FeCl3 Injury Research Articles

ADAMTS13 exerts a thrombolytic effect in microcirculation

Recombinant tissue plasminogen activator (r-tPA) is the drug of choice for thrombolysis, but it is associated with a significant risk of bleeding and is not always successful. By cleaving von Willebrand factor (VWF), the metalloprotease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats-13) down-regulates thrombus formation in injured vessels. We investigated whether recombinant ADAMTS13 (r-ADAMTS13) induces thrombolysis in vivo in mice. Thrombosis was produced by ferric chloride-induced (FeCl(3)) injury in the venules of a dorsal skinfold chamber. Phosphate-buffered saline (PBS, vehicle), r-tPA or r-ADAMTS13, supplemented with hirudin (to stop on-going thrombin generation), was directly applied onto the occluded vessel, and thrombus dissolution was evaluated by intravital microscopy. The incidence of blood flow restoration significantly increased 30 minutes (min) after r-ADAMTS13 vs. PBS treatment (60% vs. 0%, p<0.05) and 60 min after r-tPA treatment (75% vs. 17%, p<0.05). Both r-tPA and r-ADAMTS13 significantly reduced thrombus size 60 min after their superfusion (53.2% and 62.3% of the initial thrombus size, p<0.05 and p<0.01, respectively). Bleeding occurred in all r-tPA-treated chambers, while it was absent in mice treated with r-ADAMTS13 or PBS. We observed that, similar to r-tPA, r-ADAMTS13 can dissolve occlusive thrombi induced by FeCl(3) injury in venules. In contrast to r-tPA, the in vivo thrombolytic effect of ADAMTS13 was not associated with any signs of haemorrhage. ADAMTS13 could represent a new therapeutic option for thrombolysis.

Distinct Roles of the Extrinsic and the Intrinsic Coagulation Pathways In Different Murine Thrombosis Models,

Abstract 3278In vivo vascular lesion models are used to elucidate the pathophysiological mechanisms of thrombosis. We found that the intrinsic and extrinsic coagulation pathways exhibit a distinct relative contribution to arterial occlusion initiated by a FeCl3- or nitrogen dye pulse laser (NDPL)-induced injury evaluated comparatively in the carotid artery (CA), the femoral artery (FA) or the femoral vein (FV). Methods. C57Bl/6J (BL6), IL4Rα/GPIbα-tg (IL4) and VWF−/− mice between 8 and 12 weeks of age were anesthetized with 2% isofluorane, and the right jugular vein was cannulated for IV delivery of fluids. Injury in the exposed left CA was induced by applying a 0.7 ml drop of 7 or 8 % FeCl3 for 3 minutes; variations of blood flow in the injured artery were then monitored with a miniaturized ultrasound flow probe for an additional 30 min. In all experiments, FeCl3 concentrations refer to the hexahydrate form of the salt. The time to first occlusion (flow rate <0.1 ml/min) and the normalized area under the flow rate curve, defined as the Flow Index (FI), were used as endpoints for statistical analysis. In the FA and FV, respectively, vascular injury by FeCl3 was induced by applying a 9% drop for 2.5 minutes or 4% drop for 1 minute; then, platelet and fibrin deposition in forming thrombi were visualized by epifluorescence microscopy of injected green calcein-labeled platelets and AlexaFluor 546-labelled anti-fibrin antibody. The NDPL injury was induced in both FV and FA with two series of 30 s laser light pulses, the second upstream of and 30 s after the first. Images selected at regular time intervals from a 20 min real-time video recording were used for measuring the integrated density of formed thrombi with the ImageJ software. Results. In the CA, 8% FeCl3 reliably caused a stable occlusion that was completely abolished by lepirudin, a thrombin inhibitor, at a calculated 42 μM concentration in blood. Also, IL4 mice, lacking a functional extracellular GPIbα domain, and VWF−/− mice failed to develop a normal thrombus. In C57Bl/6J mice, injection of the anti-mouse factor XI (mFXI) monoclonal antibody (mAB) 14E11 inhibited thrombus formation in a dose dependent manner, and completely when injected at a dose of 0.5 μg/g. In contrast, the anti-mouse tissue factor (mTF) mAB 21E10 had no effect when injected at 25 μg/g; the latter amount of anti-mTF mAB was variably inhibitory only when a presumably milder lesion was induced by a reduced 7% FeCl3 concentration. Of note, however, even at the lower dosage of 9 μg/g the anti-mTF mAB completely prevented CA occlusion when co-injected with an individually non-effective dose (0.065 μg/g) of anti-mFXI mAB. Essentially the same results, albeit obtained with a different methodology, were observed by evaluating thrombus formation in the FA and FV after FeCl3-induced injury. Findings were remarkably different in the NDPL model of FA and FV thrombosis. In this case, platelet aggregate formation was transient and almost completely reversed by 20 minute post-injury, while fibrin deposition at the site of injury increased progressively in time. Fibrin deposition was essentially absent in the IL4 mouse but, contrary to what we observed in the same vascular beds after FeCl3 injury, it was only partially inhibited by injecting the anti-mFXI mAB at dosage as high as 1 μg/g. Moreover, and again in contrast with the results after FeCl3 injury, the anti-mTF mAB injected at 25 μg/g by itself significantly inhibited fibrin deposition. Conclusions. Our findings indicate that distinct thrombogenic vascular lesions elicit a varied response by the extrinsic and intrinsic coagulation pathways, as reflected by the different effects of inhibiting initiating events mediated by TF or FXI activation by FXIIa (the reaction blocked by mAB 14E11), respectively. Moreover, and again in a lesion-specific manner, the two coagulation pathways exhibit a variably relevant synergistic function in leading to thrombin generation as evidenced by fibrin deposition. These results, and the underlying mechanisms presently under investigation, suggest that the thrombogenic potential of different pathological lesions in human vascular disease is also likely varied and may be influenced by lesion-specific characteristics. Consequently, more direct mechanistic evidence must be generated to establish how a given animal thrombosis model may reflect the pathogenesis of acute vascular occlusion in human disease. Disclosures:Gruber:Aronora, LLC: Consultancy, Equity Ownership.

Infusion of Recombinant Full-Length ADAMTS13 and Carboxyl-Terminal Truncated Variant Alters Composition of Ferric Chloride-Induced Arterial Thrombi in Adamts13−/− mice

Abstract 2313Proteolytic cleavage of ultra large von Willebrand factor (ULVWF) released from endothelial cells by ADAMTS13 metalloprotease is critical for maintaining normal hemostasis. However, the effect of infusing ADAMTS13 on thrombus composition remains poorly understood. In this study, we determined the morphology and composition of thrombi formed in carotid arteries after topical application of FeCl3 in Adamts13−/− mice receiving PBS, recombinant human full-length ADAMTS13 (FL) and carboxyl-terminal truncated variant after spacer domain (S), using scanning electron microscopy and quantitative image analysis. We showed that in Adamts13−/− mice 5 min after FeCl3 injury, formed arterial thrombi were comprised ∼39% platelets, ∼26% red blood cells, and ∼35% fibrin. The arterial thrombi in these mice were structurally deformed. An infusion of recombinant FL (final concentration of 10 nM) significantly reduced the accumulation of platelets (∼18%) but increased the fibrin network (57%) without affecting the composition of red blood cells (∼25%) in the arterial thrombi formed at the same time point after FeCl3 injury. Similar effects on the morphology and composition of FeCl3-induced arterial thrombi were observed after infusion of recombinant S (10 nM) into Adamts13−/− mice. Kinetic analysis showed that there was a decrease in platelet accumulation over the time of 30 min during thrombus formation with a slight increase in accumulation of red blood cells and formation of fibrin in Adamts13−/− mice. But, the infusion of recombinant FL and S into Adamts13−/− mice restored the kinetics of platelet/red blood cell accumulation and fibrin formation to those observed in wild-type mice. Our findings, revealing the apparent difference in thrombus composition, provide novel insight into the mechanism of ADAMTS13 function in vivo, which may shed more light on the pathogenesis of thrombotic thrombocytopenic purpura and other arterial thrombotic disorders associated with deficiency of plasma ADAMTS13 activity. Disclosures:No relevant conflicts of interest to declare.

High Affinity Binding of FVIII to VWF Is Not Required for the Haemostatic Effect of FVIII In Vivo

Abstract 1182VWF protects FVIII from clearance in the circulation and is believed to ensure location of platelets to the site of injury. However, it is unknown if binding of FVIII to VWF has a role in localizing and thereby also facilitating the effect of FVIII in vivo. In the present study, a FVIII variant, FVIII-Y1680F, lacking the high affinity binding to VWF (Leyet et al. JCB 1991; 15; 740) was used to evaluate the binding of FVIII to VWF in clot formation hemophilia A mice in vivo.Binding of the FVIII variant to immobilized VWF was evaluated by surface plasmon resonance showing a 50–25 fold reduction in the Kd for FVIII-Y1680F compared to wt FVIII. Furthermore, pharmacokinetic studies in hemophilia A mice indicated that FVIII-Y1680F is basically devoid of VWF binding in vivo. The circulating half-life decreased from 7–8 hours for wt FVIII to 0.5 hours for FVIII-Y1680F (see figure) which is comparable to the half-life of wt FVIII in VWF knockout mice (0.5 hours). Using a chromogenic assay the specific activity of FVIII-Y1680F was 9200 IU/mg similar to that of wt FVIII confirming normal activity FVIII-Y1680F after cleavage with thrombin and removal of VWF. [Display omitted] As the short half-life may influence the haemostatic effect of FVIII-Y1680F in vivo, a 40 kDa PEG moiety was attached to the O-glycan in the B-domain of the FVIII variant. This re-establishes the circulating half-life (6.1 hours) to that of wt FVIII without affecting the specific activity in vitro. The haemostatic effect of 40K-O-PEG FVIII-Y1680F was subsequently used to investigate if high affinity VWF binding of FVIII influences its haemostatic effect in vivo.The acute haemostatic effect of 40K-O-PEG-FVIII-Y1680F was compared to wt FVIII (Advate®) in the tail bleeding model in hemophilia A mice at doses equivalent to the 50% of the maximal effect and at maximal efficacy (20 and 280 IU/kg; Elm et al., Hemophilia 2011 epub). The blood loss was significantly reduced at both doses with comparable effect of 40K-O-PEG-FVIII-Y1680F and wt FVIII (see see figure, * indicates significant differences compared to vehicle treated hemophilia A mice). This indicates that the lack of VWF binding does not interfere with the haemostatic properties of FVIII in this particular model. To further support these data, the haemostatic effect of 40K-O-PEG-FVIII-Y1680F was tested in the FeCl3 injury model (2.5, 5 and 10 IU/kg) in hemophilia A mice. No clot formation was observed in vehicle mice and 40K-O-PEG-FVIII-Y1680F normalized dose dependently the clot formation time comparable to wt FVIII.In conclusion, the current data suggest that the haemostatic effect of FVIII in vivo is not dependent on high affinity binding of FVIII to VWF. Disclosures:Holmberg:Novo Nordisk A/S: Employment. Kjalke:Novo Nordisk A/S: Employment. Karpf:Novo NOrdisk A/S: Employment. Hilden:Novo Nordisk A/S: Employment. Pelzer:Novo Nordisk A/S: Employment. Koefoed-Hansen:Novo Nordisk A/S: Employment. Johnsen:Novo Nordisk A/S: Employment. Thim:Novo Nordisk A/S: Employment. Karlsson:Novo Nordisk A/S: Employment. Jespersgaard:Novo Nordisk A/S: Employment. Bolt:Novo Nordisk: Employment. Stennicke:Novo Nordisk A/S: Employment.

In Vivo Monitoring of Venous Thrombosis In Mice Using Ultrasonography

AbstractAbstract 4214Deep venous thrombosis is an important cause of morbidity and mortality in clinical medicine. There has been extensive research dedicated to the clinical aspects of venous thrombosis, especially with regards to its diagnosis and treatment. However, animal models studying this phenomenon are scarce and, in most cases, very crude, relying on sacrificing animals to excise the formed thrombi. Developing an in vivo murine model of venous thrombosis, detecting and monitoring thrombi non-invasively as is done in humans can be a powerful tool given our ability to genetically modify the murine genome. Therefore, we developed such a murine model using the Vevo770®, a microimaging ultrasound system previously developed to study the arterial circulation of mice. Two different thrombosis models were employed to generate clots in the inferior vena cava (IVC) of wild type C57Bl6 mice: 1) ligation of the IVC to generate venous stasis and 2) application of Ferric Chloride (FeCl3) to the outer layer of the IVC to injure the endothelium. Using both of these techniques, adequate thromboses were generated in the IVCs of mice as determined pathologically. Other mice were allowed to recover after surgery, and the development of venous thrombosis was assessed by ultrasonography using the Vevo 770®. In order to assess the precision of clot measurements using this novel technique, we then sacrificed the mice and excised the clots. In both models, the measurement of the clot pathologically correlates favorably (R2= 0, 9116 for the ligation model, and R2 = 0,905 for the FeCl3 model) with measurements done by ultrasonography (n=20 for the ligation model, and n=5 for the FeCl3 injury model). In the ligation model, a thrombus develops less than an hour after ligation of the IVC, and the size of the clot increases over time. For example, five hours after the ligation of the IVC, a clot develops and has a cross sectional area of 4,5 mm2. The clot size increases significantly (p=0.001) over time to 6.2 mm2 at 24 hours post ligation (n=20). Treatment of these mice with an anticoagulant (dalteparin at a dose of 200 u/kg) prior to the procedure prevented the development of IVC thrombosis as determined by ultrasonagraphy. These data suggest that the Vevo770® can be used as a reliable technique for the non-invasive assessment of venous thrombosis in mice. Developing a murine model for thrombosis using more accurate, and clinically more relevant techniques such as ultrasonography, is a step towards better understanding the pathophysiology of venous thromboembolism. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Deletion of the p110β isoform of phosphoinositide 3-kinase in platelets reveals its central role in Akt activation and thrombus formation in vitro and in vivo

AbstractDuring platelet activation, phosphoinositide 3-kinases (PI3Ks) produce lipid second messengers participating in the regulation of functional responses. Here, we generated a megakaryocyte-restricted p110β null mouse model and demonstrated a critical role of PI3Kβ in platelet activation via an immunoreceptor tyrosine-based activation motif, the glyco-protein VI-Fc receptor γ-chain complex, and its contribution in response to G-protein–coupled receptors. Interestingly, the production of phosphatidylinositol 3,4,5-trisphosphate and the activation of protein kinase B/Akt were strongly inhibited in p110β null platelets stimulated either via immunoreceptor tyrosine-based activation motif or G-protein–coupled receptors. Functional studies showed an important delay in fibrin clot retraction and an almost complete inability of these platelets to adhere onto fibrinogen under flow condition, suggesting that PI3Kβ is also acting downstream of αIIbβ3. In vivo studies showed that these mice have a normal bleeding time and are not protected from acute pulmonary thromboembolism but are resistant to thrombosis after FeCl3 injury of the carotid, suggesting that PI3Kβ is a potential target for antithrombotic drugs.

Protein Kinase Cδ Differentially Regulates Platelet Functional Responses.

Protein Kinase Cδ (PKCδ), a novel PKC isoform is expressed and activated in platelets downstream of PARs and GPVI receptors. In the current study, the role of PKCδ in regulating platelet functional responses was investigated using a pharmacological inhibitor, (δV1-1)TAT (a PKCδ inhibitor) in human platelets. These studies were further confirmed by a knockout approach using PKCδ+/+ and PKCδ−/− mice. In both human and murine platelets, PAR4-mediated dense granule secretions were inhibited, whereas GPVI-mediated dense granule secretions were potentiated. Furthermore, α-granule secretions and thromboxane A2 (TXA2) generation were differentially regulated in murine platelets.. These data suggest a differential role for this isoform in regulating dense granule secretion, α-granule secretion and TXA2 generation. Previous studies have shown that PAR-mediated fibrinogen receptor activation is regulated by a Calcium-dependent and a PKC-dependent pathway. The contribution of PKCδ to PAR-mediated fibrinogen receptor activation was studied by pretreating human and murine platelets with BAPTA. Our results showed a inhibition of AYPGKF-induced aggregation in human and murine platelets in the presence of BAPTA and fibrinogen. These results suggest a small contribution of PKCδ to PAR-4- mediated platelet aggregation and aIIbb3 activation. The in vivo significance of PKCδ was tested using a FeCl3 injury model. While the wildtype mice occluded in 7 minutes, PKCδ −/− mice occluded after 4 minutes of injury with 10 % FeCl3. Therefore, we conclude that PKCδ regulates platelet functional responses such as dense, α-granule secretions, TXA2 generation downstream of both PARs and GPVI receptors, contributes to PAR-4-mediated fibrinogen receptor activation ex vivo and plays a critical role in the thrombus formation in vivo. This study is supported by predoctoral fellowships to Ramya Chari and Swaminathan Murugappan from American Heart Association, Great Rivers affiliate.

Antithrombotic Effects of a FXI Antisense Oligonucleotide in Mice

FXI, a component of the intrinsic pathway of coagulation, contributes to the generation of thrombin and is also activated by thrombin on the surface of platelets, resulting in further amplification of the coagulation process. Reports have shown that FXI deficiency in mice or in humans is generally not associated with major bleeding yet is associated with reduced risk for thromboembolic disease. To determine whether targeting FXI with antisense oligonucleotides (ASOs) can produce antithrombotic activity without increasing risk of bleeding, a series of 2nd Generation 2′-O-methoxyethyl modified ASOs were identified that specifically reduce levels of FXI in mice and were evaluated for antithrombotic activity and bleeding potential. Mice were dosed with FXI ASO (ISIS 404071) subcutaneously for three weeks. FXI ASO treatment resulted in a dosedependent and specific reduction in FXI mRNA levels in liver and FXI protein levels in plasma with an ED50 of 3.2 mg/kg. These reductions in FXI levels correlated well with a prolongation of aPTT with no effects on PT prolongation. The anticoagulant effect of FXI ASO treatment also correlated well with antithrombotic activity in a 10% FeCl3 induced IVC thrombosis mouse model across a wide dose range. Furthermore, FXI ASO treatment was generally well tolerated and no prolongation of tail bleeding time was observed at any dose tested, indicating a broad safety margin for FXI ASO targeting. Additional studies using intravital microscopy to study the effects of FXI ASO treatment on thrombosis formation in the mesenteric vein following 10% FeCl3 injury demonstrated that FXI ASO treatment significantly impeded platelet aggregation at the site of vessel injury, thrombus growth and vessel occlusion. Studies to further evaluate the safety of FXI targeting were conducted using a mouse model of intracerebral hemorrhage induced by intracranial injection of collagenase. In this model, FXI ASO treatment had no effect on hemorrhagic size, neurologic deficit score, or mortality at doses well above that needed to produce maximal antithrombotic activity. These results strongly suggest that FXI is an effective and safe target for antithrombotic therapy and that antisense drug technology could be an attractive approach to specifically target FXI for the treatment of thrombotic disorders.

CD36 Plays a Role in Murine Thrombosis Induced by Vascular Injury

We previously showed that the Type B scavenger receptor, CD36 mediates platelet responses to oxidized phospholipids. Using a FeCl3‐induced injury model and CD36 knockout (KO) and wild type (WT) mice, we examined the role of CD36 in thrombosis by in vivo video‐microscopy after injection of Rhodamine 6G to label platelets. While there was no difference in time to thrombosis (TTT) in carotid arteries using 12.5% FeCl3, when a less severe injury was induced by 7.5% FeCl3, there was increased TTT in KOs (p=0.006). We also observed increased TTT in both arterioles and venules of KOs (p&lt;0.05). Mechanistically, we previously showed that platelet signaling via CD36 was mediated by MAP kinases. Here we demonstrate a significantly elongated TTT in the carotid artery in irradiated thrombocytopenic WT mice transfused with JNK inhibitor pre‐treated WT platelets. No effect was observed using KO platelets. Isolated platelets from KOs were less sensitive to activation in vitro by low dose ADP. Immunohistochemical analysis of carotid thrombi showed a significant decrease in the endothelial cell marker CD105 in KOs, suggesting less accumulation of endothelial cell‐derived microparticles. Plasma urokinase type plasminogen activator levels were increased in KO mice after induction of thrombi. By direct injection of the superoxide indicator hydroethidine, we also found that the level of FeCl3 injury induced reactive oxygen species was lower in KOs. We conclude that CD36 plays a complex role in thrombosis via MAP kinase mediated modulation of platelets activation, promotion of reactive oxygen species formation after endothelial injury, and regulation of activation of the fibrinolytic system.

Factor V Leiden mutation is associated with enhanced arterial thrombotic tendency in lean but not in obese mice

The homozygous factor V Leiden mutation is associated with enhanced venous thrombotic risk. Obesity is a major risk factor for development of thrombotic cardiovascular disease. It was the objective of this study to investigate whether obesity affects the thrombotic risk associated with the mutation. Male mice with homozygous factor V Leiden mutation (Arg 504 to Gln) (FVQ/Q) and corresponding wild-type (WT) mice were kept on a standard fat diet (SFD) or high fat diet (HFD) for 14 weeks, and femoral artery thrombosis was induced by FeCl3 treatment. As compared to SFD, HFD feeding for 14 weeks resulted in significantly higher body weight and fat mass associated with adipocyte hypertrophy, which were, however, similar for both genotypes. In the FeCl3-induced arterial thrombosis model, FVQ/Q mice kept on SFD had a 40% shorter occlusion time (p = 0.015) and 40% lower blood flow (p = 0.03), as compared to WT mice. However, on HFD the occlusion time and blood flow were not significantly different for both genotypes. This finding could not be explained by differential changes of coagulation factors in either genotype fed on SFD or HFD. In conclusion, on SFD, but not on HFD, the factor V Leiden mutation is associated with enhanced thrombotic tendency after FeCl3 injury of the femoral artery, suggesting that in this model obesity rescues the increased thrombotic risk associated with the factor V Leiden mutation.

In Vivo Relevance for Platelet Glycoprotein Ibα Residue Tyr276 in Thrombus Formation.

Background: GPIbα is a critically important platelet receptor best recognized for its ability to bind vWF and mediate platelet adhesion. In addition, a GP Ibα binding site for thrombin has been described and characterized for several decades. However, the physiologic relevance of GPIbα/thrombin binding has remained elusive. Site-directed mutagenesis has shown that a sulfated GP Ibα residue, Tyr276, is essential for thrombin binding to GP Ibα but not critical for vWF binding. The importance of Tyr276 was further substantiated in the crystal structure of a GP Ibα/thrombin complex confirming the sulfated Tyr276 GP Ibα residue directly participates with other neighboring negatively charged residues in establishing a first contact with the anion-binding exosite II of an α-thrombin molecule. To determine the physiologic relevance, if any, of the GP Ibα Tyr276 residue we generated mice with platelets containing a mutant human GP Ibα subunit containing a Tyr276 to Phe276 substitution (Y276F) and did comparative studies with mice expressing a normal human (WT) GP Ibα subunit.Methods: Mouse colonies expressing the normal or variant GP Ibα subunits were established by transgenic technology and bred into a mouse colony devoid of murine GP Ibα. Surface expression of WT or Y276F GP Ibα subunits was measured by flow cytometry. Relevant assays included platelet counts, FeCl3-induced thrombus formation, tail bleeding times, hemoglobin content following tail resection (measured as 575 nm absorbance of lysed erythrocytes), and washed platelet aggregation induced by different agonists (fibrillar type I collagen, thrombin, ristocetin, arachidonic acid, and PAR activating peptides).Results: Founder mice were selected with nearly identical levels of surface-expressed WT and Y276F GPIbα subunits. Circulating platelet counts were similar between the 2 colonies. Stirred platelet aggregation assays induced by 0.05, 0.1, 0.5 units/ml thrombin, 10 and 20 mg/ml fibrillar collagen, 1.25 mg/ml ristocetin, 0.25 mM arachidonic acid, 100 mM PAR3 activating peptide and 100 mM PAR4 activating peptide revealed no significant differences between WT and Y276F samples. No statistically significant values were obtained in tail bleeding time assays or hemoglobin loss comparing WT and Y276F animals. However, the time required for a reduction in carotid artery blood flow following FeCl3 injury was significantly prolonged in Y276F animals (10.6±1.6 min vs. 5.8±1.4 min). All WT animals displayed complete occlusion following the initial reduction in blood flow. In contrast, 3 out of 6 Y276F animals did not show complete occlusion and had evidence of fluctuating blood flow suggestive of embolization.Discussion: FeCl3 injury of the mouse carotid artery revealed an essential role for GP Ibα residue Tyr276. In contrast, no differences were seen in tail bleeding time assays or in any platelet aggregation assay. These results are suggestive that thrombin binding to human GPIbα may be relevant for the pathological development of a thrombus. This conclusion is based on both the failure of some carotid arteries from Y276F animals to fully occlude and our observation that all Y276F animals showed an increased length of time for the reduction of blood flow. The results suggest thrombin binding to GPIbα could be significant in terms of enhancing fibrinogen cleavage and therefore stabilizing the development of a platelet-rich thrombus.

Long-Term Expression of ADAMTS13 at Therapeutic Levels in Mice by In Utero Administration of Lentiviral Vector Encoding the Human ADAMTS13 Gene.

Thrombotic thrombocytopenic purpura (TTP), characterized by profound thrombocytopenia and microangiopathic hemolytic anemia, may occur in neonates, children and adults. Two types of TTP: hereditary and acquired, have been described. Hereditary TTP is caused by mutations in the ADAMTS13 gene, whereas the acquired TTP is mainly caused by autoantibodies against ADAMTS13 protease. Despite of low incidence of hereditary TTP, patients with hereditary TTP tend to recur or relapse after an initial episode, which may result in sustained neurological and/or renal complications or death. Therefore, early diagnosis and treatment of hereditary TTP are crucial to prevent catastrophic complications. Gene therapy may provide an attractive alternative therapeutic approach to plasma infusion or exchange for long-term correction of ADAMTS13 deficiency. Lentiviral vector encoding the full-length human ADAMTS13 gene tagged with V5-His at the carboxyl terminus and an enhanced green fluorescent protein (eGFP) reporter or PBS was administered to day 14 mouse fetuses (C57/BL6) via direct injection into yolk sac vessels. The blood was collected into sodium citrate at birth, 1, 4, 8, 12, 18, 24, 28 and 36 weeks following the injection. The plasma ADAMTS13 activity, VWF antigen, VWF activity and VWF multimers were determined by FRETS-VWF73, enzyme-linked immunoassay, type III collagen binding and agarose gel electrophoresis, respectively. The transduction of lentiviral vector and expression of human ADAMTS13 in vivo were determined by direct visualization of eGFP reporter by fluorescence stereomicroscopy and immunohistochemistry with monoclonal anti-V5 antibody. We showed that liver appeared to be the predominant organ expressing human ADAMTS13 and eGFP reporter. Other organs including brain, heart, kidney, lung, intestines and testes were also positive for the transgene products, particularly in the vessels. Full-length human ADAMTS13-V5-His (~195kDa) protein was detectable in mouse plasma after immunoprecipitation with mouse anti-V5 and Western blot with rabbit anti-ADAMTS13 IgG. The plasma ADAMTS13 activity in lentiviral vector-injected mice (n=8) was approximately 15~20% higher on average than that in the buffer-injected controls (n=8). The elevated levels of ADAMTS13 persisted for at least 9 months tested. Furthermore, the ratio of VWF activity to VWF antigen levels and the multimer sizes in the lentiviral vector-injected mice were significantly smaller than those in the PBS-injected control. The mice overexpressing human ADAMTS13 had normal complete blood count and did not show signs of spontaneous bleeding. To our surprise, these mice exhibited a significant prolongation of occlusion time in the carotid arteries after 10% FeCl3 injury (28.6 ± 13.0 min, n=8) compared to the control mice (8.5 ± 1.9 min, n=8) (p<0.001). These data demonstrate for the first time that long-term expression of human ADAMTS13 is achieved in mice by in utero administration of lentiviral vector and that the over expression of human ADAMTS13 may offer a systemic anti-thrombotic effect in vivo. Our study supports the feasibility of successful gene therapy for hereditary TTP and/or other arterial thrombotic disorders that are mediated by “unusually large” or “hyperactive” VWF.

Glycoprotein VI–dependent and –independent pathways of thrombus formation in vivo

The role of the collagen receptor glycoprotein VI (GPVI) in arteriolar thrombus formation was studied in FcRgamma-null mice (FcRgamma(-/-)) lacking platelet surface GPVI. Thrombi were induced with severe or mild FeCl(3) injury. Collagen exposure was significantly delayed and diminished in mild compared with severe FeCl(3) injury. Times to initial thrombus formation and vessel occlusion were delayed in FcRgamma(-/-) compared with wild-type mice after severe injury. Platelet accumulation in wild-type mice was decreased after mild compared with severe injury. However, there was little difference between platelet accumulation after severe or mild injury in FcRgamma(-/-). These data indicate a significant role for GPVI in FeCl(3)-induced thrombus formation. Pretreatment of wild-type mice with lepirudin further impaired mild FeCl(3)-induced thrombus formation, demonstrating a role for thrombin. Laser-induced thrombus formation in wild-type and FcRgamma(-/-) was comparable. Collagen exposure to circulating blood was undetectable after laser injury. Normalized for thrombus size, thrombus-associated tissue factor was 5-fold higher in laser-induced thrombi than in severe FeCl(3)-induced thrombi. Thus, platelet activation by thrombin appears to be more important after laser injury than platelet activation by GPVI-collagen. It may thus be important when considering targets for antithrombotic therapy to use multiple animal models with diverse pathways to thrombus formation.

Regulated Phosphatidylserine Exposure on Platelets Mediates Fibrin Formation in Hemostasis and Thrombosis.

Thrombin-stimulated platelets support activity of phosphatidylserine (PS)-dependent blood coagulation reactions. However, only 2–6% of stimulated platelets expose sufficient PS to bind annexin V, leading to the supposition that procoagulant reactions are localized to the annexin V positive platelets and to microparticles shed by platelets. We hypothesized that thrombin-stimulated platelets expose sufficient PS to support the prothrombinase and factor Xase complexes but insufficient to meet the threshold for annexin V binding. We evaluated lactadherin, a PS-binding milk protein, as a reagent to detect platelet PS exposure. Thrombin or TRAP-treated platelets bound lactadherin with 3200 ± 700 sites/plt, but did not bind annexin V, as detected by flow cytometry. To confirm that lactadherin binding truly reported PS exposure, we performed activation experiments upon platelets loaded with 1-Palmitoyl-2- [6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-Glycero-3-Phospho-L-Serine (NBD-PS). Stimulation of platelets with 10 μM TRAP led to 8 ± 2 % PS exposure vs > 90% PS exposure for 10 μM A23187. PS exposure was maximal within 1 min of exposure to TRAP and was reversible with >70% of exposed PS re-internalized within 30 min. In situ platelet fibrin deposition was monitored utilizing a novel flow cytometry assay. Platelet rich plasma was recalcified and supplemented with 50 pM factor Xa, 100 μM GPRP. Platelet-bound fibrin, lactadherin and annexin V binding sites were measured at time intervals. Fibrin accumulated on lactadherin + platelets at a rate greater than or equal to the rate on platelet microparticles or annexin V + platelets. Lactadherin inhibited > 98% of prothrombinase and factor Xase activity on platelets while annexin V inhibition reached a plateau of ~ 80%. The localization and importance of regulated PS exposure in vivo was evaluated in mouse models. Anesthetized mice were injected intravenously with 1 μg of lactadherin and 1 μg annexin V. Three minutes after FeCl3 injury of exposed mesentery, mice were perfused with fixative. Immunohistochemistry showed veins edged with fibrin and decorated with platelets. Lactadherin co-localized with CD41+ platelets along the vascular wall but was less intense on platelets lodged within fibrin aggregates that extended into the vessel lumen and did not stain platelets in sequestered blood. Annexin V stained only scattered platelets and endothelial cells and did not correlate well to sites of fibrin deposition. To evaluate the hemostatic importance of platelet PS, 10 μg of lactadherin was injected intravenously prior to tail snip; bleeding volumes increased from 33 ± 29 μl to 128 ± 39 μl, n experimental = 8. Rose bengal/laser-induced carotid thrombosis delayed from 34 ± 9 min to 80 ± 20 min, n = 10 after 8–21 μg lactadherin. Four treated animals did not develop a thrombosis. In summary, use of lactadherin as a PS probe has revealed that the capacity for regulated PS exposure, at levels below the annexin V binding threshold, is a general platelet property and that low level PS exposure is sufficient to support thrombin and fibrin generation in vitro and in vivo. These results have implications about the mechanism through which PS exposure is regulated as well as for the potential value of PS exposure as a diagnostic or therapeutic target.

Effect of Factor XI or Factor IX Deficiency on an Arterial Occlusion Model.

Factor XI (fXI) and factor IX (fIX) are zymogens of plasma proteases that are required for normal formation and maintenance of a blood clot. Recent work has implicated these proteins in the pathogenesis of vascular thrombosis. Epidemiologic studies indicate that high levels (top 10% of normal distribution) of fXI or fIX are independent risk factors for venous thromboembolism, increasing risk ~2-fold. Recently, it was shown that fXI deficiency protects mice from carotid artery occlusion in a ferric chloride (FeCl3 ) injury model. FeCl3-induced thrombus formation involves thrombin generation, in addition to platelet activation and von Willebrand factor. We used a modified version of the FeCl3 model to study the antithrombotic effects of complete fXI or fIX deficiency. In wild type C57Bl/6 mice, carotid artery flow measured by Doppler flow probe is completely blocked within 10 minutes of applying 3.5% FeCl3 to the vessel. 3.0% FeCl3 induced occlusion in some (5 of 8) mice by 30 minutes, while no animal treated with 2.5% FeCl3 experienced occlusion. FXI and fIX deficient mice were fully protected from occlusion induced by 3.5% or 5% FeCl3. Some fXI (4 of 8) and fIX (4 of 6) deficient animals developed occlusion with 7.5% FeCl3, while occlusion occurred in all mice at 10% FeCl3. To put the effect of fXI or fIX deficiency on this model into perspective, it requires a very high dose of heparin (1000 U/kg) to produce similar protection. With 5% FeCl3, heparin at 200 U/kg only protects 50% of wild type mice from occlusion, despite prolonging the activated partial thromboplastin time beyond the upper limit of the assay (> 500 secs). High dose aspirin (100 mg/kg) did not prevent occlusion induced by 5% FeCl3, despite producing a nearly complete block of arachidonic acid-induced platelet aggregation in vitro. While fXI and fIX deficiency affect the FeCl3 model similarly, they have significantly different impacts on a tail bleeding time (TBT) assay. FXI deficient and wild type mice have similar mean TBTs (265 ± 68 and 287 ± 92 secs, respectively), while fIX deficiency causes prolonged bleeding (1561 ± 125 secs, p < 0.01). In comparison, heparin (200 units/kg) causes the TBT to exceed the upper limit of the assay (1800 seconds), while aspirin (30 mg/kg) modestly increases the TBT (~2.2-fold). The data indicate that fXI and fIX are involved in thrombus formation in the FeCl3 model, and support a growing body of evidence that thrombin formation through the fIX/fXI axis contributes to thrombotic disease. Given the mild bleeding diathesis associated with fXI deficiency, inhibition of fXI may be a useful component of therapy for treating or preventing thrombus formation, and would be associated with a relatively low risk of bleeding.