Thrombotic Risk Markers

The ACTICHROME® TF is a chromogenic assay intended for the measurement of human Tissue Factor procoagulant activity in human plasma and cell lysates. Tissue Factor (TF) is a 45 kD transmembrane cell surface glycoprotein known for its role in initiating coagulation. Comprised of three domains: an extracellular domain (aa 1-219), followed by a hydrophilic spanning domain (aa 220-242) and a cytoplasmic tail (aa 243-263), it functions as a receptor and cofactor for Factor VII (FVII) and Factor VIIa (FVIIa). Tissue Factor is released into the blood stream following disruption of the endothelium. Contact between TF and blood is sufficient to initiate the extrinsic pathway of coagulation. In vitro studies reveal that once TF complexes
with FVII, FVII is efficiently activated by Factor Xa (FXa). Factor VIIa possesses little proteolytic activity by itself; only when bound to TF does it possess sufficient proteolytic activity to activate Factor IX and Factor X. The TF/FVIIa complex efficiently activates both Factor IX and Factor IX, thus initiating both the intrinsic and extrinsic coagulation pathways. The extrinsic pathway is quickly dampened by Tissue Factor Pathway Inhibitor (TFPI). TFPI is the only effective inhibitor of the TF/FVIIa complex. ACTICHROME TF measures the activity of human Tissue Factor via a two stage assay. In the first stage, Tissue Factor in the sample complexes with human Factor VIIa to generate the TF/FVIIa complex and convert human Factor X to Factor Xa. In the second stage, Factor Xa cleaves SPECTROZYME® FXa, a highly specific chromogenic substrate for Factor Xa. The cleaved substrate releases a para-nitroaniline (pNA) chromophore into the reaction solution. The solution absorbance is read at 405 nm and compared to those values obtained from a standard curve generated using known amounts of human Tissue Factor.

The ACTICHROME® TFPI is a chromogenic assay intended for the measurement of Tissue Factor Pathway Inhibitor (TFPI) activity in human plasma where TFPI exhibits an inhibitory effect on the Tissue Factor/FVIIa complex. TFPI circulates in human plasma as complexes with LDL, HDL and VLDL and
can be found in several forms: a 36,000 D molecule, a 43,000 D molecule and as truncated moieties. Approximately 10% of the total amount of TFPI is carried by platelets, which release TFPI once they are activated by thrombin. At the site of a wound, where platelets aggregate, elevated levels of TFPI are present. Based on the initial isolation of the inhibitor, it was found that TFPI inhibits Tissue Factor (TF) procoagulant activity; i.e., the TF/FVIIa complex, and directly inhibits Factor Xa by binding at or near its serine active site. The inhibitory mechanism of TFPI is a two-step process. In the first step TFPI binds to Factor Xa via its Kunitz-2 domain, followed by a second step in which the TFPI/FXa complex binds to the TF/FVIIa complex via its Kunitz-1 domain, forming an inactive quaternary TFPI/FXa/TF/FVIIa complex. The direct inhibition of Factor Xa is based on a 1:1 stoichiometry and is not calcium dependent. Furthermore, Factor Xa inhibition does not rely solely on TFPI binding through its Kunitz-2 domain. The C-terminal region of TFPI is required for a high affinity binding between TFPI and Factor Xa and the subsequent Factor Xa inhibition. It has been found that TFPI is released into blood following administration of heparin and that heparin enhances TFPI inhibition of Factor Xa. ACTICHROME TFPI measures the ability of TFPI to inhibit the catalytic activity of the TF/FVIIa complex as it activates Factor X to Factor Xa. It is a two stage assay. In the first stage, samples incubate with TF/FVIIa and the residual TF/VIIa activity converts FX to FXa. In the second stage, the FXa activity generated cleaves SPECTROZYME® FXa, a highly specific chromogenic substrate for Factor Xa. The cleaved substrate releases a para-nitroaniline (pNA) chromophore into the reaction solution. The solution absorbance is read at 405 nm and compared to those values obtained from a standard curve constructed using known TFPI activity levels.

ACTICHROME® PLG is intended for the measurement of plasminogen in human plasma. The assay is For Research Use Only, not for use in diagnostic procedures. Plasminogen is synthesized in the liver as an 88,000 D molecular weight single chain glycoprotein, circulating in plasma at a concentration of approximately 200 μg/mL. The conversion of plasminogen to plasmin occurs via a variety of mechanisms yielding an 83,000 D two chain protease. A complex is formed between plasminogen and streptokinase in the presence of excess streptokinase. This plasminogen-streptokinase complex possesses plasmin activity that can be measured using SPECTROZYME® PL, a highly specific chromogenic substrate for thrombin.

ACTICHROME® AT-III is an amidolytic chromogenic assay intended for the quantitative determination of antithrombin III in human plasma. Antithrombin III (AT-III) is an inhibitor of plasma serine proteases, including thrombin. AT-III levels of 30-60% of normal may be observed in patients with hereditary AT-III deficiency. Pathological conditions associated with acquired ATIII deficiency include liver disease, DIC, nephrotic syndrome, pulmonary embolism, stroke and thrombophlebitis. AT-III inhibition of thrombin is slow under normal conditions, however, the rate of inhibition can be enhanced several thousand- fold in the presence of heparin (heparin cofactor activity). Heparin Cofactor II is another rapid heparin-dependent thrombin inhibitor in human plasma. Heparin Cofactor II can interfere with AT-III measurements especially at high heparin concentrations (2 USP units/mL). To impart specificity to antithrombin III, ACTICHROME AT-III utilizes a lower final heparin concentration (1 USP units/mL) where heparin-enhanced inactivation of thrombin by heparin cofactor II is negligible. In addition, human heparin cofactor II reacts more readily with human thrombin than with bovine thrombin, thus further specificity for antithrombin III is imparted by the use of bovine thrombin. ACTICHROME AT-III is a two-stage assay. In the first stage, thrombin is added to a diluted plasma sample in the presence of excess heparin. In the
second stage, residual thrombin activity is measured using SPECTROZYME® TH, a highly specific with thrombin chromogenic substrate. The residual thrombin activity is inversely proportional to the antithrombin III concentration in the plasma.

ACTICLOT® Protein C Resistance is a plasma based functional assay for the determination of resistance to activated protein C caused by the Factor V Leiden mutation (FV:Q506). The assay is for in-vitro diagnostic use. Activated Protein C (APC) resistance is the most frequent hereditary defect associated with deep vein thrombosis. Over 95% of the APC resistance phenotype can be explained by the Factor V Leiden mutation. This defect is caused by point mutation in the factor V gene resulting in a replacement of the amino acid Arg 506 by a Gln residue. The heterozygous defect is associated with a 5 to 10 fold increased thrombotic risk, the homozygous defect is associated with a 50 to 100 fold increased thrombosis risk. There are two possibilities of detecting factor V (FV) Leiden: plasma based functional assays identifying the phenotype expression of the defect or genotype determination performed by PCR technology. ACTICLOT® Protein C Resistance differs from other functional APC resistance tests by acting specifically on the prothrombinase complex level. It is based on a FVa-dependent prothrombin activator isolated from snake venom. Specificity of the assay is enhanced by eliminating interference by factors upstream of the coagulation cascade, as the assay is not dependent on the presence of calcium. Interference from unfractionated heparin, lowmolecular weight heparin and pentasaccharide in the blood sample is precluded by inclusion of a heparin inhibitor. Clot times of plasma samples in the presence of APC and in the absence of APC are recorded and the ratio calculated. Differentiation of homozygous, heterozygous and negative samples is based on the typical ratio ranges measured with genotyped patient plasma samples.

ACTICLOT® Protein S is a functional clotting assay intended for the quantitative determination of Protein S activity in human plasma. The assay is for in vitro diagnostic use. Protein S is a Vitamin K-dependent primarily synthesized in the liver, but also in the endothelium and possibly in megakaryocytes. It functions as a cofactor for activated Protein C (APC), mediating APC in its ability to inhibit Factor Va in the prothrombinase complex. Therefore, there is an association between Protein S deficiencies and thrombotic events.

In the assay, dilutions of patient plasma are mixed with Protein S deficient plasma. A reagent containing factor Xa, activated Protein C and phospholipids is added to the mixture. Following a 5-minute incubation period, calcium chloride is added to initiate clot formation. Under these conditions, the prolongation of the clotting time is directly proportional to the concentration of Protein S in the patient plasma.

ACTICLOT® C is a functional clotting assay intended for the quantitative determination of Protein C activity in human plasma. The assay is for in vitro diagnostic use. Protein C is a vitamin K-dependent anticoagulant protein that normally circulates in plasma as an inactive zymogen. It is activated when it binds to thrombin in the thrombin-thrombomodulin complex on the surface of endothelial cells. Following activation and release from the cell surface, Protein C exerts its anticoagulant effect by inactivating Factor Va and its ability to form the prothrombinase complex and generate thrombin. Due to the anticoagulant effects of Protein C, people with Protein C deficiency, or who possess Activated Protein C Resistance, are at increased risk for suffering from a thrombotic event. The venom of the copperhead snake Agkistrodon contortrix is a rapid activator of Protein C. Under the assay conditions of ACTICLOT C, the ACTICLOT Activator, formulated with the venom from Agkistrodon contortrix, converts human Protein C to its active protease within 5 minutes. The activator reagent is formulated to activate both Protein C and the contact factors of the intrinsic pathway. Using this reagent, the clotting time of normal plasma is very long, greater than 100 seconds, while the clotting time of a Protein C deficient plasma is essentially the same as the clotting time of an APTT test, approximately 30 – 40 seconds. When an unknown test plasma is mixed with Protein C deficient plasma, the Protein C level is proportional to the prolongation of the clotting time.

IMUBIND® Tissue PAI-1 ELISA is an enzyme-linked immunosorbent assay for the quantitative measurement of human Plasminogen Activator Inhibitor Type 1 (PAI-1) in tissue extracts and cell culture supernatants. The assay detects latent (inactive) and active forms of PAI-1 and PAI-1 complexes and is insensitive to Plasminogen Activator Inhibitor Type 2 (PAI-2). This assay is For Research Use Only, not for use in diagnostic procedures. PAI-1 is a serine protease inhibitor that serves as the principal inhibitor of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), the activators that cleave plasminogen to form plasmin, and therefore the fibrinolytic process. As a primary regulator of fibrinolysis, PAI-1 has been found in a number of different tissues, and cell types including macrophages/monocytes, hepatocytes, vascular endothelia, adipose tissue of the heart and lungs, and in platelets.

IMUBIND® Plasma PAI-1 ELISA is an enzymelinked immunosorbent assay for the quantitative measurement of human Plasminogen Activator Inhibitor Type-1 (PAI-1) antigen in plasma. This assay is for in vitro diagnostic use. As a primary regulator of fibrinolysis Plasminogen Activator Inhibitor Type-1 (PAI-1), has been found in a number of different tissues, and cell types including macrophages/monocytes, hepatocytes, vascular endothelia, adipose tissue of the heart and lungs, and in platelets. The clinical interest in measuring PAI-1 in plasma is based on case studies in which levels of this serine protease inhibitor are associated with various thrombotic and fibrinolytic complications. Deficiency of PAI-1 activity is associated with bleeding disorders wherein the routine haemostatic screening tests are normal. High levels of PAI-1 activity are found in patients suffering from myocardial infarction, haemolytic uremic syndrome, and stroke. Levels of PAI-1 in the plasma of pregnant women are also correlated with gestational diabetes, reduced placental blood flow and preeclampsia. Patients with cirrhosis may also have elevated levels of PAI-1.

The IMUBIND® Thrombomodulin ELISA is an enzyme linked immunosorbent assay for the measurement of thrombomodulin in human plasma, serum and cell culture supernatants. The ELISA measures whole and truncated forms of thrombomodulin as well as thrombomodulin/thrombin complexes, but is less sensitive to non-functional or degraded fragments. The assay is For Research Use Only, not for use in diagnostic procedures. Thrombomodulin (TM) is the cell surface receptor for thrombin. When occupied, thrombomodulin converts thrombin from a procoagulant protein into the activator of Protein C. The thrombomodulinthrombin complex enhances the catalytic activation of Protein C over 1,000 fold. Once activated Protein C (APC) has been generated, thrombomodulin acts as a major anticoagulant through its ability to inactivate various blood factors (Va, VIIIa, Xa and XIIIa). In competing for thrombin binding, thrombomodulin inhibits the proteolytic effect of thrombin in its conversion of fibrinogen to fibrin, the inactivation of
Protein S and the induction of platelet aggregation. Platelets, monocytes and neutrophils contain small amounts of TM in comparison to cultured endothelial cells. TM is present in human plasma and urine in a truncated form, lacking the transmembrane and cytoplasmic domains of TM found on the cell surface. A detailed analysis of thrombomodulin circulating in human plasma revealed smaller fragments or degraded forms that are considered to possess only limited function. Plasma levels of TM have been used as a marker for in vivo endothelial cell injury.

The IMUBIND® Tissue Factor ELISA is intended for the measurement of human tissue factor (TF, thromboplastin, factor III) in human plasma, tumor tissue extracts and cell culture supernatants (e.g. LPS stimulated monocytes). Tissue Factor (TF) is a 45 kD transmembrane cell surface glycoprotein known for its role in initiating coagulation. Comprised of three domains: an extracellular domain (aa 1-219), followed by a hydrophilic spanning domain (aa 220-242) and a cytoplasmic tail (aa 243-263), it functions as a receptor and cofactor for Factor VII and Factor VIIa. Tissue Factor is released into the blood stream following disruption of the endothelium. Contact between TF and blood is sufficient to initiate the extrinsic pathway of coagulation. In vitro studies reveal that once TF complexes with Factor VII, Factor VII is efficiently activated by Factor Xa. Factor VIIa possesses little proteolytic activity by itself; only when bound to TF does it possess sufficient proteolytic activity to activate Factor IX and Factor X. The TF/FVIIa complex efficiently activates both Factor IX and Factor IX, thus initiating both the intrinsic and extrinsic coagulation pathways. The extrinsic pathway is quickly dampened by Tissue Factor Pathway Inhibitor (TFPI). TFPI is the only effective inhibitor of the TF/FVIIa complex.

IMUBIND® uPA ELISA is an enzyme-linked immunoassay for the quantitative determination of human urokinase-type plasminogen activator (uPA) in tissue extracts, plasma and cell culture supernatants. Single chain uPA (sc-uPA, prouPA) and HMW-uPA forms of urokinase-type plasminogen activator are all recognized by the assay, as is receptor-bound uPA and uPA complexed with PAI-1 and PAI-2. This assay is For Research Use Only, not for use in diagnostic procedures. uPA was originally isolated from human urine, it is also present in blood and in the extracellular matrix of various tissues. It is a serine protease with plasminogen serving as its primary physiological substrate. Activation of plasminogen to plasmin triggers the fibrinolytic cascade and fibrin degradation. uPA is also involved in the degradation of the extracellular tumor matrix and has been implicated in the mechanics of cell proliferation, invasion and metastasis. Secreted by tumor cells in an enzymatically inactive single-chain form, sc-uPA binds to receptors on the surface of tumor cells. Sc-uPA is converted into an enzymatically active two-chain molecule by serine proteases (plasmin, plasma kallikrein, trypsin), metalloproteases (thermolysin) or cysteine proteases (Cathepsin B and L).

The IMUCLONE™ PAP ELISA is intended for the measurement of plasmin/alpha-2-antiplasmin (PAP) complexes in human plasma. The assay measures only PAP complexes. It neither measures nor is affected by free plasminogen, alpha-2-antiplasmin or other plasmin complexes. Alpha-2-antiplasmin (α-2-antiplasmin) is a single chain, 70,000 molecular weight inhibitor of plasmin which reacts rapidly with plasmin to form the inactive plasmin/alpha-2 antiplasmin complex. Synthesized by the liver, alpha-2-antiplasmin circulates in plasma at a concentration of approximately 1 μM (70 μg/mL), with 20
being cross-linked when blood clots. The formation of the PAP complex is a two-step process. First, the lysine binding sites of plasmin and the arboxylterminal region of alpha-2-antiplasmin form a reversible complex. In the second step, cleavage of the peptide bond of the inhibitor forms the
irreversible complex. Increased PAP complex formation is accompanied by increased fibrin formation. Accordingly, a correlation between the level of fibrin split products and the level of PAP complexes exists.

The IMUCLONE™ FPA ELISA is a competitive enzyme-linked immunosorbent assay (CELIA) for measuring human (FPA) on bentonite adsorbed human plasma or in any fluid where FPA may be present. FPA, Fibrinopeptide A, is a 1536 D molecular weight, 16 amino acid peptide released from the amino terminus of fibrinogen A alpha chains by thrombin cleavage. Two molecules of FPA are released per molecule of fibrinogen. Elevated blood levels of FPA are indicative of excess thrombin activity.

The IMUCLONE™ Free Protein S is a an enzymelinked immunosorbent assay for measuring human Free Protein S (the activated Protein C cofactor) in plasma or any biological fluid where Free Protein S may be present. The assay is For Research Use Only, not for use in diagnostic procedures. Protein S is an 80,000 D molecular weight, vitamin K dependent glycoprotein synthesized in the liver. The concentration of Protein S in normal human plasma is approximately 25 μg/mL and is found in two forms: Free Protein S comprises approximately 40% (10 μg/mL) of the total amount while approximately 60% (15 μg/mL) circulates in blood as a non-covalent complex with C4b Binding Protein (C4b-BP). Only the Free Protein S possesses anticoagulant activity as the cofactor of Activated Protein C. The balance between the Free and C4b-BP complexed forms of Protein S plays an important role as only the Free Protein S is active.

The IMUCLONE™ Total TAFI ELISA is an in vitro assay for the measurement of TAFI antigen in human plasma, or in any fluid where TAFI may be present. The assay is For Research Use Only, not for use in diagnostic procedures. TAFI, Thrombin Activatable Fibrinolytic Inhibitor, (also known as carboxypeptidase U and plasma pro-carboxypeptidase B) is a 60,000 D molecular weight glycoprotein (proenzyme form) present in human plasma that modulates fibrinolysis in vivo. This proenzyme is converted to a 35,000 D molecular ratio active form, TAFIa, following proteolytic cleavage by the thrombin/thrombomodulin complex. TAFIa possesses carboxypeptidase activity with a preference for cleaving lysine and arginine residues from the c terminus of proteins. Modulation of fibrinolysis occurs when TAFIa cleaves C-terminal arginine and lysine residues of partially degraded fibrin. The removal of the c-terminus arginine and lysine residues from fibrin inhibits the continued degradation of fibrin by tPA activated plasmin. TAFI may play a central role in thrombosis and fibrinolysis due to its ability to retard fibrin clot lysis.

SPECTROLYSE® PAI-1 is intended for the quantitative determination of Plasminogen Activator Inhibitor Type-1 (PAI-1) activity in human plasma. The test is for in vitro diagnostic use. As a primary regulator of fibrinolysis, PAI-1 is found in a variety of different tissues and cell types including macrophages and monocytes, hepatocytes, vascular endothelia cells, adipose tissue of the heart and lungs and platelets. Clinical interest in measuring PAI-1 in plasma is based on case studies in which levels of this serine protease inhibitor are associated with various thrombotic and fibrinolytic complications. One unit of PAI-1 activity (IU) is defined as the amount of PAI-1 that inhibits one International Unit (IU) of human tPA. SPECTROLYSE PAI 1 is a twostage, indirect chromogenic assay. In the first stage, a fixed amount of human tPA is added to a plasma sample and allowed to react with the PAI-1 present in the sample. Next, the sample is acidified to destroy alpha-2-antiplasmin that would otherwise interfere with the assay. In the second stage, the residual tPA activity is measured by adding the sample to a mixture of human glu-plasminogen, poly-D-lysine and a chromogenic substrate for plasmin (PAR). The residual tPA activity in the plasma sample catalyzes the conversion of plasminogen to plasmin, which in turn hydrolyzes the chromogenic substrate. Poly-D-lysine is present as a stimulator of this tPA catalyzed conversion of plasminogen to plasmin. As the PAI-1 activity measured in the sample is based upon the residual tPA activity, the absorbance of the assayed is inversely proportional to the amount of PAI-1 activity in the sample.