Laser-induced Thrombus Formation Research Articles

Cysteine Sulfenylation of Protein Disulfide Isomerase Links Oxidative Stress to Thrombosis

Oxidative stress increases the risk of clinically significant thrombosis in the setting of inflammation, malignancy, infection, and dyslipidemia. Oxidants generated during oxidative stress are prothrombotic; however, the mechanisms by which oxidants induce thrombus formation is poorly understood. Protein disulfide isomerase (PDI) is a thiol isomerase that serves an important role in thrombus formation. It has a domain structure of a-b-b'-a', in which the aand a' domains are catalytic and the b and b' are substrate binding. A flexible x linker is between the b' and a'domains. PDI is uniquely sensitive to oxidative cysteine modification within the CGHC catalytic motif of its a and a'domains. Yet whether PDI cysteine modifications mediates thrombosis in an oxidative environment has not previously been studied. We hypothesized that oxidized PDI links oxidative stress to a prothrombotic phenotype through post-translational modification of proteins. We have previously shown that dyslipidemia promotes hydrogen peroxide generation from platelets and oxidizes proteins. Using hydrogen peroxide as a model oxidant, we found that oxidation of recombinant PDI promoted the loss of free thiols and inhibited reductase activity in a dose-dependent manner (IC50= 3.3 ± 0.12 µM). To understand how free thiols were lost, we used a benzothiazine-based nucleophilic probe, termed BTD, to label sulfenic acid. The transient cysteine sulfenic acid (S-OH) was detected by BTD labeling in wildtype PDI following exposure to hydrogen peroxide, but not in a PDI mutant in which all active site cysteines had been mutated to alanine. PDI exposure to oxidized lipoprotein particles (OxLDL) or 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor, also promoted its sulfenylation. Using single cysteine mutants of PDI to identify the sulfenylation site, we found that Cys56 was preferentially sulfenylated as mutation of this cysteine prevented further labeling of the sulfenic acid probe. To assess whether interdomain interactions influenced sulfenylation, we evaluated BTD labeling in isolated fragments of PDI. Sulfenylation was identified in the a domain, consistent with Cys56 labeling, and was significantly enhanced in the ab fragment, implicating an interaction between the a and b domains in sulfenylation. No labeling was observed in an isolated b'xa' fragment. Further analysis of structural elements revealed that a conserved Arg120 within the a domain regulates Cys56 reactivity. Indeed, mutation of Arg120 to Ala prevented sulfenylation by peroxides compared to the wildtype control. Maleimide labeling showed that sulfenylated PDI is an intermediary that resolves into disulfided PDI. To determine whether this disulfided PDI promotes oxidase activity, we used a denatured and reduced RNAse that refolds when disulfides are transferred from oxidized PDI to the RNAse. PDI oxidized by hydrogen peroxide promoted RNAse refolding, whereas reduced PDI did not. A selective inhibitor of sulfenylation, arsenite, prevented RNAse refolding, indicating that hydrogen peroxide-induced oxidation of PDI proceeds through a sulfenic acid intermediate. Since PDI is secreted from cells, we evaluated whether activated cells release sulfenylated PDI using both human vein endothelial cells (HUVECs) and platelets. Sulfenylated PDI was increased in the cultured media of HUVECs stimulated with thrombin and in the releasate of platelets stimulated via PAR1 or glycoprotein VI. OxLDL exposure sensitizes platelets to activation by low doses of agonists. Inhibition of PDI using a neutralizing antibody inhibited augmentation of platelet aggregation by oxLDL, suggesting that oxidation of PDI by oxLDL promotes platelet activation. The ability of oxLDL to enhance the prothrombotic effect of PDI was tested in vivo in a murine model of laser-induced thrombus formation. Mice were infused with either control IgG or IgG directed at PDI, followed by the infusion of oxLDL. OxLDL significantly enhanced thrombus formation in this model. The effect of oxLDL was inhibited by anti-PDI IgG but not control IgG antibody or buffer, implicating PDI as an effector of oxLDL-augmented thrombus formation. In conclusion, our study demonstrates sulfenylation of PDI and shows that PDI sulfenylation retains PDI oxidase activity. Oxidation of PDI by oxidants such as oxLDL occurs in platelets and in endothelium and promotes thrombosis in the setting of oxidative stress.

Transient Thermal Response of Blood Vessels during Laser Irradiation Monitored by Laser Speckle Contrast Imaging

Real-time monitoring of blood flow and thrombosis formation induced by laser irradiation is critical to reveal the thermal-damage mechanism and successfully implement vascular-dermatology laser surgery. Laser speckle contrast imaging (LSCI) is a non-invasive technique to visualize perfusion in various tissues. However, the ability of the LSCI to monitor the transient thermal response of blood vessels, especially thrombus formation during laser irradiation, requires further research. In this paper, an LSCI system was constructed and a 632 nm He-Ne laser was employed to illuminate a Sprague Dawley rat dorsal skin chamber model irradiated by a 1064 nm Nd: YAG therapy laser. The anisotropic diffusion filtering (ADF) technique is implemented after temporal LSCI (tLSCI) processing to improve the SNR and temporal resolution. The speckle flow index is used to characterize the blood-flow velocity to reduce the computational cost. The combination of the tLSCI and ADF increases the temporal resolution by five times and the SNR by 17.2 times and 16.14 times, without and with laser therapy, respectively. The laser-induced thrombus formation and vascular damage during laser surgery can be visualized without any exogenous labels, which provides a powerful tool for thrombus monitoring during laser surgery.

GRK2 Regulates ADP Signaling in Platelets Via P2Y 1 and P2Y 12

AbstractVenous thromboembolism (VTE), heart attack, and stroke are all diseases in which platelets play a role, through inappropriate platelet activation and subsequent thrombus formation. Most platelet agonists activate platelets via G protein-coupled receptors (GPCRs), which are targeted by many antiplatelet drugs. Along with thrombin and TxA 2, ADP has long been recognized for its important role in hemostasis and thrombosis. It activates platelets via GPCRs, P2Y 1 and P2Y 12. However, little is known about the negative feedback mechanisms governing P2Y receptor-mediated platelet activation and thrombus formation.Here, we provide the first evidence that GPCR kinase 2 (GRK2) serves this regulatory role during platelet activation and thrombus formation by using a platelet-specific GRK2 deletion mouse model and a GRK2-specific inhibitor in human platelets. Deletion of GRK2 in mouse platelets causes increased platelet accumulation following laser-induced injury in cremaster muscle arterioles, particularly in the shell region of thrombi. In addition, this deletion increases ADP-induced pulmonary thromboembolism. GRK2 -/- platelets also have increased platelet aggregation in response to ADP, but not to PAR4 receptor agonist, TxA 2, or convulxin. Underlying these changes in GRK2 -/- platelets is an increase in Ca 2+ mobilization, Akt phosphorylation, and Rap1 activation in response to ADP, and an attenuated rise of cAMP levels in response to ADP in the presence of prostaglandin I 2. Furthermore, platelet aggregation can be restored in GRK2 -/- platelets in response to ADP re-stimulation, indicating that GRK2 contributes to ADP receptor desensitization. To further assess the role of GRK2 in the P2Y 12 signaling pathway in vivo, we examine laser-induced thrombus formation in WT and GRK2 -/- mice treated with the P2Y 12 antagonist, cangrelor. Cangrelor treatment eliminates the phenotypic difference in platelet accumulation between WT and GRK2 -/- mice in response to injury. Using a specific GRK2 inhibitor, pharmacologic inhibition of GRK2 activity in human platelets results in an increase in platelet activation in response to ADP. Finally, our biochemical studies show that GRK2 binds to endogenous Gβγ subunits during platelet activation.Taken together, we have demonstrated for the first time that 1) GRK2 plays a negative regulatory role in platelet activation by attenuating ADP-dependent signaling, 2) it does this by limiting P2Y 1 and P2Y 12-mediated signaling, 3) GRK2 interacts with Gβγ and functions as a signaling hub in platelets for fine-tuning GPCR signaling, and 4) although the potential inhibition of GRK2 can be beneficial for treatment of heart diseases, maintaining GRK2 activity in platelets could be beneficial for prevention of thrombotic diseases. DisclosuresNo relevant conflicts of interest to declare.

Protein disulfide isomerase regulation by nitric oxide maintains vascular quiescence and controls thrombus formation

Essentials Nitric oxide synthesis controls protein disulfide isomerase (PDI) function. Nitric oxide (NO) modulation of PDI controls endothelial thrombogenicity. S-nitrosylated PDI inhibits platelet function and thrombosis. Nitric oxide maintains vascular quiescence in part through inhibition of PDI. SUMMARY: Background Protein disulfide isomerase (PDI) plays an essential role in thrombus formation, and PDI inhibition is being evaluated clinically as a novel anticoagulant strategy. However, little is known about the regulation of PDI in the vasculature. Thiols within the catalytic motif of PDI are essential for its role in thrombosis. These same thiols bind nitric oxide (NO), which is a potent regulator of vessel function. To determine whether regulation of PDI represents a mechanism by which NO controls vascular quiescence, we evaluated the effect of NO on PDI function in endothelial cells and platelets, and thrombus formation in vivo. Aim To assess the effect of S-nitrosylation on the regulation of PDI and other thiol isomerases in the vasculature. Methods and results The role of endogenous NO in PDI activity was evaluated by incubating endothelium with an NO scavenger, which resulted in exposure of free thiols, increased thiol isomerase activity, and enhanced thrombin generation on the cell membrane. Conversely, exposure of endothelium to NO+ carriers or elevation of endogenous NO levels by induction of NO synthesis resulted in S-nitrosylation of PDI and decreased surface thiol reductase activity. S-nitrosylation of platelet PDI inhibited its reductase activity, and S-nitrosylated PDI interfered with platelet aggregation, α-granule release, and thrombin generation on platelets. S-nitrosylated PDI also blocked laser-induced thrombus formation when infused into mice. S-nitrosylated ERp5 and ERp57 were found to have similar inhibitory activity. Conclusions These studies identify NO as a critical regulator of vascular PDI, and show that regulation of PDI function is an important mechanism by which NO maintains vascular quiescence.

The Antithrombotic Effects of 12‐LOX Derived Metabolites of DPA, ω‐6

IntroductionPlatelet‐mediated thrombosis is the primary underlying mechanism leading to cardiovascular life‐threatening clinical events. Regulating excessive platelet reactivity is an essential aspect of antithrombotic therapy. A number of anti‐platelet drugs have been developed to target specific signaling pathways or endpoints involved in platelet activation. Despite the effectiveness of current anti‐platelet therapies, uncontrolled thrombosis or bleeding complications still persist, warranting the development of novel anti‐thrombotic strategies. Previously, we had presented that 12‐HETrE, an oxylipin generated by 12‐lipoxygenase (12‐LOX) oxidation of ω‐6 polyunsaturated fatty acid (PUFA), dihomo‐g‐linolenic acid (DGLA), could be an approach to modulate platelet reactivity through the prostacyclin receptor. Here, we demonstrate that the elongated form of DGLA, docosapentaenoic acid (DPA; 22:5n‐6) can also be endogenously oxidized by platelet 12‐LOX to generate two distinct oxylipins, 11‐oxy‐DPA and 14‐oxy‐DPA, to regulate platelet reactivity.ObjectiveTo determine the efficacy of and the mechanistic target of 12‐LOX derived metabolites of DPA 11‐ and 14‐oxy‐DPA, ω‐6 in the regulation of platelet function.ResultsBoth 11‐ and 14‐oxy‐DPA potently inhibited platelet aggregation in an aggregometer measuring optical density. Treatment of whole blood with 11‐ and 14‐oxy‐DPA, ω‐6 also significantly reduced platelet adhesion as well as thrombus growth on an ex vivo collagen‐coated perfusion chamber under arterial shear. In support of these findings, using flow cytometry, calcium mobilization was attenuated in platelets treated with 11‐ and 14‐oxy‐DPA, ω‐6. Interestingly, both 11‐ and 14‐oxy‐DPA inhibited platelet activation in a Gas independent manner. Instead, 11‐oxy‐DPA and 14‐oxy‐DPA exerted their anti‐platelet effects through a peroxisome proliferated‐activated receptor (PPAR), by which pharmacological antagonism of the PPARα isoform reversed the ability of the metabolites to inhibit platelet aggregation. In supplement of these ex vivo findings, ongoing experiments are being performed to measure laser‐induced thrombus formation (platelet and fibrin) in the cremaster vessel of wild‐type mice intravenously administered with either 11‐ and 14‐oxy‐DPA.ConclusionThis is the first study to demonstrate the novel metabolites of DPA, ω‐6 generated by 12‐LOX also have a role in regulating platelet activation through the PPAR pathway. Our findings suggest that 11‐ and 14‐oxy‐DPA, ω‐6 shows great promise as a potential anti‐platelet therapeutic for the treatment for cardiovascular‐related diseases.Support or Funding InformationThis work was funded in part through the National Institutes of Health grants R01 GM105671 (MH), R01 HL114405 (MH), R01 MD007880 (MH).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Uremic Toxin Indoxyl Sulfate Accelerates Thrombotic Response after Vascular Injury in Animal Models.

Chronic kidney disease (CKD) patients are at high risk for thrombotic events. Indoxyl sulfate (IS) is one of the most potent uremic toxins that accumulates during CKD. Even though IS is associated with an increased risk for cardiovascular disease, its impact on thrombotic events still remains not fully understood. The purpose of the study was to evaluate the direct effect of IS on thrombotic process. We examined the impact of acute exposure to IS on thrombus development induced by electric current in Wistar rats, intravital thrombus formation after laser-induced injury in the mice endothelium, coagulation profile, clot formation dynamics, platelet aggregations, and erythrocyte osmotic resistance. IS doses: 10, 30 and 100 mg/kg body weight (b.w.) increased weight of thrombus induced by electric current in dose-dependent manner (p < 0.001). Furthermore, two highest IS doses increased laser-induced thrombus formation observed via confocal system (increase in fluorescence intensity and total thrombus area (p < 0.01)). Only the highest IS dose decreased clotting time (p < 0.01) and increased maximum clot firmness (p < 0.05). IS did not affect blood morphology parameters and erythrocyte osmotic resistance, but augmented collagen-induced aggregation. Obtained data indicate that IS creates prothrombotic state and contributes to more stable thrombus formation. Thus, we concluded that IS may be one of crucial uremic factors promoting thrombotic events in CKD patients.

Abstract 54: ML355 in Prevention of Thrombosis in vivo with Minimal Effects on Hemostasis

12-lipoxygenase (12-LOX) has been demonstrated to regulate platelet function, hemostasis, and thrombosis ex vivo , supporting a key role for 12-LOX in regulation of in vivo thrombosis. While pharmacologically targeting 12-LOX in vivo has been a challenge to date, the recent development of the 12-LOX selective inhibitor, ML355, as an effective antiplatelet therapeutic in vivo was assessed. ML355 potently inhibited thrombin and other agonist-induced platelet aggregation ex vivo in washed human platelets and inhibited downstream oxylipin production of platelet 12-LOX as confirmed by Mass spectrometry analysis. Ex vivo flow chamber assays confirmed that human platelet adhesion and thrombus formation at arterial shear over collagen was attenuated in human whole blood treated with ML355 to a greater extent compared to aspirin. In vivo , PK assessment of ML355 showed reasonable 12-LOX plasma levels 12 hours following administration of ML355. FeCl 3 -induced injury of the mesenteric arterioles resulted in less stable thrombi in 12-LOX -/- mice and ML355-treated WT mice resulting in impairment of vessel occlusion. Additionally, ML355 dose-dependently inhibited laser-induced thrombus formation in the cremaster arteriole thrombosis model in WT, but not in 12-LOX -/- mice. Importantly, hemostatic plug formation and bleeding following treatment with ML355 were not affected in response to laser ablation on the saphenous vein or in a cremaster microvasculature laser-induced rupture model. Our data strongly supports 12-LOX as a key determinant of platelet reactivity in vivo and inhibition of platelet 12-LOX with ML355 may represent a new class of antiplatelet therapeutics.

TMEM16F is required for phosphatidylserine exposure and microparticle release in activated mouse platelets

Phosphatidylserine (PtdSer) exposure on the surface of activated platelets requires the action of a phospholipid scramblase(s), and serves as a scaffold for the assembly of the tenase and prothrombinase complexes involved in blood coagulation. Here, we found that the activation of mouse platelets with thrombin/collagen or Ca(2+) ionophore at 20 °C induces PtdSer exposure without compromising plasma membrane integrity. Among five transmembrane protein 16 (TMEM16) members that support Ca(2+)-dependent phospholipid scrambling, TMEM16F was the only one that showed high expression in mouse platelets. Platelets from platelet-specific TMEM16F-deficient mice exhibited defects in activation-induced PtdSer exposure and microparticle shedding, although α-granule and dense granule release remained intact. The rate of tissue factor-induced thrombin generation by TMEM16F-deficient platelets was severely reduced, whereas thrombin-induced clot retraction was unaffected. The imaging of laser-induced thrombus formation in whole animals showed that PtdSer exposure on aggregated platelets was TMEM16F-dependent in vivo. The phenotypes of the platelet-specific TMEM16F-null mice resemble those of patients with Scott syndrome, a mild bleeding disorder, indicating that these mice may provide a useful model for human Scott syndrome.

Both platelet- and endothelial cell–derived ERp5 support thrombus formation in a laser-induced mouse model of thrombosis

AbstractProtein disulfide isomerase (PDI) and endoplasmic reticulum protein 57 (ERp57) are emerging as important regulators of thrombus formation. Another thiol isomerase, endoplasmic reticulum protein 5 (ERp5), is involved in platelet activation. We show here the involvement of ERp5 in thrombus formation using the mouse laser-injury model of thrombosis and a specific antibody raised against recombinant ERp5. Anti-ERp5 antibody inhibited ERp5-dependent platelet and endothelial cell disulfide reductase activity in vitro. ERp5 release at the thrombus site was detected after infusion of Alexa Fluor 488–labeled anti-ERp5 antibody at 0.05 μg/g body weight, a dose that does not inhibit thrombus formation. Anti-ERp5 at 3 μg/g body weight inhibited laser-induced thrombus formation in vivo by causing a 70% decrease in the deposition of platelets and a 62% decrease in fibrin accumulation compared to infusion of control antibody (P < .01). ERp5 binds to β3 integrin with an equilibrium dissociation constant (KD) of 21 µM, measured by surface plasmon resonance. The cysteine residues in the ERp5 active sites are not required for binding to β3 integrin. These results provide evidence for a novel role of ERp5 in thrombus formation, a function that may be mediated through its association with αIIbβ3.

Short- and long-term treatment with folic acid suppresses thrombus formation in atherogenic mice in vivo

In the present study, we examined the effects of short- and long-term treatment with folic acid (FA) on thrombus formation in vivo in atherogenic mice to explore a novel agent for the prevention of atherothrombotic disease. Apolipoprotein E and low-density lipoprotein receptor double deficient (ApoE−/−LDLR−/−) mice were orally administrated a single bolus of FA (20mg/kg) or fed an atherogenic diet with or without FA (0.02, 0.5, and 1.5mg/kg) for 12 weeks. Thrombus formation and endothelial function were assessed in vivo using the He–Ne laser-induced carotid artery thrombus formation test and the flow-mediated vasodilation method. Platelet reactivity was assessed ex vivo using haemostatometry. Short-term treatment with FA markedly increased plasma folate levels and significantly suppressed laser-induced thrombus formation in apoE−/−LDLR−/− mice. Short-term treatment with FA suppressed platelet reactivity in apoE−/−LDLR−/− mice, but FA treatment did not affect endothelial function or plasma homocysteine levels. Long-term treatment with FA increased plasma folate levels dose-dependently. Thrombus formation and endothelial dysfunction were suppressed by treatment with 0.5 and 1.5mg/kg of FA, respectively, but not with 0.02mg/kg of FA, whereas platelet reactivity was not altered by treatment with any dose of FA. Long-term treatment with all doses of FA decreased the plasma homocysteine levels in apoE−/−LDLR−/− mice, although this result was not consistent with its anti-thrombotic action. In conclusion, our data showed that short- and long-term treatment with FA could suppress in vivo thrombus formation in an atherogenic setting, independent of its hypohomocysteinemic action.

G protein–dependent basal and evoked endothelial cell vWF secretion

Abstractvon Willebrand factor (vWF) secretion by endothelial cells (ECs) is essential for hemostasis and thrombosis; however, the molecular mechanisms are poorly understood. Interestingly, we observed increased bleeding in EC-Gα13−/−;Gα12−/− mice that could be normalized by infusion of human vWF. Blood from Gα12−/− mice exhibited significantly reduced vWF levels but normal vWF multimers and impaired laser-induced thrombus formation, indicating that Gα12 plays a prominent role in EC vWF secretion required for hemostasis and thrombosis. In isolated buffer-perfused mouse lungs, basal vWF levels were significantly reduced in Gα12−/−, whereas thrombin-induced vWF secretion was defective in both EC-Gαq−/−;Gα11−/− and Gα12−/− mice. Using siRNA in cultured human umbilical vein ECs and human pulmonary artery ECs, depletion of Gα12 and soluble N-ethylmaleimide-sensitive-fusion factor attachment protein α (α-SNAP), but not Gα13, inhibited both basal and thrombin-induced vWF secretion, whereas overexpression of activated Gα12 promoted vWF secretion. In Gαq, p115 RhoGEF, and RhoA-depleted human umbilical vein ECs, thrombin-induced vWF secretion was reduced by 40%, whereas basal secretion was unchanged. Finally, in vitro binding assays revealed that Gα12 N-terminal residues 10-15 mediated the binding of Gα12 to α-SNAP, and an engineered α-SNAP binding-domain minigene peptide blocked basal and evoked vWF secretion. Discovery of obligatory and complementary roles of Gα12 and Gαq/11 in basal vs evoked EC vWF secretion may provide promising new therapeutic strategies for treatment of thrombotic disease.

Laser-induced thrombus formation in mouse brain microvasculature: effect of clopidogrel

Antiplatelet drugs have been evaluated by measuring platelet aggregation ex vivo, but in vivo studies were scanty. The purpose of this study was to observe the effects of an antiplatelet agent (clopidogrel) on the process of laser-induced thrombus formation in mice using intravital fluorescence microscopy. C57 BL/6J mice (n = 19) were anesthetized using chloral hydrate. The head of each mouse was fixed with a head holder, and a cranial window was made in the parietal region. Platelets were labeled in vivo by intravenous administration of carboxyfluorescein diacetate succinimidyl ester. Clopidogrel (1 mg/kg, n = 6; 10 mg/kg, n = 6) was administered orally for 2 days before the experiment. Another seven mice were used as controls. Laser irradiation (1,000 mA, 9.8 mW, diode-pumped solid-state (DPSS) laser 532 nm) was directed for 4 s at pial arteries to induce thrombus formation. Labeled platelets and thrombus were observed continuously under fluorescence microscopy. We recorded the area of thrombus after 30 min and determined the complete occlusion rate. After laser irradiation to the pial artery, complete occlusion rate was significantly lower in the clopidogrel (10 mg/kg) group (16%, 4/25 vessels) than in the control group (60%, 12/20 vessels) or clopidogrel (1 mg/kg) group (55%, 11/20 vessels). Area of platelet thrombus at 30 min after laser irradiation was significantly smaller in the clopidogrel (10 mg/kg) group (209 ± 128 μm(2)) than in the control group (358 ± 256 μm(2)) or clopidogrel (1 mg/kg) group (355 ± 57 μm(2)). The apparatus which we developed is convenient for inducing thrombus formation by causing endothelial cell damage to the brain surface vasculature in small animals without damage of extravascular tissue. Clopidogrel significantly inhibited laser-induced thrombus formation in pial arteries of mice in a dose-dependent manner.

Thrombotic Events Associated to Aspirin Therapy

Acetyl salicylic acid (ASA) is widely used in clinical practice. Previous studies done in rats showed unexpected thrombotic potencies of this drug used at ultra-low doses. This review is the first report in which the effects of a wide range of ASA concentration on a microvessel model of laser-induced thrombus formation and Induced Hemorrhagic Time in animals were largely studied.

Active Site-labeled Prothrombin Inhibits Prothrombinase in Vitro and Thrombosis in Vivo

Mouse and human prothrombin (ProT) active site specifically labeled with D-Phe-Pro-Arg-CH(2)Cl (FPR-ProT) inhibited tissue factor-initiated thrombin generation in platelet-rich and platelet-poor mouse and human plasmas. FPR-prethrombin 1 (Pre 1), fragment 1 (F1), fragment 1.2 (F1.2), and FPR-thrombin produced no significant inhibition, demonstrating the requirement for all three ProT domains. Kinetics of inhibition of ProT activation by the inactive ProT(S195A) mutant were compatible with competitive inhibition as an alternate nonproductive substrate, although FPR-ProT deviated from this mechanism, implicating a more complex process. FPR-ProT exhibited ∼10-fold more potent anticoagulant activity compared with ProT(S195A) as a result of conformational changes in the ProT catalytic domain that induce a more proteinase-like conformation upon FPR labeling. Unlike ProT and ProT(S195A), the pathway of FPR-ProT cleavage by prothrombinase was redirected from meizothrombin toward formation of the FPR-prethrombin 2 (Pre 2)·F1.2 inhibitory intermediate. Localization of ProT labeled with Alexa Fluor® 660 tethered through FPR-CH(2)Cl ([AF660]FPR-ProT) during laser-induced thrombus formation in vivo in murine arterioles was examined in real time wide-field and confocal fluorescence microscopy. [AF660]FPR-ProT bound rapidly to the vessel wall at the site of injury, preceding platelet accumulation, and subsequently to the thrombus proximal, but not distal, to the vessel wall. [AF660]FPR-ProT inhibited thrombus growth, whereas [AF660]FPR-Pre 1, lacking the F1 membrane-binding domain did not bind or inhibit. Labeled F1.2 localized similarly to [AF660]FPR-ProT, indicating binding to phosphatidylserine-rich membranes, but did not inhibit thrombosis. The studies provide new insight into the mechanism of ProT activation in vivo and in vitro, and the properties of a unique exosite-directed prothrombinase inhibitor.

Laser-induced primary and secondary hemostasis dynamics and mechanisms in relation to selective photothermolysis of port wine stains

Superficial vascular anomalies such as port wine stains are commonly treated by selective photothermolysis (SP). The endovascular laser-tissue interactions underlying SP are governed by a photothermal response (thermocoagulation of blood) and a hemodynamic response (thrombosis). Currently it is not known whether the hemodynamic response encompasses both primary and secondary hemostasis, which platelet receptors are involved, and what the SP-induced thrombosis kinetics are in low-flow venules. To (1) define the role and kinetics of primary and secondary hemostasis in laser-induced thrombus formation and (2) determine which key platelet surface receptors are involved in the hemodynamic response. 532-nm laser-irradiated hamster dorsal skin fold venules were studied by intravital fluorescence microscopy following fluorescent labeling of platelets with 5(6)-carboxyfluorescein. Heparin and fluorescently labeled anti-glycoprotein Ib-α (GPIbα) and anti-P-selectin antibodies were administered to investigate the role of coagulation and platelet receptors, respectively. Lesional sizes were quantified by software. Laser irradiation consistently produced sub-occlusive thermal coagula. Thrombosis was triggered in all irradiated venules in a thermal coagulum-independent manner and peaked at 6.25min post-irradiation. Heparin decreased the maximum thrombus size and caused thrombosis to reach a maximum at 1.25min. Immunoblocking of GPIbα abated the extent of thrombosis, whereas immunoblocking of P-selectin had no effect. The hemodynamic response ensues the photothermal response in a thermal coagulum-independent manner and involves primary and secondary hemostasis. Primary hemostasis is mediated by constitutively expressed GPIbα but not by activation-dependent P-selectin.

Functional Genomics Approaches to Platelet Signaling

AbstractAbstract SCI-37The count, volume and function of platelets varies in the population and there is ample evidence that all 3 quantitative traits (QT) are highly heritable. The control of platelet function is complex with a large number of forward and reverse regulatory pathways that promote and inhibit thrombus formation, respectively. We used 4 parallel genomics approaches with the aim to identify novel regulators of platelet function. First we established a genome-wide expression (GWE) HaemAtlas of all 8 haematological elements, including erythroblasts and megakaryocytes (MKs)(1). Mining of this data identified 279 transcripts that were relatively over-expressed in MKs if compared with the results obtained with the 7 remaining blood cell types. Of these, 75 transcripts encode membrane proteins, many with known and several with a hitherto unknown function (2). Second we determined the platelet response to ADP and a collagen mimetic in 500 healthy subjects of the Platelet Function Cohort (PFC)(3). WGE studies of platelet RNA samples from 37 PFC subjects, selected to be representative of the observed variation, identified 63 transcripts that were correlated with function. Third the 500 PFC samples were typed for 1536 SNPs tagging 108 candidate genes for sequence variation and this identified 19 associations (p-value ≤ 0.005) (3). All 19 QT loci (QTLs), but the GP6 gene, were novel. Based on the above 9 genes were selected for functional studies with platelets from genotyped healthy subjects and by morpholino-based gene knockdown in a model of laser-induced thrombus formation in Danio rerio (2,3). This identified BAMBI, COMMD7, LRRC32 and LRRFIP1 playing a role in the promotion and DCBLD2, ESAM, G6B and GTF2A2 in the inhibition of thrombus formation. Finally 2 of the 15 QTLs for platelet volume and count identified in about 15,000 healthy subjects with genome-wide typing information showed an effect on platelet function demonstrating that sequence variants that modify platelet volume may also exert an effect on function (4). (1) Macaulay, I. C. et al., Comparative gene expression profiling of in vitro differentiated megakaryocytes and erythroblasts identifies novel activatory and inhibitory platelet membrane proteins. Blood 109 (8), 3260 (2007); Watkins, N. A. et al., A HaemAtlas: characterizing gene expression in differentiated human blood cells. Blood 113 (19), e1 (2009); (2) O'Connor, M. N. et al., Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins. Blood 113 (19), 4754 (2009).; (3) Jones, C. I. et al., A functional genomics approach reveals novel quantitative trait loci associated with platelet signaling pathways. Blood 114 (7), 1405 (2009); (4) Soranzo, N. et al., A genome-wide meta-analysis identifies 22 loci associated with eight hematological parameters in the HaemGen consortium. Nat Genet 41 (11), 1182 (2009). Disclosures:No relevant conflicts of interest to declare.

The Signaling Molecule CalDAG-GEFI Represents a Novel Target for Antithrombotic Therapy.

Abstract 1077Poster Board I-99 Background/Objective:The signaling molecule Ca2+ and diacylglycerol regulated guanine nucleotide exchange factor I (CalDAG-GEFI) plays a crucial role in the immediate, Ca2+ dependent activation of Rap1 and intergrin αaIIbβ3 in stimulated platelets. Our previous studies demonstrated that signaling by protein kinase C and the Gi-coupled receptor for ADP, P2Y12, provides an alternative pathway that facilitates the delayed but prolonged activation of Rap1. P2Y12 inhibitors such as clopidogrel bisulfate (Plavix) are currently considered the gold standard for the prevention and treatment of thrombotic complications. The aims of the study were to: 1) evaluate CalDAG-GEFI signaling as a potential new target for antithrombotic therapy and 2) to compare the contribution of CalDAG-GEFI and P2Y12 signaling to thrombus formation in pre-clinical models of models of thrombotic disease. Methods/Results:Thrombus formation was compared in: wildtype (WT), CalDAG-GEFI-/-, WT treated with clopidogrel (clopidogrel was administered orally 24h and 3h prior to the experiment at a dosage of 0,075mg/g bodyweight) and CalDAG-GEFI-/- mice treated with clopidogrel. Thrombosis was studied in three different models: 1) laser-induced thrombosis in the microcirculation of the cremasteric muscle (thrombin-dependent, localized), tissue factor-induced pulmonary thromboembolism (thrombin-dependent, systemic) and FeCl3-induced thrombosis in the mesentery (collagen/thrombin-dependent, localized). As shown previously, clopidogrel treatment significantly reduced but did not abolish laser-induced thrombus formation in WT mice. Laser induced thrombosis was completely inhibited in arterioles of CalDAG-GEFI -/- mice, while significant thrombus formation was observed in venules. Clopidogrel treatment of CalDAG-GEFI-/- mice further reduced venous thrombus formation, providing in vivo confirmation that platelet aggregation in the absence of CalDAG-GEFI requires P2Y12 signaling. In line with these findings, we observed that CalDAG-GEF-/- mice were partially protected from tissue factor-induced pulmonary thromboembolism when compared to clopidogrel-treated CalDAG-GEF-/- mice. In the model of FeCl3-induced thrombosis, CalDAG-GEFI-/- platelets adhered to the damaged vascular wall but failed to form thrombi in both venules and arterioles. In contrast clopidogrel-treated WT mice were protected from FeCl3-induced vessel occlusion but continuously formed embolizing thrombi of considerable size that contained activated platelets. This phenomenon was detectable in both arterioles and venules and might be of clinical relevance. Conclusion:Our studies identify CalDAG-GEFI as a promising new target for antiplatelet therapy. CalDAG-GEFI inhibition will have a strong antithrombotic effect as it is a critical component of the near-immediate platelet response to exposed extracellular matrix and/or soluble agonists. However, backup by PKC/P2Y12 signaling should allow for the formation of small but stable mural thrombi, especially under low flow conditions as found in veins and in venules, thus limiting the risk of bleeding complications. Disclosures:Poncz:Diagnostica Stago: Patents & Royalties.

Par4 is required for platelet thrombus propagation but not fibrin generation in a mouse model of thrombosis

Thrombin, a central mediator of hemostasis and thrombosis, converts fibrinogen to fibrin and is a potent platelet activator. Activated platelets provide a surface for assembly of the tenase and prothrombinase complexes required for thrombin generation. The role of thrombin-induced platelet activation in platelet accumulation and its interplay with fibrin deposition during thrombus assembly has not been fully defined. We studied these processes during laser-induced thrombus formation by using real-time digital fluorescence microscopy in mice lacking protease-activated receptor-4 (Par4), which is necessary for thrombin responsiveness in mouse platelets. Juxtamural platelet accumulation immediately after laser injury was not different in wild-type and Par4(-/-) mice. However, subsequent growth of platelet thrombi was markedly diminished in Par4(-/-) mice. At the time of maximal thrombus size in wild type, platelet accumulation was more than 10-fold higher in wild type than in Par4(-/-) mice. P-selectin expression, a marker of platelet activation, was reduced and delayed in Par4(-/-) thrombi. In contrast to platelet activation and accumulation, the rate and amount of fibrin deposition, predominantly intramural and juxtamural in this model, were indistinguishable in Par4(-/-) and wild-type mice. These results suggest that platelet activation by thrombin is necessary for normal propagation of a platelet thrombus at a distance from the injured vessel wall and hence for normal thrombus growth. However, platelet activation by thrombin is unnecessary for initial and limited accumulation of platelets at or near the vessel wall, and this limited accumulation of platelets and/or platelet-independent mechanism(s) of thrombin generation are sufficient for normal fibrin deposition in this model.

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.

Antithrombic and anti-atherogenic effects of partially defatted flaxseed meal using a laser-induced thrombosis test in apolipoprotein E and low-density lipoprotein receptor deficient mice.

Atherothrombosis can be regarded as a 'life-style related disease' of which diet is one of the important risk factors. The prophylactic effect of partially defatted flaxseed meal (PDFM) on atherothrombosis has not yet been studied. The aim of this study was to assess the effect of PDFM and a lignan from flaxseed, secoisolariciresinol diglucoside (SDG), on thrombosis and atherogenesis. An earlier developed test, the quantitative assessment of laser-induced thrombus formation in the carotid artery of apolipoprotein E and low-density lipoprotein receptor deficient mice was used in this study. Thrombotic and atherosclerotic status was assessed in mice kept on a high-fat diet for 8 weeks (40% in energy). The diet contained 0.05% cholesterol alone (control) or the same cholesterol with added PDFM (5% w/w; 8.3 g/kg body weight per day) or SDG (0.06% w/w; 100 mg/kg body weight per day). PDFM showed antithrombotic (P < 0.01) and anti-atherogenic effect (P < 0.01). SDG did not affect either atherogenesis or thrombosis. This study suggests that dietary intake of PDFM can be beneficial in reducing the risk of high-fat-induced atherothrombosis.