bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of PEGylated Factor VIII Molecules.
Authors: Severs Joanne , Samuels Nicole , Teare John , Tollner Amy , Regan Lisa
Published: April 2020

Patients with hemophilia A typically require frequent treatment with replacement Factor VIII (FVIII) products due to the relatively short circulating half-life of FVIII. Increasing the half-life of FVIII would profound impact the lives of patients with hemophilia A, especially children, by reducing the frequency of infusions. The addition of polyethylene glutamate (PEG) to therapeutic molecules or their carriers has been demonstrated to increase the survival of such molecules in the circulation. To create a longer circulating product, we have developed novel FVIII molecules using site specific PEGylation techniques. We present a characterization of these PEGylated FVIII molecules in terms of the site of PEGylation, the binding of von Willebrand Factor (vWF), and the effect of PEGylation on thrombin digestion, and we compare the one-stage and chromogenic potency test results. The site of PEGylation was determined using MALDI-mass spectrometry. A vWF binding assay was developed using surface plasmon resonance technology (Biacore). The effect of PEGylation on thrombin digestion of the novel constructs was evaluated using MALDI-mass spectrometry, thrombin activation, and potency decay curves following thrombin activation. All novel constructs were PEGylated on the expected target site based on the site of introduced mutation. PEGylation of FVIII appeared to have a slight reduction on vWF-binding capability. The site of PEGylation reduced the ability of thrombin to digest FVIII, as assayed by MALDI-mass spectrometry analysis and by thrombin digestion and decay curves. The size and position of the PEG molecule influenced the extent of FVIII thrombin sensitivity. The one-stage potency assay was more sensitive to the size and position of the attached PEG molecule than the chromogenic potency assay. We report that some PEGylated FVIII molecules are less sensitive to thrombin activation than unmodified FVIII, and that the size and position of the PEG molecule can significantly affect the potency of FVIII. The findings of this study suggest that site-directed PEGylation of FVIII may be a useful technique for the development of FVIII molecules with extended activity in circulation and that careful choice of size and position of the PEG molecule is critical.
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

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 (<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 < 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

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 (<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 (>100 nM) resulted in loss of the metalloprotease activity towards VWF73aa FRET (2).

In this study, we found that low concentrations (<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 (>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

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 (< 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

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

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

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

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

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 >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 (<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.
Derived/Clauss Fibrinogen Ratio as a Potential Adjunct to D-Dimer in the Diagnosis of DVT.
Authors: Wakeman Lisa J. , Munro Roger C. , Blundell Victoria , Al-Ismail Saad , Benton Ann , Dorward Nick , Beddall Andrew , Morris Keith
Published: April 2020

Introduction

Along with clinical assessment, D-dimer (D-d) assays are routinely used to exclude DVT. It has been suggested that measurement of derived fibrinogen (DF) may be an effective reflection of endogenously-derived thrombin generation and that ratios between this and Clauss-derived fibrinogen (CF) may be useful in determining whether patients have experienced, or are vulnerable to thromboembolic events. Some studies indicate that a D-d/fibrinogen ratio is significantly higher among patients with confirmed DVT. The purpose of this study was to determine whether DF/CF ratios in individuals are a useful adjunct to D-d assays in the detection of DVT compared to D-d assay in isolation.

Methods

Venous samples were collected into glass B-D Vacutainers containing tri-sodium citrate (Becton-Dickinson, Plymouth, UKRef: 367691) from 162 out-patients presenting to the medical admissions unit with suspected DVT. Laboratory staff (N=100) served as the normal control group. D-dimer (MiniVidas, BioMerieux), DF and CF (Dade-Behring reagents in combination with a CA1500 coagulometer) were measured on all patients and normal control samples within two hours of collection.

Results

Following clinical assessment (Wells scoring), 85 patients were considered not to have had a DVT. Doppler scanning confirmed DVT in 38 of the remaining patients and 39 were shown to be negative. Two sample t-test analysis of the data showed significant differences between DF and CF levels in the normal group (n=100), patients who did not have a DVT (n=124) and those who did have a DVT (n=38), (p = <0.05 in each group). There was a significant difference in the DF/CF ratios between the three groups (p = 0.014, one-way ANOVA). The mean DF/CF ratios in the normal control group, patient negative group and patient positive group were 1.16, 1.22 and 1.24 respectively. There was no overall correlation between D-d and DF (correlation co-efficient = 0.646), D-d and CF (correlation co-efficient = 0.581) nor between D-d and DF/CF. Of the DVT positive patients, 26 had a raised DF whereas only 12 had a raised CF.

Conclusion

Our data suggest that DF/CF ratios cannot be used as an adjunctive marker of DVT when used in combination with D-d values. DF/CF ratios are significantly higher in patients attending for clinical assessment irrespective of whether they are DVT positive or DVT negative. This may be because fibrinogen is an acute phase protein which increases with various pathological states and stressful events such as the experience of attending hospital for investigation and assessment. Further work is required to determine whether DF/CF ratios can be used adjunctively when the D-d cut off value is significantly higher than that used conventionally.
Homozygosity in the Single Nucleotide Polymorphism Ser128Arg in the E-Selectin Gene Is Associated with Recurrent Venous Thromboembolism.
Authors: Hron Gregor , Jilma Bernd , Marsik Claudia L. , Endler Georg , Eichinger Sabine , Kyrle Paul Alexander
Published: April 2020

Objective: The single nucleotide polymorphism (SNP) Ser128Arg in the E-selectin gene is over-represented in certain patient populations with atherosclerosis or restenosis. As this SNP enhances tissue factor triggered coagulation in humans during systemic inflammation, we hypothesized that it may also predispose for the development of recurrent venous thromboembolism (VTE).

Methods and Results: Patients (n=585) after first VTE were prospectively followed up for recurrent, objectively documented, symptomatic VTE. Patients with secondary VTE, homozygous Factor V Leiden, natural inhibitor deficiencies, lupus anticoagulant, or long term anticoagulation were excluded. The S128R SNP was genotyped by mutagenically separated PCR. One-hundred and two patients (17.4%) were heterozygous and 11 were homozygous (1.9%) for the Ser128Arg mutation. Ninety patients (15.4%) suffered from recurrent VTE during follow-up. Homozygosity for the Ser128Arg SNP increased the cumulative likelihood particularly for early recurrent VTE (log rank test p<0.05) and was an independent predictor of recurrent VTE (hazard ratio: 4.1; 95% CI 1.5–11.4) in a multivariate Cox Regression model. In contrast, heterozygosity for the polymorphism was associated with an unaltered hazard ratio (HR: 1.1; 95% CI 0.6–1.9) for recurrent VTE.

Conclusion: Homozygosity for the S128R E-selectin allele appears to increase the risk for recurrent VTE several-fold, and -if confirmed- may represent a novel risk factor for recurrent VTE. These results also expand our knowledge on the association of this SNP with thrombotic disorders.
Factor V Leiden and Prothrombin G20210A Gene Mutations Should Not Be Routinely Screened among Venous Thromboembolism (VTE) Patients in Singapore - This and Patterns of Other Thrombophilic Conditions among Our VTE Patients.
Authors: Ng Heng Joo , Lee Lai Heng , Liu Te-Chih , Tan Lip Kun , Kuperan Ponnudurai
Published: April 2020

Introduction: Emerging good quality evidence has suggested that the prevalence of VTE among Asians is significant and was largely underestimated previously.

Objective and methods: We conducted a study of thrombophilic markers among our population of VTE patients to determine their prevalence and help determine the best screening strategy among our increasing number of VTE patients. Three major hospitals in the country were involved in this study.

Results: Of 254 patients recruited, ethnic distribution was as follows: Chinese 68.1%, Malays 17.3%, Indians 12.6%, Others 2%. The ratio of males to females was 2:3. Major risk factors such as surgery, trauma and malignancy was recordedand identified in about 80% of the population. Twenty-one percent of patients had pulmonary embolism. Positive results of thrombophilic markers were as follows: protein C deficiency, 4 patients (1.52%); protein S deficency, 26 (10.24%), antithrombin III deficiency, 11 (4.33%); lupus anticoagulant, 9 (3.54%); anticardiolipin antibody IgG, 3 (1.18%); anticardiolipin antibody IgM, 6 (2.36%); hyperhomocysteinemia, 32 (12.6%); factor V Leiden gene mutation, 0 (0%); prothrombin 20210 gene mutation, 0 (0%). The prevalence of at least one thrombophilic condition was found in about one third of our patients. The relative risks of these thrombophilic conditions will be compared with a healthy control population.

Conclusion: Among our VTE patients, the most commonly associated thrombophilic conditions were protein S and antithrombin III deficiencies. The factor V Leiden and prothrombin G20210A mutation did not feature at all in our patient population and is consistent with other studies on Asian populations. These tests should not be included as part of our thrombophilic screening.
Preemptive Immunotherapy with Highly Purified CD56+/CD3− Natural Killer Cells after Haploidentical Stem Cell Transplantation. A Prospective Phase II Study in 2 Centers.
Authors: Passweg Jakob R. , Koehl Ulrike , Stern Martin , Tonn Torsten , Meyer-Monard Sandrine , Bader Peter , Rischewski Johannes , Soerensen Jan , Paulussen Michael , Gratwohl Alois , Klingebiel Thomas , Tichelli Andre , Schwabe Dirk
Published: April 2020

Allogeneic natural killer cells may exert cytotoxic activity against HLA-nonidentical tumor cells. This effect may be exploited after T-cell depleted haploidentical hematopoetic stem cell transplantation (HSCT) by using donor NK-cell infusions (NK-DLI) as adoptive immunotherapy.

In a prospective phase II study in 2 centers we treated 15 patients with NK-DLI preemptively with the goals to demonstrate feasibility and to explore whether such infusions would contribute to stabilize engraftment and exert graft versus leukemia activity without causing GvHD.

Patients were adults (6) or children (8) receiving transplants from haploidentical donors to treat AML (7) ALL (5) Hodgkin lymphoma (2) or sarcoma (1). Donors were haplotype mismatched siblings in 3 and parents in 12.

Donor NK cells were selected from unstimulated leukapheresis using immunomagnetic T cell depletion with anti-CD3 and NK cell enrichment with anti-CD56 coated microbeads on the CliniMACS ® device. Per protocol, patients in center A received NK-DLI on days +40 and +100 and in center B on days +3, +40 and +100. NK-DLI were either freshly infused or cryopreserved and thawed.

Center A Center B N 8 7 Adults / Children 6/2 0/7 Disease: AML/ALL/HD/oth 6/1/1/0 1/4/1/1 Number of NK-DLI per patient 1/2/3 2/5/1 2/3/2 Median NK-cell recovery (%) 62 37 NK-cell dose (10e7/kg) (median, range) 1.2 (0.5–3.4) 1.4 (0.66–3.23) T-cell dose (10e4/kg) (median, range) 0.2 (0–7.2) 0.23 (0–5.3) Events: graft failure / progression / GvHD (III–IV) 2/2/1 1/2/1 Alive / Dead 3/5 5/2 Cause of death: graft failure/progression/GvHD 2/2/1 0/2/0

NK-cell collection and ex vivo purification was successful in all donors. Outcome is shown in the Table. Of note: One patient in each center developed severe (grade III / grade IV) GvHD respectively. These 2 patients had received the highest T-cell dose. The remaining patients tolerated NK-DLI well and did not develop GvHD. Two patients in Center A experienced graft failure after NK-DLI in spite of NK-alloreactivity in GvHD direction.

In conclusion: highly purified NK cells may be infused in recipients of haploidentical HSCT recipients. None of the patients receiving < 10e4/kg CD3+ cells developed GvHD. Further studies are needed to determine the optimal dose and timing of NK-DLI for anti-tumor activity in this setting.
Treatment of Deep Vein Thrombosis with Continuous I.V. Infusion of LMWH in Children - Safe and Efficient Alternative to S.C. Application.
Authors: Fiamoli Veronika , Blatny Jan , Zapletal Ondrej , Kohlerova Svetlana , Penka Miroslav
Published: April 2020

Incidence of the thrombosis is age dependent with the lowest risk is in the childhood. Children mostly suffer from vein thrombosis (DVT/PE). Occurrence of thrombosis in children is 0,07/10 000, among hospitalized children is higher 3,5/10 000. In comparison with adults, these numbers are very low. LMWH is preferred in the treatment of DVT in children. UFH is used as second choice medication. LMWH is widely used in prevention of recurrence of DVT and sometimes it substitutes (especially in very young children) vitamin K antagonists. The most frequent form of application of LMWH is via subcutaneous injection. A few articles can be found in literature about treatment by intravenous continuous infusion in infants and in several cases in older children. The treatment by continuous infusion has certain advantages, especially for hospitalized patient with permanent i.v. access (painless, shorter half-life consecutively lower risk of bleeding, easier control). We present group of 27 children with DVT, who were treated with LMWH for first thrombosis from 2003 till 2005. We did the screening of thrombophilia in all of those children. We found FVleiden heterozygous in 38,1%, FII G2021OA heterozygous in 4,76, inherited deficiency of AT in 14,29%, BCP in 9,52 % and CVL was inserted in 52,38% of our patients.All patients had at least one of the risk factors of thrombophilia. We started to treat all patients with LMWH and the dose was adjusted to reach the level of antiXa between 0,5–1 IU/ml. We treated 6 patients (22,22%) by subcutaneous injection (average LMWH dose 215,54IU/kg/d) and 21 patients (77,78%) by continuous infusion (average dose 254,94IU/kg/d). Duration of the treatment was modified in accordance with the course of thrombosis (monitored by Doppler ultrasound with compression), but it went on for at least 7 days and was followed by the prophylaxis with LMWH or vitamin K antagonists. The treatment with LMWH (both i.v. or s.c.) lead to recanalisation of the vein in 26 cases (96,29%), 58% of them without any residue or mural thrombosis. We did not notice any bleeding as adverse event of the treatment in our patients.We would like to point out that treatment of DVT with continuous infusion of LMWH in children might be efficient and safe alternative of to s.c. application in certain circumstances. When administered by continuous infusion, LMWH has shorter half-life and it could be useful especially in hospitalized children and in children who are endangered by risk of bleeding complications.
Transplantation without Hemolysis in a Kell Antigen Positive Patient Undergoing Peripheral Hematopoietic Stem Cell Transplant from a Donor with Anti-Kell Antibodies.
Authors: Crawford Regina D. , Krugh David , Blum William , Devine Steven
Published: April 2020

Red blood cell (RBC) antibody formation, with or without hemolysis, is a well known post-transplant complication of ABO and non-ABO mismatched patients and is seen in both solid organ and hematopoietic stem cell transplants (HSCT). Hemolysis secondary to donor lymphocytes transfused during transplant, which produce antibodies directed toward the patient’s remaining red blood cells, is most commonly reported among HSCT donor-recipients with minor ABO blood group mismatching. Passenger Lymphocyte Syndrome (PLS) has been noted to occur in non-ABO group mismatched HSCTs involving the following blood group systems: Rh, Kidd, Lewis, MNS and Duffy. We present a case of a patient with acute myeloid leukemia (AML) in first complete remission who was Kell antigen positive and underwent a non-myeloablative peripheral blood HSCT from a 10/10 HLA matched, related donor who had anti-Kell antibodies. The patient was a 69-year-old male with AML in first remission who consented for transplant with non-myeloablative conditioning on CALGB 100103. At the time of transplantation, the patient was ABO type A positive, had a negative antibody screen, and was positive for the Kell (K1) antigen. The patient had not been transfused RBC or platelets for 4 months prior to HSCT. The related donor was found to be A positive and had anti-Kell that was reactive 1+ at antihuman globulin phase (GEL) prior to filgrastim mobilized peripheral blood HSC collection. The patient received a conditioning regimen of fludarabine 30mg/m2 daily (days -7 to -3) and bulsulfan 0.8mg/kg IV every 6 hours for 8 doses (days -4 to -3). GVHD prophylaxis consisted of methotrexate (5mg/m2) on day +1, +3, and +6 and tacrolimus starting on day -2. After HSCT, no RBC or platelet transfusions were required throughout the patient’s course. Neutrophil and platelet engraftment occurred by day +20. Chimerism studies on peripheral blood[c1] at day +30 revealed 61% donor CD3 T cells, 99% CD14 and CD15, myeloid cells, 90% CD19, B cells, and 96% donor whole blood. Weekly direct antiglobulin tests were performed (day +7 through day +21) with monitoring of hemoglobin, hematocrit, LDH, total and direct bilirbuin, BUN, and creatinine to evaluate for hemolysis. No evidence of hemolysis was seen in the immediate post-transplant period, and the patient’s antibody screen has remained negative to date through day +100. In conclusion, this case demonstrates that peripheral blood HSCT may be done safely in the setting of non-ABO incompatibility due to Kell in the donor-recipient direction. Given the increasing use of non-myeloablative conditioning regimens and peripheral blood HSCT, both of which increase the risk of PLS, the case highlights that the risks of hemolysis for HSCT are evolving and demonstrates the successful implementation of one strategy for monitoring at risk patients.
Coagulopathy from Severe Postpartum Hemorrhage.
Authors: James Andra H. , Paglia Michael , Grotegut Chad , Thames Betty , Gernsheimer Terry
Published: April 2020

OBJECTIVE: The purpose of this study was to estimate the incidence and risk factors associated with coagulopathy from severe postpartum hemorrhage.

STUDY DESIGN: After approval by the Institutional Review Board at Duke University Medical Center, records with the ICD-9 code 666.12 for immediate postpartum hemorrhage were reviewed for the 5-year period from January 1, 2000, to December 31, 2004. Severe postpartum hemorrhage was defined as postpartum hemorrhage requiring transfusion. Coagulopathy from postpartum hemorrhage was defined as severe postpartum hemorrhage requiring factor replacement.

RESULTS: During this period there were 12,476 deliveries. 108 had severe postpartum hemorrhage. 30/108 became coagulopathic. They received a total of 108 units of fresh frozen plasma (FFP) and 12 units of cryoprecipitate (cryo). 28/30 also received 1 to 20 units of packed red blood cells and 5/30 received 1 to 3 units of platelets. All of the women who received cryo had also received FFP. The trigger for administration of factor replacement was clinical (oozing, continued bleeding) in 13/30 cases and a prolonged partial thromboplastin time (PTT) in 17/30 cases. Peak PTTs ranged from 27 to > 150 seconds. Lowest fibrinogen levels ranged from 27 to 580. In 2 cases, however, no fibrinogen levels were obtained. Hemorrhage was attributed to uterine atony in 11/30 cases, placenta accreta in 6/30, lacerations or incisions in 6/30, HELLP syndrome in 2/30, acute fatty liver of pregnancy in 1/30, amniotic fluid embolism in 1/30, fibroids, in 1/30, hemorrhage into an ovarian fibroma in 1/30, and chronic hepatitis B in 1/30. 22/30 cases were associated with cesarean delivery. In 12/30 cases hysterectomy was performed. Hospital length of stay ranged from 2 to 11 days with a mean of 5.4. 9/30 women spent from 1 to 3 days in the ICU. There were no deaths. 2 women had evidence of organ dysfunction from HELLP syndrome or acute fatty liver of pregnancy, but none developed organ dysfunction as a consequence of severe postpartum hemorrhage.

CONCLUSION: 2.4 per 1000 women developed coagulopathy as a result of severe postpartum hemorrhage. They received an average of 4 units of either FFP or cryo. 90% of the units were FFP and 10% were cryo. While 30% of the women required intensive care, all survived with no sequelae.
Dynamin 3 Is Differentially Expressed during Human Megakaryocyte Development.
Authors: Reems Jo Anna , Wang Wenjing , Tsubata Ken , Sundell Birgitta , Gilligan Diana M.
Published: April 2020

We found that expression of dynamin 3 (DNM3) is increased significantly when human CD34+/CD38lo cells are induced to develop along the megakaryocyte (MK) lineage. DNM3 is a member of a family of mechanochemical enzymes which participate in membrane dynamics. Of the 3 family members (DNM1, DNM2 and DNM3), cDNA microarray analysis showed that only DNM3 significantly increased by 9.5±1.8-fold (mean±SEM; p≤0.001). mRNAs for DNM1 and DNM2 were detectable, but their expression levels remained essentially unchanged during MK differentiation. Consistent with our microarray data, real time quantitative RT-PCR confirmed that DNM3 expression levels increased by as much as 20.0±7.3 fold during megakaryocytopoiesis. Western Blot analysis showed that protein expression patterns for each of the 3 dynamins differ in MKs/platelets. DNM3 protein expression levels are greater in MKs than in platelets. DNM1 was not detectable in MKs, but was present in platelets. Although DNM2 protein was detectable in both MKs and platelets, the relative mobility for DNM2 was slightly lower in platelets than MKs. Indirect immunofluorescent microscopy showed that DNM3 was predominantly in the cytoplasm with localization to sites that appear to be regions of pro-platelet formation. The cDNA encoding DNM3 was amplified from culture derived human MKs, cloned and sequenced. Sequence analysis revealed that the cloned product matched a DNM3 transcriptional splice variant in the Ensemble Human GeneView database (i.e. ENST00000367731). Together these results are the first evidence indicating that members of the dynamin family of mechanochemical proteins are present in human MKs and suggest that they may play an important role in MK membrane dynamics.
Continuous Erythroblastosis Is Observed during Early Recovery Period after Allogeneic Umbilical Cord Blood Transplantation.
Authors: Noguchi Mitsuho , Tashiro Haruko , Shirasaki Ryosuke , Gotoh Moritaka , Kawasugi Kazuo , Shirafuji Naoki
Published: April 2020

Objectives: The repopulation characteristics on human hematopoietic cells are crucial for clinical performance after allogeneic hematopoietic stem cell transplantation. We observed and compared the changes of blood cells during the early recovery period after allogeneic HSCT.

Method: The study included patients from 19 to 62 years of age who were admitted to our hospital during Dec. 2000 to Mar. 2005 for the allogeneic HSCT, and in whom no serious complications were observed on transplantation. A total of 25 patients were eligible (10 patients of U-CBT with stable engraftment, 2 patients of U-CBT with no engraftment and autologous recovery thereafter, 8 patients of BMT and 5 patients of PBSCT). All the patients received G-CSF. From day 0 of transplantation, the number of blood cells was calculated mechanically and microscopically, and their morphology was microscopically determined with the stained smear-slide. Five smear slide glasses were made by hand on each of the two days, and the cells were stained with May-Giemsa solution.

Result: Continuous erythroblastosis was observed in all the engrafted U-CBT cases, which was firstly observed between the same days as when blood WBC was increased in number, and five days later (on day 15 on average). Neither correlation of the ABO incompatibilities was observed to erythroblastosis, nor prominent hemolysis was observed. However, in BMT cases and in PBSCT cases, continuous erythroblastosis was observed in a few cases. Neither continuous erythroblastosis was observed in U-CBT cases that showed no engraftment of the transplanted graft.

Discussion: These observations indicate that continuous erythroblastosis during early recovery period is specific for U-CBT, and may reflect the difference in the characters of hematopoietic stem cells.
Elimination of Platelet Factor 4 (PF4) from Platelets Reduces Atherosclerosis and Increases HDL Cholesterol in C57Bl/6 and ApoE−/− Mice.
Authors: Sachais Bruce S. , Turrentine Tiffany , Dawicki-McKenna Jeanine M. , Rader Daniel J. , Kowalska M. Anna
Published: April 2020

There is a presence of circulating, activated platelets in blood of patients with atherosclerosis, coronary disease and hypercholesterolemia. Upon activation, platelets release a large amount of platelet factor 4 (PF4), a platelet specific chemokine. Our laboratory has previously demonstrated several potentially proatherogenic properties of PF4 including alteration of LDL metabolism and cellular trafficking, and activation of NFkB, a proinflammatory transcription factor involved in atherosclerosis. We have also localized PF4 to human atherosclerotic lesions. However, to date, no direct in vivo evidence for the involvement of PF4 in atherogenesis. In the current study, we have bred PF4−/− mice onto two athero-susceptible backgrounds, WT-C57Bl/6(WT) and apoE−/−, to examine the importance of PF4 in atherogenesis. PF4−/− and PF4−/−apoE−/− (DKO) mice are viable and healthy, with no spontaneous bleeding disorders. In order to induce atherosclerosis, WT and PF4−/− mice were fed an atherogenic Paigen diet for 30 weeks (Study 1), while apoE−/− and DKO mice were fed a high fat Western style diet for 10 weeks (Study 2). Examination of lesions in the aortic roots of Study 1 animals demonstrated a 5-fold reduction in PF4−/− compared to WT mice (p = 0.008). Measurement of cholesterol levels demonstrated similar total and non-HDL cholesterol levels in WT and PF4−/− mice. However, HDL cholesterol was significantly increased in PF4−/− mice compared to WT (2.5-fold, p = 0.001). Examination of apoE−/− mice (Study 2) demonstrated similar changes, with DKO mice demonstrating a 2.7-fold reduction in aortic atherosclerosis (measured by the en face method; p = 0.03) and a 1.7-fold increase in HDL cholesterol (p = 0.02) compared to apoE−/− mice. Although platelet counts were increased by ~30% in mice lacking PF4, the activation state of the platelets in our mice at sacrifice (WT vs PF4−/− and apoE−/− vs DKO) were similar as measured by both p-selectin expression and annexin V binding. These data demonstrate, for the first time, that the platelet specific chemokine PF4 promotes atherosclerotic lesion development in vivo.