A mouse model of the protease-activated receptor 4 Pro310Leu variant has reduced platelet reactivity.

BACKGROUND: Protease-activated receptor 4 (PAR4) mediates thrombin signaling on platelets and other cells. Our recent structural studies demonstrated that a single nucleotide polymorphism in extracellular loop 3 and PAR4-P310L (rs2227376) leads to a hyporeactive receptor. OBJECTIVES: The goal of this study was to determine how the hyporeactive PAR4 variant in extracellular loop 3 impacts platelet function in vivo using a novel knock-in mouse model (PAR4-322L). METHODS: A point mutation was introduced into the PAR4 gene F2rl3 via CRISPR/Cas9 to create PAR4-P322L, the mouse homolog to human PAR4-P310L. Platelet response to PAR4 activation peptide (AYPGKF), thrombin, ADP, and convulxin was monitored by αIIbß3 integrin activation and P-selectin translocation using flow cytometry or platelet aggregation. In vivo responses were determined by the tail bleeding assay and the ferric chloride-induced carotid artery injury model. RESULTS: PAR4-P/L and PAR4-L/L platelets had a reduced response to AYPGKF and thrombin measured by P-selectin translocation or αIIbß3 activation. The response to ADP and convulxin was unchanged among genotypes. In addition, both PAR4-P/L and PAR4-L/L platelets showed a reduced response to thrombin in aggregation studies. There was an increase in the tail bleeding time for PAR4-L/L mice. The PAR4-P/L and PAR4-L/L mice both showed an extended time to arterial thrombosis. CONCLUSION: PAR4-322L significantly reduced platelet responsiveness to AYPGKF and thrombin, which is in agreement with our previous structural and cell signaling studies. In addition, PAR4-322L had prolonged arterial thrombosis time. Our mouse model provides a foundation to further evaluate the role of PAR4 in other pathophysiological contexts.

PARIN5, a Novel Thrombin Receptor Antagonist Modulates a Streptozotocin Mice Model for Diabetic Encephalopathy.

Diabetic encephalopathy (DE) is an inflammation-associated diabetes mellitus (DM) complication. Inflammation and coagulation are linked and are both potentially modulated by inhibiting the thrombin cellular protease-activated receptor 1 (PAR1). Our aim was to study whether coagulation pathway modulation affects DE. Diabetic C57BL/6 mice were treated with PARIN5, a novel PAR1 modulator. Behavioral changes in the open field and novel object recognition tests, serum neurofilament (NfL) levels and thrombin activity in central and peripheral nervous system tissue (CNS and PNS, respectively), brain mRNA expression of tumor necrosis factor α (TNF-α), Factor X (FX), prothrombin, and PAR1 were assessed. Subtle behavioral changes were detected in diabetic mice. These were accompanied by an increase in serum NfL, an increase in central and peripheral neural tissue thrombin activity, and TNF-α, FX, and prothrombin brain intrinsic mRNA expression. Systemic treatment with PARIN5 prevented the appearance of behavioral changes, normalized serum NfL and prevented the increase in peripheral but not central thrombin activity. PARIN5 treatment prevented the elevation of both TNF-α and FX but significantly elevated prothrombin expression. PARIN5 treatment prevents behavioral and neural damage in the DE model, suggesting it for future clinical research.

Repeated platelet activation and the potential of previously activated platelets to contribute to thrombus formation.

BACKGROUND: Especially in disease conditions, platelets can encounter activating agents in circulation. OBJECTIVES: To investigate the extent to which previously activated platelets can be reactivated and whether in-and reactivation applies to different aspects of platelet activation and thrombus formation. METHODS: Short-and long-term effects of glycoprotein VI (GPVI) and G protein-coupled receptor (GPCR) stimulation on platelet activation and aggregation potential were compared via flow cytometry and plate-based aggregation. Using fluorescence and electron microscopy, we assessed platelet morphology and content, as well as thrombus formation. RESULTS: After 30 minutes of stimulation with thrombin receptor activator peptide 6 (TRAP6) or adenosine diphosphate (ADP), platelets secondarily decreased in PAC-1 binding and were less able to aggregate. The reversibility of platelets after thrombin stimulation was concentration dependent. Reactivation was possible via another receptor. In contrast, cross-linked collagen-related peptide (CRP-XL) or high thrombin stimulation evoked persistent effects in αIIbß3 activation and platelet aggregation. However, after 60 minutes of CRP-XL or high thrombin stimulation, when αIIbß3 activation slightly decreased, restimulation with ADP or CRP-XL, respectively, increased integrin activation again. Compatible with decreased integrin activation, platelet morphology was reversed. Interestingly, reactivation of reversed platelets again resulted in shape change and if not fully degranulated, additional secretion. Moreover, platelets that were previously activated with TRAP6 or ADP regained their potential to contribute to thrombus formation under flow. On the contrary, prior platelet triggering with CRP-XL was accompanied by prolonged platelet activity, leading to a decreased secondary platelet adhesion under flow. CONCLUSION: This work emphasizes that prior platelet activation can be reversed, whereafter platelets can be reactivated through a different receptor. Reversed, previously activated platelets can contribute to thrombus formation.

TRAP-induced PAR1 expression with its mechanism during AMI in a rat model.

BACKGROUND AND OBJECTIVE: Protease-activated receptor 1 (PAR1) is crucial in individuals with acute myocardial infarction (AMI). The continuous and prompt PAR1 activation mainly dependent on PAR1 trafficking is essential for the role of PAR1 during AMI in which cardiomyocytes are in hypoxia. However, the PAR1 trafficking in cardiomyocytes specially during the hypoxia is still unclear. METHODS AND RESULT: A rat AMI model was created. PAR1 activation with thrombin-receptor activated peptide (TRAP) had a transient effect on cardiac function in normal rats but persistent improvement in rats with AMI. Cardiomyocytes from neonatal rats were cultured in a normal CO2 incubator and a hypoxic modular incubator chamber. The cells were then subjected to western blot for the total protein expression and staining with fluorescent reagent and antibody for PAR1 localization. No change in total PAR1 expression following TRAP stimulation was observed; however, it led to increased PAR1 expression in the early endosomes in normoxic cells and decreased expression in the early endosomes in hypoxic cells. Under hypoxic conditions, TRAP restored the PAR1 expression on both cell and endosomal surfaces within an hour by decreasing Rab11A (8.5-fold; 179.93 ± 9.82% of the normoxic control group, n = 5) and increasing Rab11B (15.5-fold) expression after 4 h of hypoxia. Similarly, Rab11A knockdown upregulated PAR1 expression under normoxia, and Rab11B knockdown downregulated PAR1 expression under both normoxic and hypoxic conditions. Cardiomyocytes knocked out of both Rab11A, and Rad11B lost the TRAP-induced PAR1 expression but still exhibited the early endosomal TRAP-induced PAR1 expression under hypoxia. CONCLUSIONS: TRAP-mediated activation of PAR1 in cardiomyocytes did not alter the total PAR1 expression under normoxic conditions. Instead, it triggers a redistribution of PAR1 levels under normoxic and hypoxic conditions. TRAP reverses the hypoxia-inhibited PAR1 expression in cardiomyocytes by downregulating Rab11A expression and upregulating Rab11B expression.

PARs in the inflammation-cancer transformation of CRC.

Colorectal cancer (CRC) is one of the common malignancies with a global trend of increasing incidence and mortality. There is an urgent need to identify new predictive markers and therapeutic targets for the treatment of CRC. Protease-activated receptors (PARs) are a class of G-protein-coupled receptors, with currently identified subtypes including PAR1, PAR2, PAR3 and PAR4. Increasingly, studies suggest that PARs play an important role in the growth and metastasis of CRC. By targeting multiple signaling pathways may contribute to the pathogenesis of CRC. In this review, we first describe recent studies on the role of PARs in CRC inflammation-cancer transformation, focusing on the important role of PARs in signaling pathways associated with inflammation-cancer transformation, and summarize the progress of research on PARs-targeted drugs.

PH-Binding Motif in PAR4 Oncogene: From Molecular Mechanism to Drug Design.

While the role of G-protein-coupled receptors (GPCR) in cancer is acknowledged, their underlying signaling pathways are understudied. Protease-activated receptors (PAR), a subgroup of GPCRs, form a family of four members (PAR1-4) centrally involved in epithelial malignancies. PAR4 emerges as a potent oncogene, capable of inducing tumor generation. Here, we demonstrate identification of a pleckstrin-homology (PH)-binding motif within PAR4, critical for colon cancer growth. In addition to PH-Akt/PKB association, other PH-containing signal proteins such as Gab1 and Sos1 also associate with PAR4. Point mutations are in the C-tail of PAR4 PH-binding domain; F347 L and D349A, but not E346A, abrogate these associations. Pc(4-4), a lead backbone cyclic peptide, was selected out of a mini-library, directed toward PAR2&4 PH-binding motifs. It effectively attenuates PAR2&4-Akt/PKB associations; PAR4 instigated Matrigel invasion and migration in vitro and tumor development in vivo. EGFR/erbB is among the most prominent cancer targets. AYPGKF peptide ligand activation of PAR4 induces EGF receptor (EGFR) Tyr-phosphorylation, effectively inhibited by Pc(4-4). The presence of PAR2 and PAR4 in biopsies of aggressive breast and colon cancer tissue specimens is demonstrated. We propose that Pc(4-4) may serve as a powerful drug not only toward PAR-expressing tumors but also for treating EGFR/erbB-expressing tumors in cases of resistance to traditional therapies. Overall, our studies are expected to allocate new targets for cancer therapy. Pc(4-4) may become a promising candidate for future therapeutic cancer treatment.

Proteinase-activated receptors regulate intestinal functions in a segment-dependent manner in rats.

Proteinases released e.g. during inflammatory or allergic responses affect gastrointestinal functions via proteinase-activated receptors such as PAR1 and PAR2. As the gastrointestinal tract exerts pronounced gradients along its longitudinal axis, the present study focuses on the effect of PAR1 and PAR2 agonists on electrogenic ion transport (measured as short-circuit current; Isc), tissue conductance (Gt) and contractility of the longitudinal muscle layer of rats. In Ussing chamber experiments, the PAR1 agonist TFLLR-NH2, which mimics the tethered ligand liberated after cleavage of the receptor, evoked only a modest increase in Isc (<0.5 µEq·h-1·cm-2) in small intestine, but a strong increase (3-4 µEq·h-1·cm-2) in colon. Pretreatment with tetrodotoxin reduced the response of the colonic segments to the level of the small intestine. Thrombin, the natural activator of PAR1, was much less effective suggesting biased activation by this peptidase. A similar gradient along the longitudinal axis of the intestine was observed with trypsin, the endogenous activator of PAR2. Divergent actions of PAR1 activation by enzymatic cleavage or a mimetic peptide were also observed when recording isometric contractions of longitudinal muscle. For example, in the jejunum TFLLR-NH2 concentration-dependently induced a contractile response, whereas thrombin showed only inconsistent effects. The PAR2 activator AC264613 induced a concentration-dependent decrease in muscle tone combined with an inhibition of phasic spontaneous contractions. PCR experiments and immunohistochemical stainings confirmed the expression of PAR1 and PAR2. The data implies that PAR1 and PAR2 functions vary depending on the intestinal segment.

Identification of a Distinct Platelet Phenotype in the Elderly: ADP Hypersensitivity Coexists With Platelet PAR (Protease-Activated Receptor)-1 and PAR-4-Mediated Thrombin Resistance.

BACKGROUND: Thrombin (via PAR [protease-activated receptor]-1 and PAR-4) and ADP (via P2Y12 receptors) are potent endogenous platelet activators implicated in the development of cardiovascular disease. We aimed to assess whether platelet pathways alter with aging. METHODS: We characterized platelet activity in community-dwelling volunteers (n=174) in the following age groups: (1) 20 to 30 (young); (2) 40 to 55 (middle-aged); (3) ≥70 years (elderly). Platelet activity was assessed by aggregometry; flow cytometry (surface markers [P-selectin: alpha granule release, CD63: dense granule release, PAC-1: measure of conformationally active GPIIb/IIIa at the fibrinogen binding site]) measured under basal conditions and after agonist stimulation [ADP, thrombin, PAR-1 agonist or PAR-4 agonist]); receptor cleavage and quantification; fluorometry; calcium flux; ELISA. RESULTS: The elderly had higher basal platelet activation than the young, evidenced by increased expression of P-selectin, CD63, and PAC-1, which correlated with increasing inflammation (IL [interleukin]-1ß/IL-6). The elderly demonstrated higher P2Y12 receptor density, with greater ADP-induced platelet aggregation (P<0.05). However, elderly subjects were resistant to thrombin, achieving less activation in response to thrombin (higher EC50) and to selective stimulation of both PAR-1 and PAR-4, with higher basal PAR-1/PAR-4 cleavage and less inducible PAR-1/PAR-4 cleavage (all P<0.05). Thrombin resistance was attributable to a combination of reduced thrombin orienting receptor GPIbα (glycoprotein Ibα), reduced secondary ADP contribution to thrombin-mediated activation, and blunted calcium flux. D-Dimer, a marker of in situ thrombin generation, correlated with platelet activation in the circulation, ex vivo thrombin resistance, and circulating inflammatory mediators (TNF [tumor necrosis factor]-α/IL-6). CONCLUSIONS: Aging is associated with a distinctive platelet phenotype of increased basal activation, ADP hyperreactivity, and thrombin resistance. In situ thrombin generation associated with systemic inflammation may be novel target to prevent cardiovascular disease in the elderly.

Triggering receptor expressed on myeloid cells (TREM) like transcript-1 (TLT-1) reveals platelet activation in preeclampsia.

Triggering receptor expressed on myeloid cells (TREM) like transcript-1 (TLT-1) is a membrane protein receptor found in α-granules of megakaryocytes and platelets. Upon platelet activation TLT-1 is rapidly relocated to the surface of platelets. In plasma, a soluble form of TLT-1 (sTLT-1) is present. Plasma levels of sTLT-1 are significantly elevated in thrombotic diseases. In the present study, we investigated to whether TLT-1 reflects platelet activation in pregnant women with preeclampsia. We studied 30 preeclamptic patients who were matched with 30 normotensive pregnant women and 30 non-pregnant controls. Basal TLT-1, P-selectin, and CD63 expressions on platelets were analyzed with the use of flow-cytometry (FCM). Platelet reactivity was induced by thrombin receptor activation peptide and determined by FCM. Plasma concentrations of sTLT-1 and soluble P-selectin (sP-selectin) were measured by an enzyme-linked immunosorbent assay. Results show that basal platelet expression of TLT-1, P-selectin and CD63 were increased in women with preeclampsia (PE) compared with normotensive pregnant women (NP). Platelets from PE women and NP women were more responsive compared to from nonpregnant women controls (NC), and which was demonstrated by increased expression of TLT-1, P-selectin, and CD63 upon stimulation in vitro. Plasma concentration of sTLT-1 was greater in PE women compared to NP women and NC women. Plasma sP-selectin level was higher in pregnant women than in nonpregnant women, but there were no significant differences between PE and NP women. In summary, our results revealed that platelet activation is prominent in preeclampsia, TLT-1 reflects platelet activation and may be a useful indicator for preeclampsia.

Peripheral versus central venous blood sampling does not influence the assessment of platelet activation in cirrhosis.

Cirrhotic patients have an increased risk of bleeding and thromboembolic events, with platelets being involved as key players in both situations. The impact of peripheral versus central blood sampling on platelet activation remains unclear. In 33 cirrhotic patients, we thus analyzed platelet function in peripheral (P) and central (C) blood samples. Platelet surface expression of P-selectin, activated glycoprotein (GP) IIb/IIIa, and leukocyte-platelet aggregate formation were measured by flow cytometry in response to different agonists: thrombin receptor-activating peptide-6, adenosine diphosphate, collagen-related peptide (CrP), epinephrine, AYPGKF, Pam3CSK4, and lipopolysaccharide. Unstimulated platelet surface expression of P-selectin (p = .850) and activated GPIIb/IIIa (p = .625) were similar in peripheral and central blood samples. Stimulation with various agonists yielded similar results of platelet surface expression of P-selectin and activated GPIIb/IIIa in peripheral and central samples, except for CrP-inducible expression of activated GPIIb/IIIa (median fluorescence intensity, MFI in P: 7.61 [0.00-24.66] vs. C: 4.12 [0.00-19.04], p < .001). The formation of leukocyte-platelet aggregate was similar in central and peripheral blood samples, both unstimulated and after stimulation with all above-mentioned agonists. In conclusion, peripheral vs. central venous blood sampling does not influence the assessment of platelet activation by flow cytometry in cirrhosis.Abbreviations: ACLD: advanced chronic liver disease; ADP: adenosine diphosphate; ALD: alcoholic liver disease; AYPGKF: PAR-4 agonist AYPGKF; CrP: collagen related protein; EPI: epinephrine; FACS: fluorescence-activated cell sorting; GP: glycoprotein; HVPG: hepatic venous pressure gradient; IQR: interquartile range; LPS: lipopolysaccharide; LSM: liver stiffness measurement; MFI: median fluorescence intensity; NAFLD: nonalcoholic fatty liver disease; PAM: lipopeptide Pam3CSK4; PAR: protease-activated receptor; PBS: phosphate-buffered saline; PH: portal hypertension; TIPS: transjugular intrahepatic portosystemic stent shunt; TLR: toll-like receptor; TRAP-6: thrombin receptor-activator peptide-6; vWF: von Willebrand factor.

PAR4-Mediated PI3K/Akt and RhoA/ROCK Signaling Pathways Are Essential for Thrombin-Induced Morphological Changes in MEG-01 Cells.

Thrombin stimulates platelets via a dual receptor system of protease-activated receptors (PARs): PAR1 and PAR4. PAR1 activation induces a rapid and transient signal associated with the initiation of platelet aggregation, whereas PAR4 activation results in a prolonged signal, required for later phases, that regulates the stable formation of thrombus. In this study, we observed differential signaling pathways for thrombin-induced PAR1 and PAR4 activation in a human megakaryoblastic leukemia cell line, MEG-01. Interestingly, thrombin induced both calcium signaling and morphological changes in MEG-01 cells via the activation of PAR1 and PAR4, and these intracellular events were very similar to those observed in platelets shown in previous studies. We developed a novel image-based assay to quantitatively measure the morphological changes in living cells, and observed the underlying mechanism for PAR1- and PAR4-mediated morphological changes in MEG-01 cells. Selective inhibition of PAR1 and PAR4 by vorapaxar and BMS-986120, respectively, showed that thrombin-induced morphological changes were primarily mediated by PAR4 activation. Treatment of a set of kinase inhibitors and 2-aminoethoxydiphenyl borate (2-APB) revealed that thrombin-mediated morphological changes were primarily regulated by calcium-independent pathways and PAR4 activation-induced PI3K/Akt and RhoA/ROCK signaling pathways in MEG-01 cells. These results indicate the importance of PAR4-mediated signaling pathways in thrombin-induced morphological changes in MEG-01 cells and provide a useful in vitro cellular model for platelet research.

Quantification of platelet function - a comparative study of venous and arterial blood using a novel flow cytometry protocol.

Studies of platelet function in surgical patients often involve both arterial and venous sampling. Possible effects of different sampling sites could be important, but have not been thoroughly investigated. We aimed to compare platelet function in arterial and venous blood samples using a novel flow cytometry protocol and impedance aggregometry. Arterial and venous blood was collected before anesthesia in 10 patients undergoing cardiac surgery of which nine was treated with acetylsalicylic acid until the day before surgery. Flow cytometry included simultaneous analysis of phosphatidylserine exposure, active conformation of the fibrinogen receptor (PAC-1 binding), α-granule and lysosomal release (P-selectin and LAMP-1 exposure) and mitochondrial membrane integrity. Platelets were activated with ADP or peptides activating thrombin receptors (PAR1-AP/PAR4-AP) or collagen receptor GPVI (CRP-XL). Leukocyte-platelet conjugates and P-selectin exposure were evaluated immediately in fixated samples. For impedance aggregometry (Multiplate®), ADP, arachidonic acid, collagen and PAR1-AP (TRAP) were used as activators. Using impedance aggregometry and in 27 out of 37 parameters studied with flow cytometry there was no significant difference between venous and arterial blood sampling. Arterial blood showed more PAC-1 positive platelets when activated with PAR1-AP or PAR4-AP and venous blood showed more monocyte-platelet and neutrophil-platelet conjugates and higher phosphatidylserine exposure with CRP-XL alone and combined with PAR1-AP or PAR4-AP. We found no differences using impedance aggregometry. In conclusion, testing of platelet function by flow cytometry and impedance aggregometry gave comparable results for most of the studied parameters in venous and arterial samples. Flow cytometry identified differences in PAC-1 binding when activated with PAR1-AP, exposure of phosphatidyl serine and monocyte/neutrophil-platelet conjugates, which might reflect differences in blood sampling technique or in flow conditions in this patient cohort with coronary artery disease. These differences might be considered when comparing data from different sample sites, but caution should be exercised if a different protocol is used or another patient group is studied.

Epigenetic Regulation of F2RL3 Associates With Myocardial Infarction and Platelet Function.

BACKGROUND: DNA hypomethylation at the F2RL3 (F2R like thrombin or trypsin receptor 3) locus has been associated with both smoking and atherosclerotic cardiovascular disease; whether these smoking-related associations form a pathway to disease is unknown. F2RL3 encodes protease-activated receptor 4, a potent thrombin receptor expressed on platelets. Given the role of thrombin in platelet activation and the role of thrombus formation in myocardial infarction, alterations to this biological pathway could be important for ischemic cardiovascular disease. METHODS: We conducted multiple independent experiments to assess whether DNA hypomethylation at F2RL3 in response to smoking is associated with risk of myocardial infarction via changes to platelet reactivity. Using cohort data (N=3205), we explored the relationship between smoking, DNA hypomethylation at F2RL3, and myocardial infarction. We compared platelet reactivity in individuals with low versus high DNA methylation at F2RL3 (N=41). We used an in vitro model to explore the biological response of F2RL3 to cigarette smoke extract. Finally, a series of reporter constructs were used to investigate how differential methylation could impact F2RL3 gene expression. RESULTS: Observationally, DNA methylation at F2RL3 mediated an estimated 34% of the smoking effect on increased risk of myocardial infarction. An association between methylation group (low/high) and platelet reactivity was observed in response to PAR4 (protease-activated receptor 4) stimulation. In cells, cigarette smoke extract exposure was associated with a 4.9% to 9.3% reduction in DNA methylation at F2RL3 and a corresponding 1.7-(95% CI, 1.2-2.4, P=0.04) fold increase in F2RL3 mRNA. Results from reporter assays suggest the exon 2 region of F2RL3 may help control gene expression. CONCLUSIONS: Smoking-induced epigenetic DNA hypomethylation at F2RL3 appears to increase PAR4 expression with potential downstream consequences for platelet reactivity. Combined evidence here not only identifies F2RL3 DNA methylation as a possible contributory pathway from smoking to cardiovascular disease risk but from any feature potentially influencing F2RL3 regulation in a similar manner.

Platelet Surface Protein Expression and Reactivity upon TRAP Stimulation after BNT162b2 Vaccination.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces a coagulopathy characterized by platelet activation and a hypercoagulable state with an increased incidence of cardiovascular events. The viral spike protein S has been reported to enhance thrombosis formation, stimulate platelets to release procoagulant factors, and promote the formation of platelet-leukocyte aggregates even in absence of the virus. Although SARS-CoV-2 vaccines induce spike protein overexpression to trigger SARS-CoV-2-specific immune protection, thrombocyte activity has not been investigated in this context. Here, we provide the first phenotypic platelet characterization of healthy human subjects undergoing BNT162b2 vaccination. Using mass cytometry, we analyzed the expression of constitutive transmembrane receptors, adhesion proteins, and platelet activation markers in 12 healthy donors before and at five different time points within 4 weeks after the first BNT162b2 administration. We measured platelet reactivity by stimulating thrombocyte activation with thrombin receptor-activating peptide. Activation marker expression (P-selectin, LAMP-3, LAMP-1, CD40L, and PAC-1) did not change after vaccination. All investigated constitutive transmembrane proteins showed similar expressions over time. Platelet reactivity was not altered after BNT162b2 administration. Activation marker expression was significantly lower compared with an independent cohort of mild symptomatic COVID-19 patients analyzed with the same platform. This study reveals that BNT162b2 administration does not alter platelet protein expression and reactivity.

Thrombin induces pro-inflammatory and anti-inflammatory cytokines secretion from human mast cell line (HMC-1) via protease-activated receptors.

Thrombin-induced mast cell activation represents cross-talk between coagulation and inflammation. However, there is still controversy concerning the pro- or anti-inflammatory effects mast cells have in response to thrombin signaling. Human mast cell HMC-1 was incubated with 0.2 U/mL thrombin. Cells and supernatants were collected. Production of pro- and anti-inflammatory mediators was determined by quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). Expression of proteinase-activated receptor-1 (PAR1) and -4 (PAR4) mRNA in HMC-1 cells was analyzed by qPCR. Activation of mitogen-activated protein kinases (MAPKs) was measured by immunoblotting. Furthermore, the impact of PAR1 inhibitor (SCH79797) and agonist (TFLLR-NH2), PAR4 inhibitor (BMS986120) and agonist (AYPGKF-NH2), and MAPK inhibitors (SB203580, PD98059, and SP600125) on the production of mediators was evaluated using qPCR and ELISA. Thrombin exposure increased pro- and anti-inflammatory mediators, expression of PAR1 and PAR4 mRNA, and phosphorylation of JNK, p38, and ERK1/2 MAPKs in HMC-1 cells. SCH79797, BMS986120, and MAPK inhibitors (SB203580, PD98059, and SP600125) were inhibited, while TFLLR-NH2 and AYPGKF-NH2 promoted pro- and anti-inflammatory cytokines in this process. HMC-1 produces pro- and anti-inflammatory cytokines after thrombin incubation, namely PAR1 and PAR4. Alongside HMC-1, MAPK signaling pathways are involved in the production of these mediators. The mast cells showed dual activation after thrombin stimulation.

PAR-4/Ca2+-calpain pathway activation stimulates platelet-derived microparticles in hyperglycemic type 2 diabetes.

BACKGROUND: Patients with type 2 diabetes (T2DM) have a prothrombotic state that needs to be fully clarified; microparticles (MPs) have emerged as mediators and markers of this condition. Thus, we investigate, in vivo, in T2DM either with good (HbA1c ≤ 7.0%; GGC) or poor (HbA1c > 7.0%; PGC) glycemic control, the circulating levels of MPs, and in vitro, the molecular pathways involved in the release of MPs from platelets (PMP) and tested their pro-inflammatory effects on THP-1 transformed macrophages. METHODS: In 59 T2DM, and 23 control subjects with normal glucose tolerance (NGT), circulating levels of CD62E+, CD62P+, CD142+, CD45+ MPs were determined by flow cytometry, while plasma levels of ICAM-1, VCAM-1, IL-6 by ELISA. In vitro, PMP release and activation of isolated platelets from GGC and PGC were investigated, along with their effect on IL-6 secretion in THP-1 transformed macrophages. RESULTS: We found that MPs CD62P+ (PMP) and CD142+ (tissue factor-bearing MP) were significantly higher in PGC T2DM than GGC T2DM and NGT. Among MPs, PMP were also correlated with HbA1c and IL-6. In vitro, we showed that acute thrombin exposure stimulated a significantly higher PMP release in PGC T2DM than GGC T2DM through a more robust activation of PAR-4 receptor than PAR-1 receptor. Treatment with PAR-4 agonist induced an increased release of PMP in PGC with a Ca2+-calpain dependent mechanism since this effect was blunted by calpain inhibitor. Finally, the uptake of PMP derived from PAR-4 treated PGC platelets into THP-1 transformed macrophages promoted a marked increase of IL-6 release compared to PMP derived from GGC through the activation of the NF-kB pathway. CONCLUSIONS: These results identify PAR-4 as a mediator of platelet activation, microparticle release, and inflammation, in poorly controlled T2DM.

Drupin, a thrombin-like protease prompts platelet activation and aggregation through protease-activated receptors.

Hemostasis is a proteolytically regulated process that requires activation of platelets and the blood coagulation cascade upon vascular injury. Activated platelets create a thrombogenic environment and amplify the coagulation process. Plant latex proteases (PLPs) have been used as therapeutic components to treat various ailments by folk healers. One of the main applications of plant latices is to stop bleeding from minor injuries and to enhance wound healing activity. Although many studies have reported the pro-coagulant activities of PLPs, an in-depth investigation is required to understand the mechanism of action of PLPs on platelets. Here, the effect of PLPs on platelet aggregation was studied systematically to validate the observed pharmacological effect by folk healers. Among 29 latices from the Ficus genus tested, Ficus drupacea exhibited potent pro-coagulant and thrombin-like activity. Drupin, a thrombin-like cysteine protease responsible for platelet aggregation was purified from F. drupacea latex. Drupin exhibits pro-coagulant activity and reduces the bleeding time in mice tail. It induces platelet aggregation by activating mitogen-activated protein kinases and the nuclear factor-κB and PI3K/Akt signalling cascade, which, in turn, phosphorylats, cytosolic phospholipase A2  leading to the release of thromboxane A2 from the granules to activate the nearby platelets to aggregate. Furthermore, we investigated the involvement of protease-activated receptors in drupin-induced platelet aggregation using specific protease activated receptor 1 (PAR1) and PAR4 receptor antagonists. The results confirmed that the drupin-induced platelet aggregation was mediated by both PAR1 and PAR4, synergistically. Overall, drupin reduces the bleeding time by exerting pro-coagulant activity and induces platelet aggregation by activating the intracellular signalling cascade.

Enhanced endothelial barrier function by monoclonal antibody activation of vascular endothelial cadherin.

Excessive vascular permeability occurs in inflammatory disease processes. Vascular endothelial cadherin (VE-cadherin) is an adhesion protein that controls vascular permeability. We identified monoclonal antibodies (mAbs) to human VE-cadherin that activate cell adhesion and inhibit the increased permeability of endothelial cell monolayers induced by thrombin receptor activator peptide-6 (TRAP-6). Two mAbs, 8A12c and 3A5a, reduce permeability, whereas an inhibitory mAb, 2E11d, enhances permeability. Activating mAbs also reduce permeability induced by tumor necrosis factor-α (TNF-α) and vascular endothelial cell growth factor (VEGF). The activating mAbs also stabilize the organization of the adherens junctions that are disrupted by TRAP-6, VEGF, or TNF-α. The activating mAbs act directly on the adhesive function of VE-cadherin because they did not block the accumulation of actin filaments stimulated by TRAP-6 and enhance physical cell-cell adhesion of VE-cadherin-expressing tissue culture cells. Therefore, VE-cadherin function can be regulated at the cell surface to control endothelial permeability.NEW & NOTEWORTHY Excessive vascular permeability is a serious complication of many inflammatory disease conditions. We have developed monoclonal antibodies that inhibit increases in endothelial monolayer permeability induced by several signaling factors by activating VE-cadherin mediated adhesion and stabilizing cell junctions. These antibodies and/or the mechanisms they reveal may lead to important therapeutics to treat vascular leakiness and inflammation.

Protamine stimulates platelet aggregation in vitro with activation of the fibrinogen receptor and alpha-granule release, but impairs secondary activation via ADP and thrombin receptors.

Heparin and protamine are fundamental in the management of anticoagulation during cardiac surgery. Excess protamine has been associated with increased bleeding. Interaction between protamine and platelet function has been demonstrated but the mechanism remains unclear. We examined the effect of protamine on platelet function in vitro using impedance aggregometry, flow cytometry, and thrombin generation. Platelets were exposed to protamine at final concentrations of 0, 20, 40, and 80 µg/mL, alone or together with adenosine diphosphate (ADP) or thrombin PAR1 receptor-activating peptide (TRAP). We found that in the absence of other activators, protamine (80 µg/mL) increased the proportion of platelets with active fibrinogen receptor (binding of PAC-1) from 3.6% to 97.0% (p < .001) measured with flow cytometry. Impedance aggregometry also increased slightly after exposure to protamine alone. When activated with ADP or TRAP protamine at 80 µg/mL reduced aggregation, from 73.8 ± 29.4 U to 46.9 ± 21.1 U (p < .001) with ADP and from 126.4 ± 16.1 U to 94.9 ± 23.7 U (p < .01) with TRAP. P-selectin exposure (a marker of alpha-granule release) measured by median fluorescence intensity (MFI) increased dose dependently with protamine alone, from 0.76 ± 0.20 (0 µg/mL) to 10.2 ± 3.1 (80 µg/mL), p < .001. Protamine 80 µg/mL by itself resulted in higher MFI (10.16 ± 3.09) than activation with ADP (2.2 ± 0.7, p < .001) or TRAP (5.7 ± 2.6, p < .01) without protamine. When protamine was combined with ADP or TRAP, there was a concentration-dependent increase in the alpha-granule release. In conclusion, protamine interacts with platelets in vitro having both a direct activating effect and impairment of secondary activation of aggregation by other agonists.

Protease-activated receptor 4 causes Akt phosphorylation independently of PI3 kinase pathways.

PI-3 Kinase plays an important role in platelet activation mainly through regulation of RASA3. Akt phosphorylation is an indicator for the activity of PI3 kinase. The aim of this study is to characterize the pathways leading to Akt phosphorylation in platelets. We performed concentration response curves of LY294002, a pan-PI3 kinase inhibitor, on platelet aggregation and Akt phosphorylation, in washed human and mouse platelets. At concentrations as low as 3.12 µM, LY294002 abolished Akt phosphorylation induced by 2MeSADP and SFLLRN, but not by AYPGKF. It required much higher concentrations of LY294002 (12.5-25 µM) to abolish AYPGKF-induced Akt phosphorylation, both in wild type and P2Y12 null mouse platelets. We propose that 3.12 µM LY294002 is sufficient to inhibit PI3 kinase isoforms in platelets and higher concentrations might inhibit other pathways regulating Akt phosphorylation by AYPGKF. We conclude that Protease-activated receptor 4 (PAR4) might cause Akt phosphorylation through pathways distinctly different from those of Protease-activated receptor 1 (PAR1).