A maize semidwarf mutant reveals a GRAS transcription factor involved in brassinosteroid signaling.

Brassinosteroids (BR) and gibberellins (GA) regulate plant height and leaf angle in maize (Zea mays). Mutants with defects in BR or GA biosynthesis or signaling identify components of these pathways and enhance our knowledge about plant growth and development. In this study, we characterized three recessive mutant alleles of GRAS transcription factor 42 (gras42) in maize, a GRAS transcription factor gene orthologous to the DWARF AND LOW TILLERING (DLT) gene of rice (Oryza sativa). These maize mutants exhibited semi-dwarf stature, shorter and wider leaves, and more upright leaf angle. Transcriptome analysis revealed a role for GRAS42 as a determinant of BR signaling. Analysis of the expression consequences from loss of GRAS42 in the gras42-mu1021149 mutant indicated a weak loss of BR signaling in the mutant, consistent with its previously demonstrated role in BR signaling in rice. Loss of BR signaling was also evident by the enhancement of weak BR biosynthetic mutant alleles in double mutants of nana plant1-1 and gras42-mu1021149. The gras42-mu1021149 mutant had little effect on GA-regulated gene expression, suggesting that GRAS42 is not a regulator of core GA signaling genes in maize. Single cell expression data identified gras42 expressed among cells in the G2/M phase of the cell cycle consistent with its previously demonstrated role in cell cycle gene expression in Arabidopsis (Arabidopsis thaliana). Cis-acting natural variation controlling GRAS42 transcript accumulation was identified by expression genome-wide association study (eGWAS) in maize. Our results demonstrate a conserved role for GRAS42/SCARECROW-LIKE 28 (SCL28)/DLT in BR signaling, clarify the role of this gene in GA signaling, and suggest mechanisms of tillering and leaf angle control by BR.

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.

Plasma growth factors maintain constitutive translation in platelets to regulate reactivity and thrombotic potential.

ABSTRACT: Mechanisms of proteostasis in anucleate circulating platelets are unknown and may regulate platelet function. We investigated the hypothesis that plasma-borne growth factors/hormones (GFHs) maintain constitutive translation in circulating platelets to facilitate reactivity. Bio-orthogonal noncanonical amino acid tagging (BONCAT) coupled with liquid chromatography-tandem mass spectrometry analysis revealed constitutive translation of a broad-spectrum translatome in human platelets dependent upon plasma or GFH exposure, and in murine circulation. Freshly isolated platelets from plasma showed homeostatic activation of translation-initiation signaling pathways: phosphorylation of p38/ERK upstream kinases, essential intermediate MNK1/2, and effectors eIF4E/4E-BP1. Plasma starvation led to loss of pathway phosphorylation, but it was fully restored with 5-minute stimulation by plasma or GFHs. Cycloheximide or puromycin infusion suppressed ex vivo platelet GpIIb/IIIa activation and P-selectin exposure with low thrombin concentrations and low-to-saturating concentrations of adenosine 5'-diphosphate (ADP) or thromboxane analog but not convulxin. ADP-induced thromboxane generation was blunted by translation inhibition, and secondary-wave aggregation was inhibited in a thromboxane-dependent manner. Intravenously administered puromycin reduced injury-induced clot size in cremaster muscle arterioles, and delayed primary hemostasis after tail tip amputation but did not delay neither final hemostasis after subsequent rebleeds, nor final hemostasis after jugular vein puncture. In contrast, these mice were protected from injury-induced arterial thrombosis and thrombin-induced pulmonary thromboembolism (PE), and adoptive transfer of translation-inhibited platelets into untreated mice inhibited arterial thrombosis and PE. Thus, constitutive plasma GFH-driven translation regulates platelet G protein-coupled receptor reactivity to balance hemostasis and thrombotic potential.

A Mouse Model of the Protease Activated Receptor 4 (PAR4) 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 a single nucleotide polymorphism in extracellular loop 3 (ECL3), PAR4-P310L (rs2227376) leads to a hypo-reactive receptor. Objectives: The goal of this study was to determine how the hypo-reactive PAR4 variant in ECL3 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. Conclusions: 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.

Aspirin pharmacokinetics and pharmacodynamics through gestation.

BACKGROUND: Low dose aspirin is recommended for prevention of preeclampsia, however there is not consensus on the appropriate dose. Pregnancy specific changes have the potential to impact the pharmacology of aspirin in pregnancy, however there are very limited studies on aspirin pharmacokinetics in pregnancy and none linking pharmacokinetics (PK, drug dose and drug level) to pharmacodynamics (PD, drug dose and physiologic response) in pregnancy. As a result, we do not have a good understanding of the pharmacologic response to aspirin in pregnancy, which has important implications for clinical efficacy. We sought to describe the PK and PD of aspirin through pregnancy and to identify individual covariates that impacted aspirin PK/PD. OBJECTIVE: We sought to describe the PK and PD of aspirin through pregnancy (first and third trimester), to identify covariates that significantly impact aspirin PK and to identify the relationship between aspirin PK and PD. STUDY DESIGN: This is a prospective study of patients at high risk for preeclampsia recommended to take 81 mg aspirin daily. This study involved 3 visits as follows: (1) baseline, first trimester (10-16 weeks of gestation) 6-hour PK visit, done before initiation of aspirin; (2) follow-up 1: 2 to 4 weeks after aspirin initiation; and (3) follow-up 2: third trimester 6-hour PK visit (28-32 weeks of gestation). The following were assessed at each visit: weight or body mass index, platelet function analysis-100 (Siemens), urinary thromboxane B2, serum thromboxane B2, and plasma salicylic acid. The PK visits consisted of blood work done at baseline (predose), administration of 81 mg nonenteric coated aspirin, and then plasma blood level of salicylic acid assessed at 30 minutes and then hourly 1 to 6 hours after dose. Pearson correlation and multivariable regression were used to identify associations between parameters and identify relevant covariates. Log-adjusted values were used for regression analysis. P<.05 was considered statistically significant. RESULTS: Nineteen participants were included with first trimester data, and 16 with third trimester data. There was no statistically significant change in mean PK parameters between the first and third trimester, although there was a trend to lower peak concentration in the third than in the first trimester (P=.08). In multivariable regression, baseline obesity and current body mass index as a continuous measures were negatively associated with log-adjusted peak salicylic acid concentration (-0.28 [-0.46 to -0.11], P=.003 and -0.02 [-0.03 to -0.009], P=.001, respectively) and log-adjusted plasma salicylic acid area under the curve 0 to 6 hours postdose (-0.25 [-0.45 to 0.05], P=.02, -0.04 [-0.07 to -0.01], P=.008 respectively). There was a significant decrease in urinary thromboxane 2 to 4 weeks after aspirin initiation compared with baseline, which correlated with a concomitant increase in platelet function analysis-100 closure time. In multivariable regression model, there was a strong association between plasma salicylic acid concentration (area under the curve 0-6 hours postdose) and urinary thromboxane (B=-3.12 [-5.38 to -1.04], P=.006), and with urinary thromboxane suppression and platelet inhibition, platelet function analysis-100 (-0.23 [-0.31 to -0.14], P<.001). With progressive thromboxane suppression, platelet inhibition (platelet function analysis-100 closure time) increased. Individual comorbidities, including weight, hypertension, and pregestational diabetes (Type I or II), also impacted aspirin response. CONCLUSION: We have demonstrated the relationship between individual factors, plasma concentrations of salicylic acid, thromboxane suppression, and platelet inhibition at a single dose (81 mg) of aspirin taken through pregnancy. Our findings suggest that dose modification of aspirin in pregnancy may help to achieve the optimal response. Our results may be used to facilitate computational modeling to identify optimal dosing, taking into consideration individual factors.

Activation of coagulation and proinflammatory pathways in thrombosis with thrombocytopenia syndrome and following COVID-19 vaccination.

Thrombosis with thrombocytopenia syndrome (TTS) is a rare but potentially severe adverse event following immunization with adenovirus vector-based COVID-19 vaccines such as Ad26.COV2.S (Janssen) and ChAdOx1 (AstraZeneca). However, no case of TTS has been reported in over 1.5 million individuals who received a second immunization with Ad26.COV2.S in the United States. Here we utilize transcriptomic and proteomic profiling to compare individuals who receive two doses of Ad26.COV2.S with those vaccinated with BNT162b2 or mRNA-1273. Initial Ad26.COV2.S vaccination induces transient activation of platelet and coagulation and innate immune pathways that resolve by day 7; by contrast, patients with TTS show robust upregulation of these pathways on days 15-19 following initial Ad26.COV2.S vaccination. Meanwhile, a second immunization or a reduced initial dose of Ad26.COV2.S induces lower activation of these pathways than does the full initial dose. Our data suggest a role of coagulation and proinflammatory pathways in TTS pathogenesis, which may help optimize vaccination regimens to reduce TTS risk.

The predominant PAR4 variant in individuals of African ancestry worsens murine and human stroke outcomes.

Protease-activated receptor 4 (PAR4) (gene F2RL3) harbors a functional dimorphism, rs773902 A/G (encoding Thr120/Ala120, respectively) and is associated with greater platelet aggregation. The A allele frequency is more common in Black individuals, and Black individuals have a higher incidence of ischemic stroke than White individuals. However, it is not known whether the A allele is responsible for worse stroke outcomes. To directly test the in vivo effect of this variant on stroke, we generated mice in which F2rl3 was replaced by F2RL3, thereby expressing human PAR4 (hPAR4) with either Thr120 or Ala120. Compared with hPAR4 Ala120 mice, hPAR4 Thr120 mice had worse stroke outcomes, mediated in part by enhanced platelet activation and platelet-neutrophil interactions. Analyses of 7,620 Black subjects with 487 incident ischemic strokes demonstrated the AA genotype was a risk for incident ischemic stroke and worse functional outcomes. In humanized mice, ticagrelor with or without aspirin improved stroke outcomes in hPAR4 Ala120 mice, but not in hPAR4 Thr120 mice. P selectin blockade improved stroke outcomes and reduced platelet-neutrophil interactions in hPAR4 Thr120 mice. Our results may explain some of the racial disparity in stroke and support the need for studies of nonstandard antiplatelet therapies for patients expressing PAR4 Thr120.

Novel Strategy to Combat the Procoagulant Phenotype in Heparin-Induced Thrombocytopenia Using 12-LOX Inhibition.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a major concern for all individuals that undergo cardiac bypass surgeries or require prolonged heparin exposure. HIT is a life- and limb-threatening adverse drug reaction with an immune response following the formation of ultra-large immune complexes that drive platelet activation through the receptor FcγRIIA. Thrombotic events remain high following the standard of care treatment with anticoagulants, while increasing risk of bleeding complications. This study sought to investigate a novel approach to treatment of HIT. Recent reports demonstrate increased procoagulant activity in HIT; however, these reports required analysis ex vivo, and relevance in vivo remains unclear. METHODS: Using human and mouse model systems, we investigated the cooperativity of PARs (protease-activated receptors) and FcγRIIA in HIT. We challenged humanized FcγRIIA transgenic mice with or without endogenous mouse Par4 (denoted as IIA-Par4+/+ or IIA-Par4-/-, respectively) with a well-established model IgG immune complex (anti [α]-CD9). Furthermore, we assessed the procoagulant phenotype and efficacy to treat HIT utilizing inhibitor of 12-LOX (12[S]-lipoxygenase), VLX-1005, previously reported to decrease platelet activation downstream of FcγRIIA and PAR4, using the triple allele HIT mouse model. RESULTS: IIA-Par4+/+ mice given αCD9 were severely thrombocytopenic, with extensive platelet-fibrin deposition in the lung. In contrast, IIA-Par4-/- mice had negligible thrombocytopenia or pulmonary platelet-fibrin thrombi. We observed that pharmacological inhibition of 12-LOX resulted in a significant reduction in both platelet procoagulant phenotype ex vivo, and thrombocytopenia and thrombosis in our humanized mouse model of HIT in vivo. CONCLUSIONS: These data demonstrate for the first time the need for dual platelet receptor (PAR and FcγRIIA) stimulation for fibrin formation in HIT in vivo. These results extend our understanding of HIT pathophysiology and provide a scientific rationale for targeting the procoagulant phenotype as a possible therapeutic strategy in HIT.

Species Differences in Platelet Protease-Activated Receptors.

Protease-activated receptors (PARs) are a class of integral membrane proteins that are cleaved by a variety of proteases, most notably thrombin, to reveal a tethered ligand and promote activation. PARs are critical mediators of platelet function in hemostasis and thrombosis, and therefore are attractive targets for anti-platelet therapies. Animal models studying platelet PAR physiology have relied heavily on genetically modified mouse strains, which have provided ample insight but have some inherent limitations. The current review aims to summarize the notable PAR expression and functional differences between the mouse and human, in addition to highlighting some recently developed tools to further study human physiology in mouse models.

Perioperative Outcomes of Patients with Bleeding Disorders Undergoing Major Surgery at an Academic Hemophilia Treatment Center.

Persons with bleeding disorders (PwBD) are at high risk for bleeding with invasive procedures. However, the risk of bleeding in PwBD undergoing major surgery and outcomes of patients managed perioperatively at a hemophilia treatment center (HTC) are not well described. We performed a retrospective review of surgical outcomes among PwBD undergoing major surgery between January 1st, 2017 and December 31st, 2019 at the Cardeza Foundation Hemophilia and Thrombosis Center in Philadelphia, PA. The primary outcome was postoperative bleeding, assessed according to the ISTH-SSC's 2010 definition. Secondary outcomes included use of unplanned postoperative hemostatic therapy, LOS, and 30-day readmission rate. Results were compared to non-PwBD population from a surgical database, matched for surgery, age, and sex. During the study period, 50 PwBD underwent 63 major surgeries. The most common diagnoses were VWD (64%) and hemophilia A (20.0%). The most common surgical procedure category was orthopedic (33.3%), predominantly arthroplasties. Postoperatively,4.8% of procedures were complicated by major bleeding and 1.6% by non-major bleeding. The mean LOS was 1.65 days, and 30-day readmission rate was 1.6%. In comparison to matched, non-PwBD patients in a national surgical database undergoing the same procedures, study patients had a similar rate of bleeding complications per procedure (5.0% vs 1.04% P = .071, Fisher's exact test). PwBD undergoing major surgeries have low rates of major bleeding when receiving comprehensive care at an HTC. Bleeding and hospital readmission rates were similar to non-PwBD baseline in a large database.

Quantification of energy-converting protein complexes in plant thylakoid membranes.

Knowledge about the exact abundance and ratio of photosynthetic protein complexes in thylakoid membranes is central to understanding structure-function relationships in energy conversion. Recent modeling approaches for studying light harvesting and electron transport reactions rely on quantitative information on the constituent complexes in thylakoid membranes. Over the last decades several quantitative methods have been established and refined, enabling precise stoichiometric information on the five main energy-converting building blocks in the thylakoid membrane: Light-harvesting complex II (LHCII), Photosystem II (PSII), Photosystem I (PSI), cytochrome b6f complex (cyt b6f complex), and ATPase. This paper summarizes a few quantitative spectroscopic and biochemical methods that are currently available for quantification of plant thylakoid protein complexes. Two new methods are presented for quantification of LHCII and the cyt b6f complex, which agree well with established methods. In addition, recent improvements in mass spectrometry (MS) allow deeper compositional information on thylakoid membranes. The comparison between mass spectrometric and more classical protein quantification methods shows similar quantities of complexes, confirming the potential of thylakoid protein complex quantification by MS. The quantitative information on PSII, PSI, and LHCII reveal that about one third of LHCII must be associated with PSI for a balanced light energy absorption by the two photosystems.

The Function of ASK1 in Sepsis and Stress-Induced Disorders.

Apoptosis signal-regulating kinase 1 (ASK1) is a serine-threonine kinase that is ubiquitously expressed in nucleated cells and is responsible for the activation of multiple mitogen-activated protein kinases (MAPK) to regulate cell stress. Activation of ASK1 via cellular stress leads to activation of downstream signaling components, activation of transcription factors, and proinflammatory cytokine production. ASK1 is also expressed in anucleate platelets and is a key player in platelet activation as it is important for signaling. Interestingly, the mechanism of ASK1 activation is cell type-dependent. In this review we will explore how ASK1 regulates a variety of cellular processes from innate immune function to thrombosis and hemostasis. We will discuss how ASK1 influences FcγRIIA-mediated platelet reactivity and how that reactivity drives platelet clearance. Furthermore, we will explore the role of ASK1 in thromboxane (TxA2) generation, which highlights differences in the way ASK1 functions in mouse and human platelets.

Platelet protease activated receptor 4 (PAR 4) receptor genotype is associated with an increased risk of preterm birth.

BACKGROUND: Platelet protease activated receptor-4 (PAR4) Thr120 is a common genetic variant associated with increased platelet activity. Increased platelet activity is implicated in the pathogenesis of preeclampsia and preterm birth. OBJECTIVE: Compare the rate of preeclampsia and preterm birth in pregnant individuals homozygous for PAR4 Thr120 variant vs not. STUDY DESIGN: This is a prospective cohort study of patients who delivered November 2020-July 2021. Maternal blood collected on admission for PAR4 genotyping. The primary outcome was the rate of preeclampsia/gestational hypertension in those with Thr/Thr genotype compared with Ala/Thr or Ala/Ala. Secondary outcomes included rates of preterm birth and placental pathology. RESULTS: Three hundred and twenty singletons were included and 52 (16.3%) were PAR4 Thr/Thr. Those PAR4 Thr/Thr were more likely to be Black (67.3% vs 29.5%, p < .001), younger (28 ± 6 vs 31 ± 6, p = .004), and have higher body mass index (35.2 ± 6.8 vs 33.1 ± 7.4, p = .047). There was no difference in preeclampsia/gestational hypertension (19.2% vs 22.8%, p = .705). Those Thr/Thr had a significantly higher rate of preterm birth (15.4% vs 3.7%, adjusted odds ratio [aOR] 4.04 [1.47-11.10], p = .007), indicated preterm birth because of fetal growth restriction or preeclampsia (5.8% vs 0.4%, aOR 10.03 [1.48-67.87], p = .02), spontaneous preterm birth (7.7% vs 2.2%, aOR 4.81 [1.27-18.27], p = .02), and placental intervillous thrombosis (18.5% vs 7.9%, aOR 4.12 [1.14-14.92], p = .03). CONCLUSION: Platelet receptor PAR4 Thr120 is a common variant associated with an increased risk of placental vascular pathology and preterm birth in homozygous individuals. Although a cohort study cannot establish causation, this strong association warrants further exploration.

GRK2 regulates ADP signaling in platelets via P2Y1 and P2Y12.

The critical role of G protein-coupled receptor kinase 2 (GRK2) in regulating cardiac function has been well documented for >3 decades. Targeting GRK2 has therefore been extensively studied as a novel approach to treating cardiovascular disease. However, little is known about its role in hemostasis and thrombosis. We provide here the first evidence that GRK2 limits platelet activation and regulates the hemostatic response to injury. Deletion of GRK2 in mouse platelets causes increased platelet accumulation after laser-induced injury in the cremaster muscle arterioles, shortens tail bleeding time, and enhances thrombosis in adenosine 5'-diphosphate (ADP)-induced pulmonary thromboembolism and in FeCl3-induced carotid injury. GRK2-/- platelets have increased integrin activation, P-selectin exposure, and platelet aggregation in response to ADP stimulation. Furthermore, GRK2-/- platelets retain the ability to aggregate in response to ADP restimulation, indicating that GRK2 contributes to ADP receptor desensitization. Underlying these changes in GRK2-/- platelets is an increase in Ca2+ mobilization, RAS-related protein 1 activation, and Akt phosphorylation stimulated by ADP, as well as an attenuated rise of cyclic adenosine monophosphate levels in response to ADP in the presence of prostaglandin I2. P2Y12 antagonist treatment eliminates the phenotypic difference in platelet accumulation between wild-type and GRK2-/- mice at the site of injury. Pharmacologic inhibition of GRK2 activity in human platelets increases platelet activation in response to ADP. Finally, we show that GRK2 binds to endogenous Gßγ subunits during platelet activation. Collectively, these results show that GRK2 regulates ADP signaling via P2Y1 and P2Y12, interacts with Gßγ, and functions as a signaling hub in platelets for modulating the hemostatic response to injury.

Photosystem stoichiometry adjustment is a photoreceptor-mediated process in Arabidopsis.

Plant growth under spectrally-enriched low light conditions leads to adjustment in the relative abundance of the two photosystems in an acclimatory response known as photosystem stoichiometry adjustment. Adjustment of photosystem stoichiometry improves the quantum efficiency of photosynthesis but how this process perceives light quality changes and how photosystem amount is regulated remain largely unknown. By using a label-free quantitative mass spectrometry approach in Arabidopsis here we show that photosystem stoichiometry adjustment is primarily driven by the regulation of photosystem I content and that this forms the major thylakoid proteomic response under light quality. Using light and redox signaling mutants, we further show that the light quality-responsive accumulation of photosystem I gene transcripts and proteins requires phytochrome B photoreceptor but not plastoquinone redox signaling as previously suggested. In far-red light, the increased acceptor side limitation might deplete active photosystem I pool, further contributing to the adjustment of photosystem stoichiometry.

Human and mouse PAR4 are functionally distinct receptors: Studies in novel humanized mice.

BACKGROUND: Human and mouse platelets both express protease-activated receptor (PAR) 4 but sequence alignment reveals differences in several functional domains. These differences may result in functional disparities between the receptors which make it difficult to translate PAR4 studies using mice to human platelet physiology. OBJECTIVES: To generate transgenic mice that express human, but not mouse, PAR4 and directly compare human and mouse PAR4 function in the same platelet environment. METHODS: Transgenic mice were made using a genomic clone of the F2RL3 gene (encoding PAR4) and backcrossed with Par4 KO mice. For certain experiments, mice were bred with GRK6 KO mice. Tail bleeding time and platelet function in response to PAR4-activating peptide were assessed. RESULTS: Human F2RL3 was successfully integrated into the mouse genome, transgenic mice were crossed to the mPar4 KO background (PAR4 tg/KO), and PAR4 was functionally expressed on platelets. Compared to WT, PAR4 tg/KO mice exhibited shortened tail bleeding time and their platelets were more responsive to PAR4-AP as assessed by α-granule release and integrin activation. The opposite was observed with thrombin. Knocking out GRK6 had no effect on human PAR4-expressing platelets, unlike mouse Par4-expressing platelets. PAR4 tg/KO platelets exhibited greater Ca2+ area under the curve and more robust extracellular vesicle release than WT stimulated with PAR4-AP. CONCLUSION: These data suggest that (1) human PAR4- and mouse Par4-mediated signaling are different and (2) the feedback regulation mechanisms of human and mouse PAR4 are different. These functional differences are important to consider when interpreting PAR4 studies done with mice.

G protein-coupled receptor kinase 5 regulates thrombin signaling in platelets via PAR-1.

The interindividual variation in the functional response of platelets to activation by agonists is heritable. Genome-wide association studies (GWASs) of quantitative measures of platelet function have identified fewer than 20 distinctly associated variants, some with unknown mechanisms. Here, we report GWASs of pathway-specific functional responses to agonism by adenosine 5'-diphosphate, a glycoprotein VI-specific collagen mimetic, and thrombin receptor-agonist peptides, each specific to 1 of the G protein-coupled receptors PAR-1 and PAR-4, in subsets of 1562 individuals. We identified an association (P = 2.75 × 10-40) between a common intronic variant, rs10886430, in the G protein-coupled receptor kinase 5 gene (GRK5) and the sensitivity of platelets to activate through PAR-1. The variant resides in a megakaryocyte-specific enhancer that is bound by the transcription factors GATA1 and MEIS1. The minor allele (G) is associated with fewer GRK5 transcripts in platelets and the greater sensitivity of platelets to activate through PAR-1. We show that thrombin-mediated activation of human platelets causes binding of GRK5 to PAR-1 and that deletion of the mouse homolog Grk5 enhances thrombin-induced platelet activation sensitivity and increases platelet accumulation at the site of vascular injury. This corroborates evidence that the human G allele of rs10886430 is associated with a greater risk for cardiovascular disease. In summary, by combining the results of pathway-specific GWASs and expression quantitative trait locus studies in humans with the results from platelet function studies in Grk5-/- mice, we obtain evidence that GRK5 regulates the human platelet response to thrombin via the PAR-1 pathway.