Sites Of Laser-induced Injury Research Articles

DNAse-dependent, NET-independent pathway of thrombus formation in vivo

The contribution of NETs (neutrophil extracellular traps) to thrombus formation has been intensively documented in both arterial and venous thrombosis in mice. We previously demonstrated that adenosine triphosphate (ATP)-activated neutrophils play a key role in initiating the tissue factor-dependent activation of the coagulation cascade, leading to thrombus formation following laser-induced injury. Here, we investigated the contribution of NETs to thrombus formation in a laser-induced injury model. In vivo, treatment of mice with DNase-I significantly inhibited the accumulation of polymorphonuclear neutrophils at the site of injury, neutrophil elastase secretion, and platelet thrombus formation within seconds following injury. Surprisingly, electron microscopy of the thrombus revealed that neutrophils present at the site of laser-induced injury did not form NETs. In vitro, ATP, the main neutrophil agonist present at the site of laser-induced injury, induced the overexpression of PAD4 and CitH3 but not NETosis. However, compared to no treatment, the addition of DNase-I was sufficient to cleave ATP and adenosine diphosphate (ADP) in adenosine. Human and mouse platelet aggregation by ADP and neutrophil activation by ATP were also significantly reduced in the presence of DNase-I. We conclude that following laser-induced injury, neutrophils but not NETs are involved in thrombus formation. Treatment with DNase-I induces the hydrolysis of ATP and ADP, leading to the generation of adenosine and the inhibition of thrombus formation in vivo.

Role of a TRIM72 ADP-ribosylation cycle in myocardial injury and membrane repair.

Mono-ADP-ribosylation of an (arginine) protein catalyzed by ADP-ribosyltransferase 1 (ART1) - i.e., transfer of ADP-ribose from NAD to arginine - is reversed by ADP-ribosylarginine hydrolase 1 (ARH1) cleavage of the ADP-ribose-arginine bond. ARH1-deficient mice developed cardiomyopathy with myocardial fibrosis, decreased myocardial function under dobutamine stress, and increased susceptibility to ischemia/reperfusion injury. The membrane repair protein TRIM72 was identified as a substrate for ART1 and ARH1; ADP-ribosylated TRIM72 levels were greater in ARH1-deficient mice following ischemia/reperfusion injury. To understand better the role of TRIM72 and ADP-ribosylation, we used C2C12 myocytes. ARH1 knockdown in C2C12 myocytes increased ADP-ribosylation of TRIM72 and delayed wound healing in a scratch assay. Mutant TRIM72 (R207K, R260K) that is not ADP-ribosylated interfered with assembly of TRIM72 repair complexes at a site of laser-induced injury. The regulatory enzymes ART1 and ARH1 and their substrate TRIM72 were found in multiple complexes, which were coimmunoprecipitated from mouse heart lysates. In addition, the mono-ADP-ribosylation inhibitors vitamin K1 and novobiocin inhibited oligomerization of TRIM72, the mechanism by which TRIM72 is recruited to the site of injury. We propose that a mono-ADP-ribosylation cycle involving recruitment of TRIM72 and other regulatory factors to sites of membrane damage is critical for membrane repair and wound healing following myocardial injury.

Effects Of Biased PAR1 Ligands On Platelets and Endothelial Cells

Protease-activated receptor 1 (PAR1) is a widely expressed G protein coupled receptor (GPCR) that monitors the extracellular protease environment with high sensitivity. Activation of PAR1 by thrombin on platelets links the coagulation cascade to platelet activation and contributes to thrombus formation. Activation of endothelial cell PAR1 by thrombin results in loss of barrier function and apoptosis. In contrast, activation of PAR1 by activated protein C (APC) stimulates a genetic program that protects endothelial cells from barrier dysfunction and apoptosis induced by chemokines or cytotoxins. To identify new strategies for modulating PAR1 activity, we screened a 300,000 compound library in a platelet-based assay to identify compounds that inhibit PAR1-mediated activation. Four structurally unrelated compounds, termed parmodulins, inhibited platelet activation mediated through PAR1 without inhibiting platelet stimulation through other receptors including PAR4. We compared the activity of parmodulins to three known orthosteric inhibitors (i.e., compounds that act at the ligand binding site) of PAR1, including SCH79797, BMS-200261, and FR-171113. While orthosteric inhibitors blocked both platelet aggregation and platelet shape change induced by the PAR1 agonist SFLLRN, parmodulins inhibited PAR1-mediated aggregation, but spared shape change. PAR1 inhibition by orthosteric inhibitors was irreversible, while that of parmodulins was readily reversible upon washing. Orthosteric inhibitors had no activity against mouse platelets stimulated with the PAR4 agonist AYPGKF. In contrast, parmodulins inhibited activation of mouse platelets stimulated with AYPGKF even though they failed to inhibit activation of human platelets by AYPGKF. Since mouse PAR4 (mPAR4) and human PAR4 (hPAR4) differ in their cytoplasmic tail distal to the DPxxY motif, we evaluated the activity of all compounds in COS7 cells transfected with wild-type hPAR1 or with a hPAR1/hPAR4 chimera in which the cytoplasmic tail of hPAR1 was replaced with that of hPAR4. All compounds inhibited stimulation of [Ca2+]i flux mediated through wild-type hPAR1. However, only orthosteric inhibitors blocked the hPAR1/hPAR4 chimera, indicating that parmodulins act at the cytosolic tail of hPAR1. When tested in human umbilical vein endothelial cells (HUVECs), all orthosteric PAR1 inhibitors elicited apoptosis as detected by YEO-PRO-1 staining by approximately as much as TNF-a. In contrast, parmodulins failed to elicit apoptosis. In fact, incubation with parmodulins protected HUVECs from apoptosis induced by TNF-a, thrombin, or staurosporine. Parmodulin-mediated protection from apoptosis was completely reversed in HUVECs in which PAR1 had been knocked down using siRNA. When tested in vivo, infusion of 5 mg/kg parmodulin 2 resulted in a 72% reduction in platelet accumulation at sites of laser-induced injury in cremaster arterioles (P<0.05). In contrast, parmodulin 2 had no effect on bleeding times in a tail snip assay. Recent clinical trials of orthosteric PAR1 inhibitors as antiplatelet agents have shown that these compounds are associated with increased major bleeding. Our results demonstrate that this class of PAR1 inhibitors induces apoptosis in endothelium. In contrast, PAR1 ligands that target the cytosolic face of the receptor are cytoprotective in endothelium. This new class of biased PAR1 ligands block prothrombotic platelet activation, but simultaneously induce cytoprotective signaling in endothelial cells. The strategy of targeting the cytosolic face of receptors to achieve biased signaling could be broadly applicable to GPCRs. Disclosures:No relevant conflicts of interest to declare.

RNAi-Mediated Inhibition of Activated Protein C- A New Approach for Hemophilia Treatment

Abstract 1204Protein C is a major anticoagulation factor that serves as a key regulator of the clotting cascade. Activated protein C (APC), which is formed by limited proteolysis of the zymogen protein C by the thrombin–thrombomodulin complex, modulates thrombin generation. The anticoagulant effect of APC is due to subsequent inactivation of both activated factors V (FVa) and VIII (FVIIIa). The important role of APC in coagulation is highlighted in FV Leiden (FVL), the most common inherited form of thrombophilia. FVL is caused by a mutation in the FV gene at the initial APC cleavage site, which results in FVL being inactivated at approximately one tenth the rate of normal FVa, leading to high thrombin levels that create a procoagulant state. Several clinical studies have suggested that the severity or onset of bleeding phenotype in hemophilia patients is substantially reduced in association with impaired inactivation of FVa by APC in the presence of the FVL mutation. Transgenic animal studies also show that hemophilic mice, either heterozygous or homozygous for FVL, have improved clotting times with the ability to form clots at sites of laser-induced injury in a microvascular bed injury model, supporting the role of the FVL mutation in enhancing hemostasis. Therapeutic strategies aimed at reducing the level of protein C and activated protein C, thereby increasing levels of FVa and thrombin could thus prove efficacious in hemophilia A and B. Here we investigate the systemic administration of lipid nanoparticle (LNP) formulated siRNA directed against Protein C (encoded by Proc). We have designed potent siRNA sequences against human, cynomologous monkey, dog, mouse and rat Proc mRNA. The lead human/cynomologous candidate has an IC50 of 5pM. The lead mouse/rat candidate has an IC50 of 30 pM and an ED50 of 20 ug/kg. We demonstrate robust and durable inhibition of Proc mRNA in normal mouse liver- a single injection of 0.3 mg/kg resulted in 90% silencing of Proc mRNA and reduction of more than 75% which was sustained for more than two weeks. We also demonstrate significant reduction of circulating protein as measured by Western blot. Further testing using genetic models of hemophilia is being conducted to provide proof-of-concept for developing a Proc siRNA as a novel therapeutic agent in hemophilia management. Disclosures:Toudjarska:Alnylam Pharmaceuticals, Inc.: Employment. Cai:Alnylam Pharmaceuticals, Inc.: Employment. Racie:Alnylam Pharmaceuticals, Inc.: Employment. Hettinger:Alnylam Pharmaceuticals, Inc.: Employment. Milstein:Alnylam Pharmaceuticals, Inc.: Employment. Bettencourt:Alnylam Pharmaceuticals, Inc.: Employment. Sah:Alnylam Pharmaceuticals, Inc.: Employment. Bumcrot:Alnylam Pharmaceuticals, Inc.: Employment. Maraganore:Alnylam pharmaceuticals, Inc.: Employment. Vaishnaw:Alnylam Pharmaceuticals, Inc.: Employment.

Visualizing the Molecular and Cellular Basis of Heparin Induced Thrombocytopenia.

Abstract 228▪▪This icon denotes an abstract that is clinically relevant.Heparin-induced thrombocytopenia (HIT) is an iatrogenic complication of heparin therapy caused by antibodies that recognize the platelet chemokine, Platelet Factor 4 (PF4), complexed to heparin or to cellular glycosaminoglycans (GAG). The origin of the sentinel feature of HIT - high propensity for thrombosis in the setting of mild thrombocytopenia - remains undefined. We have shown previously that monocytes are particularly susceptible to activation by HIT antibody because unlike platelets, whose surface glycosaminoglycans (GAGs) consist almost entirely of chondroitin sulfates (CS), monocytes express heparan and dermatan sulfates that bind to PF4 with greater affinity. Therefore, antigenic complexes form at lower concentrations of PF4 on monocytes than on platelets and are more resistant to removal by exogenous heparin. We now provide 2 lines of visual evidence in support of this model of the prothrombotic state in HIT: 1) We demonstrate by scanning electron microscopy that spheroidal particles indicative of PF4/GAG complexes are present on the surface of human monocytes upon addition of PF4. 2) Thrombus formation has been visualized in a previously described murine HIT model involving the infusion of the HIT-like monoclonal antibody KKO into mice double-transgenic for human PF4 (hPF4+) and FcγRIIA receptor (FcγRIIA+) on their platelets. After KKO infusion, hPF4+/FcγRIIA+ mice developed vigorous and concurrent accumulation of HIT antigenic complexes and fibrin at sites of laser-induced endothelial injury, not present in similarly treated single transgenic mice. Accumulation of PF4 and fibrin at sites of injury was not increased by a polyclonal anti-hPF4 antibody that does not induce thrombocytopenia in hPF4+/FcγRIIA+ mice. Monocytes and/or monocyte microparticles accumulated at sites of laser-induced injury in KKO-infused hPF4+/FcγRIIA+ mice, but not in KKO-infused single transgenic mice or hPF4+/FcγRIIA+ mice infused with an isotype control, beginning 2.5 min after formation of HIT antigenic complexes and fibrin deposition, accumulating preferentially at the upstream end of developing thrombi. These results provide new insights into the potential role of monocytes in the prothrombotic sequelae of HIT. Antigenic complexes form on monocytes exposed to PF4 and accumulate in HIT antibody-induced thrombi. Monocytes appear to reinforce thrombus formation initiated by injured endothelial cells and activated platelets. Disclosures:Poncz:Diagnostica Stago: Patents & Royalties.

Thrombin-Dependent Platelet Activation Is VWF-Independent during Thrombus Formation In Vivo.

Adhesion to and activation of platelets at an injured vessel wall are critical events in the formation of a thrombus. Calcium mobilization is one marker of platelet activation. Of different agonists capable of activating platelets in vitro, thrombin, collagen and vWF have been described to induce calcium mobilization, leading to the formation of aggregates. Using high speed digital multichannel intravital microscopy, we characterized calcium mobilization during platelet activation and thrombus formation in genetically modified mice. The kinetics of platelet activation and accumulation after laser-induced injury in cremaster muscle arterioles of living mice were analyzed. In wild type mice, platelets adhered and accumulated rapidly at the site of laser-induced injury. Thrombi increased in size, reached a maximum size at 90–120 sec, decreased in size and then stabilized within 3 to 4 min post-injury. In vWF−/− mice, the kinetics of platelet accumulation followed the same pattern as in wild type mice. However, a significant albeit modest reduction in the size of each thrombus was observed in these genetically deficient mice in comparison with wild type mice. By ranking the thrombi by size, we observed that 40% of the thrombi formed in vWF−/− mice were present in the quadrant containing the smallest thrombi versus 18% for the wild type mice. Only 8% of the thrombi formed in vWF−/− mice were distributed in the quadrant containing the largest thrombi versus 32% for the wild type mice. In wild type mice treated with lepirudin, a specific inhibitor of thrombin activity, a small early accumulation of platelets was observed at about 16 sec whereas in untreated wild type mice this early accumulation is often obscured by subsequent thrombin-mediated platelet accumulation and activation. However, at the time of maximal thrombus size in wild-type mice, platelet accumulation in wild type mice was more than ten-fold greater than in wild type mice treated with lepirudin. The kinetics of platelet accumulation were similar in FcRγ−/− mice lacking GPVI, GPVI-depleted mice and wild type mice. Furthermore, depletion of GPVI from the platelet surface of vWF−/− mice or platelets of wild type mice treated with lepirudin did not alter the kinetics of platelet accumulation in those mice. By monitoring calcium mobilization per platelet engaged in the growing thrombus, we observed that elevated calcium levels in each platelet were similar in FcRγ−/−, GPVI depleted, vWF−/− and wild type mice. However in wild type mice treated with lepirudin, platelet calcium mobilization was almost completely inhibited in comparison with those observed in wild type mice. Our results indicate that thrombin is the major agonist leading to platelet activation after laser-induced injury. Collagen, as previously reported (Dubois, Blood. 2006; 107:3902) does not play a role in platelet thrombus formation after laser injury and, based on data reported here, does not play a role in platelet activation in this model. vWF is important for the growth of the platelet thrombus but is not required for initial platelet accumulation or platelet activation in vivo in this thrombosis model. The platelet agonist or ligand responsible for initial early platelet accumulation after laser-induced injury is unknown, and does not require GPVI, thrombin or vWF.

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.