keyword(s):

Platelet Activation ◽

Ex Vivo ◽

Thrombus Formation ◽

Receptor Activation ◽

Clot Retraction ◽

Integrin Αiibβ3 ◽

Genetic Ablation ◽

Fibrinogen Receptor ◽

Inside Out

Abstract Mounting evidence suggests that agonist-initiated signaling in platelets is closely regulated to avoid excessive responses to injury. A variety of physiologic agonists induce a cascade of signaling events termed as inside-out signaling that culminate in exposure of high-affinity binding sites on integrin αIIbβ3. Once platelet activation has occurred, integrin αIIbβ3 stabilizes thrombus formation by providing agonist-independent “outside-in” signals mediated in part by contractile signaling. Junctional adhesion molecule A (JAM-A), a member of the cortical thymocyte marker of the Xenopus (CTX) family, was initially identified as a receptor for a platelet stimulatory mAb. Here we show that JAM-A in resting platelets functions as an endogenous inhibitor of platelet function. Genetic ablation of Jam-A in mice enhances thrombotic function of platelets in vivo. The absence of Jam-A results in increase in platelet aggregation ex vivo. This gain of function is not because of enhanced inside-out signaling because granular secretion, Thromboxane A2 (TxA2) generation, as well as fibrinogen receptor activation, are normal in the absence of Jam-A. Interestingly, integrin outside-in signaling such as platelet spreading and clot retraction is augmented in Jam-A–deficient platelets. We conclude that JAM-A normally limits platelet accumulation by inhibiting integrin outside-in signaling thus preventing premature platelet activation.

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Phosphatidylinositol-3,4-Bisphosphate-Akt Signaling Pathway Promotes Platelet Activation

Blood ◽

10.1182/blood-2018-99-115066 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1131-1131

Author(s):

Jasna Marjanovic ◽

Brad Rumancik ◽

Luke Weber ◽

Felix Wangmang ◽

Dane Fickes ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Conflicts Of Interest ◽

Glycogen Synthase ◽

Thrombus Formation ◽

Type I ◽

Dependent Manner ◽

Wild Type ◽

Phosphorylated Akt

Abstract Phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) is a messenger that accumulates in platelets in a phosphoinositide 3-kinase and platelet aggregation-dependent manner. PtdIns(3,4)P2 is broken down by inositol polyphosphate 4-phosphatases, type I (INPP4A) and type II (INPP4B). These enzymes do not catalyze hydrolysis of phosphoinositides other than PtdIns(3,4)P2, and therefore provide unique means for studying the role of this lipid in platelet activation. We have found that the dominant isoform of 4-phosphatases expressed in platelets is INPP4A and we have generated radiation chimera mice with the deficiency in INPP4A restricted to hematopoietic cell lineage. Compared to wild type platelets, agonist-stimulated INPP4A-deficient platelets accumulated higher levels of PtdIns(3,4)P2. An increase in platelet aggregation in INPP4A-deficient platelets was observed with all tested agonists. To study platelet function in vivo, we performed carotid artery injury mouse thrombosis model experiments. Time to occlusion was dramatically reduced in mice with INPP4A deficiency. These data support the hypothesis that by regulating PtdIns(3,4)P2 levels, INPP4A downregulates platelet aggregation and thrombus formation. To investigate mechanisms mediating INPP4A-dependent signals, we compared levels of phosphorylated Akt and phosphorylated glycogen synthase kinase (GSK) in wild type and INPP4A-deficient platelets in response to agonist stimulation. An increase in phospho-Akt levels was observed in INPP4A-deficient platelets, suggesting that in addition to its well-characterized regulator, PtdIns(3,4,5)P3, PtdIns(3,4)P2 can promote Akt activation. Interestingly, this was not accompanied by a significant increase in phospho-GSK levels, suggesting a possible novel mechanism involved in platelet aggregation. Disclosures No relevant conflicts of interest to declare.

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Plasma Fibronectin In Thrombosis and Hemostasis: Exploring the Fibrin Dependent and Independent Mechanisms

Blood ◽

10.1182/blood.v116.21.484.484 ◽

2010 ◽

Vol 116 (21) ◽

pp. 484-484

Author(s):

Yiming Wang ◽

Adili Reheman ◽

Jalil Kalantari ◽

Wuxun Jin ◽

Peter L. Gross ◽

...

Keyword(s):

Platelet Aggregation ◽

Intravital Microscopy ◽

Bleeding Time ◽

Thrombus Formation ◽

Conditional Knockout ◽

Dependent Manner ◽

Plasma Fibronectin ◽

Tail Vein ◽

Vessel Injury ◽

Covalently Linked

Abstract Abstract 484 Background: Plasma fibronectin (pFn) is an abundant protein in the blood. It has long been suspected that pFn plays a role in thrombosis/hemostasis, but this has remained controversial. Our previous study using pFn deficient mice demonstrated that pFn supports thrombosis (PNAS. 2003; 100: 2415-9). Unexpectedly, depletion of pFn in fibrinogen (Fg) and von Willebrand factor (VWF) double deficient (Fg/VWF−/−) mice enhanced, rather than abolished, platelet aggregation and thrombus formation, revealing a functional switch of pFn in the presence and absence of Fg and VWF (Blood. 2009;113:1809-17). However, the mechanism that controls this switch is not known. Furthermore, the hemostatic function of pFn in VW disease (VWD) or afibrinogenemia is unclear. Methods: To address these questions, we bred pFn conditional knockout mice with VWF−/− or Fg−/− mice, establishing 2 new strains of mice: Fg/pFn−/− and VWF/pFn−/−. We also extended our studies of pFn in the triple knockout (TKO, Fg/VWF/pFn−/−) mice. PolyI-polyC was injected into Cre+ and Cre- mice, which resulted in the depletion of plasma pFn (>98%) and platelet pFn (>80%) in Cre+ mice but not in Cre- littermate controls. Aggregometry, a perfusion chamber system, thromboelastography (TEG), tail vein bleeding assay and intravital microscopy were used to study these mice. Results: We first observed a significantly higher mortality in TKO (25%, P<0.05) mice than their Cre- Fg/VWF−/− littermates within 1–2 weeks following the depletion of pFn. Autopsy of these mice revealed severe subcutaneous or abdominal bleeding at the sites of injection. The tail vein bleeding time in TKO mice was also prolonged (P<0.05). Using a laser injury model of intravital microscopy, we observed rapid deposition of fluorescently-labeled pFn at sites of vessel injury in Fg/VWF−/− mice prior to significant platelet deposition. This suggested pFn is a quick/efficient factor contributing to hemostasis in the absence of Fg/VWF. We further found that the mortality rate in Fg/pFn−/− mice was also higher than their Cre- Fg−/− littermates (29%, P<0.05), demonstrating that pFn is a critical hemostatic factor that prevents fatal hemorrhage in afibrinogenemic mice via a fibrin-independent mechanism. We also found that pFn supports hemostasis in VWF−/− mice, although no significant mortality difference was observed (P>0.05). The tail vein bleeding time was longer in VWF/pFn−/− mice than in Cre- VWF−/− littermates (P<0.05), and significantly smaller thrombi were observed when VWF/pFn−/− whole blood was perfused over a collagen surface under shear rate of 1800s-1 (P<0.05). This suggests that pFn may play a role in VWF deficiency (i.e. in type 3 VWD). pFn was also found to support hemostasis in a fibrin-dependent manner. We first demonstrated with TEG that fibrin clot strength was significantly stronger in Cre- littermates than in pFn−/− mice (P<0.05). Platelet aggregation in gel-filtered platelets induced by thrombin, which converts Fg to fibrin on the platelet surface, was greater in Cre- VWF−/− than VWF/pFn−/− platelets (P<0.05). Very interestingly, in keeping with our earlier observation in TKO mice, pFn also inhibited platelet aggregation when fibrin was absent. In Fg−/− mice, we found that pFn depletion enhanced gel-filtered platelet aggregation induced by both thrombin and thrombin receptor activating peptide (TRAP, AYPGKF; P<0.05). In Cre- VWF−/− mice where Fg is present, pFn depletion also enhanced gel-filtered platelet aggregation induced by TRAP (which cannot convert Fg to fibrin) (P<0.05). pFn therefore plays a dual role in platelet aggregation based on the presence of fibrin (i.e. covalently linked fibrin-pFn supports platelet aggregation, while pFn alone inhibits aggregation). Conclusion: Our data demonstrated that pFn is a critical factor for the survival of Fg−/− mice and supports hemostasis in VWF−/− mice via both fibrin-independent and dependent pathways. We clearly showed that fibrin, likely in the form of covalently-linked fibrin-pFn complexes, is required for pFn to support platelet aggregation. Through inhibition of platelet aggregation, non-fibrin-linked soluble pFn may play an important role in the prevention of excessive thrombus formation at the site of vessel injury and thus maintains blood circulation. pFn is therefore likely a crucial supportive factor in hemostasis (for afibrinogenemic and VWD patients), and an important regulator in thrombosis. Disclosures: No relevant conflicts of interest to declare.

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