A description of the coagulopathy characteristics in amniotic fluid embolism: a case report.

Amniotic fluid embolism is frequently associated with coagulopathy. However, the exact nature and evolution of the bleeding disorder is incompletely understood. We report a case of clinically diagnosed amniotic fluid embolism associated with major haemorrhage and coagulopathy. We measured sequential levels of all individual clotting factors, thrombin generation, fibrinogen, and D-dimer levels over the course of the event, beginning shortly after the patient's initial collapse and during the subsequent resuscitation, to identify the specific abnormalities of coagulation from stored blood samples. A better understanding of amniotic fluid embolism and the associated coagulopathy is an important area of research to inform targeted treatment of the coagulopathy and improve outcomes for patients.

The sensitivity and specificity of rotational thromboelastometry (ROTEM) to detect coagulopathy during moderate and severe postpartum haemorrhage: a prospective observational study.

INTRODUCTION: Point-of-care viscoelastic haemostatic assays such as rotational thromboelastometry (including ROTEM and TEG) have been used in the management of postpartum haemorrhage (PPH). This study compared results obtained from the automated ROTEM Sigma with laboratory tests of coagulation and platelet count during PPH. METHODS: A prospective observational cohort study recruited women with PPH ≥1000 mL (or clinical concern of bleeding). The Fibtem A5, Extem CT and Pltem (Extem A5 - Fibtem A5) results were compared with laboratory tests of fibrinogen, prothrombin time (PT), activated partial thromboplastin time (APTT) and platelet count. RESULTS: 521 women were recruited, including 274/277 (98.9%) of women with PPH ≥1500 mL. Fibtem A5 results were matched with laboratory fibrinogen in 552/644 (85.7%) samples. The incidence of abnormal laboratory results was low: fibrinogen ≤2 g/L 23/464 (5.0%), PT or APTT >1.5 × midpoint of reference range 4/464 (0.9%), and platelet count <75 × 109/L 11/477 (2.3%). Area-under-the-receiver operator characteristic curve for Fibtem A5 to detect fibrinogen ≤2 g/L was 0.96 (95% Confidence Interval (CI) 0.94 to 0.98, P<0.001), with sensitivity and specificity of Fibtem A5 ≤11 mm to detect fibrinogen ≤2 g/L of 0.76 and 0.96. Prolonged Extem CT results improved after treatment of hypofibrinogenaemia alone. Intervention points for platelet and fresh frozen plasma (FFP) transfusion based on ROTEM Sigma parameters could not be established. CONCLUSION: During PPH (≥1000 mL or cases of clinical concern about bleeding), ROTEM Sigma Fibtem A5 can detect fibrinogen ≤2 g/L and guide targeted fibrinogen replacement. Laboratory results should continue to be used to guide platelet and FFP transfusion.

Thrombin generation in haemophilia A patients with mutations causing factor VIII assay discrepancy.

Up to 40% of patients with mild haemophilia A have a discrepancy whereby factor VIII (FVIII) measurements by a two-stage chromogenic assay (FVIII:C(CH)) are disproportionately reduced compared with the FVIII one-stage clotting value (FVIII:C). Which assay best reflects the coagulation potential and clinical phenotype in this patient group is of clinical significance, yet remains unclear. We have assessed the global coagulant ability of haemophilia patients with FVIII assay discrepancy using calibrated automated thrombography (CAT). A total of 18 patients with mutations Arg531His/Cys or Arg698Trp causing FVIII discrepancy were investigated, together with 12 haemophilia patients with concordant FVIII values and 15 normal controls. Factor VIII levels in all patients and controls were measured using both one-stage clotting assay and two-stage chromogenic assay. Thrombin generation was assessed in platelet-poor plasma by CAT using a low tissue factor concentration (1 pm). FVIII:C(CH) values were below normal in all patients, and in the discrepant group were between 1.5- and 8-fold lower than FVIII:C values. CAT parameters were affected in all haemophilia patients. The endogenous thrombin potential (ETP) was reduced to 58-67% of the mean normal value (1301 nm min(-1)), whereas peak thrombin was further reduced to 27-30% of the mean normal value (178 nm) in both discrepant and concordant patient groups. Analysis of the discrepant patient group showed the most significant correlation between the one-stage FVIII:C assay and ETP (r(2) = 0.44) and peak thrombin parameters (r(2) = 0.27).

Factor VIII and von Willebrand factor interaction: biological, clinical and therapeutic importance.

The interaction of factor VIII (FVIII) with von Willebrand Factor (VWF) is of direct clinical significance in the diagnosis and treatment of patients with haemophilia A and von Willebrand disease (VWD). A normal haemostatic response to vascular injury requires both FVIII and VWF. It is well-established that in addition to its role in mediating platelet to platelet and platelet to matrix binding, VWF has a direct role in thrombin and fibrin generation by acting as a carrier molecule for the cofactor FVIII. Recent studies show that the interaction affects not only the biology of both FVIII and VWF, and the pathology of haemophilia and VWD, but also presents opportunities in the treatment of haemophilia. This review details the mechanisms and the molecular determinants of FVIII interaction with VWF, and the role of FVIII-VWF interaction in modulating FVIII interactions with other proteases, cell types and cellular receptors. The effect of defective interaction of FVIII with VWF as a result of mutations in either protein is discussed.

Mutation analysis of haemophilia B in the Irish population: increased prevalence caused by founder effect.

Haemophilia B generally arises as a result of unique mutations within the F9 gene and occurs with a prevalence of approximately one case per 30 000 males worldwide. The population prevalence of haemophilia B in Ireland at one per 12 500 males is particularly high. To identify the mutations responsible for haemophilia B and to define the biological basis underlying the increased prevalence in the Irish population, we performed sequence analysis of the F9 gene in 51 apparently unrelated kindred. In 18 kindred with severe or moderate haemophilia B, we identified 14 different mutations; these occurred throughout the F9 gene and included small deletions, missense, non-sense and splice-site mutations and included four novel candidate mutations. In contrast to the variety of different causative mutations with moderate or severe haemophilia B, we found three common mutations accounted for 83% (24/29) of Irish kindred with mild haemophilia B. The mutation n-6 G>A in the promoter region of F9 (which results in the characteristic haemophilia B Leyden phenotype) was found in 10 unrelated kindred. The mutation C>T 30933 in exon 8 (Ala271Val) was identified in a further 10 apparently unrelated kindred. Finally, 10430 G>A mutation (Gly60Ser) was observed in four different kindred. Haplotype analysis was performed on the index cases with the most common mutations and supported the hypothesis that the increased population prevalence of mild haemophilia B in the Irish population arose as a result of founder effect rather than an increased incidence of de-novo F9 mutations.

Clustered basic residues within segment 484-510 of the factor VIIIa A2 subunit contribute to the catalytic efficiency for factor Xa generation.

Residues 484-510 of factor (F)VIIIa A2 subunit comprise a prominent epitope for inhibitor antibodies, suggesting that this region is critical for cofactor function. To address the role of this region in catalysis, FVIIIa forms were evaluated following conversion of conserved charged residues to Ala, either in clusters or individually. The two cluster mutants, Lys496Ala/Lys499Ala/Asp500Ala and Glu507Ala/Lys510Ala, were indistinguishable from wild type. The mutation Arg489Ala/Arg490Ala/Lys493Ala (489-3A) possessed near-normal affinity for FIXa and showed no effect on the Km for FX, but exhibited approximately 3-fold and approximately 30-fold reduced kcat values for FXase in the presence and absence of surface, respectively. However, the single-site mutants Arg489Ala, Arg490Ala and Lys493Ala exhibited affinity and kcat values similar to wild type. Furthermore, the 489-3A mutant showed a marked reduction in the positive electrostatic potential within this region of A2, consistent with the hypothesis that the cumulative basic charge in this region of A2 subunit modulates cofactor function.

Aberrant dimerization of von Willebrand factor as the result of mutations in the carboxy-terminal region: identification of 3 mutations in members of 3 different families with type 2A (phenotype IID) von Willebrand disease.

The 3' end of the VWF gene was screened in the affected members of 3 different families with type 2A (phenotype IID) von Willebrand disease (vWD). Exons 49 to 52 of the VWF gene were amplified and screened for mutations by chemical cleavage mismatch detection. Mismatched bands were detected in exon 52 of 2 patients and in exon 51 of a third patient. Using direct DNA sequencing, a heterozygous G8562A transition leading to a Cys2008Tyr substitution was found in all the patients in family 1, and a T8561A transversion leading to a Cys2008Ser substitution was found in both patients from family 2. In a patient from a third family, an 8-base deletion from nucleotide 8437 to 8444 was identified in exon 51. The 2 mutations in exon 52 were reproduced by in vitro site-directed mutagenesis of full-length von Willebrand factor (vWF) cDNA and transiently expressed in COS-7 cells. The corresponding recombinant VWFs for these 2 mutations exhibited the typical aberrant vWF:Ag multimer pattern seen in the plasma of the patients. These 3 mutations demonstrate the importance of other carboxy-terminal cysteines in addition to the reported Cys2010 residue, in the normal dimerization of vWF, and their essential role in the assembly of normal multimeric vWF. (Blood. 2001;98:674-680)

The effects of the 32-bp CCR-5 deletion on HIV transmission and HIV disease progression in individuals with haemophilia.

The chemokine receptor CCR5 is an important co-receptor for cell fusion. A 32-bp deletion of the CCR5 gene, leading to complete absence of functional CCR5 expression, has been associated with resistance to human immunodeficiency virus (HIV) infection in homozygotes and slower HIV disease progression in heterozygotes. The objectives of this study were to assess the effects of this 32-bp deletion on transmission of HIV infection and on HIV disease progression in haemophilic individuals. Six HIV-negative patients from our centre, known to have been exposed to infectious factor VIII concentrates, have been analysed. Three of these patients possess the CCR5 32-bp deletion, two patients being homozygous. The presence of the CCR5 32-bp gene deletion has also been analysed in 71 HIV-positive patients. In this group of patients, there was a lower than expected incidence of the 32-bp deletion. Those who possess the 32-bp deletion progress to AIDS more slowly than those who do not (P = 0.05, log-rank test). Rates of CD4 loss were slower in those heterozygous for the gene deletion. We confirm that heterozygosity for the 32-bp gene deletion in CCR5 is partially protective against initial infection with HIV. In those heterozygous patients who became infected with HIV, disease progression was slower.

Homozygous 2bp deletion in the human factor VII gene: a non-lethal mutation that is associated with a complete absence of circulating factor VII.

We report the case of a 5-year-old boy with severe factor VII deficiency. The affected child presented at the age of 8 months and again at 18 months with bleeding from the gastrointestinal tract but the diagnosis of factor VII deficiency was not made until the age of 3 years. He was treated with fresh frozen plasma and subsequently factor VII concentrates and to date remains well. To identify the causative mutation, the factor VII gene was screened by SSCP and direct sequence analysis. A single homozygous 2 bp deletion (-CT) mutation was identified in exon 1a removing nucleotides 27/28 (codons 52/53). Both parents, who were first cousins, were heterozygous for the mutation. The mutation located in the prepropeptide of factor VII, results in a complete absence of factor VII in plasma. This case indicates that a complete absence of plasma factor VII is not necessarily a lethal condition.

Type 2M vWD resulting from a lysine deletion within a four lysine residue repeat in the A1 loop of von Willebrand factor.

Type 1 von Willebrand disease is characterized by a decreased plasma concentration of functionally normal von Willebrand factor (vWF) whereas type 2M is characterised by an abnormal vWF displaying decreased affinity for platelets. In these two types of patients, the multimeric structure of vWF is normal. We report here the identification, in two unrelated families from the UK and Algeria, of an in-frame 3 bp deletion, at the heterozygous state, resulting in the deletion of a lysine residue within a four lysine repeat at position 642-645 of the mature vWF subunit (del K 1405-1408 in pre-pro vWF). The patients who have a discrepancy between vWF antigen level and vWF ristocetin cofactor activity exhibited decreased ristocetin-induced binding but only a slight decrease in the percentage of high molecular weight (HMW) multimers in plasma. Recombinant vWF harbouring this deletion did not bind to platelet GPIb in the presence of ristocetin or botrocetin although the protein is multimerized. Consequently, this lysine deletion was considered as a type 2M vWD mutation.

Molecular characterisation and three-dimensional structural analysis of mutations in 21 unrelated families with inherited factor VII deficiency.

Factor VII (FVII) is a four-domain glycoprotein that plays a critical role in the initiation of blood coagulation. Hereditary deficiencies of this plasma protein results in a bleeding diathesis that varies in severity amongst affected patients. We have analysed the FVII gene in 27 patients with FVII deficiency from 21 unrelated families predominantly of Middle-Eastern extraction. A total of 19 different mutations were identified, of which 12 were novel and 7 had been previously reported. Nine of the 12 novel mutations were missense mutations located in the Gla domain (Ser23Pro), the second epidermal growth factor domain (Cys135Arg) and the catalytic serine protease domain (Arg247Cys, Arg277Cys, Ser282Arg, Pro303Thr, Ser363Ile, Trp364Cys, Trp364Phe), of which five are homozygous. Three novel splice mutations were identified in intron 1a (IVS1a+5), intron 2 (IVS2+1) and intron 6 (IVS6+1). Of the seven previously reported mutations, five were missense mutations of which three are homozygous (Gln100Arg, Arg152Gln, Arg304Gln, Cys310Phe and Thr359Met), one was a 17 bp deletion (10585del117bp) and one was a splice site mutation within intron 7 (IVS7+7). This study has significantly extended the current database of FVII mutations, including the number of known homozygous mutations. Conformational analyses of crystal structures for FVIIa and the FVIIa-tissue factor complex provided likely explanations for the effect of the missense mutations on FVIIa secretion or function. In particular, since 23 missense mutations were located to the serine protease domain, mostly to the region between the catalytic triad and the contact surface with tissue factor, this showed that the orientation of the serine protease domain relative to bound tissue factor in the complex is crucial for functional activity.

Type 1 von Willebrand disease - a clinical retrospective study of the diagnosis, the influence of the ABO blood group and the role of the bleeding history.

This clinical retrospective study investigated the difficulties in diagnosing type 1 von Willebrand disease (VWD). A total of 246 patients previously diagnosed with type 1 VWD were reclassified into 'possible' type 1 VWD (patients with low levels of VWF adjusted for the blood group and either a significant bleeding history or family history) and 'definite' type 1 VWD, requiring low levels of von Willebrand factor (VWF), a bleeding history and inheritance. On reclassification, only 144/246 (59%) patients had low VWF levels adjusted for blood group, 88/246 (36%) patients met all the criteria for 'definite' type 1 VWD and 51/246 (21%) patients were 'possible' type 1 VWD. A significant proportion of patients, 102/246 (42%), remained an indeterminate group with blood type O, VWF levels between 35 and 50 U/dl and personal and/or family bleeding history. This subgroup might require reclassification as 'not VWD'. However, a similar bleeding tendency was found in two matched groups of patients of blood groups O and non-O and VWF levels between 35 and 50 U/dl. These results suggest that the use of ABO adjusted ranges for VWF levels might not be essential for diagnosis, because bleeding symptoms may depend on the VWF level regardless of the ABO type. Of the diagnostic criteria, the bleeding history was of prime importance in the clinical decision to diagnose and treat type 1 VWD. These observations could help in the reconsideration of how the criteria for diagnosing type 1 VWD could be adjusted in order to maximize their clinical relevance.

Identification and three-dimensional structural analysis of nine novel mutations in patients with prothrombin deficiency.

Prothrombin deficiency is an autosomal recessive disorder associated with a moderately severe bleeding tendency. In this study, 13 patients with prothrombin deficiency were screened for the presence of alterations in the prothrombin gene, and nine novel candidate mutations were identified. Of 11 patients with hypoprothrombinemia, ten are homozygous for five mutations and one patient is a compound heterozygote. The two patients with dysprothrombinemia are homozygous for two mutations. Eight of nine mutations are missense ones associated with single amino acid substitutions in the propeptide (Arg-1Gln, Arg-2Trp), the kringle-1 (Asp118Try) and kringle-2 (Arg220Cys) domains and the catalytic serine protease domain (Gly330Ser, Ser354Arg. Arg382His and Arg538Cys). The ninth mutation is an in-frame deletion of 3 bp that results in the omission of one amino acid (del Lys 301/302). The combination of these missense mutations with crystal structures for alpha-thrombin and the prothrombin fragments 1 and 2 resulted in new insight into the function of alpha-thrombin. The hypoprothrombinemia mutations were inferred to affect either the cleavage of the propeptide from the Gla domain, the stability of the kringle-1 and -2 domains, or the close association of the A and B chains of the serine protease domain. The dysprothrombinemia mutations were inferred to directly affect catalytic function through their location at the active site crevice or exosite 1 within the serine protease domain.

Identification of type 2 von Willebrand disease in previously diagnosed type 1 patients: a reappraisal using phenotypes, genotypes and molecular modelling.

In order to investigate the possibility that qualitative type 2 defects in von Willebrand factor (VWF) occurred in patients previously diagnosed with quantitative type 1 von Willebrand disease (VWD), the phenotypes and genotypes were reanalysed in 30 patients who exhibited discrepant VWF activity/VWF:Ag ratios of less than 0.7. The capacity of VWF to bind to glycoprotein Ib (GpIb) was reassessed using the ristocetin co-factor activity (VWF:RiCo) assay compared to an in-house and a commercial ELISA assay (based on a mAb directed against the GpIb binding site on VWF). This was supplemented by multimeric analysis and the amplification and sequencing of a 936 bp fragment of exon 28 of the VWF gene with the aim of identifying mutations in the A1 domain. On reappraisal, using the VWF:RiCo assay all patients demonstrated a disproportionately reduced VWF:RiCo/VWF:Ag ratio, indicative of a qualitative defect, while abnormal ratios were detected in only seven kindreds using the in-house ELISA assay and in only one kindred with the commercial ELISA assay. Eight single amino acid substitutions were found in nine kindreds, four of which were novel candidate VWF mutations and four previously described in association with type 2 VWD. In agreement with the phenotype, the novel VWF mutations were located in the VWF-A1 crystal structure at positions that corresponded to potential type 2M defects. This study underlines the difficulties of correct diagnosis of the subtype of VWD and emphasises the importance of using sensitive phenotypic assays, the relevance of the VWF:RiCo/ VWF:Ag ratio, multimeric analysis and molecular modelling analysis.

Detection of mutations causing hemophilia a using an in vitro coupled transcription and translation system.

Mutation detection in the factor VIII gene is complicated by the size and complexity of the gene-186 kb spanning 26 exons. The exons vary in size from 69 bp to 3106 bp and the introns from 207 bp to 32.4 kb (1). The first mutations to be identified in the factor VIII gene involved mutations at Taq I restriction sites (an enzyme that contains the mutational CpG dinucleotide within its recognition sequence) or were large deletions detected by Southern blotting (2). Small deletions, substitution, and missense mutations proved more difficult to detect as these appear to be randomly distributed throughout the factor VIII gene. For these reasons, therefore, many laboratories involved in carrier detection in Hemophilia A have used an indirect procedure known as gene tracking or linkage analysis. This involves the use of various polymorphic markers (RFLPs and VNTRs) to follow the segregation of the defective factor VIII gene through individuals in a family (This is not covered in this chapter, but an excellent review on the subject is available [3]). Several factors limit this technique, primarily the need for intervening family members, the need for a proband to be present, the occasional need for paternity testing, and the frequent occasions where all polymorphic markers prove to be uninformative. Furthermore, it adds little to our understanding of the mutations that underlie Hemophilia A.