Activated protein C resistance test

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Activated protein C resistance test
Synonyms APC resistance test; Activated protein C resistance assay; APC resistance assay; APCR test; APCR assay
Test of Activated protein C resistance, coagulation, hypercoagulability

The activated protein C resistance (APCR) test is a coagulation test used in the evaluation and diagnosis of activated protein C (APC) resistance, a form of hypercoagulability. [1] [2] Hereditary APC resistance is usually caused by the factor V Leiden mutation, whereas acquired APC resistance has been linked to antiphospholipid antibodies, pregnancy, and estrogen therapy. [3] [4] [5] [6] APC resistance can be measured using either an activated partial thromboplastin time (aPTT)-based test or an endogenous thrombin potential (ETP)-based test. [5] [4] [2]

Contents

Methodology

The aPTT-based APC resistance test involves a modified aPTT test performed in the presence and absence of activated protein C (APC). [1] [5] The ratio of these aPTT values is calculated and is called the APC sensitivity ratio (APCsr) or simply APC ratio (APCr). [1] [5] This ratio is inversely related to the degree of APC resistance. [7] The ETP-based APC resistance test involves the addition of APC to a thrombin generation assay (TGA). [5] This results in an inhibition of thrombin generation as measured by reduction of the endogenous thrombin potential (ETP; area under the thrombin generation curve). [5] The result is expressed as a normalized APC sensitivity ratio (nAPCsr), which corresponds to the ratio of the ETP measured in the presence and absence of APC divided by the same ratio in reference plasma. [5] nAPCsr values range from 0 to 10. [5] Opposite to the case of the APCsr with the aPTT-based APC resistance test, higher nAPCsr values indicate greater APC resistance. [5] [8] This is the result of the fact that APC prolongs the aPTT but inhibits thrombin generation. [8]

Whereas the aPTT-based APC resistance test only measures the initiation phase of coagulation, the ETP-based test is a global assay and measures the initiation, propagation, and termination phases of coagulation. [5] [9] The initiation phase accounts for less than 5% of total thrombin generation, making aPTT-based tests poorly indicative of hypercoagulability in general. [10] [11] The aPTT-based assay is more sensitive to levels of prothrombin and factor VIII, whereas the ETP-based test is more sensitive to levels of tissue factor pathway inhibitor (TFPI) and protein S. [5] The ETP-based test has traditionally been performed using methods such as the calibrated automated thrombogram (CAT) and has been limitedly available due to its technical difficulty. [2] Recently however, a fully automated commercial test system called the ST Genesia has been introduced, and it has been said that this should allow for adoption of TGAs and ETP-based APC resistance tests in routine clinical settings. [2] [12]

Influences

Thrombin generation curves in the absence (HPP -APC) and presence (HPP +APC) of activated protein C (APC) in healthy pooled plasma and in plasma of women on various combined oral contraceptives. The contraceptives are estetrol/drospirenone (E4/DRSP), ethinylestradiol/levonorgestrel (EE/LNG), ethinylestradiol/desogestrel (EE/DSG), and ethinylestradiol/drospirenone (EE/DRSP). Thrombin generation curves with and without activated protein C and with various oral contraceptives with activated protein C.png
Thrombin generation curves in the absence (HPP -APC) and presence (HPP +APC) of activated protein C (APC) in healthy pooled plasma and in plasma of women on various combined oral contraceptives. The contraceptives are estetrol/drospirenone (E4/DRSP), ethinylestradiol/levonorgestrel (EE/LNG), ethinylestradiol/desogestrel (EE/DSG), and ethinylestradiol/drospirenone (EE/DRSP).

Estrogens are well known to increase APC resistance, which has been described as acquired APC resistance. [2] [5] [4] [13] [14] However, the aPTT-based APC resistance test is much less sensitive to the procoagulatory effects of estrogens than is the ETP-based test. [13] [14] [5] [4] [2] [15] Pregnancy [7] and ethinylestradiol (EE)-containing combined birth control pills increase APC resistance as measured by either the aPTT- or ETP-based test. [4] [5] [15] EE-containing birth control pills show different degrees of influence on the ETP-based test depending on the progestin, which may be due to varying degrees of androgenic antagonism of ethinylestradiol-mediated procoagulation. [5] [4] In contrast to EE-containing birth control pills, studies have not found increased APC resistance with menopausal hormone therapy or with estetrol- or estradiol-containing birth control pills using the aPTT-based test, though increased APC resistance has been shown with the ETP-based test. [14] The increase in APC resistance is much greater with oral estrogens than with transdermal estradiol. [14] Increased APC resistance with both the aPTT-based and ETP-based tests has been observed with feminizing hormone therapy in transgender women, which involves higher doses of estradiol than are used in other contexts. [16] [17] EE produces a much stronger increase in APC resistance than does estradiol. [18] [17] In relation to this, ethinylestradiol is associated with a higher risk of venous thromboembolism (VTE) than is estradiol. [18] [19] [20]

History

The aPTT-based APC resistance test was developed in 1993, while the ETP-based test was developed in 1997. [5] For many years, the ETP-based APC resistance test suffered from a lack of standardization which hampered study-to-study comparison. [21] By 2020 however, a validated methodology was developed aiming to propose a standardized and harmonized scale for ETP-based APC resistance, the normalized activated protein C sensitivity ratio (nAPCsr). [21]

Related Research Articles

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">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.

<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.

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">Activated protein C resistance</span> Medical condition

Activated protein C resistance (APCR) is a hypercoagulability characterized by a lack of a response to activated protein C (APC), which normally helps prevent blood from clotting excessively. This results in an increased risk of venous thrombosis, which resulting in medical conditions such as deep vein thrombosis and pulmonary embolism. The most common cause of hereditary APC resistance is factor V Leiden mutation.

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.

<span class="mw-page-title-main">Nomegestrol acetate/estradiol</span> Combination drug

Nomegestrol acetate/estradiol (NOMAC-E2), sold under the brand names Naemis and Zoely among others, is a fixed-dose combination medication of nomegestrol acetate, a progestogen, and estradiol, an estrogen, which is used in menopausal hormone therapy and as a birth control pill to prevent pregnancy in women. It is taken by mouth.

Combined birth control pills that contain natural estradiol or an estradiol ester include:

Drospirenone/estetrol, sold under the brand name Nextstellis, Drovelis, and Lydisilka, among others, is a fixed-dose combination medication containing drospirenone, a progestin, and estetrol, an estrogen, which is used as a combined birth control pill for the prevention of pregnancy in women. It is taken by mouth.

Thrombin–antithrombin complex (TAT) is a protein complex of thrombin and antithrombin. It is a marker of net activation of coagulation.

Prothrombin fragment 1+2 (F1+2), also written as prothrombin fragment 1.2 (F1.2), is a polypeptide fragment of prothrombin generated by the in vivo cleavage of prothrombin into thrombin by the enzyme prothrombinase. It is released from the N-terminus of prothrombin. F1+2 is a marker of thrombin generation and hence of coagulation activation. It is considered the best marker of in vivo thrombin generation.

The Estrogen in Venous Thromboembolism Trial (EVTET) was a randomized controlled trial (RCT) of menopausal hormone therapy in 140 postmenopausal women with previous history of venous thromboembolism (VTE). It was a double-blind RCT of the estrogen, oral estradiol 2 mg/day, plus the progestogen, norethisterone acetate (NETA) (n=71) 1 mg/day versus placebo (n=69). The results of the trial were published in 2000 and 2001. The incidence of VTE was 10.7% in the hormone therapy group and 2.3% in the placebo group, with all events occurring within 261 days after study inclusion. The difference did not reach statistical significance in the sequential analysis, but was statistically significant if the sequential design was ignored. Markers of coagulation were likewise increased by hormone therapy. As a result of the high incidence of VTE in the treatment group, the trial was terminated prematurely. The researchers concluded on the basis of their findings that menopausal hormone therapy should not be used in women with a previous history of VTE.

The calibrated automated thrombogram is a thrombin generation assay (TGA) and global coagulation assay (GCA) which can be used as a coagulation test to assess thrombotic risk. It is the most widely used TGA. The CAT is a semi-automated test performed in a 96-well plate and requires specialized technologists to be performed. As a result, it has seen low implementation in routine laboratories and has been more limited to research settings. Lack of standardization with the CAT has also led to difficulties in study-to-study comparisons in research. However, efforts have recently been made towards standardization of the assay. An example of a specific commercial CAT is the Thrombinoscope by Thrombinoscope BV.

A thrombin generation assay (TGA) or thrombin generation test (TGT) is a global coagulation assay (GCA) and type of coagulation test which can be used to assess coagulation and thrombotic risk. It is based on the potential of a plasma to generate thrombin over time, following activation of coagulation via addition of phospholipids, tissue factor, and calcium. The results of the TGA can be output as a thrombogram or thrombin generation curve using computer software with calculation of thrombogram parameters.

<span class="mw-page-title-main">Fibrinopeptide</span> Chemical compound

The fibrinopeptides, fibrinopeptide A (FpA) and fibrinopeptide B (FpB), are peptides which are located in the central region of the fibrous glycoprotein fibrinogen and are cleaved by the enzyme thrombin to convert fibrinogen into covalently-linked fibrin monomers. The N-terminal FpA is cleaved from the Aα chains of fibrinogen and FpB from the Bβ chains of fibrinogen, with FpA released before FpB. Subsequent to their formation, fibrin monomers are converted to cross-linked fibrin polymers by the action of thrombin-activated factor XIII, and these fibrin polymers form the backbone of a thrombus. Hence, the fibrinopeptides are sensitive markers of fibrinogenesis, thrombin activity, and coagulation.

Plasmin-α2-antiplasmin complex (PAP) is a 1:1 irreversibly formed inactive complex of the enzyme plasmin and its inhibitor α2-antiplasmin. It is a marker of the activity of the fibrinolytic system and a marker of net activation of fibrinolysis.

The ST Genesia is a fully automated commercial analyzer system for performing thrombin generation assays (TGAs) and hence for coagulation testing. It was developed by Diagnostica Stago and was introduced by the company in 2018.

Fibrin monomers are monomers of fibrin which are formed by the cleavage of fibrinogen by thrombin. Levels of fibrin monomers can be measured using blood tests and can serve as a marker of in vivo fibrinogenesis and coagulation activation. They may be useful in the evaluation hypercoagulability.

Coagulation activation markers are biomarkers of net activation of coagulation and fibrinolysis. Examples include prothrombin fragment 1+2 (F1+2), thrombin–antithrombin complex (TAT), fibrinopeptide A (FpA), fibrin monomers (FMs), plasmin-α2-antiplasmin complex (PAP), activated protein C–protein C inhibitor (APC-PCI), and D-dimer (DD). These compounds are markers of thrombin generation, fibrin generation, and fibrinolysis. Coagulation activation markers, particularly D-dimer, are useful in the diagnosis of acute venous thromboembolism. They may also be useful in the assessment of hypercoagulability and venous thromboembolism risk.

Activated protein C–protein C inhibitor (APC-PCI) is a complex of activated protein C (APC) and protein C inhibitor (PCI). It has been measured in coagulation testing to evaluate coagulation, thrombosis, and other cardiovascular complications. It is a marker of thrombin generation and indicates hypercoagulability. The half-life of APC-PCI is either 40 minutes or 140 minutes.

References

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  12. ↑ Favaloro EJ, Lippi G (April 2019). "Recent Advances in Mainstream Hemostasis Diagnostics and Coagulation Testing". Semin Thromb Hemost. 45 (3): 228–246. doi:10.1055/s-0038-1676579. PMID   30912101. S2CID   85517668.
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