Targeting Phosphotyrosine in Native Proteins with Conditional, Bispecific Antibody Traps.

Engineering sequence-specific antibodies (Abs) against phosphotyrosine (pY) motifs embedded in folded polypeptides remains highly challenging because of the stringent requirement for simultaneous recognition of the pY motif and the surrounding folded protein epitope. Here, we present a method named phosphotyrosine Targeting by Recombinant Ab Pair, or pY-TRAP, for in vitro engineering of binders for native pY proteins. Specifically, we create the pY protein by unnatural amino acid misincorporation, mutagenize a universal pY-binding Ab to create a first binder B1 for the pY motif on the pY protein, and then select against the B1-pY protein complex for a second binder B2 that recognizes the composite epitope of B1 and the pY-containing protein complex. We applied pY-TRAP to create highly specific binders to folded Ub-pY59, a rarely studied Ub phosphoform exclusively observed in cancerous tissues, and ZAP70-pY248, a kinase phosphoform regulated in feedback signaling pathways in T cells. The pY-TRAPs do not have detectable binding to wild-type proteins or to other pY peptides or proteins tested. This pY-TRAP approach serves as a generalizable method for engineering sequence-specific Ab binders to native pY proteins.

PAR4 activation involves extracellular loop 3 and transmembrane residue Thr153.

Protease-activated receptor 4 (PAR4) mediates sustained thrombin signaling in platelets and is required for a stable thrombus. PAR4 is activated by proteolysis of the N terminus to expose a tethered ligand. The structural basis for PAR4 activation and the location of its ligand binding site (LBS) are unknown. Using hydrogen/deuterium exchange (H/D exchange), computational modeling, and signaling studies, we determined the molecular mechanism for tethered ligand-mediated PAR4 activation. H/D exchange identified that the LBS is composed of transmembrane 3 (TM3) domain and TM7. Unbiased computational modeling further predicted an interaction between Gly48 from the tethered ligand and Thr153 from the LBS. Mutating Thr153 significantly decreased PAR4 signaling. H/D exchange and modeling also showed that extracellular loop 3 (ECL3) serves as a gatekeeper for the interaction between the tethered ligand and LBS. A naturally occurring sequence variant (P310L, rs2227376) and 2 experimental mutations (S311A and P312L) determined that the rigidity conferred by prolines in ECL3 are essential for PAR4 activation. Finally, we examined the role of the polymorphism at position 310 in venous thromboembolism (VTE) using the International Network Against Venous Thrombosis (INVENT) consortium multi-ancestry genome-wide association study (GWAS) meta-analysis. Individuals with the PAR4 Leu310 allele had a 15% reduction in relative risk for VTE (odds ratio, 0.85; 95% confidence interval, 0.77-0.94) compared with the Pro310 allele. These data are consistent with our H/D exchange, molecular modeling, and signaling studies. In conclusion, we have uncovered the structural basis for PAR4 activation and identified a previously unrecognized role for PAR4 in VTE.

Protease-Activated Receptor 4 (PAR4): A Promising Target for Antiplatelet Therapy.

Cardiovascular diseases (CVDs) are currently among the leading causes of death worldwide. Platelet aggregation is a key cellular component of arterial thrombi and major cause of CVDs. Protease-activated receptors (PARs), including PAR1, PAR2, PAR3 and PAR4, fall within a subfamily of seven-transmembrane G-protein-coupled receptors (GPCR). Human platelets express PAR1 and PAR4, which contribute to the signaling transduction processes. In association with CVDs, PAR4 not only contributes to platelet activation but also is a modulator of cellular responses that serve as hallmarks of inflammation. Although several antiplatelet drugs are available on the market, they have many side effects that limit their use. Emerging evidence shows that PAR4 targeting is a safer strategy for preventing thrombosis and consequently may improve the overall cardiac safety profile. Our present review summarizes the PAR4 structural characteristics, activation mechanism, role in the pathophysiology of diseases and understanding the association of PAR4 targeting for improved cardiac protection. Conclusively, this review highlights the importance of PAR4 antagonists and its potential utility in different CVDs.

Activated protein C protects from GvHD via PAR2/PAR3 signalling in regulatory T-cells.

Graft-vs.-host disease (GvHD) is a major complication of allogenic hematopoietic stem-cell(HSC) transplantation. GvHD is associated with loss of endothelial thrombomodulin, but the relevance of this for the adaptive immune response to transplanted HSCs remains unknown. Here we show that the protease-activated protein C (aPC), which is generated by thrombomodulin, ameliorates GvHD aPC restricts allogenic T-cell activation via the protease activated receptor (PAR)2/PAR3 heterodimer on regulatory T-cells (Tregs, CD4+FOXP3+). Preincubation of pan T-cells with aPC prior to transplantation increases the frequency of Tregs and protects from GvHD. Preincubation of human T-cells (HLA-DR4-CD4+) with aPC prior to transplantation into humanized (NSG-AB°DR4) mice ameliorates graft-vs.-host disease. The protective effect of aPC on GvHD does not compromise the graft vs. leukaemia effect in two independent tumor cell models. Ex vivo preincubation of T-cells with aPC, aPC-based therapies, or targeting PAR2/PAR3 on T-cells may provide a safe and effective approach to mitigate GvHD.Graft-vs.-host disease is a complication of allogenic hematopoietic stem cell transplantation, and is associated with endothelial dysfunction. Here the authors show that activated protein C signals via PAR2/PAR3 to expand Treg cells, mitigating the disease in mice.

Protease-activated receptor-4 and purinergic receptor P2Y12 dimerize, co-internalize, and activate Akt signaling via endosomal recruitment of ß-arrestin.

Vascular inflammation and thrombosis require the concerted actions of several different agonists, many of which act on G protein-coupled receptors (GPCRs). GPCR dimerization is a well-established phenomenon that can alter protomer function. In platelets and other cell types, protease-activated receptor-4 (PAR4) has been shown to dimerize with the purinergic receptor P2Y12 to coordinate ß-arrestin-mediated Akt signaling, an important mediator of integrin activation. However, the mechanism by which the PAR4-P2Y12 dimer controls ß-arrestin-dependent Akt signaling is not known. We now report that PAR4 and P2Y12 heterodimer internalization is required for ß-arrestin recruitment to endosomes and Akt signaling. Using bioluminescence resonance energy transfer, immunofluorescence microscopy, and co-immunoprecipitation in cells expressing receptors exogenously and endogenously, we demonstrate that PAR4 and P2Y12 specifically interact and form dimers expressed at the cell surface. We also found that activation of PAR4 but not of P2Y12 drives internalization of the PAR4-P2Y12 heterodimer. Remarkably, activated PAR4 internalization was required for recruitment of ß-arrestin to endocytic vesicles, which was dependent on co-expression of P2Y12. Interestingly, stimulation of the PAR4-P2Y12 heterodimer promotes ß-arrestin and Akt co-localization to intracellular vesicles. Moreover, activated PAR4-P2Y12 internalization is required for sustained Akt activation. Thus, internalization of the PAR4-P2Y12 heterodimer is necessary for ß-arrestin recruitment to endosomes and Akt signaling and lays the foundation for examining whether blockade of PAR4 internalization reduces integrin and platelet activation.

A cluster of aspartic residues in the extracellular loop II of PAR 4 is important for thrombin interaction and activation of platelets.

Thrombin activates platelets via proteolytic cleavage of protease-activated receptors (PARs) 1 and 4. The two PARs have distinct but complementary roles. The mechanisms responsible for PAR1 activation by thrombin have been extensively studied. However, much less is known regarding thrombin activation of PAR4, especially the potential involvement of regions of PAR4 other than the N-terminal, which is bound to the catalytic site of thrombin. We have studied PAR4 in S. cerevisiae strain MMY12, an expression system in which the GPCR receptors are connected to a Lac Z reporter gene resulting in increased ß-galactosidase activity. This approach was used to assess PAR4 mutants to evaluate the contribution of different aspartic residues in facilitating PAR4 activation. Furthermore, peptides mimicking parts of the PAR4 N-terminal and the second extracellular loop (ECLII) were tested for their ability to inhibit platelet activation by thrombin. Binding of these peptides to γ-thrombin was studied by monitoring the decrease in tryptophan fluorescence intensity of thrombin. We conclude that not only the N-terminal but also the electronegative aspartic residues D224, D230 and D235 (located in ECLII) are be important for PAR4 binding to thrombin. We further suggest that they play a role for the tethered ligand binding to the receptor, as mutations also affected activation in response to a PAR4-activating peptide mimicking the new N-terminal formed after cleavage. This agrees with previous results on PAR1 and thrombin binding. We suggest that the ECLII of PAR4 could be a potential target for antithrombotic drug development.

Targeting a Proteinase-Activated Receptor 4 (PAR4) Carboxyl Terminal Motif to Regulate Platelet Function.

Thrombin initiates human platelet aggregation by coordinately activating proteinase-activated receptors (PARs) 1 and 4. However, targeting PAR1 with an orthosteric-tethered ligand binding-site antagonist results in bleeding, possibly owing to the important role of PAR1 activation on cells other than platelets. Because of its more restricted tissue expression profile, we have therefore turned to PAR4 as an antiplatelet target. We have identified an intracellular PAR4 C-terminal motif that regulates calcium signaling and ß-arrestin interactions. By disrupting this PAR4 calcium/ß-arrestin signaling process with a novel cell-penetrating peptide, we were able to inhibit both thrombin-triggered platelet aggregation in vitro and clot consolidation in vivo. We suggest that targeting PAR4 represents an attractive alternative to blocking PAR1 for antiplatelet therapy in humans.

Function of Platelet-Induced Epithelial Attachment at Titanium Surfaces Inhibits Microbial Colonization.

The aim of this study was to evaluate the barrier function of platelet-induced epithelial sheets on titanium surfaces. The lack of functional peri-implant epithelial sealing with basal lamina (BL) attachment at the interface of the implant and the adjacent epithelium allows for bacterial invasion, which may lead to peri-implantitis. Although various approaches have been reported to combat bacterial infection by surface modifications to titanium, none of these have been successful in a clinical application. In our previous study, surface modification with protease-activated receptor 4-activating peptide (PAR4-AP), which induced platelet activation and aggregation, was successful in demonstrating epithelial attachment via BL and epithelial sheet formation on the titanium surface. We hypothesized that the platelet-induced epithelial sheet on PAR4-AP-modified titanium surfaces would reduce bacterial attachment, penetration, and invasion. Titanium surface was modified with PAR4-AP and incubated with platelet-rich plasma (PRP). The aggregated platelets released collagen IV, a critical BL component, onto the PAR4-AP-modified titanium surface. Then, human gingival epithelial cells were seeded on the modified titanium surface and formed epithelial sheets. Green fluorescent protein (GFP)-expressing Escherichia coli was cultured onto PAR4-AP-modified titanium with and without epithelial sheet formation. While Escherichia coli accumulated densely onto the PAR4-AP titanium lacking epithelial sheet, few Escherichia coli were observed on the epithelial sheet on the PAR4-AP surface. No bacterial invasion into the interface of the epithelial sheet and the titanium surface was observed. These in vitro results indicate the efficacy of a platelet-induced epithelial barrier that functions to prevent bacterial attachment, penetration, and invasion on PAR4-AP-modified titanium.

Extracellular vesicles from activated platelets: a semiquantitative cryo-electron microscopy and immuno-gold labeling study.

Cells release membrane vesicles in their surrounding medium either constitutively or in response to activating signals. Two main types of extracellular vesicles (EVs) are commonly distinguished based on their mechanism of formation, membrane composition and size. According to the current model, EVs shed from the plasma membrane, often called microvesicles, expose phosphatidylserine (PS) and range in size from 100 nm to 1 µm, while EVs originating from endosomal multi-vesicular bodies, called exosomes, contain tetraspanin proteins, including CD63, and range in size from 50 to 100 nm. Heijnen et al. [1] have shown that activated platelets release EVs corresponding to these two types of vesicles, using negative staining electron microscopy (EM) and immuno-gold labeling. Here, we apply cryo-EM and immuno-gold labeling to provide a quantitative analysis of EVs released by platelets activated by thrombin, TRAP and CRP-XL, as well as EVs from serum. We show that EVs activated by these three agonists present a similar size distribution, the majority of them forming a broad peak extending from 50 nm to 1 µm, about 50% of them ranging from 50 to 400 nm. We show also that 60% of the EVs from TRAP or CRP-XL activation expose CD41, a majority of them exposing also PS. To explain the presence of large EVs CD41-negative or PS-negative, several alternative mechanisms of EV formation are proposed. We find also that the majority of EVs in activated platelet samples expose CD63, and distinguish two populations of CD63-positive EVs, namely large EVs with low labeling density and small EVs with high labeling density.

The use of regression analysis in determining reference intervals for low hematocrit and thrombocyte count in multiple electrode aggregometry and platelet function analyzer 100 testing of platelet function.

Low platelet counts and hematocrit levels hinder whole blood point-of-care testing of platelet function. Thus far, no reference ranges for MEA (multiple electrode aggregometry) and PFA-100 (platelet function analyzer 100) devices exist for low ranges. Through dilution methods of volunteer whole blood, platelet function at low ranges of platelet count and hematocrit levels was assessed on MEA for four agonists and for PFA-100 in two cartridges. Using (multiple) regression analysis, 95% reference intervals were computed for these low ranges. Low platelet counts affected MEA in a positive correlation (all agonists showed r2 ≥ 0.75) and PFA-100 in an inverse correlation (closure times were prolonged with lower platelet counts). Lowered hematocrit did not affect MEA testing, except for arachidonic acid activation (ASPI), which showed a weak positive correlation (r2 = 0.14). Closure time on PFA-100 testing was inversely correlated with hematocrit for both cartridges. Regression analysis revealed different 95% reference intervals in comparison with originally established intervals for both MEA and PFA-100 in low platelet or hematocrit conditions. Multiple regression analysis of ASPI and both tests on the PFA-100 for combined low platelet and hematocrit conditions revealed that only PFA-100 testing should be adjusted for both thrombocytopenia and anemia. 95% reference intervals were calculated using multiple regression analysis. However, coefficients of determination of PFA-100 were poor, and some variance remained unexplained. Thus, in this pilot study using (multiple) regression analysis, we could establish reference intervals of platelet function in anemia and thrombocytopenia conditions on PFA-100 and in thrombocytopenia conditions on MEA.

Establishment of Epithelial Attachment on Titanium Surface Coated with Platelet Activating Peptide.

The aim of this study was to produce epithelial attachment on a typical implant abutment surface of smooth titanium. A challenging complication that hinders the success of dental implants is peri-implantitis. A common cause of peri-implantitis may results from the lack of epithelial sealing at the peri-implant collar. Histologically, epithelial sealing is recognized as the attachment of the basement membrane (BM). BM-attachment is promoted by activated platelet aggregates at surgical wound sites. On the other hand, platelets did not aggregate on smooth titanium, the surface typical of the implant abutment. We then hypothesized that epithelial BM-attachment was produced when titanium surface was modified to allow platelet aggregation. Titanium surfaces were coated with a protease activated receptor 4-activating peptide (PAR4-AP). PAR4-AP coating yielded rapid aggregation of platelets on the titanium surface. Platelet aggregates released robust amount of epithelial chemoattractants (IGF-I, TGF-ß) and growth factors (EGF, VEGF) on the titanium surface. Human gingival epithelial cells, when they were co-cultured on the platelet aggregates, successfully attached to the PAR4-AP coated titanium surface with spread laminin5 positive BM and consecutive staining of the epithelial tight junction component ZO1, indicating the formation of complete epithelial sheet. These in-vitro results indicate the establishment of epithelial BM-attachment to the titanium surface.

[THE REFERENCE VALUES OF AGGREGATION OF PLATELETS IN ADULT POPULATION OF THE ASTRAKHAN OBLAST USING AGGREGOMETER MULTIPLATE].

The modern international standards recommend each laboratory to develop or to confirm available in literature the reference intervalsfor every laboratory indicator In the Astrakhanskaia oblast, sampling of128 healthy males andfemales were examinedfor aggregation function of thrombocytes using impedance technique and applying aggregometer Multiplate ("Verum Diagnostica", Germany). The study used as inductors peptide activating receptor of thrombin; arachidonic and adenosine diphosphoric acids. The reference range of aggregation of thrombocytes with peptide activating receptor of thrombin, at aggregometer Multiplate, in healthy population of theAstrakhanskaia oblast made up to 815.2-1498.4 AU/min, with arachidonic acid--660-1341 AU/min. with adenosine diphosphoric acid--598-1120 AU/min.

Protease-Activated Receptor 4 Variant p.Tyr157Cys Reduces Platelet Functional Responses and Alters Receptor Trafficking.

OBJECTIVE: Protease-activated receptor 4 (PAR4) is a key regulator of platelet reactivity and is encoded by F2RL3, which has abundant rare missense variants. We aimed to provide proof of principle that rare F2LR3 variants potentially affect platelet reactivity and responsiveness to PAR1 antagonist drugs and to explore underlying molecular mechanisms. APPROACH AND RESULTS: We identified 6 rare F2RL3 missense variants in 236 cardiac patients, of which the variant causing a tyrosine 157 to cysteine substitution (Y157C) was predicted computationally to have the greatest effect on PAR4 structure. Y157C platelets from 3 cases showed reduced responses to PAR4-activating peptide and to α-thrombin compared with controls, but no reduction in responses to PAR1-activating peptide. Pretreatment with the PAR1 antagonist vorapaxar caused lower residual α-thrombin responses in Y157C platelets than in controls, indicating greater platelet inhibition. HEK293 cells transfected with a PAR4 Y157C expression construct had reduced PAR4 functional responses, unchanged total PAR4 expression but reduced surface expression. PAR4 Y157C was partially retained in the endoplasmic reticulum and displayed an expression pattern consistent with defective N-glycosylation. Mutagenesis of Y322, which is the putative hydrogen bond partner of Y157, also reduced PAR4 surface expression in HEK293 cells. CONCLUSIONS: Reduced PAR4 responses associated with Y157C result from aberrant anterograde surface receptor trafficking, in part, because of disrupted intramolecular hydrogen bonding. Characterization of PAR4 Y157C establishes that rare F2RL3 variants have the potential to markedly alter platelet PAR4 reactivity particularly after exposure to therapeutic PAR1 antagonists.

Protease-activated receptor 4: from structure to function and back again.

Protease-activated receptors are a family of four GPCRs (PAR1-PAR4) with a number of unique attributes. Nearly two and a half decades after the discovery of the first PAR, an antagonist targeting this receptor has been approved for human use. The first-in-class PAR1 antagonist, vorapaxar, was approved for use in the USA in 2014 for the prevention of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. These recent developments indicate the clinical potential of manipulating PAR function. While much work has been aimed at uncovering the function of PAR1 and, to a lesser extent, PAR2, comparatively little is known regarding the pharmacology and physiology of PAR3 and PAR4. Recent studies have begun to develop the pharmacological and genetic tools required to study PAR4 function in detail, and there is now emerging evidence for the function of PAR4 in disease settings. In this review, we detail the discovery, structure, pharmacology, physiological significance and therapeutic potential of PAR4. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

Platelet-rich plasma stimulated by pulse electric fields: Platelet activation, procoagulant markers, growth factor release and cell proliferation.

Therapeutic use of activated platelet-rich plasma (PRP) has been explored for wound healing, hemostasis and antimicrobial wound applications. Pulse electric field (PEF) stimulation may provide more consistent platelet activation and avoid complications associated with the addition of bovine thrombin, the current state of the art ex vivo activator of therapeutic PRP. The aim of this study was to compare the ability of PEF, bovine thrombin and thrombin receptor activating peptide (TRAP) to activate human PRP, release growth factors and induce cell proliferation in vitro. Human PRP was prepared in the Harvest SmartPreP2 System and treated with vehicle, PEF, bovine thrombin, TRAP or Triton X-100. Platelet activation and procoagulant markers and microparticle generation were measured by flow cytometry. Released growth factors were measured by ELISA. The releasates were tested for their ability to stimulate proliferation of human epithelial cells in culture. PEF produced more platelet-derived microparticles, P-selectin-positive particles and procoagulant annexin V-positive particles than bovine thrombin or TRAP. These differences were associated with higher levels of released epidermal growth factor after PEF than after bovine thrombin or TRAP but similar levels of platelet-derived, vascular-endothelial, and basic fibroblast growth factors, and platelet factor 4. Supernatant from PEF-treated platelets significantly increased cell proliferation compared to plasma. In conclusion, PEF treatment of fresh PRP results in generation of microparticles, exposure of prothrombotic platelet surfaces, differential release of growth factors compared to bovine thrombin and TRAP and significant cell proliferation. These results, together with PEF's inherent advantages, suggest that PEF may be a superior alternative to bovine thrombin activation of PRP for therapeutic applications.

Development and characterization of monoclonal antibodies against Protease Activated Receptor 4 (PAR4).

BACKGROUND: Protease activated receptor 4 (PAR4) is a G protein coupled receptor (GPCR) which is activated by proteolytic cleavage of its N-terminal exodomain. This generates a tethered ligand that activates the receptor and triggers downstream signaling events. With the current focus in the development of anti-platelet therapies shifted towards PARs, new reagents are needed for expanding the field's knowledge on PAR4. Currently, there are no PAR4 reagents which are able to detect activation of the receptor. METHODS: Monoclonal PAR4 antibodies were purified from hybridomas producing antibody that were generated by fusing splenocytes with NS-1 cells. Immunoblotting, immunofluorescence, and flow cytometry were utilized to detect the epitope for each antibody and to evaluate the interaction of the antibodies with cells. RESULTS: Here, we report the successful generation of three monoclonal antibodies to the N-terminal extracellular domain of PAR4: 14H6, 5F10, and 2D6. We mapped the epitope on PAR4 of 14H6, 5F10, and 2D6 antibodies to residues (48-53), (41-47), and (73-78), respectively. Two of the antibodies (14H6 and 5F10) interacted close to the thrombin cleavage and were sensitive to α-thrombin cleavage of PAR4. In addition, 5F10 was able to partially inhibit the cleavage of PAR4 expressed in HEK293 cells by α-thrombin. CONCLUSIONS: These new antibodies provide a means to monitor endogenous PAR4 expression and activation by proteases on cells.

Diagnosis of Glanzmann thrombasthenia by whole blood impedance analyzer (MEA) vs. light transmission aggregometry.

BACKGROUND: Glanzmann thrombasthenia (GT) is a rare inherited platelet disorder that is characterized by spontaneous or postprocedural bleeding. The diagnosis of GT depends on identifying the dysfunction of the platelets. AIM: The aim of this study was to compare a whole blood impedance Multiplate analyzer (MEA) with the standard method, light transmission aggregometry (LTA) in diagnosis of GT. METHODS: Fifteen patients with GT were assessed on MEA and LTA using arachidonic acid (ASPI: 15 mm), (TRAP: 1 mm), collagen (100 µg/mL), ADP (0.2 mm), and ristocetin (Risto: 10 mg/mL). Whole blood samples were collected in sodium citrate and hirudin vacuum, blood collection tubes and tested within 4 h. Platelet-rich plasma was used for LTA using platelet agonists (ristocetin 1.5 mg/mL) (arachidonic acid 0.5 mg/mL) (ADP 2.5 mg/mL) and (collagen 1 mg/mL). RESULTS: The platelet count and PFA-100 results were (average and SD) 319 ± 93 × 10(9) L and 252 ± 34 s, respectively. Flow cytometry analysis showed that all samples are positive for CD42a and CD42b, whereas 9/15 samples were negative for CD61 and CD41. The other six patients had either partial or full expression of CD61/CD41. Aggregation analysis using both methods showed that all samples had no aggregation response to any of the agonists used apart from six samples which, using only the MEA, showed minimal aggregation in response to collagen (average = 14.3 ± 7 µg, which may suggest ability to detect qualitative abnormality of GPIIb/IIIa). CONCLUSION: These results suggest that the MEA is sensitive for the detection of Glanzmann thrombasthenia. Furthermore, MEA may also be able to differentiate between the subtypes of Glanzmann thrombasthenia.

Thrombin-induced platelet activation via PAR4: pivotal role for exosite II.

Thrombin-induced platelet activation via PAR1 and PAR4 is an important event in haemostasis. Although the underlying mechanisms responsible for ensuring efficient PAR1 activation by thrombin have been extensively studied, the potential involvement of recognitions sites outside the active site of the protease in thrombin-induced PAR4 activation is largely unknown. In this study, we developed a new assay to assess the importance of exosite I and II for PAR4 activation with α - and γ-thrombin. Surprisingly, we found that exosite II is critical for activation of PAR4. We also show that this dependency on exosite II likely represents a new mechanism, as it is unaffected by blockage of the previously known interaction between thrombin and glycoprotein Ibα.

Ligand binding to anion-binding exosites regulates conformational properties of thrombin.

Thrombin participates in coagulation, anticoagulation, and initiation of platelet activation. To fulfill its diverse roles and maintain hemostasis, this serine protease is regulated via the extended active site region and anion-binding exosites (ABEs) I and II. For the current project, amide proton hydrogen-deuterium exchange coupled with MALDI-TOF mass spectrometry was used to characterize ligand binding to individual exosites and to investigate the presence of exosite-active site and exosite-exosite interactions. PAR3(44-56) and PAR1(49-62) were observed to bind to thrombin ABE I and then to exhibit long range effects over to ABE II. By contrast, Hirudin(54-65) focused more on ABE I and did not transmit influences over to ABE II. Although these three ligands were each directed to ABE I, they did not promote the same conformational consequences. D-Phe-Pro-Arg-chloromethyl ketone inhibition at the thrombin active site led to further local and long range consequences to thrombin-ABE I ligand complexes with the autolysis loop often most affected. When Hirudin(54-65) was bound to ABE I, it was still possible to bind GpIbα(269-286) or fibrinogen γ'(410-427) to ABE II. Each ligand exerted its predominant influences on thrombin and also allowed interexosite communication. The results obtained support the proposal that thrombin is a highly dynamic protein. The transmission of ligand-specific local and long range conformational events is proposed to help regulate this multifunctional enzyme.

Mapping human protease-activated receptor 4 (PAR4) homodimer interface to transmembrane helix 4.

Thrombin activates platelets by binding and cleaving protease-activated receptors 1 and 4 (PAR1 and PAR4). Because of the importance of PAR4 activation on platelets in humans and mice and emerging roles for PAR4 in other tissues, experiments were done to characterize the interaction between PAR4 homodimers. Bimolecular fluorescence complementation and bioluminescence resonance energy transfer (BRET) were used to examine the PAR4 homodimer interface. In bimolecular fluorescence complementation experiments, PAR4 formed homodimers that were disrupted by unlabeled PAR4 in a concentration-dependent manner, but not by rhodopsin. In BRET experiments, the PAR4 homodimers showed a specific interaction as indicated by a hyperbolic BRET signal in response to increasing PAR4-GFP expression. PAR4 did not interact with rhodopsin in BRET assays. The threshold maximum BRET signal was disrupted in a concentration-dependent manner by unlabeled PAR4. In contrast, rhodopsin was unable to disrupt the BRET signal, indicating that the disruption of the PAR4 homodimer is not due to nonspecific interactions. A panel of rho-PAR4 chimeras and PAR4 point mutants has mapped the dimer interface to hydrophobic residues in transmembrane helix 4. Finally, mutations that disrupted dimer formation had reduced calcium mobilization in response to the PAR4 agonist peptide. These results link the loss of dimer formation to a loss of PAR4 signaling.