Adding MYC/BCL2 double expression to NCCN-IPI may not improve prognostic value to an acceptable level.

BACKGROUND: MYC/BCL2 double expression (DE) is associated with poor prognosis in patients with diffuse large B-cell lymphoma (DLBCL) receiving rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP). This study aimed to determine whether the addition of DE to the National Comprehensive Cancer Network Internal Prognostic Index (NCCN-IPI) could improve the prediction of disease progression in patients with DLBCL treated with R-CHOP. METHODS: This confirmatory prognostic factor study retrospectively recruited patients with newly diagnosed DLBCL between January 1, 2014, and January 31, 2018, at Ramathibodi Hospital (RA) and Thammasat University Hospital (TU). The follow-up period ended on July 1, 2022. Tumors expressing MYC ≥ 40% and BCL2 ≥ 50% were classified as DE. We calculated the hazard ratios (HR) for progression-free survival (PFS) from the date of diagnosis to refractory disease, relapse, or death. Discrimination of the 5-year prediction was based on Cox models using Harrell's concordance index (c-index). RESULTS: A total of 111 patients had DE (39%), NCCN-IPI (8%), and disease progression (46%). The NCCN-IPI adjusted HR of DE was 1.6 (95% confidence interval [CI]: 0.9-2.8; P = 0.117). The baseline NCCN-IPI c-index was 0.63. Adding DE to the NCCN-IPI slightly increased Harrell's concordance index (c-index) to 0.66 (P = 0.119). CONCLUSIONS: Adding DE to the NCCN-IPI may not improve the prognostic value to an acceptable level in resource-limited settings. Multiple independent confirmatory studies from a large cohort of lymphoma registries have provided additional evidence for the clinical utility of DE.

Cytotoxicity and exhaustion markers of chimeric antigen receptor T cells targeting BCMA in multiple myeloma cell lines between patients and healthy donors.

OBJECTIVES: Multiple myeloma (MM) accounts for 10% of hematologic malignancies. However, most of the patients suffered from relapsed/refractory disease. We would like to expand CAR T cell therapy to treat MM using our current platform. METHODS: BCMA CAR T lymphocytes were generated for volunteers or MM patients. The transduction efficiency was detected by the ddPCR technique. Immunophenotyping and exhaustion markers were monitored by flow cytometry. The efficacy of BCMA CAR T cells was tested using coculturing with BCMA CAR or mock, and the positive and negative targets, K562/hBCMA-ECTM and K562, respectively. RESULTS: BCMA CAR T cells were generated from consented volunteers or MM patients and could be detected CAR BCMA expression at a mean of 4.07 ± 1.95 or 4.65 ± 1.21 copies/cell, respectively. Those modified T cells were primarily effector memory T cells. Our BCMA CAR T cells could explicitly eradicate the K562/hBCMA-ECTM cell line while the K562 cell line survived. Interestingly, the BCMA CAR, mock T cells, and peripheral blood mononuclear cells from MM patients expressed similar levels of the exhaustion makers, TIM-3, LAG-3, and PD1. CONCLUSIONS: Our BCMA CAR T cells, mainly effector/effector memory, could eliminate BCMA-expressing cells in vitro and had similar levels of exhaustion markers among different populations.

Incidence of anti-platelet factor4/polyanionic antibodies, thrombocytopenia, and thrombosis after COVID-19 vaccination with ChAdOx1 nCoV-19 in Thais.

BACKGROUND: The prevalence of anti-platelet factor 4 (PF4)/polyanionic antibodies occurring after vaccination with ChAdOx1 nCoV-19 is low. Most of these antibodies are not associated with vaccine-induced thrombotic thrombocytopenia. It remains unknown whether these antibodies are preexisting or occur as a result of vaccination. In this study, we demonstrated the incidence of anti-PF4/polyanionic antibodies, thrombocytopenia, and thrombosis after vaccination with ChAdOx1 nCoV-19 in a large cohort of Thais. METHODS: We conducted a prospective study in a cohort of health care workers and members of the general population who received COVID-19 vaccination with ChAdOx1 nCoV-19. Blood collection for complete blood count, D-dimer, and anti-PF4/polyanionic antibodies was performed before vaccination (day 0), day 10, and day 28 after vaccination. Anti-PF4/polyanionic antibodies were detected using enzyme-link immunosorbent assay (ELISA). Functional assay was performed for all positive ELISA tests. RESULTS: A total of 720 participants were included in the study. 214 participants received both the first and second doses, 91 participants received only the first, 51 received only the second, and 364 received the third booster dose of ChAdOx1 nCoV-19. Median age was 42 years (IQR, 34-53). 67% of participants were female. Three participants developed seroconversion, yielding an incidence of vaccination-induced anti-PF4/polyanionic antibodies of 0.42% (95% confidence interval 0.08, 1.23). Fourteen (1.9%) participants had preexisting anti-PF4/polyanionic antibodies before the vaccination but their optical density of anti-PF4/polyanionic antibodies did not significantly increase over time. None of the anti-PF4/polyanionic positive sera induced platelet aggregation. Abnormal D-dimer levels following vaccination were not different among the positive and negative anti-PF4/polyanionic groups (11.8% vs. 13.2%, p = 0.86). Thrombocytopenia occurred in one person with negative anti-PF4/polyanionic antibodies. No clinical thrombosis or bleeding occurred. CONCLUSION: We found a low incidence of seroconversion of anti-PF4/polyanionic antibodies after vaccination with ChAdOx1 nCoV-19 in Thais. Most of the anti-PF4/polyanionic antibodies were preexisting and did not significantly increase after vaccination with ChAdOx1 nCoV-19. Following vaccination, some participants with anti-PF4/polyanionic antibodies had elevated D-dimer levels, while only one developed thrombocytopenia and no thrombotic events were observed.

External validation and comparison of IPI, R-IPI, and NCCN-IPI in diffuse large B-cell lymphoma patients treated with R-CHOP to predict 2-year progression-free survival.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive lymphoma. The standard first-line therapy for DLBCL consists of rituximab cyclophosphamide doxorubicin vincristine and prednisone (R-CHOP). About 50-70% of patients may be cured by R-CHOP. There was no data on external validation and comparison of the international prognostic index, revised-IPI (R-IPI), and enhanced-IPI (NCCN-IPI) to predict treatment outcomes in the middle-income country with a resourced-limited setting. OBJECTIVES: We aimed to externally validate and compare IPI, R-IPI, and NCCN-IPI in predicting 2-year progression-free survival (2-y PFS) of newly diagnosed DLBCL patients treated with R-CHOP. METHODS: This ambispective observational study recruited consecutive patients diagnosed between 1 January 2014 and 30 June 2020, with the last follow-up on 1 July 2022 from Thammasat University Hospital and Ramathibodi Hospital. We assessed discrimination by Harrell's concordance index (c-index), calibration by calibration plot, and absolute difference in survival (ADS) between the lowest-and the highest-risk groups. RESULTS: The cohort of 292 patients (median age 63 years and median follow-up 3.6 years) had 131 progressions and 96 deaths. The 2-y PFS was 63%. The c-indices were NCCN-IPI 0.6216, R-IPI 0.6004 (P = 0.215), and IPI 0.6104 (P = 0.463). The calibration plots of NCCN-IPI and R-IPI showed nearly perfect agreement (moderate strength), while IPI had miscalibrations. The ADSs were NCCN-IPI 52%, R-IPI 42%, and IPI 25%. CONCLUSION: NCCN-IPI is the best prognostic index compared to IPI and R-IPI in prior studies. However, the prognostic model for DLBCL patients treated with R-CHOP requires updating or integrating biomarkers to improve discrimination to the acceptable level (c-index 0.7).

A 46-Year-Old Thai Woman with Secondary Acquired Pure Red Cell Aplasia Due to Treatment with Recombinant Erythropoietin While on Dialysis for End-Stage Renal Disease Who Recovered Following ABO-Incompatible Kidney Transplantation.

BACKGROUND Pure red cell aplasia (PRCA) is an uncommon cause of anemia in end-stage kidney disease (ESKD). It is attributed to recombinant human erythropoietin (rHuEPO) administration. Although immunosuppression is the mainstay therapy, its effectiveness varies from 30% to 70%. PRCA in ESKD has been reported to improve following kidney transplantation. CASE REPORT A 46-year-old woman with ESKD secondary to lupus nephritis was treated for uremia at our center. She developed severe anemia. Bone marrow aspiration and biopsy revealed a reduction of erythroid precursors, consistent with PRCA. Because she had no sibling's blood group matched with her, ABO-incompatible kidney transplantation was an option for treatment. She underwent a desensitization protocol consisting of rituximab 375 mg/m2, tacrolimus, mycophenolate mofetil, and prednisolone 4 weeks before surgery, in addition to 3 sessions of double-filtration plasmapheresis (DFPP) every other day followed by intravenous immunoglobulin (IVIG) and 1 session of specific immunoadsorption (Glycosorb® B column) at pre-transplant day -1. She also received low-dose rabbit anti-thymocyte globulin (rATG) (Thymoglobulin®) (total 2.0 mg/kg). Maintenance therapy included tacrolimus, mycophenolate mofetil, and prednisolone. Allograft function normalized a few days after transplantation and her Hb gradually increased. CONCLUSIONS We report a rare case of PRCA in a patient with ESKD undergoing ABO-incompatible kidney transplantation. The outcome was satisfactory, with complete correction of anemia and kidney function.

Treatment Outcomes and Clinical Relevance in Patients with Double Expressor DLBCL.

BACKGROUND: Double-expressor lymphoma (DEL) was found to account for 20-30% of DLBCL. We conducted this study to analyze the survival, the clinical presentation, and the factors associated with treatment outcomes in DEL-DLBCL. METHODS: A retrospective study of 291 patients diagnosed with DLBCL during January 2015 - December 2018 was conducted. RESULTS: Of the 291 patients, the median age was 63 years, germinal center B cell-like DLBCL (GCB) and non-GCB subtypes were found in 32% and 68%, respectively. DEL was found in 46% of 264 patients with available immunohistochemistry staining for MYC protein. Patients with DEL was significantly more common in elderly patients (p= 0.017) and non-GCB subtype (p= 0.006). High serum lactate dehydrogenase (LDH) levels and high Ki-67 index were significantly found in DEL patients than non-DEL patients (p= 0.024 and p= 0.04, respectively). The 3y-OS rate was shorter in the DEL group than in the non-DEL group, 58.7% versus 78.9% (p=0.026), whereas no significant difference in 3y-DFS was identified between these groups (58.4% versus 67.7%, p= 0.343). Independent factors affecting OS and DFS in DEL patients were ECOG 3-4, high LDH levels, extranodal involvement> 1 site, high IPI, and stage III-IV in univariate analysis. CONCLUSIONS: High incidence of DEL was observed in this study, especially in patients aged 60 years or older and non-GCB subtype. Patients with DEL showed dismal DFS and OS.

Prevalence of anti-platelet factor 4/polyanionic antibodies after COVID-19 vaccination with ChAdOx1 nCoV-19 and CoronaVac in Thais.

INTRODUCTION: Vaccine-induced thrombotic thrombocytopenia (VITT) has been reported after vaccination with the adenoviral vector coronavirus disease 2019 (COVID-19) vaccine ChAdOx1 nCoV-19 in European countries. To date, two cases of VITT have been reported in Thais after COVID-19 vaccination. We determined the frequency of anti-platelet factor 4 (PF4)/polyanionic antibodies in the Thai population receiving the COVID-19 vaccines. METHODS: We conducted a cross-sectional study to evaluate the prevalence of anti-PF4/polyanionic antibodies in health care workers who received COVID-19 vaccination with ChAdOx1 nCoV-19 or CoronaVac within 7 to 35 days. A control population who had not been vaccinated was also included. Anti-PF4/polyanionic antibodies were detected using ELISA. Functional assay with platelet aggregation was performed for all positive anti-PF4/polyanionic antibody ELISA tests. RESULTS: A total of 646 participants were included in the study; 221 received ChAdOx1 nCoV-19, 232 received CoronaVac, and 193 participants were in the control group. The prevalence of anti-PF4 antibodies was 2.3% (95% confidence interval [CI], 0.7-5.2), 1.7% (95% CI, 0.5-4.4) in the ChAdOx1 nCoV-19 and CoronaVac groups, respectively. There was no positive test in the control group. None of the PF4/polyanionic positive sera induced platelet aggregation. CONCLUSION: We found a low prevalence of anti-PF4 antibodies in Thais after vaccination with ChAdOx1 nCoV-19 and CoronaVac. None of the antibodies were functional and lacked an association with VITT.

Incidence of thromboembolism in patients with COVID-19: a systematic review and meta-analysis.

BACKGROUND: Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, the incidence of thromboembolism has been increasingly reported. The aim of this systematic review was to explore the incidence of venous and arterial thromboembolism among COVID-19 patients requiring hospitalization. METHODS: Medline, Embase, Scopus, and grey literature were searched until June 2020. Observational studies reported on the incidence of venous thromboembolism (VTE), including pulmonary embolism (PE) and deep vein thrombosis (DVT) or arterial thromboembolism (ATE) were included. The pool incidences and their 95% confidence intervals (CI) were calculated using the random-effects model. RESULTS: A total of 36 studies were included. In the intensive care unit (ICU) setting, the pooled incidence of VTE was 28% (95% CI, 22-34%). Subgroups based on compression ultrasound (CUS) screening revealed a higher incidence of DVT in the CUS screening group than in the no CUS screening group (32% [95% CI, 18-45%] vs. 6% [95% CI, 4-9%]). The pooled incidence of ATE in ICU was 3% (95% CI, 2-5%). In the non-ICU setting, the pooled incidence of VTE was 10% (95% CI, 6-14%,). CONCLUSIONS: The incidence of VTE in COVID-19 patients was higher in the ICU setting than in the non-ICU setting, and also significantly higher in studies that incorporated the CUS screening protocol. The incidence of ATE in the ICU setting was low. VTE prophylactic measures should be given to all hospitalized patients diagnosed with COVID-19.

Left ventricular thrombus and pulmonary embolism: A case series of thrombosis in COVID-19 in Thai patients.

Thrombosis in COVID-19 is increasingly recognized and is generally associated with a high mortality rate. The key clinical question of this report was whether COVID-19 could be complicated with cardiac thrombus and pulmonary embolism in Asian population. We demonstrated the case series of thrombosis in Thai patients with confirmed severe acute respiratory syndrome coronavirus 2 infection. One patient had the first case of a large left ventricular thrombus, and three other patients had pulmonary embolism. All patients were male and had low absolute lymphocyte count, while lactate dehydrogenase level and d-dimer were markedly elevated, especially at the time when the thrombosis was diagnosed. All patients had severe COVID-19 with pneumonia. Two patients who needed mechanical ventilation were successfully extubated. After hospitalization for 13-49 days, pneumonia and thrombosis improved and all of them could be discharged from the hospital. Thrombosis is common in COVID-19 and could present in both arterial and venous sites even in Asian populations. d-dimer is a strong marker to predict thrombosis and could be a prognostic predictor for severity of COVID-19.

Prognostic Value of Concurrent Expression of C-MYC and BCL2 in Intravascular Large B-Cell Lymphoma: A 10-Year Retrospective Study.

BACKGROUND: Intravascular large B-cell lymphoma (IVLBCL) is a variant of extranodal diffuse large B-cell lymphoma (DLBCL), characterized by the presence of a B-lymphoma cell in the lumina of small blood vessels or capillaries. Due to its extremely variable clinical manifestations, IVLBCL typically results in a delayed diagnosis and poor disease prognosis. Skin biopsy, particularly random skin biopsy, has shown a potential role in the diagnosis of IVLBCL. However, information of clinicopathological features in patients with IVLBCL diagnosed by skin biopsy is limited. OBJECTIVES: To study the clinicopathological features in relation to immunohistochemical features and to identify prognostic factors in IVLBCL patients diagnosed by skin biopsy. MATERIALS AND METHODS: Clinical characteristics; laboratory, histological, and immunohistochemical findings; and therapeutic response of all biopsy-confirmed IVLBCL patients during the years 2008-2017 were retrospectively reviewed. RESULTS: The mean age was 67.4 (±9.8) years. Fever was the most common presenting symptom, accounting for 64.7%. Cutaneous and bone marrow involvement was found in 23.5% and 35.3% of patients, respectively. Patients receiving R-CHOP showed more favorable therapeutic outcome. C-MYC/BCL2 double expressors showed significantly higher incidence rate to mortality compared with nondouble expressors (p = 0.042). One-year and two-year overall survival rates were 67.2% and 53.8%, respectively. CONCLUSIONS: Skin biopsy is an effective diagnostic method for IVLBCL. Concurrent expression of C-MYC and BCL2 may be a useful prognostic indicator and should be performed in order to predict the prognosis in IVLBCL patients.

Etiologies and Treatment Burden in Adult Patients with Pure Red Cell Aplasia: A Single-Center Experience and Review of Literature.

BACKGROUND: Pure red cell aplasia (PRCA) is less common blood disorder; the causes and the treatments of PRCA are varied. METHODS: We conducted a retrospective study during January 2010-December 2017, to explore the etiologies and to evaluate the response and treatment burden in adult patients with PRCA. RESULTS: Of 32 PRCA patients, median age was 57 years (18-90 years). Median hemoglobin level and reticulocyte count at the time of diagnosis were 5.6 g/dL (3.3-7.3 g/dL) and 0.3% (0.1-0.7%), respectively. Median time to hematologic recovery was 12 weeks (3-72 weeks), and median number of red blood cell transfusion (RBC) was 20 units (4-100 units). Causes of PRCA were erythropoiesis-stimulating agent (ESA) (47%), parvovirus B19 infection (19%), thymoma (13%), zidovudine (6%), primary autoimmune PRCA (6%), Kaposi's sarcoma (3%), systemic lupus erythematosus (3%), and ABO-mismatched stem cell transplantation (3%). Only 9 out of 24 treated patients achieved hematologic response within 8 weeks of treatment. Intravenous immunoglobulin therapy provided 100% response rate in patients with parvovirus B19-associated PRCA and primary autoimmune PRCA. Low response rate was found in patients receiving immunosuppressants and chemotherapy for the treatment of ESA and thymoma-associated PRCA, respectively. CONCLUSIONS: Treatment outcome of PRCA depended upon the causes and the types of treatment, and the burden of RBC transfusion was very high in patients with ESA and thymoma-associated PRCA.

Treatment outcome and prognostic factors in PCNSL.

OBJECTIVES: Standard treatment with a thiotepa-based regimen in countries with a limited resource is less feasible. Aims of the study were to evaluate the treatment outcome, and identify the prognostic factors in patients with primary central nervous system lymphoma (PCNSL). METHODS: We conducted a retrospective study of 43 patients diagnosed with PCNSL, DLBCL subtype, who were treated with either HDMTX-based regimen, whole brain radiotherapy (WBRT), or both between 2010 and 2017. RESULTS: There were 43 patients with a median age of 65 years (range 34-89 years). Protein expression of CD10, Bcl6, MUM1, Bcl2 and MYC were found in 19, 86, 91, 91 and 23%, respectively. Both germinal center B cell (GCB) and double-expressor (MYC+/Bcl2+) lymphomas were found in 21%. Multiple brain lesions and maximum tumor diameter (MTD) ≥5 cm were seen in 27 and 10 patients, respectively. Chemotherapy combined with WBRT, chemotherapy and WBRT were given to 20, 14 and 9 patients, respectively. Overall complete remission (CR) rate was 55.8%. Those receiving a combined-modality therapy had a higher CR rate than those treated with either chemotherapy (75% versus 36%, p = 0.036) or WBRT (75% versus 44%, p = 0.109). Median follow-up time was 17 months, and a 7-year overall survival (OS) was 40%. Features associated with a prolonged OS were an ECOG score ≤ 2 (p = 0.001), multiple brain lesions (p = 0.010), multiple area of brain involvement (p = 0.023), MTD < 5 cm (p = 0.004), GCB subtype (p = 0.003) and positive CD10 staining (p = 0.007). Expression of Bcl2 protein was associated with a significantly worse OS in the non-GCB DLBCL patients. DISCUSSION: The factors affecting treatment outcomes in PCNSL were cell of origin of DLBCL, lesion characteristics, patients' status and treatment regimen.

Cytogenetics and FLT3-ITD mutation predict clinical outcomes in non transplant patients with acute myeloid leukemia.

BACKGROUND: Cytogenetic abnormalities and mutated genes indicate the role of consolidation therapy with hematopoietic stem cell transplantation (HSCT) or chemotherapy in acute myeloid leukemia (AML). In this study, we conducted a retrospective study in adult AML patients with newly diagnosed with de novo AML who did not undergo HSCT, to study long term relapse free survival (RFS) and overall survival (OS) after consolidation chemotherapy. METHODS: We recruited 141 consecutive AML patients during January 2010-June 2017, the patients received induction chemotherapy with standard dose Ara-C and Idarubicin (7 + 3 or 5 + 2 regimen) followed by intermediate (IDAC) or high dose Ara-c (HiDAC) consolidation therapy. RESULTS: Normal karyotype, complex, favorable, intermediate and adverse chromosomal aberrations were found in 59%, 16%, 5%, 14% and 6%, respectively. Mutated NPM1, FLT3-ITD and CEBPA genes in CN-AML were seen in 33%, 18% and 19%, respectively. A 5 year follow up, 5y-RFS was 16% and 5y-OS was 14% in the whole study population. 5y-RFS and 5y-OS in patients completed 4 cycles of consolidation therapy were 25% and 40%, respectively. Adverse cytogenetic risk and mutated FLT3-ITD were significantly associated with poor RFS (9 and 15 months, respectively) and OS (14 and 16 months, respectively), whereas patients with mutant NPM1 had favorable outcomes (RFS/OS = 51/63 months). Patients receiving 4 cycles of consolidation therapy had significantly impacts on median RFS and OS compared with those treated with 1 or 2 cycles; 15 versus 11 months (p = 0.006) and 31 versus 15 months (p < 0.001), respectively. CONCLUSIONS: Cytogenetic and mutation tests for FLT3-ITD, NPM1 and CEBPA genes were meaningful for predicting outcomes in adult AML patients. Adverse cytogenetic abnormalities and FLT3-ITD mutation showed dismal RFS and OS.

Unusual manifestation of vitamin A deficiency presenting with generalized xerosis without night blindness.

Vitamin A deficiency from malabsorption syndromes, including bariatric surgery, has become an emerging problem in developed countries. Early detection and prompt treatment lead to rapid and complete recovery. Nevertheless, it may result in irreversible blindness or death if left untreated. Health care personnel should be aware of this condition.

Excess of heme induces tissue factor-dependent activation of coagulation in mice.

An excess of free heme is present in the blood during many types of hemolytic anemia. This has been linked to organ damage caused by heme-mediated oxidative stress and vascular inflammation. We investigated the mechanism of heme-induced coagulation activation in vivo. Heme caused coagulation activation in wild-type mice that was attenuated by an anti-tissue factor antibody and in mice expressing low levels of tissue factor. In contrast, neither factor XI deletion nor inhibition of factor XIIa-mediated factor XI activation reduced heme-induced coagulation activation, suggesting that the intrinsic coagulation pathway is not involved. We investigated the source of tissue factor in heme-induced coagulation activation. Heme increased the procoagulant activity of mouse macrophages and human PBMCs. Tissue factor-positive staining was observed on leukocytes isolated from the blood of heme-treated mice but not on endothelial cells in the lungs. Furthermore, heme increased vascular permeability in the mouse lungs, kidney and heart. Deletion of tissue factor from either myeloid cells, hematopoietic or endothelial cells, or inhibition of tissue factor expressed by non-hematopoietic cells did not reduce heme-induced coagulation activation. However, heme-induced activation of coagulation was abolished when both non-hematopoietic and hematopoietic cell tissue factor was inhibited. Finally, we demonstrated that coagulation activation was partially attenuated in sickle cell mice treated with recombinant hemopexin to neutralize free heme. Our results indicate that heme promotes tissue factor-dependent coagulation activation and induces tissue factor expression on leukocytes in vivo. We also demonstrated that free heme may contribute to thrombin generation in a mouse model of sickle cell disease.

Differential contribution of FXa and thrombin to vascular inflammation in a mouse model of sickle cell disease.

Activation of coagulation and vascular inflammation are prominent features of sickle cell disease (SCD). Previously, we have shown that inhibition of tissue factor (TF) attenuates activation of coagulation and vascular inflammation in mouse models of SCD. In this study, we examined the mechanism by which coagulation proteases enhance vascular inflammation in sickle BERK mice. To specifically investigate the contribution of FXa and thrombin, mice were fed chow containing either rivaroxaban or dabigatran, respectively. In addition, we used bone marrow transplantation to generate sickle mice deficient in either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) on nonhematopoietic cells. FXa inhibition and PAR-2 deficiency in nonhematopoietic cells attenuated systemic inflammation, measured by plasma levels of interleukin-6 (IL-6). In contrast, neither thrombin inhibition nor PAR-1 deficiency in nonhematopoietic cells affected plasma levels of IL-6 in sickle mice. However, thrombin did contribute to neutrophil infiltration in the lung, independently of PAR-1 expressed by nonhematopoietic cells. Furthermore, the TF-dependent increase in plasma levels of soluble vascular cell adhesion molecule-1 in sickle mice was not mediated by FXa or thrombin. Our data indicate that TF, FXa, and thrombin differentially contribute to vascular inflammation in a mouse model of SCD.

Tissue factor promotes activation of coagulation and inflammation in a mouse model of sickle cell disease.

Sickle cell disease (SCD) is associated with a complex vascular pathophysiology that includes activation of coagulation and inflammation. However, the crosstalk between these 2 systems in SCD has not been investigated. Here, we examined the role of tissue factor (TF) in the activation of coagulation and inflammation in 2 different mouse models of SCD (BERK and Townes). Leukocytes isolated from BERK mice expressed TF protein and had increased TF activity compared with control mice. We found that an inhibitory anti-TF antibody abrogated the activation of coagulation but had no effect on hemolysis or anemia. Importantly, inhibition of TF also attenuated inflammation and endothelial cell injury as demonstrated by reduced plasma levels of IL-6, serum amyloid P, and soluble vascular cell adhesion molecule-1. In addition, we found decreased levels of the chemokines MCP-1 and KC, as well as myeloperoxidase in the lungs of sickle cell mice treated with the anti-TF antibody. Finally, we found that endothelial cell-specific deletion of TF had no effect on coagulation but selectively attenuated plasma levels of IL-6. Our data indicate that different cellular sources of TF contribute to activation of coagulation, vascular inflammation, and endothelial cell injury. Furthermore, it appears that TF contributes to these processes without affecting intravascular hemolysis.

Studies on the mechanism of action of the aptamer BAX499, an inhibitor of tissue factor pathway inhibitor.

INTRODUCTION: Promoting thrombin generation by inhibiting tissue factor pathway inhibitor (TFPI) is a potentially viable therapeutic approach to the prevention and/or treatment of bleeding in hemophilia. In this report, we studied the interaction between an aptamer (BAX499; formerly ARC19499) and TFPI that resulted in inhibition of TFPI-mediated regulation of the tissue factor pathway. MATERIALS AND METHODS: Enzyme kinetic analyses were performed to study the interaction between BAX499 and recombinant TFPI against factor Xa, the extrinsic Xase and prothrombinase activities. Diluted prothrombin time assay was used to investigate the effects of BAX499 on factor VIII-deficient plasma collected from hemophilia patients. RESULTS: Our results indicate that after binding of BAX499 to TFPI, the TFPI/ BAX499 complex retains factor Xa inhibitory activity, albeit with reduced affinity. When tested in an extrinsic Xase activity assay, BAX499 delayed TFPI-mediated inhibition of extrinsic Xase activity. In addition, BAX499 reversed TFPI inhibition of the prothrombinase complex. BAX499 shortened the dilute prothrombin time in factor VIII-deficient plasma, and when added to freshly drawn hemophilia A blood either with or without a factor VIII inhibitor, the whole blood clotting time was also shortened. These results suggest that BAX499 may be a useful addition to the armamentarium of bypassing agents to control bleeding in hemophilic patients with inhibitors.