Geoffrey F. Savidge

Novel Vascular Endothelial Growth Factor Binding Domains of Fibronectin Enhance Vascular Endothelial Growth Factor Biological Activity

Interactions between integrins and growth factor receptors play a critical role in the development and healing of the vasculature. This study mapped two binding domains on fibronectin (FN) that modulate the activity of the angiogenic factor, vascular endothelial growth factor (VEGF). Using solid-phase assays and surface plasmon resonance analysis, we identified two novel VEGF binding domains within the N- and C-terminus of the FN molecule. Native FN bound to VEGF enhanced endothelial cell migration and mitogen-activated protein (MAP) kinase activity, but FN that is devoid of the VEGF binding domains failed to do so. Coprecipitation studies confirmed a direct physical association between VEGF receptor-2 (Flk-1) and the FN integrin, &agr;5&bgr;1, which required intact FN because FN fragments lacking the VEGF binding domains failed to support receptor association. Thrombin-activated platelets released intact VEGF/FN complexes, which stimulated endothelial cell migration and could be inhibited by soluble high affinity VEGF receptor 1 and antibodies to &agr;5&bgr;1 integrin. This study demonstrates that FN is potentially a physiological cofactor for VEGF and provides insights into mechanisms by which growth factor receptors and integrins cooperate to influence cellular behavior.

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.

Primary thrombocythaemia: diagnostic criteria and a simple scoring system for positive diagnosis.

Summary Forty new patients with elevated platelet counts (>600 × 1O9/1) and without palpable splenomegaly were assigned to diagnostic groups defined by essentially conventional criteria after 3 months follow up: Proliferative (17), reactive (17) or unclassified (six). Mean platelet volume (MPV), platelet distribution width (PDW). platelet nucleotide ratio (ATP:ADP), unstimulated BFU‐E derived colony formation from peripheral blood. spleen scan and clinical ischaemia were assessed at the outset, with a view to defining diagnostic criteria for distinguishing primary thrombocythaemia from reactive thrombocytosis. All except the first variable were significantly associated with diagnostic group (P< 0.05). A simple scoring system was devised: enlarged spleen on scan, or presence of BFU‐E, each scored 2: elevated PDW (> 2 SD from mean), elevated ATP:ADP (>4 SD from mean) or presence of clinical ischaemia, each scored 1. Score totals ± 3 predicted primary thrombocythaemia, and totals < 3 suggested reactive thrombocytosis (predictive value 89%). The system correctly predicted diagnosis in four out of four (probably six out of six) patients whose diagnosis was not apparent initially, and thus whose results were not used in constructing the scoring system. Exclusion of BFU‐E from the system resulted in only one incorrect prediction in this group.

THE ORIGIN AND PHYSIOLOGICAL RELEVANCE OF α‐GRANULE ADHESIVE PROTEINS

Platelet a-granules are the principal intracellular reservoirs of proteins destined for release during primary haemostasis at the site of vessel wall injury. Despite the identification of many adhesive proteins within the a-granules, the biological significance and origin of these moieties have been subject to much speculation, particularly since high concentrations are also present in the plasma and within extracellular matrices.

Effectiveness in controlling haemorrhage after dental scaling in people with haemophilia by using tranexamic acid mouthwash

Aims To compare the effectiveness of tranexamic acid mouthwash (TAMW) in controlling gingival haemorrhage after dental scaling with that of using factor replacement therapy (FRT) prior to dental scaling in people with haemophilia.Design Double-blind cross-over randomised control trial.Setting Dedicated hospital dental practice for patients with inherited bleeding disorders.Method Sixteen patients with haemophilia who required dental scaling participated in this pilot study. The experimental treatment regime (ETR) involved transfusing each patient with saline before scaling both quadrants on one side of the mouth followed by oral rinsing with TAMW four times daily for up to eight days. The control regime (CR) involved giving each patient FRT before scaling the opposite side of the mouth followed by use of a placebo TAMW. Each patient underwent both treatments in a random-ised sequence. Both the operator and the patients were unaware of which were the ETR and CR episodes. On both occasions the patient kept a log book of the rinsing regime and any post-operative bleeding. Additionally, a structured post-treatment telephone interview was conducted to assess the effectiveness and the patient acceptability of the ETR.Results Thirteen patients completed the study. No statistically significant difference was found in gingival bleeding and mouthwashing frequencies between the ETR and the CR (p > 0.05). Five patients reported no gingival bleeding with either the ETR or the CR. No patient, using either regime, required extra FRT due to gingival haemorrhage. All subjects found the ETR acceptable and easy and reported feeling safe in using TAMW alone to control gingival bleeding after dental scaling.Conclusion TAMW use after dental scaling was as effective as using FRT beforehand in controlling gingival haemorrhage for people with haemophilia.

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.

Acquired haemophilia A: Errors in the diagnosis

The distinction between a specific factor inactivator and a non-specific inhibitor is important when confronted by a patient with a history of bleeding and abnormal in-vitro coagulation tests. We report on two patients who presented with bleeding and a prolonged activated partial thromboplastin time. Initial factor assays suggested combined deficiency of factors VIII and IX as a result of the presence of inactivators. The use of dilution studies, chromogenic assays, a novel in-house enzyme-linked-immunosorbent-assay-based technique and phospholipid neutralization, demonstrated that Case 1 had a genuine factor VIII inactivator resulting in factor VIII levels of less than 1 IU/dl but no factor IX deficiency. Case 2 had normal levels of factor VIII on further testing and no specific inactivator to either factor VIII or IX but a potent antiphospholipid antibody which had interfered with the phospholipid-dependent in-vitro assays. Care must be taken in the interpretation of laboratory assays in the presence of antiphospholipid antibodies to ensure that the correct diagnosis is made and inappropriate treatment avoided.