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.

RGS10 and RGS18 differentially limit platelet activation, promote platelet production, and prolong platelet survival.

G protein-coupled receptors are critical mediators of platelet activation whose signaling can be modulated by members of the regulator of G protein signaling (RGS) family. The 2 most abundant RGS proteins in human and mouse platelets are RGS10 and RGS18. While each has been studied individually, critical questions remain about the overall impact of this mode of regulation in platelets. Here, we report that mice missing both proteins show reduced platelet survival and a 40% decrease in platelet count that can be partially reversed with aspirin and a P2Y12 antagonist. Their platelets have increased basal (TREM)-like transcript-1 expression, a leftward shift in the dose/response for a thrombin receptor-activating peptide, an increased maximum response to adenosine 5'-diphosphate and TxA2, and a greatly exaggerated response to penetrating injuries in vivo. Neither of the individual knockouts displays this constellation of findings. RGS10-/- platelets have an enhanced response to agonists in vitro, but platelet count and survival are normal. RGS18-/- mice have a 15% reduction in platelet count that is not affected by antiplatelet agents, nearly normal responses to platelet agonists, and normal platelet survival. Megakaryocyte number and ploidy are normal in all 3 mouse lines, but platelet recovery from severe acute thrombocytopenia is slower in RGS18-/- and RGS10-/-18-/- mice. Collectively, these results show that RGS10 and RGS18 have complementary roles in platelets. Removing both at the same time discloses the extent to which this regulatory mechanism normally controls platelet reactivity in vivo, modulates the hemostatic response to injury, promotes platelet production, and prolongs platelet survival.

High-efficiency unassisted transfection of platelets with naked double-stranded miRNAs modulates signal-activated translation and platelet function.

We sought novel approaches to improve transfection efficiencies of microRNAs (miRNAs) in platelets, and to apply these approaches to investigate the roles of miRNAs in regulating signal-activated protein translation and functional effects. We found that ex vivo human platelets support gymnosis---internalization of ectopic miRNAs following co-incubation in the absence of conventional transfection reagents or schemes---and subsequently incorporate transfected miRNA into ARGONAUTE2 (AGO2)-based RNA-induced silencing complexes (RISC). Thrombin/fibrinogen stimulation activated translation of miR-223-3p target SEPTIN2, which was suppressed by miR-223-3p transfection in an AGO2/RISC-dependent manner. Thrombin/fibrinogen-induced exosome and microvesicle generation was inhibited by miR-223-3p transfection, and this effect was reversed with a RISC inhibitor. Platelet gymnosis of naked miRNAs appeared to be mediated in part by endocytic pathways including clathrin-dependent and fluid-phase endocytosis and caveolae. These results demonstrate the ability of ex vivo platelets to internalize ectopic miRNAs by unassisted transfection, and utilize them to modulate signal-activated translation and platelet function. Our results identify new roles for miR-223-3p in extracellular vesicle generation in stimulated platelets. High-efficiency gymnotic transfection of miRNAs in ex vivo platelets may be a broadly useful tool for exploring molecular genetic regulation of platelet function.

The Roles of GRKs in Hemostasis and Thrombosis.

Along with cancer, cardiovascular and cerebrovascular diseases remain by far the most common causes of death. Heart attacks and strokes are diseases in which platelets play a role, through activation on ruptured plaques and subsequent thrombus formation. Most platelet agonists activate platelets via G protein-coupled receptors (GPCRs), which make these receptors ideal targets for many antiplatelet drugs. However, little is known about the mechanisms that provide feedback regulation on GPCRs to limit platelet activation. Emerging evidence from our group and others strongly suggests that GPCR kinases (GRKs) are critical negative regulators during platelet activation and thrombus formation. In this review, we will summarize recent findings on the role of GRKs in platelet biology and how one specific GRK, GRK6, regulates the hemostatic response to vascular injury. Furthermore, we will discuss the potential role of GRKs in thrombotic disorders, such as thrombotic events in COVID-19 patients. Studies on the function of GRKs during platelet activation and thrombus formation have just recently begun, and a better understanding of the role of GRKs in hemostasis and thrombosis will provide a fruitful avenue for understanding the hemostatic response to injury. It may also lead to new therapeutic options for the treatment of thrombotic and cardiovascular disorders.

Modulating platelet reactivity through control of RGS18 availability.

Most platelet agonists activate platelets by binding to G-protein-coupled receptors. We have shown previously that a critical node in the G-protein signaling network in platelets is formed by a scaffold protein, spinophilin (SPL), the tyrosine phosphatase, Src homology region 2 domain-containing phosphatase-1 (SHP-1), and the regulator of G-protein signaling family member, RGS18. Here, we asked whether SPL and other RGS18 binding proteins such as 14-3-3γ regulate platelet reactivity by sequestering RGS18 and, if so, how this is accomplished. The results show that, in resting platelets, free RGS18 levels are relatively low, increasing when platelets are activated by thrombin. Free RGS18 levels also rise when platelets are rendered resistant to activation by exposure to prostaglandin I2 (PGI2) or forskolin, both of which increase platelet cyclic adenosine monophosphate (cAMP) levels. However, the mechanism for raising free RGS18 is different in these 2 settings. Whereas thrombin activates SHP-1 and causes dephosphorylation of SPL tyrosine residues, PGI2 and forskolin cause phosphorylation of SPL Ser94 without reducing tyrosine phosphorylation. Substituting alanine for Ser94 blocks cAMP-induced dissociation of the SPL/RGS/SHP-1 complex. Replacing Ser94 with aspartate prevents formation of the complex and produces a loss-of-function phenotype when expressed in mouse platelets. Together with the defect in platelet function we previously observed in SPL(-/-) mice, these data show that (1) regulated sequestration and release of RGS18 by intracellular binding proteins provides a mechanism for coordinating activating and inhibitory signaling networks in platelets, and (2) differential phosphorylation of SPL tyrosine and serine residues provides a key to understanding both.

A newly identified complex of spinophilin and the tyrosine phosphatase, SHP-1, modulates platelet activation by regulating G protein-dependent signaling.

Platelets are essential for normal hemostasis, but close regulation is required to avoid the destructive effects of either inappropriate platelet activation or excessive responses to injury. Here, we describe a novel complex comprising the scaffold protein, spinophilin (SPL), and the tyrosine phosphatase, SHP-1, and show that it can modulate platelet activation by sequestering RGS10 and RGS18, 2 members of the regulator of G protein signaling family. We also show that SPL/RGS/SHP1 complexes are present in resting platelets where constitutive phosphorylation of SPL(Y398) creates an atypical binding site for SHP-1. Activation of the SHP-1 occurs on agonist-induced phosphorylation of SHP-1(Y536), triggering dephosphorylation and decay of the SPL/RGS/SHP1 complex. Preventing SHP-1 activation blocks decay of the complex and produces a gain of function. Conversely, deleting spinophilin in mice inhibits platelet activation. It also attenuates the rise in platelet cAMP normally caused by endothelial prostacyclin (PGI(2)). Thus, we propose that the role of the SPL/RGS/SHP1 complex in platelets is time and context dependent. Before injury, the complex helps maintain the quiescence of circulating platelets by maximizing the impact of PGI(2). After injury, the complex gradually releases RGS proteins, limiting platelet activation and providing a mechanism for temporal coordination of pro thrombotic and antithrombotic inputs.

Revolutionizing vascular imaging: trends and future directions of 4D flow MRI based on a 20-year bibliometric analysis.

Background: Four-dimensional flow magnetic resonance imaging (4D flow MRI) is a promising new technology with potential clinical value in hemodynamic quantification. Although an increasing number of articles on 4D flow MRI have been published over the past decades, few studies have statistically analyzed these published articles. In this study, we aimed to perform a systematic and comprehensive bibliometric analysis of 4D flow MRI to explore the current hotspots and potential future directions. Methods: The Web of Science Core Collection searched for literature on 4D flow MRI between 2003 and 2022. CiteSpace was utilized to analyze the literature data, including co-citation, cooperative network, cluster, and burst keyword analysis. Results: A total of 1,069 articles were extracted for this study. The main research hotspots included the following: quantification and visualization of blood flow in different clinical settings, with keywords such as "cerebral aneurysm", "heart", "great vessel", "tetralogy of Fallot", "portal hypertension", and "stiffness"; optimization of image acquisition schemes, such as "resolution" and "reconstruction"; measurement and analysis of flow components and patterns, as indicated by keywords "pattern", "KE", "WSS", and "fluid dynamics". In addition, international consensus for metrics derived from 4D flow MRI and multimodality imaging may also be the future research direction. Conclusions: The global domain of 4D flow MRI has grown over the last 2 decades. In the future, 4D flow MRI will evolve towards becoming a relatively short scan duration with adequate spatiotemporal resolution, expansion into the diagnosis and treatment of vascular disease in other related organs, and a shift in focus from vascular structure to function. In addition, artificial intelligence (AI) will assist in the clinical promotion and application of 4D flow MRI.

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.

Multi-parameter cardiac magnetic resonance imaging detects anthracycline-induced cardiotoxicity in rabbits model.

Background: Cardiac magnetic resonance (CMR) quantitative T1 and T2 mapping offers a non-invasive means to evaluate early cardiotoxicity changes. This study aimed to pinpoint the earliest CMR indicators of myocardial injury in Anthracycline-induced cardiotoxicity (AIC) and to elucidate the connections between these CMR indicators and associated pathological indicators. Methods: A total of 34 rabbits were administered doxorubicin at a dosage of 1 mg/kg/weekly. The study incorporated six 3T CMR scan time points: baseline, and at intervals of four, six, eight, twelve, and sixteen weeks. Cine, T1 and T2 mapping sequences assessed the left ventricular ejection fraction (LVEF), native T1, extracellular volume fraction (ECV), and T2 values. Following each time point, three rabbits were sacrificed for histological analysis involving Hematoxylin and eosin (H&E), Masson, TUNEL, and microvascular density (MVD) stains. Spearman correlations and linear mixed model analysis served in the statistical analysis. Results: Diverse degrees of alternation were recorded in LVEF, native T1, T2, and ECV over time. LVEF declined to 49.0 ± 2.6 % at 12 weeks from the baseline of 53.4 ± 3.2 %, p < 0.001. Native T1 values increase from the baseline (1396.5 ± 79.2 ms) until 8 weeks (1498.8 ± 95.4 ms, p < 0.001). T2 values increased from the baseline (36.6 ± 3.3 ms) within 4 weeks of initiation (37.5 ± 3.4, p = 0.02) and remained elevated through 16 weeks (42.8 ± 0.3, p < 0.01). ECV was elevated at 8 weeks (33.9 ± 3.8 %, p = 0.005) compared to the baseline (30.2 ± 2.5 %). By week 12, myocardial edema and increased CVF were apparent (p = 0.04 and = 0.001, respectively). The area under ROC curve for positive CMR presence and the gold standards were 0.87 (T2-ROC, 4 weeks) and 0.92 (LVEF&BNP-ROC, 12 weeks). Conclusion: T1 and T2 mapping are effective tools for cardiotoxicity detection and monitoring. The prolongation of T2 value emerged as the most consistent and early-onset indicator.

A regulatory node involving Gαq, PLCß, and RGS proteins modulates platelet reactivity to critical agonists.

BACKGROUND: Most platelet agonists work through G protein-coupled receptors, activating pathways that involve members of the Gq, Gi, and G12/G13 families of heterotrimeric G proteins. Gq signaling has been shown to be critical for efficient platelet activation. Growing evidence suggests that regulatory mechanisms converge on G protein-coupled receptors and Gq to prevent overly robust platelet reactivity. OBJECTIVES: To identify and characterize mechanisms by which Gq signaling is regulated in platelets. METHODS: Based on our prior experience with a Gαi2 variant that escapes regulation by regulator of G protein signaling (RGS) proteins, a Gαq variant was designed with glycine 188 replaced with serine (G188S) and then incorporated into a mouse line so that its effects on platelet activation and thrombus formation could be studied in vitro and in vivo. RESULTS AND CONCLUSIONS: As predicted, the G188S substitution in Gαq disrupted its interaction with RGS18. Unexpectedly, it also uncoupled PLCß-3 from activation by platelet agonists as evidenced by a loss rather than a gain of platelet function in vitro and in vivo. Binding studies showed that in addition to preventing the binding of RGS18 to Gαq, the G188S substitution also prevented the binding of PLCß-3 to Gαq. Structural analysis revealed that G188 resides in the region that is also important for Gαq binding to PLCß-3 in platelets. We conclude that the Gαq signaling node is more complex than that has been previously understood, suggesting that there is cross-talk between RGS proteins and PLCß-3 in the context of Gαq signaling.

RGS/Gi2alpha interactions modulate platelet accumulation and thrombus formation at sites of vascular injury.

Although much is known about extrinsic regulators of platelet function such as nitric oxide and prostaglandin I(2) (PGI(2)), considerably less is known about intrinsic mechanisms that prevent overly robust platelet activation after vascular injury. Here we provide the first evidence that regulators of G-protein signaling (RGS) proteins serve this role in platelets, using mice with a G184S substitution in G(i2α) that blocks RGS/G(i2) interactions to examine the consequences of lifting constraints on G(i2)-dependent signaling without altering receptor:effector coupling. The results show that the G(i2α)(G184S) allele enhances platelet aggregation in vitro and increases platelet accumulation after vascular injury when expressed either as a global knock-in or limited to hematopoietic cells. Biochemical studies show that these changes occur in concert with an attenuated rise in cyclic adenosine monophosphate levels in response to prostacyclin and a substantial increase in basal Akt activation. In contrast, basal cyclic adenosine monophosphate (cAMP) levels, agonist-stimulated increases in [Ca(++)](i), Rap1 activation, and α-granule secretion were unaffected. Collectively, these observations (1) demonstrate an active role for RGS proteins in regulating platelet responsiveness, (2) show that this occurs in a pathway-selective manner, and (3) suggest that RGS proteins help to prevent unwarranted platelet activation as well as limiting the magnitude of the normal hemostatic response.

Platelet signaling.

This chapter summarizes current ideas about the intracellular signaling that drives platelet responses to vascular injury. After a brief overview of platelet activation intended to place the signaling pathways into context, the first section considers the early events of platelet activation leading up to integrin activation and platelet aggregation. The focus is on the G protein-mediated events utilized by agonists such as thrombin and ADP, and the tyrosine kinase-based signaling triggered by collagen. The second section considers the events that occur after integrin engagement, some of which are dependent on close physical contact between platelets. A third section addresses the regulatory events that help to avoid unprovoked or excessive platelet activation, after which the final section briefly considers individual variations in platelet reactivity and the role of platelet signaling in the innate immune response and embryonic development.

Histologic validation of stress cardiac magnetic resonance T1-mapping techniques for detection of coronary microvascular dysfunction in rabbits.

BACKGROUND: To investigate the diagnostic performance of stress cardiac magnetic resonance (CMR) T1-mapping for the detection of coronary microvascular dysfunction (CMD) by correlating microvascular density (MVD) and collagen volume fraction (CVF) with T1 response to adenosine triphosphate (ATP) stress (stress ΔT1) in rabbits. METHODS: Twenty-four New Zealand white rabbits were randomly divided into the CMD group induced by microembolization spheres (n = 10), sham-operated group (n = 5), and control group (n = 9). All rabbits underwent 3.0 T CMR, both rest and ATP stress T1-maps were obtained, and first-pass perfusion imaging was performed. Stress ΔT1 and myocardial perfusion reserve index (MPRI) were calculated. For the histologic study, each rabbit was sacrificed after CMR scanning. Left ventricular myocardial tissue was stained with Hematoxylin-eosin (H&E), Masson, and CD31, from which MVD and CVF were extracted. Pearson correlation analyses were performed to determine the strength of the association between the stress ΔT1 and both MVD and CVF. RESULTS: The stress ΔT1 values (CMD, 2.53 ± 0.37% vs. control, 6.00 ± 0.64% vs. Sham, 6.07 ± 0.97%, p < 0.001) and MPRI (CMD, 1.45 ± 0.13 vs. control, 1.94 ± 0.23, vs. sham, 1.89 ± 0.15, p < 0.001) were both lower in CMD rabbits compared with sham-operated and control rabbits. Further, the stress ΔT1 showed a high correlation with CVF (r = -0.806, p < 0.001) and MVD (r = 0.920, p < 0.001). CONCLUSIONS: Stress T1 response strongly correlates with pathological MVD and CVF, indicating that stress CMR T1 mapping can accurately detect microvascular dysfunction.

Linear late gadolinium enhancement in the basal anterior septum and lateral wall may represent the contrast enhancement of vessels: A CMR and CCTA comparison study.

BACKGROUND: The purpose of this paper was to verify that the linear high-intensity signal on late gadolinium enhancement-cardiac magnetic resonance (LGE-CMR) may represent the contrast enhancement of vessels rather than scars or fibrosis, and to assess whether this linear high-intensity signal will affect the quantification of myocardial fibrosis in patients with hypertrophic cardiomyopathy (HCM). METHODS: A total of 58 patients who underwent both coronary computed tomography angiography (CCTA) and LGE-CMR in our hospital were ultimately enrolled. The definitions of positive linear LGE (LLGE+) were as follows: (1) LLGE in the basal anterior septum or lateral wall, and (2) LLGE observable at 10 mm or more. All other patients were regarded as negative LLGE (LLGE-). In LLGE+ patients, the length of the LLGE located in the anterior septum and lateral wall was compared with the length of the septal perforator artery and the circumflex artery on CCTA, respectively. For nine patients with HCM, the LGE% was measured before and after removal of LLGE. RESULTS: Among the 58 patients, 40 showed LLGE+ and 18 showed LLGE-. For patients with LLGE in the anterior septum, there was a strong correlation between LLGE and anterior septal perforator arteries in length (r=0.887, p<0.001). For patients with LLGE in the lateral wall, LLGE also correlated well with the circumflex arteries in length (r=0.962, p<0.001). In nine patients with HCM, the LGE% decreased significantly after the removal of LLGE [9.50 (7.70 - 17.35)% vs. 8.80 (6.20 - 15.55)%, p<0.05]. CONCLUSIONS: The LLGE in the anterior septum and lateral wall may represent contrast enhancement of the anterior septal perforator artery and the circumflex artery, respectively. This LLGE may overestimate the extent of myocardial fibrosis in patients with HCM.

Stress CMR T1-mapping technique for assessment of coronary microvascular dysfunction in a rabbit model of type II diabetes mellitus: Validation against histopathologic changes.

Background: Coronary microvascular dysfunction (CMD) is an early character of type 2 diabetes mellitus (T2DM), and is indicative of adverse events. The present study aimed to validate the performance of the stress T1 mapping technique on cardiac magnetic resonance (CMR) for identifying CMD from a histopathologic perspective and to establish the time course of CMD-related parameters in a rabbit model of T2DM. Methods: New Zealand white rabbits (n = 30) were randomly divided into a control (n = 8), T2DM 5-week (n = 6), T2DM 10-week (n = 9), and T2DM 15-week (n = 7) groups. The CMR protocol included rest and adenosine triphosphate (ATP) stress T1-mapping imaging using the 5b(20b)3b-modified look-locker inversion-recovery (MOLLI) schema to quantify stress T1 response (stress ΔT1), and first-pass perfusion CMR to quantify myocardial perfusion reserve index (MPRI). After the CMR imaging, myocardial tissue was subjected to hematoxylin-eosin staining to evaluate pathological changes, Masson trichrome staining to measure collagen volume fraction (CVF), and CD31 staining to measure microvascular density (MVD). The associations between CMR parameters and pathological findings were determined using Pearson correlation analysis. Results: The stress ΔT1 values were 6.21 ± 0.59%, 4.88 ± 0.49%, 3.80 ± 0.40%, and 3.06 ± 0.54% in the control, T2DM 5-week, 10-week, and 15-week groups, respectively (p < 0.001) and were progressively weakened with longer duration of T2DM. Furthermore, a significant correlation was demonstrated between the stress ΔT1 vs. CVF and MVD (r = -0.562 and 0.886, respectively; p < 0.001). Conclusion: The stress T1 response correlated well with the histopathologic measures in T2DM rabbits, indicating that it may serve as a sensitive CMD-related indicator in early T2DM.

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.

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.

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.

Heparanase deglycanation of syndecan-1 is required for binding of the epithelial-restricted prosecretory mitogen lacritin.

Cell surface heparan sulfate (HS) proteoglycans are carbohydrate-rich regulators of cell migratory, mitogenic, secretory, and inflammatory activity that bind and present soluble heparin-binding growth factors (e.g., fibroblast growth factor, Wnt, Hh, transforming growth factor beta, amphiregulin, and hepatocyte growth factor) to their respective signaling receptors. We demonstrate that the deglycanated core protein of syndecan-1 (SDC1) and not HS chains nor SDC2 or -4, appears to target the epithelial selective prosecretory mitogen lacritin. An important and novel step in this mechanism is that binding necessitates prior partial or complete removal of HS chains by endogenous heparanase. This limits lacritin activity to sites where heparanase appears to predominate, such as sites of exocrine cell migration, secretion, renewal, and inflammation. Binding is mutually specified by lacritin's C-terminal mitogenic domain and SDC1's N terminus. Heparanase modification of the latter transforms a widely expressed HS proteoglycan into a highly selective surface-binding protein. This novel example of cell specification through extracellular modification of an HS proteoglycan has broad implications in development, homeostasis, and disease.

Restricted epithelial proliferation by lacritin via PKCalpha-dependent NFAT and mTOR pathways.

Renewal of nongermative epithelia is poorly understood. The novel mitogen "lacritin" is apically secreted by several nongermative epithelia. We tested 17 different cell types and discovered that lacritin is preferentially mitogenic or prosecretory for those types that normally contact lacritin during its glandular outward flow. Mitogenesis is dependent on lacritin's C-terminal domain, which can form an alpha-helix with a hydrophobic face, as per VEGF's and PTHLP's respective dimerization or receptor-binding domain. Lacritin targets downstream NFATC1 and mTOR. The use of inhibitors or siRNA suggests that lacritin mitogenic signaling involves Galpha(i) or Galpha(o)-PKCalpha-PLC-Ca2+-calcineurin-NFATC1 and Galpha(i) or Galpha(o)-PKCalpha-PLC-phospholipase D (PLD)-mTOR in a bell-shaped, dose-dependent manner requiring the Ca2+ sensor STIM1, but not TRPC1. This pathway suggests the placement of transiently dephosphorylated and perinuclear Golgi-translocated PKCalpha upstream of both Ca2+ mobilization and PLD activation in a complex with PLCgamma2. Outward flow of lacritin from secretory cells through ducts may generate a proliferative/secretory field as a different unit of cellular renewal in nongermative epithelia where luminal structures predominate.