bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Rapid Sensitive Assay for Measuring ADAMTS13 Activity in Plasma Utilizing a Novel Recombinant Alexa488-vWF86 (Q1599\P1611C) FRET Substrate.
Authors: Guinto Enriqueta R. , Grafos Nicholas , Owen John , Lawson Heather L. , Greenfield Robert S. 
Published: April 2020

Abstract
ADAMTS13 (von Willebrand Factor cleaving protease) is a zinc metalloprotease that cleaves the Tyr(1605)/Met(1606) bond within the A2 domain of von Willebrand Factor (vWF). Low levels of ADAMTS13 activity (&lt;5%) are strongly associated with the life threatening hemostatic condition thrombotic thrombocytopenic purpura (TTP). A rapid clinical assay for ADAMTS13 is necessary to provide critical information for the early diagnosis of TTP and to guide and monitor the treatment of TTP.
Present methods for measuring ADAMTS13 activity (e.g. collagen binding, vWF multimer analysis and ELISA) are cumbersome, take a long time to complete and lack sensitivity. A fluorescence resonance energy transfer (FRET) assay for ADAMTS13 has been described, which uses the costly and difficult to synthesize truncated A2 domain VWF73aa peptide as substrate (1). However, the lower limit of detection of ADAMTS13 assays using VWF73 FRET substrate is only about 5–10% of normal ADAMTS13 activity levels (1,2). Recently, Zhang et al (3) reported a novel recombinant VWF A2 FRET substrate of 86 amino acids in which cysteine residues were substituted for residues Q1599 and P1611, respectively. The cysteine residues were chemically labeled with fluorescein maleimide to produce a FRET substrate whereby the two fluorescein dyes flanking the ADAMTS13 Tyr/Met cleavage site interact sufficiently such that auto-quenching of fluorescence occurs.
In this study, we developed a rapid more sensitive fluorescent assay for ADAMTS13 using a novel recombinant VWF86(Q1599C/P1611C) FRET substrate. VWF86(Q1599C/P1611C) peptide was chemically coupled via the cysteine residues to the brighter, more photostable Alexa488 fluorochrome. Assay conditions were optimized to obtain higher analytical sensitivity &lt; 5% normal ADAMTS13 activity (0.5 ng/ml) and shorter assay times (20–30 minutes). ADAMTS13 activity in 50 normal individuals as well as subjects with TTP, thrombosis and lupus anticoagulant will be presented. The rapid FRET assay using the novel recombinant Alexa488-vWF86 (Q1599\P1611C) substrate should prove clinically useful for quantitation of ADAMTS13 activity and the rapid diagnosis of TTP.
Recombinant ADAMTS13 Processing by FXIa Is Distinct from Thrombin.
Authors: Guinto Enriqueta R. , Grafos Nicholas , Motto David G. , Greenfield Robert S. 
Published: April 2020

Abstract
ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type1 domain 13) is associated with processing monomeric and multimeric or ultralarge von Willebrand factor (vWF), cleaving between Tyr1605 and Met1606 in the A2 domain. Low levels of ADAMTS13 activity (&lt;5%) are strongly associated with the life threatening hemostatic condition, thrombotic thrombocytopenic purpura (TTP). It is being recognized that not all cases of TTP can be attributed exclusively to the deficiency in ADAMTS13 or presence of ADAMTS13 autoantibodies. Other biological factor(s) or cofactor(s) may play a role in the pathogenesis of TTP.
We initially reported (1) that purified FXIa (1–40 nM) also cleaved purified recombinant A2 domain fragment (100 ug/mL) and FXIa at 200 nM also cleaves purified vWF protein (100 ug/mL) at still unknown sites. We reported (1) that FXIa cleaved purified recombinant (r) ADAMTS13 and that high concentrations of FXIa (&gt;100 nM) resulted in loss of the metalloprotease activity towards VWF73aa FRET (2).
In this study, we found that low concentrations (&lt;20 nM ) of purified FXIa will cleave purified rADAMTS13 which resulted in about 2 fold enhancement of the metalloprotease activity towards the fluorescently labeled VWF73aa peptide substrate using either VWF73aa FRETs (2) or the newly developed Alexa488-VWF86 FRET substrate from American Diagnostica Inc (3). However, at high concentrations (&gt;100nM) of purified FXIa, inactivation of rADAMTS13 metalloprotease activity was observed. The proteolytic patterns at high and low amounts of FXIa will be presented. Since thrombin was reported (4) as possible regulator of ADAMTS13, we used various polyclonal and monoclonal antibodies, recognizing different regions of ADAMTS13 to compare the ADAMTS13 processing by the two serine proteases. The results showed that the proteolytic patterns by FXIa and thrombin are different. In addition, specific monoclonal anti FXI antibodies but not hirudin inhibits the FXIa processing of ADAMTS13. The identification of cleavage sites by FXIa on ADAMTS13 and VWF are in progress. The physiological relevance of FXIa processing of ADAMTS13 and VWF protein and possible link to the etiology of TTP and FXIa related pathophysiology (5) are under investigation.
Regular Prophylactic Treatment with Factor IX P® in a Patient with Severe Factor X Deficiency.
Authors: Gerhardt Andrea , Araba Fatima , Zotz Rainer B. , Scharf Rudiger E. 
Published: April 2020

Abstract
Background: Congenital factor X deficiency, a rare coagulation disorder with variable severity, is an inherited autosomal recessive disorder. The incidence of homozygous factor X deficiency is ~ 1 in 1 million of the general population. The gene encoding for factor X is found adjacent to that encoding for factor VII on chromosome 13q34. Bleeding sites vary according to the severity of the deficiency. Mucocutaneous soft tissue hemorrhages, including menorrhagia in women, are common. Hemarthros, exsanguinating postoperative hemorrhage, pseudotumors, and hemorrhages of the central nervous system have been reported in severely affected patients. Mildly affected patients experience easy bruising and excessive bleeding after trauma or surgery. Treatment options consist of fresh frozen plasma (FFP), prothrombin complex concentrates (PCC) containing factor X or pasteurized Factor IX P® (ZLB Behring). Disadvantage of FFP is the large infusion volume, potential viral transmission, and no standardized factor X content. These aspects, in addition to the thrombotic risk, also need to be addressed for the PCCs. Factor IX P®, which is virus inactivated, contains almost equal amounts of factor IX (1200 IU) and X (800 IU) and suits therefore well for the treatment of factor X deficiency.
Case report: We report on our experience of prophylactic treatment with Factor IX P® in a 31-year-old male with severe factor X deficiency (&lt; 1%) associated with a homozygous Cys350Phe mutation in exon 8 on chromosome 13. After birth the patient experienced severe mucosal bleedings and haematomas and later on various joint bleedings with consecutive hemophilic arthropathy. Initially he received FFP on demand and later regular prophylaxis with PCC (containing 600 IU factor X) 2 to 3 times a week (~ 20–25 IU/kg/bw), age at onset of prophylaxis ~ 7 years. The patient is positive for HIV, HCV, and HBV (known since 1984). He is now on regular prophylaxis with Factor IX P® since 7 months. The prophylaxis is given 2 times a week in doses of ~ 20 IU/kg bw. The trough level after 72 hours was 12% using PCC and 20% using Factor IX P®. The patient reported on joint pain when factor X activities were below 20%. The rate of joint pain episodes is lower when using Factor IX P® two times a week as compared to PCC two to three times a week. Orthopedic and dental surgery were performed using Factor IX P® concentrate with excellent hemostatic effect, no thromboembolic complications, and no adverse drug reactions.
In conclusion, prophylactic treatment with Factor IX P® in severe factor X deficient patients appears to be an effective and safe therapeutic option.
Acquired Factor IX (fIX) Deficiency Associated with a Multiple Myeloma.
Authors: Lissandre Severine , Ternisien Catherine , Rousselet Marie-Christine , Tanguy-Schmidt Aline , Ifrah Norbert , Hunault-Berger Mathilde 
Published: April 2020

Abstract
Multiple myeloma is associated with bleeding disorders in about 15 % of patients. The pathophysiology is multifactorial and may be related to the paraprotein level. The bleeding tendency has been reported to be caused by thrombocytopenia, paraprotein-induced qualitative platelet dysfunction, inhibition of polymerization of fibrin monomers, monoclonal thrombin inhibitor, factor X deficiency, acquired von Willebrand syndrome and local tissue fragility secondary to amyloidosis. Here, we report the first case of acquired fIX deficiency associated with a multiple myeloma (MM) in a 54 year-old Caucasian man.
The patient presented with a Monoclonal Gammapathy of Undetermined Significance (MGUS) (IgA lambda 15 g/L). His laboratory evaluation then revealed an activated prothrombin time (aPTT) of 37 s (normal 29–36 seconds) without any bleeding disorder. Three years later, the MGUS evolved into a stage III multiple myeloma (IgA lambda 55 g/L) and the patient presented a hemorrhage after a bone marrow biopsy. At this time, the coagulation tests showed an aPTT of 54 s, a thrombin time of 27 s (normal 20–25 seconds), a normal platelet count without any other abnormalities. Clotting factors assay revealed a fIX deficiency of 10 %. No inhibitor was found using the Bethesda method and no fIX mutation was detected in the patient DNA as well as in his sister’s. After receiving chemotherapy (VAD-type chemotherapy and high-dose of Melphalan with autologous stem cells transplantation), the paraprotein concentration decreased to 9 g/L and the coagulation results returned to normal. Sixteen months later, the MM recurred (IgA lambda 78 g/L) and the patient presented hemorrhagic manifestations associated with an aPTT of 70 s and a fIX deficiency at 11 %. Factor IX deficiency and bleeding diathesis were detected in every MM relapses. He died during a severe bleeding event (epistaxis, hematuria and rectorrhagia) despite fIX concentrate treatments.
The etiology of this acquired fIX deficiency is not clear. The simultaneous evolution of MM and fIX deficiency, and the absence of congenital hemophilia mutation confirm that it is an acquired deficiency. Nevertheless there is no fIX inhibitor. Until now no case of acquired fIX deficiency is described in the literature apart from Gaucher disease. Our hypothesis is that fIX can be adsorbed by the malignant plasma cells. We are currently performing immunostaining of fIX in the bone marrow of this patient to assess whether fIX is adsorbed by the medullar malignant cells.
Efficacy, Safety and Economic Evaluation of Recombinant Activated Factor VII (rFVIIa) in the Management of Mild and Moderate Bleedings in Haemophilia Patients with Inhibitors in Brazil.
Authors: Ozelo Margareth C. , Villaça Paula R. , Bueno Telma , Almeida Jussara , Miranda Paulo A. , Hart Warren M. 
Published: April 2020

Abstract
Background: In Brazil treatment option for mild and moderate bleeding episodes in high responding inhibitors patients is Activated Prothrombin Complex Concentrates (aPCC). rFVIIa is only used as second line therapy in severe bleeds or for elective surgeries.
Objectives: To evaluate efficacy, safety and economic impact of rFVIIa as first line treatment for mild and moderate bleeds in high responding inhibitors patients.
Methods: Efficacy and safety of rFVIIa were based on a prospective evaluation of 22 mild and moderate bleeds treated between July 2004 and February 2006. Resource utilization was based on retrospective and prospective analysis of 66 bleeding episodes treated in less than 12 hours of beginning of symptoms with rFVIIa and aPCC during the period of August 2002 to February 2006. Clinical outcomes were derived from the analysis of the data obtained from the review of patients’ records and prospective evaluation. Results were validated through a consensus of four Brazilian hematologists and a decision analyis model was constructed to assess total direct health care costs using the software package DATATM 3.5 (TreeAge Software, Willianstown - MA, USA).
Results: From July 2004 to February 2006, nine mild (40.9%) and thirteen moderate (59.1%) bleeding episodes were prospectively treated with rFVIIa. All bleedings resolved with 1–4 doses (average 2.05). Haemostasis was maintained in 18/22 bleedings for five days (81.8%). No adverse events were reported. From August 2002 to May 2004 other 44 mild and moderate bleeding episodes were retrospectively reviewed for the economic evaluation, 33 with aPCC and 11 with rFVIIa. Overall, mean time to bleed resolution in hours, mean number of doses and efficacy rate were 56.8, 3.7, 63.6% and 2.7, 1.6 and 100% for aPCC and rFVIIa, respectively. Using the decision analysis model constructed, total direct costs were US$ 12,134 and US$ 7,307 for aPCC and rFVIIa, respectively. When sensitivity analysis was carried out using first-line efficacy rates based on literature data (aPCC: 80% and rFVIIa: 92%)1,2, the total process costs were lower for rFVIIa (US$ 7,850) as compared with aPCC (US$ 10,328) as initial treatment.
Conclusion: On the basis of the results of this study it could be concluded that the use of rFVIIa as first-line treatment for mild and moderate bleeds in haemophilia patients with inhibitors in Brazil is not only justified on a clinical basis but also from and economic point of view.
Minor Surgical Procedures - Survey of Hematologists To Determine Preferences Regarding Management of Hemophilia Patients.
Authors: Saxena Kapil , Nabi Sumbal , Vesely Sara K. , Pethe Kalpana , Hawk Sarah M. , Johnson Kristina , Stine Kimo 
Published: April 2020

Abstract
Objective: To determine individual/institutional practices at hemophilia treatment centers (HTCs) regarding management of severe hemophilia patients during minor surgery (e.g., central venous line insertion, cataract surgery, lymph node biopsy), both in the absence and presence of low and high titer inhibitors to Factor VIII (FVIII).
Background: The management of hemophilia patients undergoing surgery has changed over the past 20 years as product choice and availability has evolved. Individual preference and experience may affect treatment decisions in the absence of data from clinical trials.
Methods: The survey was distributed to both adult and pediatric hematologists at HTCs listed on the CDC website. 93 surveys were successfully emailed. Survey questions evaluated management of minor surgery, both in the absence and presence of a low and high titer inhibitor to FVIII.
Results: Surveys from 32 HTCs were received. Twenty-two respondents were affiliated with a University Hospital. For patients without inhibitor, the majority of respondents prefer a pre-operative bolus dose of FVIII 50 U/kg (73%, 22/30; range 25–50 U/kg); only 3 respondents reported a continuous infusion (range 4–8 u/kg/hr). For the post-operative dose, most (n=22) prefer a bolus dose (25–50 U/kg); only 6 reported using a continuous dose (range 2–8 U/kg/hr). Total number of days treated pre-operatively was reported as less than a day in 88% (23/26) respondents; days of post operative treatment ranged from 1–14 with a median of 4 days (n=22). Desired FVIII level ranged from 50–100% for 1–10 days. Fifty percent (15/30) reported adjuvant use of Amicar. For patients with high inhibitor levels no one reported a pre or post operative FVIII bolus or continuous infusion rate. Fifty percent (9/18) reported adjuvant Amicar use. The reported rFVIIa dose in patients with high titer inhibitors, ranged from 90–200 mcg/kg (most commonly 90 mcg/kg), usual frequency of every 2 hours, for a range of 1–10 days. The reported FEIBA dose in patients with high titer inhibitors, ranged from 50–100 U/kg (most commonly 75–100 U/kg with a frequency of every 12 hours, for 1–10 days.
Conclusion: Substantial practice variations exist in managing minor surgeries in hemophilia patients, both with and without low and high titer inhibitors, as is evident with this survey.
Major Surgical Interventions - Survey of Hematologists To Determine Personal/Institutional Preferences Regarding Management of Hemophilia Patients.
Authors: Saxena Kapil , Vesely Sara K. , Nabi Sumbal , Pethe Kalpana , Hawk Sarah M. , Johnson Kristina , Stine Kimo 
Published: April 2020

Abstract
Objective: To determine individual/institutional practices at hemophilia treatment centers (HTCs) regarding management of severe hemophilia patients during major surgery (e.g., knee replacement, appendectomy), both in the absence and presence of low and high titer inhibitors to Factor VIII (FVIII).
Background: There are considerable variations in the management of hemophilia patients undergoing surgery. The use of alternative products like Amicar, recombinant Factor VIIa (rFVIIa) and FEIBA is also growing.
Methods: The survey was distributed to both adult and pediatric hematologists at HTCs listed on the CDC website. Ninety-three surveys were successfully emailed. Survey questions evaluated management of hemophilia patients during major surgical procedures, both in the absence and presence of a low and high titer inhibitor.
Results: Surveys from 32 HTCs were received. Twenty-two respondents were affiliated with a University Hospital. For patients without inhibitor, the majority of respondents prefer a pre-operative bolus dose of 50 U/kg (84%, 22/28, range 40–70 U/kg); 15 respondents prefer a continuous dose (range 2–8 U/kg/hr). For the post-operative dose, 11 reported a bolus dose, usually 25–50 u/kg; 17 reported using a continuous dose (range 2–8 U/kg/hr). Total number of days treated pre-operatively was reported as less than a day by 90% (18/20) respondents; days of post operative treatment ranged from 7–42 with a median of 13 days (n=22). Desired FVIII level ranged from 50–100% for 3– 15 days. Median number of days inpatient was 6 (n=21). Eight percent (2/25) reported adjuvant use of Amicar. Thirty-nine percent (11/28) reported use of thrombotic prophylaxis after surgery. For patients with high titer inhibitor levels, only 1 respondent reported a pre or post operative FVIII bolus or continuous dose. The reported rFVIIa dose in patients with high titer inhibitor, ranged from 90–270 mcg/kg (most reporting 90 mcg/kg), with most reporting a frequency of every 2 hours for a range of 1–14 days. The reported FEIBA dose in patients with high titer inhibitors ranged from 50–100 U/kg (most 75 U/kg) with most reporting a frequency of every 12 hours for 6–14 days. Sixty four percent (7/11) reported use of thrombotic prophylaxis after surgery.
Conclusion: Substantial practice variations exist in managing major surgeries in hemophilia patients, both with and without low and high titer inhibitors. This data will assist in developing future clinical trials and practice guidelines for management of hemophilia patients undergoing surgical procedures.
High Incidence of Thromboembolic Complications and PF4 Positivity in Patients with Ventricular Assist Devices.
Authors: McMahon Brandon J. , Ifthikharuddin Jainulabdeen J. , Dib Elie , Chen Leway , Francis Charles W. , Massey Todd H. 
Published: April 2020

Abstract
Patients with ventricular assist devices (VAD) are at high risk for thromboembolic complications (TEC) probably due to platelet activation and excessive thrombin generation once the device is implanted. This warrants prophylactic use of full dose anticoagulation, usually with unfractionated heparin (UFH), which increases the risk for development of heparin-induced thrombocytopenia (HIT). We retrospectively evaluated the incidence of TEC and PF4 positivity in 50 sequential patients who underwent VAD placement for severe ventricular dysfunction between November 2002 and April 2005. Median age at VAD placement was 52 years (range 18–72), 68% were men, and all patients were on full dose UFH post-VAD insertion. TEC were observed in 22/50 patients (44%), with 8 patients experiencing &gt;1 event. CNS embolic phenomena occurred in 11/22 (50% of TEC), with watershed/non-embolic CNS infarcts being identified in 2 additional patients. Splenic infarcts were identified in 6/22 (27%). Two patients had lower extremity deep vein thromboses (DVT), and two patients had pulmonary emboli. One patient died from complications of multiple infarcts involving his spleen and gut that were attributed to HIT. All patients became thrombocytopenic (&lt;150K) at some point during VAD/UFH use. 44 patients (88%) dropped the platelet count below 100K with an average nadir of 56K. Antibodies to the PF4/heparin complex were checked using a standard enzyme immunoassay (EIA) in 38 (76%) patients, and found to be positive in 17 (44.7%). PF4 antibodies were detected using the EIA in 50% of patients with TEC and in 25% of patients with no documented TEC. A functional antibody assay was checked in a total of 17 patients, the serotonin release assay (SRA) in 13 and heparin-induced agglutination (HIA) in 4 cases. The SRA was only positive in 2/13 cases, and concordant with the PF4 result both times. The HIA was positive once, and concordant with the PF4 result. These results highlight the high incidence of TEC in patients with a VAD, despite anticoagulation therapy. There is a particularly high rate of embolic stroke in this patient population, and better ways of preventing this complication need to be explored. Thrombocytopenia is also extremely common post-VAD placement, prompting evaluation of HIT in over ¾ of the patients, often necessitating use of direct thrombin inhibitors. The frequent detection of PF4 antibodies in this patient group and lack of correlation with functional assays illustrates the need for more specific laboratory means to quickly confirm those patients with HIT.