Technozym® tPA-PAI-1 Complex ELISA is a research assay kit for determination of t-PA-PAI-1 complexes in plasma. The t-PA-PAI-1 Complex kit is based on a monoclonal antibody directed against t-PA as a catching antibody for t-PA-PAI-1 complexes and a peroxidase-labeled monoclonal anti-PAI-1 antibody used for the detection of bound t-PA-PAI-1 complexes. The Technozym® t-PA-PAI-1 test is a solid phase enzyme immunoassay.
- PLATE + PLATE COVER: 12×8 well microtitre strips precoated with a monoclonal anti t-PA coating antibody in bicarbonate buffer and blocked with 1% bovine serum albumin (BSA) (TC-Code GN).
- STANDARD: 1 x lyophilized t-PA-PAI-1 complexes (TC-Code BT)
- POX-ANTIBODY: 1 x conjugated monoclonal anti-PAI-1 antibody (concentrated) (TC-Code KL)
- DILUTION BUFFER: (white cap) 1 x 20 ml 2.5x concentrated (PBS, 1% BSA, 5mM EDTA (TC-Code AD).
- POX DILUTION BUFFER: (white cap) 1 x 12ml PBS, 1% BSA (TC-Code DD).
- SUBSTRATE: (green cap) 1 x 12 mL TMB (Tetramethylbenzidine) in substrate buffer containing H2O2. Ready to use. (TC-Code KN)
- STOP SOLUTION: 1 x 15 ml sulfuric acid 0,45 mol/l (TC-Code KK)
- WASH BUFFER: (blue cap) 1 x 20 ml 12×5 x concentrated (PBS 0.5%, Tween 20) (TC-Code BE)
Plasminogen Activator Inhibitor Type I (PAI-1) is the major inhibitor of tissue type plasminogen activator (t-PA). In the resting state, the body’s plasma concentration of PAI-1 greatly exceeds the t-PA plasma level and even following physical exertion or venous occlusion, conditions which lead to increased t-PA levels, PAI-1 is usually available in adequate amounts to compensate for increased activator levels. Thus, increases in t-PA-PAI-1 complexes as long as active, non-complexed PAI-1 is available. Certain pathological conditions are associated with increased t-PA values. The liver is the major site of t-PA degradation and hepatic disorders are often accompanied by increased t-PA levels and, thus, increased t-PA-PAI-1 complex values.
In healthy persons, the t-PA level increases with age and complex levels rise accordingly. The quantitation of t-PA-PAI-1 complexes aids in the interpretation of alterations in the levels of t-PA and/or PAI-1. Since the liver, in addition to being a site of t-PA degradation, is an organ of PAI-1 synthesis the quantitation of activator-inhibitor complexes may serve as and indicator of the extent to which active PAI-1 is available to compensate for a reduction in the hepatic degradation of t-PA, i.e. elevation of t-PA plasma values.
The detection of elevated t-PA-PAI-1 complex values may be useful for the research of liver dysfunctions which may result in clotting disorders.
|What is the importance of t-PA and PAI in clinical research?|
|Elevated t-PA and PAI-1 antigen and reduced t-PA activity may be associated with cardiovascular disease. t-PA is given to stroke and heart attack victims shortly after the event to help break up the clot.|
|Explain the function of t-PA.|
|tPA activates plasminogen to plasmin, and also binds to fibrin. It is synthesized mainly in vascular endothelial cell and is secreted into the plasma continuously, and also through the acute release of tPA. tPA is a relatively poor activator of plasminogen in the absence of fibrin due to the low affinity of tPA for its substrate. However, tPA has a high affinity for fibrin, and binding increases its activating capacity up to 1000 fold. This dramatic increase is attributed to specific binding sites on the fibrin surface that concentrate and correctly orientate tPA with its substrate, as well as promote efficient clot lysis.|
|What is the difference between single and two-chain tPA?|
|Single-chain tPA is the native form of tPA secreted from endothelial cells, whereas the two-chain form is the result of the proteolytic activity of plasmin. Both forms are catalytically active and have similar enzymatic properties in the presence of fibrin.|
|What factors cause increased and decreased levels of t-PA and PAI?|
|There are numerous physiological factors that influence tPA and PAI antigen and activity level. For a complete list, please request the tPA monograph from DiaPharma. One interesting feature of the fibrinolytic system is the circadian variation in tPA and PAI-1level. Free tPA levels are lowest in the morning, increase during the day, and reach their peak activity level in the late afternoon. tPA and PAI-1 antigen are highest in the early morning and decrease during the day. This may help explain the high incidence of MI and stroke in the morning hours. Other factors that influence tPA and PAI-1 include alcohol, drugs, oral contraceptives, exercise, food, heparin administration, pregnancy, smoking, etc.|
|What are some of the inhibitors to t-PA?|
|PAI-1, PAI-2, PAI-3, protease nexin, a2-macroglobulin, trypsin inhibitor, and C1 Inhibitor all inhibit tPA. Plasminogen activator inhibitor 1 (PAI-1) is the most efficient inhibitor of tPA in plasma. It is a serine protease inhibitor (serpin) that acts as a pseudo-substrate for its target protease, with which it forms an inactive complex. PAI-1 is synthesized by several cell types including endothelial cells and hepatocytes and is present in platelets, placenta, and serum. The normal concentration range of PAI-1 in plasma is 5-40 mg/l and the normal activity is 0-20 AU/ml.
PAI-2 is a serpin with a higher affinity for u-PA (urinary-type plasminogen activator, or urokinase) than for t-PA. It is often only detectable during pregnancy, specifically in the third trimester.
PAI-3 is also called Protein C inhibitor, and inhibits u-PA and thrombin, and is present in plasma and urine.
|Won’t tPA be inhibited by PAI-1?|
|tPA is inhibited in vitro by plasminogen activator inhibitor, so something must be done to avoid this. Acidification of whole blood is therefore performed immediately after withdrawal. This can be done by mixing 1 ml of the citrated blood with 1 ml acetate buffer.|
|How does a specific gene polymorphism, PAI-1 4G genotype, relate to CVD risk?|
|For a detailed description of the 4G polymorphism, read Kohler et al. PAI-1 and Coronary Artery Disease. NEJM 2000; 342 (24): 1792-1801. In some studies, the 4G allele (four guanine bases) was significantly associated with high plasma PAI-1 concentrations, and was most strongly associated with previous MI, as well as risk of future MI. Studies have shown that subjects who are homozygous for the 4G allele have plasma PAI-1 concentrations approximately 25% higher than those with the 5G allele (5 guanine bases). Similarly, among patients with hypertriglyceridemia, those with the 4G allele also have higher plasma PAI-1 concentrations than those with the 5G allele. There are still conflicting data on the strength of the relation between PAI-1 gene polymorphism and MI, but it is suggested that the 4G allele is more likely to contribute to MI, particularly in the presence of hypertriglyceridemia.|
|Which substrate is best suited for measuring two-chain tPA, and why?|
|S-2765™, S-2366™, and S-2288™, S-2403™ are suitable for single-chain tPA, and S-2251™ was used in the discontinued Chromogenix Coaset® tPA kit, but there are no substrates specifically for two-chain tPA. A paper by Verheijen et al. (Thromb Res, 1985; 39: 281 – 288), however, describes a method comparing the direct amidolytic activity of tPA on S-2366™ and the plasminogen activating activity. Also, the substrate S-2288™ is suitable for measuring double-chain tPA because it has a slightly higher sensitivity than S-2366™. S-2288™ should be used with purified systems, though, since this substrate is sensitive for several proteases.|
|Protein concentrations in plasma|