Douglas A. Triplett

Evaluation of lupus anticoagulants: Antiphospholipid antibodies, endothelium associated immunoglobulin, endothelial prostacyclin secretion, and antigenic protein S levels

Plasma samples from nineteen patients with well characterized lupus anticoagulants (LA) were evaluated using a series of test systems. An ELISA was used to determine if the plasmas contained antiphospholipid antibodies (APA); fifteen of nineteen LA plasmas contained APA, with five exhibiting IgG only, two exhibiting IgM, and eight plasmas containing both IgG and IgM. Anti-phosphatidyl serine (PS) was the predominant IgG specificity and all IgM APA-containing plasmas reacted with phosphatidyl inositol (PI). An ELISA was developed to determine if LA plasmas contained immunoglobulin which would associate with cultured human umbilical cord vein-derived endothelial cells (HUV); ten of nineteen plasmas contained endothelium associated immunoglobulin (EAI). There was significant concordance between the occurrence of EAI and IgM anti-PI. The occurrence of EAI or APA, either singly or in combination, did not correlate with a past history of thrombosis. Patient plasmas were incubated with HUV and examined for effects on HUV prostacyclin (PGI2) secretion; six plasmas significantly stimulated PGI2 secretion and one plasma was inhibitory. Finally, plasma levels of free and total antigenic protein S were determined by EID. Five plasmas contained significantly reduced levels of free antigenic protein S, and total antigenic protein S was reduced in ten plasmas. Patient histories were examined for evidence of thrombotic episodes; six patients had a history of either arterial or venous thrombosis, with five of these six patients having drug-induced LA. Thus, unlike previous studies, drug-induced LA were associated with thrombosis.

Antiphospholipid Antibodies: Proposed Mechanisms of Action

ABSTRACT: Antiphospholipid antibodies (APA) are a family of immunoglobulins that react with anionic phospholipids, or anionic phospholipids‐protein complexes. Recent evidence would support the latter definition. Lupus anticoagulants (LA) inhibit in vitro phospholipid dependent coagulation tests [e.g., activated partial thromboplastin time (APTT), prothrombin time (PT), and dilute Russell viper venom time (dRVVT)]. This inhibition appears to be specific for reagent phospholipids. The addition of freeze‐thawed platelets or activated platelets will result in correction of the LA‐induced abnormality. Anticardiolipin antibodies (ACA) are related to LA but appear to be distinct. ACA are detected by solid phase assays (ELISA, RIA) and require a plasma cofactor: β2 Glycoprotein‐I (β2 GPI). ACA and LA activities can be separated in individual patient plasmas by affinity chromatography. In some instances they are of differing isotypes. Preliminary evaluation of β2 GPI in coagulation assays suggests it may function as a cofactor for LA activity. Recent work also suggests human prothrombin may represent a necessary cofactor for in vitro LA activity. Paradoxically, patients with LA/ACA may sustain thromboembolic events involving both venous and arterial sites. The prothrombotic properties of LA/ ACA have not been satisfactorily characterized. A number of proposals have been reported, including inhibition of prostacyclin (PGI2) generation by endothelial cells, decreased activity of the protein C system, impaired fibrinolysis, and inhibition of β2 GPI. Among these various hypotheses, down regulation of the protein C system appears most plausible. Also, LA/ACA may interfere with the phospholipase A2‐phospholipid substrate complex involved in the generation of arachidonic acid from membrane phospholipids.

CURRENT RECOMMENDATIONS FOR WARFARIN THERAPY: Use and Monitoring

With the aging population, the use of warfarin will continue to increase. The introduction of new thromboplastins with International Sensitivity Indices (ISI) of 1.0 to 1.5 has improved the efficacy of monitoring warfarin therapy with the prothrombin time (PT). Increasingly, outpatient oral anticoagulant clinics and home testing are the sites for PT monitoring.

New Diagnostic Strategies for Lupus Anticoagulants and Antiphospholipid Antibodies

Laboratories are currently challenged with the problem of ruling out APA in a patient plasmas. In order to optimize the identification of these antibodies, it is necessary to institute a well-planned

A clinical study on the use of a fluorogenic substrate assay of plasminogen.

A fluorogenic substrate assay of plasminogen was compared with acceptable methodologies, i.e., caseinolytic and radial immunodiffusion assays. Reference intervals based on a population of 25 members of the laboratory staff were 2.5-3.8 CTA u/ml (fluorogenic), 2.2-4.0 CTA u/ml (caseinolytic) and 8.7-14.3 mg/dl (RID). Ninety eight determinations were performed on 65 patients. Regression analysis showed linear correlation between the fluorometric and caseinolytic assays (r = 0.8046, p less than 0.03) and between the fluorometric and immunologic assays (r = 0.8572, p less than 0.005). Pre-operative and post-operative determinations were performed on 33 patients undergoing coronary artery bypass surgery and there was a consistent and significant (10%-76%) decrease in the plasminogen levels post-operatively (p less than 0.01). Twenty-five patients with various malignancies were compared with the normal population; no significant difference in the plasminogen levels was observed between the two groups.

Fluorogenic substrate assay of antithrombin III: a clinical study

The fluorogenic substrate assay of antithrombin III (AT III) was compared with a thrombin inhibition (two-stage) clotting test and a radial immunodiffusion assay. Normal ranges based on a population of 25 individuals were 88-121% (fluorogenic substrate), 72-111% (clotting) and 22.5-35.3 mg/dl (RID). A clinical comparison of the methods showed a linear relationship between the fluorogenic substrate and immunologic methods (r = 0.69, p less than 0.005), as well as between the fluorogenic substrate and clotting methods (r = 0.75, p less than 0.005). The fluorogenic substrate assay reflected the highest incidence of low AT III levels in all three clinical groups included (36 patients with arteriosclerosis, 18 with fractures and 37 with thrombosis/embolism).

Comprehensive hypercoagulable state testing is indicated in patients with a first idiopathic deep venous thrombosis

A 45-year-old man presents with a 3-day history of right leg swelling and pain. He is diagnosed with an acute right common femoral vein thrombosis. He takes no medications, has an unremarkable medical history, no recent trauma or surgery, no recent travel, and no known cancer. Performance of a comprehensive hypercoagulable state panel is contemplated.

Laboratory testing for lupus anticoagulants

Abstract The diagnosis of LA requires special attention to specimen processing and careful determination of a reference interval for the screening procedures. A sensitive APTT reagent should be used as the routine screen with appropriate secondary screening procedures available for the difficult patient (e.g., KCT and dRVVT). Appropriate attention to the ratio of patient to normal plasma in mixing studies is imperative. The ratio of four parts patient to one part normal is recommended. In some instances, confirmatory tests utilizing either the PNP or other test systems may be ambiguous. In these cases it is necessary to perform factor assays for a definitive diagnosis. There is a need for reference plasmas and a common means of expressing LA titers.