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.

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.

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.

A quantitative thrombin time for determining levels of hirudin and Hirulog.

The anticoagulant effect of recombinant hirudin (rHir) and Hirulog has been monitored in patients with the activated partial thromboplastin time. Accurate monitoring with this test cannot be achieved if plasmas contain heparin, lupus anticoagulants, low concentrations of fibrinogen or other factors, or elevated fibrinogen-fibrin degradation products (FDP). We have therefore developed a simple, rapid, sensitive clot-based method, the quantitative thrombin time (QTT), to measure levels of rHir and Hirulog in patient plasma (or whole blood). The QTT is performed by mixing a 1:10 dilution of patient plasma (50 microliters) with human fibrinogen (50 microliters, 128 mg/dl) at 37 degrees C; the clotting time is initiated by adding human thrombin (50 microliters, 5-7.5 U/ml). The concentration of Hirulog or rHir in plasma can be determined by comparing the QTT in patient plasma with a standard curve that is generated by adding different concentrations of anticoagulant to pooled normal plasma. Studies with whole blood using the same procedure yield similar results. In the absence of Hirulog or rHir, the baseline QTT is the same in normal and abnormal plasmas (fibrinogen < 150 mg/dl and FDP as high as 1024 micrograms/ml, elevated FDP alone, lupus anticoagulant, or heparin < 0.9 U/ml). When known concentrations of either rHir or Hirulog are added to abnormal plasmas, the mean observed concentrations as determined by the QTT deviate from the expected values by less than 10% (range 0-19%). The data indicate that the QTT is a simple, rapid, and accurate test for the determination of levels of rHir and Hirulog in plasma or whole blood.

Effect of plasminogen activator inhibitor-1 on tissue-type plasminogen activator-induced fibrinolysis.

The effect of fibrin on the interaction of human recombinant single-chain tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) was studied in normal rabbit plasma and in plasma with high levels of native PAI-1. t-PA was added to diluted plasma containing calcium (10 mM) and 125I-fibrinogen at 37 degrees C. Clotting was initiated with human thrombin, and lysis was monitored both turbidimetrically and by release of 125I-fibrin degradation products (fdp). The activity of t-PA (50 IU/ml) was rapidly reduced to 15% of the initial value in plasma containing PAI-1 (23 AU/ml). When thrombin and t-PA were added simultaneously to the plasma, more than 70% of the activity was retained through incorporation of t-PA into the fibrin clot. t-PA-induced fibrinolysis in PAI-1 enriched plasma was further delayed when the temperature was reduced from 37 to 25 degrees C. Turbidimetric and 125I-fdp release data provided complementary information. The former technique traced fiber dissolution, while the latter reflected network integrity. These results indicate that t-PA-induced fibrinolysis in PAI-1 enriched plasma is modulated by the presence of fibrin and by temperature.

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.

The dilute phospholipid APTT: a sensitive assay for verification of lupus anticoagulants.

A simple sensitive method for verification of lupus anticoagulants utilizing dilution of phospholipid in the activated partial thromboplastin time (APTT) system is described. Patient plasma, mixed with an equal volume of normal plasma, is activated with micronized silica. To this mixture are added different dilutions of Thrombofax and then calcium chloride. Clotting times are plotted linearly against the logarithm of the phospholipid dilutions and slopes are calculated by regression analysis. In this assay the mean negative slope of 19 plasmas that contained anti-phospholipid activity was five times greater than those of normal plasma or those obtained from patients having single or multiple coagulation factor deficiencies such as those induced by warfarin. The assay can be modified to test heparinized plasmas. Thus, it is a sensitive means by which to verify the presence of lupus anticoagulants in patients who have congenital or acquired factor deficiencies or who are receiving anticoagulant therapy.

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.

Diagnosis of heparin-induced thrombocytopenia in the vascular surgery patient.

BACKGROUND: Recent studies have shown that patients with heparin-induced thrombocytopenia (HIT) form immunoglobulin G (IgG) and/or IgM antibodies directed against a complex of platelet factor 4 (PF4) and heparin. This recognition has resulted in the development of enzyme-linked immunosorbent assays (ELISAs) that use the heparin/PF4 complex as the antigen. This study describes the use of a standardized ELISA to assess antibody formation in five patients suspected of having HIT. METHODS: Five patients received heparin for treatment of arterial or venous thrombotic disorders. All patients had the ELISA performed to detect IgG or IgM antibodies directed against heparin-PF4, as well as the 14C serotonin release assay, when HIT was clinically suspected. RESULTS: HIT was diagnosed in four patients and ruled out in a fifth by using the ELISA. All patients had a 40% decrease in platelet count that returned to normal after heparin cessation. Only one of the four patients who tested positive by ELISA for IgG antibodies also tested positive by the 14C serotonin release assay. Treatment was significantly altered by the ELISA results in all five patients. CONCLUSIONS: It is likely that the ELISA is more sensitive in the diagnosis of HIT than the more traditional aggregation tests, and it may emerge as a new gold standard. Prospective studies in which serial laboratory testing is combined with measurement of clinical outcomes are needed and will eventually provide a greater understanding of the full spectrum of HIT and the clinical settings that precipitate thrombosis in the vascular surgery patient.

Stimulation of plasminogen activator inhibitor activity in human monocytes infected with dengue virus.

Human monocytes, in an essentially serum-free culture medium, were infected with dengue 2 virus in the presence of sub-neutralizing concentrations of antibody. Changes in procoagulant activity (PCA), in the plasminogen activator urokinase (UK), and the plasminogen activator inhibitor-2 (PAI-2) were quantitated. One day after exposure to dengue virus, the cell-associated PAI-2 activity in the infected monocytes was 562 +/- 9 mU/10(6) cells (mean +/- SE) compared to 206 +/- 56 mU/10(6) cells for uninfected monocytes. Supernatants of the infected cells also showed greater than 2-fold increase in PAI-2 activity. This increase in cell-associated and supernatant PAI-2 activity was maintained during 4 days of culture. UK activity was not detected in control and infected cells nor in their supernatants. PCA activity was the same in control and dengue virus infected monocytes when measured during 4 days of culture. These data suggest that dengue infected monocytes may affect fibrinolysis at a localized level through increased production of PAI-2.

Heparinoid anticoagulation and topical fibrin sealant in heparin-induced thrombocytopenia.

The development of heparin-induced thrombocytopenia in patients who require systemic anticoagulation for cardiac and vascular operations poses a therapeutic dilemma because no alternative anticoagulants are generally available. Heparinoid (Org 10172) has been used as an alternative anticoagulant under protocol or on a compassionate use basis, and has recently been approved by the Food and Drug Administration. There is, however, no heparinoid antagonist to reverse the anticoagulation. This report describes the combined use of heparinoid anticoagulation and adjunctive fibrin sealant for topical hemostasis in a patient with heparin-induced thrombocytopenia. Recommendations for perioperative monitoring of heparinoid anticoagulation are provided.

Inhibitory effects of lysine analogues on t-PA induced whole blood clot lysis.

The lysine analogues epsilon-aminocaproic acid (EACA) and trans-4-amino-methyl cyclohexane carboxylic acid (AMCA) are used to prevent excessive bleeding in patients with coagulopathies, such as hemophilia and thrombocytopenia, or in those who have received tissue plasminogen activator (t-PA). However, their relative efficacy in inhibiting lysis of clots that have been formed in the presence of exogenous t-PA or that have been formed and then exposed to exogenous t-PA has not been well characterized. The present study utilized blood from normal volunteers and 125I-fibrinogen in a dilute whole blood clot assay to determine the relative concentrations of lysine analogues required for inhibition of clot lysis induced by exogenous t-PA. AMCA (0.06 mM) and EACA (0.6 mM) were effective in prolonging clot lysis if (1) whole blood clots were formed and then exposed to a lysine analogue and exogenous t-PA or if (2) whole blood clots were formed in the presence of exogenous t-PA and a lysine analogue. However, their inhibitory effect was markedly reduced if clots were formed in the presence of t-PA and then exposed to either of the lysine analogues. The analogues did not inhibit the initial binding of t-PA to fibrin. They did inhibit binding of plasminogen to fibrin as well as the activation of plasminogen by t-PA in the absence of fibrin. The data suggest that lysine analogues, even at low concentrations, reduce the rate of t-PA induced whole blood clot lysis by several mechanisms.

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.

Preparation of beta-XIIa (Hageman factor fragment) from human plasma.

beta-Factor XIIa (beta-XIIa, Mr approximately 30,000) was isolated from human plasma by a procedure which utilized, as an initial step, the adsorption of Factor XII to celite. Activation of Factor XII and subsequent release of beta-XIIa was brought about by the proteolytic action of co-adsorbed kallikrein. Two successive chromatographic procedures were then used to achieve a final purification of 4,420-fold over plasma and an overall field of 2.3 mg of beta-XIIa per liter.

Rabbit fibrin: characterization of the separated chains.

In preparation for studies of the time course of production in vivo of the constituent chains of rabbit fibrin, we characterized the structural features of the fibrin molecule. Fibrin was isolated from plasma and reduced and alkylated. The alpha, beta, and gamma chains were separated by CM-cellulose chromatography and their molecular weights and amino acid compositions were determined. The gamma chain was sequenced 36 steps with 32 positive identifications and the beta chain, 12 steps with 12 identifications. No major differences between the sequences of these chains and those of man, chicken, and dog were noted.

Stimulated production of urokinase and plasminogen activator inhibitor-2 by the human promyelocytic leukemia cell line HL-60.

The effects of maturation inducing agents on the production of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) by the human promyelocytic leukemia cell line HL-60 were examined. PA activity, which was calibrated with a urokinase standard, was 3-6 mU/10(6) cells when measured in supernatants from control cells. This activity increased at least two-fold after dimethylformamide (DMF) or retinoic acid (RA) was added to cell cultures, and as much as ten to thirty-fold when cells were exposed to 12-O-tetradecanoylphorbol-13-acetate (PMA), an agent that induces monocytoid differentiation in HL-60 cells. The PA activity produced by control and induced cells had the same molecular weight as urokinase (UK), and was completely inhibited by antibodies to UK. Cells that were induced with PMA but not with RA or DMF also produced an inhibitor to UK that was identified as PAI-2, the plasminogen activator inhibitor that is produced by monocytes. Because of its dual capacity to produce both UK and PAI, the HL-60 cell line represents a useful model for studies of the fibrinolytic mediators that are generated and released by leukemia cells.

Human plasma prekallikrein and high molecular weight kininogen decrease during parturition.

Prekallikrein and high molecular weight kininogen were measured in plasma taken from nine women during parturition. Prekallikrein decreased significantly (p less than 0.01) from 1.49 +/- 0.15 S-2302 U/ml (mean +/- SEM) in early labor to 1.26 +/- 0.13 S-2302 U/ml in the immediate postpartum period. Immunoreactive high molecular weight kininogen also significantly decreased from 76 +/- 5 micrograms/ml to 68 +/- 5 micrograms/ml one day postpartum (p less than 0.01). Both proteins rose to normal levels within two days. The data suggest that the kallikrein-kinin system is utilized during parturition.

Heparin-associated thrombocytopenia in the neurosurgical patient.

Although thrombocytopenia occurs in approximately 10% of medical patients who receive heparin, this complication has not been described in neurosurgical patients. We report the clinical course of two patients who in the immediate postoperative period developed heparin-associated thrombocytopenia that resulted in significant morbidity. In these two cases, the origin of the heparin was in "flush" solutions used to maintain the patency of indwelling vascular catheters and was infused at a dose of 250 to 500 units/day. The minimal daily dose previously reported to result in thrombocytopenia is 9000 units/day administered in divided doses subcutaneously. The case reports indicate that heparin in "flush" solutions should be considered as a cause for unexpected thrombocytopenia and that platelet counts should be monitored in patients receiving heparin in any amount.

Effect of fibrin structure on plasmin-mediated dissolution of plasma clots.

Previous studies in purified systems have demonstrated that fibrin structure influences the rate of conversion of plasminogen to plasmin by t-PA as well as the rate of plasmin-mediated clot digestion. The present study extended these observations to a plasma system in which fibrin structure was altered by varying the thrombin concentration, varying the plasma ionic strength, or by adding dextran 40. The effect of fibrin structure on the rate of fibrinolysis was assessed by adding plasminogen activators (t-PA or urokinase (UK)) either before or after clot formation. Gel formation and dissolution were monitored optically (turbidity) and isotopically (125I-fibrinogen). Clots formed under conditions of high ionic strength and/or high thrombin concentration were composed of thin fibrin fibres that dissolved slowly. Clots formed at lower ionic strengths, at lower thrombin concentrations or in the presence of dextran were composed of thicker fibres and dissolved more rapidly. The difference in fibrinolytic rate between thin and thick fibres was noted when t-PA or UK was added before or after clot formation. These data indicate that even in a plasma milieu fibre diameter is a factor in determining fibrinolytic rate induced by either UK or t-PA. The method by which fibre diameter is altered does not influence the conclusion that fibrinolytic rate is increased with increasing diameter.

Fibrin sealant: current and potential clinical applications.

There are few well-controlled studies of the clinical efficacy of fibrin sealant, defined by lives saved or reduced need for blood transfusions. Evaluation of fibrin sealant in trauma situations, e.g. liver laceration, has been difficult to perform. Only recently has fibrin sealant been actively promoted by US manufacturers as a commercially valuable alternative to the relatively inexpensive crude bovine thrombin and cryoprecipitate that are in current use. Regulatory agencies and manufacturers are aware that patients in the USA are receiving a suboptimal form of fibrin glue since cryoprecipitate is not virally inactivated and has a variable fibrinogen concentration. In addition, bovine thrombin is not regulated with respect to factor V content or any other impurities. During the past year regulatory agencies, together with manufacturers and clinicians, have begun to define clinically valid endpoints for efficacy of a commercially prepared fibrin sealant. These may include improvement in hemostasis compared with a placebo or agents considered to be 'standard of care'. Thus, the regulatory agencies may be willing to consider studies in animals that demonstrate efficacy as well as surrogate endpoints, such as reduced factor concentrate requirements in patients with severe hemophilia requiring dental extraction. As fibrin sealant becomes available in a liquid and potentially in a bandage form, it may also become an essential matrix for recombinant factors that can affect endothelial function.