M P Smith

A prospective study of recombinant activated factor VII administered by continuous infusion to inhibitor patients undergoing elective major orthopaedic surgery: a pharmacokinetic and efficacy evaluation

Summary. After surgery in haemophilia, haemostasis is difficult to maintain in the presence of an antifactor VIII antibody. This study assessed the pharmacokinetics of recombinant activated factor VII (rFVIIa) and its efficacy in securing post‐operative haemostasis in haemophiliacs with inhibitors. Continuous infusion of rFVIIa was evaluated for elective major orthopaedic surgery in nine patients with neutralizing antibodies to FVIII and at high risk of bleeding. After an initial preoperative bolus of 90 µg/kg, rFVIIa was infused at a fixed rate of 50 µg/kg/h for a median of 20 d (range 7–20 d). The median plasma FVII coagulant activity (FVII:C) at 24 h, 72 h and 20 d after surgery was 38 IU/ml (range 22–169 IU/ml), 45 IU/ml (range 17–88 IU/ml) and 31 IU/ml (range 27–46 IU/ml) respectively. The median plasma FVIIa:C at the same time points was 51 (range 24–211), 63 (range 22–99) and 44 (range 28–76) IU/ml respectively. Median total rFVIIa clearance remained stable during the rFVIIa continuous infusion period and was 40 (range 9–70), 34 (range 17–86) and 48 (range 32–55)ml/kg/h at the end of 24 h, 72 h and 20 d infusion respectively. Post‐operatively, there were bleeds in six patients, which settled readily after a single bolus of rFVIIa (60 µg/kg). There was a good clinical outcome for all patients. These data indicate that rFVIIa infusion at50 µg/kg/h achieves continuous plasma FVII procoagulant activity in excess of 30 IU/ml (12–15 nmol/l) and provides adequate haemostatic control for inhibitor patients during major orthopaedic surgery.

Tinzaparin sodium for thrombosis treatment and prevention during pregnancy.

OBJECTIVE This study was undertaken to assess the pharmacodynamic profile, safety, and efficacy of tinzaparin during pregnancy. STUDY DESIGN Fifty-four pregnant women, 12 for treatment of thrombosis and 42 for thromboprophylaxis, received tinzaparin by once daily injection. Four-hour postdose anti-Xa results were analyzed by use of repeated measures statistical methods. RESULTS One woman (3.4%) on the 175 anti-Xa U/kg dose and three women (20%) on the 50 anti-Xa U/kg dose required a dose increase during the initial dose titration phase to achieve target anti-Xa activity. No thrombotic events occurred. CONCLUSION The 175 anti-Xa U/kg dose is appropriate for treatment and for high-risk thromboprophylaxis throughout pregnancy. In pregnant women at moderate risk of thrombosis, a higher tinzaparin dose is required than in the nonpregnant state and 75 anti-Xa U/kg appears to be appropriate. The majority of women do not need a dose increase with advancing gestation.

Immune Tolerance Therapy for Haemophilia

The development of anti-factor VIII and anti-factor IX allo-antibodies in haemophilia A and B, respectively, remains a serious complication of treatment for these two X-linked haemostatic disorders, with major clinical and economic consequences. Treatment of this potentially fatal complication remains one of the greatest challenges facing haematologists at the beginning of the 21st century. Immune tolerance induction (ITI) therapy has been generally accepted as the best available treatment, extinguishing the inhibitor and permitting a resumption of standard dosing schedules. Although there have been several established protocols for ITI therapy developed over the last quarter century, the optimal scheme in terms of safety, clinical efficacy and pharmacoeconomic considerations has yet to be determined.

7 Megakaryocytes and platelets in α-granule disorders

This chapter summarizes research data contributing to current understanding of disorders affecting α-granules of megakaryocytes and platelets. Diagnostic features of the gray platelet syndrome are well defined. Combined evidence suggests a defect, specific to the megakaryocyte cell lineage, causing a cytoskeletal abnormality and defective targeting of endogenously synthesized proteins to the α-granule. The abnormalities linked by signal transduction pathways. von Willebrand disease and afibrinogenaemia are disorders which highlight the functional importance of platelet storage pools of von Willebrand factor and fibrinogen, essential ligands in the process of adhesion and aggregation. The abnormality in the factor V Quebec disorder leads to a degradation of most proteins contained within the α-granule. The familial platelet disorder Paris-Trousseau thrombocytopenia is the only α-granule disorder associated with a cytogenetic abnormality, and it presents a useful model for exploring the genetic influence on regulation of thrombopoiesis. Study of these syndromes has elucidated aspects of the physiology of normal megakaryocyte maturation and platelet formation, including storage organelle biosynthesis.

Low molecular weight heparin (tinzaparin) therapy for moderate risk thromboprophylaxis during pregnancy - A pharmacokinetic study

Low molecular weight heparin (LMWH) is used increasingly for prophylaxis and treatment of venous thromboembolism during pregnancy. However, the prophylactic dose for patients at moderate risk varies between centers, and the recommended LMWH dose for the non pregnant patient is frequently used in pregnant women. The aim of this study was to investigate the effects of pregnancy on the pharmacokinetics of anti-Xa levels during moderate risk thromboprophylaxis with the LMWH, tinzaparin. In 24 pregnant women, one of three doses of tinzaparin (50, 75 or 100 IU/kg) were given according to the treating physicians assessment of their risk profile. Four-hour peak anti-Xa levels were measured throughout pregnancy and 24-hour profiles were measured at 28 and 36 weeks gestation. Doses were adjusted when peak anti-Xa levels fell below 0.1 IU/ml and, in some cases, when levels at 10 and 18 hours post injection were undetectable (<0.05 IU/ml). Our results showed that women receiving tinzaparin (50 IU/kg) frequently had peak (4 hour) anti-Xa levels below 0.1IU/ml and that 46% of these patients required dose adjustment. Similarly anti-Xa levels were also found to be low over the 24-hour period. A starting dose of 75 IU/kg, once daily, gave greater anti-Xa cover over the 24-hour period and may avoid the need for dose adjustment. The results suggest that the pharmacokinetics of tinzaparin are affected by pregnancy. Larger studies are required to determine whether an increased tinzaparin dose (75 IU/kg) would be more effective in the prevention of thrombosis during pregnancy than 50 IU/kg.

Heterozygous factor XI deficiency associated with three novel mutations

To determine the utility of single‐stranded conformation polymorphism (SSCP) analysis for screening mutations in the factor XI (fXI) gene, we investigated three patients with heterozygous factor XI deficiency. DNA sequence analysis confirmed three novel mutations; a CGC → TGC (Arg308Cys) mutation in exon 9, a GCT→GTT (Ala412Val) mutation in exon 11 and an AGC → AGA (Ser576Arg) mutation in exon 15. We postulated on the structural implications of these missense mutations. Our results demonstrated that genotypic analysis is a useful tool for conclusive differentiation between heterozygous factor XI deficiency and normal subjects.

The Ecarin time is an improved confirmatory test for the Taipan snake venom time in warfarinized patients with lupus anticoagulants

&NA; The Taipan snake venom time using dilute phospholipid as a screening test with a platelet neutralization procedure as a confirmatory test has been shown to be a sensitive and specific approach to detection of lupus anticoagulants. Taipan venom is largely insensitive to the effects of ongoing warfarin anticoagulation and this can be useful in detection of lupus anticoagulants in patients receiving this treatment. This study compared the use of the platelet neutralization procedure with the Ecarin time as confirmatory tests for the Taipan snake venom time, the Ecarin venom fraction being insensitive to both lupus anticoagulants and the effects of oral anticoagulants. Screening and confirmatory test data were assessed for phospholipid dependence by three different mathematical methods and there was no advantage in using the Ecarin time in detection of ‘uncomplicated’ lupus anticoagulants. In lupus anticoagulant‐positive warfarinized patients, the Ecarin time achieved higher detection rates than the platelet neutralization procedure irrespective of the method used to assess correction. The Ecarin time confirmed lupus anticoagulants in all of those samples that generated elevated Taipan snake venom time ratios whereas the platelet neutralization procedure identified only 33%. Taipan snake venom time plus Ecarin time offers good diagnostic precision for lupus anticoagulant detection in a group of patients where lupus anticoagulant identification is difficult due to ongoing anticoagulation. Blood Coagul Fibrinolysis 14:307‐312

Identification of a novel mutation in a non-Jewish factor XI deficient kindred

The role of factor XI (FXI) in blood coagulation has been clarified in recent years by descriptions of FXI‐ deficient patients who are prone to excessive bleeding after haemostatic challenge. We have studied a large kindred of an Italian FXI‐deficient patient with a previously undescribed mutation. The propositus, a 68‐year‐old woman, presented with a cerebral thromboembolic event but had no history of bleeding (FXI activity 1.6 U/dl). A sensitive ELISA failed to detect FXI antigen in the propositus. Sequence analysis of the entire FXI gene revealed a TGG to TGC transversion in codon 228 of exon 7 (FXI‐W228C). This missense mutation results in a Trp to Cys substitution within the third apple domain of FXI. We conclude that this novel mutation occurred in a structurally conserved region and may therefore have interfered with either chain folding and secretion or stability of FXI and was responsible for the inherited abnormality seen in this kindred. It is unclear why this kindred does not exhibit a bleeding tendency but it may correlate with a FXI‐like antigen and factor IX binding activity expressed on platelets.

Differential identification of a rare form of platelet-type (pseudo-) von Willebrand disease (VWD) from Type 2B VWD using a simplified ristocetin-induced-platelet-agglutination mixing assay and confirmed by genetic analysis

(HA) with a negative DAT. If the patients had at least two indicators of haemolysis (increased indirect bilirubin, absolute reticulocyte count, lactase dehydroxinase or low haptoglobin), the authors classified them as having AIHA. These criteria are inadequate for a diagnosis of AIHA. DAT-negative AIHA is a well-described subgroup of the AIHAs and is difficult to diagnose, but there are suggested approaches (Petz & Garratty, 2004; Garratty, 2005). If a haematologist has a patient with HA and suspects an immune aetiology, the DAT is the first step (the positive predictive value of a positive DAT in this setting is 83%, and the negative PV is 99% for a negative DAT) (Kaplan & Garratty, 1985). If the DAT is negative, then extensive testing has to be performed to exclude the multiple other causes for haemolytic anaemia. Following this, a workup for DAT-negative AIHA can be performed [e.g. detection of small amounts of red blood cell (RBC)-bound IgG; detection of RBC-bound IgA, IgM; detection of low affinity IgG autoantibodies; studies on patient’s serum and eluates from the patient’s RBCs] (Petz & Garratty, 2004; and Garratty, 2005). If these tests are non-productive and there are haematological suggestions of immune HA (e.g. spherocytes on peripheral blood smear), then a possible diagnosis of DATnegative AIHA can be entertained. None of these investigations were initiated by Borthakur et al (2007); their 14 patients were classified as AIHA on the basis of criteria that only suggested the presence of a haemolytic anaemia, not AIHA (i.e. haemolytic anaemia because of RBC autoantibodies). George Garratty and Lawrence D. Petz American Red Cross Blood Services, Southern California Region, Pomona, and StemCyte International Cord Blood Center, Arcadia, CA, USA. E-mail: Garratty@usa.redcross.org

Real-Time quantitative PCR analysis of factor XI mRNA variants in human platelets

Summary.  Coagulation factor XI (FXI) plays an essential role in blood coagulation. A deficiency of FXI is an unusual hemorrhagic diathesis in that the bleeding tendency can be highly variable, ranging from severe deficiencies with no symptoms to mild and moderate deficiencies requiring multiple blood transfusions for hemorrhages. This variability in bleeding has been attributed to a number of factors including the presence of a novel form of FXI associated with platelets, which ameliorates the bleeding in some cases of FXI deficiency. However, the nature of this platelet FXI molecule is controversial. Hsu et al. (J Biol Chem 1998; 273: 13787–93) suggest that it is a product of normal FXI – but lacking exon V whilst Martincic et al. (Blood 1999; 94: 3397–404) were unable to detect this alternatively spliced variant using RT‐PCR. In order to resolve this controversy, we have employed the highly sensitive technique of real‐time quantitative RT‐PCR using RNA isolated from FXI‐deficient patients. Our results indicate that the platelets of both normal and FXI deficient individuals contain FXI mRNA that is identical to the mRNA found in liver. An exon V deleted splice variant was not detected. Thus the FXI message is not alternatively spliced in platelets and therefore would not be able to produce an unusual FXI protein.