Ecarin

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Ecarin is an enzyme [1] that is derived from the venom of the Indian saw-scaled viper, Echis carinatus , [2] It is the primary reagent in the Ecarin clotting time test.

The venom of the saw-scaled viper, Echis carinatus, causes bleeding and eventually death. The venom contains a metalloprotease called ecarin that converts prothrombin to meizothrombin, a thrombin analog with increased esterase activity, and not to normal thrombin. [3] Ecarin is a glycoprotein with unique metalloproteinase properties. It has a molecular weight of 56,000 and has been found to specifically activate only prothrombin due to its strict substrate specificity. To better understand the structure-function relationships of Ecarin, researchers need to know its primary structure. This understanding is crucial for gaining insight into the related functions of the glycoprotein. To compare the covalent structures of Ecarin and RVV-X, researchers have determined the complete cDNA sequence and translated protein sequence of Ecarin. [4]

Determining the activity of the protein Ecarina is an important step in comprehending its role in initiating blood clotting. To accomplish this, researchers use a special substance called a chromogenic thrombin substrate, which is a molecule that can be cleaved by thrombin, a protein that is a critical player in the blood clotting cascade. The cleavage products that are generated by the substrate can be measured over time, and this parameter can be used to determine the activity of Ecarina. [5]

The activity of Ecarina is usually compared between different types of Ecarin. Ecarin is a purified protein that is obtained from the venom of Echis carinatus. Ecarin is known to activate prothrombin, another protein that is a critical component of the blood clotting cascade. Therefore, measuring the activity of Ecarina is essential in understanding how it participates in the regulation of blood clotting. [5]

In a recent study, the activation of human prothrombin and human prothrombin-2 was compared using two different Ecarin preparations. The chromogenic test was used at pH 8, which is an optimal pH for the activation of prothrombin. The results of this study provide valuable insights into the mechanism of blood clotting and could have implications for the development of new therapies for bleeding disorders or thrombotic diseases. [5]

Ecarin-based assays are tests that have the prospect to be clinically helpful in detecting vitamin K deficiency and lupus anticoagulant. The Ecarin clotting time (ECT) is a test that is widely used for lupus anticoagulant testing in certain regions. Dabigatran etexilate is a direct oral thrombin inhibitor that has been approved as a substitute for warfarin in preventing stroke in patients with nonvalvular atrial fibrillation. It is also used to treat deep venous thrombosis and pulmonary embolism, and to reduce the risk of recurrence. Ecarin-based testing is used to monitor dabigatran, with both clot-based and chromogenic-based methods available. [6]

In 2015, it was reported that 30% of oral anticoagulants prescribed for Medicare patients in the United States were direct oral anticoagulants (DOACs), with dabigatran being a popular choice among cardiologists and internal medicine physicians. This is largely due to the drug's safety and efficacy. It has been shown to be effective in preventing stroke and systemic embolism, and has a lower risk of bleeding compared to warfarin. However, it is important to note that monitoring the effects of dabigatran can be challenging, as traditional coagulation tests such as the prothrombin time (PT) and activated partial thromboplastin time (aPTT) are not sensitive to the drug's effects. This is where Ecarin-based testing comes in, as it provides a more accurate measure of dabigatran's anticoagulant effect. [6]

When performing Ecarin clotting testing, it's important to be aware of the two types of testing available: clot formation and chromogenic analysis. Both methods have their advantages and disadvantages, and choosing the appropriate method can help ensure accurate results.

One essential consideration when determining a testing technique is the effect of a patient's prothrombin and fibrinogen levels. If these levels are low, it can result in prolonged clotting times, which can impact the precision of the results obtained through the clot formation method. However, the Ecarin chromogenic assay eliminates the effect of low prothrombin levels by diluting the sample in prothrombin buffer. It also does not rely on fibrinogen conversion, meaning that it is not affected by low fibrinogen levels. As a result, the Ecarin chromogenic assay is often believed to be a more reliable choice.

the Ecarin clotting time technique can still be useful in certain cases. For example, in situations where the patient has high levels of heparin, which can interfere with the chromogenic assay, the clot formation method may be more appropriate. However, in most cases, the chromogenic assay is the preferred method due to its greater accuracy and reliability. [6]

In determination, when conducting Ecarin clotting testing, it's important to understand the key differences between the two available methods and choose the appropriate method based on the patient's individual circumstances. By doing so, you can help ensure that you obtain accurate and reliable results that can aid in the diagnosis and treatment of various medical disorders. [6]

Ecarin is a highly versatile drug compound that finds extensive use in blood clotting experiments. It is an invaluable tool for monitoring and treating a range of diseases, including but not limited to cancer, liver diseases, lupus, and cardiovascular disorders. [7] Its multifaceted properties make it a go-to solution for healthcare professionals seeking effective and reliable treatment options for their patients.

Related Research Articles

<span class="mw-page-title-main">Anticoagulant</span> Class of drugs

An anticoagulant, commonly known as a blood thinner, is a chemical substance that prevents or reduces coagulation of blood, prolonging the clotting time. Some of them occur naturally in blood-eating animals such as leeches and mosquitoes, where they help keep the bite area unclotted long enough for the animal to obtain some blood. As a class of medications, anticoagulants are used in therapy for thrombotic disorders. Oral anticoagulants (OACs) are taken by many people in pill or tablet form, and various intravenous anticoagulant dosage forms are used in hospitals. Some anticoagulants are used in medical equipment, such as sample tubes, blood transfusion bags, heart–lung machines, and dialysis equipment. One of the first anticoagulants, warfarin, was initially approved as a rodenticide.

<span class="mw-page-title-main">Coagulation</span> Process of formation of blood clots

Coagulation, also known as clotting, is the process by which blood changes from a liquid to a gel, forming a blood clot. It potentially results in hemostasis, the cessation of blood loss from a damaged vessel, followed by repair. The mechanism of coagulation involves activation, adhesion and aggregation of platelets, as well as deposition and maturation of fibrin.

<span class="mw-page-title-main">Disseminated intravascular coagulation</span> Medical condition where blood clots block small blood vessels

Disseminated intravascular coagulation (DIC) is a condition in which blood clots form throughout the body, blocking small blood vessels. Symptoms may include chest pain, shortness of breath, leg pain, problems speaking, or problems moving parts of the body. As clotting factors and platelets are used up, bleeding may occur. This may include blood in the urine, blood in the stool, or bleeding into the skin. Complications may include organ failure.

<span class="mw-page-title-main">Antiphospholipid syndrome</span> Medical condition

Antiphospholipid syndrome, or antiphospholipid antibody syndrome, is an autoimmune, hypercoagulable state caused by antiphospholipid antibodies. APS can lead to blood clots (thrombosis) in both arteries and veins, pregnancy-related complications, and other symptoms like low platelets, kidney disease, heart disease, and rash. Although the exact etiology of APS is still not clear, genetics is believed to play a key role in the development of the disease. Diagnosis is made based on symptoms and testing, but sometimes research criteria are used to aid in diagnosis. The research criteria for definite APS requires one clinical event and two positive blood test results spaced at least three months apart that detect lupus anticoagulant, anti-apolipoprotein antibodies, and/or anti-cardiolipin antibodies.

Factor V Leiden is a variant of human factor V, which causes an increase in blood clotting (hypercoagulability). Due to this mutation, protein C, an anticoagulant protein that normally inhibits the pro-clotting activity of factor V, is not able to bind normally to factor V, leading to a hypercoagulable state, i.e., an increased tendency for the patient to form abnormal and potentially harmful blood clots. Factor V Leiden is the most common hereditary hypercoagulability disorder amongst ethnic Europeans. It is named after the Dutch city of Leiden, where it was first identified in 1994 by Rogier Maria Bertina under the direction of Pieter Hendrick Reitsma. Despite the increased risk of venous thromboembolisms, people with one copy of this gene have not been found to have shorter lives than the general population. It is an autosomal dominant genetic disorder with incomplete penetrance.

<span class="mw-page-title-main">Thrombin</span> Enzyme involved in blood coagulation in humans

Thrombin is a serine protease, an enzyme that, in humans, is encoded by the F2 gene. During the clotting process, prothrombin is proteolytically cleaved by the prothrombinase enzyme complex to form thrombin. Thrombin in turn acts as a serine protease that converts soluble fibrinogen into insoluble strands of fibrin, as well as catalyzing many other coagulation-related reactions.

<span class="mw-page-title-main">Prothrombin time</span> Assay for evaluating the extrinsic pathway & common pathway of coagulation

The prothrombin time (PT) – along with its derived measures of prothrombin ratio (PR) and international normalized ratio (INR) – is an assay for evaluating the extrinsic pathway and common pathway of coagulation. This blood test is also called protime INR and PT/INR. They are used to determine the clotting tendency of blood, in such things as the measure of warfarin dosage, liver damage, and vitamin K status. PT measures the following coagulation factors: I (fibrinogen), II (prothrombin), V (proaccelerin), VII (proconvertin), and X.

<span class="mw-page-title-main">Partial thromboplastin time</span> Test for coagulation of blood

The partial thromboplastin time (PTT), also known as the activated partial thromboplastin time, is a blood test that characterizes coagulation of the blood. A historical name for this measure is the kaolin-cephalin clotting time (KCCT), reflecting kaolin and cephalin as materials historically used in the test. Apart from detecting abnormalities in blood clotting, partial thromboplastin time is also used to monitor the treatment effect of heparin, a widely prescribed drug that reduces blood's tendency to clot.

Mixing studies are tests performed on blood plasma of patients or test subjects to distinguish factor deficiencies from factor inhibitors, such as lupus anticoagulant, or specific factor inhibitors, such as antibodies directed against factor VIII. The basic purpose of these tests is to determine the cause of prolongation of Prothrombin Time (PT), Partial Thromboplastin Time, or sometimes of thrombin time (TT). Mixing studies take advantage of the fact that factor levels that are 50 percent of normal should give a normal Prothrombin time (PT) or Partial thromboplastin time (PTT) result. Factor deficient plasmas are used in mixing studies. Plasma with known factor deficiencies are commercially available but are very expensive, so they are often prepared in the laboratory and can then be used for mixing experiments.

Lupus anticoagulant is an immunoglobulin that binds to phospholipids and proteins associated with the cell membrane. Its name is a partial misnomer, as it is actually a prothrombotic antibody in vivo. Lupus anticoagulant in living systems causes a decrease in clotting time. The name derives from their properties in vitro, as these antibodies increase coagulation times in laboratory tests such as the activated partial thromboplastin time (aPTT). Investigators speculate that the antibodies interfere with phospholipids used to induce in vitro coagulation. In vivo, the antibodies are thought to interact with platelet membrane phospholipids, increasing adhesion and aggregation of platelets, which accounts for the in vivo prothrombotic characteristics.

<span class="mw-page-title-main">Thrombophilia</span> Abnormality of blood coagulation

Thrombophilia is an abnormality of blood coagulation that increases the risk of thrombosis. Such abnormalities can be identified in 50% of people who have an episode of thrombosis that was not provoked by other causes. A significant proportion of the population has a detectable thrombophilic abnormality, but most of these develop thrombosis only in the presence of an additional risk factor.

<span class="mw-page-title-main">Argatroban</span> Pharmaceutical drug

Argatroban is an anticoagulant that is a small molecule direct thrombin inhibitor. In 2000, argatroban was licensed by the Food and Drug Administration (FDA) for prophylaxis or treatment of thrombosis in patients with heparin-induced thrombocytopenia (HIT). In 2002, it was approved for use during percutaneous coronary interventions in patients who have HIT or are at risk for developing it. In 2012, it was approved by the MHRA in the UK for anticoagulation in patients with heparin-induced thrombocytopenia Type II (HIT) who require parenteral antithrombotic therapy.

<span class="mw-page-title-main">Dilute Russell's viper venom time</span>

Dilute Russell's viper venom time (dRVVT) is a laboratory test often used for detection of lupus anticoagulant (LA).

The prothrombinase enzyme complex consists of factor Xa (a serine protease) and factor Va (a protein cofactor). The complex assembles on negatively charged phospholipid membranes in the presence of calcium ions. The prothrombinase complex catalyzes the conversion of prothrombin (factor II), an inactive zymogen, to thrombin (factor IIa), an active serine protease. The activation of thrombin is a critical reaction in the coagulation cascade, which functions to regulate hemostasis in the body. To produce thrombin, the prothrombinase complex cleaves two peptide bonds in prothrombin, one after Arg271 and the other after Arg320. Although it has been shown that factor Xa can activate prothrombin when unassociated with the prothrombinase complex, the rate of thrombin formation is severely decreased under such circumstances. The prothrombinase complex can catalyze the activation of prothrombin at a rate 3 x 105-fold faster than can factor Xa alone. Thus, the prothrombinase complex is required for the efficient production of activated thrombin and also for adequate hemostasis.

<span class="mw-page-title-main">Hypoprothrombinemia</span> Medical condition

Hypoprothrombinemia is a rare blood disorder in which a deficiency in immunoreactive prothrombin, produced in the liver, results in an impaired blood clotting reaction, leading to an increased physiological risk for spontaneous bleeding. This condition can be observed in the gastrointestinal system, cranial vault, and superficial integumentary system, affecting both the male and female population. Prothrombin is a critical protein that is involved in the process of hemostasis, as well as illustrating procoagulant activities. This condition is characterized as an autosomal recessive inheritance congenital coagulation disorder affecting 1 per 2,000,000 of the population, worldwide, but is also attributed as acquired.

<span class="mw-page-title-main">Tirofiban</span> Antiplatelet drug

Tirofiban, sold under the brand name Aggrastat, is an antiplatelet medication. It belongs to a class of antiplatelets named glycoprotein IIb/IIIa inhibitors. Tirofiban is a small molecule inhibitor of the protein-protein interaction between fibrinogen and the platelet integrin receptor GP IIb/IIIa and is the first drug candidate whose origins can be traced to a pharmacophore-based virtual screening lead.

<span class="mw-page-title-main">Thrombin time</span>

The thrombin time (TT), also known as the thrombin clotting time (TCT), is a blood test that measures the time it takes for a clot to form in the plasma of a blood sample containing anticoagulant, after an excess of thrombin has been added. It is used to diagnose blood coagulation disorders and to assess the effectiveness of fibrinolytic therapy. This test is repeated with pooled plasma from normal patients. The difference in time between the test and the 'normal' indicates an abnormality in the conversion of fibrinogen to fibrin, an insoluble protein.

<span class="mw-page-title-main">Ecarin clotting time</span>

Ecarin clotting time (ECT) is a laboratory test used to monitor anticoagulation during treatment with hirudin, an anticoagulant medication which was originally isolated from leech saliva. Ecarin, the primary reagent in this assay, is derived from the venom of the saw-scaled viper, Echis carinatus.

Direct thrombin inhibitors (DTIs) are a class of anticoagulant drugs that can be used to prevent and treat embolisms and blood clots caused by various diseases. They inhibit thrombin, a serine protease which affects the coagulation cascade in many ways. DTIs have undergone rapid development since the 90's. With technological advances in genetic engineering the production of recombinant hirudin was made possible which opened the door to this new group of drugs. Before the use of DTIs the therapy and prophylaxis for anticoagulation had stayed the same for over 50 years with the use of heparin derivatives and warfarin which have some well known disadvantages. DTIs are still under development, but the research focus has shifted towards factor Xa inhibitors, or even dual thrombin and fXa inhibitors that have a broader mechanism of action by both inhibiting factor IIa (thrombin) and Xa. A recent review of patents and literature on thrombin inhibitors has demonstrated that the development of allosteric and multi-mechanism inhibitors might lead the way to a safer anticoagulant.

Blood clotting tests are the tests used for diagnostics of the hemostasis system. Coagulometer is the medical laboratory analyzer used for testing of the hemostasis system. Modern coagulometers realize different methods of activation and observation of development of blood clots in blood or in blood plasma.

References

  1. ↑ Pötzsch B, Hund S, Madlener K, Unkrig C, Müller-Berghaus G (June 1997). "Monitoring of recombinant hirudin: assessment of a plasma-based ecarin clotting time assay". Thromb. Res. 86 (5): 373–83. doi:10.1016/S0049-3848(97)00082-0. PMID   9211628.
  2. ↑ ecarin at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
  3. ↑ Paine, Mark J. I.; Laing, Gavin D. (2013-01-01), Rawlings, Neil D.; Salvesen, Guy (eds.), "Chapter 240 - Ecarin", Handbook of Proteolytic Enzymes (Third Edition), Academic Press, pp. 1064–1067, doi:10.1016/b978-0-12-382219-2.00240-4, ISBN   978-0-12-382219-2 , retrieved 2023-10-05
  4. ↑ Nishida, Shinji; Fujita, Taizo; Kohno, Noriatsu; Atoda, Hideko; Morita, Takashi; Takeya, Hiroyuki; Kido, Isao; Paine, Mark J. I.; Kawabata, Shun-ichiro; Iwanaga, Sadaaki (1995-02-07). "cDNA cloning and deduced amino acid sequence of prothrombin activator (ecarin) from Kenyan Echis carinatus venom" . Biochemistry. 34 (5): 1771–1778. doi:10.1021/bi00005a034. ISSN   0006-2960. PMID   7849037.
  5. 1 2 3 Jonebring, Anna; Lange, Ute; Bucha, Elke; Deinum, Johanna; Elg, Margareta; Lövgren, Ann (June 2012). "Expression and characterization of recombinant ecarin". The Protein Journal. 31 (5): 353–358. doi:10.1007/s10930-012-9409-6. ISSN   1875-8355. PMC   3380252 . PMID   22528138.
  6. 1 2 3 4 Gosselin, Robert C.; Douxfils, Jonathan (July 2020). "Ecarin based coagulation testing". American Journal of Hematology. 95 (7): 863–869. doi: 10.1002/ajh.25852 . ISSN   0361-8609. PMID   32350907. S2CID   217549415.
  7. ↑ Mohammadi, Nasrin; Bandehpour, Mojgan; Sotoodehnejadnematalahi, Fattah; Kazemi, Bahram (March 2022). "Prokaryotic expression, evaluation, and prediction of the structure and function of the ecarin metalloproteinase domain" . Proteins: Structure, Function, and Bioinformatics. 90 (3): 802–809. doi:10.1002/prot.26275. ISSN   0887-3585. PMID   34739152. S2CID   243761342.
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