Thrombin Receptor Research Articles

Platelet dysfunction persists after trauma despite balanced blood product resuscitation

BackgroundPlatelet activation and aggregation are critical to the initiation of hemostasis after trauma with hemorrhage. Platelet dysfunction is a well-recognized phenomenon contributing to trauma-induced coagulopathy. The goal of this study was to evaluate the timing and severity of platelet dysfunction in massively transfused, traumatically injured patients during the first 72 hours after injury and its association with 30-day survival. MethodsA retrospective secondary cohort study of platelet count and function was performed using samples from the Pragmatic Randomized Optimal Platelet and Plasma Ratios trial. Platelet characteristics were measured at 8 timepoints during the first 72 hours of hospitalization and compared between 30-day survivors and nonsurvivors. Platelet counts were assessed via flow cytometry. Platelet function was analyzed with the use of serial thrombelastography and impedance aggregometry with agonists arachidonic acid, adenosine diphosphate, collagen, thrombin receptor activating peptide, and ristocetin. ResultsIn total, 680 patients were included for analysis. Platelet counts were significantly lower from baseline to 72 hours after hospital admission with further 1.3 to 2-fold reductions noted in nonsurvivors compared to survivor patients. Platelet aggregation via adenosine diphosphate, arachidonic acid, collagen, thrombin receptor activating peptide, and ristocetin was significantly lower in nonsurvivors at all time points. The nadir of platelet aggregation was 2 to 6 hours after admission with significant improvements in viscoelastic maximum clot formation and agonist-induced aggregation by 12 hours without concomitant improvement in platelet count. ConclusionPlatelet aggregability recovers 12 hours after injury independent of worsening thrombocytopenia. Failure of platelet function to recover portends a poor prognosis.

Platelet function testing using the Multiplate analyzer after administration of aspirin in Aachen minipigs.

Knowledge of platelet function in pigs and the effectiveness of antiplatelet therapy is important to ensure proper transferability from animal studies to humans. Our aim was to (1) characterize baseline platelet function of Aachen minipigs using the bedside Multiplate analyzer, (2) compare baseline platelet function with Göttingen minipigs, and (3) characterize platelet inhibition within the first 5 minutes after intravenous administration of acetylsalicylic acid (ASA). We characterized the baseline platelet function and hematological parameters in 9 Aachen minipigs. Historical data of 8 unmedicated Göttingen minipigs were used for comparison of baseline values. Platelet inhibition in Aachen minipigs was tested 1–5 minutes after intravenous administration of 500 mg ASA. Multiplate examinations included the following tests: ASPI test (to assess the effect of ASA), adenosine-diphosphate-test (ADP test) and thrombin receptor activating peptide test (TRAP test). Median values and interquartile range (IQR) of the Multiplate baseline tests in Aachen minipigs were as follows: ASPI: 39 U (IQR = 21–71), ADP: 70 U (IQR = 48–73), and TRAP: 8 U (IQR = 6–9), whereas the values in Göttingen minipigs were as follows: ASPI: 70.5 U (IQR = 60–78), ADP: 51 U (IQR = 45–66), and TRAP: 6.5 U (IQR = 4–8). ASPI values of Göttingen minipigs were significantly higher than those of Aachen minipigs (p = 0.046). Intravenous administration of ASA in Aachen minipigs resulted in significant platelet inhibition after 1 minute, which remained stable over a period of 5 minutes (p≤0.038). Aachen minipigs appeared to have a high variance in arachidonic acid-mediated platelet aggregation. In Aachen minipigs, intravenous ASA administration resulted in immediate platelet inhibition.

Thrombin receptor protease-activated receptor 1 (PAR1) related cytotoxic CD8+ T cell activity is associated with atrial myopathy and pro-inflammatory immune response in early atrial fibrillation

Abstract Background and aims Cytotoxic CD8+ T cells mediate myocardial damage in patients with virus induced myocarditis, plaque erosion during acute coronary syndrome, and promote adverse post-ischemic cardiac remodeling. There is emerging evidence to support a link between inflammation and atrial fibrillation (AF). Protease-activated receptor 1 (PAR1) is the thrombin receptor expressed on platelets, but it is also expressed in endothelial cells, vascular smooth muscle cells, cardiac fibroblasts, and CD8+ T cells. The role of CD8+ T cells and PAR1 in atrial myopathy and early AF have not been studied. Methods In 80 non-anticoagulated patients with a first documented episode of AF, and 20 control subjects without AF but with a comparable cardiovascular risk profile (e.g. SCORE2, CHA2DS2-VASc, p>0.05), we studied PAR1-related cytotoxic T cell activity in the first hours (up to 24 h) of AF clinical manifestation. The Apolipoprotein E–knock out (ApoEko) mouse is a commonly used model of metabolic syndrome that develops atrial myopathy (structural correlate of AF). ApoEko male mice (Apoetm1Unc with a C57BL/6 genetic background) were fed for six weeks with a standard chew, western-type diet (high fat and high sucrose = WD) ± the specific PAR1 inhibitor vorapaxar. Results High levels of circulating, activated CD8+ T cells (CD8+CD57+ T cells with pro-inflammatory and high cytotoxic properties) were present in patients with early AF (flow cytometry). Stimulation of cytotoxic T cells, that expressed the thrombin receptor PAR1, that were isolated from patients with new onset of AF, released pro-inflammatory cytokines (e.g. IFN-γ, TNF-α). Enhanced cytotoxic activity of CD8+ T cells was suggested by elevated plasma levels of effector molecules (e.g. granulysin and granzyme). In ApoEko mice fed a WD, atrial myopathy as the structural correlate for AF, was paralleled by increased expression of effector molecules that were associated with CD8+ T cell activity (like perforin, granzyme A, granzyme B, IFN-γ). Cytotoxic T cell activity disrupted the release of the potective atrial natriuretic peptide, ANP, and increased signaling pathways associated with atrial fibrosis (e.g. TGF-β, cTGF, TGF-β receptor). The specific PAR1 inhibitor, vorapaxar, reduced inflammatory T cell activity, transcription of fibrotic mediators, and reconstituted ANP secretion in atria of ApoEko mice. Conclusions Targeting the FXa/FIIa-PAR1-CD8+ axis might be a promising approach to reduce atrial fibrosis and inflammation. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): BIH Charité Clinician Scientist Program

Effect of coagulation factors FXIa, FXIIa, F alpha-XIIa, F beta-XIIa and FXIIIa on platelet function

Abstract Background FXa has recently been shown to active platelets via protease activated receptor. Direct inhibition of FXa leads to reduced platelet reactivity and arterial thrombosis. It is not known, if other coagulation factors besides FXa and FIIa directly activate platelets. In this study, we hypothesized that FXIa, FXIIa, Fα-XIIa, Fβ-XIIa and FXIIIa directly active platelets. Purpose Direct inhibitors of above mentioned factors are currently in clinical trials. Hence, investigation of coagulation factors' non-canonical effects beyond activation of the coagulation cascade are of great importance. Methods Platelet reactivity was measured using light transmission aggregometry (LTA) and flow cytometry. Ex-vivo stimulation of washed platelets was conducted using adenosine diphosphate (ADP) and thrombin receptor activator peptide (TRAP). Results FXIa, FXIIa, Fα-XIIa, Fβ-XIIa and FXIIIa have no direct effect on platelet function in terms of aggregation (Maximum of aggregation: FXIa: 2.799±1,823%, FXIIa: 1,174±1,080%, Fα-XIIa: 1,663±1,680%, Fβ-XIIa: 1,060±1,356%, Fα-XIIa + Fβ-XIIa 1,790±2,327% and FXIIIa: 1,345±1,060%). Also, incubation of platelets does not cause expression of P-selectin (FXIa: 4,346±4,650%, FXIIa: 7,072±6,098%, Fα-XIIa: 9,124±6,075%, Fβ-XIIa: 4,556±4,693%, Fα-XIIa + Fβ-XIIa 8,070±4,342% and FXIIIa: 5,166±5,512%). Conclusion FXIa, FXIIa, Fα-XIIa, Fβ-XIIa and FXIIIa have no direct effect on platelet function in terms of aggregation and P-selectin expression. Thus, existing pharmacological inhibitors of these factors are to be classified as purely anticoagulant and do not exert non-canonical effects on platelet activation. Funding Acknowledgement Type of funding sources: None.

Effect of low-dose rivaroxaban with low-dose aspirin vs low-dose aspirin on platelet and oxidative biomarkers: a randomized study in diabetes patients with stable peripheral or coronary artery disease

Abstract Background Rivaroxaban (Riva), a direct FXa inhibitor, at 2.5 mg twice-daily (bid) combined with low-dose aspirin (ASA, 100 mg once daily-od) reduced major vascular events vs. ASA alone in subjects with stable coronary artery (CAD) or symptomatic peripheral artery disease (PAD). Whether this benefit is due to the anticoagulant effect or additional FXa-mediated effects through the platelet and endothelial cell thrombin receptors is unknown. Type 2 diabetes mellitus (T2DM) is characterized by high platelet activation and oxidative stress that may contribute to increased cardiovascular risk. Purpose We investigated the effects of Riva (2.5 mg bid) + ASA (100 mg od) vs. ASA (100 mg od) alone on platelet and oxidative biomarkers in subjects with T2DM and stable vascular disease (stable CAD, symptomatic PAD and/or significant carotid stenosis). Methods In this randomized, open-label, cross-over trial, patients were randomized to continue ASA for 4 weeks and then add Riva for 4 weeks, or add Riva in the first 4 weeks and then continue with ASA alone for 4 weeks. Primary endpoints were: in vivo platelet activation and lipid peroxidation, assessed by the urinary excretion of 11-dehydro-TXB2 (TXM) and 8-iso-PGF2alpha (ISOP), respectively. Secondary endpoints included: routine coagulation tests, D-dimer, thrombin generation, serum TXB2, and Riva plasma concentrations. Results Seventy-6 subjects (10 females) were recruited: age 68±7 years (mean±SD); BMI 27.1±3.5 kg/m2; fasting glucose 129±31 mg/dL; HbA1c 6.8±0.9%; serum creatinine 1±0.25 mg/dL; LDL-cholesterol 77±31 mg/dL. Two patients dropped out: one for benign, self-limiting hematuria, one for unwillingness to continue, 8 subjects are completing the study leaving 66 who completed the 8-week randomized treatment and showed no sequence effect. Urinary TXM and ISOP were significantly reduced by Riva+ASA vs. ASA alone: TXM was 260 [195–398] vs. 335 [225–441] pg/mg creatinine and ISOP 722 [601–991] vs. 827 [648–1350] pg/mg creatinine (median [IQR]) on Riva+ASA vs. ASA alone, respectively (p<0.001 for paired samples). Riva plasma concentrations were 48±1.9 ng/ml at peak and 21±1.4 ng/ml at trough. The velocity of thrombin formation significantly decreased with Riva+ASA vs. ASA alone (velocity index, 46±3% vs. 83±3%; peak-height, 66±2% vs. 83±1%, respectively). aPTT levels were slightly but significantly prolonged by Riva vs. ASA alone (44±1 vs. 39±1 sec). Serum TXB2, D-dimer, von Willebrand factor, PT, fibrinogen and endogenous thrombin potential values were similar between treatments. Conclusions In ASA-treated subjects with T2DM and stable vascular disease, the addition of very low-dose Riva restrained incompletely-suppressed lipid peroxidation and platelet activation and modified the kinetics of thrombin formation. These changes may contribute to the beneficial effects of the Riva+ASA combination. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Investigator-initiated study funded by Bayer AG

Parameters of coagulation and platelet hyperreactivity reflect an increased mortality in patients with moderate to severe COVID-19

Abstract Background Hemostasis is dysregulated in patients with moderate-to-severe coronavirus disease 2019 (COVID-19). However, patients with respiratory diseases other than COVID-19 can also show disturbed coagulation reactions during the acute inflammatory process. Parameters of coagulation and platelet function are here compared between patients with upper respiratory infections with and without COVID-19 and are related to the clinical outcome. Methods Hospitalized patients with acute respiratory symptoms and with severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)-positive (COVpos) and SARS-CoV2-negative (COVneg) status were included. We assessed several parameters of coagulation and fibrinolysis as well as adenosine diphosphate (ADP)-, thrombin receptor activator peptide 6 (TRAP)-, and arachidonic acid (AA)-induced platelet reactivity by impedance aggregometry, as well as leukocyte subtype spectrum and platelet-leukocyte aggregates by flow cytometry and inflammatory cytokines by cytometric bead array. The SOFA score was assessed as marker of the clinical outcome. Results 87 patients were included in the study of which 50 were COVpos and 37 were COVneg. Von Willebrand factor was significantly higher in COVID positive patients compared to the control group (4456,4 mU/ml [2701,4; 9067,0] vs. 2528,0 mU/ml [1301,2; 3693,8], p<0,001). COVID-positive patients exhibited also more tissue plasminogen activator in the circulating blood than COVID-negative patients with an respiratory infection (11,4 mU/ml [7,2; 24,6] vs. 7,3 mU/ml [4,9; 10,5], p=0,001). ADP, TRAP-, and AA-induced platelet reactivity was significantly higher in COVpos than in COVneg patients. The SOFA score was higher in COVpos than in COVneg patients and again higher in deceased COVpos patients than in surviving COVpos. The SOFA score correlated significantly with parameters of coagulation and platelet function. A larger percentage of classical and intermediate monocytes, and of CD4pos T cells (TH) aggregated with platelets in COVpos than in COVneg patients. Interleukin (IL)-1 receptor antagonist (RA) and IL-6 levels were higher in COVpos than in COVneg patients and again higher in deceased COVpos patients than in surviving COVpos. IL-1RA and IL-6 levels correlated with the SOFA score in COVpos but not in COVneg patients, indicating a COVID-19-specific mechanism. Conclusion In moderate-to-severe COVID-19, but not in other respiratory diseases, disease severity was associated with parameters of coagulation and platelet function. Dysregulated coagulation and platelet hyperreactivity were associated to a worse clinical outcome in patients with COVID-19, pointing to the importance of antithrombotic therapy for reducing disease severity. Funding Acknowledgement Type of funding sources: None.

A Comprehensive Literature Review on Cardioprotective Effects of Bioactive Compounds Present in Fruits of Aristotelia chilensis Stuntz (Maqui).

Some fruits and vegetables, rich in bioactive compounds such as polyphenols, flavonoids, and anthocyanins, may inhibit platelet activation pathways and therefore reduce the risk of suffering from CVD when consumed regularly. Aristotelia chilensis Stuntz (Maqui) is a shrub or tree native to Chile with outstanding antioxidant activity, associated with its high content in anthocyanins, polyphenols, and flavonoids. Previous studies reveal different pharmacological properties for this berry, but its cardioprotective potential has been little studied. Despite having an abundant composition, and being rich in bioactive products with an antiplatelet role, there are few studies linking this berry with antiplatelet activity. This review summarizes and discusses relevant information on the cardioprotective potential of Maqui, based on its composition of bioactive compounds, mainly as a nutraceutical antiplatelet agent. Articles published between 2000 and 2022 in the following bibliographic databases were selected: PubMed, ScienceDirect, and Google Scholar. Our search revealed that Maqui is a promising cardiovascular target since extracts from this berry have direct effects on the reduction in cardiovascular risk factors (glucose index, obesity, diabetes, among others). Although studies on antiplatelet activity in this fruit are recent, its rich chemical composition clearly shows that the presence of chemical compounds (anthocyanins, flavonoids, phenolic acids, among others) with high antiplatelet potential can provide this berry with antiplatelet properties. These bioactive compounds have antiplatelet effects with multiple targets in the platelet, particularly, they have been related to the inhibition of thromboxane, thrombin, ADP, and GPVI receptors, or through the pathways by which these receptors stimulate platelet aggregation. Detailed studies are needed to clarify this gap in the literature, as well as to specifically evaluate the mechanism of action of Maqui extracts, due to the presence of phenolic compounds.

Retraction: Coagulation factor 2 thrombin receptor promotes malignancy in glioma under SOX2 regulation.

Glioma is the most common human primary brain cancer with high mortality and unfavorable clinical outcome. Coagulation factor 2 thrombin receptor (F2R), is a key component in the thrombosis process and has been demonstrated upregulated in various cancers. However, the effect and molecular mechanisms of F2R in glioma remains unclear. In our study, we confirmed that the expression of F2R was upregulated in glioma and predicted poor prognosis. Gene Set Enrichment Analysis (GSEA) and function assays demonstrated that F2R overexpression promoted glioma cell proliferation, metastasis and epithelial-mesenchymal transition (EMT) in vitro and tumor growth in vivo. Then, we identified and validated F2R was the target gene of SRY-box 2 (SOX2) by dual luciferase reporter assay and chromatin immunoprecipitation assay. Besides, High expression of F2R in malignant glioma was associated with β-catenint signaling pathway activation. Our findings conclude that F2R promotes glioma cell proliferation and metastasis under SOX2 and actives WNT/β-catenin Signaling pathway, which provides novel insight to the therapeutic regimen in glioma.

Platelet-Derived Procoagulant Microvesicles Are Elevated in Patients with Retinal Vein Occlusion (RVO).

The etiopathogenesis of retinal vein occlusion (RVO) is multifactorial, and the contribution of platelets to RVO development has not been fully elucidated. We aimed to analyze platelet function in RVO patients (n = 35) and controls (n = 35). We found a higher (p < 0.05) level of soluble P-selectin in RVO group vs. controls. Additionally, in RVO patients, the concentration of platelet-derived microvesicles was higher (p < 0.05), and the difference between groups was deeper for the fraction of platelet-derived microvesicles with the procoagulant phenotype (p < 0.0001) and for overall procoagulant microvesicles level (p < 0.0001). The results were similar for the total RVO group and for both RVO types (central- and branched-retinal vein occlusion). We did not find differences in simple platelet parameters (platelet count, mean platelet volume, platelet distribution width, platecrit, reticulated platelets) and inflammatory markers (platelet-lymphocyte ratio, neutrophil-lymphocyte ratio). Similarly, no differences were found for platelet aggregation-stimulated byadenosine diphosphate; collagen; arachidonic acid; and in multiparametric flow cytometry evaluation of P-selectin, PAC-1, and fibrinogen binding for both unstimulated and adenosine diphosphate-, collagen-, and thrombin receptor activating peptide-stimulated platelets. Our results suggest that platelets can contribute to developing RVO by enhancing procoagulant activity through providing a procoagulation surface via platelet-derived microvesicles. The direct role of platelets’ hyperreactivity in developing RVO is less apparent, which is consistent with the complexity and multifactorial background of this disorder.

Delayed administration of nafamostat mesylate inhibits thrombin-mediated blood–spinal cord barrier breakdown during acute spinal cord injury in rats

BackgroundNafamostat mesylate (nafamostat, NM) is an FDA-approved serine protease inhibitor that exerts anti-neuroinflammation and neuroprotective effects following rat spinal cord injury (SCI). However, clinical translation of nafamostat has been limited by an unclear administration time window and mechanism of action.MethodsTime to first dose of nafamostat administration was tested on rats after contusive SCI. The optimal time window of nafamostat was screened by evaluating hindlimb locomotion and electrophysiology. As nafamostat is a serine protease inhibitor known to target thrombin, we used argatroban (Arg), a thrombin-specific inhibitor, as a positive control in the time window experiments. Western blot and immunofluorescence of thrombin expression level and its enzymatic activity were assayed at different time points, as well its receptor, the protease activated receptor 1 (PAR1) and downstream protein matrix metalloproteinase-9 (MMP9). Blood–spinal cord barrier (BSCB) permeability leakage indicator Evans Blue and fibrinogen were analyzed along these time points. The infiltration of peripheral inflammatory cell was observed by immunofluorescence.ResultsThe optimal administration time window of nafamostat was 2–12 h post-injury. Argatroban, the thrombin-specific inhibitor, had a similar pattern. Thrombin expression peaked at 12 h and returned to normal level at 7 days post-SCI. PAR1, the thrombin receptor, and MMP9 were significantly upregulated after SCI. The most significant increase of thrombin expression was detected in vascular endothelial cells (ECs). Nafamostat and argatroban significantly downregulated thrombin and MMP9 expression as well as thrombin activity in the spinal cord. Nafamostat inhibited thrombin enrichment in endothelial cells. Nafamostat administration at 2–12 h after SCI inhibited the leakage of Evans Blue in the epicenter and upregulated tight junction proteins (TJPs) expression. Nafamostat administration 8 h post-SCI effectively inhibited the infiltration of peripheral macrophages and neutrophils to the injury site.ConclusionsOur study provides preclinical information of nafamostat about the administration time window of 2–12 h post-injury in contusive SCI. We revealed that nafamostat functions through inhibiting the thrombin-mediated BSCB breakdown and subsequent peripheral immune cells infiltration.

Outcomes After Acute Coronary Syndrome in Patients With Diabetes Mellitus and Peripheral Artery Disease (from the TRACER, TRILOGY-ACS, APPRAISE-2, and PLATO Clinical Trials)

Patients with acute coronary syndrome (ACS) are at risk for recurrent adverse events, and multiple reports suggest that this risk is increased in patients with concomitant diabetes mellitus (DM) and peripheral artery disease (PAD). The aim of this article was to investigate cardiovascular outcomes in patients with DM presenting with ACS, stratified by PAD status. Data were derived from 4 randomized post-ACS trials (PLATO [Platelet Inhibition and Patient Outcomes], APPRAISE-2 p Apixaban for Prevention of Acute Ischemic Events 2], TRILOGY [Targeted Platelet Inhibition to Clarify the Optimal Strategy to Medically Manage], and TRACER [Thrombin Receptor Agonist for Clinical Event Reduction in Acute Coronary Syndrome]). Using Cox regression analysis, we investigated major adverse cardiovascular events (MACEs), a composite of cardiovascular mortality, myocardial infarction (MI), or stroke and the individual components of MACE and all-cause mortality in patients with DM, presenting with ACS, stratified by PAD status as the risk modifier. This study included 15,387 patients with a diagnosis of DM and ACS, of whom 1,751 had an additional diagnosis of PAD. PAD was associated with more than doubled rates of MACE (hazard ratio [HR] 2.03, 95% confidence interval [CI] 1.81 to 2.27), all-cause mortality (HR 2.48, 95% CI 2.14 to 2.87), cardiovascular mortality (HR 2.42, 95% CI 2.04 to 2.86), and MI (HR 2.07, 95% CI 1.79 to 2.38). Patients with both PAD and DM were also more optimally treated with antihypertensive, antidiabetic, and statin medication at baseline. In conclusion, this analysis of 4 major post-ACS trials showed that patients with DM and PAD had a substantially higher risk of MACE, cardiovascular mortality, all-cause mortality, and MI despite being optimally treated with guideline-based therapies.

Induction of endothelial barrier dysfunction by serum factors in rats subjected to traumatic brain injury and hemorrhagic shock.

Traumatic brain injury (TBI) has been associated with the development of indirect acute respiratory distress syndrome (ARDS). However, the causative relationship between TBI and lung injury remains unclear. To explore potential mechanisms linking TBI with the development of ARDS, we characterized the effects of serum factors released following TBI and hemorrhagic shock (HS) in a rat model on the pulmonary endothelial cell (EC) barrier dysfunction, a key feature of ARDS. We found that serum samples from animals exposed to both controlled cortical impact (CCI) and HS, but not from sham‐operated rats induced significant barrier dysfunction in human pulmonary artery EC monolayers at 2 days post injury. Thrombin inhibitor and thrombin receptor antagonist attenuated the acute phase of the serum‐induced trans‐endothelial resistance (TER) decline caused by CCI‐HS serum, but not in later time points. However, both the early and late phases of CCI‐HS‐induced EC permeability were inhibited by heparin. The barrier disruptive effects of CCI‐HS serum were also prevented by serum preincubation with heparin‐sepharose. Pulmonary EC treated for 3 h with serum from CCI‐HS rats demonstrated a significant decline in expression of EC junctional protein, VE‐Cadherin, and disassembly of peripheral EC adherens junction complexes monitored by immunostaining with VE‐cadherin antibody. These results suggest that exposure to CCI‐HS causes early and late‐phase barrier disruptive effects in vascular endothelium. While thrombin‐PAR1 signaling has been identified as a mechanism of acute EC permeability increase by CCI‐HS serum, the factor(s) defining long‐term EC barrier disruption in CCI‐HS model remains to be determined.

Cleavage-Responsive Biofactory T Cells Suppress Infectious Diseases-Associated Hypercytokinemia.

Severe infectious diseases, such as coronavirus disease 2019 (COVID‐19), can induce hypercytokinemia and multiple organ failure. In spite of the growing demand for peptide therapeutics against infectious diseases, current small molecule‐based strategies still require frequent administration due to limited half‐life and enzymatic digestion in blood. To overcome this challenge, a strategy to continuously express multi‐level therapeutic peptide drugs on the surface of immune cells, is established. Here, chimeric T cells stably expressing therapeutic peptides are presented for treatment of severe infectious diseases. Using lentiviral system, T cells are engineered to express multi‐level therapeutic peptides with matrix metallopeptidases‐ (MMP‐) and tumor necrosis factor alpha converting enzyme‐ (TACE‐) responsive cleavage sites on the surface. The enzymatic cleavage releases γ‐carboxyglutamic acid of protein C (PC‐Gla) domain and thrombin receptor agonist peptide (TRAP), which activate endothelial protein C receptor (EPCR) and protease‐activated receptor‐1 (PAR‐1), respectively. These chimeric T cells prevent vascular damage in tissue‐engineered blood vessel and suppress hypercytokinemia and lung tissue damages in vivo, demonstrating promise for use of engineered T cells against sepsis and other infectious‐related diseases.

Discovery of 7, 4′-dimethoxy-3-hydroxyflavone as a protease-activated receptor 4 antagonist with antithrombotic activity and less bleeding tendency in mice

There is growing evidence of the importance of protease-activated receptor 4 (PAR4), one of thrombin receptors, as a therapeutic target in thrombotic cardiovascular diseases. In the present study, we utilized ligand-based virtual screening, bioassay, and structure–activity relationship study to discover PAR4 antagonists with new chemical scaffolds from natural origin, and examined their application as antiplatelet agents. By using these approaches, we have identified a flavonoid, 7, 4′-dimethoxy-3-hydroxyflavone, that exhibits anti-PAR4 activity. 7, 4′-Dimethoxy-3-hydroxyflavone inhibited PAR4-mediated human platelet aggregation, GPIIb/IIIa activation, and P-selectin secretion. Also, it inhibited PAR4 downstream signaling pathways, including Ca2+/protein kinase C, Akt, and MAP kinases ERK and p38, in human platelets, and suppressed PAR4-mediated β-arrestin recruitment in CHO-K1 cells exogenously expressed human PAR4. In a microfluidic system, 7, 4′-dimethoxy-3-hydroxyflavone reduced thrombus formation on collagen-coated chambers at an arterial shear rate in recalcified whole blood. Furthermore, mice treated with 7, 4′-dimethoxy-3-hydroxyflavone were significantly protected from FeCl3-induced carotid arterial occlusions, without significantly affecting tail bleeding time. In conclusion, 7, 4′-dimethoxy-3-hydroxyflavone represents a new class of nature-based PAR4 antagonist, it shows effective in vivo antithrombotic properties with less bleeding tendency, and could be a potential candidate for developing new antiplatelet agents.

Protease-activated receptor-mediated platelet aggregation in patients with type 2 diabetes on potent P2Y12 inhibitors.

BackgroundAntiplatelet therapy is a cornerstone in the secondary prevention of ischemic events following percutaneous coronary intervention (PCI). The new P2Y12 receptor inhibitors prasugrel and ticagrelor have been shown to improve patients' outcomes. Whether or not these drugs have equal efficacy in individuals with or without diabetes is disputed. Furthermore, platelets can be activated by thrombin, which is, at least in part, independent of P2Y12‐mediated platelet activation. Protease‐activated receptor (PAR)‐1 and −4 are thrombin receptors on human platelets. We sought to compare the in vitro efficacy of prasugrel (n = 121) and ticagrelor (n = 99) to inhibit PAR‐mediated platelet aggregation in individuals with type 2 diabetes (prasugrel n = 26, ticagrelor n = 29).Materials and MethodsWe compared P2Y12‐, PAR‐1‐ and PAR‐4‐mediated platelet aggregation as assessed by multiple electrode platelet aggregometry between prasugrel‐ and ticagrelor‐treated patients without and with type 2 diabetes who underwent acute PCI.ResultsOverall, there were no differences of P2Y12‐, PAR‐1‐ and PAR‐4‐mediated platelet aggregation between prasugrel‐ and ticagrelor‐treated patients. However, both drugs inhibited P2Y12‐mediated platelet aggregation stronger, and thereby to a similar extent in patients with type 2 diabetes than in those without diabetes. There was no correlation between either P2Y12‐, or PAR‐1‐ or PAR‐4‐mediated platelet aggregation and levels of HbA1c or the body mass index (BMI). However, we observed patients with high residual platelet reactivity in response to PAR‐1 and PAR‐4 stimulation in all cohorts.ConclusionPrasugrel and ticagrelor inhibit P2Y12‐ and PAR‐mediated platelet aggregation in individuals with diabetes to a similar extent, irrespective of HbA1c levels and BMI.

PAR2, not PAR1, regulates endothelium‐dependent vascular tone in resistance arteries

Protease activated receptors (PARs), a subfamily of G‐protein‐coupled receptors, are activated by cleavage of its extracellular domains. Four mammalian PAR members have been identified (PAR1‐4). PAR1 is the thrombin receptor, while PAR2 is activated physiologically by peptides or N‐terminal proteolytic cleavage. The resulting cleaved N‐termini acts as a tethered ligand that initiates receptor signaling. PARs may regulate vascular tone, cell permeability and cell proliferation. However, the precise roles of PAR1 and PAR2 in regulating vascular tone via the endothelium, and how PAR‐evoked signals transmit extracellularly and intracellularly, are incompletely understood.To explore the physiological functions, and the underlying signalling pathways in native endothelium, PAR1 (TFLLR‐NH2) and PAR2 (2‐Furoyl‐LIGRLO‐amide; 2fLI) activating peptides were used, and changes in intracellular Ca2+ and contractility measured in en face rat mesenteric arteries. Surprisingly, PAR1 activation (TFLLR‐NH2or trypsin) neither triggered Ca2+ signals in endothelial cells or alter vascular tone. On the other hand, PAR2 activation (2fLI) evoked large global propagating Ca2+ waves in endothelial cells and endothelium‐dependent dilation of mesenteric arteries. The Ca2+ signals evoked by PAR2 activation were kinetically distinctive when compared to those of muscarinic receptor activation. PAR2 activation (2fLI) also evoked responses in different clusters of endothelial cells from those of muscarinic receptor (acetylcholine) activation. PAR2 activation with 2fLI or trypsin failed to evoke Ca2+ signals after depletion of the internal store with the Ca2+‐ATPase (SERCA) inhibitor, CPA. This result suggests that PAR2 evokes Ca2+ release from internal stores. Pharmacological block of inositol 1,4,5‐trisphosphate (IP3) with either 2‐APB or caffeine, also blocked PAR2‐evoked Ca2+ signals. Phospholipase C inhibition (U73122) also blocked the Ca2+ signals evoked by PAR2 activation. Therefore, PAR2 is a PLC linked G protein coupled receptor which evokes Ca2+ release from the internal store via IP3 receptors in endothelial cells. The TRPC3 blocker, PYR3 and store‐operated Ca2+ entry (SOCE) blocker, YM58483 each significantly supressed PAR2 mediated Ca2+ signals, suggesting internal store was replenished via TRPC3 channel and SOCE.In conclusion, PAR2 rather than PAR1, regulates vascular tone in mesenteric arteries by evoking Ca2+ release from the internal store via IP3 receptors to generate propagating Ca2+ waves in distinct sub‐populations of endothelial cells.

Serum from rats subjected to traumatic brain injury and hemorrhagic shock induces profound endothelial barrier dysfunction

IntroductionTraumatic brain injury (TBI) has been associated with development of indirect acute respiratory distress syndrome (ARDS). However, the causative relationship between TBI and lung injury remains unclear. To explore potential mechanism linking TBI with development of ARDS, we characterized effects of serum factors released during TBI and hemorrhagic shock (HS) in a rat model on the pulmonary endothelial cell (EC) barrier dysfunction, a key feature of ARDS.MethodsThe adult male rat polytrauma model consisted of controlled cortical impact (CCI)–induced TBI followed by 30 minutes of hemorrhage shock (HS) induced by blood withdrawal. The HS phase was followed by a 1‐hour “prehospital” Hextend fluid resuscitation phase and then a 1‐hour “hospital phase” when shed blood was reinfused. 48 hours after the TBI, blood was drawn from right ventricle and serum samples were obtained. Serum was collected from the TBI or sham rats, and its effects on barrier properties of pulmonary endothelial cell culture were assessed by measurements of transendothelial electrical resistance (TER) using the electrical cell impedance sensor and visualization of fluorescent tracer penetration through EC monolayer using XPerT assay.ResultsWe found pronounced barrier‐disruptive effects of serum samples from animals exposed to controlled cortical impact (CCI) and HS, but not from sham operated rats. Rapid and sustained decrease in EC monolayer transendothelial resistance was observed within minutes after serum addition and remained below basal TER levels after incubation with CCI‐HS sera. Thrombin has been implicated in the early‐phase barrier disruptive activity of CCI‐HS serum, as both thrombin inhibitor and thrombin receptor antagonist attenuated the acute phase of serum‐induced TER decline, but did not affect TER decline at later time points. These findings suggest that thrombin elevation may play a role in early‐phase barrier disruptive activity of CCI‐HS serum. However, both the early and late phases of CCI‐HS‐induced EC permeability were inhibited by heparin. The late phase barrier disruptive effect of CCI‐HS was also prevented by serum preincubation with heparin‐sepharose. Pulmonary EC treated with serum from CCI‐HS rats for 3 hours have demonstrated significant decline in expression of EC junctional protein, VE‐Cadherin, and disassembly of peripheral EC adherens junction complexes monitored by immunostaining with VE‐cadherin antibody.ConclusionThese results suggest that exposure to CCI‐HS leads to production of humoral factors causing early and late‐phase barrier disruptive effects in lung vascular endothelium. While thrombin‐PAR1 signaling has been identified as mechanism of acute EC permeability increase by CCI‐HS serum, the factor(s) defining long‐term EC barrier disruption in CCI‐HS model remain to be determined.

Abstract 337: Contribution Of Platelet Par4 To Influenza A Virus Induced Lung Pathology In Mice

The influenza A virus (IAV) causes a seasonal respiratory tract infection and is characterized by excessive inflammation and tissue pathology in the lungs. Protease-activated receptor 4 (PAR4) is expressed megakaryocytes/platelets, immune cells, and lung epithelial cells and it is the only functional thrombin receptor on murine platelets. Studies showed that platelets contribute to the pathogenesis of IAV. However, the mechanisms are unknown. While platelet inhibition in IAV infection was protective, a global PAR4 deficiency in mice was associated with increased IAV pathology including mortality. The objective of this study is to investigate the contribution of platelet PAR4 to IAV pathology. Mice (both sex) with PAR4 deficiency on megakaryocytes/platelets (PAR4 fl/fl ;PF4 Cre+ ) and controls (PAR4 fl/fl ) were infected with mouse adapted IAV (H1N1/PR8) at a dose, which induces 20% mortality in control mice. Inflammatory mediators and cellular inflammation in bronchoalveolar lavage fluid (BALF) was analyzed before, 3, 5 and 7 days post infection (dpi). Body weight changes were analyzed over 14 dpi and survival calculated with a human endpoint of a body weight loss of ≥25% initial weight. Lastly, lung function was analyzed by noninvasive whole-body plethysmography at 0 and 7 dpi. There were no significant differences in the analyzed inflammatory mediators, except for IFNγ, IL6, and protein, or any immune cell numbers in the BALF of both genotypes at any time point after infection. Both genotypes exhibited similar body weight changes over the course of infection which resulted that mice of both genotypes exhibited a mortality of approx. 20%. However, the lung function parameters calculated as PenH (airway resistance) and EF50 (flow rate at 50% expired volume) were significant higher in PAR4 fl/fl ;PF4 Cre+ compared to PAR4 fl/fl mice 7 dpi. Platelet PAR4 does not influence pathology in a mild IAV infection model. However, the more severe pulmonary dysfunction in PAR4 fl/fl ;PF4 Cre+ compared to PAR4 fl/fl mice suggest a protective role of platelet PAR4 in maintaining lung function during IAV infection. More studies are needed to investigate if the contribution of platelet PAR4 to IAV pathology is dependent on disease severity as recently suggested.

Thrombin induces morphological and inflammatory astrocytic responses via activation of PAR1 receptor

Spinal cord injury (SCI) will result in the significant elevation of thrombin production at lesion site via either breakage of blood-spinal cord barrier or upregulated expression within nerve cells. Thrombin-induced activation of the protease activated receptors (PARs) evokes various pathological effects that deteriorate the functional outcomes of the injured cord. The cellular consequences of thrombin action on the astrocytes, as well as the underlying mechanism are not fully elucidated by far. In the present study, SCI model of rats was established by contusion, and primary astrocytes were isolated for culture from newborn rats. The expression levels of thrombin and PAR1 receptor at lesion sites of the spinal cord were determined. The primary astrocytes cultured in vitro were stimulated with different concentration of thrombin, and the resultant morphological changes, inflammatory astrocytic responses, as well as PAR1-activated signal pathway of astrocytes were accordingly examined using various agonists or antagonists of the receptor. Thrombin was found to reverse astrocytic stellation, promote proliferation but inhibit migration of astrocytes. Furthermore, the serine protease was shown to facilitate inflammatory response of astrocytes through regulation of MAPKs/NFκB pathway. Our results have provided the morphological evidence of astrocytic reactivity in response to thrombin stimulation and its neuroinflammatory effects following SCI, which will be indicative for the fundamental insights of thrombin-induced neuropathology.

207 Omega-3 and omega-6 fatty acids attenuate platelet reactivity in postmenopausal women

OBJECTIVES/GOALS: This study aimed to investigate the mechanistic effects of fish oil (âμ-3 fatty acids) or evening primrose oil (âμ-6 fatty acids) supplementation on platelet reactivity in postmenopausal women. METHODS/STUDY POPULATION: Postmenopausal women were recruited from the Ann Arbor community and the University of Michigan Medicine Center. All subjects were recruited under study protocols approved by the University of Michigan IRB between November 2015 and March 2017. We conducted a randomized, double-blind, two-period crossover trial, consisting of a 60-day supplementation period followed by a 14-day washout period in between and at the end of the study. Subjects were treated daily in random order with 2g of fish oil supplement and 2g of evening primrose oil. Blood was drawn at baseline, post-supplementation, and after washout. The effects of fatty acid supplementation on platelet aggregation, dense granule secretion and activation of basal integrin âºIIbÎ23 were assessed following supplementation and washout period. RESULTS/ANTICIPATED RESULTS: The study started with 90 postmenopausal women. A total of 78 subjects completed the study, with 12 subjects dropping out due to non-compliance and medical reasons. Supplementation with fish oil attenuated the thrombin receptor PAR4-induced platelet aggregation, whereas primrose oil supplementation attenuated aggregation mediated by PAR4 or collagen. Supplementation with âμ-3 or âμ-6 fatty acids decreased platelet dense granule secretion and attenuated basal levels of integrin âºIIbÎ23 activation. Post-washout following supplementation with primrose oil, the thrombin receptor PAR1-induced platelet aggregation was similarly attenuated. For either treatment, the observed effects post supplementation on dense granule secretion and basal integrin activation were sustained after the washout. DISCUSSION/SIGNIFICANCE: Postmenopausal women are at increased risk for a cardiovascular event due to platelet hyperactivity. This study indicates that supplementation with âμ-3 and âμ-6 fatty acids may offer significant protection for postmenopausal women against cardiovascular diseases and occlusive thrombotic events by reducing platelet reactivity.