Insights into platelet pharmacology from a cryo-EM structure of the ABCC4 transporter.

Structural determination of the ABCC4 transporter is a major first step in providing crucial molecular insights into the transport of platelet substrates into granules, as well as drug transport from platelets. The findings provide a framework for understanding platelet interactions and potential design of specific platelet antagonists.

Aspirin thromboprophylaxis in joint replacement surgery.

BACKGROUND: Aspirin is commonly used as the only pharmacologic agent for prevention of venous thromboembolism (VTE) after joint replacement surgery in the United States. Despite this, prospective studies investigating VTE events after aspirin-only thromboprophylaxis in joint replacement surgery are lacking in the real-world setting. OBJECTIVES: The aim of this study was to estimate the risk of VTE with aspirin-only pharmacologic prophylaxis following joint replacement surgery. METHODS: We carried out a prospective observational study of 350 low-risk patients (no prior history of VTE and low cardiovascular risk factors) who underwent total knee and total hip arthroplasty and received only aspirin for thromboprophylaxis postoperatively. RESULTS: The observed risk of symptomatic VTE was 1.7% (95% confidence interval, 0.9%-3.3%) over 3 months of follow up, with only one major bleeding event and no surgical hematomas. CONCLUSION: The risk of VTE with aspirin monotherapy for thromboprophylaxis in joint replacement surgery in this real-world cohort was higher than previously reported.

Circulating Protein Disulfide Isomerase Is Associated with Increased Risk of Thrombosis in JAK2-Mutated Myeloproliferative Neoplasms.

PURPOSE: Thromboembolic events (TE) are the most common complications of myeloproliferative neoplasms (MPN). Clinical parameters, including patient age and mutation status, are used to risk-stratify patients with MPN, but a true biomarker of TE risk is lacking. Protein disulfide isomerase (PDI), an endoplasmic reticulum protein vital for protein folding, also possesses essential extracellular functions, including regulation of thrombus formation. Pharmacologic PDI inhibition prevents thrombus formation, but whether pathologic increases in PDI increase TE risk remains unknown. EXPERIMENTAL DESIGN: We evaluated the association of plasma PDI levels and risk of TE in a cohort of patients with MPN with established diagnosis of polycythemia vera (PV) or essential thrombocythemia (ET), compared with healthy controls. Plasma PDI was measured at enrollment and subjects followed prospectively for development of TE. RESULTS: A subset of patients, primarily those with JAK2-mutated MPN, had significantly elevated plasma PDI levels as compared with controls. Plasma PDI was functionally active. There was no association between PDI levels and clinical parameters typically used to risk-stratify patients with MPN. The risk of TE was 8-fold greater in those with PDI levels above 2.5 ng/mL. Circulating endothelial cells from JAK2-mutated MPN patients, but not platelets, demonstrated augmented PDI release, suggesting endothelial activation as a source of increased plasma PDI in MPN. CONCLUSIONS: The observed association between plasma PDI levels and increased risk of TE in patients with JAK2-mutated MPN has both prognostic and therapeutic implications.

Anti-thrombotic effects mediated by dihydromyricetin involve both platelet inhibition and endothelial protection.

Classical antithrombotics and antiplatelets are associated with high frequencies of bleeding complications or treatment failure when used as single agents. The platelet-independent fibrin generation by activated endothelium highlights the importance of vascular protection in addition to platelet inhibition in thrombosis prevention. Dihydromyricetin (DHM), the most abundant flavonoid in Ampelopsis grossedentata, has unique vasoprotective effects. This study aims to characterize the antithrombotic potential of DHM. The effects of DHM on the activation of platelets and endothelial cells were evaluated in vitro. Calcium mobilization and activation of mitogen-activated protein kinases (MAPKs) were examined as the potential targets of DHM based on molecular docking analysis. The in vivo effects of DHM were determined in FeCl3-injured carotid arteries and laser-injured cremasteric arterioles. The results showed that DHM suppressed a range of platelet responses including aggregation, secretion, adhesion, spreading and integrin activation, and inhibited exocytosis, phosphatidylserine exposure and tissue factor expression in activated endothelial cells. Mechanistically, DHM attenuated thrombin-induced calcium mobilization and phosphorylation of ERK1/2 and p38 both in platelets and endothelial cells. Intravenous treatment with DHM delayed FeCl3-induced carotid arterial thrombosis. Furthermore, DHM treatment inhibited both platelet accumulation and fibrin generation in the presence or absence of eptifibatide in the laser injury-induced thrombosis model, without prolonging ex vivo plasma coagulation or tail bleeding time. DHM represents a novel antithrombotic agent whose effects involve both inhibition of platelet activation and reduction of fibrin generation as a result of endothelial protection.

VWF maturation and release are controlled by 2 regulators of Weibel-Palade body biogenesis: exocyst and BLOC-2.

von Willebrand factor (VWF) is an essential hemostatic protein that is synthesized in endothelial cells and stored in Weibel-Palade bodies (WPBs). Understanding the mechanisms underlying WPB biogenesis and exocytosis could enable therapeutic modulation of endogenous VWF, yet optimal targets for modulating VWF release have not been established. Because biogenesis of lysosomal related organelle-2 (BLOC-2) functions in the biogenesis of platelet dense granules and melanosomes, which like WPBs are lysosome-related organelles, we hypothesized that BLOC-2-dependent endolysosomal trafficking is essential for WPB biogenesis and sought to identify BLOC-2-interacting proteins. Depletion of BLOC-2 caused misdirection of cargo-carrying transport tubules from endosomes, resulting in immature WPBs that lack endosomal input. Immunoprecipitation of BLOC-2 identified the exocyst complex as a binding partner. Depletion of the exocyst complex phenocopied BLOC-2 depletion, resulting in immature WPBs. Furthermore, releasates of immature WPBs from either BLOC-2 or exocyst-depleted endothelial cells lacked high-molecular weight (HMW) forms of VWF, demonstrating the importance of BLOC-2/exocyst-mediated endosomal input during VWF maturation. However, BLOC-2 and exocyst showed very different effects on VWF release. Although BLOC-2 depletion impaired exocytosis, exocyst depletion augmented WPB exocytosis, indicating that it acts as a clamp. Exposure of endothelial cells to a small molecule inhibitor of exocyst, Endosidin2, reversibly augmented secretion of mature WPBs containing HMW forms of VWF. These studies show that, although BLOC-2 and exocyst cooperate in WPB formation, only exocyst serves to clamp WPB release. Exocyst function in VWF maturation and release are separable, a feature that can be exploited to enhance VWF release.

Dose-adjusted enoxaparin thromboprophylaxis in hospitalized cancer patients: a randomized, double-blinded multicenter phase 2 trial.

Hospitalized patients with cancer are at an increased risk of developing venous thromboembolism (VTE). The recommendation for routine pharmacologic thromboprophylaxis in hospitalized patients with cancer to prevent VTE is based on extrapolation of results from noncancer cohorts. There are limited data to support the efficacy and safety of fixed-dose low-molecular-weight heparin (LMWH) regimens in high-risk hospitalized patients with cancer. We conducted a randomized, double-blinded, phase 2 trial in hospitalized patients with active cancer at high risk of developing VTE based on Padua risk score. Patients were randomly assigned to fixed-dose enoxaparin (40 mg daily) vs weight-adjusted enoxaparin (1 mg/kg daily) during hospitalization. The primary objectives were to evaluate the safety of dose-adjusted enoxaparin and evaluate the incidence of VTE with fixed-dose enoxaparin. Blinded clinical assessments were performed at day 14, and patients randomly assigned to fixed-dose enoxaparin subsequently underwent a bilateral lower extremity ultrasound. A total of 50 patients were enrolled and randomized. The median weight of patients enrolled in weight-adjusted enoxaparin arm was 76 kg (range, 60.9-124.5 kg). There were no major hemorrhages or symptomatic VTE in either arm. At time of completion of the blinded clinical assessment, there was only 1 incidentally identified pulmonary embolus that occurred in the weight-adjusted arm. In the group randomly assigned to fixed-dose enoxaparin who subsequently underwent surveillance ultrasound, the cumulative incidence of DVT was 22% (90% binomial confidence interval, 0%-51.3%). This phase 2 trial confirms a high incidence of asymptomatic VTE among high-risk hospitalized patients with cancer and that weight-adjusted LMWH thromboprophylaxis is feasible and well-tolerated. This trial was registered at www.clinicaltrials.gov as #NCT02706249.

The life cycle of platelet granules.

Platelet granules are unique among secretory vesicles in both their content and their life cycle. Platelets contain three major granule types-dense granules, α-granules, and lysosomes-although other granule types have been reported. Dense granules and α-granules are the most well-studied and the most physiologically important. Platelet granules are formed in large, multilobulated cells, termed megakaryocytes, prior to transport into platelets. The biogenesis of dense granules and α-granules involves common but also distinct pathways. Both are formed from the trans-Golgi network and early endosomes and mature in multivesicular bodies, but the formation of dense granules requires trafficking machinery different from that of α-granules. Following formation in the megakaryocyte body, both granule types are transported through and mature in long proplatelet extensions prior to the release of nascent platelets into the bloodstream. Granules remain stored in circulating platelets until platelet activation triggers the exocytosis of their contents. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, located on both the granules and target membranes, provide the mechanical energy that enables membrane fusion during both granulogenesis and exocytosis. The function of these core fusion engines is controlled by SNARE regulators, which direct the site, timing, and extent to which these SNAREs interact and consequently the resulting membrane fusion. In this review, we assess new developments in the study of platelet granules, from their generation to their exocytosis.

Regulatory role of thiol isomerases in thrombus formation.

INTRODUCTION: The protein disulfide isomerase (PDI) family of thiol isomerases are intracellular enzymes known to catalyze the oxidation, reduction and isomerization of disulfide bonds during protein synthesis in the endoplasmic reticulum. PDI and related members of the thiol isomerase family are known to localize extracellularly where they possess various functions. Among these, the role of PDI in the initiation of thrombus formation is best characterized. PDI is secreted within seconds from activated platelets and endothelial cells at the site of vascular injury and accumulates in the developing platelet-fibrin thrombus. Inhibition of PDI by antibodies or small molecule inhibitors blocks thrombus formation. Efforts are underway to identify extracellular substrates of PDI that participate in the network pathways linking thiol isomerases to thrombus formation. ERp57, ERp5 and ERp72 also play a role in initiation of thrombus formation but their specific extracellular substrates are unknown. Areas covered: The following review gives an overview of biochemistry of vascular thiol isomerases followed by a detailed description of their role in thrombosis and its clinical implications. Expert commentary: The thiol isomerase system, by controlling the initiation of thrombus formation, provides the regulatory switch by which the normal vasculature is protected under physiologic conditions from thrombi generation.

Detection of membrane-bound and soluble antigens by magnetic levitation.

Magnetic levitation is a technique for measuring the density and the magnetic properties of objects suspended in a paramagnetic field. We describe a novel magnetic levitation-based method that can specifically detect cell membrane-bound and soluble antigens by measurable changes in levitation height that result from the formation of antibody-coated bead and antigen complex. We demonstrate our method's ability to sensitively detect an array of membrane-bound and soluble antigens found in blood, including T-cell antigen CD3, eosinophil antigen Siglec-8, red blood cell antigens CD35 and RhD, red blood cell-bound Epstein-Barr viral particles, and soluble IL-6, and validate the results by flow cytometry and immunofluorescence microscopy performed in parallel. Additionally, employing an inexpensive, single lens, manual focus, wifi-enabled camera, we extend the portability of our method for its potential use as a point-of-care diagnostic assay.

Detection and quantification of subtle changes in red blood cell density using a cell phone.

Magnetic levitation has emerged as a technique that offers the ability to differentiate between cells with different densities. We have developed a magnetic levitation system for this purpose that distinguishes not only different cell types but also density differences in cells of the same type. This small-scale system suspends cells in a paramagnetic medium in a capillary placed between two rare earth magnets, and cells levitate to an equilibrium position determined solely by their density. Uniform reference beads of known density are used in conjunction with the cells as a means to quantify their levitation positions. In one implementation images of the levitating cells are acquired with a microscope, but here we also introduce a cell phone-based device that integrates the magnets, capillary, and a lens into a compact and portable unit that acquires images with the phone's camera. To demonstrate the effectiveness of magnetic levitation in cell density analysis we carried out levitation experiments using red blood cells with artificially altered densities, and also levitated those from donors. We observed that we can distinguish red blood cells of an anemic donor from those that are healthy. Since a plethora of disease states are characterized by changes in cell density magnetic cell levitation promises to be an effective tool in identifying and analyzing pathologic states. Furthermore, the low cost, portability, and ease of use of the cell phone-based system may potentially lead to its deployment in low-resource environments.

Extracellular Thiol Isomerases and Their Role in Thrombus Formation.

SIGNIFICANCE: The mammalian endoplasmic reticulum (ER) houses a large family of twenty thioredoxin-like proteins of which protein disulfide isomerase (PDI) is the archetypal member. Although the PDI family is best known for its role in oxidative protein folding of secretory proteins in the ER, these thioredoxin-like proteins fulfill ever-expanding roles, both within the secretory pathway and beyond. RECENT ADVANCES: Secreted PDI family proteins have now been shown to serve a critical role in platelet thrombus formation and fibrin generation. Utilizing intravital microscopy to visualize thrombus formation in mice, we have demonstrated the presence of extracellular PDI antigen during thrombus formation following injury of the vascular wall. Inhibition of PDI abrogates thrombus formation in vivo (16, 26, 46, 55). These observations have been extended to other PDI family members, including ERp57 (39, 116, 118, 123) and ERp5 (77). The vascular thiol isomerases are those PDI family members secreted from platelets and/or endothelium (40): PDI, ERp57, ERp5, ERp72, ERp44, ERp29, and TMX3. We focus here on PDI (16, 46, 55), ERp57 (39, 116, 118, 123), and ERp5 (77), which have been implicated in thrombus formation in vivo. CRITICAL ISSUES: It would appear that a system of thiol isomerase redox catalysts has been hijacked from the ER to regulate thrombus formation in the vasculature. FUTURE DIRECTIONS: How this redox system is trafficked to and regulated at the cell surface, the identity of extracellular substrates, why so many thiol isomerases are required, and which thiol isomerase functions are necessary are critical unanswered questions in understanding the role of thiol isomerases in thrombus formation.

Quercetin-3-rutinoside Inhibits Protein Disulfide Isomerase by Binding to Its b'x Domain.

Quercetin-3-rutinoside inhibits thrombus formation in a mouse model by inhibiting extracellular protein disulfide isomerase (PDI), an enzyme required for platelet thrombus formation and fibrin generation. Prior studies have identified PDI as a potential target for novel antithrombotic agents. Using a fluorescence enhancement-based assay and isothermal calorimetry, we show that quercetin-3-rutinoside directly binds to the b' domain of PDI with a 1:1 stoichiometry. The binding of quercetin-3-rutinoside to PDI induces a more compact conformation and restricts the conformational flexibility of PDI, as revealed by small angle x-ray scattering. The binding sites of quercetin-3-rutinoside to PDI were determined by studying its interaction with isolated fragments of PDI. Quercetin-3-rutinoside binds to the b'x domain of PDI. The infusion of the b'x fragment of PDI rescued thrombus formation that was inhibited by quercetin-3-rutinoside in a mouse thrombosis model. This b'x fragment does not possess reductase activity and, in the absence of quercetin-3-rutinoside, does not affect thrombus formation in vivo. The isolated b' domain of PDI has potential as an antidote to reverse the antithrombotic effect of quercetin-3-rutinoside by binding and neutralizing quercetin-3-rutinoside.

Defective PDI release from platelets and endothelial cells impairs thrombus formation in Hermansky-Pudlak syndrome.

Protein disulfide isomerase (PDI), secreted from platelets and endothelial cells after injury, is required for thrombus formation. The effect of platelet and endothelial cell granule contents on PDI-mediated thrombus formation was studied by intravital microscopy using a mouse model of Hermansky-Pudlak syndrome in which platelet dense granules are absent. Platelet deposition and fibrin generation were nearly absent, and extracellular PDI was significantly reduced in HPS6(-/-) mice after vascular injury. HPS6(-/-) platelets displayed impaired PDI secretion and impaired exocytosis of α granules, lysosomes, and T granules due to decreased sensitivity to thrombin, but these defects could be corrected by addition of subthreshold amounts of adenosine 5'-diphosphate (ADP). Human Hermansky-Pudlak syndrome platelets demonstrated similar characteristics. Infusion of wild-type platelets rescued thrombus formation in HPS6(-/-) mice. Human umbilical vein endothelial cells in which the HPS6 gene was silenced displayed impaired PDI secretion and exocytosis of Weibel-Palade bodies. Defective thrombus formation in Hermansky-Pudlak syndrome, associated with impaired exocytosis of residual granules in endothelial cells and platelets, the latter due to deficiency of ADP, is characterized by a defect in T granule secretion, a deficiency in extracellular PDI secretion, and impaired fibrin generation and platelet aggregation. Hermansky-Pudlak syndrome is an example of a hereditary disease whereby impaired PDI secretion contributes to a bleeding phenotype.

Measurement of shear-activated platelet aggregate formation in non-anticoagulated blood: utility in detection of clopidogrel-aspirin-induced platelet dysfunction.

We studied the ability of a new instrument, the PlaCor PRT that measures shear-induced platelet aggregation in fingerstick, non-anticoagulated blood without added agonists, to detect platelet dysfunction ex vivo. Platelet reactivity time (PRT) and whole blood aggregation (WBA) were measured in 160 healthy volunteers, before and after aspirin and in 170 participants with established vascular disease or risk factors thereof treated with aspirin ± clopidogrel. Pretreatment PRT and WBA were significantly correlated (collagen r = -.63; arachidonate r = -.65; P < .0001). Following aspirin, the mean PRT increased from 82 to 142 seconds (P < .0001), and in participants treated with clopidogrel-aspirin, the mean PRT (286 seconds, n = 65) was significantly longer than with aspirin alone (166 seconds, n = 105; P < .001). Only 13% of PRTs of participants treated with clopidogrel and aspirin were within the normal range. We conclude that the PlaCor PRT is a simple, rapid, point-of-care instrument that compares favorably with published descriptions of other platelet function instruments.