Scotopic vision deficits in young monkeys exposed to lead.

Rhesus monkeys were reared on diets designed to produce blood lead concentrations of 14 (untreated), 55, or 85 micrograms per 100 milliliters for the first year of life. Eighteen months later, blood lead levels were normal in all animals. At this time, however, visual discrimination performance in the 85-microgram group was impaired under dim light relative both to their own performance under bright light and to the performance of the other groups under all light levels used. We interpret these results to reflect a deleterious, enduring impairment of scotopic visual function (night blindness) as a result of early lead intoxication.

Advantages, disadvantages and optimization of one-stage and chromogenic factor activity assays in haemophilia A and B.

Haemophilia A and B diagnosis and disease severity classification are determined on the basis of results from factor VIII (FVIII) and factor FIX (FIX) activity assays, respectively. These assays are also used for potency labelling, postinfusion monitoring of factor replacement products and testing for FVIII/FIX inhibitors. This review focuses on activated partial thromboplastin time (APTT)-based one-stage assays (OSAs) and two-stage chromogenic substrate assays (CSAs). Currently, there is considerable inter-laboratory variability in the results obtained using OSAs, which can be intensified in a reagent-specific manner by the presence of the new modified recombinant factor replacement products that are entering the market. Furthermore, the use of CSAs, which tend to show less variability, especially with the new modified products, is recommended in a number of clinical scenarios. Clinical laboratories may, therefore, need to establish CSAs for routine use. In this review, we aim to improve understanding and help establish best practices by describing the methodology behind OSAs and CSAs and highlighting assay advantages and limitations. We argue that there can be value in offering both assay methodologies in clinical laboratories that contribute to the care of patients with haemophilia A or B. Educating both laboratory scientists and clinicians about the strengths and weaknesses of each type of assay will help to establish the necessary dialogue that is key to ensuring not only that the appropriate assays are used in the right clinical situations, but also that the results are interpreted correctly.

Deficiency of protein C inhibitor in combined factor V/VIII deficiency disease.

Activated protein C is an anticoagulant plasma protease enzyme that inactivates Factors V and VIII in plasma. Normal plasma contains a protein that inhibits activated protein C and that is distinct from previously described plasma protease inhibitors. Protein C inhibitory activity is not detectable in plasmas from four unrelated patients with combined Factor V/VIII deficiency but is present in normal amounts in plasmas from patients with simple factor V deficiency or Factor VIII deficiency. It is suggested that the molecular basis for combined Factor V/VIII deficiency that exhibits simple autosomal recessive inheritance is a deficiency of protein C inhibitor.

The PiCT® test is a reliable alternative to the activated partial thromboplastin time in unfractionated heparin therapy management: results from a multicenter study.

Essentials Activated partial thromboplastin time (APTT) or anti-Xa tests are used to monitor heparin. Prothrombinase-induced Clotting Time (PiCT) was compared to APTT in a clinical study. PiCT shows higher correlation to anti-Xa than APTT does and is more comparable between centers. PiCT demonstrates significantly higher accuracy and reliability than APTT in heparin monitoring. SUMMARY: Background Unfractionated heparin (UFH) is still a commonly used anticoagulant for prevention and treatment of thromboembolism in a variety of situations. Increasingly, chromogenic anti-Xa assays are used for UFH monitoring given the high variability of the activated partial thromboplastin time (APTT) in this setting. On the other hand, and despite the known variability, the APTT test remains the most frequently used monitoring tool in UFH therapy because of its broad availability, lower costs and wide acceptance. Various guidelines continue to recommend the use of the APTT as an anti-Xa surrogate, but this approach remains controversial. Objective To assess the prothrombinase-induced clotting time (PiCT® ) test, reported in seconds, as an alternative to the APTT in the management of UFH-mediated anticoagulation. Methods Plasma samples from patients receiving UFH were obtained in three different centers in the USA and Europe. Samples were analyzed for PiCT, APTT and anti-Xa activities with conditions set to allow comparability. Target-ranges in seconds for PiCT and APTT were established for a UFH concentration of 0.3-0.7 IU mL-1 , derived from anti-Xa results as suggested by the ACCP guidelines. Results PiCT demonstrated better correlation with anti-Xa IU mL-1 than APTT, higher ability to identify samples within target range and, importantly, comparable target-ranges between different centers. Conclusion Accuracy and reliability of PiCT are significantly better than those of APTT in monitoring UFH for anticoagulant therapy.

Thrombomodulin activity and localization.

An overview on the properties, actions and localization of thrombomodulin (TM) in situations of tissue injury and in selected tumors is presented. The localization and activity of TM after injury to vascular endothelium shows that following balloon catheter denudation of the endothelium of the rabbit aorta, the activity and immunohistochemical staining is markedly reduced. The functional and antigenic levels approach the control levels approximately one week after the initial injury. The results suggest that the neointimal smooth muscle cells express TM. This phenotypic plasticity of the neointimal smooth muscle cells may be important in conferring thrombo-resistance to the lumenal lining cells of vessels after injury. Studies are also reviewed on the use of soluble recombinant TM to prevent thrombosis after ligature of vessels in an experimental model. Further characterization on the immunohistochemical distribution of TM in normal tissues and tumors shows that staining with a monoclonal anti TM antibody can be very useful in separating mesotheliomas from pulmonary adenocarcinomas. These studies may lead to insights concerning the role of TM in tissue-injury-repair and tissue differentiation.

Homozygous protein C deficiency: observations on the nature of the molecular abnormality and the effectiveness of warfarin therapy.

An infant with severe homozygous protein C deficiency was brought to medical attention because of purpura fulminans and severe bilateral vitreous hemorrhages in the neonatal period. Infusions of fresh frozen plasma were given for 8 months. On two occasions, attempts to decrease the frequency of fresh frozen plasma infusions to less than twice a day led to episodes of microangiopathic hemolysis, fibrinolysis, and acute renal failure. Infarction of skin and subcutaneous tissues did not recur. Both episodes were controlled after reinstitution of fresh frozen plasma. Complications of therapy with fresh frozen plasma included hyperproteinemia and hypertension. Warfarin therapy was instituted when the baby was 8 months of age, followed by a gradual withdrawal of fresh frozen plasma therapy. The dose of warfarin required to maintain the prothrombin time in a range of 1.8 to 2.2 times normal varied considerably during short periods, a phenomenon that may have been due to several factors: hypercatabolism of the drug with prolonged administration, abnormality of liver function, variation in levels of serum albumin, fluctuations in drug dosage secondary to oral administration, and variations in dietary vitamin K. Protein C determinations by immunologic and functional assays consistently showed detectable but reduced protein C antigen levels with undetectable activity levels, suggesting that a dysproteinemia rather than a deficiency of synthesis is responsible for the child's coagulopathy.

Thrombomodulin gene defects in families with thromboembolic disease--a report on four families.

It has been suggested that an impaired thrombomodulin (TM) function could constitute an abnormality leading to thromboembolic disease (TED). The TM gene from 51 unrelated American patients with TED and 100 American blood donors was screened for mutations. Four heterozygous point mutations in the TM gene were detected. The mutations are distributed throughout the TM gene and predict amino acid changes 1) Pro483 to Leu, 2) Gly61 to Ala, 3) Asp468 to Tyr (earlier described) and 4) a silent mutation not predicting any amino acid change at Glu163. Family studies reveal that the occurrence of the different TM mutations is associated with a history of TED, but there are indications of multiple risk factors and no perfect co-segregation of the TM defects and TED. Among the controls. three individuals carried heterozygous TM variants predicting either a Pro477-Ser mutation (two cases) or an Asp468-Tyr mutation. Our results thus demonstrate that a previously undocumented abnormality in the protein C anticoagulant pathway, a defect in the TM gene, to a certain extent co-segregates with familial thrombophilia. Further studies are needed to prove the causality of these TM mutations.

Thrombomodulin gene variations and thromboembolic disease.

Thrombomodulin (TM) is the endothelial cell cofactor for protein C activation. Since deficiencies of other protein C system proteins are known to cause thrombotic disease, then defects in the gene coding for TM could be responsible for inherited thrombophilia. We have searched for mutations in the TM gene among healthy controls as well as patients with thrombophilia and identified eight patients heterozygous for TM mutations that are distributed throughout the TM gene. We have shown that the respective TM mutation co-segregates with thromboembolic disease (TED) in four families. Moreover, we have demonstrated that the C allele in a common C/T dimorphism in the TM gene is significantly more frequent among survivors of premature myocardial infarction (MI) than in matched controls. We suggest that TM defects should be added to the list of risk factors in TED, and after further evaluation possibly be included in a routine laboratory evaluation of thrombophilia.

Contribution of plasma proteinase inhibitors to the regulation of activated protein C in plasma.

Activated protein C (APC), a serine protease, is regulated in plasma by protease inhibitors. This study was undertaken to determine the role of the major plasma inhibitors in regulating APC in plasma. Kinetic analysis and specific immunoassays for APC-inhibitor complexes were used to determine the inhibitors that form complexes with APC. Of the eight plasma inhibitors investigated, four interact with APC: protein C inhibitor (PCI), alpha 1-proteinase inhibitor (PI), alpha 2-antiplasmin (AP) and C1 esterase inhibitor (C1 Inh). The second order rate constants are: 1.3 x 10(4) M-1 s-1 (PCI); 15 M-1 s-1 (PI); 410 M-1 s-1 (AP); and < 6 M-1 s-1 (C1 Inh), with a relative effectiveness of each inhibitor to inactivate APC in plasma: 49:36:15: < 1, respectively. PCI, PI and AP are the major inhibitors of APC in plasma. Low concentrations of APC will be inhibited by PCI with PI and AP playing a secondary role. However, as increasing APC is generated, PI and AP begin to play more important roles as the PCI is consumed.

Molecular forms of human protein C: comparison and distribution in human adult plasma.

Protein C (PC) is a vitamin K-dependent protein which functions as both an anticoagulant and profibrinolytic. It is synthesized as a single chain protein (SC-PC) and post-translationally modified into a two chain form (2C-PC). Two chain PC consists of a light chain (LC) and a heavy chain (HC). The present study was undertaken to determine the composition of the molecular forms of PC in plasma. PC was immunoprecipitated, subjected to SDS-PAGE and Western blotting. The blots were scanned by densitometry to determine the distribution of the various forms. The percentage of SC-PC and 2C-PC was found to be 10% and 90% respectively. This is in agreement with previous work. SC-PC and the heavy chain of 2C-PC consisted of three molecular forms ("alpha", "beta", and "gamma"). The "alpha" form of HC is the standard 2C form with a MW of 40 Kd. The "beta" form of HC has also been described and has MW which is 4 Kd less than the "alpha" form. The "gamma" species of the SC and 2C-PC has not been previously described. However, its 3 Kd difference from the "beta" form could be due to modification of the "beta" species or to a separate modification of the alpha-HC. The LC of PC was shown to exist in two forms (termed form 1 and form 2). The difference between these two forms is unknown. The molecular forms of PC are most likely due to a post-translational modification (either loss of a carbohydrate or a peptide) rather than from plasma derived degradation.

Hereditary dysfunctional protein C molecules (type II): assay characterization and proposed classification.

Protein C (PC) deficiency is among the increasing number of recognized causes of hereditary thrombotic disease. Two types of PC deficiency have been described: 1) Type I, which is characterized by a concomitant decrease in PC activity and antigen, and 2) Type II, characterized by disproportionately low activity compared to antigen (i.e. a dysfunctional molecule). To date, only a small number of Type II patients have been described. This study was undertaken to evaluate a number of dysfunctional PC molecules by comparing PC clotting and amidolytic activities with antigen levels. For these studies, an automated PTT-based clotting PC assay was developed. This assay was sensitive to 1% of a normal plasma pool, specific, accurate, and reproducible (+/- 12%). A good correlation (r = 0.918) of the clotting activity to antigen was found in normal individuals and Type I heterozygous and homozygous patients. To classify Type II PC deficient patients, the antigen, amidolytic and clotting PC levels were compared in ten affected families. The clotting activities were decreased in all affected members, whereas the antigen levels were within the normal limits. In four of the 10 families, the amidolytic activity was normal and similar to the antigen levels. This suggests that in certain families, defects in the PC molecule occur in regions not associated with amidolytic functions. From these studies, the molecular basis of Type II PC deficiency is varied and complex, involving different functional domains of the PC molecule. Therefore, we have suggested a nomenclature algorithm for Type II PC deficiency based on the location of the defect within the specific domains of the PC molecule.

Specificity of the thrombin-induced release of tissue plasminogen activator from cultured human endothelial cells.

The addition of thrombin (9 nM) to primary cultures of human endothelial cells induces a 6- to 7-fold increase in the rate of release of tissue plasminogen activator (tPA). Several other serine proteases which specifically interact with endothelial cells were also analyzed for their effect on tPA release. Gamma-thrombin, an autocatalytic product of alpha-thrombin, promoted tPA release but was less effective than alpha-thrombin. A maximum increase of 5.5-fold was observed, although a concentration of gamma-thrombin 20 times greater than alpha-thrombin was required. The response to Factor Xa was similar to alpha-thrombin, although the stimulation was significantly reduced by the addition of hirudin or DAPA suggesting that prothrombin activation was occurring. The simultaneous addition of prothrombin with Factor Xa resulted in enhanced tPA release equal to that observed with an equimolar concentration of active alpha-thrombin. Thus, under these conditions, Factor Xa-cell surface mediated activation of prothrombin can lead to a secondary effect resulting from cell-thrombin interaction. Activated protein C, which has been implicated as a profibrinolytic agent, was also tested. No change in tPA release occurred after the addition of up to 325 nM activated protein C in the presence or absence of proteins. Factor IXa and plasmin were also ineffective. The effect of thrombin on the endothelial cell derived plasminogen activator specific inhibitor was also studied. Thrombin produced a small but variable release of the inhibitor with an increase of less than twice that of non-thrombin treated controls.

Homozygous protein C deficiency: description of a new mutation and successful treatment with low molecular weight heparin.

We present a kindred with a new mutation of the protein C gene, in which the proband had an unusual clinical presentation. The relationship between warfarin induced skin necrosis and level of anticoagulation was investigated. The pharmacokinetics of protein C concentrate was assessed to determine frequency of replacement therapy. The clinical and biochemical efficacy of therapy with low molecular weight heparin (LMWH) was assessed. The effect of long-term LMWH on bone density in the growing child was monitored using whole body densitometry. Warfarin therapy required an INR of greater than 3.5 to avoid skin necrosis. If protein C replacement was to be used, doses of 100 U/kg/day would have been required to maintain protein C levels consistently at or above 0.20 U/ml. While receiving prophylactic therapy with LMWH for almost 3 years, there were no episodes of recurrent thrombosis, no skin necrosis and no bleeding. Biochemical markers of in vivo thrombin generation were suppressed and within the normal range. Bone density continued to increase at the normal rate throughout the treatment period. LMWH is an effective form of long-term therapy for homozygous protein C deficient patients with measurable protein C levels.

Clinical evaluation of protein C: a comparative review of antigenic and functional assays.

Thromboembolic disease is a major complication of protein C (PC) deficiency, which is among the increasing number of recognized causes of hereditary thrombotic disease. The laboratory evaluation of PC is of the utmost importance in the accurate diagnosis of this deficiency. In this review, we describe the various types of clinical antigenic and activity assays of PC. The attributes of each type of assay are discussed, as well as the value of each assay in diagnosing the various acquired and hereditary deficiencies of PC. This basic review is intended for laboratories planning to institute PC assays.

Coagulation and fibrinolytic profiles in patients with severe pulmonary hypertension.

STUDY OBJECTIVES: Although in situ thrombosis is a prominent finding in lung vessels from patients with primary and secondary pulmonary hypertension, to our knowledge, plasma coagulation factors that might contribute to a hypercoagulable state have not been fully investigated. We hypothesized that the local coagulation environment in the lung vasculature is important to progression if not initiation of pulmonary hypertension. DESIGN: Quasi-experimental cross-sectional design with concurrent controls. SETTING: Referral clinics and inpatient services of a University Hospital and a Veterans Administration Medical Center. PARTICIPANTS: To investigate the role of plasma coagulation factors in severe pulmonary hypertension, we sampled plasma from patients with primary pulmonary hypertension, patients with pulmonary hypertension secondary to a discernible etiology, and normal adult control subjects. RESULTS: We detected abnormalities of the thrombomodulin/protein C anticoagulant system, evidenced by a decrease in soluble thrombomodulin, in patients with primary pulmonary hypertension. In the patients with primary pulmonary hypertension, we found impaired fibrinolytic activity, with a rise in the fibrinolytic inhibitor plasminogen activator 1 and elevated euglobulin lysis time. Lower fibrinolytic activity correlated with high mean pulmonary artery pressure. In contrast, in patients with secondary pulmonary hypertension, von Willebrand factor antigen and fibrinogen levels were increased, and fibrinolytic activity decreased. CONCLUSIONS: Different patterns of coagulation and fibrinolytic abnormalities are apparent in plasma from patients with primary and secondary pulmonary hypertension. Although we are unable to address causality with this study, we speculate that abnormalities of these coagulation mechanisms may initiate or play a role in perpetuation of pulmonary hypertension.

A laboratory approach to the evaluation of hereditary hypercoagulability.

The concept of hypercoagulability and especially its evaluation in the clinical laboratory has changed dramatically during the last few years. The genetic basis and the mechanisms of the various factors responsible for hypercoagulability are briefly reviewed with emphasis on the most common genetic deficiencies. The major thrust of this review centers on the cost-effective approach to examining patients with a personal or family history of venous thrombosis. Several new concepts dealing with thrombotic risk are presented with a focus on the theory that multiple factors cause thrombosis in affected patients. A proposal for a cost-effective sequential testing scheme for the accurate diagnosis of hereditary hypercoagulability is discussed. The knowledge of thrombotic risk factors is evolving rapidly, requiring the clinical laboratory to remain flexible. Ultimately, the clinical laboratory must take a leading role in the diagnosis of hereditary thrombotic disease by serving as the consultant to the primary caregiver by providing an up-to-date and cost-effective evaluation.

Dysfunctional protein C deficiency (type II). A report of 11 cases in 3 American families and review of the literature.

A cause of recurrent venous thrombosis is discernible in about 30% of symptomatic patients. Type I protein C (PC) deficiency (concomitant decrease of activity and antigen) is a well-described cause of venous thrombosis. Dysfunctional PC or type II PC deficiency (a disproportionate decrease in activity compared with antigen), however, is less well understood. Eleven subjects from three American families had dysfunctional PC. The patient base was moderately sized. These 11 subjects are compared with the 67 patients (39 symptomatic and 28 asymptomatic) that have been reported with dysfunctional PC at this time. Dysfunctional protein C deficiency is a more common cause of venous thrombosis than previously was recognized. Protein C activity should be determined in evaluating a patient with recurrent venous thromboses or thrombosis in early adult life. If the PC activity is low, repeat PC activity and a PC antigen levels should be determined so that patients with Type II PC deficiency will be identified. Further testing must include family studies to rule out an acquired deficiency and confirm the hereditary basis of the Type II PC deficiency.

Effect of 3.2% vs 3.8% sodium citrate concentration on routine coagulation testing.

The effects of 3.2% and 3.8% sodium citrate concentration on the results of routine coagulation assays (prothrombin time [PT] and activated partial thromboplastin time [aPTT]) were evaluated by means of two sets of reagents, one responsive and the other nonresponsive. Five groups were entered in the study: healthy volunteers; outpatients receiving stable oral anticoagulant therapy; and hospitalized patients receiving intravenous (i.v.) heparin therapy, both i.v. heparin and oral anticoagulant therapy, or no anticoagulant therapy. With use of nonresponsive PT and aPTT reagents, varying the citrate concentration has little clinical significance except in patients receiving i.v. heparin therapy. In contrast, when responsive PT and aPTT reagents are used, the concentration of sodium citrate anticoagulant has a significant effect on assay results. Eighteen percent of samples from patients receiving stable oral anticoagulant therapy demonstrated a change of less than 0.7 INR (International Normalized Ratio) units between citrate concentrations. Nineteen percent of patients receiving i.v. heparin therapy had a greater than 7-second difference when aPTT results were compared. These data demonstrate that citrate concentration affects the results of coagulation tests. On the basis of these data, it is recommended that 3.2% citrate be used for all coagulation tests.

Minimum specimen volume requirements for routine coagulation testing: dependence on citrate concentration.

We evaluated the effect of sample volume and citrate concentration on results of routine coagulation assays (prothrombin time [PT] and activated partial thromboplastin time [APTT]). The study was performed on samples obtained from healthy persons and patients receiving oral anticoagulant therapy. Standard evacuated tubes (3.2% and 3.8% sodium citrate) were filled to varying total sample volumes ranging from 3.0 to 5.0 mL, and results of routine coagulation tests were compared. Underfilling may significantly affect the APTT and PT, resulting in artifactual prolongation of results. This effect is most pronounced in samples drawn into 3.8% citrate. By using 3.8% citrate, there is a statistically significant difference in the results of PT assays in the samples less than 80% filled compared with those that are 100% filled. For APTT assays performed on samples drawn into 3.8% citrate, a statistical difference occurred at less than 90% filled. This effect was less pronounced when samples were drawn into 3.2% sodium citrate. We found no statistically significant difference in PT results from a 3.2% citrate tube between fill volumes of 60% and 100% and none for APTT results between fill volumes of 70% and 100%. This study further supports the recommendation to use 3.2% sodium citrate concentration, because 60% of the optimum filled volume for PT and 70% of the optimum filled volume for APTT are acceptable.

Comparison of the sensitivity of commercial APTT reagents in the detection of mild coagulopathies.

This study was undertaken to evaluate the precision and sensitivity of three different commercial APTT reagents containing the activators kaolin, micronized silica, or ellagic acid. These reagents varied greatly in their ability to detect mild coagulopathies. The ellagic acid reagent was able to detect the mildest deficiencies for the most common coagulopathies. This reagent was sensitive to 50% levels of Factor VIIIC, whereas the APTT with the kaolin reagent was not prolonged until levels of 35% or less were attained. The micronized silica reagent was the least sensitive to Factor IX deficiency, detecting levels of 12% or less. Precision was similar for all reagents when tested with normal and slightly abnormal plasmas. Since methods and instrumentation vary, each laboratory should evaluate their APTT reagent to determine its precision and sensitivity.