Translatability of In Vitro Stress for Predicting Deamidation and Oxidation Biotransformation on Biotherapeutics.

Biotransformation leading to single residue modifications (e.g., deamidation, oxidation) can contribute to decreased efficacy/potency, poor pharmacokinetics, and/or toxicity/immunogenicity for protein therapeutics. Identifying and characterizing such liabilities in vivo are emerging needs for biologics drug discovery. In vitro stress assays involving PBS for deamidation or AAPH for oxidation are commonly used for predicting liabilities in manufacturing and storage and are sometimes considered a predictive tool for in vivo liabilities. However, reports discussing their in vivo translatability are limited. Herein, we introduce a mass spectrometry workflow that characterizes in vivo oxidation and deamidation in pharmacokinetically relevant compartments for diverse protein therapeutic modalities. The workflow has low bias of <10% in quantitating degradation in the relevant pharmacokinetic concentration range for monkey and rabbit serum/plasma (1-100 µg/mL) and allows for high sequence coverage (∼85%) for discovery/monitoring of amino acid modifications. For oxidation and deamidation, the assay was precise, with percent coefficient of variation of <8% at 1-100 µg/mL and ≤6% method-induced artifacts. A high degree of in vitro and in vivo correlation was observed for deamidation on the six diverse protein therapeutics (seven liability sites) tested. In vivo translatability for oxidation liabilities were not observed for the 11 molecules tested using in vitro AAPH stress. One of the molecules dosed in eyes resulted in a false positive and a false negative prediction for in vivo oxidation following AAPH stress. Finally, peroxide stress was also tested but resulted in limited success (1 out of 4 molecules) in predicting oxidation liabilities.

A Spike-Control Approach that Evaluates High Resolution Mass Spectrometry-Based Sequence Variant Analytical Method Performance for Therapeutic Proteins.

An amino acid sequence variant (SV) is defined as an unintended amino acid substitution in protein drug products. SVs contribute to product heterogeneity and can potentially impact product quality, safety, immunogenicity, and efficacy. The analysis of biotherapeutics for SVs is important throughout the product life cycle including clone selection, development of nutrient feed strategies, commercial manufacturing process, and post-approval changes to monitor product quality. The proposed analytical procedure for SVs consists of both qualitative (identification of SVs) and quantitative (quantitation of identified SVs) components. The complexities of SV analysis and the variety of current procedures highlight the need for a systematic approach for assessing the capability of these methodologies to reliably identify and quantitate SVs in biotherapeutics. We described here a "spike-control" approach for evaluating SV analytical procedure. The concept was adopted from quality control samples routinely used in analytical procedure validation. One FDA approved monoclonal antibody (mAb) was spiked with accurate amounts of highly homologous mAb to create mAb samples containing low yet accurate levels of "artificial" SVs. Spike-control samples were denatured, reduced, alkylated, digested and then analyzed by high resolution Orbitrap mass spectrometry. In silico analysis revealed four single amino acid differences between the two mAbs that could be used to represent SVs in the spike-control samples. All four "artificial" SVs were reliably identified by the current workflow. Analytical range (0.01% to 2%), accuracy and precision of identified SVs have also been evaluated. Overall, spike-control sample(s) helped to demonstrate that the SV analytical procedure (i.e., sample preparation, LC separation, mass spectrometry determinations and bioinformatic software) was fit for purpose and suitable for the identification and quantitation of SVs at a pre-determined threshold.

Development of a Highly Sensitive Hybrid LC/MS Assay for the Quantitative Measurement of CTLA-4 in Human T Cells.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a check point protein expressed on the surface of T cells and plays a central role in regulating the immune response. In recent years, CTLA-4 has become a popular target for cancer immunotherapy in which blocking CTLA-4 can restore T-cell function and enhance the immune response against cancer. Currently, there are many CTLA-4 inhibitors in a variety of modalities, including cell therapies, which are being developed in both preclinical and clinical stages to further harness the potential of the target for the treatment of certain types of cancer. In drug discovery research, measuring the level of CTLA-4 in T cells is important for drug discovery and development because it provides key information for quantitative assessment of the pharmacodynamics, efficacy, and safety of the CTLA-4-based therapies. However, to our best knowledge, there is still no report of a sensitive, specific, accurate, and reliable assay for CTLA-4 measurement. In this work, an LC/MS-based method was developed to measure CTLA-4 in human T cells. The assay demonstrated high specificity with an LLOQ of 5 copies of CTLA-4 per cell when using 2.5 million T cells for analysis. As shown in the work, the assay was successfully used to measure CTLA-4 levels in subtype T-cell samples from individual healthy subjects. The assay could be applied in supporting the studies of CTLA-4-based cancer therapies.

Ethnic, geographic, and seasonal differences of vitamin D status among adults in south-west China.

BACKGROUND: There are limited data on vitamin D status of Sichuan province, and no investigation has been carried out on the correlations of 25(OH)D and BTMs between healthy Hans and Tibetans of Sichuan province. This study aimed to examine 25(OH)D levels around Sichuan province and to assess differences by ethnicity, age, gender, sunlight exposure, geographic location, and seasons. METHODS: Blood samples from 2317 healthy adults aged of 18 to 75 years and of Han and Tibetan ethnicities were collected in six regions and during four seasons. Serum 25(OH)D2 and 25(OH)D3 levels were measured by LC-MS/MS method. Serum total P1NP and ß-CTX were measured by immunoassay. RESULTS: Participants aged 18-40 years showed significantly lower 25(OH)D levels than participants aged 41-75 years old (P < .0001). The median serum 25(OH)D level for males was significantly higher than that of females (P < .0001). Serum 25(OH)D levels among four seasons and different districts varied significantly (P < .0001). In addition, the 25(OH)D level of Tibetans was significantly lower than that of Hans, while the serum total P1NP and ß-CTX levels of Tibetans were significantly higher than those of Hans (P < .0001). CONCLUSION: Adult population was more common to have vitamin D deficiency/insufficiency among Tibetans, females, north regions and in spring and winter.

The influence of porcine epidemic diarrhea virus on pig small intestine mucosal epithelial cell function.

Porcine epidemic diarrhea (PED) is a highly contagious, acute enteric tract infectious disease of pigs (Sus domesticus) caused by porcine epidemic diarrhea virus (PEDV). PED is characterized by watery diarrhea, dehydration, weight loss, vomiting and death. PEDV damages pig intestinal epithelial tissue, causing intestinal hyperemia and atrophy of intestinal villi, with formation of intestinal epithelial cell cytoplasmic vacuoles. Since pig small intestinal epithelial cells (IECs) are target cells of PEDV infection, IEC cells were utilized as a model for studying changes in cellular activities post-PEDV infection. Monitoring of Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities demonstrated that PEDV infection decreased these activities. In addition, IECs proliferation was shown to decrease after PEDV infection using an MTT assay. Moreover, IECs apoptosis detected by flow cytometry with propidium iodide (PI) staining was clearly shown to increase relative to the control group. Meanwhile, animal experiments indicated that PEDV virulence for IEC cells was greater than viral virulence for Vero cells, although this may be due to viral attenuation after numerous passages in the latter cell line. Collectively, these studies revealed viral pathogenic mechanisms in PEDV-infected IECs and offer a theoretical basis for PEDV prevention and control.

Tunable magnetic and half-metallic properties of the two-dimensional electron gas in LaAlO3/SrTiO3(111) heterostructures.

Half-metallic materials have gained a lot of attention because of their unique properties and applications in spintronic devices. Despite the fact that these materials have been studied by several research groups there are very limited studies on their heterostructure (HS) systems. In the current study we have investigated the electronic and magnetic properties of (LaAlO3)6.5/(SrTiO3)2.5(111) HS using density functional theory (DFT) calculations. We demonstrate that the system exhibits a 100% spin-polarized two-dimensional electron gas (2DEG) which is extremely confined to the Ti 3d orbitals of the SrTiO3 layers. In particular, this system can keep its half-metallic properties under different in-plane strains from -3 to 2%. This property proves that this material has relatively stable half-metallic properties. In addition, the conducting and magnetic ground states of the system can also be tailored by changing in-plane strain and interfacial cation intermixing of La and Sr (Sr ⇔ La intermixing). By increasing the in-plane lattice parameters, this system has the ability to evolve from a nonmagnetic to a ferromagnetic metal and then to a half-metal and by further increasing the in-plane lattice parameter it becomes a ferromagnetic insulator. Sr ⇔ La intermixing can destroy the original half-metallic properties and the system exhibits an AFM Mott-type insulator phase. Our results demonstrate that the system has high potential for application in the field of spintronics, and opens the prospect of using LaAlO3/SrTiO3(111) HSs to explore quantum phase transitions.

The diosgenin prodrug nanoparticles with pH-responsive as a drug delivery system uniquely prevents thrombosis without increased bleeding risk.

Thrombosis is the leading cause of death in patients with cardiovascular disease in the world. Current antithrombotic agent aspirin has serious side effects such as higher bleeding risk and serious gastrointestinal ulcers. Diosgenin reported in clinical research could prevent thrombosis without side effects. However, poor bioavailability and low knowledge on its molecular targets limit its clinical application. A novel prodrug with antithrombotic effect was prepared based on conjugating diosgenin derivatives to PEG with Schiff-base bond. The prodrug with long blood circulation time and satisfying safety could self-assemble into micelles in water. The prodrug micelles with pH-responsibility could targetedly release diosgenin in position of thrombus in vivo. The results indicate that the prodrug micelles without bleeding risk and histological damages prevent thrombosis by inhibiting platelet activation and apoptosis. Our studies demonstrate that the prodrug micelles could obviously enhance the efficacy in the prevention of arterial thrombus and venous thrombus than aspirin.

Preventive effect of a novel diosgenin derivative on arterial and venous thrombosis in vivo.

Current therapy for blood vessel thrombosis has the risk of leading to gastrointestinal bleeding and thrombocytopenia. We previously reported that a new derivative of diosgenin, compound 5, had significant anti-inflammatory activity superior to that of aspirin, prolonged bleeding time, and inhibited platelet aggregation in vitro. In the present study, we investigated the in vivo efficacy and safety of compound 5 using the ferric chloride (FeCl3)-induced arterial and venous thrombosis models in rats as well as its toxicity in mice. Compared with the control rats, those treated with compound 5 showed significantly less adenosine diphosphate (ADP)-induced platelet aggregation and a prolonged activated partial thromboplastin time mediated by the specific regulation of factor VIII. Furthermore, compound 5 significantly reduced the average length and weight of thrombi in both arteries and veins. These findings were similar to those of aspirin at the same dose. The safety evaluation revealed a much lower risk of bleeding and lesser gastric mucosal damage with compound 5 than with the same dose of aspirin. An oral dose of up to 575.5mg/kg showed no toxicity in mice. In conclusion, consistent with our in vitro findings, compound 5 exhibited an in vivo antithrombotic activity that was comparable to aspirin mainly by reducing platelet aggregation and regulating factor VIII, but with fewer side effects.

Reference Intervals for Hemoglobin and Age- and Gender-Related Trends in the Population of Southwest China.

BACKGROUND: The establishment of a proper reference range is necessary for both the prognosis and diagnosis of clinical disease. Our study aimed to establish the hemoglobin reference interval for the population of Southwest of China and to compare this with published data for other races. METHODS: A total of 21085 healthy individuals (11561 men and 9524 women, age range 18 - 100 years) living in Southwest China were selected. CBC was performed on an XE-2100 hematology analyzer. The medians were calculated and reference values were determined at the 2.5th and 95th percentiles. RESULTS: There was a statistically significant gender difference for RBC count, Hb, and MCV (p = 0.000). The comparison of Hb by gender showed higher values among males, with lower values in females and this difference was similar to the values for RBC count and MCV. For males, mean values for Hb and RBC decreased with age. However, mean values for Hb in females increased after middle age. CONCLUSIONS: Reference intervals for major red blood cell parameters established in this study showed significant gender differences. For males, mean values for Hb and RBC decreased with age. This is the first region-wide population study on hematologic reference values among people in Southwest China.

Deglycosylation induces extensive dynamics changes in α-amylase revealed by hydrogen/deuterium exchange mass spectrometry.

RATIONALE: N-Linked glycosylation plays important roles in modulating protein structure and function. The direct impact of the modification on protein conformation is not yet well understood. METHODS: Here we probed the dynamic changes following Endo H trimming of high mannose glycans in α-amylase by means of amide hydrogen/deuterium exchange mass spectrometry. RESULTS: The results revealed that deglycosylation elicited extensive alterations in backbone dynamics, affecting regions both adjacent to and distal from the glycosylation site. CONCLUSIONS: The overall exchange rate is reduced in the glycosylated state, which indicates rigidity enhancement due to the attached carbohydrates.

In-depth size and charge variants characterization of monoclonal antibody with native mass spectrometry.

Although the reversed-phase liquid chromatography (RPLC) is the most used separation front for mass spectrometry, many other separation modes are critical for enabling characterization of the protein therapeutics. Specifically, chromatographic separations under native conditions, such as those based on size exclusion chromatography (SEC) and ion-exchange chromatography (IEX), are used for characterizing important biophysical properties of protein variants in drug substance and drug product. Because most native state separation modes use non-volatile buffers with high salt concentration, optical detection has been traditionally used. However, there is an increasing need to understand and identify the optical underlying peaks by mass spectrometry for structure elucidation. For size variant separation by SEC, the native MS helps to understand the nature of the high molecular weight species, as well as clipping sites for low molecular weight fragments. For charge variant separation by IEX, native MS can reveal the post-translational modifications or other important factors contributing to charge heterogeneity at the intact level. Here, we demonstrate the power of native MS by direct coupling of SEC and IEX eluent to a time-of-flight mass spectrometer to characterize bevacizumab and NISTmAb. Our studies exemplify the effectiveness of native SEC-MS for characterizing bevacizumab's high molecular weight species at less than 0.3% (based on SEC/UV peak area%) and analyzing the fragment pathway with single amino acid difference for its low molecular weight species at less than 0.05%. Good IEX charge variant separation was obtained with consistent UV and MS profiles. The identity of separated acidic and basic variants were elucidated by native MS at intact level. We successfully differentiated several charge variants including glycoform variants that have not been reported before. In addition, native MS allowed identification of higher molecular weight species as late eluted variants. Overall, the SEC and IEX separation combined with high resolution and high sensitivity native MS, which is significantly different from the traditional RPLC-MS workflows, can be an effective tool that offers valuable insights for us to understand protein therapeutics at native state.

An X-ray inactivated vaccine against Pseudomonas aeruginosa Keratitis in mice.

Pseudomonas aeruginosa (P. aeruginosa) is one of the most prevalent pathogens of bacterial keratitis. Bacterial keratitis is a major cause of blindness worldwide. The rising incidence of multidrug resistance of P. aeruginosa precludes treatment with conventional antibiotics. Herein, we evaluated the protective efficiency and explored the possible underlying mechanism of an X-ray inactivated vaccine (XPa) using a murine P. aeruginosa keratitis model. Mice immunized with XPa exhibit reduced corneal bacterial loads and pathology scores. XPa vaccination induced corneal macrophage polarization toward M2, averting an excessive inflammatory reaction. Furthermore, histological observations indicated that XPa vaccination suppressed corneal fibroblast activation and prevented irreversible visual impairment. The potency of XPa against keratitis highlights its potential utility as an effective and promising vaccine candidate for P. aeruginosa.

Discovery and multimerization of cross-reactive single-domain antibodies against SARS-like viruses to enhance potency and address emerging SARS-CoV-2 variants.

Coronaviruses have been the causative agent of three epidemics and pandemics in the past two decades, including the ongoing COVID-19 pandemic. A broadly-neutralizing coronavirus therapeutic is desirable not only to prevent and treat COVID-19, but also to provide protection for high-risk populations against future emergent coronaviruses. As all coronaviruses use spike proteins on the viral surface to enter the host cells, and these spike proteins share sequence and structural homology, we set out to discover cross-reactive biologic agents targeting the spike protein to block viral entry. Through llama immunization campaigns, we have identified single domain antibodies (VHHs) that are cross-reactive against multiple emergent coronaviruses (SARS-CoV, SARS-CoV-2, and MERS). Importantly, a number of these antibodies show sub-nanomolar potency towards all SARS-like viruses including emergent CoV-2 variants. We identified nine distinct epitopes on the spike protein targeted by these VHHs. Further, by engineering VHHs targeting distinct, conserved epitopes into multi-valent formats, we significantly enhanced their neutralization potencies compared to the corresponding VHH cocktails. We believe this approach is ideally suited to address both emerging SARS-CoV-2 variants during the current pandemic as well as potential future pandemics caused by SARS-like coronaviruses.

In vitro evaluation of hepatic transporter-mediated clinical drug-drug interactions: hepatocyte model optimization and retrospective investigation.

To assess the feasibility of using sandwich-cultured human hepatocytes (SCHHs) as a model to characterize transport kinetics for in vivo pharmacokinetic prediction, the expression of organic anion-transporting polypeptide (OATP) proteins in SCHHs, along with biliary efflux transporters, was confirmed quantitatively by liquid chromatography-tandem mass spectrometry. Rifamycin SV (Rif SV), which was shown to completely block the function of OATP transporters, was selected as an inhibitor to assess the initial rates of active uptake. The optimized SCHH model was applied in a retrospective investigation of compounds with known clinically significant OATP-mediated uptake and was applied further to explore drug-drug interactions (DDIs). Greater than 50% inhibition of active uptake by Rif SV was found to be associated with clinically significant OATP-mediated DDIs. We propose that the in vitro active uptake value therefore could serve as a cutoff for class 3 and 4 compounds of the Biopharmaceutics Drug Disposition Classification System, which could be integrated into the International Transporter Consortium decision tree recommendations to trigger clinical evaluations for potential DDI risks. Furthermore, the kinetics of in vitro hepatobiliary transport obtained from SCHHs, along with protein expression scaling factors, offer an opportunity to predict complex in vivo processes using mathematical models, such as physiologically based pharmacokinetics models.

Capillary Isoelectric Focusing: Mass Spectrometry Method for the Separation and Online Characterization of Monoclonal Antibody Charge Variants at Intact and Subunit Levels.

Monoclonal antibodies (mAbs) are one of the most widely used types of protein therapeutics. Charge variants are important quality attributes for evaluating developability, activity, and safety for mAb therapeutics. Here, we report a novel online capillary isoelectric focusing-mass spectrometry (CIEF-MS) method for mAb charge variant analysis using an electrokinetically pumped sheath-flow nanospray ion source on a time-of-flight (TOF) MS with a pressure-assisted chemical mobilization. Key factors that enable online CIEF-MS include effective capillary electrophoresis-MS (CE-MS) interface with enhanced sensitivity, utilization of MS-friendly electrolytes, beneficial effects of glycerol that reduces non-CIEF electrophoretic mobility and limits band broadening, appropriate ampholyte type and concentration selection for balanced separation resolution and MS detection sensitivity, optimized sheath liquid composition to realize high-resolution CIEF separation and effective MS electrospray ionization, as well as judiciously selected CIEF running parameters. The fundamental premise of CIEF has been verified by the linear correlation between isoelectric point (pI) values and migration time using a mixture of pI markers. By achieving high separation resolutions that are similar as those obtained from imaged CIEF (iCIEF), this method successfully provides highly sensitive MS identification for intact mAb charge variants. Furthermore, a middle-up sample treatment workflow can be adopted to provide in-depth charge variant analysis at subunit level for mAbs with complex charge heterogeneity. The mAb subunit CIEF-MS reveals the source of charge variant with enhanced resolution on both CIEF separation and MS spectra. This novel CIEF-MS method is a valuable tool with distinct advantage for objective and accurate assessment of charge heterogeneity of protein therapeutics.

Cold-hot nature identification of Chinese herbal medicines based on the similarity of HPLC fingerprints.

The nature theory of Chinese herbal medicines (CHMs) is the core theory of traditional Chinese medicine (TCM). Cold-hot nature is an important part of CHM nature. It is found that the material basis of cold-hot nature is CHM ingredients. To test the scientific hypothesis that "CHMs with similar cold-hot nature should have similar material basis," we explored an intelligent method for cold-hot nature identification of CHMs based on the feature similarity of CHM ingredients in this work. Sixty one CHMs were selected for cold-hot nature identification. High performance liquid chromatography (HPLC) was used to separate the chemical ingredients of CHMs and extract the feature information of CHM ingredients. A distance metric learning algorithm was then learned to measure the similarity of HPLC fingerprints. With the learned distance metric, cold-hot nature identification scheme (CHNIS) was proposed to build an identification model to evaluate the cold-hot nature of CHMs. A number of experiments were designed to verify the effectiveness and feasibility of the proposed CHNIS model. The total identification accuracy rate of 61 CHMs is 80.3%. The performance of the proposed CHNIS algorithm outperformed that of the compared classical algorithms. The experimental results confirmed our inference that CHMs with similar cold-hot nature had similar composition of substances. The CHNIS model was proved to be effective and feasible.

An update on the role of tumor necrosis factor alpha stimulating gene-6 in inflammatory diseases.

At present, the occurrence and development of inflammatory diseases are closely related to the abnormal changes of the content and function of many cytokines. At the same time, targeting related cytokines to prevent and treat diseases has also achieved good results. Therefore, it is very important to explore the role of various cytokines in inflammatory diseases. As an inflammation related protein, Tumor necrosis factor alpha stimulating gene-6 (TSG-6) has attracted more and more attention. In the process of disease, it's like a double-edged sword, showing different responses. It is constitutively expressed in some tissues with high metabolic activity and barrier protection. The diversity of its functions depends on the binding of TSG-6 with a variety of ligands, including matrix molecules, autoimmune regulatory factors and growth factors, participating in extracellular matrix remodeling and regulating protease network. This paper reviews the structure, biological function and research progress of TSG-6 in inflammatory diseases, in order to provide reference for drug development in the future.

Chronic treatment with epoxyeicosatrienoic acids modulates insulin signaling and prevents insulin resistance in hepatocytes.

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites produced by cytochrome P450 epoxygenases which are highly expressed in hepatocytes. The functions of EETs in hepatocytes are not well understood. In this study, we investigated the effects of 14,15-EETs treatment on the insulin signal transduction pathway in hepatocytes. We report that chronic treatment, not acute treatment, with 30 µM 14,15-EETs prevents palmitate induced insulin resistance and potentiates insulin action in cultured HepG2 hepatocytes. 14,15-EETs increase Akt phosphorylation at S473, activating Akt, in an insulin dependent manner in HepG2 cells. Under insulin resistant conditions induced by palmitate, 14,15-EETs restore the insulin response by increasing S473-phosphorylated Akt. 8,9-EETs and 11,12-EETs demonstrated similar effects to 14,15-EETs. Furthermore, 14,15-EETs potentiate insulin-suppression of gluconeogenesis in cultured H4IIE hepatocytes. To elucidate the mechanism of EETs function, we analyzed the insulin signaling factors upstream of Akt. Inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 attenuated the 14,15-EETs-induced activating phosphorylation of Akt. 14,15-EETs reduced palmitate-stimulated phosphorylation of IRS-1 on S312 and phosphorylation of c-Jun N-terminal kinase (JNK) at threonine 183 and tyrosine 185 residues. The regulation of insulin sensitivity in cultured hepatocytes by chronic 14,15-EETs treatment appears to involve the JNK-IRS-PI3K pathway. The requirement of chronic treatment with EETs suggests that the effects of EETs on insulin response may be indirect.

Piezo1 initiates platelet hyperreactivity and accelerates thrombosis in hypertension.

BACKGROUND: Thrombosis is the pathological basis of cardiovascular and cerebrovascular diseases, which seriously threaten human life and health. Among them, nearly half of cardiovascular disease patients suffer from severe hypertension complications. Hypertension is thought to cause abnormal platelet activation and increases the risk of thrombosis, but the related mechanism is still vague. OBJECTIVES: This study hypothesized that the abnormal hemodynamics of blood under hypertension might affect platelet function and accelerate thrombosis by activating mechanoreceptor Piezo1. METHODS: To assess the activation effect of hypertension on mechanoreceptor Piezo1, we injected Piezo1 agonist Yoda1 and antagonist GsMTx-4 through the tail vein, then examined the platelet activation status and thrombosis. RESULTS: Our results displayed that antagonist GsMTx-4 effectively inhibited calcium influx caused by hypertension and agonist Yoda1. Antithrombotic studies proved that the inhibition of Piezo1 effectively inhibited arterial thrombosis and reduced the infarct size of stroke in hypertensive mice. CONCLUSIONS: Our study explains the activation of mechanoreceptor Piezo1 under hypertension is the key to abnormal platelet activation and thrombosis while providing novel platelet intervention strategies to prevent thrombosis.