Severe and moderate haemophilia A and B in US females.

Haemophilia A and B are rare X-lined hemorrhagic disorders that typically affect men. Women are usually asymptomatic carriers, but may be symptomatic and, rarely, also express severe (factor VIII (FVIII) or factor IX (FIX) <0.01 U mL(-1)) or moderately severe (FVIII/FIX 0.01-0.05 U mL(-1)) phenotypes. However, data on clinical manifestations, genotype and the psychosocial ramifications of illness in severely affected females remain anecdotal. A national multi-centre retrospective study was conducted to collect a comprehensive data set on affected US girls and women, and to compare clinical observations to previously published information on haemophilic males of comparable severity and mildly affected haemophilic females. Twenty-two severe/moderate haemophilia A/B subjects were characterized with respect to clinical manifestations and disease complications; genetic determinants of phenotypic severity; and health-related quality of life (HR-QoL). Clinical data were compared as previously indicated. Female patients were older than male patients at diagnosis, but similarly experienced joint haemorrhage, disease- and treatment-related complications and access to treatment. Gynaecological and obstetrical bleeding was unexpectedly infrequent. F8 or F9 mutations, accompanied by extremely skewed X-chromosome inactivation pattern (XIP), were primary determinants of severity. HR-QoL was diminished by arthropathy and viral infection. Using systematic case verification of participants in a national surveillance registry, this study elucidated the genetics, clinical phenotype and quality of life issues in female patients with severe/moderate haemophilia. An ongoing international case-controlled study will further evaluate these observations. Novel mechanistic questions are raised about the relationship between XIP and both age and tissue-specific FVIII and FIX expression.

Changes in international normalized ratio (INR) and model for endstage liver disease (MELD) based on selection of clinical laboratory.

Priority for liver transplantation is based on the Model for Endstage Liver Disease (MELD) score, a mathematical function which includes international normalized ratio (INR). We present an analysis to determine the lab-to-lab variation in INR at 14 clinical laboratories across the United States. We performed a survey to identify representative clinical laboratories across the United States, where INR was measured in the determination of MELD score. Five 'standard' samples for INR were formulated and were sent to the 14 clinical laboratories to determine variation in INR and MELD score. Among the 14 clinical laboratories, the range in INR for the five samples was: sample 1 (1.2-2.0), sample 2 (1.4-2.5), sample 3 (1.7-3.4), sample 4 (1.9-3.7) and sample 5 (2.4-5.1). The range in calculated MELD score was: sample 1 (8-14), sample 2 (10-17), sample 3 (12-20), sample 4 (14-21) and sample 5 (16-25). The selection of the clinical laboratory used to determine INR may result in substantial changes in MELD score independent of severity-of-illness. These data suggest that further review of interlaboratory variation in MELD should be undertaken because of the potential impact on prioritization for liver transplantation.

Fibrinogen Denver: a dysfibrinogenemia associated with an abnormal Reptilase time and significant bleeding.

This paper reports a new dysfibrinogenemia with an unusual pattern of laboratory assays. The patient, a 51-year-old female with a lifelong moderate bleeding history, was initially diagnosed with von Willebrand disease based on routine coagulation assays and the clinical bleeding presentation. During recent testing as part of a preoperative screen and without any current history of treatment, levels of von Willebrand factor (VWF) antigen, VWF activity, and factor VIII activity were all significantly elevated, which was unexpected given her previous diagnosis. Additional testing was performed looking for other heritable causes for her considerable bleeding tendency. Interestingly, the patient had a significantly prolonged Reptilase time, minimally short thrombin time, and an abnormal fibrinogen-crossed immunoelectrophoresis pattern. Clearly, this patient had a fibrinogen abnormality that had been missed when only routine coagulation screening assays were performed. A brief review of the fibrinogen literature revealed no other dysfibrinogenemias reported with a similar pattern of test results, and thus this defect was designated fibrinogen Denver.

A common mutation, Arg457-->Gln, links prothrombin deficiencies in the Puerto Rican population.

Five unrelated families with Puerto Rican ancestry were identified as having at least one member with bleeding due to a prothrombin deficiency. Genetic prothrombin deficiencies are extremely rare, but at the University of Puerto Rico Hemophilia Center, prothrombin deficiency is the third most common congenital coagulation factor deficiency. Because Puerto Rico is relatively isolated, there was a reasonable expectation of a founder effect. Prothrombin genes from probands and their parents were directly sequenced from PCR amplified exons using forward and reverse primers. Four novel prothrombin mutations were identified. The first, a G-->A substitution at DNA position 10150 predicting an Arg457-->Gln (R457Q) replacement, is common to all five families. In two of the families, the proband children are homozygous for R457Q. In the other three families, the probands are compound heterozygotes for R457Q and one of the other three mutations, which include another point mutation (gamma16Q), a deletion and a splice junction mutation. The two point mutations have been designated Puerto Rico I and Puerto Rico II. The crystal structure of alpha-thrombin predicts that the R457Q mutation removes a salt bridge that links the A- and B-chains of thrombin. The primary effect of this defect appears to be destabilization of the circulating prothrombin, creating a moderate hypoprothrombinemia. However, prothrombin antigen/activity ratios indicate a dysprothrombinemia as well, most likely due to the inability of R457Q prothrombin to activate fully to thrombin.

An institution-specific heparin titration nomogram: development, validation, and assessment of compliance.

STUDY OBJECTIVE: To develop, validate, and assess compliance with a heparin titration nomogram. DESIGN: Prospective, open-label trial. SETTING: University teaching hospital. SUBJECTS: Patients admitted with heart failure who required therapy with intravenous unfractionated heparin. Intervention. An in vitro concentration-response was determined by measuring activated partial thromboplastin times (aPTTs) on normal pooled plasma containing known concentrations of heparin. The therapeutic aPTT range was determined from the concentration-response by using the therapeutic heparin concentration range of 0.2-0.4 U/ml (protamine neutralization). Patients were consecutively enrolled, and therapy was managed by using the heparin titration nomogram. Paired aPTT-heparin concentrations were obtained, and nomogram validation was performed by comparing the in vitro and the ex vivo concentration-responses with use of linear regression. Nomogram compliance also was assessed. MEASUREMENTS AND MAIN RESULTS: The therapeutic aPTT ranges based on in vitro and ex vivo data were determined to be 45-72 seconds and 47-61 seconds, respectively. The ranges were significantly different (p<0.001). Overall compliance with the nomogram was 88%. CONCLUSION: These results confirm that, even in a relatively homogeneous disease-state patient population, in vitro data do not accurately predict ex vivo data. If in vitro data are used to develop an institution-specific nomogram, a validation procedure should be used to ensure accuracy. Although 100% compliance to a nomogram may not be attainable, it should be expected. Therefore, a compliance rate of 88% is concerning and suggests a need for increased nursing and physician education.

Factor IX Denver, ASN 346-->ASP mutation resulting in a dysfunctional protein with defective factor VIIIa interaction.

Hemophilia B is a sex-linked recessive bleeding disorder characterized by the presence of either a decreased amount of normal factor IX (FIX) or the presence of a dysfunctional FIX. We have identified a unique mutation in a family with mild hemophilia B. DNA analysis of family members revealed a single base transition in the 8th exon of the FIX gene predicting an amino acid change of Asn 346-->Asp in the catalytic domain. The FIX variant, named FIX Denver, was purified from proband plasma. Kinetic studies of factor X (FX) interactions with normal FIXa or FIXa Denver and phospholipid (PL) showed little difference in kcat but a significant difference when factor VIIIa (FVIIIa) was included in the reaction. Using kinetic assays to infer the Kd of FIXa for FVIIIa, normal FIXa had a Kd of 0.095 nM while that of FIXa Denver was 9.85 nM. The major defect caused by this point mutation is a marked decrease in the affinity of FIXa Denver for factor VIIIa.

Fibrinogen Longmont. A heterozygous abnormal fibrinogen with B beta Arg-166 to Cys substitution associated with defective fibrin polymerization.

B beta Arg166 to Cys substitution was identified in an abnormal fibrinogen named fibrinogen Longmont. The proband, a young woman, and her mother were heterozygous; both experienced episodes of severe hemorrhage at childbirth. The neo-Cys residues were found to be disulfide-bridged to either an isolated Cys amino acid or to the corresponding Cys residue of another abnormal fibrinogen molecule, forming dimers. Thrombin and batroxobin induced fibrin polymerization were impaired, despite normal release of fibrinopeptides A and B. Moreover, the polymerization defect was not corrected by removing the dimeric species or adding calcium. Fibrinogen Longmont had normal polymerization site a, as evidenced by normal GPRP-peptide binding. Thus, the sites A and a can interact to form protofibrils, as evidenced by dynamic light scattering measurements. These protofibrils, however, do not associate laterally in a normal manner, leading to an abnormal clot formation.

The impaired polymerization of fibrinogen Longmont (Bbeta166Arg-->Cys) is not improved by removal of disulfide-linked dimers from a mixture of dimers and cysteine-linked monomers.

This study identified a new substitution in the Bbeta chain of an abnormal fibrinogen, denoted Longmont, where the residue Arg166 was changed to Cys. The variant was discovered in a young woman with an episode of severe hemorrhage at childbirth and a subsequent mild bleeding disorder. The neo-Cys residues were always found to be disulfide-bridged to either an isolated Cys amino acid or to the corresponding Cys residue of another abnormal fibrinogen molecule, forming dimers. Removing the dimeric molecules using gel filtration did not correct the fibrin polymerization defect. Fibrinogen Longmont had normal fibrinopeptide A and B release and a functional polymerization site "a." Thus, the sites "A" and "a" can interact to form protofibrils, as evidenced by dynamic light-scattering measurements. These protofibrils, however, were unable to associate in the normal manner of lateral aggregation, leading to abnormal clot formation, as shown by an impaired increase in turbidity. Therefore, it is concluded that the substitution of Arg166-->Cys-Cys alters fibrinogen Longmont polymerization by disrupting interactions that are critical for normal lateral association of protofibrils. (Blood. 2001;98:661-666)

Unusual complications of warfarin therapy: skin necrosis and priapism.

Skin necrosis and priapism are unusual complications of warfarin therapy. We report a teenager with warfarin-associated skin necrosis and priapism who was subsequently found to be a compound heterozygote for protein C deficiency and a heterozygote for the factor V Leiden mutation.

Fibrinogen Longmont: a dysfibrinogenemia that causes prolonged clot-based test results only when using an optical detection method.

A new fibrinogen variant was discovered as a result of discrepancies found in routine laboratory screening. The patient, a healthy 37-year-old woman, had a mild bleeding history. Initial coagulation studies on the patient revealed a prolonged prothrombin time (PT) and a normal activated partial thromboplastin time (APTT). Further investigation on the patient and her mother demonstrated both had a PT with no end point using an optical detection method (ACL3000+) and a normal PT using an electromechanical detection method (ST4 Clot Detection System). The APTT for both the patient and her mother were essentially normal with both optical and mechanical detection methods. The patient and her mother also had markedly prolonged thrombin time and reptilase time results on the ACL3000+, but they were normal on the ST4. Coagulation test results on the patient's father were all normal. We believe the fibrinogen defect in this family may affect fibrin polymerization only enough to effect light scatter interpretation, while there is enough polymerization to increase plasma viscosity and yield an end point using an electromechanical analyzer. This report should alert pathologists and clinicians to possible discrepancies between mechanical and spectrophotometric clot testing methods.

Short activated partial thromboplastin times are related to increased thrombin generation and an increased risk for thromboembolism.

This prospective study was designed to investigate whether patients with short activated partial thromboplastin times (aPTTs) have increased thrombin generation and are at increased risk for thromboembolism. During a 4-month period, routine coagulation specimens were screened for the presence of a short or normal aPTT, and, accordingly, 250 specimens were collected. Prothrombin fragment F1 + 2 (F1 + 2) was measured to evaluate thrombin activation, and a second aPTT was performed with a different reagent. Diagnoses were obtained from medical records after conclusion of sample collection. Five to 9 months later, patients were questioned on thromboembolic events during the previous 18 months by questionnaire and telephone interview. F1 + 2 and the incidence of venous thromboses were elevated significantly in the short aPTT group. Unexpectedly, patients with acute bleeding had short aPTTs, but 36% of these also had thromboembolic events during the 18 months proximal to blood collection. These findings were confirmed with the second aPTT reagent. Patients with short aPTTs have increased thrombin generation and are at increased risk for thromboembolism, mainly venous thromboses, despite the fact that a short aPTT can occur in the acute setting of bleeding.

The prothrombin Denver patient has two different prothrombin point mutations resulting in Glu-300-->Lys and Glu-309-->Lys substitutions.

Dysprothrombinaemia is a rare, congenital cause of bleeding. Fewer than 25 families who express a functional prothrombin (factor II) defect have been reported. The original patient with prothrombin Denver had a severe haemophilia-like bleeding disorder treated with weekly prophylactic factor replacement. Analysis of factor II activity and antigen in the patient showed a factor II activity of 5 units/dl and factor II antigen of 21 units/dl. Genomic DNA from the patient, mother and brother was obtained from peripheral blood white cells. Oligonucleotides were constructed, and prothrombin exons were amplified via polymerase chain reaction (PCR). The entire sequence of the thrombin portion of the molecule (exons VIII-XIV) and that of exons I-II and IV-VII was determined. This moderately severe dysprothrombinaemia was found to be associated with compound heterozygosity for two different Glu-->Lys point mutations, at amino acid positions 300 and 309. Assays of plasma from the prothrombin Denver proband suggested that the functional defect was in the activation of zymogen to enzyme.

Hereditary deficiency of vitamin-K-dependent coagulation factors in Rambouillet sheep.

A flock of Rambouillet sheep experienced unexpected lamb mortality associated with excessive bleeding at the time of parturition. Most lambs died of blood loss through the umbilicus or into subcutaneous tissues. Subsequently, nine ewes which had previously delivered lambs that bled to death were bred to the suspected sire of the previous bleeding lambs. Fifteen lambs were born alive the following Spring, and three males and one female bled clinically. These lambs had markedly decreased factor IX (< 16%) and factor X (< 4%) activities, with variably decreased factor II (11-36%) and factor VII (20-37%) activities. Protein C chromogenic activity was also markedly decreased (< 1%) in these lambs. The results from crossed immunoelectrophoresis and 'protein-induced-in-vitamin-K-absence' determination of the plasma of affected lambs, with antiserum directed against coagulation factor X, protein C or proteins S, suggested that these proteins were not carboxylated normally. Examination of liver from one lamb in the first batch and the four subsequent lambs did not reveal a known vitamin K antagonist. The breeding data suggested that the coagulopathy in these sheep was inherited as an autosomal recessive trait. The genetic or molecular defect that exists in these lambs is unknown, but possibilities include abnormal gamma-glutamyl carboxylase activity or abnormal metabolism of vitamin K.

Brodifacoum intoxication with marijuana smoking.

We report the case of a 17-year-old boy with a significant history of drug and alcohol abuse, which included smoking marijuana mixed with brodifacoum. As a consequence, the patient developed a prolonged coagulopathy that persisted for more than 1 year. To our knowledge, this is the first case reported in the literature in which super-warfarin intoxication has been associated with marijuana smoking. This report should increase the awareness of pathologists and clinicians when examining a patient with a history of drug abuse who exhibits persistent vitamin K1-dependent coagulopathy.

Comparison of protein S functional and antigenic assays in normal pregnancy.

OBJECTIVE: Our purpose was to determine the effect of pregnancy on the protein S functional assay (clot based), which is used to screen for all subtypes of protein S deficiency states, and to compare its behavior in pregnancy with antigenic assays. STUDY DESIGN: This was a cross-sectional study of 37 normal pregnant women without thromboembolic risks who were tested by both functional and antigenic protein S assays during the first, second, and third trimesters. RESULTS: Mean protein S functional levels decline strikingly from the first to the third trimester, all 10 third-trimester patients had functional protein S levels well below the lower limit of the reference range. In contrast, only 3 of 10 third-trimester and none of the second-trimester patients had free protein S antigenic levels below the reference range. CONCLUSIONS: The protein S functional assay should not be used in pregnancy to screen for the subtypes of protein S deficiency; misdiagnosis and inappropriate treatment could result.

Lupus anticoagulant and protein S deficiency in children with postvaricella purpura fulminans or thrombosis.

OBJECTIVE: The objective of this study was to determine the cause of purpura fulminans, disseminated intravascular coagulation, or thrombosis in seven children with varicella. All children were found to have a lupus anticoagulant and acquired protein S deficiency. Thrombosis in five children was associated with presumed or documented infection with streptococcus. STUDY DESIGN: Coagulation tests included determinations of the activated partial thromboplastin time, the prothrombin time, the dilute Russell viper venom time, the prothrombin F 1 + 2 fragment, the C4b-binding protein (C4b), total and free protein S antigen, and clotting activities of factors II, V, VII, and X and of protein C and protein S. Autoantibodies to phospholipids, cardiolipin, and protein S were determined in enzyme-linked immunosorbent assays. RESULTS: All children had a lupus anticoagulant and acquired protein S deficiency. Thrombosis in five children was associated with presumed or documented infection with streptococcus. All children transiently expressed free protein S deficiency, elevated levels of IgG, IgM, or both binding to protein S, the lupus anticoagulant, and increased concentration of the F 1+2 fragment. Four children also had antiphospholipid or anticardiolipin antibodies. In one child a purified IgG fraction cross-reacted with both protein S and a specific varicella antigen. CONCLUSIONS: A subset of children with varicella infection, some of whom are coinfected with streptococcus, are prone to development of a lupus anticoagulant and an autoantibody to protein S, which results in acquired free protein S deficiency. Such children are at risk of having life-threatening thrombotic events.

Diabetic muscle infarction: a new perspective on pathogenesis and management.

Two patients with insulin dependent diabetes mellitus developed recurrent episodes of focal muscle pain and swelling. Clinical evaluation, magnetic resonance imaging (MRI) and muscle biopsy confirmed the diagnosis of recurrent hemorrhagic muscle infarctions. Our studies suggest that muscle infarction occurred because of hypercoagulability and associated vascular endothelial damage. Based on these findings we recommend long-term anticoagulation to prevent recurrent infarction.

Comparison of the behavior of normal factor IX and the factor IX Bm variant Hilo in the prothrombin time test using tissue factors from bovine, human, and rabbit sources.

A subset of hemophilia B patients have a prolonged bovine-brain prothrombin time. These CRM+ patients are classified as having hemophilia Bm. The prolongation of the prothrombin time has been reported only with bovine brain (referred to as ox brain in some literature) as the source of thromboplastin; prothrombin times determined with thromboplastin from rabbit brain or human brain are not reported to be prolonged. Factor IX from a hemophilia Bm patient (factor IX Hilo) was isolated. The activity of factor IX Hilo was compared to that of normal factor IX in prothrombin time assays when the thromboplastin source was of bovine, rabbit, or human origin. Factor IX, either normal or Hilo, prolonged a prothrombin time regardless of the tissue factor source. However, unless thromboplastin was from a bovine source, this prolongation required high concentrations of factor IX. Further, factor IX normal was as effective as factor IX Hilo in prolonging the prothrombin time when rabbit or human thromboplastin was used. With bovine thromboplastin, factor IX Hilo was significantly better than factor IX normal at prolonging the prothrombin time. The amount of prolongation was dependent on the amount of factor IX Hilo added. In addition, the prolongation was dependent on the concentration of factor X present in the sample. The prothrombin time changed as much as 20 seconds when the factor X concentration was varied from 50% to 150% to normal (fixed concentration of factor IX Hilo). These results demonstrate the difficulty of classifying the severity of a hemophilia Bm patient based on the bovine brain prothrombin time unless both the factor IX and factor X concentrations are known.

Sudden death due to fibromuscular dysplasia of the sinoatrial nodal artery.

A case of sudden death is reported in a previously healthy 28-year-old female. No gross abnormalities of the heart or other organ systems were identified at autopsy. Routine microscopic sections of heart were unremarkable; however, sections of the cardiac conduction system revealed marked fibromuscular dysplasia of the sinoatrial node artery. The authors suggest that pathologists and clinicians index of suspicion be high when confronted with the enigma of sudden death in a patient with no apparent anatomic or toxicologic cause of death.