Ferric Chloride Injury Model Research Articles

Anti-thrombotic Effects Mediated by a Novel Dual-Target Peptide Inhibiting Both Platelet Aggregation and Thrombin Activity without Causing Bleeding.

Classical anticoagulants and antiplatelets are associated with high frequencies of bleeding complications or treatment failure when used as single agents. Thrombin plays an important role in the blood coagulation system. GP IIb/IIIa is the central receptor of platelets, which can recognize the Arg-Gly-Asp (RGD) sequence and activate platelets. Molecular simulation and homology modeling were performed to design a novel dual-target anticoagulant short peptide (PTIP ). The activities of PTIP on coagulation and platelet in vitro were analyzed. The antithrombotic activity of PTIP was determined by pulmonary thromboembolism model, ferric chloride injury model and arteriovenous bypass thrombosis model. Bleeding effect and toxicity of PTIP were evaluated. We have constructed a novel dual-target peptide (PTIP) based on the direct thrombin inhibitor peptide (DTIP). PTIP was expressed at high levels in Pichia pastoris. PTIP interfered with thrombin-mediated coagulation and ADP-induced platelet aggregation in vitro. When injected intravenously or subcutaneously, PTIP showed potent and dose-dependent extension of aPTT and PT which were similar to DTIP; but only PTIP was capable of inhibiting platelet aggregation. PTIP (1.0 mg/kg) decelerated thrombosis formation in venous and arterial vessels induced by FeCl3 injury. PTIP (1.0 mg/kg) also prevented deep venous thrombosis and increased the survival rate associated with pulmonary thromboembolism. And PTIP effectively reduced thrombus length in arteriovenous bypass thrombosis model. Moreover, the antithrombotic dose of PTIP could not induce bleeding. These data establish that PTIP represents a novel antithrombotic agent whose effects involve both inhibition of platelet activation and reduction of fibrin generation. And PTIP not only can be used in venous thrombosis and arterial thrombosis, it can also replace the combined treatment of antiplatelet and anticoagulant drugs in thrombotic diseases.

Phosphorylation of spleen tyrosine kinase at Y346 negatively regulates ITAM-mediated signaling and function in platelets

Spleen tyrosine kinase (Syk) is expressed in a variety of hemopoietic cells. Upon phosphorylation of the platelet immunoreceptor-based activation motif of the glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor, both the tyrosine phosphorylation and activity of Syk are increased leading to downstream signaling events. Although it has been established that the activity of Syk is regulated by tyrosine phosphorylation, the specific roles of individual phosphorylation sites remain to be elucidated. We observed that Syk Y346 in mouse platelets was still phosphorylated when GPVI-induced Syk activity was inhibited. We then generated Syk Y346F mice and analyzed the effect this mutation exerts on platelet responses. Syk Y346F mice bred normally, and their blood cell count was unaltered. We did observe potentiation of GPVI-induced platelet aggregation and ATP secretion as well as increased phosphorylation of other tyrosines on Syk in the Syk Y346F mouse platelets when compared to WT littermates. This phenotype was specific for GPVI-dependent activation, since it was not seen when AYPGKF, a PAR4 agonist, or 2-MeSADP, a purinergic receptor agonist, was used to activate platelets. Despite a clear effect of Syk Y346F on GPVI-mediated signaling and cellular responses, there was no effect of this mutation on hemostasis as measured by tail-bleeding times, although the time to thrombus formation determined using the ferric chloride injury model was reduced. Thus, our results indicate a significant effect of Syk Y346F on platelet activation and responses invitro and reveal its complex nature manifesting itself by the diversified translation of platelet activation into physiological responses.

Loss of α4A- and β1-tubulins leads to severe platelet spherocytosis and strongly impairs hemostasis in mice

AbstractNative circulating blood platelets present with a discoid flat morphology maintained by a submembranous peripheral ring of microtubules, named marginal band. The functional importance of this particular shape is still debated, but it was initially hypothesized to facilitate platelet interaction with the injured vessel wall and to contribute to hemostasis. The importance of the platelet discoid morphology has since been questioned on the absence of clear bleeding tendency in mice lacking the platelet-specific β1-tubulin isotype, which exhibits platelets with a thinner marginal band and an ovoid shape. Here, we generated a mouse model inactivated for β1-tubulin and α4A-tubulin, an α-tubulin isotype strongly enriched in platelets. These mice present with fully spherical platelets completely devoid of a marginal band. In contrast to the single knockouts, the double deletion resulted in a severe bleeding defect in a tail-clipping assay, which was not corrected by increasing the platelet count to normal values by the thrombopoietin-analog romiplostim. In vivo, thrombus formation was almost abolished in a ferric chloride–injury model, with only a thin layer of loosely packed platelets, and mice were protected against death in a model of thromboembolism. In vitro, platelets adhered less efficiently and formed smaller-sized and loosely assembled aggregates when perfused over von Willebrand factor and collagen matrices. In conclusion, this study shows that blood platelets require 2 unique α- and β-tubulin isotypes to acquire their characteristic discoid morphology. Lack of these 2 isotypes has a deleterious effect on flow-dependent aggregate formation and stability, leading to a severe bleeding disorder.

Multimerin 1 supports platelet function in vivo and binds to specific GPAGPOGPX motifs in fibrillar collagens that enhance platelet adhesion

BackgroundMultimerin 1 (human: MMRN1, mouse: Mmrn1) is a homopolymeric, adhesive, platelet and endothelial protein that binds to von Willebrand factor and enhances platelet adhesion to fibrillar collagen ex vivo. ObjectivesTo examine the impact of Mmrn1 deficiency on platelet adhesive function, and the molecular motifs in fibrillar collagen that bind MMRN1 to enhance platelet adhesion. MethodsMmrn1‐deficient mice were generated and assessed for altered platelet adhesive function. Collagen Toolkit peptides, and other triple‐helical collagen peptides, were used to identify multimerin 1 binding motifs and their contribution to platelet adhesion. ResultsMMRN1 bound to conserved GPAGPOGPX sequences in collagens I, II, and III (including GPAGPOGPI, GPAGPOGPV, and GPAGPOGPQ) that enhanced activated human platelet adhesion to collagen synergistically with other triple‐helical collagen peptides (P < .05). Mmrn1−/− and Mmrn1+/− mice were viable and fertile, with complete and partial platelet Mmrn1 deficiency, respectively. Relative to wild‐type mice, Mmrn1−/− and Mmrn1+/− mice did not have overt bleeding, increased median bleeding times, or increased wound blood loss (P ≥ .07); however, they both showed significantly impaired platelet adhesion and thrombus formation in the ferric chloride injury model (P ≤ .0003). Mmrn1−/− platelets had impaired adhesion to GPAGPOGPX peptides and fibrillar collagen (P ≤ .03) and formed smaller aggregates than wild‐type platelets when captured onto collagen, triple‐helical collagen mimetic peptides, von Willebrand factor, or fibrinogen (P ≤ .008), despite preserved, low shear, and high shear aggregation responses. ConclusionsMultimerin 1 supports platelet adhesion and thrombus formation and binds to highly conserved, GPAGPOGPX motifs in fibrillar collagens that synergistically enhance platelet adhesion.

The Microbiota Promotes Arterial Thrombosis in Low-Density Lipoprotein Receptor-Deficient Mice.

Atherosclerotic plaque development depends on chronic inflammation of the arterial wall. A dysbiotic gut microbiota can cause low-grade inflammation, and microbiota composition was linked to cardiovascular disease risk. However, the role of this environmental factor in atherothrombosis remains undefined. To analyze the impact of gut microbiota on atherothrombosis, we rederived low-density lipoprotein receptor-deficient (Ldlr-/- ) mice as germfree (GF) and kept these mice for 16 weeks on an atherogenic high-fat Western diet (HFD) under GF isolator conditions and under conventionally raised specific-pathogen-free conditions (CONV-R). In spite of reduced diversity of the cecal gut microbiome, caused by atherogenic HFD, GF Ldlr-/- mice and CONV-R Ldlr-/- mice exhibited atherosclerotic lesions of comparable sizes in the common carotid artery. In contrast to HFD-fed mice, showing no difference in total cholesterol levels, CONV-R Ldlr-/- mice fed control diet (CD) had significantly reduced total plasma cholesterol, very-low-density lipoprotein (VLDL), and LDL levels compared with GF Ldlr-/- mice. Myeloid cell counts in blood as well as leukocyte adhesion to the vessel wall at the common carotid artery of GF Ldlr-/- mice on HFD were diminished compared to CONV-R Ldlr-/- controls. Plasma cytokine profiling revealed reduced levels of the proinflammatory chemokines CCL7 and CXCL1 in GF Ldlr-/- mice, whereas the T-cell-related interleukin 9 (IL-9) and IL-27 were elevated. In the atherothrombosis model of ultrasound-induced rupture of the common carotid artery plaque, thrombus area was significantly reduced in GF Ldlr-/- mice relative to CONV-R Ldlr-/- mice. Ex vivo, this atherothrombotic phenotype was explained by decreased adhesion-dependent platelet activation and thrombus growth of HFD-fed GF Ldlr-/- mice on type III collagen.IMPORTANCE Our results demonstrate a functional role for the commensal microbiota in atherothrombosis. In a ferric chloride injury model of the carotid artery, GF C57BL/6J mice had increased occlusion times compared to colonized controls. Interestingly, in late atherosclerosis, HFD-fed GF Ldlr-/- mice had reduced plaque rupture-induced thrombus growth in the carotid artery and diminished ex vivo thrombus formation under arterial flow conditions.

ELMO1 deficiency enhances platelet function

AbstractPhosphatidylinositol 3-kinase is an important signaling molecule that, once activated, leads to the generation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3). We performed a proteomic screen to identify PIP3-interacting proteins in human platelets. Among these proteins, we found engulfment and cell motility 1 (ELMO1), a scaffold protein with no catalytic activity. ELMO1 is expressed in platelets and interacts with active RhoG. However, the function of ELMO1 in platelets is not known. The focus of this study was to determine the function of ELMO1 in platelets utilizing ELMO1−/− mice. Platelet aggregation, granule secretion, integrin αIIbβ3 activation, and thromboxane generation were enhanced in ELMO1−/− platelets in response to glycoprotein VI (GPVI) agonists but unaltered when a protease-activated receptor 4 agonist was used. The kinetics of spreading on immobilized fibrinogen was enhanced in ELMO1−/− platelets compared with wild-type (WT) littermate controls. This suggests that ELMO1 plays a role downstream of the GPVI and integrin αIIbβ3 pathway. Furthermore, whole blood from ELMO1−/− mice perfused over collagen exhibited enhanced thrombus formation compared with WT littermate controls. ELMO1−/− mice showed reduced survival compared with control following pulmonary embolism. ELMO1−/− mice also exhibited a shorter time to occlusion using the ferric-chloride injury model and reduced bleeding times compared with WT littermate controls. These results indicate that ELMO1 plays an important role in hemostasis and thrombosis in vivo. RhoG activity was enhanced in ELMO1−/− murine platelets compared with WT littermate controls in response to GPVI agonist. Together, these data suggest that ELMO1 negatively regulates GPVI-mediated thrombus formation via RhoG.

ELMO1 Is a Novel Negative Regulator of Glycoprotein VI Signaling in Platelets

PI3-kinase (phosphoinositide 3-kinase) is an important signaling molecule that is activated downstream of various receptors upon platelet activation. PI3-kinase activation leads to the generation of PIP3 (Phosphatidylinositol (3,4,5)-trisphosphate) subsequently leading to the recruitment of PH (pleckstrin homology) domain containing proteins to the plasma membrane. Our laboratory screened for proteins that interacted with PIP3 (Phosphatidylinositol (3,4,5)-trisphosphate) using PIP3 beads. One of the proteins that interacted with PIP3 was ELMO1 (Engulfment and cell motility-1). ELMO1 is a scaffold protein with no catalytic activity and is well known to regulate actin cytoskeletal rearrangement via Rac1 in other cells. However, it is not known whether ELMO1 is expressed in platelets and if so, does it regulate platelet functional responses. Here, we show that ELMO1 is present in both human and murine platelets. We used ELMO1-deficient (ELMO1-/-) mice to study its role in platelets. ELMO1-/- murine platelets showed enhanced platelet aggregation and dense granule secretion in response to the GPVI agonist, CRP (Figure 1 A & B), compared to the wildtype controls although there was no difference in GPVI expression levels between the two. There was no difference observed in response to AYPGKF- or 2-MeSADP. These data suggest that ELMO1 plays a specific role downstream of GPVI pathway but GPCRs. Moreover, ELMO1-/- platelets exhibited enhanced clot retraction and spreading indicating its role in Glycoprotein IIb/IIa (GPIIb/IIIa) mediated outside-in signaling. Furthermore, whole blood from ELMO1-/- mice perfused over collagen under arterial shear conditions exhibited enhanced thrombus formation. In an in vivo pulmonary thromboembolism model, ELMO1-/- mice showed reduced survival compared to the wildtype control. ELMO1-/- mice also showed shorter time to occlusion and increased thrombus stability using the ferric-chloride injury model indicating the role of ELMO1 in thrombus formation in vivo. At the molecular level, Rac1 activity was enhanced in ELMO1-/- murine platelets compared to the wildtype control in response to CRP (Figure 1C). Together, these data suggest that ELMO1 regulates Rac1 activity upon GPVI-mediated thrombus formation and it may play a negative regulator role in both inside-out and outside-in signaling, which might involve Rac1. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Use of a mouse model to elucidate the phenotypic effects of the von Willebrand factor cleavage mutants, Y1605A/M1606A and R1597W

von Willebrand Factor (VWF) is tightly regulated by the metalloproteinase ADAMTS13, which cleaves VWF to reduce VWF multimer size and binding affinity for collagen and platelets. This study examines two VWF mutations, R1597W (enhanced cleavage) and Y1605A-M1606A (decreased cleavage), to determine their impact on VWF, in addition to ADAMTS13-mediated cleavage. In vitro mouse ADAMTS13 digestions were performed on recombinant proteins. VWF knockout mice received hydrodynamic injections of mouse Vwf cDNA, following which VWF antigen, multimer profile and VWF propeptide levels were determined. A ferric chloride injury model of thrombosis was also evaluated. In vitro ADAMTS13 digestion of full-length mouse VWF required > 97-fold higher ADAMTS13 levels for Y1605A/M1606A, and 68% lower ADAMTS13 levels for R1597W compared with wild type. In vivo, R1597W had reduced VWF:Ag and both mutations exhibited increased VWF propeptide/VWF:Ag ratios. R1597W multimers show a lower molecular weight profile compared with wild type and Y1605A/M1606A mice. When co-injected with Adamts13 cDNA, Y1605A/M1606A multimers were larger compared with wild type, and R1597W showed only a single multimer band and decreased clearance via VWFpp/VWF:Ag ratio. R1597W was associated with reduced thrombus formation but normal platelet accumulation in a ferric chloride injury model while Y1605A/M1606A had a loss of occlusive thrombi but increased platelet accumulation compared with wild type. This study demonstrates that mutations that alter ADAMTS13 cleavage also can affect VWF clearance, VWF antigen level, multimer structure and thrombotic potential in the VWF knockout hydrodynamic injection model.

Pathologic mechanisms of type 1 VWD mutations R1205H and Y1584C through in vitro and in vivo mouse models

AbstractType 1 VWD is the mild to moderate reduction of VWF levels. This study examined the mechanisms underlying 2 common type 1 VWD mutations, the severe R1205H and more moderate Y1584C. In vitro biosynthesis was reduced for both mutations in human and mouse VWF, with the effect being more severe in R1205H. VWF knockout mice received hydrodynamic injections of mouse Vwf cDNA. Lower VWF antigen levels were demonstrated in both homozygous and heterozygous forms for both type 1 mutations from days 14-42. Recombinant protein infusions and hydrodynamic-expressed VWF propeptide to antigen ratios demonstrate that R1205H mouse VWF has an increased clearance rate, while Y1584C is normal. Recombinant ADAMTS13 digestions of Y1584C demonstrated enhanced cleavage of both human and mouse VWF115 substrates. Hydrodynamic-expressed VWF shows a loss of high molecular weight multimers for Y1584C compared with wild-type and R1205H. At normal physiologic levels of VWF, Y1584C showed reduced thrombus formation in a ferric chloride injury model while R1205H demonstrated similar thrombogenic activity to wild-type VWF. This study has elucidated several novel mechanisms for these mutations and highlights that the type 1 VWD phenotype can be recapitulated in the VWF knockout hydrodynamic injection model.

Arrestin-2 Differentially Regulates PAR4 and ADP Receptor Signaling in Platelets

Arrestins can facilitate desensitization or signaling by G protein-coupled receptors (GPCR) in many cells, but their roles in platelets remain uncharacterized. Because of recent reports that arrestins can serve as scaffolds to recruit phosphatidylinositol-3 kinases (PI3K)s to GPCRs, we sought to determine whether arrestins regulate PI3K-dependent Akt signaling in platelets, with consequences for thrombosis. Co-immunoprecipitation experiments demonstrate that arrestin-2 associates with p85 PI3Kα/β subunits in thrombin-stimulated platelets, but not resting cells. The association is inhibited by inhibitors of P2Y12 and Src family kinases (SFKs). The function of arrestin-2 in platelets is agonist-specific, as PAR4-dependent Akt phosphorylation and fibrinogen binding were reduced in arrestin-2 knock-out platelets compared with WT controls, but ADP-stimulated signaling to Akt and fibrinogen binding were unaffected. ADP receptors regulate arrestin recruitment to PAR4, because co-immunoprecipitates of arrestin-2 with PAR4 are disrupted by inhibitors of P2Y1 or P2Y12. P2Y1 may regulate arrestin-2 recruitment to PAR4 through protein kinase C (PKC) activation, whereas P2Y12 directly interacts with PAR4 and therefore, may help to recruit arrestin-2 to PAR4. Finally, arrestin2(-/-) mice are less sensitive to ferric chloride-induced thrombosis than WT mice, suggesting that arrestin-2 can regulate thrombus formation in vivo. In conclusion, arrestin-2 regulates PAR4-dependent signaling pathways, but not responses to ADP alone, and contributes to thrombus formation in vivo.

Seeing is believing: An update on modalities to image inflammation in vivo

Inflammation is the body’s adaptive response to noxious and potentially harmful stimuli and conditions such as infection or tissue damage (1–3). What was defined by Celsius as ‘rubor, calor, dolor, and tumor’ has now developed into a challenge for researchers in many fields including systems biology, signal transduction, and physiology. Translation of findings from basic science to clinical applications requires understanding of the underlying processes, development, evaluation, and optimisation of lead compounds as well as diagnosis and staging of patients. All of these stages require a broad spectrum of intravital imaging modalities that cover optical and non-optical, invasive and non-invasive, high resolution and deep tissue penetration techniques. In this theme issue of Thrombosis and Haemostasis we have collected five review articles and two original contributions of experts in the field of intravital imaging of inflammation covering imaging of the various aspects of the inflammatory response including acute inflammatory processes with platelet activation, permeability increases, and leukocyte recruitment as well as processes of rather chronic inflammatory diseases such as angiogenesis. In addition, each contribution also describes usage of different imaging modalities covering a broad spectrum from subcellular imaging to detection at tissue levels as well as clinical applications. This variety of herein described imaging techniques may challenge the reader to cross-bridge and adapt the illustrated methodology for application in different fields of research, or ultimately, stimulate the use of complementary imaging modalities and labelling agents for multimodal imaging. Platelet activation is among the earliest events during the inflammatory processes. Upon vascular injury the haemostatic process must rapidly stanch blood loss while avoiding obstruction of flowing blood within the vessel. In recent years, methods for studying coagulation in vivo such as the ferric chloride injury model and the laser injury model have been standardised. Bellido-Martin et al. highlight imaging modalities that have been employed over the last decade to increase our understanding of fibrin formation and platelet activation (4). Activation of platelets also induces subsequent activation of neutrophils which are of primary importance in regulation of inflammatory transendothelial permeability changes (5). Increases in vascular permeability contribute to containing and resolving the inflammatory cascade as that way complement factors and immunoglobulins may enter the tissue. On the other hand, overzealous oedema formation in e.g. brain injury or acute lung injury may result in fatal outcomes. Kenne and Lindbom review modalities for intravital imaging of inflammatory permeability changes by use of optical (e.g. fluorescence microscopy) as well as of non-optical imaging (e.g. PET and MRI) techniques (6). While the former are primarily employed in experimental setups, the latter are frequently used in clinical settings. The endothelial glycocalyx is one determinant of the endothelial barrier in the microvasculature. Its degradation is known to facilitate leukocyte adhesion and to increase vascular permeability to fluids and proteins. By use of intravital microscopy of the cremaster muscle, Constantinescu et al. show that hyperlipidaemia, a Correspondence to: Remco Megens or Oliver Soehnlein Institute for Cardiovascular Prevention Ludwig-Maximilians-University Munich Pettenkoferstr. 9, 80336 Munich, Germany Tel.: +49 89 5160 4673, Fax: +49 89 5160 4352 E-mail: remco.megens@med.uni-muenchen.de or oliver.soehnlein@med.uni-muenchen.de

Mouse Models of the Common, Recurring Type 1 von Willebrand Disease Mutations Y1584C and R1205H

AbstractAbstract 22 Introduction:Type 1 von Willebrand disease (VWD) is caused by mutations that result in moderate decreases in VWF antigen (VWF:Ag is 5–50% of normal levels) and a mild bleeding phenotype. The common recurrent VWF missense mutation Y1584C is associated with mildly decreased VWF:Ag levels, increased ADAMTS13 cleavage, as well as a possible increase in clearance. The Vicenza mutation, R1205H, exhibits a more severe phenotype (VWF:Ag ∼10%) and accelerated clearance. Although well described in patients and through in vitro studies, extensive controlled in vivo investigation of these mutations has yet to be performed. In this study, we compared both Y1584C and R1205H to wild type VWF using hydrodynamic gene delivery of mouse VWF and ADAMTS13 transgenes in the VWF knockout mouse to determine the pathological mechanisms associated with these variants. Methods:Hydrodynamic injections were performed using 100 μ g wild type (WT) or mutant mouse Vwf cDNA in Ringer's solution in 7–9 week old C57Bl6 VWF knockout mice, replacing plasma VWF. Co-injections with mouse Adamts13 cDNA were also performed. Mice were sampled at days 2, 5, 8, and then weekly. Mouse plasma was analyzed for complete blood counts, VWF:Ag, VWF propeptide, and VWF multimer structure. Thrombotic injury was induced using ferric chloride injury to the arterioles of the cremaster in VWF knockout mice expressing VWF:Ag levels from 0.5–2 U/ml. Platelets were labeled with Rhodamine-6G to evaluate platelet accumulation. Time to stable vessel occlusion and platelet accumulation by relative fluorescence intensity were compared. Results:Hydrodynamic injection caused no adverse events in any animals. Complete blood count values were unaffected for both variants compared to WT. Initial high VWF:Ag values at day 2 were similar for WT VWF (25.4 ± 2.5 U/ml, n= 12, mean U/ml±SEM, n) and Y1584C (26.8 ± 5.5, n= 10), but R1205H levels were 36% lower (16.3 ± 2.1, n= 10). Lower VWF:Ag levels were demonstrated in both “homozygous” and “heterozygous” forms for both type 1 mutations from days 14–42, when VWF expression plateaus. R1205H VWF:Ag was 34.3 ± 5.9% of WT (P < 0.001) and “heterozygous” 1:1 ratio R1205H/WT co-delivery was 27.5 ± 4.7% (p < 0.001). Y1584C was 29.4 ± 7.5% of WT (P < 0.001), and Y1584C/WT was 51.1 ± 4.6% (p < 0.001). VWF propeptide to VWF:Ag ratios (days 2–42) demonstrate that R1205H mouse VWF had an increased clearance rate (165.4 ± 13.5%, p < 0.001), while Y1584C was normal (97.1 ± 6.8 %, P > 0.05) compared to WT (100.0 ± 10.0%). The R1205H mutation showed no significant difference in multimer structure by mean multimer band numbers (days 2 to 42, 93 ± 16%, n = 4, P > 0.05) to wild type VWF (100 ± 12%, n = 4). In contrast, Y1584C had a significant decrease (66 ± 18%, n = 4, P < 0.001). This effect was exaggerated by co-delivery of mouse ADAMTS13 for Y1584C, but not R1205H. Y1584C showed reduced thrombus formation in a ferric chloride injury model while R1205H demonstrated similar thrombogenic activity to wild type VWF. Mean occlusion times were WT = 29.9 ± 2.1 minutes, n = 8, R1205H = 29.1 ± 4.0, n = 8 (p > 0.05), and Y1584C = 38.7 ± 1.1, n = 9 (p = 0.001). Total platelet accumulation was decreased for Y1584C (83.6 ± 6.3%, p = 0.043), but was similar for R1205H (103 ± 6.3, P = 0.72) and WT (100 ± 5.3%). Conclusions:This study demonstrates that these two type 1 VWD mutations have a strong observable effect in the VWF knockout mouse model. R1205H exhibits a large decrease in VWF:Ag levels and evidence of accelerated clearance with R1205H. However, there is no alteration in multimer structure and apparently normal participation in a thrombosis model. Y1584C, in contrast, shows a loss of high molecular weight multimers that is exacerbated by the additional expression of ADAMTS13, indicating that ADAMTS13 cleavage is increased. Y1584C also has an initially high VWF:Ag level that was less than WT levels from day 14 onward, but shows no alteration in clearance, suggesting that there is a biosynthetic defect. Y1584C shows a significant defect in the arteriolar thrombosis model, presenting a Type 2A VWD-like phenotype in the mouse model, which is more severe than the human phenotype. This study has elucidated several novel mechanistic details for these two mutations and highlights that the pathogenic aspects of type 1 VWD can be recapitulated in the VWF knockout hydrodynamic injection model. Disclosures:No relevant conflicts of interest to declare.

Prothrombotic Effects of Fibronectin Isoforms Containing the EDA Domain

Fibronectin (FN) plays an important role in the formation of stable arterial thrombi at the site of vascular injury. FN containing Extra Domain A (EDA+ FN) is absent from normal plasma, but elevated plasma levels of EDA+ FN are found in several pathological conditions. We hypothesized that EDA+ FN plays a special role in thrombosis. We used mouse strains constitutively including (EDA+/+) or excluding (EDA-/-) the EDA domain in all tissues and plasma. Using a flow chamber and the ferric-chloride injury model we found that EDA+ FN accelerates thrombosis both in vitro and in vivo at arterial shear rates. In EDA+/+ mice thrombi (>30 microm) grew faster when compared with EDA(WT/WT) (6.6+/-0.2 minutes versus 8.3+/-0.6 minutes, P<0.05) and the mean vessel occlusion time was shorter (9.9+/-0.4 minutes versus 14.6+/-1.7 minutes, P<0.05). However, the presence of EDA+ FN affected neither single platelet adhesion to subendothelium nor thrombosis in veins. In addition, the mortality rate of EDA+/+ mice after collagen/epinephrine infusion was twice that of EDA(WT/WT) or EDA-/- mice. Our findings reveal that EDA+ FN has prothrombotic activity, and its presence in plasma may worsen pathological conditions in which this form is elevated.

ABSENCE OF ADAMTS13 ON A VWF DEFICIENT BACKGROUND IS NO LONGER PROTHROMBOTIC IN A MURINE MODEL OF ARTERIAL THROMBOSIS

ISTH07A1_P-S-278: Contact View [P-S-278] ABSENCE OF ADAMTS13 ON A VWF DEFICIENT BACKGROUND IS NO LONGER PROTHROMBOTIC IN A MURINE MODEL OF ARTERIAL THROMBOSIS A.K. Chauhan, M.T. Walsh, D.G. Motto, D. Ginsburg, D.D. Wagner. Department of Pathology, Harvard Medical School, CBR Institute for Biomedical Research, Boston; Department of Internal Medicine, University of Iowa, Iowa; Department of Internal Medicine, University of Michigan, Michigan, United States Introduction: In circulation Ultra-Large von Willebrand factor (UL-VWF) is cleaved by ADAMTS13 into a series of smaller multimers. We have shown that ADAMTS13 deficiency in the mouse results in accelerated thrombosis in injured arterioles. We reasoned that if VWF is the only ADAMTS13 substrate relevant for this model, then VWF deficiency would result in the abrogation of the effect of ADAMTS13 deficiency. Alternatively, if we observe an effect of ADAMTS13 deficiency on thrombus formation in the absence of VWF, this would suggest the existence of an ADAMTS13 substrate (s) in addition to VWF. Methods: We crossed Adamts13-/mice on a VWF-/genetic background. Using the ferric chloride injury model we compared experimental thrombosis in arterioles of Adamts13-/-/VWF-/mice with VWF -/mice by intravital microscopy. Results: Since Adamts13-/mice of mixed genetic background were used in early studies, we first confirmed our previous findings of accelerated thrombus formation, in Adamts13 -/on C57BL6/J background. In the Adamts13-/mice, thrombi >20 μm were seen at 5.7 ± 0.5 min as compared to 7.9 ± 0.4 min in wild-type (WT) mice (P 20 μm in Adamts13-/-/ VWF-/(10.7 ± 0.4 min) was similar to VWF-/mice (10.6 ± 0.7 min). The rate of individual thrombus growth in Adamts13-/-/VWF-/was also similar to VWF-/thrombi. After 40 min of observation, thrombi failed to grow to occlusive size in either the Adamts13-/-/VWF-/or VWF-/mice. Conclusions: The absence of ADAMTS13 in the setting of VWF-deficiency is no longer prothrombotic in arterial thrombosis, suggesting that at least in this model system, VWF is the only ADAMTS13 substrate relevant to thrombus growth and stability at arterial shear. Chauhan AK, Walsh MT, Motto DG, Ginsburg D, Wagner DD. ABSENCE OF ADAMTS13 ON A VWF DEFICIENT BACKGROUND IS NO LONGER PROTHROMBOTIC IN A MURINE MODEL OF ARTERIAL THROMBOSIS. J Thromb Haemost 2007; 5 Supplement 2: P-S-278 Date: Sunday, July 8, 2007 Session Info: Poster: TTP/HUS and ADAMTS 13 Room: Exhibition Area file:///E|/working/jaymehra/Poster%20TTPHUS%20and%...3/Abstract%20ISTH07A1_P-S-278%20Contact%20View.htm (1 of 2) [1/16/2014 7:07:02 PM]

GSK3β Is a Negative Regulator of Platelet Function In Vitro and In Vivo.

GSK3β is a ser-thr kinase that is itself phosphorylated on ser9 by the kinase Akt. Because Akt has recently been shown to regulate platelet aggregation and arterial thrombosis in mice, we sought to identify Akt substrates in platelets that may play important roles in platelet function. We show here that the Akt effector, GSK3β, is present in platelets and becomes phosphorylated after treatment of mouse or human platelets with ADP or thrombin receptor-activating peptides (TRAP). Agonist-dependent phosphorylation of GSK3β is reduced by pre-treatment of mouse or human platelets with the PI3K inhibitor LY294002 and is also reduced in platelets from Akt2−/−Akt1+/− mice relative to non-littermate controls, suggesting that agonist-induced GSK3β phosphorylation is partially PI3K- and Akt-dependent. To determine whether GSK3β plays a role in platelet function, aggregation and secretion of dense granule contents were evaluated in human platelets treated with the GSK3 inhibitors, LiCl or SB216763. The dose-response curves for agonist-induced platelet aggregation and secretion were left-shifted in the presence of either inhibitor compared to untreated control platelets, suggesting that GSK3 activity suppresses platelet aggregation. Comparative immunoblots suggest that GSK3β is more highly expressed in platelets than GSK3α. Therefore, to confirm that GSKβ plays a suppressive role in platelet function, the aggregation of platelet-rich plasma (PRP) from GSK3β+/− mice was compared to that of non-littermate controls (GSK3β −/− mice die in utero). PRP from GSK3β+/− mice showed enhanced aggregation and secretion in response to U46619 or TRAP compared to control PRP. TRAP-induced binding of AlexaFluor-fibrinogen to platelet surfaces was also enhanced in washed platelets from GSK3β+/− mice compared to control platelets. Finally, the effect of GSK3β on platelet function in vivo was evaluated using two thrombosis models: a ferric chloride injury model of arterial thrombosis and a collagen-induced model of disseminated thrombosis. In the arterial thrombosis model, all GSK3β+/− mice (n=5) formed stable occlusive thrombi after ferric chloride injury to the carotid artery, whereas the majority of wildtype mice (67%) formed no thrombi, 27% formed stable occlusive thrombi, and 7% formed unstable thrombi under the same conditions (n=15). In a model of disseminated thrombosis, injection of a combination of collagen (170 μg/kg) and epinephrine (350 μM/kg) resulted in reduced survival of GSK3β+/− mice 10 minutes post-injection relative to wildtype mice (20%, n=5 versus 83%, n=6, respectively). Histological examination of lung sections suggested that all mice that died did so due to pulmonary embolism. These data suggest that removal of a single allele of GSK3β in mice confers enhanced sensitivity to thrombotic insult. Taken together, these results suggest that GSK3β is a substrate of Akt-dependent phosphorylation in platelets and, in contrast to the function of Akt, acts as a negative regulator of platelet function in vitro and in vivo.