Ancylostoma factor Xa inhibitor: partial purification and its identification as a major hookworm-derived anticoagulant in vitro.

Soluble protein extracts from adult Ancylostoma hookworms were found to contain an anticoagulant activity that markedly prolonged both the prothrombin time (PT) and partial thromboplastin time (PTT). By chromogenic peptide substrate and clotting time assays, the anticoagulant activity was attributed to a specific inhibitor of clotting factor Xa. The hookworm anticoagulant was partially purified by ion-exchange column chromatography. Those column fractions containing anti-Xa activity by chromogenic assay also prolonged the PT and PTT as well as the factor X (Stypven) clotting time. These data suggest that this potent factor Xa inhibitor is a major anticoagulant from the adult Ancylostoma hookworm.

The lupus anticoagulant. High incidence of 'negative' mixing studies in a human immunodeficiency virus-positive population.

We identified 100 patients (51 males and 49 females) as having the lupus anticoagulant. The following diagnoses were found in the patient population: human immunodeficiency virus positivity, 20%; systemic lupus erythematosus, 10%; prolonged preoperative activated partial thromboplastin time (APTT), 10%; procainamide hydrochloride-induced inhibitor, 9%; deep vein thrombosis, 6%; seizure disorders/epilepsy, 5%; and miscellaneous conditions, 40%. Identification was based on a prolonged APTT (> 40 seconds) that normalized with increased phospholipid concentrations and/or a prolonged Russell viper venom clotting time patient-control ratio of 1.20 or greater. In 68 cases (group 1), patient plasma prolonged the APTT of normal plasma in a 1:1 mixing study. However, in 32 cases (group 2), no such prolongation was observed. There was a significant difference between presenting APTTs in patients from group 1 (mean +/- SD, 58.29 +/- 13.30 seconds) compared with that in group 2 (mean +/- SD, 47.93 +/- 5.09 seconds). Furthermore, 66% of group 1 patients had elevated anticardiolipin antibody titers compared with only 41% in group 2. Of the 32 patients in group 2, 16 (50%) were positive for human immunodeficiency virus. We concluded that the investigation of a lupus anticoagulant should not be abandoned because patient plasma does not prolong the APTT of normal plasma in a mixing study, especially in a human immunodeficiency virus-positive population.

Successful childbirth by a patient with congenital factor XI deficiency and an acquired inhibitor.

Acquired inhibitors in factor XI deficiency (FXI) are rare. The presence of an inhibitor during pregnancy poses a potential haemorrhagic risk to the fetus. We report an uncomplicated pregnancy and successful childbirth by a woman with congenital FXI deficiency and an acquired inhibitor, and discuss the persistence of residual FXI activity in the presence of an inhibitor.

Fibrinogen Ledyard (A alpha Arg16----Cys): biochemical and physiologic characterization.

Fibrinogen Ledyard was discovered in a 10-year-old boy with a mild bleeding history. His father had the same defect and a bleeding history after surgery. Both patients were heterozygous. The plasma fibrinogen concentration was normal immunologically (335 mg/dL) and very low functionally (52 mg/dL). Purified fibrinogen Ledyard had a prolonged polymerization, which was somewhat corrected by addition of Ca2+ ions. High performance liquid chromatography (HPLC) analyses of the fibrinopeptides released by thrombin showed 1 mol of fibrinopeptide A (FPA) and 2 mol of fibrinopeptide B (FPB) released per mole of fibrinogen Ledyard. Steady-state kinetic parameters were evaluated for release of FPA by thrombin. When the concentration of fibrinogen Ledyard was corrected to 50% of total protein, because only 50% of fibrinogen Ledyard can release FPA, the kinetic constants were similar to those of control fibrinogen (Km = 7.5 mumol/L for A alpha chain, kcat = 54 s-1). This finding indicates that the cleavage site of the A alpha chain in these abnormal molecules may not interact with the catalytic site of thrombin. The three chains of fibrinogen Ledyard were isolated on reverse-phase C4-HPLC. The sequence of the amino terminus of A alpha chain showed that Arg in position 16 was replaced by Cys in the abnormal molecules. Approximately half of fibrinogen Ledyard (52%) was clotted by reptilase, suggesting that fibrinogen Ledyard may consist of 50% normal homodimers (A alpha Arg16 . A alpha Arg16) and 50% abnormal homodimers (A alpha Cys16 . A alpha Cys16). Abnormal molecules could form disulfide bond between the A alpha Cys16 residues. Thus, the abnormal molecules have a different structure that does not bind to thrombin. Probably the abnormality of polymerization of fibrinogen Ledyard results from the interaction of the abnormal molecules with normal fibrin monomers, so that the growth of fibrin protofibrils is inhibited. This abnormal fibrinogen supports adenosine diphosphate-induced platelet aggregation in a normal manner.

The prevalence and biologic significance of lupus anticoagulant and anticardiolipin antibodies in a general obstetric population.

Circulating antibodies to negatively-charged phospholipids have been implicated in the genesis of adverse pregnancy outcomes. However, it has yet to be established that these antibodies are causative or that they are invariably associated with untoward perinatal outcomes. To address this issue, the prevalence of lupus anticoagulant and anticardiolipin antibodies was recorded in a low-risk obstetric population, and the outcome of untreated pregnancies were evaluated. Two of 737 patients (0.27%) had lupus anticoagulant documented by a prolonged activated partial thromboplastin time that did not correct this mixing studies. In comparison, greatly elevated concentrations of immunoglobulin M-anticardiolipin antibodies or immunoglobulin G-anticardiolipin antibodies were identified in 16/737 (2.2%) patients by means of an enzyme-linked immunosorbent assay. Spontaneous abortions occurred in both lupus anticoagulant-positive patients, suggesting that the activated partial thromboplastin time used was a relatively insensitive but specific marker for antiphospholipid antibody-associated adverse pregnancy outcomes. In contrast, although 12 of 16 anticardiolipin antibodies-positive pregnancies were complicated by perinatal loss, preterm delivery, or fetal growth retardation, four patients had uncomplicated pregnancies. Moreover, the distribution of anticardiolipin antibodies concentrations in these four patients was not clustered among the lowest anticardiolipin antibodies values, and anticardiolipin antibodies concentrations correlated weakly with adverse outcomes. These findings suggest that antiphospholipid antibodies are related to adverse pregnancy outcomes in a complex fashion and that therapy is not always required for acceptable outcomes in patients without other risk factors.

Procainamide-induced circulating anticoagulants in a congenitally-deficient factor XI patient.

A 70 year old male patient admitted for coronary bypass surgery presented with a procainamide-induced lupus syndrome. This syndrome included a LLAC with a positive IgM ACA titer as well as a factor XII inhibitor. These drug-induced inhibitors were superimposed upon the patient's congenital deficiency of factor XI. The methods used to identify these abnormalities are described together with the replacement therapy employed to cover the surgical procedure. The long-term withdrawal of procainamide was associated with correction of all coagulation abnormalities except the factor XI deficiency.

Time dependency of lupuslike anticoagulants.

Fifty-two patients (29 female and 23 male) with lupuslike anticoagulants were reviewed retrospectively to determine whether their inhibitors were time dependent (TD). In 21 cases (40%), a TD pattern emerged: when patient plasma was added to normal plasma and an activated partial thromboplastin time (APTT) test was performed on the mixture, the patient/control ratio after incubation for one hour at 37 degrees C (60-minute ratio) exceeded significantly the respective preincubation ratio (zero-minute ratio). In four cases (8%), anticoagulant activity would have gone undetected if mixing studies had been restricted to the preincubation phase. The TD anticoagulants appeared to be more potent than their time-independent (TI) counterparts (mean APTT, 74.1 vs 58.5 s, respectively). An APTT greater than 63 s was 85% predictive of TD behavior. Greater overlap between the two groups was seen when zero-minute ratios were compared; an equivalent cutoff of 1.36 for the zero-minute ratio was only 65% predictive of TD behavior. The separation between the two groups was most striking when 60-minute ratios were compared. Nineteen TD patients (90%) had 60-minute ratios that exceeded the mean TI ratio of 1.33, while 30 TI patients (97%) had 60-minute ratios that were lower than the mean TD ratio of 1.89. Collectively, these findings indicate that many potent lupuslike anticoagulants require incubation to express maximal anticoagulant activity. Indeed, in some cases, anticoagulant activity might not be detected if mixing studies are restricted to the preincubation phase. The APTT can be helpful in predicting which anticoagulants will show TD behavior.

Acquired factor XI inhibitors in congenitally deficient patients.

Four factor XI (F XI)-deficient patients are described, all of whom formed circulating anticoagulants against F X1. In the three most severely affected patients (F XI 0%-6% activity), the anticoagulant appeared to have been stimulated by plasma infusion. However, in the milder case (25% F XI activity), no infusion had been documented. The findings in these cases emphasize the diversity of F XI inhibitors in congenitally deficient patients. Awareness of the potential development of these inhibitors will be helpful in both daily management and perioperative care of such patients.

Species specificity of lupus-like anticoagulants.

Mixing studies using activated partial thromboplastin time (APTT) technique were performed on 14 patients with lupus-like anticoagulants (LLACs) using human, equine, bovine, porcine and canine plasma. Eleven patients significantly prolonged the APTT of normal human plasma (patient/control ratio = greater than 1.15) but no patient inhibited bovine plasma. However, with one exception, equine APTT ratios were concordant with human ratios. Seven of fourteen patients also inhibited porcine plasma but in each case porcine APTT ratios were lower than their human or equine counterparts. None of five patients tested inhibited canine plasma. Collectively, these results suggest heterogeneity among LLACs at least with regard to species specificity.

Circulating anticoagulant to animal plasmas in a cardiac patient.

A circulating anticoagulant against bovine, equine, guinea pig, and sheep plasmas developed in a 15-year-old cardiac patient. He had been exposed to both bovine and porcine heparin over a period of 13 years, and had a porcine valve placed four years before the anticoagulant was noted. There was no anticoagulant activity detected against human, rat, or porcine plasma, and an equivocal reaction against rabbit plasma. There was no apparent clinical significance to the anticoagulant. It did, however, confuse the interpretation of coagulation factor assays based on animal substrate plasmas.

Plasma requirement for expression of lupus-like anticoagulant.

Described here are five patients with lupus-like anticoagulants, four of whom required coincubation of normal plasma in order to inhibit the pro-coagulant activity of crude brain phospholipid. It is suggested that this plasma requirement for expression of anticoagulant activity is similar or identical to the "lupus-cofactor" effect described by earlier observers.

Correction of clotting factor "deficiencies" in plasma from patients with lupus-like anticoagulants.

Identification of spurious coagulation factor deficiencies that are known to occur in association with lupus-like anticoagulants (LLACs) requires the use of cumbersome laboratory procedures. To determine whether single-stage assays employing the APTT system may be used to identify such artifacts, we measured multiple clotting factor levels by several techniques in plasma of six patients with typical LLACs. While normal activities of factors VIII, IX, XI and XII were measured in only 4/24 APTT assays (17%) employing human plasma substrate, normal factor activities were present in all 24 APTT assays employing bovine, canine or rabbit plasma substrate. Normal factor II, V and X activities were recorded in all but one case in assays that utilized a modified Stypven time, while normal factor VIII levels were determined in 5/6 plasmas when the thromboplastin generation test was employed. These results indicate that the use of heterologous plasma substrates in the APTT system may provide a simple method to identify such coagulation factor "deficiencies".

Spontaneous factor XI inhibitors. Seven additional cases and a review of the literature.

Circulating anticoagulants are endogenous blood components that inhibit the action of clotting factors. In some inhibitor conditions this inactivation in the function of the hemostatic system may lead to life-threatening hemorrhagic diathesis. Inhibitors directed against factor XI are generally associated with little or no impairment of the hemostatic system. We analyzed all reported cases of spontaneous factor XI inhibitor in the international literature, as well as cases identified at the Yale--New Haven (Conn) Hospital between 1970 and 1980, considering clinical spectrum, diagnosis, and therapy.

Circulating factor XI antibody and disseminated intravascular coagulation.

Association of a circulating factor XI anticoagulant and disseminated intravascular coagulation (DIC) is described in a 33-year-old woman. Although the patient had rheumatoid arthritis and a bacterial infection treated with antibiotics, the anticoagulant was thought to be secondary to systemic lupus erythematosus. Curiously, the low levels of factor XI did not prevent the DIC from developing.

Spontaneously acquired Factor IX inhibitors in childhood.

Two previously healthy children, ages 8 months and 35 months, developed spontaneous inhibitors to Factor IX. Brief illnesses of presumed viral origin preceeded hemorrhagic symptoms in both children. Cutaneous ecchymoses and traumatic soft tissue hemorrhage were the predominant clinical features in each case. Bleeding symptoms persisted for less than 3 days and laboratory evidence of Factor IX inhibition resolved within 3 weeks. One child required treatment with fresh frozen plasma and packed red blood cell transfusions. The other child received corticosteroid therapy. Given the transient nature of acquired Factor IX inhibitors in the nonhemophilic child, a conservative approach toward therapy is recommended unless life-threatening complications supervene.