scholarly journals Stimulated Platelets but Not Endothelium Generate Thrombin Via a Factor XIIa-Dependent Mechanism Requiring Phosphatidylserine Exposure

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 258-258 ◽  
Author(s):  
Pavan K. Bendapudi ◽  
Karen Deceunynck ◽  
Secil Koseoglu ◽  
Roelof Hendrik Bekendam ◽  
Shauna D Mason ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Thrombin Generation ◽  
Thrombus Formation ◽  
Specific Inhibitor ◽  
Unexpected Finding ◽  
Factor Xii ◽  
Fibrin Formation ◽  
Platelet Accumulation

Abstract To inhibit pathological thrombus formation without impairing hemostasis is the holy grail of anticoagulant therapy. Recent data from animal models have indicated that factor XII (FXII) may be a promising new antithrombotic target that is particularly intriguing due to the longstanding clinical observation that severe congenital FXII deficiency is not associated with a bleeding diathesis in humans. FXII is thought to participate in thrombus formation after being activated in high shear arterial environments. FXIIa then initiates downstream activation of the contact pathway, culminating in thrombin generation. However, the relevant cell surface for FXII activation remains unclear. Here we compare the role of platelets versus endothelial cells in FXII activation and study the function of surface phospholipids in this process. To explore the effect of FXII inhibition on thrombus formation in vivo, we used antibody X210-C01, a novel human IgG1 developed using phage-display technology that blocks both mouse and human FXIIa. Using a mouse laser injury model of arterial thrombosis, we showed that X210-C01 inhibited both fibrin formation and platelet accumulation at sites of vascular injury. Plasma removed from animals after completion of these experiments was used to quantify the concentrations of X210-C01 achieved in vivo at a given dose. FXII inhibition was somewhat more potent in preventing platelet accumulation (IC50 dose = 27 mg/kg, R2=0.93) than fibrin formation (IC50 dose = 43 mg/kg, R2=0.95). Importantly, treatment with X210-C01 at 100 mg/kg did not prolong bleeding times or increase total blood loss in a tail bleeding assay. To evaluate the mechanism underlying our in vivo observations, we studied the differential role of FXII in thrombin generation by stimulated platelets and endothelium. X210-C01 did not globally inhibit SFLLRN-induced platelet aggregation or granule release. We next performed a fluorogenic thrombin generation assay (TGA) using human platelets treated with the peptide agonist SFLLRN. X210-C01 inhibited platelet-based thrombin generation in a dose-dependent fashion, whereas anti-tissue factor (TF) and anti-factor VIIa (FVIIa) antibodies did not. By contrast, in a similar TGA using SFLLRN-stimulated endothelial cells, X210-C01 had no effect, while anti-TF antibodies abrogated thrombin generation. These results indicate that stimulated endothelium generates thrombin by a mechanism distinct from that of platelets. FXII is known to be activated in vitro by anionic surfaces. Because phosphatidylserine (PS) is a negatively-charged phospholipid expressed on the surface of stimulated platelets, we reasoned that PS may serve as the platelet-based activator of FXII. To test this hypothesis, we used lactadherin, a potent and specific inhibitor of PS, in the platelet-based TGA and showed that PS blockade inhibited platelet-based thrombin generation at concentrations as low as 10 nM. We then used a chromogenic FXIIa activity assay to test the ability of PS-containing liposomes to activate FXII. Liposomes containing 80% phosphatidylcholine (PC) and 20% PS (PC-PS 80/20) failed to activate FXII at concentrations as high as 100 µM. In this assay, SFLLRN-stimulated platelets led to significantly greater FXII activation than either resting platelets or the PC-PS 80/20 liposomes tested. In summary, we have made the unexpected finding that thrombin generation on the surface of stimulated platelets proceeds by a FXIIa-dependent pathway and does not require FVIIa or TF. By contrast, thrombin generation on endothelium requires TF but not FXIIa. Additionally, surface PS is necessary but not sufficient for platelet-based FXII activation and thrombin generation, pointing to the involvement of a second platelet component. Further studies will be directed towards investigating the in vivo role of platelet-based FXII activation in arterial thrombus formation. Disclosures Mason: Shire Pharmaceuticals: Employment. Kenniston:Shire Pharmaceuticals: Employment.

Dual role of collagen in factor XII–dependent thrombus formation

Blood ◽  
2009 ◽  
Vol 114 (4) ◽  
pp. 881-890 ◽  
Author(s):  
Paola E. J. van der Meijden ◽  
Imke C. A. Munnix ◽  
Jocelyn M. Auger ◽  
José W. P. Govers-Riemslag ◽  
Judith M. E. M. Cosemans ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Type I Collagen ◽  
Factor Viia ◽  
Thrombus Formation ◽  
Dual Role ◽  
Type I ◽  
Factor Xii ◽  
Glycoprotein Vi

Abstract In vivo mouse models have indicated that the intrinsic coagulation pathway, initiated by factor XII, contributes to thrombus formation in response to major vascular damage. Here, we show that fibrillar type I collagen provoked a dose-dependent shortening of the clotting time of human plasma via activation of factor XII. This activation was mediated by factor XII binding to collagen. Factor XII activation also contributed to the stimulating effect of collagen on thrombin generation in plasma, and increased the effect of platelets via glycoprotein VI activation. Furthermore, in flow-dependent thrombus formation under coagulant conditions, collagen promoted the appearance of phosphatidylserine-exposing platelets and the formation of fibrin. Defective glycoprotein VI signaling (with platelets deficient in LAT or phospholipase Cγ2) delayed and suppressed phosphatidylserine exposure and thrombus formation. Markedly, these processes were also suppressed by absence of factor XII or XI, whereas blocking of tissue factor/factor VIIa was of little effect. Together, these results point to a dual role of collagen in thrombus formation: stimulation of glycoprotein VI signaling via LAT and PLCγ2 to form procoagulant platelets; and activation of factor XII to stimulate thrombin generation and potentiate the formation of platelet-fibrin thrombi.

scholarly journals Tissue factor–positive neutrophils bind to injured endothelial wall and initiate thrombus formation

Blood ◽  
2012 ◽  
Vol 120 (10) ◽  
pp. 2133-2143 ◽  
Author(s):  
Roxane Darbousset ◽  
Grace M. Thomas ◽  
Soraya Mezouar ◽  
Corinne Frère ◽  
Rénaté Bonier ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Blood Coagulation ◽  
Thrombus Formation ◽  
Coagulation Cascade ◽  
Factor Xii ◽  
Long Time ◽  
Platelet Accumulation

AbstractFor a long time, blood coagulation and innate immunity have been viewed as interrelated responses. Recently, the presence of leukocytes at the sites of vessel injury has been described. Here we analyzed interaction of neutrophils, monocytes, and platelets in thrombus formation after a laser-induced injury in vivo. Neutrophils immediately adhered to injured vessels, preceding platelets, by binding to the activated endothelium via leukocyte function antigen-1–ICAM-1 interactions. Monocytes rolled on a thrombus 3 to 5 minutes postinjury. The kinetics of thrombus formation and fibrin generation were drastically reduced in low tissue factor (TF) mice whereas the absence of factor XII had no effect. In vitro, TF was detected in neutrophils. In vivo, the inhibition of neutrophil binding to the vessel wall reduced the presence of TF and diminished the generation of fibrin and platelet accumulation. Injection of wild-type neutrophils into low TF mice partially restored the activation of the blood coagulation cascade and accumulation of platelets. Our results show that the interaction of neutrophils with endothelial cells is a critical step preceding platelet accumulation for initiating arterial thrombosis in injured vessels. Targeting neutrophils interacting with endothelial cells may constitute an efficient strategy to reduce thrombosis.

Effect Of Selective Inhibition Of Platelet Thromboxane On Platelet-Vessel Wall Interaction In Vivo

1981 ◽  
Author(s):  
Y C Chen ◽  
K K Wu ◽  
E R Hall ◽  
D L Venton ◽  
G C Le Breton
Keyword(s):  
Vessel Wall ◽  
Platelet Reactivity ◽  
Thrombus Formation ◽  
Balloon Catheter ◽  
Specific Inhibitor ◽  
Constant Infusion ◽  
Catheter Technique ◽  
Platelet Thrombus ◽  
Platelet Accumulation

It is well recognized that thromboxane A2(TXA2) plays an important role in platelet reactivity. To determine the role of TXA2 in platelet-vessel wall (P-V) interaction, the effect of 1-benzylimidazole (1-BI), a specific inhibitor of thromboxane synthetase, and 13-azaprostanoic acid (APA), a TXA2 antagonist, on platelet thrombus formation was evaluated in vivo in NZW male rabbits using the autologous indium-111 (111In) labeled platelet technique. Rabbits were treated with intravenous 1-BI or APA or vehicles. After injection of autologous 111In-platelets, de-endothelialization of the abdominal aorta was created by a balloon catheter technique. At 3 hrs, blood samples were obtained and the animals were sacrificed. The aortae were removed and the injured and uninjured segments were dissected. Radioactivity counts and dry weight of the tissues and blood were determined. The vascular radioactivity counts were converted to platelet numbers by using a standard linear calibration curve. As small numbers of platelets adhered to normal vessel wall nonspecifically, this number was subtracted to obtain specific platelet accumulation at the injured sites. 1-BI at 10mg/kg reduced the specific platelet accumulation significantly (n=5, 12.3±S.D.I.5×106 pl/gm tissue; p<0.01) when compared with the controls (n=10, 33.0±5.1×106 pl/gm tissue). Platelet accumulation was further reduced by increasing the dosage to 30mg/kg. By contrast, APA injection (10mg/kg) had no significant effect. However, when APA was given by constant infusion at 250μg/kg/min 1 hr prior to injury, the APA-treated animals had an 80% reduction of platelet accumulation relative to controls. These findings indicate that TXA2 plays an important role in P-V interaction and specific inhibition of TXA2 appears to be efficacious in eliminating platelet thrombus formation.

Endobrevin/VAMP-8-Mediated Dense Granule Release Is Required for Efficient Thrombus Formation in Vivo.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1836-1836
Author(s):  
Price S. Blair ◽  
Qiansheng Ren ◽  
Gwenda J. Graham ◽  
James R. Dilks ◽  
Sidney W. Whiteheart ◽  
...  
Keyword(s):  
Vascular Injury ◽  
Thrombus Formation ◽  
Dense Granule ◽  
Wild Type ◽  
Platelet Accumulation ◽  
Induced Aggregation ◽  
Kinetics Of ◽  
Granule Release

Abstract Individuals whose platelets lack dense core or alpha-granules suffer varying degrees of abnormal bleeding, implying that granule cargo contributes to hemostasis. Despite these clinical observations, little is known regarding the effects of impaired platelet granule secretion on thrombus formation in vivo. The release of cargo from platelet granules requires a group of membrane proteins called SNAREs (Soluble NSF Attachment Protein Receptors) that mediate fusion of granule membranes to the plasma membrane and open canalicular system. Endobrevin/VAMP-8 is the primary vesicular-SNARE (v-SNARE) responsible for efficient release of dense core and a-granule contents. To evaluate the importance of VAMP-8-mediated secretion on the kinetics of thrombus formation in vivo, we measured platelet accumulation following laser-induced vascular injury in VAMP-8−/− mice. Three different phases of thrombus formation - initiation, maximal accumulation, and stabilized platelet accumulation - were tested. Analysis of initial thrombus formation from wild-type and VAMP-8−/− mice showed that average platelet accumulation in VAMP- 8−/− mice was 23% of accumulation in wild-type mice (P=0.009) at 30 sec following injury. There was a trend towards smaller maximal thrombus size in VAMP-8−/− mice, but the difference was not statistically significant (P=0.1). Average stabilized platelet accumulation at 180 sec in VAMP-8−/− mice was 40% of wild-type mice (P=0.05). Thus, thrombus formation is delayed and decreased in VAMP-8−/− mice, but not absent. Dense granule release occurs more rapidly than alpha-granule release, which does not occur for 2–3 min following laser-induced vascular injury. Agonist-induced dense granule release from VAMP-8−/− platelets is defective. To directly evaluate the role of dense granule release on the kinetics of thrombus formation, we assessed thrombus formation in the mouse model of Hermansky-Pudlak syndrome, ruby-eye, which lack dense granules. Thrombus formation following laser-induced vascular injury was nearly abolished in ruby-eye mice such that maximal platelet accumulation was 15% that of wild-type mice. In vitro, the thrombin doses required to induce irreversible aggregation in wild-type, VAMP-8−/−, and ruby-eye platelets were 25 mU, 50 mU, and 150 mU, respectively. Incubation with apyrase had little effect on thrombin-induced aggregation of VAMP-8−/− or ruby-eye platelets. In contrast, incubation of wild-type platelets with apyrase reduced their thrombin sensitivity compared to that of ruby-eye platelets. Supplementation with a substimulatory ADP concentration reversed the thrombin-induced aggregation defect in VAMP-8−/− and ruby-eye mice. Thus, defective ADP release is the primary abnormality leading to impaired aggregation in VAMP-8−/− and ruby-eye mice. Tail bleeding times were assessed in VAMP- 8−/− mice to evaluate the role of VAMP-8 in hemostasis. In contrast to ruby-eye mice, which have a markedly prolonged bleeding time, tail bleeding times in VAMP-8−/− mice were not significantly prolonged compared to those in wild-type mice. These results demonstrate the importance of VAMP-8 and dense granule release in the initial phases of thrombus formation and validate the distal platelet secretory machinery as a potential target for anti-platelet therapies.

Extracellular Protein Disulfide Isomerase Regulates Vitronectin during the Initiation of Thrombus Formation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 15-15
Author(s):  
Chao Fang ◽  
Sheryl R. Bowley ◽  
Barbara C. Furie ◽  
Bruce Furie
Keyword(s):  
Endothelial Cells ◽  
Protein Disulfide Isomerase ◽  
Vessel Wall ◽  
Plasma Sample ◽  
Thrombus Formation ◽  
Disulfide Isomerase ◽  
Platelet Accumulation ◽  
Protein Disulfide ◽  
Kinetic Trapping

Abstract Protein disulfide isomerase (PDI), secreted by platelets and endothelial cells upon vascular injury, is required for thrombus formation. However, the precise mechanism by which PDI regulates thrombosis remains elusive. Using PDI variants that form stable mixed disulfide complexes with their substrates, we performed kinetic trapping experiment in platelet rich plasma and identified multiple substrate proteins for PDI, including vitronectin. Importantly, when using variants of endoplasmic reticulum protein 57 (ERp57), a thiol isomerase that has a similar domain structure as PDI and is also important for thrombus formation, the trapping mutants of ERp57 do not interact with vitronectin. This result has demonstrated the substrate specificity of PDI during our kinetic trapping experiment. Further study using polyethylene glycol (PEG)-based gel mobility shift assay combined with mass spectrometry has identified the redox reaction between PDI and vitronectin occurs on two disulfide bonds Cys 137-161 and Cys 274-453 in the hemopexin-like domains of plasma vitronectin. Vitronectin, as a substrate of extracellular PDI, has been shown to be important for thrombus formation. Vitronectin null mice have reduced platelet accumulation and fibrin deposition in the cremaster arterioles following laser injury. Vitronectin null mice also have significantly prolonged large-vessel thrombosis in the carotid artery using the ferric chloride thrombosis model. Using intravital microscopy we showed that vitronectin rapidly accumulates in a growing thrombus following vessel injury. When mice are treated with eptifibatide to eliminate platelet accumulation, we still observe significant amount of vitronectin accumulation on the vessel wall in the absence of platelet thrombus. This observation was further confirmed using confocal intravital microscopy. After 3D reconstruction of a growing thrombus in mouse cremaster arteriole, vitronectin was identified to locate primarily on the CD31 stained vessel wall. These combined studies suggest that plasma-derived vitronectin and not platelet-derived vitronectin is the primary substrate of PDI. Our study further showed that the indispensable role of vitronectin to a growing thrombus depends on extracellular PDI. Native plasma vitronectin does not bind to αvβ3 or αIIbβ3-integrins on endothelial cells and platelets. On solid phase binding assay, plasma sample pre-treated with wild-type PDI showed significantly increased binding of vitronectin to its ligand αvβ3 or αIIbβ3-integrins. However, this increase was not observed in plasma pre-treated with dead-mutant PDI or ERp57. In addition, using immunofluorescent staining, PDI treated plasma sample also showed significantly increased binding of vitronectin to activated human umbilical vein endothelial cells (HUVECs) and this binding was abrogated by RGD peptides or an αvβ3 blocking antibody. The critical role of extracellular PDI for the regulation of vitronectin in a growing thrombus was further confirmed in our in vivo studies. When mice were treated with quecetin-3-rutinoside or two different inhibitory antibodies that selectively block PDI activity, the accumulation of vitronectin and platelets was significantly reduced. These combined results demonstrate that extracellular PDI regulates vitronectin in a growing thrombus to promote platelet accumulation and fibrin generation. In summary, our studies have revealed a novel regulatory mechanism during the initiation of thrombus formation. Under normal physiologic conditions in the absence of secreted PDI, thrombus formation is suppressed and maintains a quiescent, patent vasculature. The release of PDI during vascular injury serves as a novel regulatory switch that allows activation of proteins, including vitronectin, which are critical for the following platelet accumulation and fibrin generation. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Extracellular Protein Disulfide Isomerase Cleaves Allosteric Disulfides in Histidine-Rich Glycoprotein to Regulate Thrombus Formation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 11-12
Author(s):  
Shuai Chen ◽  
Xu-Lin Xu ◽  
Joyce Chiu ◽  
Sheryl Bowley ◽  
Yi Wu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Heparan Sulfate ◽  
Disulfide Bonds ◽  
Thrombus Formation ◽  
Anticoagulant Activity ◽  
Laser Injury ◽  
Protein Disulfide ◽  
Kinetics Of

Introduction The fine-tuning of thrombus formation is influenced by multiple factors among which extracellular protein disulfide isomerase (PDI) released by activated platelets and endothelial cells plays critical roles. However, the precise mechanisms whereby PDI modulates the kinetics of thrombosis remain elusive. Using mechanism-based kinetic trapping strategy, we identified plasma histidine-rich glycoprotein (HRG) as a substrate of extracellular PDI during thrombus formation. HRG exerts both anticoagulant and procoagulant functions. On one hand, HRG inhibits the contact pathway by binding to activated factor XII (fXIIa); on the other hand, HRG attenuates the anticoagulant activity of antithrombin (AT) by competing with AT binding to endothelial heparan sulfate. Both functions are dependent on zinc ions. In this study, we characterized the effects of PDI-mediated disulfide bond cleavage on HRG functions in the context of thrombosis. Methods Recombinant PDI variant with the C-terminal catalytic Cys of the CGHC motif replaced with Ala (PDI-CA) was used to trap its redox substrates in platelet rich plasma (PRP). Dual fluorescent immunoblotting was utilized to detect the stabilized intermediate complex between PDI-CA and HRG. Differential cysteine alkylation and mass spectrometry was performed using purified plasma HRG to identify the disulfide bonds targeted by PDI. ELISA was performed to determine the effects of PDI treatment on HRG binding to heparin, an analog of endothelial heparan sulfate, and fXIIa. Cell-based ELISA, immunofluorescent imaging, and immunohistochemistry were employed to examine in vitro and in vivo binding of HRG and AT on endothelial cells. HRG-mediated inhibition of fXIIa activity was determined using the chromogenic substrate S-2302. The kinetics of HRG accumulation during thrombus formation were examined using high-speed intravital microscopy in the cremasteric arterioles. The effects of HRG on thrombus formation were examined in the laser injury thrombosis model in the presence (wild-type mice) or absence of fXII (f12-/- mice). Results The trapping mutant PDI-CA, but not variants of endoplasmic protein 57 (ERp57), a close member in the PDI family with similar domain structure, formed disulfide-linked complexes with HRG in PRP. Mass spectrometry showed that PDI cleaves three disulfide bonds, C306-C309, C390-C434 and C409-C410, in the histidine-rich region of HRG that is important for its binding to heparan sulfate and fXIIa. Compared to inert-PDI (PDI-AA), where both catalytic Cys were substituted with Ala, wild-type PDI (PDI-CC) increased HRG binding to heparin in a Zn2+-dependent manner. Plasma treated with PDI-CC had increased HRG binding but decreased AT binding to cultured endothelial cells compared to PDI-AA treated control. Further, PDI-CC increased HRG binding to fXIIa and enhanced its inhibitory effect on fXIIa activity. Following laser injury of cremaster arterioles, plasma HRG accumulates rapidly at the injury site preceding the main platelet signal. When mice were treated with Eptifibatide, an integrin αIIbβ3 antagonist that eliminates platelet deposition and Zn2+release, plasma HRG accumulation at the site of vessel injury was reduced, indicating a critical role of Zn2+ for HRG binding in vivo. Intravenous treatment with a PDI inhibitor, isoquercetin, also inhibited HRG accumulation in the growing thrombus. In addition, following FeCl3-induced carotid injury, PDI inhibition by isoquercetin was found to reduce HRG binding but sustain AT binding on the injured artery as determined by immunohistochemistry. Finally, knockdown of plasma HRG with vivo-siRNA in f12-/- mice attenuated thrombus formation compared to scramble siRNA treatment thus suggesting a procoagulant role of HRG independent of fXIIa. Conclusion PDI cleavage of allosteric disulfide bonds in HRG represents a novel regulatory mechanism that fine-tunes the kinetics of thrombus formation. Our results indicate that at the early stage of thrombosis, PDI promotes HRG binding to endothelial cells to suppress the anticoagulant activity of AT and allow the rapid initiation of thrombosis; at the later stage, PDI reduction of HRG enhances its binding to fXIIa leading to inhibition of fXIIa activity to prevent excessive clot formation. Disclosures Bowley: Pfizer: Current Employment.

The Principal Eosinophil Peroxidase Oxidant, HOSCN, Is a Uniquely Potent Phagocyte Oxidant Inducer of E-Selectin, ICAM-1 and VCAM-1 Expression on Endothelial Cells and Promotes Leukocyte-Endothelial Cell Adhesion. A Potential Mechanism Regulating Leukocyte Trafficking in Eosinophilic Inflammatory States.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3085-3085
Author(s):  
Jianguo Wang ◽  
Rong Tang ◽  
Yuqing Huo ◽  
Arne Slungaard
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Western Blotting ◽  
Hypereosinophilic Syndrome ◽  
Specific Inhibitor ◽  
Fold Increase ◽  
In Vivo Model ◽  
Pcr Analysis ◽  
Unexpected Finding

Abstract We have shown that SCN- is the principal substrate for EPO in vivo and that its product, HOSCN, a weak sulfhydryl-reactive oxidant, is a singularly effective oxidant inducer of tissue factor activity in human umbilical vein endothelial cells (HUVEC) through activation of the NF-κB transcription factor. Because their genes contain upstream NF-κB binding sites, we hypothesized that HOSCN would induce expression of the cell adhesion molecules E-selectin, ICAM-1 and VCAM-1 and thereby enhance leukocyte-endothelium adhesion. HUVEC monolayers were exposed to various phagocyte-derived oxidants or 10 μg/ml LPS in M199 medium with 10% FCS and assayed by western blotting and flow cytometry. We find that 150 μM HOSCN induces VCAM-1 and ICAM-1 expression starting at 2 h that peaks stably at up to 10-fold from 4–12 h while upregulation of E-selectin is first detectable by 2h, peaks up to 10-fold at 4 hours, then rapidly diminishes to baseline. This induction is dose-dependent and does not occur in the presence of the other phagocyte oxidants HOCl, HOBr and H2O2. HOSCN inducton of adhesion molecule expression is transcriptionally mediated as documented by semi-quantitative RT-PCR analysis. Moreover, HOSCN, but not HOCl, HOBr or H2O2 strongly activates the NF-κB p65/p50 heterodimer assayed by EMSA and cytoplasmic IκB-α degradation assayed by western blotting. To test the functional significance and specificity of these findings we performed neutrophil/HUVEC static adhesion assays with blocking monoclonal antibodies. 150 μM HOSCN induces a 6-fold increase in neutrophil adhesion that is totally blocked by 10 μg/ml of andrographolide, a specific inhibitor of the NF-κB pathway. Adhesion peaks at 4 h then wanes, consistent with the kinetics of E-selectin expression. Neutrophil/HUVEC adhesion was decreased 30–40% by blocking antibodies to either E-selectin or ICAM-1 but not VCAM-1. In an in vivo model of neutrophil rolling and adhesion, mice were intraperitoneally injected with reagent HOSCN (10 nmol/g) or buffer control 2 h before the cremaster muscle was externalized and leukocyte/endothelial interactions in post-capillary venules were quantitated by intravital microscope using a CCD video camera. Peritoneal cavities were lavaged and leukocytes enumerated and identified. HOSCN treatment induces a 4.6-fold (n = 8, p = 0.006) increase in total endothelium-adherent leukocytes but no significant change in leukocyte rolling flux. HOSCN also stimulates a 20-fold increase in peritoneal neutrophils. These findings show that HOSCN is a uniquely potent oxidant inducer of HUVEC E-selectin, ICAM-1 and VCAM-1 expression and promotes neutrophil-HUVEC adhesion. We propose that HOSCN generated by eosinophils adhering to and infiltrating endothelium may stimulate the recruitment and activation of neutrophils, eosinophils and other leukocytes to sites of eosinophilic inflammation and thereby participate in the pathogenesis of allergic diseases and the hypereosinophilic syndrome. The recent unexpected finding that neutrophil myeloperoxidase generates equimolar amounts of HOSCN and HOCl as its principal physiologic products raises the possiblility that neutrophils also utilize HOSCN as an inflammatory amplification mechanism.

scholarly journals Inhibition of Sars-Cov-2 Viral Replication and In Vivo Thrombus Formation By a Novel Plant Flavonoid

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3144-3144
Author(s):  
Moua Yang ◽  
Lin Lin ◽  
Christina Scartelli ◽  
Da-Yuan Chen ◽  
Anika Patel ◽  
...  
Keyword(s):  
Viral Replication ◽  
In Silico ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Fibrin Formation ◽  
In Silico Studies ◽  
Main Protein ◽  
Platelet Accumulation

Abstract Plant-based flavonoids have been examined as inhibitors of β-coronavirus replication and as potential therapeutics for COVID19 based on their safety profile and widespread availability. SARS-CoV-2 viral replication is dependent on a cysteine protease known as 3CL protease, or main protease (Mpro), which cleaves the polyprotein translated from SARS-CoV-2 ssRNA into 11 functional proteins. This protease is highly conserved among β-coronaviruses and is intolerant of mutation. The main protein (Mpro) of SARS-CoV, SARS-CoV-2, and MERS has been identified as a target of flavonoids both by in silico and in vitro approaches. We have previously showed that select flavonoids inhibit protein disulfide isomerase (PDI), which is essential for normal thrombosis. These flavonoid PDI inhibitors block thrombus formation in vivo and have shown efficacy as antithrombotics in clinical studies. Given the substantial morbidity and mortality caused by COVID19-associated coagulopathy, we sought to identify a flavonoid that inhibits both SARS-CoV-2 Mpro and PDI, potentially blocking both viral replication and thrombus formation. While in silico studies identified many flavonoids as SARS-CoV-2 main protein (Mpro) inhibitors, no comprehensive in vitro testing of flavonoids against SARS-CoV-2 has previously been performed. We therefore evaluated 1,020 diverse flavonoids using high throughput screening for their ability to inhibit SARS-CoV-2 Mpro in a fluorescence-based Mpro substrate cleavage assay. This analysis identified four new flavonoid inhibitors of Mpro that had IC 50s ranging from 5-15 µM: amentoflavone, 3,8'-biapigenin, jaceidin triacetate, and pinocembrin 7-O-(3''-galloyl-4'',6''-(S)-hexahydroxydiphenoyl)-beta-D-glucose (PGHG). These compounds were equally or more potent than previously identified flavonoid inhibitors of SARS-CoV-2 Mpro, baicalein and myricetin. Structure activity relationships identified apigenin as an additional Mpro inhibitor. In a Vero-E6-based assay of SARS-CoV-2 replication, PGHG inhibited with an IC 50 = 4.9 µM. At 50 µM, apigenin showed 94±2.1% inhibition and baicalein 65±8.0% inhibition, while myricetin, amentoflavone, and 3,8'-biapigenin did not inhibit viral replication. Jaceidin triacetate was too toxic for further analysis. We next evaluated novel Mpro inhibitors for their ability to inhibit PDI. The most potent PDI inhibitor was PGHG, which blocked PDI reductase activity in an insulin turbidimetric assay with an IC 50 = 3.99±1.14 µM and in a di-eosin-GSSG assay with an IC 50 = 1.50±0.60 µM. When tested against isolated fragments of PDI, PGHG inhibited isolated a and a' fragments as well as ab, b'xa' and abb'x fragments, indicating that it acts on the a and a' domains of PDI. Since PDI is essential for thrombosis, we evaluated whether PGHG blocks platelet accumulation and fibrin formation following vascular injury. We infused mice with 25 mg/kg PGHG or vehicle and subsequently induced thrombus formation via laser-induced injury of an arteriole within the cremaster circulation. Infusion of PGHG resulted in a 82±6.2% inhibition of platelet accumulation and a 79±3.7% inhibition of fibrin formation. In contrast 25 mg/kg had no significant effect on tail bleeding in mice compared to vehicle control. Targeted therapies remain an important component of the armamentarium against COVID19. Our results show that a naturally occurring flavonoid, PGHG, found in Penthorum chinense Pursh , inhibits both SARS-CoV-2 replication and thrombosis without enhancing bleeding. This observation provides proof-of-principle for the development of plant-based flavonoid therapies for inhibition of β-coronaviruses and supports the further evaluation of PGHG for therapeutic use in COVID19. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1510-1510
Author(s):  
Christophe Dubois ◽  
Laurence Panicot-Dubois ◽  
Justin F. Gainor ◽  
Barbara C. Furie ◽  
Bruce Furie
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
Specific Inhibitor ◽  
Calcium Mobilization ◽  
Wild Type ◽  
Platelet Surface ◽  
Platelet Thrombus ◽  
Platelet Accumulation ◽  
Kinetics Of

Abstract 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.

Rapid Activation of Unstimulated Endothelial Cells Containing Tissue Factor Following Laser-Induced Injury as Monitored Via Calcium Mobilization.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1786-1786 ◽  
Author(s):  
Ben T. Atkinson ◽  
Cynthia A. Kos ◽  
Barbara C. Furie ◽  
Bruce Furie
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Intracellular Calcium ◽  
Tissue Factor ◽  
Vessel Wall ◽  
Thrombus Formation ◽  
Fibrin Formation ◽  
Thrombosis Model ◽  
Calcium Elevation

Abstract The induction of thrombus formation in vivo through a laser-induced injury to the vessel wall in the living mouse has demonstrated the rapid expression of tissue factor antigen, thrombin generation and fibrin formation during thrombus formation. This thrombosis model is dominated by a tissue factor-dependent mechanism of platelet activation and fibrin formation. Specifically, this model does not lead to exposure of detectable sub-endothelial collagen, and the growth of the resulting thrombus is not dependent upon collagen-induced signaling. In light of prior in vivo results suggesting the rapid expression of active tissue factor on the vessel wall, we have determined if endothelial cells are activated in response to the injury induced by a pulsed laser. Laser-induced activation of individual cultured endothelial cells and cell populations were monitored through elevation of intracellular calcium and fluorescence microscopy. Cultured human umbilical vein endothelial cells (HUVECs) were loaded with the calcium-sensitive dye Fluo-4 and subjected to a single pulse of the nitrogen dye-tuned laser. HUVEC activation, was characterized by rapid elevation of intracellular calcium, with a sustained peak observed within 10 sec. Comparable calcium elevation could be achieved by exogenous addition of either ADP (10 μM) or thrombin (1 U/ml). Although reduced, significant laser-induced elevation of intracellular calcium remained when cells were bathed in calcium-free media, thus suggesting that both calcium influx and calcium mobilization play a part in the total calcium elevation observed. In addition, targeting of single cells within a confluent culture of endothelial cells initiated calcium elevation of the targeted cell and was followed by a wave of calcium elevation in surrounding cells. These results imply either a release of secondary mediators or cell-cell communication. Using both isolated and confluent cultured HUVECs, we performed widefield immunofluorescence and differential interference contrast microscopy to detect tissue factor (TF) in unstimulated HUVECs using an anti-TF antibody of high affinity and specificity. Tissue factor was not detected in intact endothelial cells but was localized to abundant small granules within the cytoplasm in Triton X-100-permeabilized cultured HUVECs. The J82 bladder carcinoma cell line, which constitutively expresses TF on the plasma membrane, was used as a positive control for surface expression of TF and an isotype-matched non-immune antibody used as a negative control. These results indicate the presence of a preformed intracellular pool of TF within HUVECs that under resting conditions is not detectable on the cell surface. Given the rapid time course of TF expression and platelet accumulation in vivo following laser-induced injury of the endothelium, any TF expression on the vessel wall must be preformed and not derived via endothelial protein synthesis. Although to date, we have not been able to detect TF antigen on the plasma membrane of laser-activated HUVECs it is possible that the amount of TF antigen is below the limits of sensitivity of immunodetection. Alternatively, TF, like P-selectin, may be rapidly recycled from the plasma membrane to the cell interior. Nonetheless, we suspect that tissue factor is translocated to the endothelial cell surface following cell activation and that preformed endothelial cell tissue factor, following laser-induced injury of the endothelium, plays a critical role in thrombin generation and fibrin formation in this thrombosis model.

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