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1.  Targeting the anionic region of human protease activated receptor 4 (PAR4) inhibits platelet aggregation and thrombosis without interfering with hemostasis 
Summary
Background
Human platelet activation and aggregation is a complex process. To date, many therapies have been developed targeting proteins that mediate this process to prevent unwanted activation. However, the current standard of care for acute coronary syndromes still has limitations including bleeding risk.
Objective
The aim of the current study is to evaluate the PAR4 anionic cluster as a viable antiplatelet target using a polyclonal antibody (CAN12).
Methods
We used western blotting, aggregation, and secretion ex vivo to evaluate the ability of CAN12 to interact with PAR4 and inhibit platelet activation. The effects of CAN12 in vivo were evaluated with the Rose Bengal arterial thrombosis model and two models of hemostasis.
Results
We show that CAN12 is able to interact with human PAR4 and delay PAR4 cleavage. In addition, CAN12 inhibits thrombin induced human platelet aggregation and secretion in a dose dependent manner. We next determined that the specificity of CAN12 is agonist dependent. In vivo, we determined that CAN12 is able to inhibit arterial thrombosis and using two independent methods, we found that CAN12 does not influence hemostasis.
Conclusion
Targeting the extracellular anionic cluster on PAR4 is a viable novel strategy as an anti-platelet therapy.
doi:10.1111/jth.12619
PMCID: PMC4127092  PMID: 24888424
Protease-Activated Receptor 4 - human; Platelet Aggregation Inhibitors; Antibodies; Thrombosis; Hemostasis; G-Protein-Coupled Receptors
2.  In vivo enrichment of genetically manipulated platelets corrects the murine hemophilic phenotype and induces immune tolerance even using a low MOI 
Summary
Background
Our previous studies have demonstrated that platelet-specific gene delivery to hematopoietic stem cells can induce sustained therapeutic levels of platelet-FVIII expression in hemophilia A (HA) mice.
Objective
In this study, we aimed to enhance platelet-FVIII expression while minimizing potential toxicities.
Methods
A novel lentiviral vector (LV), which harbors dual genes, the FVIII gene driven by the αIIb promoter (2bF8) and a drug-resistance gene, the MGMTP140K cassette, was constructed. Platelet-FVIII expression in HA mice was introduced by transduction of HSCs and transplantation. The recipients were treated with O6-benzylguanine followed by 1,3-bis-2 chloroethyl-1-nitrosourea monthly for 3 or 4 times. Animals were analyzed by PCR, qPCR, FVIII:C assays, and inhibitor assays. Phenotypic correction was assessed by tail clipping tests and ROTEM analysis.
Results
Even using a low MOI (multiplicity of infection) of 1 and a non-myeloablative conditioning regimen, after in vivo selection, the levels of platelet-FVIII expression in recipients increased to 4.33 ± 5.48 mU per 108 platelets (n = 16), which were 19.7-fold higher than the levels obtained from the recipients before treatment. qPCR results confirmed that 2bF8/MGMT-LV-transduced cells were effectively enriched after drug-selective treatment. Fifteen of 16 treated animals survived tail clipping. Blood loss and whole blood clotting time were normalized in the treated recipients. Notably, no anti-FVIII antibodies were detected in the treated animals even after rhF8 challenge.
Conclusion
we have established an effective in vivo selective system that allows us to enrich 2bF8LV-transduced cells, enhancing platelet-FVIII expression while reducing the potential toxicities associated with platelet gene therapy.
doi:10.1111/jth.12633
PMCID: PMC4127102  PMID: 24931217
Blood platelet; Factor VIII; Genetic therapy; Hemophilia A; Immune tolerance
3.  PAI-1 over-expression decreases experimental post-thrombotic vein wall fibrosis by a non-vitronectin dependent mechanism 
SUMMARY
Background
Factors associated with post-thrombotic syndrome are known clinically, but the underlying cellular processes at the vein wall are not well-delineated. Prior work suggests that vein wall damage does not correlate with thrombus resolution, but rather with plasminogen activator-1 (PAI-1) and matrix metalloproteinase (MMP) activity.
Objective
We hypothesized that PAI-1 would confer post venous thrombosis (VT) vein wall protection via a Vitronectin (Vn) dependent mechanism.
Methods
A stasis model of VT was used with harvest over 2 weeks, in wild type (WT), Vn−/−, and PAI-1 overexpressing mice (PAI-1 Tg).
Results
PAI-1 Tg mice had larger VT at 6 and 14 days, compared to controls, but Vn−/−mice had no alteration of VT resolution. Gene deletion of Vn resulted in increased, rather than expected decrease in circulating PAI-1 activity. While both Vn−/− and PAI-1 Tg had attenuated intimal fibrosis, PAI-1 Tg had significantly less vein wall collagen and a compensatory increase in collagen III gene expression. Both Vn−/− and PAI-1 Tg vein wall had less monocyte chemotactic factor-1, and fewer macrophages (F4/80), with significantly less MMP-2 activity and decreased TIMP-1 antigen. Ex vivo assessment of TGFβ mediated fibrotic response showed that PAI-1 Tg vein walls had increased profibrotic gene expression (collagen I, III, MMP-2 and α-SMA) as compared with controls, opposite of the in vivo response.
Conclusions
The absence of Vn increases circulating PAI-1, which positively modulates vein wall fibrosis in a dose-dependent manner. Translationally, PAI-1 elevation may decrease vein wall damage after DVT, perhaps by decreasing macrophage-mediated activities.
doi:10.1111/jth.12644
PMCID: PMC4127110  PMID: 24943740
fibrosis; PAI-1; postthrombotic syndrome; venous thrombosis; vitronectin
4.  Platelets: The Unsung Hero of the Immune Response 
doi:10.1111/jth.12806
PMCID: PMC4369386  PMID: 25471925
5.  Utility of Multiplex Ligation-Dependent Probe Amplification (MLPA) for Hemophilia Mutation Screening 
doi:10.1111/j.1538-7836.2012.04843.x
PMCID: PMC4521386  PMID: 22759210
F8 mutations; F9 mutations; Hemophilia; Multiplex Ligation-Dependent Probe Amplification
6.  Manipulating megakaryocytes to manufacture platelets ex vivo 
Journal of Thrombosis and Haemostasis  2015;13(Suppl 1):S47-S53.
Historically, platelet transfusion has proven a reliable way to treat patients suffering from thrombocytopenia or similar ailments. An undersupply of donors, however, has demanded alternative platelet sources. Scientists have therefore sought to recapitulate the biological events that convert hematopoietic stem cells into platelets in the laboratory. Such platelets have shown good function and potential for treatment. Yet the number manufactured ex vivo falls well short of clinical application. Part of the reason is the remarkable gaps in our understanding of the molecular mechanisms driving platelet formation. Using several stem cell sources, scientists have progressively clarified the chemical signaling and physical microenvironment that optimize ex vivo platelets and reconstituted them in synthetic environments. Key advances in cell reprogramming and the ability to propagate self-renewal have extended the lifetime of megakaryocytes to increase the pool of platelet progenitors.
doi:10.1111/jth.12946
PMCID: PMC4501322  PMID: 26149050
bioreactors; blood platelets; induced pluripotent stem cells; megakaryocytes; polyploidy
7.  Evidence that α2-antiplasmin becomes covalently ligated to plasma fibrinogen in the circulation: a new role for plasma factor XIII in fibrinolysis regulation 
Summary
Background
Plasma alpha2-antiplasmin (α2AP) is a rapid and effective inhibitor of the fibrinolytic enzyme plasmin. Congenital α2AP deficiency results in a severe hemorrhagic disorder due to accelerated fibrinolysis. It is well established that in the presence of thrombin-activated factor XIII (FXIIIa), α2AP becomes covalently ligated to the distal α chains of fibrin or fibrinogen at lysine 303 (two potential sites per molecule). Some time ago we showed that α2AP is covalently linked to plasma fibrinogen. That singular observation led to our hypothesis that native plasma factor XIII (FXIII), which is known to catalyze covalent cross-linking of fibrinogen in the presence of calcium ions, can also incorporate α2AP into fibrinogen in the circulation.
Results and Conclusions
We now provide evidence that FXIII incorporates I125-labelled α2AP into the Aα-chain sites on fibrinogen or fibrin. We also measured the content of α2AP in isolated plasma fibrinogen fractions by ELISA and found that substantial amounts were present (1.2–1.8 moles per mole fibrinogen). We propose that α2AP becomes ligated to fibrinogen while in the circulation through the action of FXIII, and that its immediate presence in plasma fibrinogen contributes to regulation of in vivo fibrinolysis.
doi:10.1111/j.1538-7836.2008.03056.x
PMCID: PMC4489681  PMID: 18564219
factor XIII; fibrinogen; fibrinolysis; serpin; α2-antiplasmin
8.  Abnormal plasma clot structure and stability distinguish bleeding risk in patients with severe factor XI deficiency 
Background
Factor XI (FXI) deficiency is a rare autosomal recessive disorder. Many patients with even very low FXI levels (<20 IU/dL) are asymptomatic or exhibit only mild bleeding, whereas others experience severe bleeding usually following trauma. Neither FXI antigen nor activity predicts bleeding risk in FXI-deficient patients.
Objectives
1) Characterize the formation, structure and stability of plasma clots from patients with severe FXI deficiency, and 2) Determine whether these assays can distinguish asymptomatic patients (“non-bleeders”) from those with a history of bleeding (“bleeders”).
Methods
Platelet-poor plasmas were prepared from 16 severe FXI-deficient patients who were divided into bleeders or non-bleeders, based on bleeding associated with at least two tooth extractions without prophylaxis. Clot formation was triggered by recalcification and addition of tissue factor and phospholipids in the absence or presence of tissue plasminogen activator and/or thrombomodulin. Clot formation and fibrinolysis were measured by turbidity, and fibrin network structure by laser scanning confocal microscopy.
Results
Non-bleeders and bleeders had similarly low FXI levels, normal prothrombin times, normal levels of fibrinogen, factor VIII, von Willebrand factor, factor XIII, and normal platelet number and function. Compared to non-bleeders, bleeders exhibited lower fibrin network density and lower clot stability in the presence of tissue plasminogen activator. In the presence of thrombomodulin, 7 of 8 bleeders failed to form a clot, whereas only 3 of 8 non-bleeders did not clot.
Conclusions
Plasma clot structure and stability assays distinguished non-bleeders from bleeders. These assays may reveal hemostatic mechanisms in FXI-deficient patients and have clinical utility for assessing bleeding risk.
doi:10.1111/jth.12600
PMCID: PMC4107079  PMID: 24815347
bleeding; Factor XI; Factor XI deficiency; plasma clot lysis time; fibrin clot lysis time
9.  Integrin αIIb Tail Distal of GFFKR Participates in Inside-out αIIbβ3 Activation 
Background
Increases in ligand binding to integrins (“activation”) play critical roles in platelet and leukocyte function. Integrin activation requires talin and kindlin binding to integrin β cytoplasmic tails. Much research has focused on the conserved GFFKR motif in integrin αIIb tails for its importance in keeping integrins inactive, integrin β cytoplasmic tails and the binding partners of β tails. However, the roles of αIIb tail distal of GFFKR motif are unexplored.
Objective
To investigate the role of αIIb tail distal of GFFKR in talin-mediated inside-out integrin signaling.
Methods
We used model cell systems to examine the role of αIIb tail distal of GFFKR in bi-directional αIIbβ3 signaling and αIIbβ3-talin interactions.
Results
Deletion of amino acid residues after the GFFKR motif in αIIb tail moderately decreased β3(D723R)-induced activation, abolished talin-induced αIIbβ3 activation in model cells and inhibited agonist-induced αIIbβ3 activation in megakaryocytic cells. Furthermore, residues in αIIb tail distal of GFFKR did not affect outside-in αIIbβ3 signaling or αIIbβ3-talin interaction. Addition of non-homologous or non-specific amino acids to the GFFKR motif restored αIIbβ3 activation in model cells and in megakaryocytic cells. Molecular modeling indicates that β3-bound talin sterically clashes with the αIIb tail in the αIIbβ3 complexes, potentially disfavoring the α-β interactions that keep αIIbβ3 inactive.
Conclusion
The αIIb tail sequences distal of GFFKR participate in talin-mediated inside-out αIIbβ3 activation through its steric clashes with β3-bound talin.
doi:10.1111/jth.12610
PMCID: PMC4107134  PMID: 24837519
Integrin alphaIIbbeta3; talin; kindlin-2 protein; human; signal transduction; cell adhesion
10.  CENTRAL VENOUS CATHETER-RELATED THROMBOSIS AND THROMBOPROPHYLAXIS IN CHILDREN: A SYSTEMATIC REVIEW AND META-ANALYSIS 
SUMMARY
OBJECTIVES
In preparation for a pediatric randomized controlled trial on thromboprophylaxis, we determined the frequency of catheter-related thrombosis in children. We also systematically reviewed the pediatric trials on thromboprophylaxis to evaluate its efficacy and to identify possible pitfalls in the conduct of these trials.
PATIENTS/METHODS
We searched MEDLINE, EMBASE, Web of Science and Cochrane Central Register for Controlled Trials for articles published until December 2013. We included cohort studies and trials on patients 0–18 years old with central venous catheter actively surveilled for thrombosis with radiologic imaging. We estimated the pooled frequency of thrombosis and the pooled risk ratio (RR) with thromboprophylaxis using random effects model.
RESULTS
Of 2,651 articles identified, we analyzed 37 articles with 3,128 patients. The pooled frequency of thrombosis was 0.20 (95% confidence interval [CI]: 0.16–0.24). Of 10 trials, we did not find evidence that heparin-bonded catheter (RR: 0.34; 95% CI: 0.01–7.68), unfractionated heparin (RR: 0.93; 95% CI: 0.57–1.51), low molecular weight heparin (RR: 1.13; 95% CI: 0.51–2.50), warfarin (RR: 0.85; 95% CI: 0.34–2.17), antithrombin concentrate (RR: 0.76; 95% CI: 0.38–1.55) and nitroglycerin (RR: 1.53; 95% CI: 0.57–4.10) reduced the risk for thrombosis. Most of the trials were either not powered for thrombosis or powered to detect large, likely unachievable, reductions in thrombosis. Missing data on thrombosis also limited these trials.
CONCLUSIONS
Catheter-related thrombosis is common in children. An adequately powered multicenter trial that can detect a modest, clinically significant, reduction in thrombosis is critically needed. Missing outcome data should be minimized in this trial.
doi:10.1111/jth.12598
PMCID: PMC4107177  PMID: 24801495
anticoagulants; heparin; pediatrics; prevention; prophylaxis
11.  Validation of Nijmegen-Bethesda Assay Modifications to Allow Inhibitor Measurement during Replacement Therapy and Facilitate Inhibitor Surveillance 
Summary
Background
As part of a pilot U.S. inhibitor surveillance project initiated at the Centers for Disease Control and Prevention (CDC) in 2006, centralized inhibitor measurement was instituted.
Objective
To validate a modified method for inhibitor measurement suitable for surveillance of treated and untreated patients.
Methods/Results
710 subjects with hemophilia A were enrolled; 122 had history of inhibitor (HI). Nijmegen-Bethesda assay (NBA) results on 50 split specimens shipped on cold packs and frozen were equivalent (r=0.998). Because 55% of 228 initial specimens had factor VIII (FVIII) activity (VIII:C) present, a heat treatment step was added. Heating specimens to 56°C for 30 minutes and centrifuging removed FVIII, as demonstrated by reduction of VIII:C and FVIII antigen to <1 U/dL in recently treated patients. Among specimens inhibitor-negative before heating, 1 of 159 with negative HI and 5 of 30 with positive HI rose to ≥0.5 Nijmegen Bethesda units (NBU) after heating. Correlation of heated and unheated inhibitor-positive specimens was 0.94 (P=0.0001). The modified method had a CV for a 1 NBU positive control of 10.3% and for the negative control of 9.8%. Based on results on 710 enrollment specimens, a positive CDC inhibitor was defined as ≥ 0.5 NBU. Results were similar when 643 post-enrollment specimens were included. Of 160 enrolled hemophilia B patients, 2 had HI. All others had NBU ≤ 0.2 at enrollment.
Conclusion
The CDC experience demonstrates that this modified NBA can be standardized to be within acceptable limits for clinical tests and can be used for national surveillance.
doi:10.1111/j.1538-7836.2012.04705.x
PMCID: PMC4477703  PMID: 22435927
factor VIII; factor IX; inhibitor
12.  Comparison of Clot-based, Chromogenic, and Fluorescence Assays for Measurement of Factor VIII Inhibitors in the U.S. Hemophilia Inhibitor Research Study 
Summary
Background
Detection and validation of inhibitors (antibodies) to hemophilia treatment products are important for clinical care, evaluation of product safety, and assessment of population trends.
Methods
Centralized monitoring for factor VIII (FVIII) inhibitors was conducted for patients in the Hemophilia Inhibitor Research Study using a previously reported modified Nijmegen-Bethesda clotting assay (NBA), a chromogenic Bethesda assay (CBA), and a novel fluorescence immunoassay (FLI).
Results
NBA and CBA were performed on 1005 specimens and FLI on 272 specimens. CBA was negative on 880/883 specimens (99.7%) with Nijmegen-Bethesda units (NBU)<0.5 and positive on 42/42 specimens (100%) with NBU≥2.0 and 43/80 specimens (53.8%) with NBU 0.5–1.9. Among specimens with positive NBA and negative CBA, 58.1% were FLI-negative, 12.9% had evidence of lupus anticoagulant, and 35.5% had non-time-dependent inhibition. CBA and FLI were positive on 72.4% and 100% of 1.0–1.9 NBU specimens and 43.1% and 50.0% of 0.5–0.9 NBU specimens. FLI detected antibodies in 98.0% of CBA-positive and 81.6% of NBA-positive specimens (P=0.004). Among 21 new inhibitors detected by NBA, 5 (23.8%) with 0.7–1.3 NBU did not react in CBA or FLI. Among previously positive patients with 0.5–1.9 NBU, 7/25 (28%) were not CBA or FLI positive. FLI was positive on 36/169 NBU-negative specimens (21.3%).
Conclusions
FVIII specificity could not be demonstrated by CBA or FLI for 26% of inhibitors of 0.5–1.9 NBU; such results must be interpreted with caution. Low titer inhibitors detected in clot-based assays should always be repeated, with consideration given to evaluating their reactivity with FVIII using more specific assays.
doi:10.1111/jth.12259
PMCID: PMC4477744  PMID: 23601690
factor VIII inhibitor; Nijmegen-Bethesda assay; chromogenic factor VIII assay; fluorescence immunoassay
13.  rFVIIa transported from blood stream into tissues is functionally active 
doi:10.1111/j.1538-7836.2010.04010.x
PMCID: PMC4472316  PMID: 20695977
14.  Associations of Pentraxin 3 with Cardiovascular Disease: The Multi-Ethnic Study of Atherosclerosis 
Objective
Pentraxin 3 (PTX3) is likely a specific marker of vascular inflammation. However, associations of PTX3 with cardiovascular disease (CVD) risk have not been well studied in healthy adults or multi-ethnic populations. We examined associations of PTX3 with CVD risk factors, measures of subclinical CVD, coronary artery calcification (CAC) and CVD events in the Multi-Ethnic Study of Atherosclerosis (MESA).
Approach and Results
2838 participants free of prevalent CVD with measurements of PTX3 were included in the present study. Adjusting for age, sex and ethnicity, PTX3 was positively associated with age, obesity, insulin, systolic blood pressure, C-reactive protein (CRP) and carotid intima media thickness (all p<0.045). A one standard deviation increase in PTX3 (1.62 ng/ml) was associated with the presence of CAC in fully adjusted models including multiple CVD risk factors (relative risk; 95% confidence interval 1.05; 1-01-1.08). In fully adjusted models, a standard deviation higher level of PTX3 was associated with an increased risk of myocardial infarction (hazard ratio; 95% confidence interval 1.51; 1.16-1.97), combined CVD events (1.23; 1.05-1.45) and combined CHD events (1.33; 1.10-1.60) but not stroke, CVD-related mortality or all cause death.
Conclusions
In these apparently healthy adults, PTX3 was associated with CVD risk factors, subclinical CVD, CAC and incident coronary heart disease events independent of CRP and CVD risk factors. These results support the hypothesis that PTX3 reflects different aspects of inflammation than CRP and may provide additional insight into the development and progression of atherosclerosis.
doi:10.1111/jth.12557
PMCID: PMC4055511  PMID: 24628740
Atherosclerosis; Cardiovascular Diseases; Epidemiology; Inflammation; Pentraxin 3
15.  Neutrophil extracellular traps form predominantly during the organizing stage of human venous thromboembolism development 
Background
A growing health problem, venous thromboembolism (VTE), including pulmonary embolism (PE) and deep vein thrombosis (DVT), requires refined diagnostic and therapeutic approaches. Neutrophils contribute to thrombus initiation and development in experimental DVT. Recent animal studies recognized neutrophil extracellular traps (NETs) as an important scaffold supporting thrombus stability. However, the hypothesis that human venous thrombi involve NETs has not undergone rigorous testing.
Objective
To explore the cellular composition and the presence of NETs within human venous thrombi at different stages of development.
Patients and Methods
We examined sixteen thrombi obtained from 11 patients during surgery or at autopsy using histomorphological, immunohistochemical and immunofluorescence analyses.
Results
We classified thrombus regions as unorganized, organizing, and organized according to their morphological characteristics. We then evaluated them focusing on neutrophil and platelet deposition as well as micro-vascularization of the thrombus body. We observed evidence of NET accumulation, including the presence of citrullinated histone H3 (H3Cit)-positive cells. NETs, defined as extracellular diffuse H3Cit areas associated with myeloperoxidase and DNA, localized predominantly during the phase of organization in human venous thrombi.
Conclusions
NETs are present in organizing thrombi in patients with VTE. They are associated with thrombus maturation in humans. Dissolution of NETs might thus facilitate thrombolysis. This finding provides new insights into the clinical development and pathology of thrombosis and provides new perspectives for therapeutic advances.
doi:10.1111/jth.12571
PMCID: PMC4055516  PMID: 24674135
blood platelets; clinical pathology; histones; neutrophils; venous thromboembolism
16.  BLOOD GLUCOSE AS A MARKER OF VENOUS THROMBOEMBOLISM IN CRITICALLY ILL CHILDREN 
SUMMARY
Background
The ability to predict the development of venous thromboembolism is highly desirable.
Objective
We aim to determine the association between hyperglycemia and venous thromboembolism in non-diabetic critically ill children.
Patients/Methods
We conducted a retrospective cohort study that included children in the pediatric intensive care unit on vasopressor or on mechanical ventilator and without history of diabetes mellitus or prior diagnosis of thrombosis. Based on maximum blood glucose >150 mg/dl while admitted to the unit, children were categorized as hyperglycemic or non-hyperglycemic. Primary outcome was development of venous thromboembolism while admitted to the unit. We determined the association between hyperglycemia and venous thromboembolism using logistic regression models adjusting for selected subject characteristics.
Results
Of the 789 subjects analyzed, 34 subjects developed venous thromboembolism (incidence: 4.3%; 95% confidence interval: 3.0%–6.0%). Venous thromboembolism was more likely to develop in hyperglycemic subjects compared with non-hyperglycemic subjects. A total of 31 subjects (6.2%; 95% confidence interval: 4.2%–8.7%) developed venous thromboembolism after becoming hyperglycemic compared with 3 non-hyperglycemic subjects with venous thromboembolism (1.0%, 95% confidence interval: 0.2%–3.0%). When adjusted for age, diagnosis, presence of central venous catheter, prophylactic antithrombotic use and severity of illness, the odds ratio of venous thromboembolism with hyperglycemia was 4.1 (95% confidence interval: 1.2–14.1). For every 10 mg/dl increase in maximum blood glucose, adjusted odds ratio of venous thromboembolism was 1.04 (95% confidence interval: 1.01–1.06).
Conclusion
Hyperglycemia is associated with venous thromboembolism in critically ill non-diabetic children. Maximum blood glucose is a potential predictor of venous thromboembolism in this population.
doi:10.1111/jth.12583
PMCID: PMC4055532  PMID: 24708410
blood glucose; deep venous thrombosis; hyperglycemia; pulmonary embolism; risk factors
17.  Long-term tolerance to factor VIII is achieved by administration of IL-2/IL-2mAb complexes and low dosages of factor VIII 
Summary
Background
Regulatory T cells (Tregs) play a pivotal role in regulating anti-factor VIII (FVIII) immune responses. Interleukin (IL)-2 mixed with a particular IL-2 monoclonal antibody (mAb; JES6-1) can induce selective expansion of Tregs in vivo.
Methods
In the prevention experiments, we treated hemophilia A mice with IL-2/IL-2mAb complexes (3x/week), and concurrently with FVIII protein (80U/kg/week) for 4 weeks. Generation of anti-FVIII immune responses was examined afterwards. Next, in order to induce long-term tolerance to FVIII, a series of treatment dosages and schedules for administering IL-2/IL-2mAb complexes and FVIII protein in hemophilia A mice was evaluated.
Results
Compared to control FVIII only treated mice which produced high-titer anti-FVIII antibodies, mice treated with IL-2/IL-2mAb complexes + FVIII produced no antibodies. A marked 7 fold increase in CD4+CD25+Foxp3+Helios+ natural Tregs was maintained for 4 weeks in blood, spleen and lymph nodes, and dropped to normal levels within the next 10 days. The suppressive functions of expanded Tregs were demonstrated by suppressive, proliferative and cytokine assays. Administration of anti-CD25 mAb (PC-61) blocked this protective effect, confirming the involvement of Tregs in suppressing anti-FVIII immune responses. Importantly, administration of IL-2/IL-2mAb complexes (3x/week for 8 weeks) combined with contiguous weekly injections of low dosage FVIII protein (20U/kg/week for 18 weeks) not only abrogated the formation of anti-FVIII antibodies, but also induced long-term tolerance to FVIII.
Conclusions
Treatment of IL-2/IL-2mAb complexes is highly promising for induction and maintenance of FVIII-specific tolerance following FVIII protein replacement therapy.
doi:10.1111/jth.12576
PMCID: PMC4055525  PMID: 24684505
IL-2; anti-IL2 antibody; factor VIII; hemophilia; immune tolerance; immunomodulation; regulatory T cell; protein replacement therapy
18.  Comparative performance of gene-based warfarin dosing algorithms in a multiethnic population 
Summary
Background
Gene-based warfarin dosing algorithms have largely been developed in homogeneous populations, and their generalizability has not been established.
Objectives
We sought to assess the performance of published algorithms in a racially diverse and multiethnic sample, and determine if additional clinical variables or genetic variants associated with dose could enhance algorithm performance.
Patients and methods
In 145 compliant patients on warfarin with a goal international normalized ratio (INR) of 2–3, stable, therapeutic doses were compared with predicted doses using 12 reported algorithms that incorporated CYP2C9 and VKORC1 variants. Additional covariates tested with each model included race, concurrent medications, medications known to interact with warfarin and previously described CYP4F2, CALU and GGCX variants.
Results
The mean patient age was 67 ± 14 years; 90 (62%) were male. Eighty-two (57%) were Caucasian, 28 (19%) African-American, 20 (14%) Hispanic and 15 (10%) Asian. The median warfarin dose was 35 mg per week (interquartile range 23–53 mg per week). Gene-based dosing algorithms explained 37–55% of the variation in warfarin dose requirements. Neither the addition of race, number of concurrent medications nor the number of concurrent medications interacting with warfarin enhanced algorithm performance. Similarly, consideration of CYP4F2, CALU or GGCX variant genotypes did not improve algorithms.
Conclusions
Existing gene-based dosing algorithms explained between approximately one-third and one-half of the variability in warfarin dose requirements in this racially and ethnically diverse cohort. Additional clinical and recently described genetic variants associated with warfarin dose did not enhance prediction in our patient population.
doi:10.1111/j.1538-7836.2010.03792.x
PMCID: PMC4441275  PMID: 20128861
CYP2C9; CYP4F2; pharmacogenetics; prediction; VKORC1; warfarin
19.  Endothelial cell specific adhesion molecule (ESAM) localizes to platelet–platelet contacts and regulates thrombus formation in vivo 
Summary
Background
In resting platelets, endothelial cell specific adhesion molecule (ESAM) is located in alpha granules, increasing its cell surface expression following platelet activation. However, the function of ESAM on platelets is unknown.
Objective
To determine whether ESAM has a role in thrombus formation.
Methods and results
We found that following platelet activation ESAM localizes to the junctions between adjacent platelets, suggesting a role for this protein in contact-dependent events that regulate thrombus formation. To test this hypothesis we examined the effect of ESAM deletion on platelet function. In vivo, ESAM−/− mice achieved more stable hemostasis than wild-type mice following tail transection, and developed larger thrombi following laser injury of cremaster muscle arterioles. In vitro, ESAM−/− platelets aggregated at lower concentrations of G protein-dependent agonists than wild-type platelets, and were more resistant to disaggregation. In contrast, agonist-induced calcium mobilization, αIIbβ3 activation, alpha-granule secretion and platelet spreading, were normal in ESAM-deficient platelets. To understand the molecular mechanism by which ESAM regulates platelet activity, we utilized a PDZ domain array to identify the scaffold protein NHERF-1 as an ESAM binding protein, and further demonstrated that it associates with ESAM in both resting and activated platelets.
Conclusions
These findings support a model in which ESAM localizes to platelet contacts following platelet activation in order to limit thrombus growth and stability so that the optimal hemostatic response occurs following vascular injury.
doi:10.1111/j.1538-7836.2009.03606.x
PMCID: PMC4441405  PMID: 19740102
ESAM; NHERF-1; PDZ domain; platelets; thrombosis
20.  Towards standardization of in vivo thrombosis studies in mice 
doi:10.1111/j.1538-7836.2011.04350.x
PMCID: PMC4441407  PMID: 21585649
21.  The Y’s that bind: negative regulators of Src family kinase activity in platelets 
Summary
Members of the Src family of protein tyrosine kinases play important roles in platelet adhesion, activation, and aggregation. The purpose of this review is to summarize current knowledge regarding how Src family kinase activity is regulated in general, to describe what is known about mechanisms underlying SFK activation in platelets, and to discuss platelet proteins that contribute to SFK inactivation, particularly those that use phosphotyrosine-containing sequences to recruit phosphatases and kinases to sites of SFK activity.
doi:10.1111/j.1538-7836.2009.03369.x
PMCID: PMC4423597  PMID: 19630799
blood platelets; phosphorylation; platelet activation; protein tyrosine phosphatases; protein-tyrosine kinases; signal transduction
22.  Recommendations for the use of NOD/SCID mouse model in autoimmune- and drug-induced thrombocytopenia: communication from the SSC of the ISTH 
doi:10.1111/jth.12879
PMCID: PMC4424079  PMID: 25714467
Antibody Specificity; Platelets; Animal model; Thrombocytopenia; Autoimmune Thrombocytopenia
23.  Effects of airborne fine particles (PM2.5) on Deep Vein Thrombosis Admissions in North Eastern United States 
Background
Literature relating air pollution exposure to DVT and pulmonary embolism (PE), in spite of biological plausibility, is sparse. No comprehensive study examining associations between both short and long term exposure to Particulate matter (PM)2.5 and DVT or PE has been published to date. Using a novel PM2.5 prediction model we study whether long and short term PM2.5 exposure is associated with DVT and PE admissions among elderly across the northeastern USA.
Methods
We estimated daily exposure of PM2.5 in each zipcode. We investigated long and short-term effects of PM2.5 on DVT and PE hospital admissions. There were 453,413 DVT and 151,829 PE admissions in the study. For short term exposure, we performed a case crossover analysis matching on month and year and defined the hazard period as lag 01 (exposure of day of admission and previous day). For the long term association, we used a Poisson regression.
Results
A 10-µg/m3 increase in short term exposure was associated with a 0.63 % increase in DVT admissions (95% CI = 0.03 to 1.25) and a 6.98 % (95% CI = 5.65 to 8.33) increase in long term exposure admissions. For PE, the associated risks were 0.38 (95% CI = −0.68 to 1.25) and 2.67 % (95% CI = 5.65 to 8.33). These results persisted when analyses were restricted to location-periods meeting the current EPA annual standard of 12-µg/m3.
Conclusions
Our findings showed that PM2.5 exposure was associated with DVT and PE hospital admissions, and that current standards are not protective of this result.
doi:10.1111/jth.12873
PMCID: PMC4424156  PMID: 25678264
Air Pollution; Public health; Epidemiology; Environment; Venous Thrombosis; Deep-Venous Thrombosis
24.  Coagulation factor XII protease domain crystal structure 
Background
Coagulation factor XII is a serine protease that is important for kinin generation and blood coagulation, cleaving the substrates plasma kallikrein and FXI.
Objective
To investigate FXII zymogen activation and substrate recognition by determining the crystal structure of the FXII protease domain.
Methods and results
A series of recombinant FXII protease constructs were characterized by measurement of cleavage of chromogenic peptide and plasma kallikrein protein substrates. This revealed that the FXII protease construct spanning the light chain has unexpectedly weak proteolytic activity compared to β-FXIIa, which has an additional nine amino acid remnant of the heavy chain present. Consistent with these data, the crystal structure of the light chain protease reveals a zymogen conformation for active site residues Gly193 and Ser195, where the oxyanion hole is absent. The Asp194 side chain salt bridge to Arg73 constitutes an atypical conformation of the 70-loop. In one crystal form, the S1 pocket loops are partially flexible, which is typical of a zymogen. In a second crystal form of the deglycosylated light chain, the S1 pocket loops are ordered, and a short α-helix in the 180-loop of the structure results in an enlarged and distorted S1 pocket with a buried conformation of Asp189, which is critical for P1 Arg substrate recognition. The FXII structures define patches of negative charge surrounding the active site cleft that may be critical for interactions with inhibitors and substrates.
Conclusions
These data provide the first structural basis for understanding FXII substrate recognition and zymogen activation.
doi:10.1111/jth.12849
PMCID: PMC4418343  PMID: 25604127
active site; catalytic domain; factor XII; plasma prokallikrein; tertiary protein structure; zymogens
25.  Factor VIIa binding to endothelial cell protein C receptor protects vascular barrier integrity in vivo 
Summary
Background
Recent studies have shown that factor VIIa binds to endothelial cell protein C receptor (EPCR), a cellular receptor for protein C and activated protein C. At present, the physiologic significance of FVIIa interaction with EPCR in vivo remains unclear. Objective: To investigate whether exogenously administered FVIIa, by binding to EPCR, induces a barrier protective effect in vivo.
Methods
Lipopolysaccharide (LPS)-induced vascular leakage in the lung and kidney, and vascular endothelial growth factor (VEGF)-induced vascular leakage in the skin, were used to evaluate the FVIIa-induced barrier protective effect. Wild-type, EPCR-deficient, EPCR-overexpressing and hemophilia A mice were used in the studies.
Results
Administration of FVIIa reduced LPS-induced vascular leakage in the lung and kidney; the FVIIa-induced barrier protective effect was attenuated in EPCR-deficient mice. The extent of VEGF-induced vascular leakage in the skin was highly dependent on EPCR expression levels. Therapeutic concentrations of FVIIa attenuated VEGF-induced vascular leakage in control mice but not in EPCR-deficient mice. Blockade of FVIIa binding to EPCR with a blocking mAb completely attenuated the FVIIa-induced barrier protective effect. Similarly, administration of protease-activated receptor 1 antagonist blocked the FVIIa-induced barrier protective effect. Hemophilic mice showed increased vascular permeability, and administration of therapeutic concentrations of FVIIa improved barrier integrity in these mice.
Conclusions
This is the first study to demonstrate that FVIIa binding to EPCR leads to a barrier protective effect in vivo. This finding may have clinical relevance, as it indicates additional advantages of using FVIIa in treating hemophilic patients.
PMCID: PMC4085578  PMID: 24977291
activated protein C receptor; capillary permeability; factor VIIa; hemophilia A; protein C

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