The Resolution Mediator Annexin A1 Affords Protection Against Thromboinflammation

Abstract Stroke is a leading cause of death and disability worldwide, with the majority (~85 %) being ischemic in origin. Age is the most important non-modifiable risk factor for acute ischemic stroke (AIS). While inflammation with ageing is a well-known complication of AIS, a new model is emerging in which ageing-associated thrombosis is being viewed as a multi-step, multi-cellular process driven by inflammatory stimuli and recruitment/activation of leukocytes. The ideal outcome of inflammation is resolution, an active process involving specific endogenous mediators (e.g. annexin A1 [AnxA1]) and related pathways (e.g. formyl peptide receptor-2 [Fpr2/ALX] pathway).[1,2] The development of therapies that temper inflammation and enhance resolution offer potential therapeutic strategies for the treatment and management of thromboinflammation associated with AIS. We have shown that the AnxA1 mimetic peptide AnxA1 Ac2-26 ameliorates thrombotic responses in thromboinflammatory conditions such as Sickle Cell Disease,[3] however, the role that AnxA1 plays in age-related thrombosis is currently unknown. Here we sought to comprehensively elucidate the functional significance of targeting the AnxA1/Fpr2/ALX pathway in age-related thrombosis. Initially, to evaluate the role of AnxA1, thrombosis in cerebral vessels was induced using the light/dye thrombosis model.[2] Male and female adult (10-14 weeks) and ageing (18-24 months) wild type (WT, C57/BL6) or AnxA1 knock-out (AnxA1 -/-) mice were used. WT mice received AnxA1 (1 µg/mouse), or saline vehicle injected 20 min before the onset of thrombus formation in cerebral pial vessels. Thrombogenesis and blood flow cessation times were quantified. AnxA1 treatment was able to prolong blood flow cessation times in both cerebral arterioles and venules, an effect which was more pronounced in ageing mice (p<0.05) via regulation of the FPR2/ALX-pathway. Next, to investigate the mechanism of action of AnxA1 in an inflammatory backdrop (i.e. lipopolysaccharide [LPS]), the effect of AnxA1 on platelet P-selectin and αIIbβ3 receptor expression, following stimulation with the GPVI collagen receptor agonist convulxin (CVX), was performed. CVX treatment increased platelet activation, which was suppressed by AnxA1 co-administration (100 ng. p<0.05). CVX+LPS increased platelet αIIbβ3 or P-selectin levels, which were inhibited by the administration of AnxA1. Finally, to determine whether a deletion of AnxA1 impacts thrombosis, we performed the light/dye thrombosis model in AnxA1 −/− mice. These mice displayed accelerated cerebral microvascular thrombus formation (decrease in blood flow cessation time) compared to WT mice in both arterioles and venules (arterioles: 17.9 ± 2.3 vs 33.2 ± 1.9 min and venules: 13.2 ± 2.4 vs 20.9 ± 2.2 min. p<0.05). In conclusion, these results demonstrate the ability of AnxA1 to modify the thromboinflammatory environment, including reducing platelet activation under inflammatory conditions via GPVI. Collectively, these data show the importance of the AnxA1/Fpr2/ALX system in effecting the resolution of cerebral thromboinflammation in ageing and may provide a novel therapeutic strategy for AIS and other thromboinflammatory conditions. Disclosures No relevant conflicts of interest to declare.

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STIM1 Deficiency Results In Impaired Platelet Procoagulant Activity and Protection From Arterial Thrombosis

Blood ◽

10.1182/blood.v116.21.485.485 ◽

2010 ◽

Vol 116 (21) ◽

pp. 485-485

Author(s):

Firdos Ahmad ◽

Lucia Stefanini ◽

Timothy Daniel Ouellette ◽

Teshell K Greene ◽

Stefan Feske ◽

...

Keyword(s):

Platelet Activation ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Critical Role ◽

Phosphatidyl Serine ◽

Flow Chamber ◽

Procoagulant Activity ◽

Static Conditions

Abstract Abstract 485 Platelet activation is a central event in thrombosis and hemostasis. We recently demonstrated that most aspects of platelet activation depend on synergistic signaling by two signaling modules: 1) Ca2+/CalDAG-GEFI/Rap1 and 2) PKC/P2Y12/Rap1. The intracellular Ca2+ concentration of platelets is regulated by Ca2+ release from the endoplasmic reticulum (ER) and store-operated calcium entry (SOCE) through the plasma membrane. Stromal interaction molecule 1 (STIM1) was recently identified as the ER Ca2+ sensor that couples Ca2+ store release to SOCE. In this study, we compared the activation response of platelets lacking STIM1−/− or CalDAG-GEFI−/−, both in vitro and in vivo. To specifically investigate Ca2+-dependent platelet activation, some of the experiments were performed in the presence of inhibitors to P2Y12. The murine Stim1 gene was deleted in the megakaryocyte/platelet lineage by breeding Stim flox/flox mice with PF4-Cre mice (STIM1fl/fl). STIM1fl/fl platelets showed markedly reduced SOCE in response to agonist stimulation. aIIbβ3 activation in STIM1fl/fl platelets was significantly reduced in the presence but not in the absence of the P2Y12 inhibitor, 2-MesAMP. In contrast, aIIbb3 activation was completely inhibited in 2-MesAMP-treated CalDAG-GEFI−/− platelets. Deficiency in STIM1, and to a lesser extent in CalDAG-GEFI, reduced phosphatidyl serine (PS) exposure in platelets stimulated under static conditions. PS exposure was completely abolished in both STIM1fl/fl and CalDAG-GEFI−/− platelets stimulated in the presence of 2-MesAMP. To test the ability of platelets to form thrombi under conditions of arterial shear stress, we performed flow chamber experiments with anticoagulated blood perfused over a collagen surface. Thrombus formation was abolished in CalDAG-GEFI−/− blood and WT blood treated with 2-MesAMP. In contrast, STIM1fl/fl platelets were indistinguishable from WT platelets in their ability to form thrombi. STIM1fl/fl platelets, however, were impaired in their ability to express PS when adhering to collagen under flow. Consistently, when subjected to a laser injury thrombosis model, STIM1fl/fl mice showed delayed and reduced fibrin generation, resulting in the formation of unstable thrombi. In conclusion, our studies indicate a critical role of STIM1 in SOCE and platelet procoagulant activity, but not in CalDAG-GEFI mediated activation of aIIbb3 integrin. Disclosures: No relevant conflicts of interest to declare.

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A Locally Administered Novel Heparin Complex Attenuates Collagen-Induced Platelet Activation and Thrombosis In Vivo,

Blood ◽

10.1182/blood.v118.21.3361.3361 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3361-3361

Author(s):

Riitta Lassila ◽

Annukka Jouppila ◽

Ulla M Marzec ◽

Stephen R Hanson

Keyword(s):

Blood Flow ◽

Platelet Aggregation ◽

Thrombus Formation ◽

Ptfe Graft ◽

Thrombin Time ◽

Thrombosis Model ◽

Baboon Model ◽

Induced Aggregation

Abstract Abstract 3361 We have developed a semi-synthetic antithrombotic heparin complex, APL001, to mimic mast cell-derived natural heparin proteoglycans (HepPG). HepPG attenuate platelet-collagen interactions under blood flow by inhibiting VWF- and GPIIb/IIIa -mediated platelet aggregation. In addition, rat-derived HepPG arrest platelet thrombus growth on collagen surfaces or at vascular injury sites, both in vitro and in vivo (Lassila et al.ATVB 1997, Kauhanen et al. ATVB 2000, Olsson et al. Thromb Haemost 2002). Our objective was to study the inhibitory capacity of APL001 for preventing human platelet aggregation in vitro and acute thrombosis in a baboon model in vivo. The effects of unfractionated heparin (UFH) and APL001 were compared in relevant coagulation assays (APTT, PT, thrombin time, anti-FXa activity, fibrinogen, FVIII:C and VWF activity (VWF:RCo) and antigen). Additionally, agonist-induced (collagen, ristocetin and ADP) platelet aggregation in citrate or hirudin-anticoagulated whole blood (Multiplate®) (n=10 healthy subjects), and platelet function analysis (PFA100®) in citrated platelet rich plasma (PRP) were assessed. In a well-established baboon thrombosis model a collagen-coated PTFE graft (length 2 cm, lumen 4 mm) was placed in an arterio-venous shunt. Prior to blood contact the thrombogenic surface was treated for 10 min with UFH or APL001 (both at 4 mg/mL). Thrombus formation was initiated by exposing the surface to blood flow (100 mL/min, shear rate 265−1), and the deposition of 111-In-labeled platelets and of fibrin was quantified continuously over 1h. Fibrin thrombus accumulation was assessed from the incorporation of circulating 125-I-fibrinogen. In the heparin-relevant coagulation tests APL001 was comparable or 20–30% more potent than UFH while FVIII, fibrinogen and VWF variables remained unaltered. In contrast to UFH, APL001 (300 μg/mL) consistently inhibited collagen- and ristocetin-induced platelet aggregation, whereas UFH had only a modest effect in comparison with PBS control (Table). ADP-induced aggregation was unaffected. Comparable results were observed in the PRP aggregation assay. PFA100 testing also demonstrated inhibitory effects. In the in vivo thrombosis model (n=4) APL001 reduced platelet deposition on collagen (vs. the results with UFH) by 34% (p=0.01), while platelet accumulation in distal propagated thrombus was reduced by 61% (p=0.16). APL001-treated surfaces accumulated 45% less fibrin than the UFH-treated surfaces (p=0.008). In conclusion, when compared with UFH APL001 inhibited both collagen- and ristocetin-induced platelet aggregation in human blood, while anticoagulant properties were comparable. In the absence of systemic antithrombotic drugs, exposure of APL001 to a highly thrombogenic collagen surface arrested thrombus formation in an in vivo baboon model. This finding suggests that locally administered APL001 alone, due to its dual antiplatelet and anticoagulant effects, may limit the growth and size of thrombus and thereby prevent subsequent thrombo-occlusion.TableAnticoagulantInhibition-% of platelet aggregation ± SDConc. 300 μg/mLnColl (3.2 μg/mL)Ristocetin (0.77 mg/mL)ADP (6.4 μM)CitrateAPL0011033 ± 1543 ± 166 ± 24UFH1011 ± 1323 ± 153 ± 7p value0.0030.0100.700HirudinAPL0011032 ± 1043 ± 178 ± 10UFH108 ± 1116 ± 166 ± 9p value0.0000.0020.600 Disclosures: Lassila: Aplagon: Chief Scientific Advisor.

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Gβ1 a Component Of The Heterotrimeric G Protein Is a New Protein Phosphatase 1c Interacting Protein That Regulates Platelet Activation

Blood ◽

10.1182/blood.v122.21.3508.3508 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3508-3508

Author(s):

Subhashree Pradhan ◽

Tanvir Khatlani ◽

Satya P. Kunapuli ◽

K. Vinod Vijayan

Keyword(s):

Signal Transduction ◽

G Protein ◽

Platelet Activation ◽

Protein Interactions ◽

Protein Phosphatase ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Adaptor Protein ◽

Gpcr Signaling

Abstract Platelet activation at the site of injury is tied to signal transduction events that are mediated by protein kinases and phosphatases. Reversible tyrosine, serine/threonine (Ser/Thr) phosphorylation-dependent assembly and/or disassembly of effector (cytoskeletal, signaling and adaptor) protein complexes propagate signaling downstream of G protein coupled receptors (GPCRs). Compared to kinases, the contribution of Ser/Thr phosphatases and its effectors in GPCR signaling studies is not well explored. Our previous studies had revealed that the catalytic subunit of protein phosphatase 1γ (PP1cγ) support GPCR signaling and thrombus formation. Since cell signaling networks are dependent on protein-protein interactions, we sought to identify the potential effectors of PP1cγ. We employed yeast two-hybrid interaction studies with the full length PP1cγ fused to GAL4 activating domain as bait and screened human bone marrow library. A novel interaction of PP1cγ with a protein called Gβ1 (GNB1) was identified. Gβ1 is a component of the heterotrimeric G proteins like the Gα and couple to GPCR. However, unlike Gα subunits, Gβ1 is unexplored in platelets. Co-immunoprecipitation (co-IP) studies validated PP1cγ-Gβ1 interaction in 293 cells expressing PP1cγ-HA and Gβ1-FLAG. Importantly, Gβ1 interacted with all the PP1c isoforms, suggesting that Gβ1 could target all PP1c isoforms to the GPCR complex. Purified PP1c bound to recombinant Gβ1-GST protein but not to GST protein, indicating that the in vitro interaction of PP1c with Gβ1 was direct and independent of Gα and Gγ subunits. A small molecule inhibitor of G protein βγ, gallein decreased thrombin-induced human platelet aggregation and adhesion to immobilized fibrinogen. There is a paucity of Gβ1-/- platelets because Gβ1-/- mice die within 2 days of birth due to microencephaly. siRNA mediated depletion of Gβ1 in murine megakaryocytes reduced PAR4-activating peptide induced soluble fibrinogen binding to αIIbβ3. These studies suggest a functional role for Gβ1 in GPCR signaling. PP1c co-immunoprecipitated with Gβ1 in resting platelets and agonist (thrombin and ADP) treatment under non-stirring conditions induced dissociation of PP1c from Gβ1. These studies indicate that Gβ1-PP1c complex in platelets is responsive to agonist. Furthermore, PP1c and Gβ1 associated with P2Y12 receptor in resting but not agonist activated platelets in a co-IP assay, suggesting a role for this complex in G protein signaling. Finally, agonist induced dissociation of PP1c from Gβ1 correlated with the association of PP1c with the downstream GPCR effector phospholipase C β3 (PLCβ3) with a concomitant dephosphorylation of PLCβ3 at Ser1105. Since previous studies have revealed that PLCβ3 activity is inhibited by Ser1105 phosphorylation, our observation suggest that agonist-induced association of PP1c with PLCβ3 facilitates dephosphorylation and activation of PLCβ3. These studies highlight a coupling of GPCR signaling with the phosphatase driven signal transduction during platelet activation. Disclosures: No relevant conflicts of interest to declare.

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TLT-1 Regulates Thrombus Growth Under Non-Inflammatory Conditions

Blood ◽

10.1182/blood-2021-152975 ◽

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.

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The P2Y12 Receptor Antagonist Selatogrel Dissolves Preformed Platelet Thrombi In Vivo

Journal of Clinical Medicine ◽

10.3390/jcm10225349 ◽

2021 ◽

Vol 10 (22) ◽

pp. 5349

Author(s):

Lydie Crescence ◽

Markus Kramberg ◽

Martine Baumann ◽

Markus Rey ◽

Sebastien Roux ◽

...

Keyword(s):

Blood Flow ◽

Guinea Pigs ◽

Thrombus Formation ◽

P2y12 Receptor ◽

Early Application ◽

Acute Thrombosis ◽

Thrombosis Model ◽

Platelet Thrombus ◽

Platelet Thrombi

Selatogrel, a potent and reversible antagonist of the P2Y12 receptor, inhibited FeCl3-induced thrombosis in rats. Here, we report the anti-thrombotic effect of selatogrel after subcutaneous applications in guinea pigs and mice. Selatogrel inhibited platelet function only 10 min after subcutaneous application in mice. In addition, in a modified Folts thrombosis model in guinea pigs, selatogrel prevented a decrease in blood-flow, indicative of the inhibition of ongoing thrombosis, approximately 10 min after subcutaneous injection. Selatogrel fully normalised blood flow; therefore, we speculate that it may not only prevent, but also dissolve, platelet thrombi. Thrombus dissolution was investigated using real-time intravital microscopy in mice. The infusion of selatogrel during ongoing platelet thrombus formation stopped growth and induced the dissolution of the preformed platelet thrombus. In addition, platelet-rich thrombi were given 30 min to consolidate in vivo. The infusion of selatogrel dissolved the preformed and consolidated platelet thrombi. Dissolution was limited to the disintegration of the occluding part of the platelet thrombi, leaving small mural platelet aggregates to seal the blood vessel. Therefore, our experiments uncovered a novel advantage of selatogrel: the dissolution of pre-formed thrombi without the disintegration of haemostatic seals, suggesting a bipartite benefit of the early application of selatogrel in patients with acute thrombosis.

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Genetic Labeling of Cells Allows Identification and Tracking of Transgenic Platelets in Mice

International Journal of Molecular Sciences ◽

10.3390/ijms22073710 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3710

Author(s):

Irena Krüger ◽

Friedrich Reusswig ◽

Kim Jürgen Krott ◽

Celina Fabienne Lersch ◽

Martina Spelleken ◽

...

Keyword(s):

Platelet Activation ◽

Small Proportion ◽

Thrombus Formation ◽

Knock Out ◽

Reporter Mouse ◽

Hematopoietic Organs ◽

Real Time Visualization ◽

Genetic Labeling ◽

Knock Out Mouse

Background: The use of knock-out mouse models is crucial to understand platelet activation and aggregation. Methods: Analysis of the global double fluorescent Cre reporter mouse mT/mG that has been crossbred with the megakaryocyte/platelet specific PF4-Cre mouse. Results: Platelets show bright mT (PF4-Cre negative) and mG (PF4-Cre positive) fluorescence. However, a small proportion of leukocytes was positive for mG fluorescence in PF4-Cre positive mice. In mT/mG;PF4-Cre mice, platelets, and megakaryocytes can be tracked by their specific fluorescence in blood smear, hematopoietic organs and upon thrombus formation. No differences in platelet activation and thrombus formation was observed between mT/mG;PF4-Cre positive and negative mice. Furthermore, hemostasis and in vivo thrombus formation was comparable between genotypes as analyzed by intravital microscopy. Transplantation studies revealed that bone marrow of mT/mG;PF4-Cre mice can be transferred to C57BL/6 mice. Conclusions: The mT/mG Cre reporter mouse is an appropriate model for real-time visualization of platelets, the analysis of cell morphology and the identification of non-recombined platelets. Thus, mT/mG;PF4-Cre mice are important for the analysis of platelet-specific knockout mice. However, a small proportion of leukocytes exhibit mG fluorescence. Therefore, the analysis of platelets beyond hemostasis and thrombosis should be critically evaluated when recombination of immune cells is increased.

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Sialic Acid Deficiency Impairs GPVI-Mediated Platelet Activation in ST3Gal-IV−/− Mice.

Blood ◽

10.1182/blood.v114.22.3005.3005 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3005-3005

Author(s):

Viktoria Rumjantseva ◽

Anne Louise Sørensen ◽

Karin M Hoffmeister ◽

Hervé Falet

Keyword(s):

Flow Cytometry ◽

Sialic Acid ◽

Platelet Activation ◽

Conflicts Of Interest ◽

Receptor Expression ◽

Platelet Surface ◽

Lectin Domain ◽

Terminal Sialic Acid ◽

Fibrinogen Binding ◽

Surface Receptor

Abstract Abstract 3005 Poster Board II-972 Lack of terminal sialic acid residues on platelet surface glycoproteins results in rapid platelet clearance. Using null mice for the ST3Gal-IV sialyltransferase gene (ST3Gal-IV−/− mice), we have recently identified galactose residues on the N-terminus of the platelet Von Willebrand Factor receptor GPIbαa as a major counter receptor for the lectin domain of the asialoglycoprotein receptor on both hepatocytes and liver Kupffer cells (Sørensen et al., Blood 2009). ST3Gal-IV−/− mice have increased tail bleeding time. However, the role of terminal sialic acid residues on platelet activation is unclear. We investigated here whether loss of sialylation affects platelet activation mediated through the collagen receptor GPVI or by thrombin. Platelets were isolated from ST3Gal-IV−/− and ST3Gal-IV+/+ mouse littermates, stimulated with collagen-related peptide (CRP), convulxin (CVX), or thrombin, and platelet activation was evaluated by flow cytometry using P-selectin expression, as a marker for αa-granule secretion, and fibrinogen binding, as a marker for integrin αaIIbβ3 activation. Stimulation of ST3Gal-IV−/− platelets with CRP and CVX revealed a profound activation defect, compared to ST3Gal-IV+/+ platelets. The defect was not due to loss of surface receptor expression since ST3Gal-IV−/− and ST3Gal-IV+/+ platelets had comparable GPVI expression, as evidenced by flow cytometry. By contrast, activation of ST3Gal-IV−/− platelets with thrombin was normal. The data show that terminal sialic acid residues on GPVI are required for maximal platelet activation by CRP and CVX. Disclosures: No relevant conflicts of interest to declare.

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Coagulation Factor XIIa Activates Platelets and Is the Physiological Agonist of Protease-Activated Receptor 3.

Blood ◽

10.1182/blood.v114.22.770.770 ◽

2009 ◽

Vol 114 (22) ◽

pp. 770-770 ◽

Cited By ~ 2

Author(s):

Yingying Mao ◽

Todd M Getz ◽

Jianguo Jin ◽

Satya P. Kunapuli

Keyword(s):

Intracellular Calcium ◽

Platelet Activation ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Shape Change ◽

Coagulation Factor ◽

Calcium Mobilization ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Factor Xiia

Abstract Abstract 770 Protease-activated receptors (PARs) are G-protein coupled receptors that are activated by proteases. Thrombin is the major agonist for PAR1 and PAR4, whereas tryptase and coagulation factor Xa are the agonists for PAR2. In addition to these major agonists, PARs can be activated by other coagulation proteases. The physiological agonist of PAR3 has not been identified to date; as a result, the molecular pharmacology and physiology of PAR3 remain poorly understood. The purpose of this study is to identify a physiological agonist to PAR3. We used PAR4 null murine platelets, which are known to express only PAR3. In this study, we tested the effect of several coagulation proteases and found that only coagulation factor XIIa (FXIIa) activated PAR4-/- murine platelets, in a concentration-dependent manner. FXIIa caused murine platelet shape change, aggregation, secretion and thromboxane A2 generation and this activation was abolished by C1 esterase inhibitor, a FXIIa inhibitor. FXIIa-induced murine platelet activation was completely abolished by BMS200261, a PAR1 antagonist, without affecting the catalytic activity of FXIIa. As murine platelets do not express PAR1, these data indicate that BMS200261 acts as an antagonist of PAR3 and hence inhibits FXIIa-induced platelet activation. FXIIa also caused mobilization of intracellular calcium from murine platelets and this calcium increase is abolished by BMS200261 in the presence or absence of the PAR4. PAR1 and PAR4 couple to Gq to cause intracellular calcium increases. YM-254890, a Gq inhibitor, abrogates PAR1- or PAR4-mediated calcium mobilization. However, YM-254890 did not affect FXIIa –induced platelet calcium mobilization in murine platelets. FXIIa caused activation of Gq-/- mice platelets similar to wild -type platelets, suggesting that FXIIa -induced calcium mobilization in platelets is independent of Gq pathways. Furthermore, FXIIa-induced platelet activation was completely abolished by BAPTA-AM, which indicates that calcium is required for FXIIa-induced platelet activation. Furthermore, FXIIa caused phosphorylation of Erk and Akt in PAR4 null murine platelets and this phosphorylation was abolished by BMS200261, but not by YM-254890. These observations may explain previous reports that demonstrated lack of stable thrombus formation in FXII null mice. We conclude that FXIIa activates platelets through PAR3 independently of Gq pathways leading to calcium mobilization and activation of Erk and Akt. 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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GPIbα As a Selective Bidirectional Modulator Of α-Thrombin Prothrombotic Functions Influencing Fibrin Deposition and PAR-Dependent Platelet Activation

Blood ◽

10.1182/blood.v122.21.32.32 ◽

2013 ◽

Vol 122 (21) ◽

pp. 32-32

Author(s):

Alessandro Zarpellon ◽

Antonella Zampolli ◽

Patrizia Marchese ◽

James R. Roberts ◽

Grazia Loredana Mendolicchio ◽

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Keyword(s):

Platelet Activation ◽

Defense Mechanism ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Thrombus Formation ◽

Blood Platelets ◽

Human Platelets ◽

Fibrillar Structure ◽

Clotting Time ◽

Platelet Aggregates

Abstract Background Generation of α-thrombin (FIIa) in response to vascular injury is a key host defense mechanism influencing thrombus formation and inflammation. Blood platelets express glycoprotein (GP) Ibα as the most abundant FIIa membrane binding site, as well as different protease activated receptors (PARs) with an effector role in platelet activation after proteolytic cleavage. The functional role of GPIbα, which is not a substrate for FIIa, relative to that of different PARs remains unclear. Aims Goal of these studies was to define with mechanistic understanding whether and how binding to GPIbα can modulate FIIa prothrombotic functions in vivo and ex vivo. Methods Endogenous mouse platelet GPIbα was replaced by the human (hu) counterpart with wild type (WT) sequence; or containing the single substitution of Asp277 (mutated to Asn), which interacts selectively with a site involving FIIa exosite 2; or with the combined substitution of post-translationally sulfated Tyr276, Tyr278 and Tyr279 (each mutated to Phe), which interact with FIIa residues in proximity of exosite 1 as well as exosite 2. These mice were evaluated in intravital models of arterial thrombosis. Moreover, their platelets were tested ex vivo for the response to FIIa-induced activation measuring changes in intracytoplasmic Ca2+ levels; and for effects on fibrinogen clotting and fibrin formation. Comparative ex vivo experiments were conducted with human and huGPIbα-WT mouse platelets in which FIIa binding was similarly blocked by the anti-human GPIbα monoclonal antibody, LJ-Ib10. Ex vivo FIIa effects on platelet activation/aggregation and fibrin clot formation were also evaluated concurrently in a model of thrombus formation in blood perfused over a thrombogenic surface under controlled flow conditions. Results Genetically modified mouse platelets expressed ≈9000 WT or mutant huGPIbα molecules; platelets with huGPIbα-WT bound ≈10,000 FIIa molecules with 1:1 stoichiometry and KD of ≈3 nM. FIIa binding to mutant huGPIbα was essentially abolished. Mice with defective FIIa binding to GPIbα exhibited a pronounced prothrombotic phenotype, with a shorter time to carotid artery occlusion following ferric chloride injury (median 550.5 seconds in 18 mutant huGPIbα, vs. 1980 seconds in 19 huGPIbα-WT mice; P<0.01). Accordingly, the platelet-rich plasma (PRP) of mutant huGPIbα mice exhibited a significantly shorter clotting time in the presence of 4 nM FIIa and significantly enhanced intracytoplasmic Ca2+ transients and platelet aggregation following stimulation by 0.5 nM FIIa. Human platelets, similar to mouse platelets, bound FIIa with a 1:1 stoichiometry relative to GPIbα and KD of ≈3 nM. Remarkably, blocking FIIa binding to GPIbα with antibody LJ-Ib10 essentially abolished activation by 1 nM FIIa in human platelets, in which FIIa effects are mediated predominantly by PAR1; this was in contrast to the enhanced activation seen under the same conditions in hu GPIbα-WT mouse platelets, in which FIIa acts through PAR3 and PAR4. Accordingly, the volume of platelet aggregates and fibrin formed in huGPIbα-WT mouse blood perfused over a thrombogenic surface was enhanced by blocking FIIa binding to platelets; in contrast, the volume of platelet aggregates, but not that of fibrin clots, was decreased under the same conditions in human blood. Antibody LJ-Ib10 shortened the clotting time of both huGPIbα-WT mouse and human PRP; however, in the absence of GPIbα-bound FIIa, fibrin associated with platelet aggregates had a less ordered fibrillar structure. Conclusions Our findings identify GPIbα as a relevant FIIa activity modulator. Through distinct mechanisms influenced by the expression of specific PAR subtypes, GPIbα can modulate FIIa function in hemostasis and thrombosis both enhancing and controlling prothrombotic responses and, thus, size and structure of platelet/fibrin thrombi. The effect of GPIbα on PAR4-mediated platelet activation, as well as fibrinogen clotting, can be explained by competition for FIIa exosites required for substrate binding, but the mechanism supporting the distinct GPIbα-PAR1 functional association remains to be elucidated. Disclosures: No relevant conflicts of interest to declare.

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