Hereditary warfarin resistance. Investigation of a rare phenomenon.

A 57-year-old black woman required a daily dosage of 50 mg of warfarin sodium to maintain her prothrombin time in a therapeutic range. The central volume of distribution and clearance of warfarin were normal for this patient. These findings, combined with the patient's requirement for plasma warfarin levels four times greater than those usually required to achieve adequate anticoagulation, indicated that the relative resistance was due to altered pharmacodynamics of warfarin. The only child of the propositus, a daughter, showed a similar relative resistance, confirming that this family is the third to be reported with hereditary resistance to warfarin.

Acquired dysfibrinogenemia. Paraneoplastic syndrome in renal cell carcinoma.

Acquired dysfibrinogenemia has not been previously reported as a paraneoplastic marker for malignancy. This report describes the clinical course of a patient who at the time of diagnosis of nonmetastatic renal cell carcinoma had dysfibrinogenemia characterized by prolongation of the thrombin and Reptilase times and increased sialic acid content of the purified fibrinogen. The thrombin and Reptilase times returned toward normal values after nephrectomy but became abnormal with the development of nonhepatic metastases. It is concluded that acquired dysfibrinogenemia can be part of a paraneoplastic syndrome and is a sensitive plasma marker for tumor progression.

Correlation between lupus anticoagulants and anticardiolipin antibodies in patients with prolonged activated partial thromboplastin times.

PURPOSE: An increased incidence of thrombosis has been reported in patients with a prolonged activated partial thromboplastin time (APTT) due to a lupus anticoagulant (LA), which is an antibody to negatively charged phospholipids. The antiphospholipid antibodies can be quantitated in an enzyme-linked immunoabsorbent assay (ELISA) that utilizes cardiolipin as the antigen. With the development of the ELISA, two major areas of controversy have arisen. First, the correlation between assay results for LA and for the ELISA has varied widely among laboratories. Second, some investigators have described a correlation between high levels of anticardiolipin antibodies (ACA) and thrombotic disorders, whereas others have found no association between ACA levels and thrombosis in a general population of medical patients. To explore these issues further, the present study determined the sensitivity and specificity of an LA assay for detecting ACA in medical patients with a prolonged APTT. The association between the isotype and titer of ACA and thrombosis was examined in those patients positive for LA. PATIENTS AND METHODS: Plasma samples from 70 medical patients with a prolonged APTT by routine screening studies were tested for the presence of LA by dilution of phospholipid in an APTT system and for IgM and IgG ACA according to a standardized ELISA. Clinical records were reviewed for a history of thrombotic events by an investigator who had no knowledge of the laboratory results. RESULTS: The ACA assay gave positive results in 47 patients, 44 of whom also tested positive for LA. Thus, the sensitivity for the LA assay for detecting ACA was 94% (confidence interval, 82% to 99%). The result of the LA assay was negative in 20 of 23 patients who were ACA-negative. The specificity of the LA assay was 87% (confidence interval, 67% to 98%). Twelve of the 47 patients (26%) had a history of venous or arterial thrombosis. Of these patients, 75% tested in the high-positive range for IgG or IgM ACA, or both. Of the 35 patients without thrombosis, only 14% were in this range. Patients with thrombosis had either underlying systemic lupus erythematosus, lymphoma, or no apparent etiology for LA. There was no history of thrombosis in patients with LA associated with infection or medication. CONCLUSION: A test for LA in medical patients with a prolonged APTT can be sensitive and specific for ACA. Determination of ACA levels in patients who have LA that is not induced by medication or infection may define those patients at increased risk for thrombosis.

Comparison between a one-point dilute phospholipid APTT and the dilute Russell viper venom time for verification of lupus anticoagulants.

In the present study, the dilute Russell viper venom time (RVVT) and the dilute phospholipid activated partial thromboplastin time (PL-APTT), which are two assays used for the verification of lupus anticoagulants (LA), were modified to increase standardization. The modified assays were then compared with respect to sensitivity and specificity in detecting LA in plasmas from 72 patients with a prolonged APTT. Modifications included utilizing a single dilution of phospholipid that was either bovine brain thromboplastin (Thrombofax) or liposomes comprised of phosphatidylcholine/phosphatidylserine, and expressing the results as a ratio of the clotting times of the mixture of patient and normal plasma/clotting time of normal plasma. In the RVVT, the correlation coefficient between assay results for liposomes and Thrombofax was 0.88 and in the PL-APTT, the correlation was 0.68. A positive test for LA was defined as a ratio of greater than or equal to 1.3 for the PL-APTT with liposomes and greater than or equal to 1.2 for the PL-APTT with Thrombofax and the RVVT with Thrombofax or liposomes. Regardless of the phospholipid source in the test system, the PL-APTT demonstrated higher sensitivity and the RVVT showed greater specificity in detecting patient plasmas that contained antiphospholipid antibodies.

Plasminogen activator and plasminogen activator inhibitor activities in a reference population.

The influence of age, gender, and aspirin ingestion on plasma levels of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) activities was studied in a reference population of 35 men and 35 women between the ages of 20 and 65 years. The t-PA values (mean +/- SD) in the women before and after 5 minutes of venous occlusion were 3.8 +/- 1.4 and 7.8 +/- 4.4 micrograms/L, respectively; in men these values were 3.3 +/- 1.2 and 8.8 +/- 8.9 micrograms/L. Men had higher mean PAI levels than did women (5.0 vs. 2.5 kU/L). T-PA showed an inverse relationship to PAI in both sexes. There was a negative correlation of t-PA levels with age, whereas PAI levels were positively correlated. The ingestion of a single dose of aspirin (650 mg) did not alter PAI or t-PA activities. This study indicates that factors such as age and sex may need to be considered when reference populations are developed for clinical studies of fibrinolysis.

The rabbit as a model for studies of fibrinolysis.

When compared to man, the rabbit shows marked prolongation of the dilute whole blood clot lysis time and an attenuated increase in plasminogen activator (PA) after the infusion of desmopressin (DDAVP). The levels of specific components of the plasma fibrinolytic system of the rabbit were compared to those in human plasma to ascertain their role in the differences between species. PA activity and plasminogen levels were similar in the two species. Anti-plasmin and plasminogen activator inhibitor (PAI) activity were lower in the rabbit than in man. The rabbit PAI, apparently similar to that described in man, was not increased by DDAVP infusion. The disparity between man and rabbit with respect to the lysis times of dilute blood clots and response to DDAVP cannot be explained by differences in functional plasma levels of inhibitors or activators of the fibrinolytic system.

A note on the measurement of accuracy for subnational demographic estimates.

Mean absolute percentage error (MAPE), the measure most often used for evaluating subnational demographic estimates, is not always valid. We describe guidelines for determining when MAPE is valid. Applying them to case study data, we find that MAPE understates accuracy because it is unduly influenced by outliers. To overcome this problem, we calculate a transformed MAPE (MAPE-T) using a modified Box-Cox method. Because MAPE-T is not in the same scale as the untransformed absolute percentage errors, we provide a procedure for calculating MAPE-R, a measure in the same scale as the original observations. We argue that MAPE-R is a more appropriate summary measure of average absolute percentage error when the guidelines indicate that MAPE is not valid.

Fibrinogenolysis and fibrinolysis with strenuous exercise.

Twenty healthy young men were exercised on a treadmill according to the protocol of Balke and Ware. Mean duration of exercise was 24.9 +/- 5.7 min and mean maximum heart rate was 195 +/- 9. Fibrinolytic activity was markedly accelerated with euglobulin lysis times decreasing to 36% of control values and fibrinogen-fibrin degradation products increasing 109% after exercise. Assays for fibrin monomer were negative in all samples. In vivo fibrinogen A alpha-chin degradation was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis of reduced samples of fibrin monomer isolated by clotting plasma samples in the presence of 0.1 M epsilon-aminocaproic acid and 1% disodium ethylenediaminetetraacetate. The A alpha-chain, the fibrinogen chain most susceptible to plasmin degeneration, showed no evidence of increased degeneration after exercise. Gel scans showed no decrease in the ratio of total alpha-chain to beta- and gamma-chains after exercise. The ratio of intact alpha 1 chain (alpha 1, 67,000 mol wt) to total alpha-chain was 0.66 +/- 0.13 before exercise, 0.64 +/- 0.14 immediately after exercise, and 0.65 +/- 0.13 1 h after exercise. The rate and extent of crosslinking of the alpha-chain of fibrin formed by clotting plasma samples was unaltered by exericse. These data suggest that physiologically significant fibrinogenolysis does not occur with strenuous exercise, even when fibrinolytic activity is markedly accelerated.

Replacement therapy for congenital Factor X deficiency.

We studied a young woman with severe (less than 1%) congenital factor X deficiency during a 2-year period in order to document the levels of factor X required to provide hemostasis for vaginal bleeding, epistaxis, and hemarthroses, as well as during surgery. Factor X levels of 9 to 17 percent, achieved with fresh-frozen plasma (FFP) were satisfactory for minor bleeding. Hemostasis was achieved during emergency surgery for hemoperitoneum by increasing the factor X level to 35 percent with a Factor IX concentrate, followed with infusions of FFP to maintain levels between 10 and 20 percent for 6 days postoperatively. These data suggest that factor X levels of 10 to 20 percent are sufficient for hemostasis in factor X-deficient patients even in the immediate postoperative period.

L-asparaginase: acute effects on protein synthesis in rabbits with normal and increased fibrinogen production.

The acute effects of a single intravenous dose of L-asparaginase on protein synthesis were studied in normal rabbits and in animals that had received turpentine to stimulate fibrinogen production. Male New Zealand rabbits received L-asparaginase (500 U/kg) 16 hr before the injection of the radiolabeled amino acid [75Se]selenomethionine (75SeM). Incorporation of 75SeM into fibrinogen and serum proteins in the L-asparaginase-treated rabbits was the same as for saline-treated controls, with fibrinogen representing approximately 5% of the labeled plasma proteins. In turpentine-treated rabbits, the maximal incorporation of 75SeM into serum proteins remained unchanged, whereas 75SeM-fibrinogen increased sixfold and accounted for 25% of the labeled proteins. Animals that received L-asparaginase at the same time as turpentine or 14 hr later showed significant decreases in synthesis of both serum proteins and fibrinogen. 75SeM-fibrinogen that was purified from L-asparaginase-treated rabbits underwent normal catabolism when injected into normal recipient rabbits. These data indicate that L-asparaginase can acutely cause partial inhibition of both serum protein and fibrinogen synthesis when administered to rabbits shortly before or during a period of increased fibrinogen production. Fibrinogen that is synthesized in the presence of L-asparaginase does not have an abnormal rate of catabolism.

Enhancement of tissue plasminogen activator-induced fibrinolysis by activated protein C in endotoxin-treated rabbits.

Endotoxin-treated rabbits produce high levels of plasminogen activator inhibitor-1 (PAI-1), which inhibits fibrinolysis by neutralizing endogenous tissue-type plasminogen activator (t-PA). These animals will develop renal fibrin deposition when infused with ancrod, an enzyme that acts directly on fibrinogen. In normal rabbits with an intact fibrinolytic system, ancrod induces hypofibrinogenemia without fibrin deposition. Rabbit PAI-1 activity can be neutralized by recombinant human t-PA or by bovine activated protein C. The present study determined the efficacy of these two agents used alone or in combination in neutralizing increased PAI-1 activity and in preventing renal fibrin deposition in a rabbit model. Male New Zealand rabbits first received intravenous endotoxin to increase PAI-1 activity. Ancrod was infused intravenously during hour 4 to 5, and the kidneys were examined at hour 5.5. Renal fibrin deposition occurred in 100% (6 out of 6) of the endotoxin-treated rabbits that received ancrod; this was reduced to 14% (1 out of 7) for rabbits receiving t-PA (170 micrograms/kg) before and during the ancrod infusion. Fibrin deposition occurred in only 12% (1 out of 8) of the rabbits that received a 10-fold lower dose of t-PA (17 micrograms/kg) combined with activated protein C (1 mg/kg) before and during the ancrod. Activated protein C at this dose completely neutralized plasma PAI-1 activity. However, low-dose t-PA and activated protein C did not prevent fibrin deposition when used as single agents, with fibrin deposition occurring in 75% and 100% of rabbits, respectively. The data indicate that activated protein C can neutralize plasma PAI-1 activity in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue factor generation by human mononuclear cells: effects of endotoxin and dissociation of tissue factor generation from mitogenic response.

The effects of the presence of endotoxin in a mononuclear cell culture system have been assessed. Endotoxin was shown to be mitogenic for human peripheral blood lymphocytes and capable of stimulating the generation of tissue factor. Concentrations of endotoxin, found to contaminate many commercial mitogens and antigens, activated mononuclear cells in a time-dependent manner. Generation of tissue factor was detected in cultures harvested from 2 to 72 hours following stimulation with endotoxin. Dose-response curves relating concentrations of endotoxin to mononuclear cell stimulation were determined; as little as 0.001 microng/ml. of E. coli endotoxin was capable of stimulating the generation of tissue factor in the cell cultures. The mitogenic effect of endotoxin was modest, however, and appeared to be unrelated to the ability of endotoxin to active tissue factor. Inhibition of DNA synthesis in the cell cultures by cytosine arabinoside or nonlethal irradiation failed to impair the generation of tissue factor. Endotoxin contamination of various reagents used in cell culture was evaluated with the Limulus assay, which detected as little as 1 X 10(-4) microng/ml. of endotoxin. Endotoxin was detected in preparations of phytohemagglutinin, purified protein derivative of the tubercle bacillus, mumps vaccine, tetanus toxoid, concanavalin A, and pokeweed mitogen. Because of the broad implications of contamination by endotoxin of various reagents, we assessed the specificity of the Limulus assay for the detection of endotoxin in the lectin, concanavalin A, and determined that the reaction was specific for endotoxin. Contamination by endotoxin of mononuclear cell culture systems should be considered as a possible factor in the production of various biological effects attributed to some commonly used mitogens and antigens.

Plasminogen activator inhibitor: a regulator of ancrod-induced fibrin deposition in rabbits.

Plasma levels of a fast-acting plasminogen activator inhibitor (PAI), which neutralizes both tissue plasminogen activator (t-PA) and urokinase, are markedly increased in endotoxin-treated rabbits. The ability of this inhibitor to prevent the fibrinolysis that occurs after a thrombogenic stimulus was investigated in a rabbit model. Normal and endotoxin-treated male New Zealand rabbits were infused with ancrod, an enzyme that causes noncrosslinked fibrin formation in vivo. Ancrod stimulated t-PA activity by 90% in normal rabbits and caused hypofibrinogenemia but did not increase PAI levels or induce fibrin deposition in target organs. Rabbits injected with endotoxin (10 micrograms/kg) showed an increase in PAI from less than 1 to 32 U/mL 4 hours later. When ancrod was infused at this time, 90% of the rabbits developed renal fibrin thrombi. Fibrin deposition was recorded in 40% of the rabbits that received a lower dose of endotoxin (1.0 microgram/kg) and had a PAI level of 14 U/ml at the time of ancrod infusion 4 hours later. Fibrin deposition did not occur in the endotoxin-treated rabbits that received normal saline. These data suggest that high levels of PAI inhibit fibrinolysis in vivo, thereby promoting fibrin clot deposition following a thrombogenic stimulus.

Inhibition of platelet deposition by combined hirulog and aspirin in a rat carotid endarterectomy model.

PURPOSE: Hirulog, a thrombin-specific inhibitor, has shown efficacy in reducing arterial thrombosis in patients treated with aspirin who require angioplasty or have unstable angina. In this study, the effect of hirulog on reducing deposition of indium 111-labeled platelets was assessed in a surgical model of aspirin-treated rats undergoing carotid endarterectomy. METHODS: Animals were randomly assigned to one of five groups: control (no aspirin or hirulog); aspirin alone (10 mg/kg); aspirin plus low-dose hirulog (0.2 mg/kg bolus followed by 0.5 mg/kg/hr); aspirin plus medium-dose hirulog (0.4 mg/kg bolus followed by 1.0 mg/kg/hr); or aspirin plus high-dose hirulog (0.6 mg/kg bolus followed by 1.5 mg/kg/hr). Hirulog was infused before surgery and continued until termination of the experiment 30 minutes after endarterectomy. RESULTS: Platelet deposition in rats receiving aspirin alone was reduced by 19% +/- 23% SE (p = 0.26) compared with controls. Deposition in aspirin-treated groups receiving low-, medium-, and high-dose hirulog decreased in a dose-dependent manner by 37% +/- 20% (p = 0.048), 44% +/- 19% (p = 0.061), and 56% +/- 13% (p = 0.022), respectively. As the dose of hirulog was increased, the plasma hirulog levels and activated partial thromboplastin time ratios (final:initial) also increased in a dose-dependent manner. The mean plasma hirulog levels ranged from 0.74 +/- 0.08 micrograms/ml in the low-dose hirulog group to 2.55 +/- 0.08 micrograms/ml in the high-dose hirulog group, and the corresponding activated partial thromboplastin time ratios were 1.5 +/- 0.12 (p = 0.001) and 3.3 +/- 0.63 (p = 0.001). Bleeding was easily controlled by local hemostatic measures for all experimental groups. CONCLUSION: Hirulog causes significant decrease in 111In-labeled platelet deposition in aspirin-treated rats subjected to microsurgical endarterectomy at doses that allow surgical hemostasis to be easily established.