Inadequacy of high K+/nigericin for calibrating BCECF. I. Estimating steady-state intracellular pH.

Intracellular pH (pHi) was measured in single vascular smooth muscle (VSM) cells, cultured from rabbit abdominal aorta, using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) on a microscope-based fluorescence system. Three lines of evidence are presented that using nigericin along with high external K+ to calibrate intracellular BCECF produces systematic errors in pHi. 1) The intrinsic buffering power (beta int), measured using weak bases (e.g., ammonium), was 2.5 times smaller than that measured using weak acids (e.g., propionic acid). This discrepancy became small if pHi had really been approximately 0.2 lower than what was estimated using nigericin-calibrated pHi values. 2) Total cellular buffering power (beta tot) in the presence of CO2/HCO-3 was measured and found to be much smaller than could account for the beta int, together with the contribution of CO2/HCO3 (beta CO2: assumed to be an open system buffer). If the true pHi values were approximately 0.2-0.4 lower than our nigericin-calibrated values, then the sum of beta int and beta CO2 equals beta tot. 3) A null technique was utilized for bracketing steady-state pHi; estimates of steady-state pHi using this null technique were approximately 0.2 lower than the high K+/nigericin-calibrated estimates. Four other cell types were examined: rat hepatocytes, rat corticotrophs, human keratinocytes, and rabbit fibroblasts. These other cells also displayed discrepancies between null and nigericin estimates of steady-state pHi, as well as differences between buffering power assessed using weak bases and acids. Finally, one potential source for these discrepancies is described: selecting an inappropriate external K+ to use with nigericin can produce systematic errors in pHi of approximately 0.1.

Inadequacy of high K+/nigericin for calibrating BCECF. II. Intracellular pH dependence of the correction.

In the accompanying study [G. Boyarsky, C. Hanssen, and L.A. Clyne. Am. J. Physiol. 271 (Cell Physiol. 40): C1131-C1145, 1996], it was demonstrated that steady-state intracellular pH (pHi) determined using high K+/nigericin calibrations was systematically in error in vascular smooth muscle (VSM) cells by approximately 0.2 pH units. In this paper the possibility is explored that this correction (pHcor) to the nigericin-calibrated pHi (pHnig) might not be a constant but could vary as pHi varies. The range of pHi during exposures to "null solutions" designed to bracket pHi was extended to acidic and alkaline levels relative to the starting pHi in VSM cells. The pHcor necessary to correct pHnig was linearly dependent on pHnig, increasing from near zero at approximately 6.0 to approximately 0.2 at steady-state pHi, to approximately 0.3 at alkaline pHnig. It is shown how to retrieve previously acquired (tabulated) data using the linear relationship between pHcor and pHnig. Also examined were what corrections must be made to high K+/nigericin calibration curves to correct for this pHi-dependent pHcor. The following changes in the calibration parameters were found: the maximal fluorescence ratio increased from 16.75 to 17.28; the minimal fluorescence ratio decreased from 2.15 to 1.57; and the pK of 2',7'-bis (carboxy-ethyl)-5 (6)-carboxyfluorescein decreased from 7.13 to 6.93. Three potential explanations for these changes are discussed: external [K+] in the nigericin solutions could have been too low; internal [K+] changes during the calibration because of the finite buffering power of cells; and other acid-base transport/generation could have been contributing during the nigericin calibrations (i.e., nigericin does not overwhelm to insignificance other processes generating/consuming H+). The nonconstancy of pHcor is shown to have profound implications for measuring changes in pHi.

Superiority of in vitro over in vivo calibrations of BCECF in vascular smooth muscle cells.

We measured intracellular pH (pHi) in single vascular smooth muscle cells (VSM) cultured from rabbit abdominal aorta, using 2',7'-biscarboxyethyl-5(6)carboxyfluorescein (BCECF) on a microscope-based fluorimetric system. We previously found substantial errors introduced by using high K+/nigericin to calibrate intracellular BCECF (1). We also previously demonstrated that the necessary correction (pHcor) to the high K+/nigericin-calibrated pHi was linearly dependent on pHi, increasing with increasing pHi (2). When the nigericin calibration data were corrected using this pHcor, the new corrected calibration was similar to the result of calibrating BCECF in vitro (higher Rmax, lower Rmin, and lower pK). Therefore, in this study the possibility is considered that in vitro calibrations might provide better estimates of pHi. Our best estimate for the absolute level of pHi derives from a null method for bracketing steady-state pHi. In VSM cells, using only in vitro calibrations to estimate steady-state pHi leads to less error (only approximately 0.08 different from null estimates) than using nigericin calibrations alone (approximately 0.2 different from null estimates). Unlike high K+/nigericin calibrations, the error, pHcor, introduced by using an in vitro calibration is pHi independent. Using high K+/nigericin or in vitro calibrations, along with the respective pHcor on the same experimental days in the same cells, gave the same estimate of pHi whether in the steady state, in acid-loaded cells, or in alkali-loaded cells. Similarly, when appropriately corrected, both methods gave indistinguishable calibration curves. Thus, the two methods are entirely equivalent from the standpoint of accuracy. Because nigericin is toxic, expensive, and complicated in its use, we suggest that in vitro calibrations, along with simple null determinations to assess the small, constant correction factor, be used to calibrate intracellular BCECF.-Boyarsky, G., Hanssen, C., Clyne, L. A. Superiority of in vitro over in vivo calibrations of BCECF in vascular smooth muscle cells.

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.

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.

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.

A unique factor XIII inhibitor to a fibrin-binding site on factor XIIIA.

An 81-year-old woman, who presented with sudden episodes of spontaneous bleeding, was found to have a specific inhibitor of factor XIII. Her fibrin clots had approximately 70% gamma-gamma and no alpha polymer formation, under conditions where normal fibrin was fully cross-linked; the patient's clots were soluble in urea or monochloroacetic acid. Factor XIII activity in her plasma was 24%, measured by the dansylcadaverine incorporation assay. When mixed with normal plasma, the patient's plasma inhibited fibrin cross-linking; however, in mixtures of patient and normal plasma, there was no inhibition of factor XIII activity when assayed by the incorporation of dansylcadaverine into casein. Thus, this inhibitor was active against fibrin cross-linking but not against ligation of small molecules to casein. Consequently, gel electrophoresis of reduced, sodium dodecyl sulfate-solubilized fibrin clots was a simple, quantitative method that was used to measure inhibitor activity. This inhibitor is unique and has been designated inhibitor New Haven. It was neutralized by anti-IgG and anti-kappa. It did not inhibit the activation of factor XIII but did inhibit fibrin cross-linking. There was complex formation between the inhibitor and activated factor XIII (A', A*) but not between A2 or fibrinogen. Only A', A* and the 56-Kd fragment bound to affinity columns made with this IgG. The inhibitor significantly decreased the binding of A', A* to fibrin clots. These data indicate that the epitope for this inhibitor is in a fibrin binding site. It is hidden in the zymogen and expressed on A' and A*, indicating that the conformational change occurring with the cleavage of the activation peptide is sufficient to expose the fibrin binding site.

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.