The intron-22-inverted F8 locus permits factor VIII synthesis: explanation for low inhibitor risk and a role for pharmacogenomics.

Intron-22-inversion patients express the entire Factor VIII (FVIII)-amino-acid sequence intracellularly as 2 non-secreted polypeptides and have a positive "intracellular (I)-FVIII-CRM" status. Mutations conferring a positive I-FVIII-CRM status are associated with low inhibitor risk and are pharmacogenetically relevant because inhibitor risk may be affected by the nature of the therapeutic FVIII-protein (tFVIII), the affinity of any tFVIII-derived foreign peptide (tFVIII-fp) for any HLA class-II isomer (HLA-II) comprising individual major histocompatibility complex (MHC) repertoires, and the stability of any tFVIII-fp/HLA-II complex. We hypothesize that mutations conferring a completely or substantially negative I-FVIII-CRM status are pharmacogenetically irrelevant because inhibitor risk is high with any tFVIII and individual MHC repertoire.

Characterization of thrombosis in patients with Proteus syndrome.

Patients with overgrowth and complex vascular malformation syndromes, including Proteus syndrome have an increased risk of thromboembolism. Proteus syndrome is a mosaic, progressive overgrowth disorder involving vasculature, skin, and skeleton, and caused by a somatic activating mutation in AKT1. We conducted a comprehensive review of the medical histories and hematologic evaluations of 57 patients with Proteus syndrome to identify potential risk factors for thrombosis. We found that six of ten patients, who were deceased, died secondary to deep venous thrombosis and/or pulmonary embolism. Of the remaining 47 living patients, six had thromboembolic events that all occurred postoperatively and in an affected limb. Eleven of 21 patients had an abnormal hypercoagulable panel including Factor V Leiden heterozygotes, antithrombin III deficiency, positive lupus anticoagulant, or Protein C or S deficiencies. We observed that eight of 17 patients had an abnormal D-dimer level >0.5 mcg/dl, but deep venous thromboses occurred in only four of those with D-dimer >1.0 mcg/dl. We conclude that the predisposition to thrombosis is likely to be multifaceted with risk factors including vascular malformations, immobility, surgery, additional prothrombotic factors, and possible pathophysiologic effects of the somatic AKT1 mutation on platelet function or the vascular endothelium. The D-dimer test is useful as a screen for thromboembolism, although the screening threshold may need to be adjusted for patients with this disorder. We propose developing a registry to collect D-dimer and outcome data to facilitate adjustment of the D-dimer threshold for Proteus syndrome and related disorders, including PIK3CA-Related Overgrowth Spectrum.

Incidence and risk factors of bleeding-related adverse events in patients with chronic lymphocytic leukemia treated with ibrutinib.

Ibrutinib is associated with bleeding-related adverse events of grade ≤ 2 in severity, and infrequently with grade ≥ 3 events. To investigate the mechanisms of bleeding and identify patients at risk, we prospectively assessed platelet function and coagulation factors in our investigator-initiated trial of single-agent ibrutinib for chronic lymphocytic leukemia. At a median follow-up of 24 months we recorded grade ≤ 2 bleeding-related adverse events in 55% of 85 patients. No grade ≥ 3 events occurred. Median time to event was 49 days. The cumulative incidence of an event plateaued by 6 months, suggesting that the risk of bleeding decreases with continued therapy. At baseline, von Willebrand factor and factor VIII levels were often high and normalized on treatment. Platelet function measured via the platelet function analyzer (PFA-100™) was impaired in 22 patients at baseline and in an additional 19 patients on ibrutinib (often transiently). Collagen and adenosine diphosphate induced platelet aggregation was tested using whole blood aggregometry. Compared to normal controls, response to both agonists was decreased in all patients with chronic lymphocytic leukemia, whether on ibrutinib or not. Compared to untreated chronic lymphocytic leukemia patients, response to collagen showed a mild further decrement on ibrutinib, while response to adenosine diphosphate improved. All parameters associated with a significantly increased risk of bleeding-related events were present at baseline, including prolonged epinephrine closure time (HR 2.74, P=0.012), lower levels of von Willebrand factor activity (HR 2.73, P=0.009) and factor VIII (HR 3.73, P=0.0004). In conclusion, both disease and treatment-related factors influence the risk of bleeding. Patients at greater risk for bleeding of grade ≤ 2 can be identified by clinical laboratory tests and counseled to avoid aspirin, non-steroidal anti-inflammatory drugs and fish oils. ClinicalTrials.gov identifier NCT01500733.

Recombinant human factor VIIa for alveolar hemorrhage following allogeneic stem cell transplantation.

The mortality rate of alveolar hemorrhage (AH) after allogeneic hematopoietic stem cell transplantation is greater than 60% with supportive care and high-dose steroid therapy. We performed a retrospective cohort analysis to assess the benefits and risks of recombinant human factor VIIa (rFVIIa) as a therapeutic adjunct for AH. Between 2005 and 2012, 57 episodes of AH occurred in 37 patients. Fourteen episodes (in 14 patients) were treated with steroids alone, and 43 episodes (in 23 patients) were treated with steroids and rFVIIa. The median steroid dose was 1.9 mg/kg/d (interquartile range [IQR], 0.8 to 3.5 mg/kg/d; methylprednisolone equivalents) and did not differ statistically between the 2 groups. The median rFVIIa dose was 41 µg/kg (IQR, 39 to 62 µg/kg), and a median of 3 doses (IQR, 2 to 17) was administered per episode. Concurrent infection was diagnosed in 65% of the episodes. Patients had moderately severe hypoxia (median PaO2/FiO2, 193 [IQR, 141 to 262]); 72% required mechanical ventilation, and 42% survived to extubation. The addition of rFVIIa did not alter time to resolution of AH (P = .50), duration of mechanical ventilation (P = .89), duration of oxygen supplementation (P = .55), or hospital mortality (P = .27). Four possible thrombotic events (9% of 43 episodes) occurred with rFVIIa. rFVIIa in combination with corticosteroids did not confer clear clinical advantages compared with corticosteroids alone. In patients with AH following hematopoietic stem cell transplantation, clinical factors (ie, worsening infection, multiple organ failure, or recrudescence of primary disease) may be more important than the benefit of enhanced hemostasis from rFVIIa.

Gene therapy. Factor IX Padua: them that have, give.

In this issue of Blood, Finn et al have taken a factor IX variant with increased specific activity associated with thrombophilia and used it to improve gene therapy of hemophilia B in dogs, and Cantore et al have shown similar results in mice.

Targeting hematologic malignancies by inhibiting E-selectin: A sweet spot for AML therapy?

E-selectin, a cytoadhesive glycoprotein, is expressed on venular endothelial cells and mediates leukocyte localization to inflamed endothelium, the first step in inflammatory cell extravasation into tissue. Constitutive marrow endothelial E-selectin expression also supports bone marrow hematopoiesis via NF-κB-mediated signaling. Correspondingly, E-selectin interaction with E-selectin ligand (sialyl Lewisx) on acute myeloid leukemia (AML) cells leads to chemotherapy resistance in vivo. Uproleselan (GMI-1271) is a carbohydrate analog of sialyl Lewisx that blocks E-selectin binding. A Phase 2 trial of MEC chemotherapy combined with uproleselan for relapsed/refractory AML showed a median overall survival of 8.8 months and low (2%) rates of severe oral mucositis. Clinical trials seek to confirm activity in AML and mitigation of neutrophil-mediated adverse events (mucositis and diarrhea) after intensive chemotherapy. In this review we summarize E-selectin biology and the rationale for uproleselan in combination with other therapies for hematologic malignancies. We also describe uproleselan pharmacology and ongoing clinical trials.

Treatment of high-risk venous thrombosis patients using low-dose intraclot injections of recombinant tissue plasminogen activator and regional anticoagulation.

Seven patients with venous thrombosis and contraindications to traditional thrombolytic therapy, consisting of recent intracranial surgery, recent pineal or retroperitoneal hemorrhage, active genitourinary or gastrointestinal bleeding, epidural procedures, and impending surgery, were successfully treated with a modified thrombolytic regimen. To improve safety, prolonged continuous infusions of tissue plasminogen activator (tPA) was eliminated in favor of once-daily low-dose intraclot injections of tPA to minimize the amount and duration of tPA in the systemic circulation, and low-therapeutic or regional anticoagulation was used to reduce anticoagulant risks. These modifications may allow thrombolytic treatment for selected patients with severe venous thrombosis who are deemed to be at high risk.

Gene therapy for haemophilia: prospects and challenges to prevent or reverse inhibitor formation.

Monogenic hereditary diseases, such as haemophilia A and B, are ideal targets for gene therapeutic approaches. While these diseases can be treated with protein therapeutics, such as factor VIII (FVIII) or IX (FIX), the notion that permanent transfer of the genes encoding these factors can cure haemophilia is very attractive. An underlying problem with a gene therapy approach, however, is the patient's immune response to the therapeutic protein (as well as to the transmission vector), leading to the formation of inhibitory antibodies. Even more daunting is reversing an existing immune response in patients with pre-existing inhibitors. In this review, we will describe the laboratory and clinical progress, and the challenges met thus far, in achieving the goal of gene therapy efficacy, with a focus on the goal of tolerance induction.

Spray: single-donor plasma product for room temperature storage.

BACKGROUND: Spray-drying techniques are commonly utilized in the pharmaceutical, dairy, and animal feed industries for processing liquids into powders but have not been applied to human blood products. Spray-dried protein products are known to maintain stability during storage at room temperature. STUDY DESIGN AND METHODS: Plasma units collected at the donor facility were shipped overnight at room temperature to a processing facility where single-use spray drying occurred. After 48 hours' storage at room temperature, the spray-dried plasma product was split in two and rehydrated with 1.5% glycine or deionized water and assayed for chemistry analytes and coagulation factors. Matched fresh-frozen plasma was analyzed in parallel as controls. RESULTS: Reconstitution was achieved for both rehydration groups within 5 minutes (n = 6). There was no significant intergroup difference in recovery for total protein, albumin, immunoglobulin (Ig)G, IgA, and IgM (96% or higher). With the exception of Factor VIII (58%), the recovery of clotting factors in the glycine reconstituted products ranged from 72% to 93%. Glycine reconstitution was superior to deionized water. CONCLUSION: We documented proteins and coagulation activities were recovered in physiologic quantities in reconstituted spray-dried plasma products. Further optimization of the spray-drying method and reconstitution fluid may result in even better recoveries. Spray drying is a promising technique for preparing human plasma that can be easily stored at room temperature, shipped, and reconstituted. Rapid reconstitution of the microparticles results in a novel plasma product from single donors.

Low-dose, once-daily, intraclot injections of alteplase for treatment of acute deep venous thrombosis.

PURPOSE: To evaluate the safety and efficacy of once-daily intraclot injections of low doses (≤ 10 mg) of tissue plasminogen activator (tPA) for thrombolysis of venous thrombosis. MATERIALS AND METHODS: In prospective studies, 33 patients with subclavian, jugular, and central venous thrombosis (SJ-CVT) (all but two cases associated with central catheters) were treated once a day with ≤ 4 mg/day of tPA, and 30 patients with acute deep vein thrombosis of the lower extremity (DVT-LE) < 14 days old were treated once a day with ≤ 10 mg/leg/day of tPA by intraclot "lacing" of thrombus without continuous infusions of tPA. RESULTS: Patency was restored in 26 (79%) of 33 patients with SJ-CVT using an average total dose of 7.1 mg of tPA/per patient and average of 2.1 treatments or days of therapy. Five patients received thrombolytic therapy for SJ-CVT as outpatients. Initial patency was restored in 29 (97%) of 30 patients with acute DVT-LE using an average total dose of 20 mg of tPA per patient over an average of 2.7 treatments/or days per patient. Follow-up imaging examinations at 6 months showed continued patency in 27 (96%)/of 28 patients. There were no major bleeding complications, and no patient required a blood transfusion. CONCLUSIONS: Intraclot injection of low doses of alteplase is effective for acute venous thrombosis, and pharmacokinetic data suggest potentially greater safety.

Pseudoneutropenia in lymphangioleiomyomatosis (LAM) patients receiving sirolimus: evaluation in a 100 patient cohort.

In lymphangioleiomyomatosis patients receiving sirolimus treatment, transient leukopenia in the morning may be due to circadian rhythm, with leukocyte counts recovering later in the day, indicating that a decrease in drug dose may not be warranted http://ow.ly/jPFz30iysgV.

Low-dose, short course alteplase treatment of submassive pulmonary embolism: a case series from the National Institutes of Health Clinical Center.

: Guidelines-recommend thrombolytic therapy for pulmonary embolism in patients with severe hemodynamic compromise and low risk of bleeding. Thrombolytics in submassive pulmonary embolism have an unfavorable risk/benefit ratio and remain controversial. Based on our experience with extensive, lower extremity thrombi, nine patients with symptomatic, submassive pulmonary embolisms (five medical, four surgical) were treated with low-dose alteplase (<10 mg/day, infused over 6 h per treatment). Alteplase was delivered by pulse spray and/or directed or undirected central venous catheters depending on clot size and location. All patients improved symptomatically and as determined objectively by pulmonary artery pressures and/or imaging, though acute benefits ranged from substantial to modest. One surgical patient required re-exploration for bleeding at the site of a recent retroperitoneal lymph node dissection. This experience may help guide the design of a randomized controlled trial to determine the safety and efficacy of low-dose alteplase for submassive pulmonary embolism.

Thromboembolic adverse events after use of recombinant human coagulation factor VIIa.

CONTEXT: The US Food and Drug Administration (FDA) licensed recombinant human coagulation factor VIIa (rFVIIa) on March 25, 1999, for bleeding in patients with hemophilia A or B and inhibitors to factors VIII or IX. Use in patients without hemophilia has been increasing since licensure. OBJECTIVE: To review serious thromboembolic adverse events (AEs) reported to the FDA's Adverse Event Reporting System (AERS). DESIGN, SETTING, AND PATIENTS: The AERS database was reviewed from March 25, 1999, through December 31, 2004, for thromboembolic AE reports with rFVIIa. The AERS database includes US and non-US spontaneous AE reports from both approved (specific indications for patients with hemophilia) and unlabeled uses. It also includes serious AEs in patients enrolled in postlicensure clinical trials who received rFVIIa. Manufacturer reporting to FDA is mandatory, but primary notification from clinicians and others to FDA or manufacturers is voluntary for spontaneous reports; therefore, AERS underrepresents actual event occurrences. MAIN OUTCOME MEASURE: Reported thromboembolic events occurring in patients administered rFVIIa. RESULTS: A total of 431 AE reports for rFVIIa were found, of which 168 reports described 185 thromboembolic events. Seventeen events occurred in patients with hemophilia and 59 occurred in patients enrolled in postlicensure trials. Unlabeled indications accounted for 151 of the reports, most with active bleeding (n = 115). Reported AEs were thromboembolic cerebrovascular accident (n = 39), acute myocardial infarction (n = 34), other arterial thromboses (n = 26), pulmonary embolism (n = 32), other venous thromboses (including deep vein thrombosis) (n = 42), and clotted devices (n = 10). In 36 (72%) of 50 reported deaths, the probable cause of death was the thromboembolic event. In 144 patients with timing information, 73 events (52%) occurred in the first 24 hours after the last dose (30 events within 2 hours). Sixty-four reports (38%) noted concomitant use of hemostatic agents. Most reports lacked sufficient information to evaluate potential dosage associations. CONCLUSIONS: Most reported thromboembolic AEs followed the use of rFVIIa for unlabeled indications and occurred in arterial and venous systems, often resulting in serious morbidity and mortality. Analysis of the relationship between AEs and rFVIIa is hindered by concomitant medications, preexisting medical conditions, confounding by indication, and inherent limitations of passive surveillance. Randomized controlled trials are needed to establish the safety and efficacy of rFVIIa in patients without hemophilia.

Severe Hemophilia A in a Male Old English Sheep Dog with a C→T Transition that Created a Premature Stop Codon in Factor VIII.

Animals with hemophilia are models for gene therapy, factor replacement, and inhibitor development in humans. We have actively sought dogs with severe hemophilia A that have novel factor VIII mutations unlike the previously described factor VIII intron 22 inversion. A male Old English Sheepdog with recurrent soft-tissue hemorrhage and hemarthrosis was diagnosed with severe hemophilia A (factor VIII activity less than 1% of normal). We purified genomic DNA from this dog and ruled out the common intron 22 inversion; we then sequenced all 26 exons. Comparing the results with the normal canine factor VIII sequence revealed a C→T transition in exon 12 of the factor VIII gene that created a premature stop codon at amino acid 577 in the A2 domain of the protein. In addition, 2 previously described polymorphisms that do not cause hemophilia were present at amino acids 909 and 1184. The hemophilia mutation creates a new TaqI site that facilitates rapid genotyping of affected offspring by PCR and restriction endonuclease analyses. This mutation is analogous to the previously described human factor VIII mutation at Arg583, which likewise is a CpG dinucleotide transition causing a premature stop codon in exon 12. Thus far, despite extensive treatment with factor VIII, this dog has not developed neutralizing antibodies ('inhibitors') to the protein. This novel mutation in a dog gives rise to severe hemophilia A analogous to a mutation seen in humans. This model will be useful for studies of the treatment of hemophilia.

A1C Combined With Glycated Albumin Improves Detection of Prediabetes in Africans: The Africans in America Study.

OBJECTIVE: Slowing the diabetes epidemic in Africa requires improved detection of prediabetes. A1C, a form of glycated hemoglobin A, is recommended for diagnosing prediabetes. The glycated proteins, fructosamine and glycated albumin (GA), are hemoglobin-independent alternatives to A1C, but their efficacy in Africans is unknown. Our goals were to determine the ability of A1C, fructosamine, and GA to detect prediabetes in U.S.-based Africans and the value of combining A1C with either fructosamine or GA. RESEARCH DESIGN AND METHODS: Oral glucose tolerance tests (OGTT) were performed in 217 self-identified healthy African immigrants (69% male, age 39 ± 10 years [mean ± SD], BMI 27.6 ± 4.5 kg/m(2)). A1C, fructosamine, and GA were measured. Prediabetes was diagnosed by American Diabetes Association criteria for glucose obtained from a 2-h OGTT. The thresholds to diagnose prediabetes by A1C, fructosamine, and GA were the cutoff at the upper tertile for each variable: ≥5.7% (39 mmol/mol) (range 4.2-6.6% [22.4-48.6 mmol/mol]), ≥230 µmol/L (range 161-269 µmol/L), and ≥13.35% (range 10.20-16.07%), respectively. RESULTS: Prediabetes occurred in 34% (74 of 217). The diagnostic sensitivities of A1C, fructosamine, and GA were 50%, 41%, and 42%, respectively. The P values for comparison with A1C were both >0.3. Combining A1C with either fructosamine or GA increased sensitivities. However, the sensitivity of A1C combined with fructosamine was not better than for A1C alone (72% vs. 50%, P = 0.172). In contrast, the sensitivity of A1C combined with GA was higher than for A1C alone (78% vs. 50%, P < 0.001). CONCLUSIONS: As individual tests, A1C, fructosamine, and GA detected ≤50% of Africans with prediabetes. However, combining A1C with GA made it possible to identify nearly 80% of Africans with prediabetes.

Detection of abnormal glucose tolerance in Africans is improved by combining A1C with fasting glucose: the Africans in America Study.

OBJECTIVE: Abnormal glucose tolerance is rising in sub-Saharan Africa. Hemoglobin A1c by itself and in combination with fasting plasma glucose (FPG) is used to diagnose abnormal glucose tolerance. The diagnostic ability of A1C in Africans with heterozygous variant hemoglobin, such as sickle cell trait or hemoglobin C trait, has not been rigorously evaluated. In U.S.-based Africans, we determined by hemoglobin status the sensitivities of 1) FPG ≥5.6 mmol/L, 2) A1C ≥ 5.7% (39 mmol/mol), and 3) FPG combined with A1C (FPG ≥5.6 mmol/L and/or A1C ≥5.7% [39 mmol/mol]) for the detection of abnormal glucose tolerance. RESEARCH DESIGN AND METHODS: An oral glucose tolerance test (OGTT) was performed in 216 African immigrants (68% male, age 37 ± 10 years [mean ± SD], range 20-64 years). Abnormal glucose tolerance was defined as 2-h glucose ≥7.8 mmol/L. RESULTS: Variant hemoglobin was identified in 21% (46 of 216). Abnormal glucose tolerance occurred in 33% (72 of 216). When determining abnormal glucose tolerance from the OGTT (2-h glucose ≥7.8 mmol/L), sensitivities of FPG for the total, normal, and variant hemoglobin groups were 32%, 32%, and 33%, respectively. Sensitivities for A1C were 53%, 54%, and 47%. For FPG and A1C combined, sensitivities were 64%, 63%, and 67%. Sensitivities for FPG and A1C and the combination did not vary by hemoglobin status (all P > 0.6). For the entire cohort, sensitivity was higher for A1C than FPG and for both tests combined than for either test alone (all P values ≤ 0.01). CONCLUSIONS: No significant difference in sensitivity of A1C by variant hemoglobin status was detected. For the diagnosis of abnormal glucose tolerance in Africans, the sensitivity of A1C combined with FPG is significantly superior to either test alone.

Adenoviral vectors do not induce, inhibit, or potentiate human platelet aggregation.

Adenoviruses are commonly used as vectors in human clinical gene therapy trials. High doses of intravenous adenovirus vectors have been associated with development of thrombocytopenia of undetermined origin. Viral internalization requires the presence cell surface integrins, alpha(v)beta(3) or alpha(v)beta(5), that can blind ligands with a arginine-glycine-aspartic acid (RGD) sequence. This sequence is found in the adenovirus penton base. Platelets express the alpha(v)beta(3) integrin and other integrins that bind the RGD sequence of ligands such as fibrinogen, laminin, vitronectin, and von Willebrand factor (vWF). Platelet aggregation is mediated, in part, by the binding of the RGD sequence of fibrinogen to a platelet surface integrin, glycoprotein IIb/IIIa (GP IIb/IIIa). We investigated whether adenovirus particles could interfere with or potentiate agonist-induced platelet aggregation. Incubation of platelet-rich plasma with adenovirus under stirred conditions did not promote spontaneous aggregation. The addition of physiological platelet agonists, ADP, collagen, or epinephrine, induced platelet aggregation. However, the presence of adenovirus in a wide range of concentrations did not inhibit or potentiate agonist-induced aggregation. These results suggest that the adenovirus-associated thrombocytopenia observed in vivo is independent of a direct effect of the virus on platelet aggregation.

Toxicity of a first-generation adenoviral vector in rhesus macaques.

We constructed a first-generation adenovirus vector (AVC3FIX5) that we used to assess the rhesus macaque as a nonhuman primate model for preclinical testing of hemophilia B gene therapy vectors. Although we succeeded in our primary objective of demonstrating expression of human factor IX we encountered numerous toxic side effects that proved to be dose limiting. Following intravenous administration of AVC3FIX5 at doses of 3.4 x 10(11) vector particles/kg to 3.8 x 10(12) vector particles/kg, the animals in our study developed antibodies against human factor IX, and dose-dependent elevations of enzymes specific for liver, muscle, and lung injury. In addition, these animals showed dose-dependent prolongation of clotting times as well as acute, dose-dependent decreases in platelet counts and concomitant elevation of fibrinogen and von Willebrand factor. These abnormalities may be caused by the direct toxic effects of the adenovirus vector itself, or may result indirectly from the accompanying acute inflammatory response marked by elevations in IL-6, a key regulator of the acute inflammatory response. The rhesus macaque may be a useful animal model in which to evaluate mechanisms of adenovirus toxicities that have been encountered during clinical gene therapy trials.