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

Blood ◽  
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.

FUNDC2 regulates platelet activation through AKT/GSK-3β/cGMP axis

2018 ◽  
Vol 115 (11) ◽  
pp. 1672-1679 ◽  
Author(s):  
Qi Ma ◽  
Weilin Zhang ◽  
Chongzhuo Zhu ◽  
Junling Liu ◽  
Quan Chen
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Mitochondrial Protein ◽  
Dependent Manner ◽  
Clot Retraction ◽  
Akt Phosphorylation ◽  
Gsk 3Β

Abstract Aims AKT kinase is vital for regulating signal transduction in platelet aggregation. We previously found that mitochondrial protein FUNDC2 mediates phosphoinositide 3-kinase (PI3K)/phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent AKT phosphorylation and regulates platelet apoptosis. The aim of this study was to evaluate the role of FUNDC2 in platelet activation and aggregation. Methods and results We demonstrated that FUNDC2 deficiency diminished platelet aggregation in response to a variety of agonists, including adenosine 5′-diphosphate (ADP), collagen, ristocetin/VWF, and thrombin. Consistently, in vivo assays of tail bleeding and thrombus formation showed that FUNDC2-knockout mice displayed deficiency in haemostasis and thrombosis. Mechanistically, FUNDC2 deficiency impairs the phosphorylation of AKT and downstream GSK-3β in a PI3K-dependent manner. Moreover, cGMP also plays an important role in FUNDC2/AKT-mediated platelet activation. This FUNDC2/AKT/GSK-3β/cGMP axis also regulates clot retraction of platelet-rich plasma. Conclusion FUNDC2 positively regulates platelet functions via AKT/GSK-3β/cGMP signalling pathways, which provides new insight for platelet-related diseases.

scholarly journals TLT-1 Regulates Thrombus Growth Under Non-Inflammatory Conditions

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1050-1050
Author(s):  
Angela Doerr ◽  
Denise Pedrosa ◽  
Maria Schander ◽  
Yotis A. Senis ◽  
Alexandra Mazharian ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Type I Collagen ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Type I ◽  
Wild Type ◽  
Platelet Surface ◽  
Knock Out ◽  
Integrin Αiibβ3

Abstract Background Thrombus formation is a complex, dynamic and multistep process, based on two crucial steps: platelet adhesion and platelet aggregation that both involve the large multimeric plasma glycoprotein Von Willebrand Factor (VWF). VWF binding to the GPIb/X/V complex initiates platelet adhesion to the vessel wall at high shear stress and triggers platelet activation resulting in the generation of thrombin and activation of integrin αIIbβ3 on the platelet surface. This activation of αIIbβ3 in turn leads to outside-in signalling and promotes binding of αIIbβ3 to fibrinogen and VWF, mediating thrombus growth. Trigging receptor expressed on myeloid cells like transcript-1 (TLT-1) is a transmembrane receptor, which is targeted to α-granules of platelets and megakaryocytes. Thrombin-induced platelet activation rapidly presents TLT-1 on the platelet surface and releases a soluble form (sTLT-1) into the circulation. To date the only known ligand for TLT-1 is fibrinogen and TLT-1 has been implicated in the regulation of inflammation-associated thrombosis. Interestingly, a putative interaction of VWF with TLT-1 was indicated by a screen with known platelet receptors. Aim We aimed to evaluate the effect of TLT-1/VWF interaction on platelet aggregation and thrombus formation. Methods Recombinant TLT-1 and VWF were purified and the interaction between TLT-1 and VWF was analyzed by surface plasmon resonance. Static interaction was confirmed by an ELISA based binding assay. Flow assays assessed TLT-1 dependent thrombus formation in vitro. The effects of TLT-1 knockout on thrombus formation in vivo were examined via intravital microscopy of the flow restricted inferior vena cava (IVC) and imaging of platelet attachment and fibrin formation over 6 hours. Furthermore, thrombus formation and resolution was followed by high resolution ultrasound imaging after stenosis induction for 28 days. Integrin aIIbb3 activation was analysed by flow cytometry using the JonA antibody in murine platelet rich plasma. Results VWF bound to soluble TLT-1 with high affinity in a calcium dependent manner (K D = 1.9 nM). The binding site on VWF was mapped to the A3D4 domains and high molecular weight VWF multimers had the greatest affinity for TLT-1. Moreover, HEK293 cells transfected with TLT-1 bound to VWF and VWF strings formed specifically on TLT-1 expressing cells, confirming the interaction between the two proteins. VWF inhibited the binding of fibrinogen to TLT-1, suggesting that VWF is a preferred binding partner of TLT-1. Human platelets exhibited increased TLT-1 surface expression after TRAP-6 induced platelet activation and TLT-1 was detected throughout thrombi formed under flow. Furthermore, a TLT-1 blocking antibody inhibited the interaction of TLT-1 with VWF and reduced platelet capture to type I collagen under shear stress. Ex vivo perfusion of blood from TLT-1 knock out mice over type I collagen also resulted in reduced thrombus formation compared to blood from wild-type mice. TLT-1 knock-out platelets were activated by thrombin similar to wild-type controls, based on P-selectin expression in platelet rich plasma. However, activation of integrin αIIbβ3 determined by JonA staining was reduced in the absence of TLT-1. This phenotype of reduced integrin αIIbβ3 activation on P-selectin positive platelets was phenocopied by the thrombin platelet response in platelet rich plasma from VWF -/- mice, but not GPIbα-deficient mice, indicating that the TLT-1-VWF interaction on platelets directly influences integrin αIIbβ3 activation. Significantly, thrombus formation was markedly reduced in TLT-1 knockout mice in the IVC model in vivo in comparison to wild-type mice. Conclusions This study demonstrates that TLT-1 is a novel platelet ligand for VWF, and that TLT-1 may preferentially bind VWF over fibrinogen. We propose a TLT-1/VWF dependent integrin αIIbβ3 activation mechanism which plays a pivotal role in thrombus formation under non-inflammatory and potentially inflammatory conditions. Disclosures Ruf: ICONIC Therapeutics: Consultancy; MeruVasimmune: Current holder of individual stocks in a privately-held company; ARCA bioscience: Consultancy, Patents & Royalties.

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

Blood ◽  
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.

Role of Thiol Isomerase ERp5 in Thrombus Formation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 370-370
Author(s):  
Freda H. Passam ◽  
Lin Lin ◽  
Mingdong Huang ◽  
Jonathan M. Gibbins ◽  
Bruce Furie ◽  
...  
Keyword(s):  
Cho Cells ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Dependent Manner ◽  
Wild Type ◽  
In Vivo Models ◽  
Platelet Accumulation

Abstract Abstract 370 Protein disulfide isomerase is required for thrombus formation in various in vivo models of thrombosis. Another member of the thiol isomerase family, endoplasmic reticulum protein 5 (ERp5), is released from activated platelets and co-immunoprecipitates with beta 3 integrin (Jordan et al, 2005). We further investigated the association of ERp5 with the platelet fibrinogen receptor alpha IIb beta 3 and the significance of ERp5 release in thrombus formation in vivo. Recombinant purified ERp5 was labeled with Alexa 488 and used in direct binding assays to CHO cells expressing wild type (WT) alpha IIb beta 3, CHO cells expressing mutant alpha IIb beta 3 (containing an Asp119Tyr substitution in the beta 3 subunit) and to control CHO cells. The mutant alpha IIb beta 3 does not bind fibrinogen. ERp5 bound to CHO cells expressing wild type (WT) alpha IIb beta 3 in a dose-dependent manner but did not bind to CHO cells expressing mutant alpha IIb beta 3 or to control CHO cells. The relative increase in the geomean of Alexa 488-labeled ERp5 binding to 0.5 ×106 WT alpha IIb beta 3 CHO cells over that bound to control CHO cells was 20, 45 and 85% for ERp5 concentrations of 80, 160 and 400 nM respectively. Binding of ERp5 (160 nM) to WT alpha IIb beta 3 expressing CHO cells was further increased by 75% when the integrin was activated with 2 mM Mn2+ compared to non-activated WT alpha IIb beta 3 CHO cells. A role for ERp5 in thrombus formation was studied in the laser injury model of thrombosis in mouse cremaster arterioles using a rabbit polyclonal anti-ERp5 antibody, immunoaffinity purified against recombinant ERp5. This antibody detected ERp5 in the releasate of thrombin-activated mouse platelets in vitro by Western blot and on the surface of thrombin-activated mouse platelets by flow cytometry. Dylight 649-labeled anti-CD42b was infused into the mouse circulation to detect platelet accumulation and Alexa 488-labeled anti-ERp5 antibody at 0.05 ug/g, a dose that does not inhibit thrombus formation, was infused to detect ERp5. The fluorescent anti-ERp5 signal detected at the thrombus site was compared to the signal produced by a non-specific IgG labeled with Alexa 488 infused into a control mouse. Anti-ERp5 fluorescence was detected in the thrombus with kinetics that followed platelet accumulation whereas there was minimal signal from the control IgG. We examined whether higher doses of anti-ERp5 affect thrombus formation. Platelet and fibrin accumulation were detected using fluorescently labeled anti-CD42b antibody and monoclonal anti-fibrin-specific antibody respectively before or after the injection of unlabeled anti-ERp5 antibody or pre-immune IgG at 2.5 ug/g. Platelet and fibrin accumulation, expressed as area under the curve of the median integrated fluorescence over time, was obtained from 14 thrombi in 6 mice formed before infusion of antibody, 18 thrombi in 2 mice formed after infusion of control IgG and 29 thrombi in 3 mice formed after infusion of anti-ERp5. Anti-ERp5 infusion caused a 70% decrease in the deposition of platelets and a 62% decrease in fibrin accumulation compared to infusion of control antibody (p<0.01). There was no difference in platelet and fibrin accumulation before infusion of antibody and after infusion of control antibody. These results provide evidence for a role of a second thiol isomerase, ERp5, in thrombus formation, a function which may be mediated through its association with alpha IIb beta 3. Disclosures: No relevant conflicts of interest to declare.

Fucoidan Is a Novel Platelet Agonist for CLEC-2 Receptor

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 94-94
Author(s):  
Bhanukanth Manne ◽  
Todd M Getz ◽  
Craig Hughes ◽  
Carol T Dangelmaier ◽  
Steve P Watson ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Animal Models ◽  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Human Platelets ◽  
Lag Phase ◽  
Dependent Manner ◽  
Wild Type ◽  
Concentration Dependent Manner

Abstract Abstract 94 Fucoidan, a sulphated polysaccharide from fucus vesiculosus, decreases bleeding time and clotting time in hemophilia, possibly through inhibition of tissue factor pathway inhibitor (TFPI) (Prasad et al., Blood 111:672, 2008). The decrease in bleeding times in the hemophilia animal models by in vivo administration of fucoidan suggests the beneficial effect of fucoidan as a novel treatment. Furthermore, in vitro studies using platelet poor plasma from hemophilia animal models and human patients has shown that fucoidan inhibits TFPI thereby contributing to an increase in the extrinsic coagulation pathway activity. The effect of fucoidan on platelets however has not been studied. As it is known that the platelet count remains unaffected in hemophilia A patients and bleeding times are primarily measured to assess normal platelet function, we hypothesize that the decrease in bleeding times in the hemophilia animal models may be due to platelet activation by fucoidan. In this study, we demonstrate for the first time that fucoidan induces platelet activation in a concentration dependent manner. Fucoidan-induced platelet activation is completely abolished by the pan-Src family kinase (SFK) inhibitor, PP2, and in the absence of Syk and PLC-g2. Furthermore, fucoidan-induced platelet activation has a lag phase, which is reminiscent of platelet activation by collagen and by CLEC-2 receptor agonists. Platelet activation by fucoidan however was only slightly inhibited in FcRg-chain null mice indicating that fucoidan is not acting primarily through GPVI receptor. On the other hand, fucoidan-induced platelet activation was inhibited in CLEC-2 deficient mouse platelets revealing CLEC-2 as a physiological target of fucoidan. Thus, our data shows fucoidan as a novel CLEC-2 receptor agonist that activates platelets through an SFK-dependent signaling pathway. Further, the efficacy of fucoidan in hemophilia raises the possibility that decreased bleeding times could be achieved through activation of platelets. A) Fucoidan induces platelet activation: Washed aspirin-treated human platelets were stimulated with increasing concentrations of fucoidan at 37°C. Platelet aggregation was measured using a Lumi-aggregometer. The tracings are representative of data from at least three independent experiments. B) Effect of SFK inhibition on fucoidan-induced platelet activation: Washed aspirin-treated human platelets were pre-treated with SFK inhibitor PP2 10uM or PP3 (vehicle) at 37°C for 5 min followed by stimulation with fucoidan (50 ug/ml) for 3 minutes under stirred conditions. Platelet aggregation was measured using Lumi-aggregometer and effect on phosphorylation of Syk (Y525/26) and LAT (Y191) in the presence of SFK inhibitor PP2 an PP3 (control) were analyzed. The results are representative of data from platelets at least three independent experiments. C) Identifying a possible receptor for fucoidan on platelets: Wild type, FcRg-chain or CLEC-2 null murine platelets were stimulated with fucoidan (50 ug/ml) at 37°C under stirred conditions and aggregation was measured using Lumi-aggregometer. A) Fucoidan induces platelet activation: Washed aspirin-treated human platelets were stimulated with increasing concentrations of fucoidan at 37°C. Platelet aggregation was measured using a Lumi-aggregometer. The tracings are representative of data from at least three independent experiments. . / B) Effect of SFK inhibition on fucoidan-induced platelet activation: Washed aspirin-treated human platelets were pre-treated with SFK inhibitor PP2 10uM or PP3 (vehicle) at 37°C for 5 min followed by stimulation with fucoidan (50 ug/ml) for 3 minutes under stirred conditions. Platelet aggregation was measured using Lumi-aggregometer and effect on phosphorylation of Syk (Y525/26) and LAT (Y191) in the presence of SFK inhibitor PP2 an PP3 (control) were analyzed. The results are representative of data from platelets at least three independent experiments. . / C) Identifying a possible receptor for fucoidan on platelets: Wild type, FcRg-chain or CLEC-2 null murine platelets were stimulated with fucoidan (50 ug/ml) at 37°C under stirred conditions and aggregation was measured using Lumi-aggregometer. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
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.

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

Blood ◽  
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

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.

CalDAG-GEFI Is a Key Signal Integrator in Platelet Activation and Thrombus Formation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 326-326
Author(s):  
Wolfgang Bergmeier ◽  
Jill R. Crittenden ◽  
Crystal L. Piffath ◽  
Denisa D. Wagner ◽  
David E. Housman ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Knockout Mice ◽  
Thrombus Formation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Premature Stop Codon ◽  
Small Gtpase ◽  
Integrin Αiibβ3

Abstract Inside-out activation of platelet integrin αIIbβ3 is a key step in agonist-induced platelet aggregation. Recent studies suggested the involvement of the small GTPase Rap1b in this process as it is highly expressed in platelets and becomes activated during platelet activation. In cell lines, overexpression of the Rap activator CalDAG-GEFI increased αIIbβ3-dependent adhesion, while overexpression of RapGAP, which inactivates Rap1, reduced αIIbβ3 activity. Here we provide evidence that CalDAG-GEFI is an essential component of this pathway in vivo. To generate CalDAG-GEFI knockout mice, we engineered mouse embryonic stem (ES) cells with a deletion that results in a frameshift mutation and a premature stop codon at the position encoding the 37th amino acid of CalDAG-GEFI. These ES cells were then used to derive chimeric mice that yielded germline transmission of the CalDAG-GEFI mutation. Deficiency of CalDAG-GEFI in mutant mice was confirmed by immunohistochemistry and western blot analysis. CalDAG-GEFI−/− platelets showed impaired Rap1b activation and aggregation in response to various agonists, with aggregation being completely blocked when platelets were activated with ADP, thromboxaneA2 analog, or calcium ionophore. Under physiological flow conditions in vitro and in vivo, CalDAG-GEFI-deficient platelets showed normal tethering to basement membrane components but failed to form thrombi. Mice deficient in CalDAG-GEFI were further characterized by a greatly increased bleeding time as well as by a strong protection against collagen-induced pulmonary thrombosis. In summary, we identified CalDAG-GEFI as a key signal integrator in the cascade leading through Rap1 and integrin αIIbβ3 to platelet aggregation and thrombus formation. The fact that CalDAG-GEFI knockout mice are resistant to collagen-induced thrombosis, and do not undergo spontaneous hemorrhaging, suggests that CalDAG-GEFI may be a promising new target for antithrombotic therapy.

“M-AAA-Thrombin”, a New Thrombin Mutant with Potent Anticoagulant and Antiplatelet Properties.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4154-4154
Author(s):  
Kazuya Hosokawa ◽  
Tomoko Ohnishi ◽  
Hiroyuki Matsuda ◽  
Kousuke Kashima ◽  
Takehiko Koide
Keyword(s):  
Platelet Aggregation ◽  
Thrombus Formation ◽  
Antiplatelet Agent ◽  
Dependent Manner ◽  
In Vivo Experiments ◽  
Thrombosis Model ◽  
Venous Shunt ◽  
Prolonged Aptt ◽  
Antithrombotic Efficacy

Abstract Thrombosis is a major cause of morbidity and mortality, and thrombin is a major inducer of thrombus formation. Thus several antithrombotic agents targeting thrombin have been developed. We previously reported an anticoagulant and antiplatelet thrombin derivative, ‘M-anhydrothrombin’ prepared by chemical modifications. In this study, we prepared a new thrombin mutant, specificity of which was highly modulated with substantially improved antithrombotic efficacy. The thrombin mutant designated “AAA-Thrombin” in which Lys65, His43 and Ser205 in B-chain have been replaced by Ala revealed higher affinity and specificity for factor VIII with no enzymatic activity. AAA-Thrombin prolonged APTT much more than anhydrothrombin in a dose dependent manner without affecting PT and TT. Platelet aggregation induced by activation of PAR-1 was also effectively suppressed by AAA-Thrombin. “M-AAA-Thrombin” prepared by further chemical modification of carboxyl groups in AAA-Thrombin enhanced its antithrombotic efficacy. M-AAA-Thrombin (250nM) prolonged APTT approx. two times, and suppressed platelet aggregation by PAR-1 activation, while AAA-Thrombin did not at the same concentration. M-AAA-Thrombin also suppressed ristocetin-induced platelet aggregation. In vivo experiments, M-AAA-Thrombin demonstrated significant antithrombotic property in the arterio-venous shunt thrombosis model and the FeCl3-induced carotid artery thrombosis model in guinea pigs. These results indicate that M-AAA-Thrombin would be a candidate for quite an innovative anticoagulant and antiplatelet agent for both arterial and venous thromboses. Further optimization of mutagenesis and modification, in terms of efficacy and safety is in progress in our laboratory.

The Role of LIM Kinase-1 in the Glycoprotein Ib-IX Complex-Mediated Platelet Activation and Thrombus Formation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 112-112
Author(s):  
Aleksandra Stojanovic ◽  
Matvey Gorovoy ◽  
Tatyana Voyno-Yasenetskaya ◽  
Xiaoping Du
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Adhesion ◽  
Wild Type ◽  
Glycoprotein Ib ◽  
Lim Kinase ◽  
And Function

Abstract LIM Kinase (LIMK)-1 is a member of the LIMK family of serine-threonine protein kinases that phosphorylates actin-binding protein cofilin and regulates actin cytoskeleton organization. LIMK1 is expressed in many cell types including platelets but the exact role of LIMK1 in platelet function remains unclear. To determine the role of LIMK1 in platelet activation, wild type or LIMK1 knockout mouse platelets were stimulated with platelet agonists. Platelet aggregation and granule secretion were analyzed. Integrin-dependent second wave of platelet aggregation induced by von Willebrand factor (VWF) in the presence of VWF activator botrocetin was abolished in LIMK1 knockout platelets. In contrast, platelet aggregation in response to the agonist peptide of protease-activated receptor-4 (PAR4, thrombin receptor), ADP and collagen was either not affected or enhanced in LIMK1 knockout platelets in comparison with wild type mouse platelets. Thus, LIMK appears to play an important role in platelet activation stimulated by VWF binding to its platelet receptor, glycoprotein Ib-IX complex (GPIb-IX) but had no stimulatory effect on or negatively regulate the GPIb-IX-independent platelet activation pathways mediated by PAR-4, ADP receptors and collagen receptors. To determine whether ligand binding to GPIb-IX stimulates LIMK activation and function, platelets were stimulated with VWF in the presence of either ristocetin or botrocetin, and immunoblotted with antibodies specifically recognizing phosphorylated LIMK1 (Serine 505) or cofilin (Serine 3). VWF induced phosphorylation of LIMK1 and LIMK substrate cofilin. Thus, VWF indeed stimulates LIMK1 activation and function. An important physiological role of GPIb-IX in platelets is to mediate platelet adhesion to subendothelial-bound VWF under shear stress at sites of vascular injury. To determine whether LIMK1 is important in platelet adhesion, we investigated whether LIMK1 knockout affected platelet adhesion to VWF-coated surfaces. LIMK1 knockout platelets are defective in mediating stable platelet adhesion to vWF under shear stress, suggesting that LIMK1 plays an important role in GPIb signaling and GPIb-IX-mediated integrin activation that is required for stable platelet adhesion under shear stress. Importantly, LIMK1 knockout mice showed significant delay in the formation of occlusive thrombus following FeCl3-induced carotid artery injury in comparison with wild type mice, indicating that the role of LIMK1 in GPIb-IX-mediated platelet activation is important in in vivo thrombosis. Together, our study reveals that LIMK1 plays an important role in GPIb-IX-mediated platelet activation and arterial thrombosis in vitro and in vivo.

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