Review of the limitations of current biomarkers in acute kidney injury clinical practices.

Acute kidney injury is a prevalent disease in hospitalized patients and is continuously increasing worldwide. Various efforts have been made to define and classify acute kidney injury to understand the progression of this disease. Furthermore, deviations from structure and kidney function and the current diagnostic guidelines are not adequately placed due to baseline serum creatinine values, which are rarely known and estimated based on glomerular function rate, resulting in misclassification of acute kidney injury staging. Hence, the current guidelines are still developing to improve and understand the clinical implications of risk factors and earlier predictive biomarkers of acute kidney injury. Yet, studies have indicated disadvantages and limitations with the current acute kidney injury biomarkers, including lack of sensitivity and specificity. Therefore, the present narrative review brings together the most current evidenced-based practice and literature associated with the limitations of the gold standard for acute kidney injury diagnoses, the need for novel acute kidney injury biomarkers, and the process for biomarkers to be qualified for diagnostic use under the following sections and themes. The introduction section situates the anatomy and normal and abnormal kidney functions related to acute kidney injury disorders. Guidelines in providing acute kidney injury definitions and classification are then considered, followed by a discussion of the disadvantages of standard markers used to diagnose acute kidney injury. Characteristics of an ideal acute kidney injury biomarker are discussed concerning sensitivity, specificity, and anatomic location of injury. A particular focus on the role and function of emerging biomarkers is discussed in relation to their applications and significance to the prognosis and severity of acute kidney injury. Findings show emerging markers are early indicators of acute kidney injury prediction in different clinical settings. Finally, the process required for a biomarker to be applied for diagnostic use is explained.

The G-protein ßγ subunits regulate platelet function.

AIMS: G-protein coupled receptors (GPCRs) tightly regulate platelet function by interacting with various physiological agonists. An essential mediator of GPCR signaling is the G protein αßγ heterotrimers, in which the ßγ subunits are central players in downstream signaling. Herein, we investigated the role of Gßγ subunits in platelet function, hemostasis and thrombogenesis. METHODS: To achieve this goal, platelets from both mice and humans were employed in the context of a small molecule inhibitor of Gßγ, namely gallein. We used an aggregometer to examine aggregation and dense granules secretion. We also used flow cytometry for P-selectin and PAC1 to determine the impact of inhibiting Gßγ on α -granule secretion and αIIbß3 activation. Clot retraction and the platelet spreading assay were used to examine Gßγ role in outside-in platelet signaling, whereas Western blot was employed to examine its role in Akt activation. Finally, we used the bleeding time assay and the FeCl3-induced carotid-artery injury thrombosis model to determine Gßγ contribution to in vivo platelet function. RESULTS: We observed that gallein inhibits platelet aggregation and secretion in response to agonist stimulation, in both mouse and human platelets. Furthermore, gallein also exerted inhibitory effects on integrin αIIbß3 activation, clot retraction, platelet spreading and Akt activation/phosphorylation. Finally, gallein's inhibitory effects manifested in vivo, as documented by its ability to modulate physiological hemostasis and delay thrombus formation. CONCLUSION: Our findings demonstrate, for the first time, that Gßγ subunits directly regulate GPCR-dependent platelet function, in vitro and in vivo. Moreover, these data highlight Gßγ as a novel therapeutic target for managing thrombotic disorders.

The JUUL E-Cigarette Elevates the Risk of Thrombosis and Potentiates Platelet Activation.

BACKGROUND: Smoking is the main preventable cause of death in the United States and worldwide and is associated with serious cardiovascular health consequences, including thrombotic diseases. Recently, electronic cigarettes (e-cigarettes) and, in particular JUUL, have attained wide popularity among smokers, nonsmokers, pregnant females, and even the youth, which is alarming. Interestingly, there is/are no information/studies regarding the effect of JUUL on cardiovascular diseases, specifically in the context of modulation of platelet activation. Thus, it is important to discern the cardiovascular disease health risks associated with JUUL. METHODS AND RESULTS: We used a passive e-vape vapor inhalation system where C57BL/6J mice (10-12 weeks old) were exposed to JUUL e-cigarette vape. Menthol flavored JUUL pods containing 5% nicotine by weight were used as the e-liquid. Mice were exposed to a total of 70 puffs daily for 2 weeks; 3-second puff duration, and 25-second puff interval. The effects of JUUL relative to clean air were analyzed, on mouse platelet function in vitro (eg, aggregation) and in vivo (eg, FeCl3-induced carotid artery injury thrombosis model). Our results indicate that short-term exposure to JUUL e-cigarette causes hyperactivation of platelets and shortens the thrombus occlusion as well as hemostasis/bleeding times, relative to clean air (medians of 14 vs. 200 seconds, P < .01 and 35 vs. 295 seconds, P < .001, respectively). CONCLUSION: Our findings document-for the first time-that short-term exposure to the JUUL e-cigarette increases the risk of thrombotic events, in part by modulating platelet function, such as aggregation and secretion, in mice.

Short-Term Exposure to Waterpipe/Hookah Smoke Triggers a Hyperactive Platelet Activation State and Increases the Risk of Thrombogenesis.

OBJECTIVE: Cardiovascular disease is a major public health problem. Among cardiovascular disease's risk factors, tobacco smoking is considered the single most preventable cause of death, with thrombosis being the main mechanism of cardiovascular disease mortality in smokers. While tobacco smoking has been on the decline, the use of waterpipes/hookah has been rising, mainly due to the perception that they are less harmful than regular cigarettes. Strikingly, there are few studies on the negative effects of waterpipes on the cardiovascular system, and none regarding their direct contribution to thrombus formation. Approach and Results: We used a waterpipe whole-body exposure protocol that mimics real-life human exposure scenarios and investigated its effects, relative to clean air, on platelet function, hemostasis, and thrombogenesis. We found that waterpipe smoke (WPS)-exposed mice exhibited both shortened thrombus occlusion and bleeding times. Further, our results show that platelets from WPS-exposed mice are hyperactive, with enhanced agonist-induced aggregation, dense and α-granule secretion, αIIbß3 integrin activation, phosphatidylserine expression, and platelet spreading, when compared with clean air-exposed platelets. Finally, at the molecular level, it was found that Akt (protein kinase B) and ERK (extracellular signal-regulated kinases) phosphorylation are enhanced in the WPS and in nicotine-treated platelets. CONCLUSIONS: Our findings demonstrate that WPS exposure directly modulates hemostasis and increases the risk of thrombosis and that this is mediated, in part, via a state of platelet hyperactivity. The negative health impact of WPS/hookah, therefore, should not be underestimated. Moreover, this study should also help in raising public awareness of the toxic effects of waterpipe/hookah.

Mouse transient receptor potential channel type 6 selectively regulates agonist-induced platelet function.

While changes in intracellular calcium levels is a central step in platelet activation and thrombus formation, the contribution and mechanism of receptor-operated calcium entry (ROCE) via transient receptor potential channels (TRPCs) in platelets remains poorly defined. In previous studies, we have shown that TRPC6 regulates hemostasis and thrombosis, in mice. In the present studies, we employed a knockout mouse model system to characterize the role of TRPC6 in ROCE and platelet activation. It was observed that the TRPC6 deletion (Trpc6 -/- ) platelets displayed impaired elevation of intracellular calcium, i.e., defective ROCE. Moreover, these platelets also exhibited defects in a host of functional responses, namely aggregation, granule secretion, and integrin αIIbß3. Interestingly, the aforementioned defects were specific to the thromboxane receptor (TPR), as no impaired responses were observed in response to ADP or the thrombin receptor-activating peptide 4 (TRAP4). The defect in ROCE in the Trpc6 -/- was also observed with 1-oleoyl-2-acetyl-sn-glycerol (OAG). Finally, our studies also revealed that TRPC6 regulates clot retraction. Taken together, our findings demonstrate that TRPC6 directly regulates TPR-dependent ROCE and platelet function. Thus, TRPC6 may serve as a novel target for the therapeutic management of thrombotic diseases.

The effects of hookah/waterpipe smoking on general health and the cardiovascular system.

Hookah or waterpipe smoking or use is an emerging trend in the US population, especially among the youth. The misperception of hookah being less harmful than cigarettes and the availability of different but "appealing" flavors are considered among the main reasons for this trend. Hookah users however are exposed to many of the same toxic compounds/by-products as cigarette users, but at dramatically higher levels, which might lead to more severe negative health effects. In fact, hookah users are at risks of infections, cancers, lung disease, and other medical conditions. Moreover, because of the overlapping toxicant/chemical profile to conventional cigarettes, hookah smoke effects on the cardiovascular system are thought to be comparable to those of conventional cigarettes. A major source of tobacco addiction is nicotine, whose levels in hookah are extremely variable as they depend on the type of tobacco used. Taken together, in this review of literature, we will provide insights on the negative health effects of hookah in general, with a focus on what is known regarding its impact on the cardiovascular system.

Arhgef1 Plays a Vital Role in Platelet Function and Thrombogenesis.

Background Platelets are the cellular mediators of hemostasis and thrombosis, and their function is regulated by a number of molecular mediators, such as small GTP ases. These small GTP ases are themselves regulated by guanine nucleotide exchange factors such as Arhgefs, several of which are found in platelets, including the highly expressed Arhgef1. However, the role of Arhgef1 in platelets has not yet been investigated. Methods and Results We employed mice with genetic deletion of Arhgef1 (ie, Arhgef1-/-) and investigated their platelet phenotype by employing a host of in vivo and in vitro platelet assays. Our results indicate that Arhgef1-/- mice had prolonged carotid artery occlusion and tail bleeding times. Moreover, platelets from these mice exhibited defective aggregation, dense and α granule secretion, α II bß3 integrin activation, clot retraction and spreading, in comparison to their wild-type littermates. Finally, we also found that the mechanism by which Arhgef1 regulates platelets is mediated in part by a defect in the activation of the RhoA-Rho-associated kinase axis, but not Rap1b. Conclusions Our data demonstrate, for the first time, that Arhgef1 plays a critical role in platelet function, in vitro and in vivo.

Regulator of G-Protein Signaling 16 Is a Negative Modulator of Platelet Function and Thrombosis.

Background Members of the regulator of G-protein signaling ( RGS ) family inhibit G-protein coupled receptor signaling by modulating G-protein activity. In platelets, there are 3 different RGS isoforms that are expressed at the protein level, including RGS 16. Recently, we have shown that CXCL 12 regulates platelet function via RGS 16. However, the role of RGS 16 in platelet function and thrombus formation is poorly defined. Methods and Results We used a genetic knockout mouse model approach to examine the role(s) of RGS 16 in platelet activation by using a host of in vitro and in vivo assays. We observed that agonist-induced platelet aggregation, secretion, and integrin activation were much more pronounced in platelets from the RGS 16 knockout ( Rgs16 -/-) mice relative to their wild type ( Rgs16 +/+) littermates. Furthermore, the Rgs16 -/- mice had a markedly shortened bleeding time and were more susceptible to vascular injury-associated thrombus formation than the controls. Conclusions These findings support a critical role for RGS 16 in regulating hemostatic and thrombotic functions of platelets in mice. Hence, RGS 16 represents a potential therapeutic target for modulating platelet function.

Author Correction: Clopidogrel as a donor probe and thioenol derivatives as flexible promoieties for enabling H2S biomedicine.

The original version of this Article contained an error in the spelling of the author Bin Geng, which was incorrectly given as Bing Geng. This has been corrected in both the PDF and HTML versions of the Article.

Clopidogrel as a donor probe and thioenol derivatives as flexible promoieties for enabling H2S biomedicine.

Hydrogen sulfide has emerged as a critical endogenous signaling transmitter and a potentially versatile therapeutic agent. The key challenges in this field include the lack of approved hydrogen sulfide-releasing probes for in human exploration and the lack of controllable hydrogen sulfide promoieties that can be flexibly installed for therapeutics development. Here we report the identification of the widely used antithrombotic drug clopidogrel as a clinical hydrogen sulfide donor. Clopidogrel is metabolized in patients to form a circulating metabolite that contains a thioenol substructure, which is found to undergo spontaneous degradation to release hydrogen sulfide. Model studies demonstrate that thioenol derivatives are a class of controllable promoieties that can be conveniently installed on a minimal structure of ketone with an α-hydrogen. These results can provide chemical tools for advancing hydrogen sulfide biomedical research as well as developing hydrogen sulfide-releasing drugs.

Short-Term E-Cigarette Exposure Increases the Risk of Thrombogenesis and Enhances Platelet Function in Mice.

BACKGROUND: Cardiovascular disease is the main cause of death in the United States, with smoking being the primary preventable cause of premature death, and thrombosis being the main mechanism of cardiovascular mortality in smokers. Due to the perception that electronic/e-cigarettes are "safer/less harmful" than conventional cigarettes, their usage-among a variety of ages-has increased tremendously during the past decade. Notably, there are limited studies regarding the negative effects of e-cigarettes on the cardiovascular system, which is also the subject of significant debate. METHODS AND RESULTS: We employed a passive e-VapeTM vapor inhalation system and developed an in vivo whole-body e-cigarette mouse exposure protocol that mimics real-life human exposure scenarios/conditions and investigated the effects of e-cigarettes and clean air on platelet function and thrombogenesis. Our results show that platelets from e-cigarette-exposed mice are hyperactive, with enhanced aggregation, dense and α granule secretion, activation of the αIIbß3 integrin, phosphatidylserine expression, and Akt and ERK activation, when compared with clean air-exposed platelets. E-cigarette-exposed platelets were also found to be resistant to inhibition by prostacyclin, relative to clean air. Furthermore, the e-cigarette-exposed mice exhibited a shortened thrombosis occlusion and bleeding times. CONCLUSIONS: Taken together, our data demonstrate for the first time that e-cigarettes alter physiological hemostasis and increase the risk of thrombogenic events. This is attributable, at least in part, to the hyperactive state of platelets. Thus, the negative health consequences of e-cigarette exposure should not be underestimated and warrant further investigation.

Investigation of a Thromboxane A2 Receptor-Based Vaccine for Managing Thrombogenesis.

BACKGROUND: Despite the well-established role for the thromboxane A2 receptor (TPR) in the development of thrombotic disorders, none of the antagonists developed to date has been approved for clinical use. To this end, we have previously shown that an antibody targeted against TPR's ligand-binding domain inhibits platelet activation and thrombus formation, without exerting any effects on hemostasis. Thus, the goal of the present studies is to design a novel TPR-based vaccine, demonstrate its ability to trigger an immune response, and characterize its antiplatelet and antithrombotic activity. METHODS AND RESULTS: We used a mouse keyhole limpet hemocyanin/peptide-based vaccination approach rationalized over the TPR ligand-binding domain (ie, the C-terminus of the second extracellular loop). The biological activity of this vaccine was assessed in the context of platelets and thrombotic diseases, and using a host of in vitro and in vivo platelet function experiments. Our results revealed that the TPR C-terminus of the second extracellular loop vaccine, in mice: (1) triggered an immune response, which resulted in the development of a C-terminus of the second extracellular loop antibody; (2) did not affect expression of major platelet integrins (eg, glycoprotein IIb-IIIa); (3) selectively inhibited TPR-mediated platelet aggregation, platelet-leukocyte aggregation, integrin glycoprotein IIb-IIIa activation, as well as dense and α granule release; (4) significantly prolonged thrombus formation; and (5) did so without impairing physiological hemostasis. CONCLUSIONS: Collectively, our findings shed light on TPR's structural biological features, and demonstrate that the C-terminus of the second extracellular loop domain may define a new therapeutic target and a TPR vaccine-based approach that should have therapeutic applications.

Role of IκB kinase ß in regulating the remodeling of the CARMA1-Bcl10-MALT1 complex.

The current work investigates the notion that inducible clustering of signaling mediators of the IKK pathway is important for platelet activation. Thus, while the CARMA1, Bcl10, and MALT1 (CBM) complex is essential for triggering IKK/NF-κB activation upon platelet stimulation, the signals that elicit its formation and downstream effector activation remain elusive. We demonstrate herein that IKKß is involved in membrane fusion, and serves as a critical protein kinase required for initial formation and the regulation of the CARMA1/MALT1/Bcl10/CBM complex in platelets. We also show that IKKß regulates these processes via modulation of phosphorylation of Bcl10 and IKKγ polyubiquitination. Collectively, our data demonstrate that IKKß regulates membrane fusion and the remodeling of the CBM complex formation.

P2Y12 antibody inhibits platelet activity and protects against thrombogenesis.

Given that platelet hyperactivity is known to give rise to thrombotic disorders, new and/or novel antiplatelet therapies are constantly being developed to add to, or to complement the current arsenal of agents. To this end, adenosine diphosphate (ADP) is an important platelet activator that acts by binding to the G-protein coupled P2Y1 and P2Y12 receptors. Although the contribution of the P2Y12 receptor to the genesis of thrombosis is well established, the parenteral arsenal of drugs targeting this receptor in clinical use is limited to cangrelor. In this study, we investigated the potential antiplatelet activity of an antibody targeting the ligand-binding domain of the P2Y12 receptor (abbreviated P2Y12Ab). Our in vitro studies revealed that the P2Y12Ab could effectively inhibit aggregation induced by ADP, as well as that triggered by the thromboxane receptor agonist U46619. Additionally, using FACS analysis, we observed reduced P-selectin, phosphatidylserine exposure and integrin activation in the presence of P2Y12Ab. As for its in vivo effects, the P2Y12Ab also demonstrated protection against thrombus formation; albeit this was accompanied with a bleeding diathesis (longer bleeding time). Notably, this inhibitory profile is consistent with that observed with oral anti-P2Y12 agents. Collectively, our findings demonstrate that the P2Y12Ab functionally blocks platelet activity in vitro and in vivo, and support the notion that it can be purposed as a parenteral antiplatelet agent, to be used in conjunction with and/or as a complement to current antiplatelet therapies.

RGS10 Negatively Regulates Platelet Activation and Thrombogenesis.

Regulators of G protein signaling (RGS) proteins act as GTPase activating proteins to negatively regulate G protein-coupled receptor (GPCR) signaling. Although several RGS proteins including RGS2, RGS16, RGS10, and RGS18 are expressed in human and mouse platelets, the respective unique function(s) of each have not been fully delineated. RGS10 is a member of the D/R12 subfamily of RGS proteins and is expressed in microglia, macrophages, megakaryocytes, and platelets. We used a genetic approach to examine the role(s) of RGS10 in platelet activation in vitro and hemostasis and thrombosis in vivo. GPCR-induced aggregation, secretion, and integrin activation was much more pronounced in platelets from Rgs10-/- mice relative to wild type (WT). Accordingly, these mice had markedly reduced bleeding times and were more susceptible to vascular injury-associated thrombus formation than control mice. These findings suggest a unique, non-redundant role of RGS10 in modulating the hemostatic and thrombotic functions of platelets in mice. RGS10 thus represents a potential therapeutic target to control platelet activity and/or hypercoagulable states.

Platelet Hyperactivity in TNFSF14/LIGHT Knockout Mouse Model of Impaired Healing.

Objective: Impaired and chronic wounds occur due to defects in one or more of the overlapping stages of healing. However, problems related to the vascular system are critical for nonhealing, and chronic wounds in humans often show the presence of fibrin cuffs/clots. We hypothesized that these clots are due to alterations in platelet function; hence, we have investigated whether alterations in platelet function are present during impaired healing. Approach: Platelets were subjected to different agonists to determine the rate of aggregation and evaluate the molecules involved in adhesion and aggregation that could lead to faster thrombosis and potentially contribute to impaired wound healing. Results: We show that wounding of TNFSF14/LIGHT-/- mice, which have impaired healing, leads to an enhanced response in platelet aggregation and a faster time to blood vessel occlusion (thrombosis). In addition, after wounding, platelets from these mice have increased levels of P-selectin, integrin αIIbß3, and phosphatidylserine, molecules that contribute to platelet adhesion. They also have more extensive open canalicular system than platelets of control mice, suggesting increased surface area for interactions upon activation. Innovation: These results show a novel function for TNFSF14/LIGHT during wound healing. Conclusion: The absence of TNFSF14/LIGHT from the cell surface of platelets causes rapid platelet aggregation and thrombus formation that may contribute to impaired healing by reducing the ability of the blood vessels to transport nutrients and oxygen and other molecules needed for proper healing.

CXCL12 regulates platelet activation via the regulator of G-protein signaling 16.

The regulators of G protein signaling (RGS) protein superfamily negatively controls G protein-coupled receptor signal transduction pathways. One of the members of this family, RGS16, is highly expressed in megakaryocytes and platelets. Studies of its function in platelet and megakaryocyte biology have been limited, in part, due to lack of pharmacological inhibitors. For example, RGS16 overexpression inhibited CXC chemokine receptor 4 (CXCR4)-mediated megakaryocyte migration. More recent studies showed that the chemokine stromal cell-derived factor (SDF1α or CXCL12) regulates platelet function via CXCR4. Based on these considerations, the present study investigated the capacity of RGS16 to regulate CXCL12-dependent platelet function, using the RGS16 knockout mouse model (Rgs16(-/-)). RGS16-deficient platelets had increased protease activated receptor 4 and collagen-induced aggregation, as well as increased CXCL12-dependent agonist-induced aggregation, dense and alpha granule secretion, integrin αIIbß3 activation and phosphatidylserine exposure compared to those from WT littermates. CXCL12 alone did not stimulate aggregation or secretion in either RGS16-deficient or WT platelets. Furthermore, platelets from Rgs16(-/-) mice displayed enhanced phosphorylation of ERK and Akt following CXCL12 stimulation relative to controls. Finally, we also found that PKCδ is involved in regulating CXCL12-dependent activation of ERK and Akt, in the Rgs16-deficient platelets. Collectively, our findings provide the first evidence that RGS16 plays an important role in platelet function by modulating CXCL12-dependent platelet activation.

Arachidonic acid-derived signaling lipids and functions in impaired healing.

Very little is known about lipid function during wound healing, and much less during impaired healing. Such understanding will help identify what roles lipid signaling plays in the development of impaired/chronic wounds. We took a lipidomics approach to study the alterations in lipid profile in the LIGHT(-/-) mouse model of impaired healing which has characteristics that resemble those of impaired/chronic wounds in humans, including high levels of oxidative stress, excess inflammation, increased extracellular matrix degradation and blood vessels with fibrin cuffs. The latter suggests excess coagulation and potentially increased platelet aggregation. We show here that in these impaired wounds there is an imbalance in the arachidonic acid (AA) derived eicosonoids that mediate or modulate inflammatory reactions and platelet aggregation. In the LIGHT(-/-) impaired wounds there is a significant increase in enzymatically derived breakdown products of AA. We found that early after injury there was a significant increase in the eicosanoids 11-, 12-, and 15-hydroxyeicosa-tetranoic acid, and the proinflammatory leukotrienes (LTD4 and LTE) and prostaglandins (PGE2 and PGF2α ). Some of these eicosanoids also promote platelet aggregation. This led us to examine the levels of other eicosanoids known to be involved in the latter process. We found that thromboxane (TXA2 /B2 ), and prostacyclins 6kPGF1α are elevated shortly after wounding and in some cases during healing. To determine whether they have an impact in platelet aggregation and hemostasis, we tested LIGHT(-/-) mouse wounds for these two parameters and found that, indeed, platelet aggregation and hemostasis are enhanced in these mice when compared with the control C57BL/6 mice. Understanding lipid signaling in impaired wounds can potentially lead to development of new therapeutics or in using existing nonsteroidal anti-inflammatory agents to help correct the course of healing.