Site Of Laser Injury Research Articles

Engineered Knockout of TRPA1 Inhibits Laser-Induced Choroidal Neovascularization Along With Associated TGFβ1 Expression and Neutrophil Infiltration

We examined the effects of gene ablation and chemical inhibition of transient receptor potential ankyrin 1 (TRPA1) on the growth of experimental argon laser–induced choroidal neovascularization (CNV) in mice. CNV was induced in the eyes of 6- to 8-week-old TRPA1-null (knockout [KO]) and wild-type (WT) mice by argon laser irradiation. Gene expression analysis was performed in laser-injured tissues at days 1 and 3. CNV growth was evaluated at day 14. Reciprocal bone marrow transplantation was performed between each genotype to identify the components responsible for either recipient tissue or bone marrow–derived inflammatory cells. Our results show that laser irradiation successfully induced CNV growth at the site of laser injury. The size of induced CNV was significantly smaller in KO mice than in WT mice at day 14, as determined by angiography with fluorescein isothiocyanate-dextran. Invasion of neutrophils, but not macrophages, was suppressed in association with suppression of the expression of transforming growth factor β1 and interleukin 6 in laser-irradiated KO tissue. Bone marrow transplantation indicated that the genotype of the recipient mouse, but not of inflammatory cells, is attributable to the KO phenotype. Systemic administration of a TRPA1 antagonist also reduced the CNV in a WT mouse. In conclusion, TRPA1 signaling in local cells is involved in growth of laser-induced CNV. The phenotype was not attributable to vascular endothelial cells and inflammatory cells. Blocking TRPA1 signal may therefore be a potential treatment strategy for CNV-related ocular diseases.

WITHDRAWN: Engineered knockout of TRPA1 inhibits laser-induced choroidal neovascularization along with associated TGFb1 expression and neutrophil infiltration

We examined the effects of gene ablation and chemical inhibition of transient receptor potential ankyrin 1 (TRPA1) on the growth of experimental argon laser-induced choroidal neovascularization (CNV) in mice. CNV was induced in the eyes of 6 - 8-week-old mice by argon laser irradiation and observed expression. This reaction was compared between TRPA1-null (KO) mice and wild-type (WT) mice. Gene expression analysis was performed in laser-injured tissues on day 1 and 3. CNV growth was evaluated on day 14. Finally, reciprocal bone marrow transplantation was performed between each genotype of mice to identify the responsible components of either recipient tissue or bone marrow-derived inflammatory cells. Results showed that laser irradiation successfully induced CNV growth at the site of laser injury. The size of induced CNV was significantly smaller in KO mice as compared with WT mice at day 14, as examined by angiography with fluorescein isothiocyanate-dextran. Invasion of neutrophils, but not macrophages, was suppressed in association with suppression of expression of transforming growth factor β1 in laser-irradiated KO tissues. Bone marrow transplantation indicated that the genotype of the recipient mouse, but not of inflammatory cells, is attributable to the KO phenotype. Systemic administration of a TRPA1 antagonist also downsized the CNV in a WT mouse. In conclusion, TRPA1 signal in local tissue cells is involved in the growth of laser-induced CNV. The phenotype was not attributable to both vascular endothelial cells and inflammatory cells. Blocking TRPA1 signal could be a potential treatment strategy for CNV-related ocular diseases. (244 words).

Enhanced Thrombotic Responses Are Associated With Striatin Deficiency and Aldosterone.

BackgroundIn addition to its role on blood pressure, aldosterone (ALDO) also affects the hemostatic system leading to increased experimental thrombosis. Striatin is an intermediate in the rapid, nongenomic actions of ALDO. Striatin heterozygote knockout (Strn +/‐ ) mice have salt sensitivity of blood pressure and mildly chronically increased ALDO levels. In addition, in humans, striatin polymorphic gene variants are associated with increased salt sensitivity of blood pressure. Thus, we hypothesized that striatin deficiency would be associated with an increased prothrombotic response.Methods and Results Strn+/ ‐ mice and wild‐type littermates were maintained on a liberal sodium diet (1.6%). We measured in vivo thrombus formation following laser‐induced injury in cremaster arterioles using intravital microscopy. Mice were randomized to intravenous administration of ALDO or its vehicle. Acutely, ALDO increased thrombotic responses in wild‐type mice (P<0.01) versus controls within minutes as determined by increased platelet accumulation and fibrin deposition at the site of laser injury. We then compared thrombus formation without ALDO administration in Strn+/‐ and wild‐type mice. Strn+/‐ mice showed highly significant increases in laser‐induced thrombosis (P<0.001), as shown by increased platelet accumulation and fibrin deposition. Interestingly, the response in the Strn+/‐ mice basally was far greater than the wild‐type mice with ALDO administration, and ALDO administration produced no additional effect on thrombus responses in Strn+/‐ mice.ConclusionsThese results demonstrate a novel protective role of striatin in experimental thrombosis. Such a protective effect may be reduced in human striatin risk allele carriers, given the similar salt sensitivity of blood pressure in these individuals and Strn+/‐ mice.

Dysfunctional Endothelium Drives a Pre-DIC State in Endotoxemia

Uncontrolled pro-coagulant responses in sepsis can lead to disseminated intravascular coagulation (DIC), a complication associated with markedly increased mortality. Abnormalities of coagulation, fibrinolysis, and platelet function can lead to both microvascular thrombosis contributing to multi-organ dysfunction syndrome or hemorrhagic complications. To understand the inciting causes of DIC in sepsis, we first evaluated the time course of platelet function, coagulation parameters and markers of endothelial activation following LPS exposure. These studies demonstrated that endothelial dysfunction preceded derangement of platelet function or coagulation parameters. To evaluate the thrombotic response of the endothelium early in endotoxemia, we injured the microvascular endothelium of cremaster arterioles of mice using a laser 1-3 hours following LPS exposure. Platelet and fibrin formation at sites of injury were significantly increased following LPS exposure to respectively 190% (p=0.026) and 195% (p<0.001) of control values. No significant differences were observed in platelet counts, platelet function (aggregation and activation) or coagulation parameters (PT) in mice treated with LPS for 3 hours compared to vehicle controls. Plasma levels of the endothelial markers VWF, soluble VCAM and E-selectin, however, were significantly increased following LPS exposure, demonstrating early endothelial activation. Furthermore, even when platelet accumulation was inhibited using the anti-platelet drug eptifibatide, fibrin generation at sites of laser injury was still significantly (p<0.01) increased in LPS-treated mice. Together, these data show that endothelial activation precedes disruption of platelets and coagulation in endotoxemia. Endothelial dysfunction is associated with perturbation of the endothelial Ang1/Tie2 pathway, characterized by significantly reduced Tie2 function and Ang1 levels. Therefore, we assessed thrombus formation in Tie2+/- mice in the absence of LPS. Fibrin generation at sites of laser injury was significantly increased in Tie2+/- mice to 192% of littermate (Tie2+/+) controls (p<0.01). As loss of Tie2 mimics the LPS-induced phenotype, we next determined whether activation of the Tie2 pathway could reduce fibrin clot formation. Ang1 stimulates phosphorylation of Tie2, promoting protective signaling in endothelium. To determine whether the pro-thrombotic consequences of endotoxemia on Tie2 signaling could be reversed using Ang1, mice were injected with adenovirus expressing Ang1 or control adenovirus prior to LPS exposure. Dephoshorylation of Tie2 associated due to LPS exposure was reduced with Ang1-treatment. In our in vivo thrombosis model, Ang1 inhibited the increased fibrin accumulation at sites of laser injury in endotoxemic mice to baseline levels. Tail snip assays showed that even though elevated Ang1 levels normalized LPS-induced augmentation of thrombus formation, increased Ang1 did not affect bleeding times. These data indicate that Ang1 stimulation of Tie2 signaling can regulate pathologic clot formation in the setting of inflammation without bleeding risk. Together, our studies show that endothelial dysfunction drives a pre-DIC state in endotoxemia. Targeting these early endothelial responses might represent an novel approach for reducing thrombosis in sepsis without enhancing bleeding risks. Future studies will evaluate the mechanism involved. DisclosuresNo relevant conflicts of interest to declare.

A Chemical APC Mimetic Protects Endothelium from Thromboinflammatory Injury

Stimulation of protease-activated receptor 1 (PAR1) on endothelium by activated protein C (APC) is protective in animal models of inflammation and APC has been used clinically in sepsis and wound healing. Clinical use of APC in sepsis, however, was terminated as it was compromised by APC’s anticoagulant activity, which is associated with bleeding and limits its dosing in patients. We used a small molecule approach to circumvent this problem. With support from the Molecular Libraries Program, we screened 302,457 compounds to identify small molecules that modulated PAR1-mediated platelet activation. One class of PAR1-targeted compounds, which we termed parmodulins, was found to act at the cytosolic face of PAR1, at the G-protein binding sites. When evaluated in endothelial cell cultures,parmodulin 1 (PM1) and parmodulin 2 (PM2) inhibited apoptosis induced by thrombin, TNF-α, or staurosporine, in a manner similar to APC. PAR1 knockdown using siRNA abolished these protective effects demonstrating that parmodulins elicit a cytoprotective pathway by acting through PAR1. To assess the mechanism of action of parmodulin cytoprotection, we first evaluated proximal signaling mechanisms. Parmodulins stimulated phosphorylation of PI3-kinase and Akt in endothelium. Inhibition of Gβγ blocked parmodulin-induced phosphorylation of Akt, indicating that parmodulins act at the cytosolic face of PAR1 by releasing Gβγ. Transcriptional profiling of over 30,000 genes and specific evaluation of NF-kB transcriptional activation showed that exposure to PM2 blocked TNF-α-induced transcriptional activation. In addition to interfering with inflammatory signaling, parmodulins also stimulated the upregulation of cytoprotective proteins such as stanniocalcin-1. Since our premise was that parmodulins could achieve cytoprotective effects without anticoagulant effects, we compared dose curves of APC versus PM2 in both apoptosis assays and standard clotting assays. APC prolonged the aPTT at concentrations lower than those required to achieve cytoprotection of the endothelium. The low APC concentration used in our study was similar to plasma concentrations measured in clinical studies. Hence, these data were consistent with the fact that clinical bleeding was observed at doses of APC used for sepsis. In contrast, despite inhibiting apoptosis as effectively as APC, PM2 had no effect on plasma aPTT at any concentration. Nonetheless, PM2 was able to inhibit LPS- and TNF-α-induced thrombin generation and FXa activation on endothelium owing to its cytoprotective effect. PM2 also prevented TNF-α-induced accumulation of platelets on endothelium in bioengineered microvessels. These data demonstrate that PM2 can reduce inflammation-induced endothelial pro-thrombotic phenotype even without directly inhibiting coagulation factors. To assess the endothelial protective effects of PM2 in vivo we evaluated leukocyte rolling in cremaster venules of mice. Infusion of PM2 significantly reduced surgery-induced leukocyte rolling flux compared to vehicle-treated mice. As selectins are critically involved in leukocyte rolling we monitored soluble E-selectin levels in LPS-induced inflammation. Treatment of mice with PM2 significantly reduced the LPS-induced release of soluble E-selectin. Previously, we demonstrated that PM2 blocks platelet accumulation in a mouse laser injury model of thrombus formation. We here show that infusion of PM2 also significantly reduces fibrin accumulation to 25% of control (p<0.001) at the site of laser injury. Together our data show that PM2 exerts endothelial-mediated anti-inflammatory, anti-coagulant and anti-thrombotic effects in vitro and in vivo. These results demonstrate that modulating PAR1 at the cytosolic face could represent an alternative approach to APC in the treatment of thromboinflammatory disorders like sepsis. DisclosuresNo relevant conflicts of interest to declare.

Zymogen-like FXa Is a Potent Bypassing Agent for Reversal of Direct FXa Inhibitors in Vivo

The pharmacokinetic challenges of warfarin therapy have led to the development of new oral anticoagulants (NOACs) that directly inhibit coagulation factor Xa (FXa). While these new drugs (including rivaroxaban and apixaban) have many benefits over warfarin, no approved strategy exists to reverse their anticoagulant effects in the event of life-threatening bleeding or emergent need for surgery. The most promising reversal strategy in clinical development is a catalytically inactive form of FXa (Gla-domainless FXaS195A, GD-FXaS195A) that binds the NOAC with high affinity to relieve inhibition of endogenous FXa. In principle, removing the inhibitor through molecular engagement is attractive, especially when administered before bleeding begins (e.g. pre-surgery), and there is in vivo evidence to support the efficacy of GD-FXaS195A as a pre-injury antidote for direct FXa inhibitors. However, since GD-FXaS195A is a scavenger of the inhibitor and not a pro-hemostatic agent, GD-FXaS195A may not be effective if given after a bleeding episode has begun. Moreover, its mechanism of action necessitates a 1:1 ratio of GD-FXaS195A to neutralize the inhibitor. Thus, high doses (hundreds of milligrams of protein) will likely be required in humans.We hypothesized that a pro-hemostatic bypassing agent might be able to reverse the effects of direct FXa inhibitors no matter when it is administered, and with high potency. To evaluate this, we used a variant of FXa (FXaI16L) that is more zymogen-like than wild-type (wt)-FXa. This “zymogen-like” variant has lower catalytic activity in in vitro assays compared to wt-FXa due to impaired active site maturation. It is also resistant to active site inhibitors including plasma protease inhibitors, resulting in an extension of its plasma half-life (>30 minutes vs ~1 minute for wt-FXa). Importantly, its activity is rescued upon binding to its cofactor FVa in vivo, and we have previously shown that FXaI16L bypasses the intrinsic pathway defect in hemophilic mice. Here we evaluated whether FXaI16L might also be able to bypass direct FXa inhibitors using in vitro thrombin generation assays (TGAs) and two in vivo injury models in mice, and directly compared FXaI16L to GD-FXaS195A. In TGA experiments, 500 nM rivaroxaban decreased peak thrombin generation to 23% of that observed in normal human plasma (NHP). This decreased thrombin generation could be reversed by the addition of 3 nM human (h)FXaI16L which normalized peak thrombin generation. In comparative studies, hFXaI16L was 300-fold more potent than GD-FXaS195A in TGA assays.These data highlight that differences in mechanism of action (pro-hemostatic vs. scavenger) will have a huge impact on amounts of protein needed to revive thrombin generation. This was further reflected in in vivo studies. In invivo experiments with wild-type mice using 7.5% FeCl3 to induce carotid artery thrombosis, 1 mg/kg rivaroxaban prevented formation of occlusive thrombi. 30 minutes after the initial FeCl3 injury to rivaroxaban-treated mice, infusion of 0.25 mg/kg murine (m)FXaI16L normalized the phenotype, causing rapid occlusion at the injury site within 5 minutes. In contrast, we did not observe carotid artery occlusion with administration of up to 25 mg/kg GD-FXaS195A 30 minutes after the injury to rivaroxaban-treated mice (Fig. 1). When both rivaroxaban and the reversal agent were given prior to the injury, both mFXaI16L (0.5 mg/kg) and GD-FXaS195A (25 mg/kg) were effective at reversing the inhibition. Furthermore, using intravital microscopy to examine thrombus formation at the site of laser injury to mouse cremasteric arterioles, we found that 1 mg/kg rivaroxaban completely abrogated fibrin deposition and substantially reduced platelet accumulation. Treatment with1 mg/kg mFXaI16L substantially increased platelet and fibrin deposition at the site of laser injury. [Display omitted] Taken together, these data provide strong support for the in vivo efficacy and potency of FXaI16L as a potential pro-hemostatic bypass agent to reverse direct FXa inhibitors. The data also illustrate that antidotes like GD-FXaS195A may not be as effective as pro-hemostatic agents like FXaI16L following an injury, and we predict that effective reversal of NOACs will require a nuanced approach that uses an antidote or a bypassing agent depending on the clinical scenario. DisclosuresJasuja:Pfizer: Employment. Patel-Hett:Pfizer: Employment. Fruebis:Pfizer: Employment. Pittman:Pfizer: Employment. Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding.

Inducible Gata1 Suppression As a Novel Strategy to Expand Physiologic Megakaryocyte Production from Embryonic Stem Cells

Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent potential sources of megakaryocytes and platelets for transfusion therapy. However, most current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny, including platelet-releasing megakaryocytes. Mutations in the mouse and human genes encoding transcription factor GATA1 cause accumulation of proliferating, developmentally arrested megakaryocytes. Previously, we reported that in vitro differentiation of Gata1-null murine ES cells generated self-renewing hematopoietic progenitors termed G1ME cells that differentiated into erythroblasts and megakaryocytes upon restoration of Gata1 cDNA by retroviral transfer. However, terminal maturation of Gata1-rescued megakaryocytes was aberrant with immature morphology and no proplatelet formation, presumably due to non-physiological expression of GATA1. We now engineered wild type (WT) murine ES cells that express doxycycline (dox)-regulated Gata1 short hairpin (sh) RNAs to develop a strategy for Gata1-blockade that upon its release, restores physiologic GATA1 expression during megakaryopoiesis. In vitro hematopoietic differentiation of control scramble shRNA-expressing ES cells with dox and thrombopoietin (TPO) produced megakaryocytes that underwent senescence after 7 days. Under similar differentiation conditions, Gata1 shRNA-expressing ES cells produced immature hematopoietic progenitors, termed G1ME2 cells, which replicated continuously for more than 40 days, resulting in ~1013-fold expansion (N=4 separate experiments). Upon dox withdrawal with multi-lineage cytokines present (EPO, TPO, SCF, GMCSF and IL3), endogenous GATA1 expression was restored to G1ME2 cells followed by differentiation into erythroblasts and megakaryocytes, but no myeloid cells. In clonal methylcellulose assays, dox-deprived G1ME2 cells produced a mixture of erythroid, megakaryocytic and erythro-megakaryocytic colonies. In liquid culture with TPO alone, dox-deprived G1ME2 cells formed mature megakaryocytes in 5-6 days, as determined by morphology, ultrastructure, acetylcholinesterase staining, upregulated megakaryocytic gene expression (Vwf, Pf4, Gp1ba, Selp, Ppbp), CD42b surface expression, increased DNA ploidy and proplatelet production. Compared to G1ME cells rescued with Gata1 cDNA retrovirus, dox-deprived G1ME2 cells exhibited more robust megakaryocytic maturation, similar to that of megakaryocytes produced from cultured fetal liver. Importantly, G1ME2 cell-derived megakaryocytes generated proplatelets in vitro and functional platelets in vivo (~40 platelets/megakaryocyte with a circulating half life of 5-6 hours). These platelets were actively incorporated into growing arteriolar thrombi at sites of laser injury and subsequently expressed the platelet activation marker p-selectin (N=3-4 separate experiments). Our findings indicate that precise timing and magnitude of a transcription factor is required for proper terminal hematopoiesis. We illustrate this principle using a novel, readily reproducible strategy to expand ES cell-derived megakaryocyte-erythroid progenitors and direct their differentiation into megakaryocytes and then into functional platelets in clinically relevant numbers. DisclosuresNo relevant conflicts of interest to declare.

VAMP-7 Interacts With The Actin Cytoskeleton To Control Platelet Spreading and Mediate α-Granule Exocytosis During Thrombus Formation

Platelet granule secretion is important not only for hemostasis and thrombosis, but also for a variety of physiological processes including inflammation, angiogenesis and malignancy. Vesicle Associated Membrane Proteins (VAMPs) are a group of v-SNARE proteins resident on the platelet granule surface that participate in granule secretion. Platelets contain several VAMP isoforms including VAMP-2, VAMP-3, VAMP-7, and VAMP-8. VAMP-7 is unique in that it contains an N-terminal profilin-like longin domain. Previous work by our group demonstrated spatial segregation of granules expressing different VAMPs during platelet spreading. Granules expressing VAMP-3 and VAMP-8 localized to the granulomere of spreading platelets, while those expressing VAMP-7 moved towards the periphery. Based on this observation, we proposed that VAMP-7+ granules move to the periphery of the spreading platelet to add membrane to growing actin structures. To assess this hypothesis, platelets from VAMP-7 null mice were used to analyze the role of VAMP-7 in platelet spreading, aggregation and secretion. VAMP-7 null platelets were normal in size, shape, and number. When compared to wild-type platelets, VAMP-7 null platelets did not show any defects in aggregation upon exposure to increasing doses of the PAR4 agonist peptide, AYPGKF, or collagen. In contrast, the surface area of VAMP-7 null platelets following 15 min of spreading on poly-L-lysine was only 51% that of wild-type of platelets (P < 0.05). To assess mechanisms of the movement of VAMP-7 to the platelet periphery, the association of VAMP-7 to the Triton X-100-insoluble platelet cytoskeleton was evaluated and results showed that VAMP-7 associated with the actin cytoskeleton. Moreover, VAMP-7 null platelets showed impaired P-selectin surface expression and PF4 secretion at low concentrations of AYPGKF. TIMP-2 and VEGF localize to VAMP-7 expressing granules in the periphery of spread platelets. We therefore evaluated the secretion of TIMP-2 and VEGF from VAMP-7 null platelets. Secretion of TIMP-2 and VEGF was reduced even at saturating doses of agonist (300 mM AYPGKF). To examine the role of VAMP-7 in a-granule exocytosis during platelet activation in vivo, PF4 release was monitored following laser-induced injury of cremaster arterioles. Platelet accumulation at sites of laser injury was identical in wild-type and VAMP-7 null mice. In wild-type mice, PF4 was secreted by activated platelets and bound back to activated endothelium and platelets producing a localized concentration of PF4 that accumulated over 15 min following injury. PF4 release from platelets lacking VAMP-7 was decreased to 47% of that of control. These results demonstrate that VAMP-7 interacts with the actin cytoskeleton and functions selectively in a-granule exocytosis. VAMP-7 associates with the actin cytoskeleton and functions during platelet spreading, adding further support to the premise that membrane fusion occurring during granule secretion is an essential component of normal platelet spreading. This VAMP-7 mediated, actin-dependent mechanism of secretion is not important for platelet thrombus formation, but rather functions in the release of particular granular contents, such as PF4, at sites of vascular injury. Disclosures:No relevant conflicts of interest to declare.

Laser-targeted ablation of the zebrafish embryonic ventricle: A novel model of cardiac injury and repair

BackgroundWhile the adult zebrafish (Danio rerio) heart demonstrates a remarkable capacity for self-renewal following apical resection little is known about the response to injury in the embryonic heart.MethodsInjury to the beating zebrafish embryo heart was induced by laser using a transgenic zebrafish expressing cardiomyocyte specific green fluorescent protein. Changes in ejection fraction (EF), heart rate (HR), and caudal vein blood flow (CVBF) assessed by video capture techniques were assessed at 2, 24 and 48 h post-laser. Change in total and mitotic ventricular cardiomyocyte number following laser injury was also assessed by counting respectively DAPI (VCt) and Phospho-histone H3 (VCm) positive nuclei in isolated hearts using confocal microscopy.ResultsLaser injury to the ventricle resulted in bradycardia and mild bleeding into the pericardium. At 2 h post-laser injury, there was a significant reduction in cardiac performance in lasered-hearts compared with controls (HR 117 ± 11 vs 167 ± 9 bpm, p ≤ 0.001; EF 14.1 ± 1.8 vs 20.1 ± 1.3%, p ≤ 0.001; CVBF 103 ± 15 vs 316 ± 13μms− 1, p ≤ 0.001, respectively). Isolated hearts showed a significant reduction in VCt at 2 h post-laser compared to controls (195 ± 15 vs 238 ± 15, p ≤ 0.05). Histology showed necrosis and apoptosis (TUNEL assay) at the site of laser injury. At 24 h post-laser cardiac performance and VCt had recovered fully to control levels. Pretreatment with the cell-cycle inhibitor, aphidicolin, significantly inhibited functional recovery of the ventricle accompanied by a significant inhibition of cardiomyocyte proliferation.ConclusionsLaser-targeted injury of the zebrafish embryonic heart is a novel and reproducible model of cardiac injury and repair suitable for pharmacological and molecular studies.

“Rolling Recruitment” of Endothelial Cell (EC) Activation in the Prothrombotic Nature of Heparin-Induced Thrombocytopenia (HIT)

Abstract 536HIT is an iatrogenic, prothrombotic, immune-mediated disorder associated with antibodies directed against platelet factor 4 (PF4) and heparin complexes. Once these pathogenic anti-PF4/heparin antibodies develop, they recognize and bind to antigenic complexes formed on cell surfaces composed of PF4 released from platelets bound to cell membrane glycosaminoglycans (GAGs). Unlike platelets, which have only chondroitin sulfate on their surface, endothelial cells express mainly heparan sulfate resulting in a very high capacity to form PF4/GAG complexes and become targets for HIT antibodies. In fact, activation of endothelial cells by HIT antibodies was recognized almost 25 years ago (Cines et. al., 1987). We now show direct interaction of the HIT-like monoclonal antibody KKO with perithrombus endothelial cells, which propagates thrombosis following laser induced cremaster vessel injury in a passive immunization murine model of HIT. PF4 released from activated platelets at the site of laser injury binds rapidly and predominantly to the adjacent endothelium and is recognized by KKO in both transgenic mice expressing only human PF4 (hPF4+) and mice expressing hPF4 and human FcgRIIA on platelets (hPF4+/FcgRIIA+). These studies show that HIT-like antigenic complexes are present within thrombi prior to antibody exposure, predominantly localized to endothelial cells, which is consistent with their surface GAGs having higher affinity for PF4 than platelets. These endothelial cell surface PF4/GAG complexes, and with it KKO binding, are dissociated by high concentrations of intravenous heparin. Infusion of KKO in hPF4+/FcgRIIA+ mice but not in control mice, reinitiates growth of previously stable thrombi at sites of laser injury, leading to a significant increase in vascular occlusion (24/33 injuries, 73%, compared to 0/30 in control mice; P<0.0001). Clot extension is followed by dissemination of endothelial activation, demarcated by binding of annexin V and Factor Xa, and extension of the endothelial surface that binds KKO. Based on these studies, we propose a model of feed-forward rolling recruitment to explain how the endothelial lining contributes to the prothrombotic state in HIT: PF4 released from activated platelets at a site of injury or disease binds to the surface of perithrombus endothelial cells, which then binds HIT antibodies followed by endothelial cell activation. These activated endothelial cells then bind and activate additional platelets, leading to the next round of PF4 release, more extensive endothelial cell activation and platelet recruitment into thrombi. Disclosures:No relevant conflicts of interest to declare.

Imaging of Coagulation Reactions During Thrombus Formation In Vivo with Novel Fluorescent Protein Derivatives

AbstractAbstract 817Significant gaps remain in the understanding of how blood cells and the vasculature differentially support coagulation enzyme complex function leading to regulated thrombus formation in vivo. These gaps partly result from the lack of analytic tools with appropriate sensitivity for incisive conclusions. Here we have combined state of the art confocal fluorescent microscopy, established mouse models, and unique recombinant clotting factors as direct imaging probes to advance our understanding of this process. Hemophilic mice typically do not form stable thrombi following microscopic laser injury in the cremaster model. However, thrombus formation in either hemophilia A (HA) or hemophilia B (HB) mice can be restored by infusion of factor Va. Such rescue implies the assembly of prothrombinase and the formation of sufficient thrombin to permit thrombus formation. We employed this injury model using HB mice as a platform to assess the localization of site-specific fluorescent derivatives of Va and Xa in the growing thrombus. We have prepared a FV variant (Va-810SYA) that is constitutively Va-like with three free cysteines mutated while leaving a single free cysteine at position 539. This site was labeled with Alexa488-maleimide without affecting cofactor function. We also employed Xa bearing Alexa488 or Alexa647 tethered to the active site with a peptidyl chloromethyl ketone. The resulting Alexa488-EGR-Xa or Alexa647-EGR-Xa derivatives are inactive but retain the ability to assemble into prothrombinase. Co-infusion of Alexa555-labeled anti-CD41 antibody to visualize platelets and Alexa488-Va-810SYA (15 μg; n = 4 mice; 20 injuries) produced a robust signal indicating the accumulation of Va-810SYA and platelets at the site of microscopic laser injury. Similarly, infusion of Va-810SYA (15 μg) with Alexa488-EGR-Xa (2 μg) into HB mice (n = 3; 19 injuries) revealed a strong signal denoting the ability to directly image Xa at the site of vascular damage. Because thrombus formation in these animals requires the infusion of Va and the assembly of prothrombinase, at least part of the Va and Xa visualized in the vicinity of the thrombus must be functional rather than adventitiously bound. Similar results were obtained in wild type mice (n = 3; 21 injuries) using Alexa488-Va-810SYA or Alexa647-EGR-Xa indicating that these findings are not peculiar to HB animals. More detailed measurements were pursued by three-dimensional (3-D) confocal imaging analysis 4 min post-injury with fluorescent derivatives of Va, Xa, platelets or fibrin. As expected, platelets and fibrin were limited to the site of microscopic injury. Factors Va and Xa were also found with platelets and fibrin; however these proteins surprisingly were also distributed away from the thrombus on vascular surfaces. These results suggest that surfaces other than platelets, such as the endothelium, can accommodate the assembly and function of Va and Xa. To test this, HB mice (n=3; 15 injuries) were infused with Va or Xa along with integrilin to inhibit platelet function. Consistent with prior results in PAR4−/− mice, the reduction of platelets at the site of microscopic injury with integrilin had no obvious effect on fibrin deposition. Despite this reduction in platelets, the accumulation of Va or Xa bound in the vicinity of the injury site was not significantly altered providing an explanation for the unchanged levels of fibrin. These unexpected results question the predominant role ascribed to activated platelets in supporting the assembly and function of procoagulant enzyme complexes. Overall these studies indicate that site-specific fluorescent derivatives of Va and Xa can be visualized with appropriate sensitivity and represent powerful tools to establish their spatial distribution at the site of laser injury in the mouse cremaster model. Our approach provides new biological insights into the integrated behavior of the system of enzymic reactions and their modulation by blood cells which lead to thrombus formation in vivo. Disclosures:Camire:Pfizer: Patents & Royalties, Research Funding.

The drusenlike phenotype in aging Ccl2-knockout mice is caused by an accelerated accumulation of swollen autofluorescent subretinal macrophages.

Drusen, which are defined clinically as yellowish white spots in the outer retina, are cardinal features of age-related macular degeneration (AMD). Ccl2-knockout (Ccl2(-/-)) mice have been reported to develop drusen and phenotypic features similar to AMD, including an increased susceptibility to choroidal neovascularization (CNV). This study was conducted to investigate the nature of the drusenlike lesions in vivo and further evaluate the Ccl2(-/-) mouse as a model of AMD. The eyes of 2- to 25-month-old Ccl2(-/-) and C57Bl/6 mice were examined in vivo by autofluorescence scanning laser ophthalmoscopy (AF-SLO) and electroretinography, and the extent of laser-induced CNV was measured by fluorescein fundus angiography. The retinal morphology was also assessed by immunohistochemistry and quantitative histologic and ultrastructural morphometry. The drusenlike lesions of Ccl2(-/-) mice comprised accelerated accumulation of swollen CD68(+), F4/80(+) macrophages in the subretinal space that were apparent as autofluorescent foci on AF-SLO. These macrophages contained pigment granules and phagosomes with outer segment and lipofuscin inclusions that may account for their autofluorescence. Only age-related retinal pigment epithelium (RPE) damage, photoreceptor loss, and sub-RPE deposits were observed but, despite the accelerated accumulation of macrophages, we identified no spontaneous development of CNV in the senescent mice and found a reduced susceptibility to laser-induced CNV in the Ccl2(-/-) mice. These findings suggest that the lack of Ccl2 leads to a monocyte/macrophage-trafficking defect during aging and to an impaired recruitment of these cells to sites of laser injury. Other, previously described features of Ccl2(-/-) mice that are similar to AMD may be the result of aging alone.

Macrophage depletion inhibits experimental choroidal neovascularization.

To investigate the role of macrophages in the development of laser-induced choroidal neovascularization (CNV) by selective depletion with liposomal clodronate (Cl(2)MDP-LIP). Laser photocoagulation was used to induce CNV in wild-type C57BL/6J mice. Animals were treated with intravenous (IV) and/or subconjunctival (SC) Cl(2)MDP-LIP or PBS-LIP at the following time points: 2 days before, immediately after, 2 days before and immediately after, or 2 days after laser injury. CNV responses were compared on the basis of en masse volumetric measurements and fluorescein angiography after laser photocoagulation. Macrophages were identified by immunostaining for F4/80, and vascular endothelial growth factor (VEGF) expression was quantified by ELISA. Macrophages invaded the site of laser injury within 1 day of photocoagulation and peaked at 3 days. IV Cl(2)MDP-LIP significantly decreased the volume of CNV and angiographic leakage when administered 2 days before and/or immediately after laser injury, but not when administered 2 days after injury. SC Cl(2)MDP-LIP significantly decreased lesion volume when coadministered with IV PBS-LIP but not IV Cl(2)MDP-LIP. IV Cl(2)MDP-LIP was significantly more beneficial when administered 2 days before laser injury than immediately after, but combining SC Cl(2)MDP-LIP with IV treatment eliminated this difference. Reduction in CNV volume correlated with VEGF protein levels and number of infiltrating macrophages. Generalized macrophage depletion reduced the size and leakage of laser-induced CNV and was associated with decreased macrophage infiltration and VEGF protein. These findings define the role of the macrophage as a critical component in initiating the laser-induced CNV response.

Targeted disruption of the CD18 or ICAM-1 gene inhibits choroidal neovascularization.

To investigate the role of the leukocyte adhesion molecules CD18 and intercellular adhesion molecule (ICAM)-1 in the development of choroidal neovascularization (CNV). Laser photocoagulation was used to induce CNV in wild-type C57BL/6J mice and species-specific counterparts with targeted homozygous disruption of the CD18 or ICAM-1 gene. Expression of CD18 and ICAM-1 after laser injury was assessed by immunostaining. CNV responses were compared on the basis of en masse volumetric measurements obtained by confocal microscopy 2 weeks after laser injury and by determination of fluorescein angiographic leakage at 1, 2, and 4 weeks after laser injury. The site of laser injury showed upregulation of ICAM-1 and invasion by CD18-positive leukocytes within 1 day of laser injury. Significantly fewer lesions exhibited fluorescein leakage defined to be pathologically significant in CD18-deficient mice at weeks 1, 2, and 4 weeks and in ICAM-1-deficient mice at 1 and 4 weeks, compared with the control. There were a significantly greater number of lesions without fluorescein leakage in CD18-deficient mice than in the other two groups at all time points. The volume of CNV in CD18- and ICAM-1-deficient mice was significantly less than in wild type. These data suggest a nonredundant role for leukocyte adhesion to vascular endothelium in the development of laser-induced choroidal neovascularization.

The contact Nd-YAG laser for oral and oropharyngeal malignant tumors.

Nine oral and oropharyngeal squamous cell carcinomas were removed with a contact Nd-YAG laser. Clinical and morphological studies on wound healing and effects of the Nd-YAG laser were performed. The wounds were not sutured and patients did not complain of any severe symptoms. The wound healing was prolonged to 3-6 weeks. Cells and glands degenerated at a depth of 2-3 mm by the direct effects of the laser. The epithelium exhibited acantholysis 5 mm from the site of laser injury. Scanning electron microscopy showed no coagulation or other coated material in the wound created by a surgical knife, while the wound surface by laser consisted of carbonization, well-coated and non-coated areas. These findings varied with the laser power. The coagulation and thermal effects of the Nd-YAG laser resulted in autolysis of tumor cells at least within 5 mm of the cutting surface. The contact Nd-YAG laser was useful for the removal of the malignant tumor in the oral cavity and oropharynx where it is difficult to maintain a generous safety margin.

Measurement of growth rate of laser-induced intravascular platelet aggregation and the influence of blood flow velocity

The growth rate of platelet microthrombi induced by laser injury in ear chamber arterioles on conscious rabbits was measured. A growth rate constant was obtained for each microthrombus by plotting its volume change on a semilogarithmic plot. In most instances each platelet microthrombus formed at any one injury site conformed to a similar growth rate pattern. There was a direct positive correlation between the mean growth rate constant and the number of emboli counted for each laser injury. Blood flow velocity had a variable effect on the number of emboli from sites of laser injury. At velocities above 2.5 mm sec −1 the number of emboli remained relatively constant; between 1.0 and 2.5 cm sec −1 the number of emboli tended to increase and there was a greater variation at this level; below 1.0 mm sec −1 the number of emboli decreased. The number of platelets participating in formation of a microthrombus was expressed as a percentage of the number of available platelets to give a measure of platelet aggregability in vivo. Platelet aggregability varied with alterations in blood flow velocity. At blood flow velocities between 1.0 and 2.5 mm sec −1 the percentage of platelets participating varied between 20 and 50%, but at velocities above 3.0 mm sec −1 the percentage decreased to less than 20%. The results suggested that the number of emboli was an indirect measurement of the growth rate of microthrombi forming at sites of laser injury. Further, both the number of emboli and the percentage platelet aggregability in vivo were not significantly affected by changes in blood flow velocity over the range normally found in rabbit ear chamber arterioles.

Measurement of the Platelet Response to Laser- induced Microvascular Injury

SummaryA study has been made of the biochemical factors underlying the platelet response to laser-induced microvascular injury. A platelet aggregating substance is produced at sites of laser-induced injury which markedly stimulates platelet activity at a site of injury inflicted a short distance downstream. Distal sites of injury are not similarly influenced if the distance between the injuries is increased or if the proximal site no longer shows platelet-stimulating activity. The stimulating effect of an adjacent proximal injury on platelet activity at a distal site is inhibited by local intra-arterial infusion of adenosine. Measurements of arterial blood pressure and microvascular blood flow velocity during adenosine infusion showed that its inhibitory effect on platelet activity is largely independent of its vasodilator properties. The effect of infusion of different adenosine phosphates (AMP, ADP, ATP) was also studied. Very small amounts of ADP markedly stimulated platelet activity and the emboli formed were similar to those normally produced at sites of laser injury. At high concentration AMP inhibited while ATP stimulated platelet activity in vivo. The results emphasise the fundamental role of ADP as a mediator of the platelet response at sites of laser- induced microvascular injury.

Platelet response to laser-induced microvascular injury in the rabbit mesentery and the rabbit ear chamber. A statistical comparison

Laser-induced microvascular injury combined with rabbit ear chamber techniques offers a satisfactory means of measuring platelet reactivity over extended periods in conscious animals. A factor limiting the general applicability of the technique may be difficulty in producing suitable ear chamber preparations. Since the rabbit mesentery can be quickly and easily prepared for microcirculatory studies we have evaluated this preparation as an alternative to the ear chamber for quantitative studies of platelet aggregation in vivo. Platelet activity at sites of laser injury in mesenteric microvessels was about half that observed in the ear chamber but the standard deviation of the observation was not similarly reduced. Despite this, it was possible to demonstrate significant suppression of platelet activity in both ear chamber and mesenteric preparations after dextran administration. In contrast, a significant increase in platelet reactivity could not be detected in animals previously subjected to a standardised laparotomy at sites of injury in mesenteric vessels while such an increase was consistently observed in ear chamber preparations. Thus, while the mesenteric model can be used in studies of inhibition of platelet reactivity at sites of laser injury it does not appear suitable for examination of situations of enhanced platelet reactivity in vivo.