Influence of factor IX on overall plasma coagulability and fibrinolytic potential as measured by global assay: monitoring in haemophilia B.

We sought to determine the influence of factor IX (FIX) deficiency upon overall coagulative and fibrinolytic capacities in plasma using the clot formation and lysis (CloFAL) assay, and to investigate the role of this global assay as an adjunctive monitoring tool in haemophilia B. CloFAL assay parameters were measured in vitro in platelet-poor plasma in relation to FIX activity and antigen (FIX:Ag), and were determined ex vivo among FIX-deficient patients (n = 41) in comparison to healthy individuals (n = 48). Supplementation of FIX-deficient plasma with FIX in vitro demonstrated a non-linear concentration dependence of FIX upon overall plasma coagulability. Ex vivo, coagulability was significantly decreased in FIX-deficient vs. healthy subjects among adults [median coagulation index (CI): 4% vs. 104% respectively; P < 0.001] and children (median CI: 9% vs. 63%; P < 0.001). Fibrinolytic capacity was increased in adult FIX-deficient vs. healthy subjects (median fibrinolytic index: 216% vs. 125%, respectively, P < 0.001), and was supported by a trend in shortened euglobulin lysis time (ELT). Severe haemophilia B patients showed heterogeneity in aberrant CloFAL assay waveforms, influenced partly by FIX:Ag levels. Patients with relatively preserved FIX:Ag (i.e. dysfunctional FIX) exhibited a shorter time to maximal amplitude in clot formation than those with type I deficiency. During patient treatment monitoring, markedly hypocoagulable CloFAL assay waveforms normalized following 100% correction with infused FIX. The CloFAL global assay detects FIX deficiency, demonstrates differences in coagulability between dysfunctional FIX and type I deficiency, and appears useful as an adjunctive test to routine FIX measurement in monitoring haemophilia B treatment.

Influence of factor VIII on overall coagulability and fibrinolytic potential of haemophilic plasma as measured by global assay: monitoring in haemophilia A.

The objectives of the present study were to evaluate the analytical sensitivity of the recently developed Clot Formation and Lysis (CloFAL) global assay for factor VIII (FVIII) deficiency, both in vitro and ex vivo, to determine whether this global assay is influenced by FVIII inhibitors, and to investigate the coagulative response to FVIII replacement in haemophilia A patients using the CloFAL assay in comparison with FVIII activity. Among adults and children alike, the CloFAL assay coagulation index (CI) was significantly decreased in FVIII-deficient vs. healthy subjects (adults median CI: 2% vs. 94% respectively; children median CI: 3% vs. 63%; P < 0.001 for each), and correlated significantly with activated partial thromboplastin time-based FVIII activity across all individuals (r = 0.78; P < 0.001). The CloFAL assay was analytically sensitive to deficient FVIII activity and also influenced by the presence of von Willebrand factor. Severe haemophilia A patients without inhibitory antibodies to FVIII showed considerable heterogeneity in CloFAL assay waveforms, despite a uniformly diminished CI of 0-1%. During FVIII infusion half-life studies in patients with severe haemophilia A, the CloFAL assay demonstrated a marked rise in coagulability 30 min following infusion, with progressive decrease in coagulability towards baseline over the ensuing 48-h period. In each case, the profile of coagulative response to FVIII infusion as determined by CloFAL assay CI differed qualitatively from that measured by FVIII activity. These findings indicate that the CloFAL assay may be useful as an adjunctive test to FVIII activity measurements in the therapeutic monitoring of haemophilia A.

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.

The prolonged thrombin time of nephrotic syndrome.

PURPOSE: Little information is available that documents the incidence and possible etiology of a prolonged thrombin time (TT) found in children with nephrotic syndrome. We speculated that this prolonged TT might best be explained by an altered fibrinogen similar to that found in the newborn. PATIENTS AND METHODS: We describe 20 children with nephrosis, an unexplained prolonged TT and reptilase time (RT), and an elevated fibrinogen level. Thrombin and/or plasmin effect on plasma fibrinogen was studied by analyzing for soluble monomer, D-dimer, fibrin degradation products, B beta 1-42 peptide, and by polyacrylamide gel electrophoresis (PAGE) and agarose electrophoresis. Structural changes in the fibrinogen molecule were investigated using two-dimensional gel (2D gel) electrophoresis. Fibrinogen was purified via glycine precipitation, and the sialic acid (SA) content was determined. RESULTS: No evidence for in vivo thrombin and/or plasmin effect on fibrinogen could be detected in 13 of 20 children tested. Additionally, no fibrin/fibrinogen degradation products or soluble fibrin complexes were detected using PAGE or agarose electrophoresis in this group of patients. The B beta isoforms of nephrosis fibrinogen were similar in isoelectric point on 2D gel electrophoresis to those of fetal fibrinogen and demonstrated a greater electronegative shift when compared with normal adult fibrinogen. Also, the SA content of nephrosis and fetal fibrinogen were greater than that measured in adult fibrinogen. Both nephrosis and fetal fibrinogen were more resistant to neuraminidase treatment than was normal adult fibrinogen. CONCLUSIONS: These data support the notion that an altered fibrinogen exists in some children with nephrotic syndrome characterized by an increased TT and RT, elevated fibrinogen, and both an increased negative charge and SA content.

Thrombosis in children receiving L-asparaginase. Determining patients at risk.

PURPOSE: A prospective study of coagulation in 15 children who received L-asparaginase, vincristine, or prednisone plus or minus an anthracycline as part of a treatment program for leukemia or leukemia-lymphoma syndrome was conducted. PATIENTS AND METHODS: One patient developed a central nervous system thrombosis. RESULTS: The inhibitors of coagulation, including antithrombin-III, protein C, protein S, and plasminogen, were decreased in many individuals, but were not significantly different in the patient who had experienced the thrombotic event. Platelet aggregations to low molar ADP were performed in four patients, and in three patients showed a hyperaggregable pattern. CONCLUSIONS: The patient with thrombosis developed a transient acquired type II pattern on multimeric analysis of the von Willebrand factor, which was not seen in the other individuals studied.

Vitamin K deficiency.

Vitamin K (phylloquinone, K1; menaquinone, K2) functions as an essential cofactor for the synthesis of the coagulation protein factors II, VII, IX, X and protein C and S by promoting a unique post-translational modification of specific glutamic acid residues to gamma-carboxylglutamic acid, thus mediating calcium binding to phospholipid surfaces. Vitamin K deficiency results in a depletion of liver stores of phylloquinone, decreased plasma levels of vitamin K1, increased levels of K1 epoxide, appearance of noncarboxylated protein (PIVKA), decreased levels of functioning vitamin K-dependent clotting factors and prolongation of the APTT, PT and thrombotest. When the progression of deficiency leads to abnormal clotting tests a generalized bleeding tendency occurs. Noncarboxylated prothrombin (PIVKA-II) determinations are a sensitive indicator of vitamin K deficiency. Although Vitamin K deficiency can occur at any age (warfarin, fasting, antibiotic therapy, malabsorption syndromes) the major public health problem is related to prevention of early, classic and late hemorrhagic disease of the newborn (HDN). A single dose of oral or parenteral vitamin K prevents classic HDN but the most effective way to prevent early HDN is by giving large doses to the mother prior to delivery (2 weeks). Late HDN in breastfed infant occurs with a prevalence of about 20 per 100,000 live births when no neonatal prophylaxis is given. Parenteral (1 mg) K1 prevents late HDN and single or repeated doses of oral vitamin K reduces the incidence but does not eliminate all late HDN. The optimal (cost, feasibility, effective) mode of neonatal prophylaxis remains to be determined.

The coagulopathy of childhood leukemia. Thrombin activation or primary fibrinolysis?

Little information is available on the prevalence and etiology of the coagulopathy present in some children with acute leukemia at disease presentation. We studied 102 children with newly diagnosed acute leukemia (50 retrospective: Group A; and 52 prospective: Group B) with prothrombin time (PT), partial thromboplastin time (PTT), thrombin time (TT), fibrinogen (FIB), and fibrin degradation products (FDP). All patients in Group B also had assessment of thrombin activation by measurement of the crosslinked fibrin fragment, D-dimer, and of primary fibrinolysis with the B beta 1-42 peptide. Additionally, ten patients from Group B had Factors II, V, VII, and X measured, and eight of these patients had measurement of tissue factor from sonicated bone marrow cells. Thirty-two percent of Group A and 40% of Group B had totally normal coagulation studies, whereas 20% of Group A and 10% of Group B had a severe coagulopathy on disease presentation. A high percentage of both groups had elevated PT (Group A, 52%; Group B, 27%) and increased FDP (Group A, 39%; Group B, 25%). In Group B, 38% of the patients had a positive D-dimer, whereas only 4% of this prospective group had an elevated B beta 1-42 peptide (P less than 0.00001). Nine of ten patients with a positive D-dimer had low levels of one or more of the extrinsic pathway factors. Three of four patients with the highest tissue factor levels were of monocytoid leukemia cell type. These data indicate that the coagulopathy associated with acute leukemia of childhood is usually mediated by thrombin activation.

Use of DDAVP in inherited and acquired platelet dysfunction.

Twenty-one patients with prolonged bleeding times secondary to inherited disorders of platelet function and eight patients with prolonged bleeding times secondary to acquired platelet dysfunction were given 0.3 micrograms per kilogram of DDAVP, 1-deamino-8-D-arginine vasopressin, intravenously. Sixteen of twenty-two DDAVP trials in patients with inherited platelet dysfunction (73%) and seven of the nine DDAVP trials in patients with acquired platelet dysfunction (78%) resulted in normalization or shortening of the prolonged bleeding times by at least 4 min. The bleeding time response did not correlate with changes in the levels of von Willebrand factor (vWf) antigen or ristocetin cofactor activity, nor was it associated with changes in vWf multimeric analysis or in vitro platelet aggregations following the administration of DDAVP. Shortening of the bleeding time with DDAVP was seen in patients with a failure to release/storage pool type defect, thromboxane synthesis type defect, Bernard-Soulier syndrome, Glanzmann's thrombasthenia, the May-Hegglin anomaly, liver disease, nonuremic renal disease, myelofibrosis, and Tangier's disease.

Evaluation of the safety, recovery, half-life, and clinical efficacy of antithrombin III (human) in patients with hereditary antithrombin III deficiency. Cooperative Study Group.

Antithrombin III (Human) (AT III) was administered to 18 patients with documented hereditary AT III deficiency. In eight patients with no ongoing clinical symptoms of thrombosis, the percent increase per unit AT III infused per kilogram of body weight ranged from 1.56% to 2.74%, and the half-life from 43.3 to 77.0 hours. No significant difference was noted between patients receiving and those not receiving coumarin therapy. In clinically ill patients, the in vivo recovery was significantly lower and ranged from 0.64% to 1.90% increase per unit AT III infused/kg. Efficacy of AT III was evaluated in 13 patients for the prevention or treatment of thrombosis. AT III was efficacious as assessed by the absence of thrombotic complications after surgery and/or parturition, and the nonextension and nonrecurrence of thrombosis in patients exhibiting an acute thrombotic episode. No side effects were noted. Follow-up studies indicated no hepatitis B seroconversion and no alanine aminotransferase elevations in patients who were not transfused with other blood products.

Effect of intravenous immune globulin on the coagulopathy of Kawasaki syndrome.

We studied the effects of immune globulin and aspirin versus aspirin alone on platelet count, platelet activation, and factor-mediated coagulation in patients with Kawasaki syndrome. Coagulation tests were performed on the day of admission to the study and 4 to 6 days later. Twenty-three patients were enrolled; 12 received immune globulin intravenously plus aspirin, and 11 received aspirin alone. At initiation of the study the groups were comparable with regard to age, sex, race, and time from onset of illness to study entry. Coagulation values were similar at entry with the exception that the aspirin group had a geometric mean platelet count that was higher than the platelet count in the aspirin-immune globulin group (p = 0.02). Four days after entry there were no significant differences between the two groups in any coagulation studies. Although the immune globulin preparation used has been effective in reducing the prevalence of coronary artery aneurysms, it appears to have no early effect on reduction of platelet activation or other measures of coagulopathy. The mechanism of action of immune globulin in patients with Kawasaki syndrome remains to be elucidated.

Hyponatremia and seizures in young children given DDAVP.

Desmopressin (DDAVP), a synthetic vasopressin, temporarily corrects bleeding abnormalities associated with mild hemophilia A, von Willebrand disease, and disorders of platelet function. The side effects of DDAVP are considered benign although most of its use has been in adults and older children. We report four children under the age of 2 years who became hyponatremic after intravenous DDAVP administration (0.3 microgram/kg). Three of them developed grand mal seizures. A review of the literature and these cases indicate that associated risk factors for hyponatremia after DDAVP administration include stress, surgery, anesthesia and narcotics (endogenous release of antidiuretic hormone), vomiting (loss of Na+), liver disease (hindered metabolism of DDAVP), renal tubular acidosis (chronically low serum Na+), multiple doses of DDAVP, and overhydration with hyponatremic fluids. DDAVP is not a benign drug in this age group and shows a serious potential for hyponatremia and seizures. Fluid restriction, avoidance of hyponatremic solutions, and close monitoring of serum electrolytes and urine output for at least 15-20 hr after the administration of DDAVP, when used in children under the age of 2 years, is warranted.

Heparin cofactor II in adults and infants with thrombosis and DIC.

Heparin cofactor II (HC II) was measured by a chromogenic activity assay in normal preterm infants (gestational age, 28-36 weeks; n = 17; 29% +/- 11.5 [mean +/- 1 SD], range 11-51), normal full-term infants (n = 18; 49% +/- 6.6 [mean +/- 1 SD], range 36-58), and normal adults (n = 38; 101% +/- 14 [mean +/- 1 SD], range 73-130). Normal children attained adult levels at approximately 5 to 7 months of age. The lower values in preterm and term infants most likely reflect immature liver function. Neither adults and children with a history of thrombosis with prior negative evaluation (n = 74), patients with documented protein C and protein S deficiency (n = 4), nor sick infants without evidence of consumptive coagulopathy (n = 15) had significantly lower levels of HC II activity. Infants with disseminated intravascular coagulation (n = 2) had strikingly lower levels of HC II activity.