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Objectives: The naming convention in coagulation may cause confusion in electronic ordering systems, leading to inappropriate test orders. We implemented test utilization efforts and studied utilization before and after interventions for two specialty coagulation assays., Methods: Two interventions were implemented: test names were changed from factor assay to activity, and residents reviewed all factor V and X requests. A retrospective review of factor V and X activity orders was performed for the period 1 year before and after interventions., Results: After interventions, factor V and X activity orders decreased by approximately 40%. Resulted tests decreased by 53.8% and 47.8%, corresponding to reductions of $2,493.05 and $1,867.80 per year in laboratory charges for factor V and factor X activity, respectively. Abnormal factor V activity results increased from 45% to 59%. Factor V activity orders from outpatient clinics decreased by 21.6%., Conclusions: Simple interventions can reduce inappropriate specialty coagulation test orders and unnecessary costs., (© American Society for Clinical Pathology, 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Disturbances in the thrombotic and fibrinolytic systems are a feature of insulin resistance, obesity, and the metabolic syndrome. However, there are few studies in which these relationships have been explored in mainly asymptomatic individuals using sophisticated measures of insulin sensitivity and regional adiposity. Variables of the hemostatic system were measured in 106 men (aged 32-68 yr; body mass index, 20-34 kg/m(2)). Insulin sensitivity was measured by minimal model analysis and regional adiposity by dual energy x-ray absorptiometry. Clustering of intercorrelated variables was assessed by the statistical technique of factor analysis. Plasma levels of procoagulant factors VII and X, anticoagulant proteins C and S, and plasminogen activator inhibitor-1 correlated positively with total and percent central body fat (r = 0.25-0.38; P < 0.05) and negatively with insulin sensitivity (except protein S; r = -0.24 to -0.35; P < 0.05). On factor analysis, procoagulant factors VII and X, proteins C and S, and plasminogen activator inhibitor-1 were components of the cluster of variables that explained the greatest proportion of the variance in the data (39.2%). Other variables included in this cluster were those typical of the metabolic syndrome and also serum gamma-glutamyl transferase activity. These results suggest that factors VII and X and proteins C and S are features of the intercorrelated disturbances of the metabolic syndrome. Associations with adiposity and liver enzyme activity suggest the involvement of hepatic fat deposition.

Warfarin is a 4-hydroxycoumarin anticoagulant drug used for the prevention and management of thromboembolic and vascular diseases. It acts through the inhibition of the vitamin K-dependent transcarboxylation reactions that convert precursors of clotting factors into their active form. Appropriate use of warfarin requires patient monitoring and dosage adjustments, to ensure its safety and efficacy. The aim of this work was to clarify the relationship between traditional (prothrombin time, usually expressed as the international normalized ratio; INR) and alternative (clotting factors II and X) warfarin response markers to establish their usefulness for therapeutic drug monitoring. Seventy adult outpatients, aged between 31 and 86 years old, were involved in the study. All subjects received warfarin in a monotherapy regimen and had been on a stable dosing schedule for at least two weeks to assure a steady-state condition. A total of 81 prothrombin times (expressed as INR), and factor II and factor X activity were simultaneously determined. Eleven patients presented repeated measurements at different time periods under the same dosing regimen. The results obtained from regression and cluster analysis showed a close relationship between factors II and X (r = 0.73), a weak correlation between INR and both factor II (r = -0.35) and factor X (r = -0.36), and a very slight dependency between warfarin and the response markers used. In addition, it seems that independent of the selected response marker, in long-term warfarin therapy, reproducible responses can be obtained over time if a steady-state condition is achieved. The coefficients of variation for factors II and X were greater (35.44 and 37.93%, respectively) than INR (14.50%), indicating that INR is a more precise measure than either factor II or factor X. In conclusion, INR appears to be the most appropriate warfarin response marker for therapeutic drug monitoring due to its universality, objectivity as a direct physiological effect measurement, and the available information regarding appropriate endpoints. However, when INR values are not in accordance with patient response therapy, factor II and factor X should be considered as an alternative to optimize warfarin therapy.

A 67-year-old woman had symptoms of an upper respiratory tract infection for which she received a five-day course of erythromycin. Epistaxis and gross hematuria subsequently developed, and the patient was found to have a selective Factor X deficiency. She received supportive therapy and prothrombin complex concentrates (Factors II, VII, IX, and X), with subsequent resolution of her transient Factor X deficiency. Her hospital course, however, was complicated by the development of multiple cerebral infarctions. This is the tenth reported case of transient Factor X deficiency not associated with amyloidosis. In seven of the previous cases, as in this patient, the deficiency was associated with a preceding upper respiratory infection. This is the only case, however, with evidence of inhibitory activity in the plasma that was directed toward Factor X.

We have found that rat plasma corrected the non-activated PT of human normal or factor-X deficient plasma, and the factor Xa-like activity being constantly detected in every 1 ml of blood collected via the cannulated carotid artery of rats. The present study was undertaken to characterize the factor Xa-like activity in rat plasma by preparing rat factor X and a monoclonal antibody against it. Factor X was purified from a BaCl2 eluate of rat plasma by chromatographies on columns of DEAE-Sepharose CL-6B and Sulfate Cellulofine or on a column of Affi-Gel 10 conjugated with a monoclonal antibody against rat factor X. Factor Xa-like activity in rat plasma was eliminated by the treatment of rat plasma with a monoclonal antibody which recognized the heavy chain portions of rat factors X and Xa. A kinetical study demonstrated that rat factor Xa was strongly inhibited by rat antithrombin III, with a Ki of 2.2 x 10(-11) M, in the presence of heparin. However, in the absence of heparin, the second order rate constant for the inhibition of rat factor Xa by rat antithrombin III was 2.6 x 10(4) M-1.min-1, which was one forty-third that for the inhibition of human factor Xa by human antithrombin III. Furthermore, rat factor Xa was resistant to the inhibition by rat alpha-1-antitrypsin and alpha-2-macroglobulin.(ABSTRACT TRUNCATED AT 250 WORDS)

We describe the effect of pH and NaCl concentration on the activation of factor X in plasma by Russell's viper venom and on the amidolytic activity of the activated enzyme towards factor Xa-sensitive chromogenic substrates. An increasing NaCl concentration results in a decrease in the activation rate of factor X by Russell's viper venom, whereas in this step no pH effect is observed. Increasing NaCl concentrations decrease the Km and Vmax for both the factor Xa-sensitive chromogenic substrates, S 2222 and S 2337. Km values were lowest between pH 7.8 and 8.6; Vmax increased with increasing pH. Comparison of NaCl and KCl in the activation step as well as in the amidolytic step shows that the observed effect is specific for Na+, not just an effect of ionic strength. No difference was detected between activation by the crude venom or its purified factor X-activating enzyme. Also, values with the substrates S 2222 or S 2337 were the same in the mechanized amidolytic factor X assay.

We describe a mechanized chromogenic assay for factor X, the results of which correlate well with those for the one-stage clotting assays for factor X in which it is activated either via the extrinsic pathway by thromboplastin or directly by Russell's viper venom. We purified human factor X and raised monospecific antibodies to it in rabbits. We used our chromogenic assay for factor X to develop a factor-X-inhibitor neutralization assay for determination of factor-X antigen. Patients receiving oral anticoagulant treatment had significantly different factor-X activities after activation via thromboplastin or with Russell's viper venom. The concentration of factor-X antigen, although decreased, significantly exceeded factor-X clotting activity or chromogenic activity in this group of patients. Results of the chromogenic assay for factor X correlated well with results of "Thrombotest," a clotting test introduced by Owren (Lancet ii: 754, 1959) to control anticoagulant therapy. For patients taking oral anticoagulant drugs, the therapeutic range by our assay is 180 to 300 units/L.

Factor X was assayed using chromogenic substrate S-2222 for four patients with severe factor X deficiency and for nine patients with homozygous or heterozygous factor X Friuli disorder. Factor X Friuli disorder is characterized by the presence of an abnormal factor X that is normally activated by Russell's viper venom, but is not activated by tissue thromboplastins. The levels of factor X found in factor X deficiency varied between 2 and 10% of normal and therefore were higher than those found in the same plasmas using "clotting" methods (1% or less than 1% of normal). The levels of factor X found in homozygous factor X Friuli patients varied between 4 and 11% of normal, and therefore were practically identical to those found by means of clotting methods that employed tissue thromboplastins (7-9% of normal). These values were definitely lower than those obtained using a Russell's viper venom and cephalin mixture as thromboplastin (82-92%). A similar pattern was observed for patients heterozygous for the abnormality. These findings indicate that "amidolytic" methods are not necessarily identical to clotting methods. Furthermore, they indicate that substrate S-2222 is not specific for factor X.

Specific radioimmunoassays, sensitive to plasma levels of less than 1% of normal, were developed for protein C and Factor X. In 31 normal subjects, mean plasma antigen levels were as follows: protein C, 3.23 +/- 0.79 microgram/mL (2 SD); Factor X, 7.74 +/- 1.81 microgram/mL. In patient son chronic warfarin therapy, protein C and factor X were depressed equivalently: protein C, 42% +/- 20% (of a pooled plasma reference); Factor X, 44% +/- 24%. Protein C antigen fell much more rapidly than Factor X antigen when warfarin therapy was begun, creating an initial period of potential hypercoagulability. In patients with severe liver disease, mean protein C antigen (25% +/- 17%) was lower than Factor X antigen (51% +/- 29%). Protein C antigen levels did not appear to be a sensitive indicator of compensated intravascular coagulation or systemic fibrinolysis induced by infusion of streptokinase. Clinical implications of these findings are discussed.

Human liver cDNA coding for protein C has been synthesized, cloned and sequenced. The abundance of protein C message is approximately 0.02% of total mRNA. Three overlapping clones contain 1,798 nucleotides of contiguous sequence, which approximates the size of the protein's mRNA, based upon Northern hybridization. The cDNA sequence consists of 73 5'-noncoding bases, coding sequence for a 461 amino acid nascent polypeptide precursor, a TAA termination codon, 296 3'-noncoding bases, and a 38 base polyadenylation segment. The nascent protein consists of a 33 amino acid "signal", a 9 amino acid propeptide, a 155 amino acid "light" chain, a Lys-Arg connecting dipeptide, and a 262 amino acid "heavy" chain. Human protein C and Factor IX and X precursors possess about one third identical amino acids (59% in the gamma-carboxyglutamate domain), including two forty-six amino acid segments homologous to epidermal growth factor. Human protein C also has similar homology with prothrombin in the "leader", gamma-carboxyglutamate and serine protease domains, but lacks the two "kringle" domains found in prothrombin.

With the introduction of synthetic substrates, many newer methods have been developed for testing coagulation parameters. Some of these methods are used in clinical settings, and many have been evaluated for their diagnostic efficacy in various coagulation disorders. Clinical chemistry laboratory instrumentation has been used to accommodate these methods. Commercially available kits for antithrombin III, heparin, plasminogen, alpha 2-antiplasmin, and factor X are used by many laboratories. Individual reagents have also become available and are being used to develop assays for routine diagnostic use. The development of suitable standards is rapidly underway, and their availability will facilitate adoption of these methods by hospital laboratories. Many of the synthetic-substrate-based assays that have been introduced for routine diagnostic purposes have been published recently. Here, we provide an overview of the newer amidolytic methods for the measurement of certain coagulation variables.

The relative sensitivities of the prothrombin time, Russell's viper venom time and activated partial thromboplastin time were determined in one-stage quantitative assays for factor V and factor X. Sensitivity was defined as the slope of the reference curve. The prothrombin time was the most sensitive test for factor X and the least sensitive for factor V. The Russell's viper venom time was the most sensitive test for factor V and least sensitive for factor X. The activated partial thromboplastin time approximated the sensitivity of Russell's viper venom for factor V and had intermediate sensitivity for factor X. The data indicate that factor V is best quantitated by the Russell's viper venom time and factor X by the prothrombin time, exactly the opposite of what is customarily done.

This solid-phase colorimetric microtiter-plate clotting assay is much more sensitive than standard clotting tests. In enzyme-linked coagulation assay (ELCA), enzyme-labeled fibrinogen and solid-phase fibrinogen are the substrate for thrombin generated in the clotting cascade. We used this assay to measure the factors of the extrinsic pathway by an extrinsic pathway-specific assay (EP-ELCA) and to determine the individual factors of the extrinsic pathway (VII, X, V, II) in plasmas of coumadin-treated and heparin-treated patients, with prothrombin time (PT) values used as a reference. In the ELCA method, samples and controls are incubated on the same plate, eliminating the requirement for pre-standardization of the substrate "plasma" before the factor assay is done. Concentrations of factors are determined by serially diluting sample and control plasmas to yield equivalent activity at given dilutions, a more direct approach for measuring specific factors than determining log concentrations vs log clotting time. Changes in the concentrations of clotting factors are seen before changes are apparent by PT. For coumadin-treated patients, all vitamin K-dependent factors were significantly (P less than or equal to 0.001) less than in normal controls, whereas factor V concentrations were normal, as expected. For patients treated with heparin, concentrations of factors X and VII were less than in normal controls (P less than 0.01) and results for EP-ELCA, II, and V assays were normal. This methodology can readily be automated.

Enzyme immunoassays are very useful for the detection of low concentrations of coagulation proteins and pathological markers in plasma. Analytes in the ng/mL range are measurable with good reproducibility with intra- and interassay CVs of less than 5% to 10%. "Sandwich" methods have been developed for von Willebrand factor (plasma concentration about 8 micrograms/mL, Factor IX (5 micrograms/mL), protein C (4 micrograms/mL), and Factor X (10 micrograms/mL). However, this technique is only suitable for macromolecules; for low-molecular-mass peptides such as fibrinopeptide A a competitive method is used. Normal concentrations of fibrinopeptide A are below 3 ng/mL, with greater values suggesting in vivo generation of thrombin; thus this test is quite useful in detecting thrombosis. Reagents for both the sandwich and competitive methods are commercially available and cost effective, and have a longer shelf-life than those for radioimmunoassays.