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COVID-19 convalescent plasma (CCP) was approved under emergency authorization to treat critically ill patients with COVID-19 in the United States in 2020. We explored the demographics of donors contributing plasma for a hyperimmune, plasma-derived therapy to evaluate factors that may be associated with anti-SARS-CoV-2 antibody response variability and, subsequently, antibody titers.An electronic search of CCP donors was performed across 282 US plasma donation centers. Donations were screened for nucleocapsid protein-binding-IgG using the Abbott SARS-CoV-2 IgG assay.Overall, 52 240 donors donated 418 046 units of CCP. Donors were of various ethnicities: 43% Caucasian, 34% Hispanic, 17% African American, 2% Native American, 1% Asian, and 3% other. Females had higher initial mean anti-SARS-CoV-2 antibody titers but an overall faster rate of decline (P .0001). Initial antibody titers increased with age: individuals aged 55 to 66 years had elevated anti-SARS-CoV-2 titers for longer periods compared with other ages (P = .0004). African American donors had the lowest initial antibody titers but a slower rate of decline (P .0001), while Caucasian (P = .0088) and Hispanic (P = .0193) groups had the fastest rates of decline. Most donor antibody levels decreased below the inclusion criteria (≥1.50) within 30 to 100 days of first donation, but donation frequency did not appear to be associated with rate of decline.Several factors may be associated with anti-SARS-CoV-2 antibody response including donor age and sex. Evaluating these factors during development of future hyperimmune globulin products may help generation of therapies with optimal efficacy.

BACKGROUND Plasma contains many important proteins of therapeutic interest including albumin, clotting factors, and antibodies. Source plasma (SP) is in great demand particularly due to a shortage of immunoglobulin. To better understand how to increase supply, we examined SP donor deferrals for the previous 3 years. STUDY DESIGN This is a description of donor deferrals at 255 plasma donation centers in the United States for April 1, 2017 to March 31, 2020. RESULTS A total of 4 587 923 events were evaluated for the 3-year period 2017-2020. There were 873 227 deferrals analyzed for 2017-2018, 1 765 582 in 2018-2019, and 1 949 114 for 2019-2020. The most common deferral each year was for unacceptable blood pressure (BP) or pulse which comprised 27.9%, 28.2%, and 28.3% of deferrals in 2017-2018, 2018-2019, and 2019-2020, respectively. The second most common cause of deferral was for unacceptable hematocrit which comprised 14.1% of deferrals in 2017-2018, and 16.0% in 2018-2019 and 2019-2020. The majority of these deferred donors had low hematocrits and were predominately (~80%) female. Deferral for unacceptable total protein comprised a smaller percentage (~4%) of deferrals. DISCUSSION Most donor deferrals were due to unacceptable screening results, particularly high BP, elevated pulse, low protein, and low hematocrit. Although rates of deferrals in other categories have been slightly increasing over time, they comprise a small percentage. Donor education regarding healthy lifestyle choices may improve overall donor health, decrease deferrals, and increase SP supply.

Coagulation factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (GLA) domain, two epidermal growth factor-like (EGF) domains and a protease domain. FVIIa binds three Mg2+ ions and four Ca2+ ions in the GLA domain, one Ca2+ ion in the EGF1 domain and one Ca2+ ion in the protease domain. Further, FVIIa contains an Na+ site in the protease domain. Since Na+ and water share the same number of electrons, Na+ sites in proteins are difficult to distinguish from waters in X-ray structures. Here, to verify the Na+ site in FVIIa, the structure of the FVIIa–soluble tissue factor (TF) complex was solved at 1.8 Å resolution containing Mg2+, Ca2+ and Rb+ ions. In this structure, Rb+ replaced two Ca2+ sites in the GLA domain and occupied three non-metal sites in the protease domain. However, Rb+ was not detected at the expected Na+ site. In kinetic experiments, Na+ increased the amidolytic activity of FVIIa towards the synthetic substrate S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide) by ∼20-fold; however, in the presence of Ca2+, Na+ had a negligible effect. Ca2+ increased the hydrolytic activity of FVIIa towards S-2288 by ∼60-fold in the absence of Na+ and by ∼82-fold in the presence of Na+. In molecular-dynamics simulations, Na+ stabilized the two Na+-binding loops (the 184-loop and 220-loop) and the TF-binding region spanning residues 163–180. Ca2+ stabilized the Ca2+-binding loop (the 70-loop) and Na+-binding loops but not the TF-binding region. Na+ and Ca2+ together stabilized both the Na+-binding and Ca2+-binding loops and the TF-binding region. Previously, Rb+ has been used to define the Na+ site in thrombin; however, it was unsuccessful in detecting the Na+ site in FVIIa. A conceivable explanation for this observation is provided.

Importance Neuromyelitis optica/neuromyelitis optica spectrum disorder patients' response to therapeutic plasma exchange (TPE) is currently incompletely characterized. Objective Our study aims to understand the clinical status improvement of neuromyelitis optica/neuromyelitis optica spectrum disorder patients treated with TPE. Design, setting, and participants This is a multicenter retrospective study conducted between 1 January 2003 and 31 July 2017 at 13 US hospitals performing apheresis procedures. Subjects studied were diagnosed with neuromyelitis optica/neuromyelitis optica spectrum disorder who received TPE during presentation with acute disease. Main outcomes and measures The primary outcome was clinical status improvement in patients treated with TPE. Secondary measures were procedural and patient characteristics associated with response to treatment. Results We evaluated 114 patients from 13 institutions. There was a female predilection. The largest ethnic group affected was non-Hispanic Caucasian. The average age of diagnosis was 43.1 years. The average time to diagnosis was 3.1 years. On average, five procedures were performed during each treatment series. The most commonly performed plasma volume exchange was 1.0 to 1.25 using 5% albumin as replacement fluid. Most patients (52%) did not require an additional course of TPE and noted "mild" to "moderate" clinical status improvement. Maximal symptom improvement appeared by the fourth or fifth TPE treatment. Conclusion and relevance TPE improved the clinical status of patients. Adults responded more favorably than children. Procedural characteristics, including number of TPEs, plasma volume exchanged, and replacement fluid used, were similar between institutions. TPE was well-tolerated and had a low severe adverse event profile.

Essentials Consensus sequence and biochemical data suggest a Na+ -site in the factor (F) IXa protease domain. X-ray structure of the FIXa EGF2/protease domain at 1.37 A reveals a Na+ -site not observed earlier. Molecular dynamics simulations data support that Na+ ± Ca2+ promote FIXa protease domain stability. Sulfate ions found in the protease domain mimic heparin sulfate binding mode in FIXa. SUMMARY: Background Activated coagulation factor IX (FIXa) consists of a γ-carboxyglutamic acid domain, two epidermal growth factor-like (EGF) domains, and a C-terminal protease domain. Consensus sequence and biochemical data support the existence of a Na+ -site in the FIXa protease domain. However, soaking experiments or crystals grown in high concentration of ammonium sulfate did not reveal a Na+ -site in wild-type or mutant FIXa EGF2/protease domain structure. Objective Determine the structure of the FIXa EGF2/protease domain in the presence of Na+ ; perform molecular dynamics (MD) simulations to explore the role of Na+ in stabilizing FIXa structure. Methods Crystallography, MD simulations, and modeling heparin binding to FIXa. Results Crystal structure at 1.37-A resolution revealed that Na+ is coordinated to carbonyl groups of residues 184A, 185, 221A, and 224 in the FIXa protease domain. The Na+ -site in FIXa is similar to that of FXa and is linked to the Asp189 S1-site. In MD simulations, Na+ reduced fluctuations in residues 217-225 (Na+ -loop) and 70-80 (Ca2+ -loop), whereas Ca2+ reduced fluctuations only in residues of the Ca2+ -loop. Ca2+ and Na+ together reduced fluctuations in residues of the Ca2+ -loop and Na+ -loop (residues 70-80, 183-194, and 217-225). Moreover, we observed four sulfate ions that make salt bridges with FIXa protease domain Arg/Lys residues, which have been implicated in heparin binding. Based upon locations of the sulfate ions, we modeled heparin binding to FIXa, which is similar to the heparin binding in thrombin. Conclusions The FIXa Na+ -site in association with Ca2+ contributes to stabilization of the FIXa protease domain. The heparin binding mode in FIXa is similar to that in thrombin.

The main clinical distinction between post-transfusion purpura (PTP) and idiopathic thrombocytopenic purpura (ITP) is the sudden development of severe thrombocytopenia in the days after transfusion. Herein, we report the case of a 53-year-old Caucasian woman who developed multiple myeloma (MM) after peripheral blood-stem-cell transplant (PBSCT), along with severe thrombocytopenia (with a nadir of 1 × 109/L); she also experienced severe adverse events after each platelet transfusion, including the first one. These reactions were absent with any other transfused blood products. The results of an human leukocyte antigen (HLA) class-1 panel reactive antibody assay were 0%, and the results of a platelet-antibody screening assay were positive for HLA class-1 antibodies and glycoprotein (Gp)IIb/IIIa antibodies. Her platelet count reached 42 × 109 per L on day 50, after rituximab on day 22 and daratumumab on day 29. Her clinical scenario was most consistent with the course of PTP.

Background Factor (F) IX/IXa inactivation by plasmin has been studied; however, whether plasmin converts FIXa to a fibrinolytic enhancer is not known. Objective Investigate plasmin proteolysis site(s) in FIXa that inactivates and transforms it into a fibrinolytic enhancer. Methods NH2 -terminal sequencing, mass spectrometry analysis, and functional assays. Results Plasmin in the presence of Ca2+ /phospholipid (PL) rapidly cleaved FIXaβ at Lys316↓Gly317 to yield FIXaγ followed by a slow cleavage at Lys413↓Leu414 to yield FIXaδ. FIXaγ/FIXaδ migrated indistinguishably from FIXaβ in nondenaturing gel system indicating that C-terminal residues 317-415/317-413 of heavy chain remain noncovalently associated with FIXaγ/FIXaδ. However, as compared with FIXaβ, FIXaγ or FIXaγ/FIXaδ (25-75 mixture, 8-hour/24-hour incubation analysis by mass spectrometry) was impaired ~ 10-fold in hydrolyzing synthetic substrate CBS 31.39 (CH3-SO2-D-Leu-Gly-Arg-pNA), ~ 30-fold (~ 5-fold higher Km , ~ 6-fold lower kcat ) in activating FX in a system containing Ca2+ /PL, and ~ 650-fold in a system containing Ca2+ /PL and FVIIIa. Further, FIXaγ or FIXaγ/FIXaδ bound FVIIIa with ~ 60-fold reduced affinity compared with FIXaβ. Additionally, in ligand blots, plasminogen or diisopropylfluorophosphate-inhibited plasmin (DIP-plasmin) bound FIXaγ and FIXaδ but not FIXaβ. This interaction was prevented by e-aminocaproic acid or carboxypeptidase B treatment suggesting that plasminogen/DIP-plasmin binds to FIXaγ/FIXaδ through newly generated C-terminal Lys316 and Lys413. Importantly, FIXaγ/FIXaδ mixture but not FIXaγ enhanced tissue plasminogen activator (tPA)-mediated plasminogen activation in a concentration dependent manner. Similarly, FIXaγ/FIXaδ mixture but not FIXaγ enhanced tPA-induced clot lysis in FIX-depleted plasma. Conclusion Plasmin cleavage at Lys316↓Gly317 abrogates FIXaβ coagulant activity, whereas additional cleavage at Lys413↓Leu414 converts it into a fibrinolytic enhancer.

Platelet-transfusion refractoriness (PTR) is common in patients with hematological malignancies. The etiology of immune PTR is typically human leukocyte antigen (HLA) antibodies (Abs) from pregnancy or previous transfusion. Herein, we report PTR in the setting of induction chemotherapy for acute myelogenous leukemia (AML) from Abs against CD36/glycoprotein (GP)IV. A 66-year-old African American woman presented with anemia and thrombocytopenia. She was found to have transfusion-dependent AML, and a 7 + 3 regimen (7 days of standard-dose cytarabine and 3 days of an anthracycline antibiotic or an anthracenedione, most often daunorubicin) was initiated. The patient developed profound thrombocytopenia, with platelet nadir of 0 by day 13. The results of HLA antibody screening were negative. However, the results of a screening test for platelet-specific antibodies screen showed Abs against cluster of differentiation (CD)36. The platelets of the patient lacked expression of CD36, and DNA analysis showed mutations in the CD36 gene. HLA Ab–mediated PTR is common in patients with hematological malignancies. However, once HLA Abs are excluded, other less-frequent Abs should be considered, particularly in patient populations of Asian, African, or Middle Eastern descent.

INTRODUCTION Performing therapeutic plasma exchange (TPE) with albumin replacement decreases coagulation factor and platelet levels. No defined guidelines exist regarding laboratory testing to assess hemostasis in patients undergoing TPE. MATERIALS AND METHODS A survey to evaluate hemostasis testing with TPE was distributed using online survey software. One response per institution was analyzed based on a hierarchical algorithm, excluding membrane filtration users, resulting in a maximum of 120 respondents per question. Descriptive analysis was performed with results reported as the number and/or frequency (%) of respondents to each question. RESULTS The practices represented vary by institution type, number of apheresis procedures per year, and performance of TPE on children. Prior to TPE planned with albumin replacement, many respondents obtain laboratory studies for almost all patients (54.9% outpatients and 68.7% inpatients); however, some do not routinely obtain laboratory studies (9.7% outpatients and 4.4% inpatients). Hemoglobin/hematocrit, platelet count, fibrinogen, partial thromboplastin time (aPTT), and international normalized ratio (INR) are obtained prior to all TPE by 62.5%, 53.4%, 31.0%, 18.1%, and 17.7% of respondents, respectively; however, 1.0%, 8.7%, 29.0%, 38.3%, and 35.4%, respectively, do not routinely obtain these studies. Variation was observed in laboratory threshold values for action; the most common reported were hemoglobin/hematocrit reference range and >1.5 times reference range (tied, 28.1%), and INR >1.5 (20.7%). CONCLUSIONS Practice variation exists in hemostasis laboratory testing and threshold values for action with TPE. Further studies are needed to determine optimal hemostasis testing strategies with TPE.

Background Extracorporeal membrane oxygenation supplies oxygenated blood to the body supporting the heart and lungs. Survival rates of 20% to 50% are reported among patients receiving ECMO for cardiac arrest, severe cardiogenic shock, or failure to wean from cardiopulmonary bypass following cardiac surgery. Bleeding is one of the most common complications in ECMO patients due to coagulopathy, systemic anticoagulation, and the presence of large bore cannulas at systemic pressure. Absence of a standardized transfusion protocol in this population leads to inconsistent transfusion practices. Here, we assess a newly developed dedicated transfusion protocol in this clinical setting. Methods Data were retrospectively reviewed for the first 30 consecutive cardiac ECMO patients prior and post implementation of the ECMO transfusion protocol. Diagnoses, laboratory results, blood component utilization, and outcomes were collected and analyzed. Results Comorbidities were similar between the 2 eras, as well as the pre-ECMO ejection fraction (P = .568) and duration on ECMO (P = .278). Transfusion utilization data revealed statistically significant decreases in almost all blood components and a savings in blood component acquisition costs of 51% ($175, 970). In addition, an almost 2-fold increase in survival rate was observed in the post-ECMO transfusion protocol era (63% vs 33%; relative risk = 1.82; 95% confidence interval, 1.07-3.10; P = .028). Conclusions Our data indicate that implementation of a standardized transfusion protocol, using more restrictive transfusion indications in cardiac ECMO patients, was associated with reduced blood product utilization, decreased complications, and improved survival. This multidepartmental approach facilitates better communication and adherence to consensus clinical decision making between intensive care unit, surgery, and transfusion service and optimizes care of complicated and acutely ill patients.

Objectives We evaluated thrombosis and mortality rates of hospitalized patients receiving prophylactic platelet transfusion prior to an invasive procedure. Methods Patient age and underlying medical condition(s), preprocedure and postprocedure platelet counts, type of procedure, number of platelet products transfused, and any complications were recorded on every prophylactic platelet given prior to an invasive procedure. Results A total of 376 prophylactic transfusion recipients were identified. Nineteen (5%) thrombotic events were identified and 60 (16%) deaths occurred within 30 days of the preprocedure platelet transfusion. Most deaths were due to infection, sepsis, or organ failure, and none were due to bleeding or thrombosis. Conclusions Preprocedure platelet transfusion is associated with an increased risk of thrombosis and 30-day mortality. Whether these findings are due to higher incidences of comorbidities and confounding or to cause and effect is not determinable from these data. This study highlights an association between prophylactic platelet transfusion and thrombosis and poor outcome, including death.

Thrombocytopenia or receipt of antiplatelet drugs, with or without bleeding, is a common indication for platelet transfusions in the ICU. However, there is almost no evidence base for these practices other than expert opinion. Also common is use of platelet transfusions prior to invasive procedures or surgery in patients with thrombocytopenia. Likewise, there is no high-quality evidence that such practices are efficacious or safe. Recently, it has become clear that, whether causal or not, patients receiving prophylactic platelet transfusions experience high rates of nosocomial infection, thrombosis, organ failure, and mortality, which increase the urgency and need for randomized trials to assess these practices. Investigational methods of improving the safety and efficacy of platelet transfusions include use of alternate strategies such as antifibrinolytics; use of ABO-identical, leukoreduced, and washed platelet transfusions; and improved storage solutions.

Rivaroxaban (Xarelto; Johnson & Johnson Services, Inc) is a direct oral anticoagulant (DOAC) that works by directly inhibiting the active site of factor Xa (FXa). Rivaroxaban is metabolized and cleared via the kidney and liver. The results of various studies have shown that patients with severe renal impairment should receive reduced dosages of rivaroxaban or another anticoagulant due to impaired clearance. Although it is not required, monitoring rivaroxaban is useful in some conditions; however, the assays required for such monitoring are not readily available. Herein, we present a case of a 68-year-old Caucasian male patient who was receiving rivaroxaban (20 mg/day) for atrial flutter and had mild renal impairment. The patient was found to have increased effect of rivaroxaban due to further impairment of renal clearance caused by several renally cleared medications. This case highlights the importance of closely examining the renal function of and medication list for a patient before starting DOACs such as rivaroxaban.

ObjectivesTo provide an overview of the clot viscoelastic testing technology and to describe its utility in guiding blood product transfusions.MethodsA case scenario will be discussed as well as interpretation of thromboelastography (TEG) tracings. In addition, literature examining the utility of viscoelastic testing in guiding patient management and blood product transfusions will be reviewed.ResultsTEG/rotational thromboelastometry (ROTEM) is useful in evaluating clot kinetics in trauma and acutely bleeding patients. TEG/ROTEM parameters are reflective of values measured using standard coagulation assays; however, TEG/ROTEM parameters are more rapidly available and more costly. TEG and ROTEM are used in three main settings: cardiac surgery, liver transplantation, and trauma to assess global hemostasis and administration of blood products.ConclusionsTEG/ROTEM can be helpful in guiding resuscitation and blood product transfusion. Several studies have demonstrated a reduction in transfusion of blood components with TEG/ROTEM; however, other studies have suggested that TEG/ROTEM is not clinically effective in guiding transfusion.

Background Although many apheresis centers offer extracorporeal photopheresis (ECP), little is known about current treatment practices. Methods An electronic survey was distributed to assess ECP practice internationally. Results Of 251 responses, 137 met criteria for analysis. Most respondents were from North America (80%). Nurses perform ECP at most centers (84%) and the majority of centers treat adults only (52%). Most centers treat fewer than 50 patients/year (83%) and perform fewer than 300 procedures/year (70%). Closed system devices (XTS and/or Cellex) are used to perform ECP at most centers (96%). The most common indications for ECP are acute/chronic skin graft versus host disease (89%) and cutaneous T-cell lymphoma (63%). The typical wait time for ECP treatment is less than 2 weeks (91%). Most centers do not routinely perform quality control assessment of the collected product (66%). There are device-specific differences in treatment parameters. For example, XTS users more frequently have a minimum weight limit (P = 0.003) and use laboratory parameters to determine eligibility for treatment (P = 0.03). Regardless of device used, the majority of centers assess the clinical status of the patient before each procedure. Greater than 50% of respondents would defer treatment for hemodynamic instability due to active sepsis or heart failure, positive blood culture in the past 24 h or current fever. Conclusion This survey based study describes current ECP practices. Further research to provide evidence for optimal standardization of patient qualifications, procedure parameters and product quality assessment is recommended.

Despite dramatically improved long term outcomes seen with all-trans retinoic acid therapy, and now arsenic trioxide, in acute promyelocytic leukemia (APL), early mortality remains a substantial challenge. Recent data from a single center study and the Surveillance, Epidemiology and End Results (SEER) registry report 30day mortality rates of 26% (n=18 of 70) and 17% (n=238 of 1400), respectively. Early deaths are predominately due to hemorrhage. Patients with APL invariably have abnormal laboratory hemostasis tests. The standard of practice is to prophylactically transfuse platelets, plasma and cryoprecipitate to mitigate abnormal platelet counts, PT/PTT and fibrinogen levels. Standard blood bank practice is to transfuse platelets, plasma and cryoprecipitate largely without regard to ABO blood group (platelets, cryoprecipitate), and, in some centers, transfusing ABO non-identical universal donor group AB plasma. Evidence from observational studies suggests that use of ABO non-identical blood components may be associated with increased bleeding. We hypothesized that use of ABO identical blood components and saline washed transfusions (red cells and platelets) would be associated with reduced early mortality in APL by avoidance of transfusion induced hemostatic dysfunction.This is a single center cohort study of APL patients treated in an 800 bed university community and referral hospital. Novel approaches to transfusion support, based upon randomized trials, include implementation of ABO identical platelet transfusions for all patients with acute leukemia in 1990, use of only ABO identical cryoprecipitate in 2005, and washed transfusions of red cells and platelets for all patients with acute leukemia50years of age beginning in 2006. Plasma transfusion has always been ABO identical. Two comparison populations were recent literature reports and the New York State Cancer Registry. We characterized 30 day mortality in APL patients seen in our institution since 2000 as a convenience sample comparable to literature reports, beginning approxcimately when ATRA therapy became uniform for induction therapy. Only patients receiving their induction therapy in our hospital were included.Of 41 patients there were 2 early (30 day) deaths (5%; a 71-81% reduction from expected). Early mortality at 100 days was 7% (n=3). The 30 day mortality in the younger cohort50years of age (n=16) receiving washed transfusions was 0%. Restricting the analysis to patients treated since 2006 (ABO identical transfusions, mostly washed) (n=27; mean age 43 years; median 41 years; range 12-79), the early mortality rate at 30days was 3.7%. Long-term survival (5 years) of our APL patients was similar to New York State Cancer Registry and literature reports (80-83%).APL patients supported with transfusion regimens including ABO identical blood components, with or without washing, experienced early mortality at 30 days that was strikingly improved (71% to 86% lower) compared with that reported in the recent literature (3.7% to 5% vs. 17% to 26%). If these observed low rates of early mortality are related to transfusion practices, avoidance of ABO immune complex formation, and subsequent interference with hemostasis, is a plausible contributing mechanism. These favorable results provide a rationale for randomized trials of relatively simple and inexpensive approaches to reducing early hemorrhagic mortality in APL: use of ABO identical transfusions and washing to remove supernatant plasma.

Objectives To provide an overview of the complexities associated with the human leukocyte antigen (HLA)-mediated platelet refractoriness. HLA antibody detection technologies and limitations associated with methodologies are discussed. Methods A case scenario and review of relevant literature describing platelet refractoriness are presented, followed by a discussion of HLA antibody testing. Results Following diagnosis of HLA-mediated refractoriness, a decision is made regarding the approach to obtain the appropriate platelets. The panel reactive antibodies (PRA) % of the patient, HLA typing, and limitations of the HLA testing should be taken into account when deciding which type of product would be the best option for a given patient. Conclusions Following confirmation and review of HLA antibody testing, platelets are ordered based upon the PRA% and approach employed, HLA-matched platelets, antigen restricted platelets, or cross-matched platelets. The platelets are transfused and a posttransfusion increment count is monitored to determine transfusion success.

Critically ill patients undergo numerous laboratory tests to better understand their disease processes and track their illnesses. Many critically ill patients have a variety of conditions such as anemia, sepsis, multiorgan failure, coagulopathies, and/or infection. For the patient to receive the correct diagnosis and treatment, laboratory tests must be carefully ordered and interpreted. Laboratory testing pertaining to anemia, thrombocytopenia, sepsis, bleeding, and hypercoagulability will be discussed in this chapter. Testing interferences as well as caveats that are important to recognize in test result interpretation will be addressed. Moreover, recent advances in hematologic testing of critically ill patients for anemia and coagulopathies such as the reticulocyte hemoglobin, chromogenic factor Xa assay, chromogenic factor VIII assay, TEG platelet mapping, and anti-Xa assay will be discussed in detail.

BACKGROUND Patients undergoing therapeutic plasma exchange (TPE) may present with risks for hemorrhage or thrombosis. Use of replacement fluids devoid of coagulation factors will decrease factor levels and platelet levels. There are no established guidelines for hemostasis management in these situations. MATERIALS AND METHODS A survey to evaluate current hemostasis management practice during TPE was conducted using online survey software. One response per institution was analyzed based on a hierarchical algorithm, excluding membrane filtration users, resulting in a maximum of 107 respondents. Descriptive analysis was performed with results reported as the number and frequency (%) of respondents to each question. RESULTS Apheresis Medicine physicians, alone (59.4%) or jointly with the requesting provider (29.2%), choose the replacement fluid. Based on a theoretical patient case receiving five TPEs approximately every other day, the percent of respondents who would use albumin with or without normal saline was 94.7% with no history of a bleeding or clotting disorder, 1.1% with active bleeding, and 8.8% with hypofibrinogenemia (

Objectives Washing cellular blood products is accepted to ameliorate repeated severe allergic reactions but is associated with RBC hemolysis and suboptimal platelet function. We compared in vitro hemolysis and platelet function in blood components after washing with Plasma-Lyte A (PL-A) vs normal saline (NS). Methods RBC (n = 14) were washed/resuspended in NS or PL-A. Free hemoglobin and heme were determined at 0, 24, 48, and 72 hours. Platelet concentrates (PCs; n = 21) were washed with NS or PL-A and resuspended in same washing solution (n = 13) or ABO-identical plasma (n = 8). Platelet aggregation and spreading were evaluated. Results The 24-hour free hemoglobin and heme levels were higher in NS (P < .05). Improved platelet function was observed in PL-A-washed PCs (P < .001). Discussion PL-A showed less RBC hemolysis and better platelet function than NS. Whether such differences would occur in vivo is unknown.

Oxidation reduction potential (ORP) or Redox is the ratio of activity between oxidizers and reducers. Oxidative stress (OS) can cause cellular injury and death, and is important in the regulation of immune response to injury or disease. In the present study, we investigated changes in the redox system as a function of cardiopulmonary bypass (CPB) in pediatric patients. 664 plasma samples were collected from 162 pediatric patients having cardiac surgery of various CPB times. Lower ORP values at 12 h post-CPB were associated with poor survival rate (mean ± SD 167 ± 20 vs. 138 ± 19, p = 0.005) and higher rate of thrombotic complications (153 ± 21 vs. 168 ± 20, p

Blood transfusion is the second most used medical procedures in health care systems worldwide. Over the last few decades, significant changes have been evolved in transfusion medicine practices. These changes were mainly needed to increase safety, efficacy, and availability of blood products as well as reduce recipients' unnecessary exposure to allogeneic blood. Blood products collection, processing, and storage as well as transfusion practices throughout all patient populations were the main stream of these changes. Health care systems across the world have adopted some or most of these changes to reduce transfusion risks, to improve overall patients' outcome, and to reduce health care costs. In this article, we are going to present and discuss some of these recent modifications and their impact on patients' safety.

Although many apheresis centers offer extracorporeal photopheresis (ECP), little is known about current treatment practices.An electronic survey was distributed to assess ECP practice internationally.Of 251 responses, 137 met criteria for analysis. Most respondents were from North America (80%). Nurses perform ECP at most centers (84%) and the majority of centers treat adults only (52%). Most centers treat fewer than 50 patients/year (83%) and perform fewer than 300 procedures/year (70%). Closed system devices (XTS and/or Cellex) are used to perform ECP at most centers (96%). The most common indications for ECP are acute/chronic skin graft versus host disease (89%) and cutaneous T-cell lymphoma (63%). The typical wait time for ECP treatment is less than 2 weeks (91%). Most centers do not routinely perform quality control assessment of the collected product (66%). There are device-specific differences in treatment parameters. For example, XTS users more frequently have a minimum weight limit (P = 0.003) and use laboratory parameters to determine eligibility for treatment (P = 0.03). Regardless of device used, the majority of centers assess the clinical status of the patient before each procedure. Greater than 50% of respondents would defer treatment for hemodynamic instability due to active sepsis or heart failure, positive blood culture in the past 24 h or current fever.This survey based study describes current ECP practices. Further research to provide evidence for optimal standardization of patient qualifications, procedure parameters and product quality assessment is recommended.

In 2008, the World Health Organization (WHO) revised the classification system for myeloproliferative neoplasms (MPNs). MPNs include chronic myelogenous leukemia, essential thrombocythemia, polycythemia vera, primary myelofibrosis, and several other disorders. The newer classification system incorporates mutations discovered in the JAK2 and MPL genes. The importance of understanding the role of mutations in JAK2, MPL, and other genes that have been discovered in MPNs is highlighted by the change in the 2008 WHO MPN classification system. Moreover, the development of highly specific inhibitors of JAK2 further stresses the importance of molecular testing in MPN diagnosis and prognosis.

Aim To study the short and long term effects of transplanting hearts over positive B-cell flow crossmatches to patients with positive DSAs that become negative when DTT or molecular weight spin columns are used. Methods Recipient and donor HLA typing was performed by rSSO-Luminex. Anti-HLA class-I and II antibodies by Single Antigen ((SAB) LABScreen Single Antigen Class-I and II) with and without molecular weight spin columns (MWSCs) or DTT. For identification of complement-fixing anti-HLA antibodies, the C1q test was used. Donor crossmatching was performed within 24 h of transplantation by flow cytometry (FXM) using serum drawn immediately before transplant. Patient allograft heart biopsy results and patient health were followed. Results Nine heart allograft recipients between 2012 and 2017 were identified that had either positive FXM and/or positive SAB I/II results that became negative when either DTT/MWSC were used (Table 1). In most, at least one of the antibodies identified was DSA. In 8, the DSA was also known to be a cryptic antigen. Some of the MFI values for DSA were below our cut-off of 3000 MFI. Patients were followed for rejection. Only one patient died from unrelated causes and never had any signs or biopsy results of cellular or antibody mediated rejection (AMR). The remaining patients are all alive and doing well. No patients ever had any biopsied signs of AMR. Several did have some cellular rejection that resolved with treatment however, rejection was not higher than in other heart transplant patients. This phenomenon was also observed in kidney transplantation. Conclusions The use of MWSCs to evaluate positive FXM, SAB1, SAB2, and DSA in heart transplant candidates is effective in eliminating interference and cryptic antigens as well as determining which antibodies are “real” and pose a risk to the recipient. Here we show that use of MWSCs allows clarification of test results and gives one additional data on which to decide to transplant across DSA. Notably, all patients did well following transplant with no AMR. Download : Download high-res image (411KB) Download : Download full-size image

Tissue factor pathway inhibitor-2 (TFPI-2) inhibits factor XIa, plasma kallikrein, and factor VIIa/tissue factor; accordingly, it has been proposed for use as an anticoagulant. Full-length TFPI-2 or its isolated first Kunitz domain (KD1) also inhibits plasmin; therefore, it has been proposed for use as an antifibrinolytic agent. However, the anticoagulant properties of TFPI-2 or KD1 would diminish its antifibrinolytic function. In this study, structure-based investigations and analysis of the serine protease profiles revealed that coagulation enzymes prefer a hydrophobic residue at the P2′ position in their substrates/inhibitors, whereas plasmin prefers a positively charged arginine residue at the corresponding position in its substrates/inhibitors. Based upon this observation, we changed the P2′ residue Leu-17 in KD1 to Arg (KD1-L17R) and compared its inhibitory properties with wild-type KD1 (KD1-WT). Both WT and KD1-L17R were expressed in Escherichia coli, folded, and purified to homogeneity. N-terminal sequences and mass spectra confirmed proper expression of KD1-WT and KD1-L17R. Compared with KD1-WT, the KD1-L17R did not inhibit factor XIa, plasma kallikrein, or factor VIIa/tissue factor. Furthermore, KD1-L17R inhibited plasmin with ∼6-fold increased affinity and effectively prevented plasma clot fibrinolysis induced by tissue plasminogen activator. Similarly, in a mouse liver laceration bleeding model, KD1-L17R was ∼8-fold more effective than KD1-WT in preventing blood loss. Importantly, in this bleeding model, KD1-L17R was equally or more effective than aprotinin or tranexamic acid, which have been used as antifibrinolytic agents to prevent blood loss during major surgery/trauma. Furthermore, as compared with aprotinin, renal toxicity was not observed with KD1-L17R.

Patients with pre-leukemic bone marrow failure syndrome, severe congenital neutropenia (CN) have ~ 20% risk of developing acute myeloid leukemia (AML) (CN-AML). More than 70 % of CN-AML patients co-acquire CSF3R and RUNX1 mutations as shown by our group (Skokowa et al 2014), indicating a cooperative role of the mutations in these two genes in the development of AML in CN patients. In order to investigate the interaction between these mutations we conducted in vitro experiments on lineage negative (lin-) bone marrow mononuclear cells (BMCs) from C57BL/6-d715csf3r mice (d715-mice). These mice carry homozygous d715G CSF3R mutations, but do not develop AML. We isolated lin- BMCs from d715 mice and transduced these cells with four different lentivirus vectors carrying BFP only (CTRL), RUNX1-Wild type BFP (RUNX1-WT), RUNX1-R139G BFP (RUNX1-MUT1) and RUNX1-R174L BFP (RUNX1-MUT2). These RUNX1 mutations where found in CN-AML patients. 72 hours after transduction, we sorted BFP+ cells and compared G-CSF triggered myeloid differentiation in vitro. We found that cells transduced with each RUNX1 mutants exhibited reduced percentages of myeloid CD11b+, Gr-1+ and double positive cells compared to RUNX1-WT. We also conducted CFU re-plating experiments with transduced cells and found that cell transduced with each of RUNX1 mutants showed 7- (RUNX1-MUT1) and 8- (RUNX1-MUT2) times higher re-plating capacity than RUNX1-WT and CTRL transduced cells. To identify signaling pathways that are deregulated in G-CSFR-mutated HSCs clones after co-acquisition of RUNX1 mutations, we performed microarray study. We starved transduced and sorted lin- BMCs for 24 hours and treated cells with G-CSF for 48 hours before mRNA was collected. Expression profiles where generated by microarray (GeneChip Mouse Gene 2.0 ST Array). Pathway Analysis was conducted using IPA Software and Motif activity response analysis (MARA) was performed using ISMARA web tool. Reported Transcription factors and targets have Z-value ≥ 2 or ≤-2 and p ≤ 0,05 and are thus considered statistically significant. Interestingly, ISMARA analysis showed, that the highest active motif in RUNX1-Mutants was Irf2_Irf1_Irf8_Irf9_Irf7 motif which is essential for the regulation of the interferon pathway genes. The corresponding transcription factors are amongst others regulated by Sp1 and Stat2 that were also active. Correspondingly, IPA Pathway analysis showed, that Interferon Signaling was highly upregulated in cells transduced with each of two RUNX1 mutants, compared to overexpressed RUNX1-WT (Z = 2). Additionally, pathway analysis showed the upregulation and activation of IL-6, IL-8-, Toll like Receptor- and TREM1 signaling pathways. This data suggests that the mutated RUNX1 may cause activation of the pro-inflammatory cell state propagating proliferation, which may be emerging as a cause of clonal hematopoiesis (CH) and consequently may lead to MDS/AML (Hemmati et al 2017). Another active motif is Spi1/PU.1 mainly known as an essential transcription factor for monocytic differentiation, but also as a maintenance factor of the pre-leukemia initiating cells (pre-LICs) or even leukemia initiating cells (LICs) (Staber et al 2014). Interestingly, we recently described elevated expression of PU.1 in hematopoietic cells of CN patients. These data together with our in vitro finding indicating that RUNX1 mutation causes differentiation block and clonal proliferation of HSCs, supporting the hypothesis, that the RUNX1 mutations are the driving factor in leukemic transformation in CN. Additionally, ISMARA revealed an upregulation of the Metyl-CpG Binding Protein 2 (Mecp2) motif. Mecp2 is a proto-oncogene that represses transcription through interaction with the corepressor SIN3A and histone deacetylases. Thus, remodeling the transcriptional profile and inhibiting differentiation. Taken together, our data shows that RUNX1 mutations in combination with CSF3R mutations may cause (1) increased proliferation through the induction of a proinflammatory cell state, (2) induce self-renewal through expression of essential proteins for LIC maintenance and (3) dimineshed myeloid differentiation through demethylation inhibition and down regulation of hematopoietic differentiation pathways. We are currently validating the model of the leukemogenic transformation in CN patients using functional studies in vitro and in vivo. Disclosures No relevant conflicts of interest to declare.

In serine proteases, Gly193 (chymotrypsin numbering) is conserved with rare exception. Mutants of blood coagulation proteases have been reported with Glu, Ala, Arg or Val substitutions for Gly193. To further understand the role of Gly193 in protease activity, we replaced it with Ala or Val in coagulation factor XIa (FXIa). For comparison to the reported FXIa Glu193 mutant, we prepared FXIa with Asp (short side chain) or Lys (opposite charge) substitutions. Binding of p-aminobenzamidine (pAB) and diisopropylfluorphosphate (DFP) were impaired 1.6-36-fold and 35-478-fold, respectively, indicating distortion of, or altered accessibility to, the S1 and oxyanion-binding sites. Val or Asp substitutions caused the most impairment. Salt bridge formation between the amino terminus of the mature protease moiety at Ile16 and Asp194, essential for catalysis, was impaired 1.4-4-fold. Mutations reduced catalytic efficiency of tripeptide substrate hydrolysis 6-280-fold, with Val or Asp causing the most impairment. Further studies were directed toward macromolecular interactions with the FXIa mutants. kcat for factor IX activation was reduced 8-fold for Ala and 400-1100-fold for other mutants, while binding of the inhibitors antithrombin and amyloid beta-precursor protein Kunitz domain (APPI) was impaired 13-2300-fold and 22-27000-fold, respectively. The data indicate that beta-branching of the side chain of residue 193 is deleterious for interactions with pAB, DFP and amidolytic substrates, situations where no S2'-P2' interactions are involved. When an S2'-P2' interaction is involved (factor IX, antithrombin, APPI), beta-branching and increased side chain length are detrimental. Molecular models indicate that the mutants have impaired S2' binding sites and that beta-branching causes steric conflicts with the FXIa 140-loop, which could perturb the local tertiary structure of the protease domain. In conclusion, enzyme activity is impaired in FXIa when Gly193 is replaced by a non-Gly residue, and residues with side chains that branch at the beta-carbon have the greatest effect on catalysis and binding of substrates.

We previously reported that the first epidermal growth factor-like (EGF1) domain in factor X (FX) or factor IX (FIX) plays an important role in the factor VIIa/tissue factor (FVIIa/TF)-induced coagulation. To assess the role of gamma-carboxyglutamic acid (Gla) domains of FX and FIX in FVIIa/TF induced coagulation, we studied four new and two previously described replacement mutants: FX(PCGla) and FIX(PCGla) (Gla domain replaced with that of protein C), FX(PCEGF1) and FIX(PCEGF1) (EGF1 domain replaced with that of protein C), as well as FX(PCGla/EGF1) and FIX(PCGla/EGF1) (both Gla and EGF1 domains replaced with those of protein C). FVIIa/TF activation of each FX mutant and the corresponding reciprocal activation of FVII/TF by each FXa mutant were impaired. In contrast, FVIIa/TF activation of FIX(PCGla) was minimally affected, and the reciprocal activation of FVII/TF by FIXa(PCGla) was normal; however, both reactions were impaired for the FIX(PCEGF1) and FIX(PCGla/EGF1) mutants. Predictably, FXIa activation of FIX(PCEGF1) was normal, whereas it was impaired for the FIX(PCGla) and FIX(PCGla/EGF1) mutants. Molecular models reveal that alternate interactions exist for the Gla domain of protein C such that it is comparable with FIX but not FX in its binding to FVIIa/TF. Further, additional interactions exist for the EGF1 domain of FX, which are not possible for FIX. Importantly, a seven-residue insertion in the EGF1 domain of protein C prevents its interaction with FVIIa/TF. Cumulatively, our data provide a molecular framework demonstrating that the Gla and EGF1 domains of FX interact more strongly with FVIIa/TF than the corresponding domains in FIX.

Inherited deficiency of the trypsin-like protease factor (F) XI is associated with a mild to moderate bleeding diathesis. In most cases, FXI protein is reduced in plasma, and examples of dysfunctional circulating FXI variants are rare. We characterized the defect in one such variant with a proline to leucine substitution at residue 520. FXI Pro520 corresponds to chymotrypsin Pro161, and is conserved in most members of the chymotrypsin protease family. Recombinant FXI containing this substitution will be referred to as FXI(P161L). k(cat) for cleavage of chromogenic substrates and for activation of the natural FXIa substrate FIX is approximately 3-fold lower for activated FXI(P161L) (FXIa(P161L)) than for wild-type FXIa (FXIa(WT)), consistent with an abnormal protease active site. Inhibition of FXIa(P161L) by diisopropyl fluorophosphate is 2.4-fold slower than for FXIa(WT), suggesting distortion of the protease oxyanion hole. Binding to p-aminobenzamidine, a probe for the integrity of the S1 substrate-binding site, was similar for FXIa(WT) and FXIa(P161L). Rates of carbamylation of Ile16 were also similar for FXIa(WT) and FXIa(P161L), indicating that the critical salt bridge between Ile16 and Asp194 forms normally during protease activation. Cumulatively, the data demonstrate that Pro161 is required for normal active site oxyanion hole conformation in FXIa. Examination of the FXIa crystal structure and modeling studies indicate that Pro161 forms several hydrophobic contacts with adjacent amino acids that stabilize active site conformation. Leucine can be incorporated at position 161 in FXIa, but would not form the extensive stabilizing network of hydrophobic interactions formed by Pro161.

Platelets perform a vital role in hemostasis and their role in inflammation is becoming increasingly evident. Blood transfusion is the most common procedure performed in hospitals and platelet transfusions comprise a significant proportion. Over the past few decades, retrospective studies and randomized clinical trials have demonstrated that blood transfusion is more harmful than previously thought and is associated with numerous complications, such as transfusion-associated lung injury, transfusion-associated cardiac overload, transfusion-associated immune modulation, and infectious diseases such as human immunodeficiency virus, hepatitis C virus, and hepatitis B virus. Recent data suggest an association between platelet transfusion and thrombosis. This review will highlight the mechanistic issues that may be relevant to the epidemiologic associations of platelet transfusion with thrombosis and mortality in critically ill patients.

Factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like domains, and a protease domain. FVIIa binds seven Ca2+ ions in the Gla, one in the EGF1, and one in the protease domain. However, blood contains both Ca2+ and Mg2+, and the Ca2+ sites in FVIIa that could be specifically occupied by Mg2+ are unknown. Furthermore, FVIIa contains a Na+ and two Zn2+ sites, but ligands for these cations are undefined. We obtained p-aminobenzamidine-VIIa/soluble tissue factor (sTF) crystals under conditions containing Ca2+, Mg2+, Na+, and Zn2+. The crystal diffracted to 1.8A resolution, and the final structure has an R-factor of 19.8%. In this structure, the Gla domain has four Ca2+ and three bound Mg2+. The EGF1 domain contains one Ca2+ site, and the protease domain contains one Ca2+, one Na+, and two Zn2+ sites. 45Ca2+ binding in the presence/absence of Mg2+ to FVIIa, Gla-domainless FVIIa, and prothrombin fragment 1 supports the crystal data. Furthermore, unlike in other serine proteases, the amide N of Gly193 in FVIIa points away from the oxyanion hole in this structure. Importantly, the oxyanion hole is also absent in the benzamidine-FVIIa/sTF structure at 1.87A resolution. However, soaking benzamidine-FVIIa/sTF crystals with d-Phe-Pro-Arg-chloromethyl ketone results in benzamidine displacement, d-Phe-Pro-Arg incorporation, and oxyanion hole formation by a flip of the 192-193 peptide bond in FVIIa. Thus, it is the substrate and not the TF binding that induces oxyanion hole formation and functional active site geometry in FVIIa. Absence of oxyanion hole is unusual and has biologic implications for FVIIa macromolecular substrate specificity and catalysis.

Kunitz domain 1 (KD1) of tissue factor pathway inhibtor-2 inhibits trypsin, plasmin, and factor VIIa (FVIIa)/tissue factor with Ki values of 13, 3, and 1640 nm, respectively. To investigate the molecular specificity of KD1, crystals of the complex of KD1 with bovine β-trypsin were obtained that diffracted to 1.8 A. The P1 residue Arg-15 (bovine pancreatic trypsin inhibitor numbering) in KD1 interacts with Asp-189 (chymotrypsin numbering) and with the carbonyl oxygens of Gly-219 and Oγ of Ser-190. Leu-17, Leu-18, Leu-19, and Leu-34 in KD1 make van der Waals contacts with Tyr-39, Phe-41, and Tyr-151 in trypsin, forming a hydrophobic interface. Molecular modeling indicates that this complementary hydrophobic patch is composed of Phe-37, Met-39, and Phe-41 in plasmin, whereas in FVIIa/tissue factor, it is essentially absent. Arg-20, Tyr-46, and Glu-39 in KD1 interact with trypsin through ordered water molecules. In contrast, insertions in the 60-loop in plasmin and FVIIa allow Arg-20 of KD1 to directly interact with Glu-60 in plasmin and Asp-60 in FVIIa. Moreover, Tyr-46 in KD1 electrostatically interacts with Lys-60A and Arg-60D in plasmin and Lys-60A in FVIIa. Glu-39 in KD1 interacts directly with Arg-175 of the basic patch in plasmin, whereas in FVIIa, such interactions are not possible. Thus, the specificity of KD1 for plasmin is attributable to hydrophobic and direct electrostatic interactions. For trypsin, hydrophobic interactions are intact, and electrostatic interactions are weak, whereas for FVIIa, hydrophobic interactions are missing, and electrostatic interactions are partially intact. These findings provide insight into the protease selectivity of KD1.

During blood coagulation, factor IXa (FIXa) activates factor X (FX) requiring Ca 2+ , phospholipid, and factor VIIIa (FVIIIa). The serine protease domain of FIXa contains a Ca 2+ site and is predicted to contain a Na + site. Comparative homology analysis revealed that Na + in FIXa coordinates to the carbonyl groups of residues 184A, 185, 221A, and 224 (chymotrypsin numbering). Kinetic data obtained at several concentrations of Na + and Ca 2+ with increasing concentrations of a synthetic substrate (CH 3 -SO 2 - d -Leu-Gly-Arg- p -nitroanilide) were fit globally, assuming rapid equilibrium conditions. Occupancy by Na + increased the affinity of FIXa for the synthetic substrate, whereas occupancy by Ca 2+ decreased this affinity but increased k cat dramatically. Thus, Na + -FIXa-Ca 2+ is catalytically more active than free FIXa. FIXa Y225P , a Na + site mutant, was severely impaired in Na + potentiation of its catalytic activity and in binding to p -aminobenzamidine (S1 site probe) validating that substrate binding in FIXa is linked positively to Na + binding. Moreover, the rate of carbamylation of NH 2 of Val16, which forms a salt-bridge with Asp194 in serine proteases, was faster for FIXa Y225P and addition of Ca 2+ overcame this impairment only partially. Further studies were aimed at delineating the role of the FIXa Na + site in macromolecular catalysis. In the presence of Ca 2+ and phospholipid, with or without saturating FVIIIa, FIXa Y225P activated FX with similar K m but threefold reduced k cat . Further, interaction of FVIIIa:FIXa Y225P was impaired fourfold. Our previous data revealed that Ca 2+ binding to the protease domain increases the affinity of FIXa for FVIIIa ∼15-fold. The present data indicate that occupancy of the Na + site further increases the affinity of FIXa for FVIIIa fourfold and k cat threefold. Thus, in the presence of Ca 2+ , phospholipid, and FVIIIa, binding of Na + to FIXa increases its biologic activity by ∼12-fold, implicating its role in physiologic coagulation.

In serine proteases, Gly193 is highly conserved with few exceptions. A patient with inherited deficiency of the coagulation serine protease factor XI (FXI) was reported to be homozygous for a Gly555 → Glu substitution. Gly555 in FXI corresponds to Gly193 in chymotrypsin, which is the numbering system used subsequently. To investigate the abnormality in FXIG193E, we expressed and purified recombinant FXIaG193E, activated it to FXIaG193E, and compared its activity to wild type-activated FXI (FXIaWT). FXIaG193E activated FIX with ∼300-fold reduced kcat and similar Km, and hydrolyzed synthetic substrate with ∼10-fold reduced Km and modestly reduced kcat. Binding of antithrombin and the amyloid β-precursor protein Kunitz domain inhibitor (APPI) to FXIaG193E was impaired ∼8,000- and ∼100,000-fold, respectively. FXIaG193E inhibition by diisopropyl fluoro-phosphate was ∼30-fold slower and affinity for p-aminobenzamidine (S1 site probe) was 6-fold weaker than for FXIaWT. The rate of carbamylation of NH2-Ile16, which forms a salt bridge with Asp194 in active serine proteases, was 4-fold faster for FXIaG193E. These data indicate that the unoccupied active site of FXIaG193E is incompletely formed, and the amide N of Glu193 may not point toward the oxyanion hole. Inclusion of saturating amounts of p-aminobenzamidine resulted in comparable rates of carbamylation for FXIaWT and FXIaG193E, suggesting that the occupied active site has near normal conformation. Thus, binding of small synthetic substrates or inhibitors provides sufficient energy to allow the amide N of Glu193 to point correctly toward the oxyanion hole. Homology modeling also indicates that the inability of FXIaG193E to bind antithrombin/APPI or activate FIX is caused, in part, by impaired accessibility of the S2′ site because of a steric clash with Glu193. Such arguments will apply to other serine proteases with substitutions of Gly193 with a non-glycine residue.

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type proteinase inhibitor that regulates a variety of serine proteinases involved in coagulation and fibrinolysis through their non-productive interaction with a P(1) residue (Arg-24) in its first Kunitz-type domain (KD1). Previous kinetic studies revealed that TFPI-2 was a more effective inhibitor of plasmin than several other serine proteinases, but the molecular basis for this specificity was unclear. In this study, we employed molecular modeling and mutagenesis strategies to produce several variants of human TFPI-2 KD1 in an effort to identify interactive site residues other than the P(1) Arg that contribute significantly to its inhibitory activity and specificity. Molecular modeling of KD1 based on the crystal structure of bovine pancreatic trypsin inhibitor revealed that KD1 formed a more energetically favorable complex with plasmin versus trypsin and/or the factor VIIa-tissue factor complex primarily due to strong ionic interactions between Asp-19 (P(6)) and Arg residues in plasmin (Arg-644, Arg-719, and Arg-767), Arg-24 (P(1)) with Asp-735 in plasmin, and Arg-29 (P(5)') with Glu-606 in plasmin. In addition, Leu-26 through Leu-28 (P(2)'-P(4)') in KD1 formed strong van der Waals contact with a hydrophobic cluster in plasmin (Phe-583, Met-585, and Phe-587). Mutagenesis of Asp-19, Tyr-20, Arg-24, Arg-29, and Leu-26 in KD1 resulted in substantial reductions in plasmin inhibitory activity relative to wild-type KD1, but the Asp-19 and Tyr-20 mutations revealed the importance of these residues in the specific inhibition of plasmin. In addition to the reactive site residues in the P(6)-P(5)' region of KD1, mutation of a highly conserved Phe at the P(18)' position revealed the importance of this residue in the inhibition of serine proteinases by KD1. Thus, together with the P(1) residue, the nature of other residues flanking the P(1) residue, particularly at P(6) and P(5)', strongly influences the inhibitory activity and specificity of human TFPI-2.

Factor IX (FIX) consists of an N-terminal gamma-carboxyglutamic acid (Gla) domain followed by two epidermal growth factor (EGF)-like domains, and the C-terminal serine protease domain. During physiologic coagulation, one of the activators of FIX is the FVIIa/tissue factor (TF) complex. In this reaction, the Gla and EGF1 domains of FIX are thought to interact with TF. The FIXa that is generated then combines with FVIIIa on the platelet surface to activate FX in the coagulation cascade. In this assembly, the protease domain and possibly the EGF2 domain of FIXa are thought to provide the primary specificity in binding to FVIIIa. Disruption of the interaction of FIX/FX with TF and of the FIXa:FVIIIa interface may provide a pharmacologic target as an alternative strategy for the development of antithrombotic agents.

The serine protease domain of activated protein C (APC) contains a Na+ and a Ca2+ site. However, the number and identity of the APC residues that coordinate to Na+ is not precisely known. Further, the functional link between the Na+ and the Ca2+ site is insufficiently defined, and their linkage to the substrate S1 site has not been studied. Here, we systematically investigate the functional significance of these two cation sites and their thermodynamic links to the S1 site. Kinetic data reveal that Na+ binds to the substrate-occupied APC withK d values of ∼24 mm in the absence and ∼6 mm in the presence of Ca2+. Sodium-occupied APC has ∼100-fold increased catalytic efficiency (∼4-fold decrease in K m and ∼25-fold increase in k cat) in hydrolyzing S-2288 (H-d-Ile-Pro-Arg-p-nitroanilide) and Ca2+ further increases thisk cat slightly (∼1.2-fold). Ca2+binds to the protease domain of APC with K d values of ∼438 μm in the absence and ∼105 μmin the presence of Na+. Ca2+ binding to the protease domain of APC does not affect K m but increases the k cat ∼10-fold, and Na+ further increases this k cat∼3-fold and decreases the K m value ∼3.7-fold. In agreement with the K m data, sodium-occupied APC has ∼4-fold increased affinity in binding top-aminobenzamidine (S1 probe). Crystallographically, the Ca2+ site in APC is similar to that in trypsin, and the Na+ site is similar to that in factor Xa but not thrombin. Collectively, the Na+ site is thermodynamically linked to the S1 site as well as to the protease domain Ca2+ site, whereas the Ca2+ site is only linked to the Na+site. The significance of these findings is that under physiologic conditions, most of the APC will exist in Na2+-APC-Ca2+ form, which has 110-fold increased proteolytic activity.

Platelet transfusion refractoriness in patients with hematological malignancies ranges from 7% to 34%. The etiology of platelet refractoriness in this patient population is typically attributable to due to formation of HLA antibodies (Abs) from pregnancy or previous transfusion. We report platelet refractoriness in the setting of induction chemotherapy for AML from allo-Abs to NAK a antigen/CD36/glycoprotein (GP) IV. A 66yo AAF presented with transfusion dependent AML with 70% circulating peripheral blasts and started on 7 + 3 regimen. She developed profound thrombocytopenia and her platelet count was 0 by day 13. As part of her evaluation for refractoriness, HLA antibody screening was done, but was negative. An Immucor ELISA assay was then performed and revealed positive Abs to GPIV (NAK a antigen/CD36). Abs against GPIIb/IIIa, GPIa/IIa, GPIb/IX, and HLA Class I were all negative. GPIV Abs were confirmed by Blood Center of Wisconsin using monoclonal Ab MBC 131.7 against CD36. Cross-matching was performed at our institution with multiple single donor apheresis platelets, all showing strong positive reactivity. Because CD36 in these units may be soluble, an incompatible leukoreduced, irradiated and washed single donor platelet unit was given which was tolerated well with a resultant increase to 14,000/ μ L. Despite strong reactivity of this unit in the crossmatch, the patient’s platelet count increased and 1 week later was 16,000/μL despite no transfusion support. Platelet refractoriness in the setting of hematological malignancy is well documented. Abs develop from prior pregnancy and transfusion in the setting of prolonged pancytopenia in hematological malignancies. Typically, allo-Abs develop against platelet membrane structures including human platelet antigens, HLA antigens, and rarely NAK a antigen on the CD36 glycoprotein molecule. CD36 is expressed in almost 100% of white Europeans and is not detectably expressed by 2% of sub-Saharan Africans and 10% of Asians. CD36 is a soluble antigen and is expressed on various cells such as erythroblasts and monocytes. We propose that washing platelets may be a useful strategy in treating platelet refractoriness in patients with abs to NAK a antigen as finding CD36 negative donors is extremely difficult and few blood banks in North America test donors for this antigen.

Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in which P2′ residue Leu17 (bovine pancreatic trypsin inhibitor numbering) is mutated to Arg selectively inhibits the active site of plasmin with ∼5-fold improved affinity. Thrombin cleavage (24 h extended incubation at a 1:50 enzyme-to-substrate ratio) of the KD1 mutant (Leu17Arg) yielded a smaller molecule containing the intact Kunitz domain with no detectable change in the active-site inhibitory function. The N-terminal sequencing and MALDI-TOF/ESI data revealed that the starting molecule has a C-terminal valine (KD1L17R-VT), whereas the smaller molecule has a C-terminal lysine (KD1L17R-KT). Because KD1L17R-KT has C-terminal lysine, we examined whether it could serve as a decoy receptor for plasminogen/plasmin. Such a molecule might inhibit plasminogen activation as well as the active site of generated plasmin. In surface plasmon resonance experiments, tissue plasminogen activator (tPA) and Glu-plasminogen bound to KD1L17R-KT (Kd ∼ 0.2 to 0.3 μM) but not to KD1L17R-VT. Furthermore, KD1L17R-KT inhibited tPA-induced plasma clot fibrinolysis more efficiently than KD1L17R-VT. Additionally, compared to ε-aminocaproic acid KD1L17R-KT was more effective in reducing blood loss in a mouse liver-laceration injury model, where the fibrinolytic system is activated. In further experiments, the micro(μ)-plasmin–KD1L17R-KT complex inhibited urokinase-induced plasminogen activation on phorbol-12-myristate-13-acetate-stimulated U937 monocyte-like cells, whereas the μ-plasmin–KD1L17R-VT complex failed to inhibit this process. In conclusion, KD1L17R-KT inhibits the active site of plasmin as well as acts as a decoy receptor for the kringle domain(s) of plasminogen/plasmin; hence, it limits both plasmin generation and activity. With its dual function, KD1L17R-KT could serve as a preferred agent for controlling plasminogen activation in pathological processes.

The physiologic activator of factor X consists of a complex of factor IXa, factor VIIIa, Ca(2+) and a suitable phospholipid surface. In one study, helix 330 (162 in chymotrypsin) of the protease domain of factor IXa was implicated in binding to factor VIIIa. In another study, residues 558-565 of the A2 subunit of factor VIIIa were implicated in binding to factor IXa. We now provide data, which indicate that the helix 330 of factor IXa interacts with the 558-565 region of the A2 subunit. Thus, the ability of the isolated A2 subunit was severely impaired in potentiating factor X activation by IXa(R333Q) and by a helix replacement mutant (IXa(helixVII) in which helix 330-338 is replaced by that of factor VII) but it was normal for an epidermal growth factor 1 replacement mutant (IXa(PCEGF1) in which epidermal growth factor 1 domain is replaced by that of protein C). Further, affinity of each 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Glu-Gly-Arg-IXa (dEGR-IXa) with the A2 subunit was determined from its ability to inhibit wild-type IXa in the tenase assay and from the changes in dansyl fluorescence emission signal upon its binding to the A2 subunit. Apparent K(d(A2)) values are: dEGR-IXa(WT) or dEGR-IXa(PCEGF1) approximately 100 nm, dEGR-IXa(R333Q) approximately 1.8 micrometer, and dEGR-IXa(helixVII) >10 micrometer. In additional experiments, we measured the affinities of these factor IXa molecules for a peptide comprising residues 558-565 of the A2 subunit. Apparent K(d(peptide)) values are: dEGR-IXa(WT) or dEGR-IXa(PCEGF1) approximately 4 micrometer, and dEGR-IXa(R333Q) approximately 62 micrometer. Thus as compared with the wild-type or PCEGF1 mutant, the affinity of the R333Q mutant for the A2 subunit or the A2 558-565 peptide is similarly reduced. These data support a conclusion that the helix 330 of factor IXa interacts with the A2 558-565 sequence. This information was used to model the interface between the IXa protease domain and the A2 subunit, which is also provided herein.

The product of the human oncogene ELL encodes an RNA polymerase II transcription factor that undergoes frequent translocation in acute myeloid leukemia (AML). In addition to its elongation activity, ELL contains a novel type of RNA polymerase II interaction domain that is capable of repressing polymerase activity in promoter-specific transcription. Remarkably, the ELL translocation that is found in patients with AML results in the deletion of exactly this functional domain. Here we report that the EAP30 subunit of the ELL complex has sequence homology to the Saccharomyces cerevisiae SNF8, whose genetic analysis suggests its involvement in the derepression of gene expression. Remarkably, EAP30 can interact with ELL and derepress ELL's inhibitory activity in vitro. This finding may reveal a key role for EAP30 in the pathogenesis of human leukemia.