Search

The author's team analyzed a bicycle as a two-wheeled vehicle. (TWV) The purpose of the study was to estimate the parameters affecting the reaction time of the cyclist. Depending on the type of brake system, the mass of the two-wheeled vehicle and the cyclist, the speed, and the bicycle designs graphs were built, and their analysis was carried out. Based on the test results, factors affecting the cyclist reaction time in case of danger were disclosed.

Background and Importance: Antiphospholipid syndrome (APS) is an autoimmune disorder associated with a hypercoagulable state and increased risk of intraoperative and postoperative thrombosis. Few neurosurgical studies have examined the management of these patients, though the standard of care in most other disciplines involves the use of anticoagulation therapy. However, this is associated with risks such as hemorrhage, thrombosis due to warfarin withdrawal, and is not compatible with operative intervention., Clinical Presentation: We report the cases of 2 antiphospholipid positive patients who were on anticoagulant therapy and underwent surgical bypasses and received perioperative management with plasmapheresis. The first was a 44-yr-old woman who presented with worsening vision, recurring headaches, and a known left internal carotid artery aneurysm that was unsuccessfully treated twice via extracranial to intracranial (ECIC) bypass at another institution. Preoperative tests at our institution revealed elevated beta 2 glycoprotein 1 IgA autoantibodies. The second case was a 24-yr-old woman with previously diagnosed APS, who presented for surgical evaluation of moyamoya disease after sustaining recurrent left hemispheric strokes. Both cases were managed with perioperative plasmapheresis to avoid the need for anticoagulation during the perioperative period, and both underwent successful ECIC bypass procedures without perioperative ischemic or hemorrhagic complications., Conclusion: Management of neurosurgical patients with APS can be a precarious proposition. We describe the successful use of plasmapheresis and antiplatelet therapy to better manage patients undergoing neurosurgical procedures, specifically ECIC bypass, and feel this approach can be considered in future cases., (Copyright © 2018 by the Congress of Neurological Surgeons.)

What Is Known and Objective: Immune destruction and decreased platelet production are major components of immune thrombocytopenia (ITP) pathogenesis. The aim of this study was to critically evaluate the role of combination therapy in relapsed/refractory ITP and the concept of medication tapering/discontinuation., Comment: Although a number of combination regimens have been reported, little is published on combining immunosuppression with thrombopoietin receptor agonists (TPO-RAs). We report a case of refractory ITP successfully treated with combination immunosuppression added to eltrombopag. An aggressive combination approach resulted in complete remission and allowed for stepwise drug tapering., What Is New and Conclusion: Combination immunosuppression can potentiate the effect of TPO-RAs. This mechanistically reasonable strategy could result in a more rapid response than the more popular, sequential, single-agent strategy. Stepwise tapering can be successfully implemented. Comparing sequential single-agent therapy with early combination approach warrants a more extensive study., (© 2016 John Wiley & Sons Ltd.)

Background: Emergency Department Reliance (EDR: total emergency department [ED] visits/total ambulatory [outpatient + ED] visits) differentiates acute episodic ED users from those who may not have adequate access to outpatient care., Objective: This study's aim was to investigate age-related patterns of EDR and associated health-care costs in pediatric patients with sickle cell disease (SCD) and those transitioning from pediatric to adult care., Methods: State Medicaid data were used for this study. Patients with two or more SCD diagnoses and one or more blood transfusion were included. Quarterly rates of ED visits, EDR, SCD complications associated with ED visits, and ED visits resulting in hospitalization were evaluated. Risk factors associated with high EDR and the association between high EDR and health-care costs were explored through regression analyses., Results: A total of 3208 patients were included. The most common SCD complications associated with ED visits were pain, infection, and pneumonia. Beginning at the age of 15 years, EDR rose from 0.17 to 0.29 visits per quarter at age 22 years, and remained high throughout adulthood. Regression analyses indicated that patients were most likely to have high EDR during the post-transition period and when experiencing an SCD complication. Patients with high EDR incurred statistically significantly higher inpatient and ED costs, resulting in significantly higher total health-care costs., Conclusions: Compared to children, patients transitioning to adulthood relied more on the ED for their care. In addition, patients with high EDR incurred more days in the hospital and significantly higher health-care costs, highlighting the need to improve transition-related support, including better access to primary care and increased engagement with patients with SCD., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Sickle cell trait (SCT) occurs in about 8% of African-Americans and is often described to be of little clinical consequence. Over time, a number of risks have emerged, and among these are rare but catastrophic episodes of sudden death in athletes and other individuals associated with physical activities which is often described as exercise collapse associated with sickle trait (ECAST). Despite an epidemiologic link between SCT and sudden death as well as numerous case reports in both medical literature and lay press, no clear understanding of the key pathophysiologic events has been identified. Strategies for identification of individuals at risk and prevention of ECAST have been both elusive and controversial. Stakeholders have advocated for different approaches to this issue particularly with regard to screening for hemoglobin S. Furthermore, the recommendations and guidelines that are in place for the early recognition of ECAST and the prevention and treatment of the illness are not well defined and remain fragmented. Among the cases identified, those in collegiate football players in the United States are often highlighted. This manuscript examines these case studies and the current recommendations to identify areas of consensus and controversy regarding recommendations for prevention, recognition and treatment of ECAST.

Background: This study explored the blood transfusion patterns, SCD complications, utilization of iron chelation therapies (ICT), healthcare resource use, and costs in pediatric, transitioning (18 years old) and adult patients with SCD., Procedure: Data from Florida (1998-2009), New Jersey (1996-2009), Missouri (1997-2010), Kansas (2001-2009), and Iowa (1998-2010) state Medicaid were used. Patients with ≥2 SCD diagnoses and ≥1 transfusion event were included. Rates of transfusion events, SCD complications, and proportion of eligible patients receiving ICT were calculated. ICT eligibility was defined as receiving ≥10 transfusions over lifetime. SCD complications included pain, pulmonary event, infection event, renal, cardiovascular, stroke, leg ulcers, and avascular necrosis. Regressions were used to assess risk factors for transfusion and identify the main drivers of costs., Results: The sample included 3,208 patients. The transfusion rate increased from 1-year-old to a peak at 16 years old, then dropped until age 26 and remained stable thereafter. In contrast the frequency of diagnoses for SCD complications increased markedly after age 16. Post-transition patients (≥18 years old) were significantly associated with fewer transfusions (odds ratio: 0.80, P = 0.002). Among eligible patients for ICT, there was no statistically significant difference in total cost between the ICT and no ICT groups (adjusted cost difference, $136, P = 0.114)., Conclusions: Patients transitioning to adult care received less transfusions and hydroxyurea, less ICT when eligible for chelation therapy, had higher healthcare costs and suffered from more frequent SCD related complications than pediatric patients. These findings highlight the changes in treatment patterns corresponding to transition to adult care., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: In sickle cell disease (SCD), pulmonary hypertension (assessed by tricuspid regurgitant jet [TRJ] velocity ≥ 2.5 m/sec) is associated with increased mortality. The relationships among TRJ velocity and left ventricular (LV) and right ventricular (RV) systolic and diastolic function (i.e., relaxation and compliance) have not been well characterized in SCD., Methods: A prospective study was conducted in 53 ambulatory adults with SCD (mean age, 34 years; range, 21-65 years) and 33 African American controls to define the relationship between LV and RV function and TRJ velocity using echocardiography., Results: Subjects with SCD had larger left and right atrial volumes and increased LV mass compared with controls. When patients with SCD were compared with controls, LV and RV relaxation (i.e., E') were similar. Among subjects with SCD, pulmonary hypertension (TRJ ≥ 2.5 m/sec) was present in 40%. Higher TRJ velocity was correlated with larger left atrial volumes in patients with SCD. Additionally, some measures of LV (peak A, lateral and septal annular E/E' ratio) and RV (tricuspid valve E/E' ratio) compliance were correlated with TRJ velocity. No other measures of LV and RV systolic function or LV diastolic function (i.e., relaxation and compliance) were associated with TRJ velocity., Conclusions: Ambulatory adults with SCD exhibited structural (i.e., LV and RV chamber enlargement) and functional (i.e., higher surrogate measures of LV and RV filling pressure) abnormalities compared with the control group. In subjects with SCD, few abnormalities of LV and RV structure and function were associated with TRJ velocity., (Copyright © 2011 American Society of Echocardiography. Published by Mosby, Inc. All rights reserved.)

Prior studies of asthma in children with sickle cell disease (SCD) were based on reports of a doctor-diagnosis of asthma with limited description of asthma features. Doctor-diagnoses of asthma may represent asthma or wheezing unrelated to asthma. Objectives of this study were to determine if asthma characteristics are present in adults with a doctor-diagnosis of asthma and/or wheezing, and to examine the relationship between doctor-diagnosis of asthma, wheezing and SCD morbidity. This was an observational cohort study of 114 adults with SCD who completed respiratory symptom questionnaires and had serum IgE measurements. A subset of 79 participants completed pulmonary function testing. Survival analysis was based on a mean prospective follow-up of 28 months and data were censored at the time of death or loss to follow-up. Adults reporting a doctor-diagnosis of asthma (N = 34) were more likely to have features of asthma including wheeze, eczema, family history of asthma, and an elevated IgE level (all P < 0.05). However, there was no difference in pain or ACS rate, lung function, or risk of death between adults with and without a doctor-diagnosis of asthma. In contrast, adults who reported recurrent, severe episodes of wheezing (N = 34), regardless of asthma, had twice the rates of pain and ACS, decreased lung function and increased risk of death compared with adults without recurrent, severe wheezing. Asthma features were not associated with recurrent, severe wheezing. Our data suggest that wheezing in SCD may occur independently of asthma and is a marker of disease severity., (Copyright © 2011 Wiley-Liss, Inc.)

Idiopathic thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease mediated by autoantibodies directed against ADAMTS-13. A number of small series and case reports have shown promising results with rituximab in refractory or relapsed TTP. In this report, we present 13 patients with TTP treated with rituximab. Twelve of the 13 patients (92%) achieved complete response; no subsequent relapses occurred with median follow-up of 24 months (range, 13-84 months). The addition of rituximab to standard therapy appears to be effective in sustaining long-term remission in TTP. However, the optimal dosing and timing of rituximab warrant further investigation. Am. J. Hematol., 2009. (c) 2009 Wiley-Liss, Inc.

Baseline level of the cysteinyl leukotriene (CysLT), leukotriene E4 (LTE4), is associated with an increased pain rate in children and adults with sickle cell disease (SCD). To provide additional evidence for a role of CysLTs in the pathogenesis of vaso-occlusion, we tested the hypothesis that LTE4 levels will increase within an individual during painful episodes compared to baseline. In a cohort of 19 children and adults with SCD, median LTE4 levels increased from 82.36 pg/mg creatinine at baseline to 162.81 pg/mg creatinine during a painful episode (P < 0.001). These data further support a contribution of CysLTs to the process of vaso-occlusion., (Copyright 2009 Wiley-Liss, Inc.)

Leukotriene E(4) (LTE(4)) levels are associated with rate of pain episodes in children with sickle cell disease (SCD). Because complications of SCD manifest differently in adults than children, we examined a cohort of adults with SCD to determine the relationship between baseline LTE(4) and SCD-related morbidity. Baseline LTE(4) levels were associated with increased rates of pain and acute chest syndrome (ACS) episodes, when those with LTE(4) values in the highest tertile were compared with those in the lowest tertile (pain: risk ratio 7.1, 95% CI 1.8-27.5, P = 0.005; ACS: risk ratio 12.2, 95% CI 2.1-69.8, P = 0.005).

Under normal conditions, reticulocytes are the youngest erythrocytes released from the bone marrow into circulating blood. They mature for 1-3 days within the bone marrow and circulate for 1-2 days before becoming mature erythrocytes. Measurement of cellular hemoglobin concentration has long been reported by automated hematology analyzers as one of the red blood cell indices. The reticulocyte hemoglobin content (CHr or Ret-He) provides an indirect measure of the functional iron available for new red blood cell production over the previous 3-4 days. Measurement of reticulocyte hemoglobin content in peripheral blood samples is useful for diagnosis of iron deficiency in adults (Mast et al., Blood 2002;99:1489-1491) and children (Brugnara et al., JAMA 1999;281:2225-2230; Ullrich et al., JAMA 2005;294:924-930; Bakr and Sarette, Eur J Pediatr 2006;165:442-445). It provides an early measure of the response to iron therapy increasing within 2-4 days of the initiation of intravenous iron therapy (Brugnara et al., Blood 1994;83:3100-3101). Sequential measurements of reticulocyte hemoglobin content in patients with iron deficiency anemia provide a rapid means for assessing the erythropoietic response to iron replacement therapy (Brugnara et al., Blood 1994;83:3100-3101). It is also an early indicator or iron-restricted erythropoiesis in patients receiving erythropoietin therapy (Fishbane et al., Kidney Int 1997;52:217-222; Fishbane et al., Kidney Int 2001;60:2406-2411; Mittman et al., Am J Kidney Dis 1997;30:912-922; Tsuchiya et al., Clin Nephrol 2003;59:115-123; Chuang et al., Nephrol Dial Transplant 2003;18:370-377). Thus, reticulocyte hemoglobin content is a recent addition to an expanding list of biomarkers that can be used to differentiate iron deficiency from other causes of anemia., ((c) 2007 Wiley-Liss, Inc.)

The purpose of this study was to evaluate the results of shoulder arthroplasty for the treatment of avascular necrosis in patients with sickle cell disease. Medical records, radiographs, operative reports, and outcome scores of 8 adult patients with sickle cell disease were evaluated. The mean follow-up was 51 months (range, 2-10 years). Seven patients had a hemiarthroplasty, and one had a total shoulder arthroplasty. One patient had an intraoperative rotator cuff tear. Two had sickle cell crises in the immediate postoperative period. In one patient, stiffness developed that required arthroscopic capsular release 22 months after her arthroplasty. Another patient with a hemiarthroplasty underwent revision to a total shoulder arthroplasty 5 years after the index procedure. The mean American Shoulder and Elbow Surgeons score improved by 31.9 points. However, only 2 patients reported improvement in pain as assessed with a visual analog scale. Although shoulder arthroplasty provides improvements in range of motion and function in the majority of patients, pain relief is less predictable.

Acquired factor VIII (FVIII) inhibitors are a rare cause of coagulopathy which are associated with a high mortality rate. Treatment of bleeding episodes is often difficult and may vary with the degree of titre elevation. Individuals with very high-titre antibodies [>100 Bethesda units mL(-1) (BU)] may have difficulty achieving a complete sustained remission and, consequently, various treatments including immunosuppression, cytotoxic chemotherapy and plasmapheresis have been reported. Rituximab is an anti-CD20 monoclonal antibody which has demonstrated efficacy in the treatment of individuals with acquired FVIII inhibitors, however there is limited data in the subgroup of patients with inhibitor titres >100 BU. In this study, we present four patients with acquired FVIII inhibitor titres >100 BU who were resistant to initial therapy with cyclophosphamide, vincristine and prednisone. The patients' inhibitor titres ranged from 249 BU mL(-1) to 725 BU mL(-1) and all received 4 weekly infusions of rituximab at 375 mg m(-2). Each patient partially responded to rituximab therapy with an improvement in inhibitor titres and FVIII activity, however, three of the four patients relapsed thereafter. The individual who did not relapse achieved a partial response for 13 months and then died of causes unrelated to her coagulopathy. We conclude that in patients with acquired FVIII inhibitors and titres >100 BU, treatment with rituximab alone is effective but not sufficient to achieve a sustained response. Rituximab in combination with other therapies may provide a better result in this high-risk population.

Acute intermittent porphyria is a rare disorder of heme biosynthesis with a clinical course characterized by exacerbations of neurologic symptoms. Drugs may precipitate these exacerbations; however, little is known about the safety of most drugs used to treat the disease. We describe a patient with acute intermittent porphyria who developed bone marrow failure and was successfully treated with cyclosporine. This agent has been reported as being unsafe for patients with acute intermittent porphyria based on an in vitro model. However, cyclosporine was used in this patient based on two published case reports of successful cyclosporine therapy in patients with acute intermittent porphyria undergoing kidney transplantation. Our patient tolerated cyclosporine well, and her blood counts demonstrated improvement. To our knowledge, this is the first case report of cyclosporine used for treatment of bone marrow failure in a patient with acute intermittent porphyria. Until more data are available, clinicians should consider immunosuppressive therapy as a safe option for treating certain patients with acute intermittent porphyria.

The bleeding diathesis associated with hereditary factor XI (fXI) deficiency is prevalent in Ashkenazi Jews, in whom the disorder appears to be an autosomal recessive condition. The homodimeric structure of fXI implies that the product of a single mutant allele could confer disease in a dominant manner through formation of heterodimers with wild-type polypeptide. We studied 2 unrelated patients with fXI levels less than 20% of normal and family histories indicating dominant disease transmission. Both are heterozygous for single amino acid substitutions in the fXI catalytic domain (Gly400Val and Trp569Ser). Neither mutant is secreted by transfected fibroblasts. In cotransfection experiments with a wild-type fXI construct, constructs with mutations common in Ashkenazi Jews (Glu117Stop and Phe283Leu) and a variant with a severe defect in dimer formation (fXI-Gly350Glu) have little effect on wild-type fXI secretion. In contrast, cotransfection with fXI-Gly400Val or fXI-Trp569Ser reduces wild-type secretion about 50%, consistent with a dominant negative effect. Immunoprecipitation of cell lysates confirmed that fXI-Gly400Val forms intracellular dimers. The data support a model in which nonsecretable mutant fXI polypeptides trap wild-type polypeptides within cells through heterodimer formation, resulting in lower plasma fXI levels than in heterozygotes for mutations that cause autosomal recessive fXI deficiency.

Background: Thrombotic thrombocytopenic purpura (TTP) in adults is usually caused by autoantibody inhibitors of ADAMTS13. Treatment with plasma exchange is often effective but does not address the underlying autoimmune process., Objective: To report the efficacy of intensive immunosuppressive therapy in refractory TTP., Design: Case report., Setting: University medical center., Patient: 42-year-old woman with chronic relapsing TTP., Intervention: Immunosuppression therapy with rituximab and cyclophosphamide., Measurements: ADAMTS13 activity and inhibitors and hematologic variables for TTP., Results: For 19 months, the patient had relapsing thrombotic microangiopathy despite plasma exchange; splenectomy; and therapy with vincristine, prednisone, and cyclosporine. ADAMTS13 activity was low, and tests detected an IgG inhibitor that recognized the metalloprotease domain of recombinant ADAMTS13. After treatment with rituximab and cyclophosphamide, the disease remitted, ADAMTS13 levels normalized, and the inhibitor was undetectable. The patient has required no treatment for 13 months., Conclusion: Intensive immunosuppressive therapy can lead to sustained clinical remission in patients with refractory autoimmune TTP.

Determination of the reticulocyte hemoglobin content (CHr) provides an early measure of functional iron deficiency because reticulocytes are the earliest erythrocytes released into blood and circulate for only 1 to 2 days. The CHr in 78 patients undergoing bone marrow examination was measured to assess its clinical utility for the diagnosis of iron deficiency. Twenty-eight patients were iron deficient, based on the lack of stainable iron in the aspirate. The diagnostic power of CHr is limited in patients with high mean cellular volume (MCV) or red cell disorders such as thalassemia. However, when patients with MCV more than 100 fL are excluded, receiver operator curve analysis of CHr, ferritin, transferrin saturation, and MCV demonstrates that CHr has the highest overall sensitivity and specificity of these peripheral blood tests for predicting the absence of bone marrow iron stores.

Warfarin use is complicated by an erratic dose response. Warfarin is metabolized by two distinct subfamilies of the cytochrome P450 (CYP) complex. We describe two siblings with extreme sensitivity to warfarin who share an unusual CYP genotype. These individuals illustrate both the importance of genetics in influencing the metabolism of warfarin as well as the potential utility of genetic testing as a guide to prescribing this medication., (Copyright 2001 Wiley-Liss, Inc.)

We report 4 acute promyelocytic leukemia cases that demonstrated karyotypic abnormalities in addition to the classic t(15;17) translocation and did not contain any Auer rods in leukemic blasts and dysplastic promyelocytes, either in the peripheral blood or in the bone marrow. Morphologically, 2 cases were characterized as the common or hypergranular type, and 2 were otherwise typical of the microgranular variant. Three patients had typical clinical and laboratory signs of disseminated intravascular coagulation. Immunophenotypic analysis of the blasts and dysplastic promyelocytes by dual-color flow cytometry revealed an immunoprofile consistent with acute promyelocytic leukemia. Cytogenetic analysis of the bone marrow revealed the following karyotypes: case 1, [47,XY,t(15;17)(q22;q12),+21]; case 2, [47,XY,t(15;17)(q22;q12),-16,+2 mar]; case 3, [47,XX,t(15;17)(q22;q12)ider(17)(q10),+8]; and case 4, [47,XY,der(5)t(5;?9)(p15;q12).t(15;17)(q22;q12]. Review of an additional 7 cases with t(15;17) as the sole cytogenetic abnormality revealed Auer rods in all cases. Our findings emphasize the importance of cytogenetics in evaluating acute myeloid leukemias. Acute promyelocytic leukemia without Auer rods, which may be morphologically confused with other types of leukemia (in particular, acute myeloblastic leukemia, type M2 or M5) or agranulocytosis with maturation arrest, appears to be associated with additional chromosomal abnormalities and possibly a poorer prognosis.

We describe an HPLC-based method for the detection and quantification of fetal hemoglobin in stools of newborns. The new procedure is an alternative to the classic qualitative test for adult hemoglobin in meconium based on the differential stability of hemoglobin species in dilute base (Apt test). The HPLC method, based on a commercial device for hemoglobin characterization (Bio-Rad Variant), readily separates fetal and adult hemoglobin from non-hemoglobin components of meconium. To validate the method, blood and meconium were mixed in various proportions and then prepared for analysis with extraction in saline. The HPLC method accurately identified hemoglobin species even when the blood constituted only 5 mL per 100 g of the meconium specimen, and nearly quantitative recovery of hemoglobin was obtained at a blood content of 20 mL per 100 g of the meconium. Analysis time was 6.5 min, and preparation of sample was simple. HPLC detection of fetal blood in stools or other specimens markedly improves detection/characterization of blood in meconium.

We previously isolated a monoclonal antithrombin IgG from a patient with multiple myeloma [Colwell et al. (1997) Br. J. Haematol. 97, 219-226]. Using a panel of 55 surface mutants of recombinant thrombin, we now show that the epitope for the IgG most likely includes Arg-101, Arg-233, and Lys-236 in exosite II. The IgG affects the rate at which thrombin cleaves various peptide p-nitroanilide substrates with arginine in the P1 position, increasing the kcat for substrates with a P2 glycine residue but generally decreasing the kcat for substrates with a P2 proline. The allosteric effect of the IgG is altered by deletion of Pro-60b, Pro-60c, and Trp-60d from the 60-loop of thrombin, which lies between exosite II and the catalytic triad. The effect of the IgG, however, does not depend on the presence or absence of sodium ions, a known allosteric regulator of thrombin. The IgG does not affect the conformation of thrombin exosite I as determined by binding of a fluorescent derivative of hirudin54-65. These results provide evidence for a direct allosteric linkage between exosite II and the catalytic site of thrombin.

Soluble transferrin receptor (sTfR) and ferritin concentrations were measured in a variety of clinical settings to compare the ability of these two tests to identify iron deficiency. Among 62 anemic patients who either had a bone marrow aspirate performed or had a documented response to iron therapy, the diagnostic sensitivity and specificity of sTfR (at a diagnostic cutoff of > 2.8 mg/L) were 92% and 84%, respectively, with a positive predictive value of 42% in this population. Ferritin (< or = 12 microg/L) had a sensitivity of 25% and a specificity of 98%. However, the sensitivity and specificity of ferritin could be improved to 92% and 98%, respectively, by using a diagnostic cutoff value of < or = 30 microg/L, resulting in a positive predictive value of 92%. Ferritin and sTfR were also measured in 267 outpatient samples and 112 medical students. In the outpatient group, the two tests agreed in 73% of the samples; however, 25% of the samples had ferritin values > 12 microg/L and increased sTfR. Among the medical students, there was 91% agreement between the two tests, but 7% of the samples had ferritin < or = 12 microg/L and normal sTfR. Together, these data suggest that measurement of sTfR does not provide sufficient additional information to ferritin to warrant routine use. However, sTfR may be useful as an adjunct in the evaluation of anemic patients, whose ferritin values may be increased as the result of an acute-phase reaction.

We investigated a patient with a long-standing IgGkappa monoclonal gammopathy who developed severe haemorrhagic complications. At IgG concentrations of approximately 50g/l the patient had severe bleeding associated with prolongation of the thrombin time, activated partial thromboplastin time, and reptilase time. Plasmapheresis resulted in improvement in the thrombin time and resolution of bleeding. Depletion of the IgG by absorption of plasma with protein G-Sepharose in vitro resulted in normalization of the thrombin time and reptilase time. The purified IgG bound to immobilized thrombin and immunoprecipitated human alpha-, beta- and gamma-thrombin but not prothrombin, other vitamin K-dependent coagulation factors, or fibrinogen. Purified IgG at concentrations >1 x 10(-2) g/l decreased (approximately 50%) the rate of hydrolysis of a chromogenic substrate by thrombin. Addition of purified IgG to normal pooled plasma at concentrations >1 x 10(-2) g/l prolonged the thrombin time and activated partial thromboplastin time, but the reptilase time was prolonged only at IgG concentrations >1 g/l. This finding suggests that at low concentrations the IgG produces a specific antithrombin effect, but at higher concentrations it also affects fibrin polymerization; the combination of these effects probably produced clinical bleeding. This is the first report of a monoclonal antithrombin antibody associated with bleeding in a patient with multiple myeloma.

Acquired inhibitors of factor V are rare causes of clinical bleeding, whose severity ranges from mild to life-threatening. Optimal treatment of patients with factor V inhibitors is uncertain. We report on our successful treatment approach in a patient with spontaneous, life-threatening intracranial bleeding caused by a factor V inhibitor. The patient deteriorated after initial treatment with fresh-frozen plasma and platelet transfusions. He was subsequently treated with a combination of plasma exchange and chemotherapy, which led to complete recovery. Our experience suggests that plasma exchange may be life-saving in cases of severe bleeding caused by factor V inhibitors. The use of plasmapheresis in conjunction with chemotherapy is an efficacious and well-tolerated treatment and should be considered in patients with factor V inhibitors.

Heparin cofactor II (HC) is a plasma serine proteinase inhibitor (serpin) that inhibits alpha-thrombin in a reaction that is dramatically enhanced by heparin and other glycosaminoglycans/polyanions. We investigated the glycosaminoglycan binding site in HC by: (i) chemical modification with pyridoxal 5'-phosphate (PLP) in the absence and presence of heparin and dermatan sulfate; (ii) molecular modeling; and (iii) site-directed oligonucleotide mutagenesis. Four lysyl residues (173, 252, 343, and 348) were protected from modification by heparin and to a lesser extent by dermatan sulfate. Heparin-protected PLPHC retained both heparin cofactor and dermatan sulfate cofactor activity while dermatan sulfate-protected PLPHC retained some dermatan sulfate cofactor activity and little heparin cofactor activity. Molecular modeling studies revealed that Lys173 and Lys252 are within a region previously shown to contain residues involved in glycosaminoglycan binding. Lys343 and Lys348 are distant from this region, but protection by heparin and dermatan sulfate might result from a conformational change following glycosaminoglycan binding to the inhibitor. Site-directed mutagenesis of Lys173 and Lys343 was performed to further dissect the role of these two regions during HC-heparin and HC-dermatan sulfate interactions. The Lys343----Asn or Thr mutants had normal or only slightly reduced heparin or dermatan sulfate cofactor activity and eluted from heparin-Sepharose at the same ionic strength as native recombinant HC. However, the Lys173----Gln or Leu mutants had greatly reduced heparin cofactor activity and eluted from heparin-Sepharose at a significantly lower ionic strength than native recombinant HC but retained normal dermatan sulfate cofactor activity. Our results demonstrate that Lys173 is involved in the interaction of HC with heparin but not with dermatan sulfate, whereas Lys343 is not critical for HC binding to either glycosaminoglycan. These data provide further evidence for the determinants required for glycosaminoglycan binding to HC.

Heparin cofactor II (HCII) is a 66-kDa plasma glycoprotein that inhibits thrombin rapidly in the presence of dermatan sulfate or heparin. Clones comprising the entire HCII gene were isolated from a human leukocyte genomic library in EMBL-3 lambda phage. The sequence of the gene was determined on both strands of DNA (15,849 bp) and included 1749 bp of 5'-flanking sequence, five exons, four introns, and 476 bp of DNA 3' to the polyadenylation site. Ten complete and one partial Alu repeats were identified in the introns and 5'-flanking region. The HCII gene was regionally mapped on chromosome 22 using rodent-human somatic cell hybrids, carrying only parts of human chromosome 22, and the chronic myelogenous leukemia cell line K562. With the cDNA probe HCII7.2, containing the entire coding region of the gene, the HCII gene was shown to be amplified 10-20-fold in K562 cells by Southern analysis and in situ hybridization. From these data, we concluded that the HCII gene is localized on the chromosomal band 22q11 proximal to the breakpoint cluster region (BCR). Analysis by pulsed-field gel electrophoresis indicated that the amplified HCII gene in K562 cells maps at least 2 Mbp proximal to BCR-1. Furthermore, the HCII7.2 cDNA probe detected two frequent restriction fragment length polymorphisms with the restriction enzymes BamHI and HindIII.

Heparin cofactor II (HCII), a member of the "serpin" family of serine protease inhibitors, is a 65,600-Da plasma glycoprotein that inhibits thrombin and chymotrypsin. The rate of thrombin inhibition is stimulated approximately 1000-fold by heparin or dermatan sulfate. Thrombin and chymotrypsin cleave the Leu444-Ser445 bond (designated P1-P'1) in the reactive site of HCII, forming a stable equimolar complex in which the protease is inactive. In this study, we have determined the effects of substituting an arginine for Leu444 in recombinant HCII (rHCII). The rHCII was expressed in Escherichia coli and partially purified by heparin-Sepharose chromatography. Apparent second-order rate constants (k2) for inhibition of thrombin, coagulation factor Xa, kallikrein, plasmin, and chymotrypsin by rHCII were determined using appropriate chromogenic substrates. In the absence of a glycosaminoglycan, rHCII(Leu444----Arg) inhibited thrombin at a 98-fold higher rate (k2 = 6.2 x 10(6) M-1 min-1) than native rHCII (k2 = 6.3 x 10(4) M-1 min-1). Dermatan sulfate accelerated thrombin inhibition by both forms of rHCII, but the maximum rate constant in the presence of dermatan sulfate was only 2-fold higher for rHCII(Leu444----Arg) (k2 = 5.3 x 10(8) M-1 min-1) than for native rHCII (k2 = 2.2 x 10(8) M-1 min-1). Heparin was less effective than dermatan sulfate in stimulating both forms of rHCII. Factor Xa, kallikrein, and plasmin were inhibited more rapidly and chymotrypsin more slowly by rHCII(Leu444----Arg) than by native rHCII. These effects are qualitatively similar to those observed with the natural mutant alpha 1-antitrypsin Pittsburgh (Met358----Arg at the P1 position) and strengthen the hypothesis that the P1 residue is a major determinant of protease specificity in the serpins. Furthermore, the rapid rate of inhibition of thrombin by rHCII(Leu444----Arg) in the absence of heparin or dermatan sulfate suggests that this variant may be useful as a therapeutic agent.

Inhibition of thrombin by heparin cofactor (HCII) is accelerated approximately 1000-fold by heparin or dermatan sulfate. We found recently that the mutation Arg189----His decreases the affinity of HCII for dermatan sulfate but not for heparin (Blinder, M. A., Andersson, T. R., Abildgaard, U., and Tollefsen, D. M. (1989) J. Biol. Chem. 264, 5128-5133). Other investigators have implicated Arg47 and Lys125 of anti-thrombin (homologous to Arg103 and Lys185 of HCII) in heparin binding. To investigate the corresponding residues in HCII, we have constructed amino acid substitutions (Arg103----Leu, Gln, or Trp; Lys185----Met, Asn, or Thr) by oligonucleotide-directed mutagenesis of the cDNA and expressed the products in Escherichia coli. The recombinant HCII variants were assayed for binding to heparin-Sepharose and for inhibition of thrombin in the presence of various concentrations of heparin or dermatan sulfate. All of the Arg103 variants bound to heparin with normal affinity. Furthermore, inhibition of thrombin by the Arg103----Leu variant occurred at a normal rate in the absence of a glycosaminoglycan and was accelerated by normal concentrations of heparin and dermatan sulfate. These results indicate that HCII, unlike anti-thrombin, does not require a positive charge at this position for the interaction with heparin or dermatan sulfate. The Arg103----Gln and Arg103----Trp variants inhibited thrombin at about one-third of the normal rate in the absence of a glycosaminoglycan, suggesting that these mutations exert an effect on the reactive site (Leu444-Ser445) of HCII. All of the Lys185 variants bound to heparin with decreased affinity but inhibited thrombin at approximately the normal rate in the absence of a glycosaminoglycan. These variants required greater than 10-fold higher concentrations of heparin to accelerate inhibition of thrombin and were not stimulated significantly by dermatan sulfate, suggesting that heparin and dermatan sulfate interact with Lys185 of HCII. These results provide evidence that the glycosaminoglycan-binding site in HCII includes Lys185 but not Arg103, both of which were predicted to be involved by homology to anti-thrombin.

Of 455 acute nonlymphocytic leukemia (ANL) patients who underwent marrow transplantation, 95 (21%) relapsed a median of 6.5 months posttransplantation and 62 received further treatment. Twenty achieved remission. Success of therapy was related to the length of time from marrow transplant to relapse and to the use of cytarabine (Ara-C) and daunomycin. Aggressive chemotherapy for patients relapsing within 100 days of marrow transplant was associated with a high incidence of early death (six of 14 patients) and a low probability of remission (one of 14). Of 23 patients who relapsed in excess of 1 year from marrow transplant, 15 achieved a complete remission. The median disease-free survival is 6 months (range, 0.4 to 53+ months). Acute lymphocytic leukemia (ALL) recurred in 130 of 366 patients (36%), and 94 received further therapy. Fifty-two achieved a remission. Remissions were more common in late relapse patients (greater than 1 year from transplantation): 65% v 7% for those relapsing within 100 days from transplant (P less than .05). Testicular relapse occurred in 11 patients and was the sole site of relapse in seven. Three are alive and free of disease 58 to 109+ months after relapse. The median survival for the treated patients is 10.5 months (range, 5 to 109+ months). We propose that reinduction be attempted in all patients relapsing greater than 1 year from marrow transplantation. Ara-C and daunomycin should be employed in the treatment of ANL. The decision for treatment of patients relapsing earlier than 1 year should be made on an individual basis.

Metastatic esophageal carcinoma to the brain is a rare occurrence that may account for abnormal neurological findings.

Heparin and dermatan sulfate increase the rate of inhibition of thrombin by heparin cofactor II (HCII) approximately 1000-fold by providing a catalytic template to which both the inhibitor and the proteinase bind. A variant form of HCII that binds heparin but not dermatan sulfate has been described recently in two heterozygous individuals (Andersson, T.R., Larsen, M.L., and Abildgaard, U. (1987) Thromb. Res. 47, 243-248). We have now purified the variant HCII (designated HCIIOslo) from the plasma of ne of these individuals. HCIIOslo or normal HCII (11 nM) was incubated with thrombin (9 nM) for 1 min in the presence of heparin or dermatan sulfate. Fifty percent inhibition of thrombin occurred at 26 micrograms/ml dermatan sulfate with normal HCII and greater than 1600 micrograms/ml dermatan sulfate with HCIIOslo. In contrast, inhibition of thrombin occurred at a similar concentration of heparin (1.0-1.5 micrograms/ml) with both inhibitors. To identify the mutation in HCIIOslo, DNA fragments encoding the N-terminal 220 amino acid residues of HCII were amplified from leukocyte DNA by the Taq DNA polymerase chain reaction and both alleles were cloned. A point mutation (G----A) resulting in substitution of His for Arg-189 was found in one allele. The same mutation was constructed in the cDNA of native HCII by oligonucleotide-directed mutagenesis and expressed in Escherichia coli. The recombinant HCIIHis-189 reacted with thrombin in the presence of heparin but not dermatan sulfate, confirming that this mutation is responsible for the functional abnormality in HCIIOslo.

Heparin cofactor II (HCII) is an inhibitor of thrombin in plasma that is activated by dermatan sulfate or heparin. An apparently full-length cDNA for HCII was isolated from a human liver lambda gt11 cDNA library. The cDNA consisted of 2215 base pairs (bp), including an open-reading frame of 1525 bp, a stop codon, a 3'-noncoding region of 654 bp, and a poly(A) tail. The deduced amino acid sequence contained a signal peptide of 19 amino acid residues and a mature protein of 480 amino acids. The sequence of HCII demonstrated homology with antithrombin III and other members of the alpha 1-antitrypsin superfamily. Blot hybridization of an HCII probe to DNA isolated from sorted human chromosomes indicated that the HCII gene is located on chromosome 22. Twenty human leukocyte DNA samples were digested with EcoRI, PstI, HindIII, KpnI, or BamHI, and Southern blots of the digests were probed with HCII cDNA fragments. A restriction fragment length polymorphism was identified with BamHI. A slightly truncated form of the cDNA, coding for Met-Ala instead of the N-terminal 18 amino acids of mature HCII, was cloned into the vector pKK233-2 and expressed in Escherichia coli. The resultant protein of apparent molecular weight 54,000 was identified on an immunoblot with 125I-labeled anti-HCII antibodies. The recombinant HCII formed a complex with 125I-thrombin in a reaction that required the presence of heparin or dermatan sulfate.