European Journal of Cardiovascular Medicine

Systemic heparinization is a mandatory step to achieve the appropriate anticoagulation and reach minimum target activated clotting time (ACT) prefixed for different procedures, like cardiovascular and percutaneous coronary interventions, according to the guidelines [1-2]. There is pivotal role of antithrombin III (AT III) in this anti-coagulation process, where heparin is used and its action is antithrombin III dependent [3].

Antithrombin is included in the group of serine protease inhibitors which has a reactive center loop (RCL), that serves as the reactive site for protease interaction that creates the antithrombin-protease complex for inhibition [4]. Synthesis of antithrombin occurs primarily in the liver. The designations Antithrombin I through to Antithrombin IV originate in early studies carried out in the 1950s by Seegers, Johnson and Fell [5]. The normal functional antithrombin III (AT III) levels in human blood are 80-120% [6].

There are two types of AT III depending on the number of occupied glycosylation sites: α-Antithrombin - constitutes major part, oligosaccharides bind all four glycosylation sites. β-Antithrombin - constitutes minor part, three of the four sites occupied, with the oligosaccharide chain at Asn135 missing [7]. This property of β-Antithrombin increases its affinity to heparin at a designated heparin-binding domain [8].

There are two mechanisms by which heparin enhances AT III action - 1. By conformational change in antithrombin involving the reactive center loop (RCL). 2. Formation of a complex between AT-III, thrombin and heparin [9]. Therefore, in view of mentioned anti-coagulation action and its reversal with protamine sulphate, heparin is currently the choice of drug for systemic Anti-coagulation required for various cardiac and vascular interventions, be it on cardiopulmonary bypass (CPB) pump procedures or off CPB pump procedures or percutaneous coronary intervention (PCI) procedures. The heparin as initial bolus in a dose of 300 IU/kg followed by additional boluses of 5000 IU to maintain ACT >400 s (for on CPB pump cases) [10].

Patients undergoing cardiac and vascular intervention procedure require in-house, quick and easily performed method. Hence, ACT is well suitable to monitor unfractionated heparin induced anti-coagulation and is a widely used method in cardiac operation theater, cardiac catheterization laboratories, extra-corporeal membrane oxygenation (ECMO) and continuous dialysis [11].

During cardiopulmonary bypass, the definition of heparin resistance is based on the ACT, with at least one ACT less than 400 seconds after heparinization and/or the need for exogenous AT III administration [12]. Heparin resistance occurs in up to 22% of patients undergoing cardiopulmonary bypass and most common cause is AT III deficiency (65%) [13]. AT III deficiency would lead to excessive thrombin activation and consumptive coagulopathy which leads to depletion of coagulation factors and platelets, post-operative bleeding and enhanced inflammatory response to cardiopulmonary bypass.

There are two types of AT III deficiency: 1. Acquired antithrombin deficiency (more common type)- It occurs due to different mechanisms such as increased renal excretion in renal failure or nephrotic syndrome, decreased synthesis due to liver dysfunction and can also be due to heparin therapy, disseminated intravascular coagulation, severe trauma and severe burns, premature infancy and sepsis, or due to interventions such as major surgery or cardiopulmonary bypass [14]. 2. Inherited antithrombin deficiency- Inherited AT III deficiency is an autosomal dominant disorder in which an individual inherits one copy of the SERPINC1 (also called AT3) gene on chromosome 1q25.1, which encodes AT III. It was first reported by Egeberg in 1965 [15].

ATIII deficiency can be treated with the Whole blood, Fresh Frozen Plasma (FFP) or pooled human plasma and AT III concentrate [16].

The aim of this study was to find incidence of AT III deficiency in patients undergoing cardiovascular surgery and percutaneous coronary interventions. To evaluate the effect of inherent AT III levels on the ACT values to heparin over the physiologic range of AT III levels and co-relation of other factors (Factor V, Fibrinogen C, Protein C and Protein S) with AT III and their implications on CPB.

This observational prospective study was conducted from June 2019 to December 2021 in 267 patients undergoing cardiovascular surgery and percutaneous coronary intervention at our institute.

Inclusion criteria:

• Patients who were suffering from cardiovascular diseases - congenital or acquired of all age group undergoing surgical and PCI who required heparin.
• Patients who were suffering from peripheral vascular diseases undergoing vascular surgery who required heparin.

Exclusion criteria:

• Patients who were suffering from diseases unfit to undergo surgery or
• Patients who did not give consent for the

Measurement of Antithrombin III levels:

Antithrombin levels in the patient’s plasma was measured preoperatively automatically on Instrumentation Laboratory ACL ELITE Pro IL Coagulation System in two stages:

1. Incubation of plasma with Factor Xa reagent in the presence of an excess of
2. Quantification of the residual Factor Xa activity with a synthetic chromogenic substrate. The para nitroaniline released was monitored kinetically at 405 nm and was inversely proportional to the Antithrombin level in the test sample.

Coagulation Testing:

The ACT measurement was done by using 2 mL of celite-activated blood placed in a Hemochron 401 Coagulation Analyzer.

Statistical analysis

The collected data was analyzed by calculating the mean of variables and chi square test for comparing the values and getting the significance by p value by using SPSS software. All statistical analyses of various parameters were done using excel sheet and SPSS software. A ‘p’ value of less than 0.05 was considered significant.

The study was conducted in 267 patients, who underwent cardiovascular and percutaneous coronary interventions at our institute from June 2019 to December 2021.

Out of 267 patients, 59 patients (22%) had AT III levels < 80%. The incidence of AT III deficiency in male was 64% (n-38) and in female was 36% (n-21). The mean age of patients having subnormal AT III levels in male group was 51.8 years and in female group was 40.6 years. 

Among patients having AT III deficiency, 38 (64%) patients under went open heart surgery, out of which 2 were redo surgeries, 14 (24%) PCI and 7 (12%) were vascular surgery. 34 (58%) patients had associated comorbidities along with AT III deficiency i.e. COPD in 14 (41%) patients; Diabetes mellitus with COPD in 11 (32%) patients; Hypothyroidism in 4 (12%); liver dysfunction in 2 (5.8%); lung infection in 1 (2.9%); CKD in 1 (2.9%) and 1 (2.9%) was post Corona virus disease-19 infection.

All the patients were preoperatively evaluated for inherent anti-coagulation factors levels i.e. AT III, Factor V, Fibrinogen C, Protein C and Protein S. AT III activity was found to be <80% in 59 (22%) of patients. 34 patients who had AT III deficiency also had associated other anti-coagulation factor deficiencies like protein C deficiency in 10 (29%) patients; protein S deficiency in 6 (18%) patients; high fibrinogen levels in 3 (9%) patients; low Factor V levels in 12 (35%) patients and raised D-dimer in 3 (9%) patients [Table 1].

Table 1: Associated anti-clotting factor deficiencies along with AT III deficiency

Pre-operatively injection heparin was being administered in 25 (42%) patients. Out of which 10 patients underwent open heart surgery, of which 5 had Rheumatic valvular heart disease with LAA clot, 4 had coronary artery disease with unstable angina and 1 had coronary artery disease with peripheral vascular disease. 7 patients underwent peripheral vascular surgery and 8 underwent PCI.

Range of AT III levels in patients having its deficiency was as follows: 37 (62.7%) patients had 71-80 % AT III levels; 19 (32.2%) had 60-70 % AT III levels and 3 (5.1%) had <60% AT III levels. FFP was administered along with extra dose of injection heparin in 19 (32%) patients. In 40 (68%) patients, extra dose of heparin sufficed to achieve desired activated clotting time. 6 patients having 71-80% AT III levels, 10 patients having 60-70% AT III levels and 3 patients having <60% AT III levels required FFP in addition to extra dose of injection heparin [Table 2].

Table 2: Range of Antithrombin III levels

For patients undergoing open heart procedures the mean baseline ACT was 128.7 seconds, after standard dose of heparin mean ACT value achieved was 471.8 seconds, after extra dose of heparin mean ACT value achieved was 485.5 seconds and after administration of FFP along with extra dose of heparin mean ACT value achieved was 544.7 seconds [Figure 1a]. For patients undergoing vascular procedures the mean baseline ACT was 113.2 seconds, after standard dose of heparin mean ACT value achieved was 258.8 seconds, after extra dose of heparin mean ACT value achieved was 434.4 seconds and after administration of FFP along with extra dose of heparin mean ACT value achieved was 537 seconds [Figure 1b]. For patients undergoing PCI procedures the mean baseline ACT was 131.4 seconds, after standard dose of heparin mean ACT value achieved was 217.5 seconds, after extra dose of heparin mean ACT value achieved was 446.4 seconds [Figure 1c].

Figure 1a: Shows baseline ACT and mean ACT values reached after first dose of heparin, extra dose of heparin and FFP with extra dose of heparin in patients undergoing open heart surgery.

Figure 1b: Shows baseline ACT and mean ACT values reached after first dose of heparin, extra dose of heparin and FFP with extra dose of heparin in patients undergoing vascular surgery.

Figure 1c: Shows baseline ACT and mean ACT values reached after first dose of heparin, extra dose of heparin and FFP with extra dose of heparin in patients undergoing PCI.

The patients having cardiovascular diseases has increased prevalence of AT III deficiency i.e. 22% (n-59) which is statistically significant (p<0.05) in our study and this increased prevalence is due to various other acquired factors in this group of patients. In our study group, 34 patients had associated commodities along with AT III deficiency. COPD (chronic smokers) and Diabetes mellitus were most commonly prevalent. Smoking causes hypercoagulable state by increased circulating levels of procoagulant factors, reduction of anti-clotting factors (protein C, protein S and AT III) and increment in circulating levels of fibrinogen [17]. A study conducted in 189 patients it was found that there was incidence of protein C deficiency in smokers compared to non-smoker group [18]. In our study, 10 patients had protein C deficiency along with AT III deficiency and all of them were chronic smokers. A study of 1612 patients concluded that type 2 diabetes is associated with decreased AT III levels [19]. In liver dysfunction there is decreased synthesis of AT III and hence its deficiency occurs. Plasma concentration of AT III is decreased in patients with kidney disease as a result of increased excretion [20]. The profound hypercoagulable

and inflammatory state is associated with COVID-19 and can result in decreased AT III levels and ineffective heparin treatment [21].

Pre-operative administration of heparin in patients also causes an increased incidence of heparin resistance and reduced AT III concentrations [22]. In our study, preoperatively heparin was given in 25 (42%) patients who had AT III deficiency.

The patients having AT III deficiency were categorized into three groups - group 1 included patients having AT III levels in the range of 71-80 % (n-37); group 2 included patients having AT III levels in the range of 60-70 % (n-19) and group 3 included patients having AT III levels in the range of less than 60 % (n-3). Our main strategy of management of AT III deficiency in these patients was as depicted in figure 4. In the patients having 71-80 % AT III levels initial dose of heparin is given then ACT is estimated, it did not reach the target value in these patients and the extra dose of heparin had to be given. In 31 patients, extra dose of heparin was enough to reach the target ACT but in 6 patients FFP was administered along with the extra dose of heparin and target ACT was achieved. Similarly, in 9 patients having 60-70% AT III levels extra dose of heparin was enough to reach the target ACT but in 10 patients FFP was administered along with the extra dose of heparin and target ACT was achieved.

In 3 patients having <60 % AT III levels, FFP transfusion had to be given along with extra dose of heparin. Hence, we suggest that in patients having AT III levels <60% FFP should be transfused to achieve desired target ACT.

AT III concentrate was not administered in our patients as it is expensive compared to FFP. Administration of FFP was enough to reach the target ACT. 

LIMITATIONS OF THE STUDY

There were a few limitations of our study. There were small numbers of cases and follow up of cases was not done. Further studies with a greater number of cases and simultaneous cytogenetic evaluation will provide definitive evidence of genetic alteration in patients with cardiovascular diseases.

The incidence of AT III deficiency is significant (i.e. 22%) among patients having cardiovascular diseases. Hence, it is important to routinely measure the AT III and other associated coagulation factors before the patient undergoes any cardiovascular intervention. This alters the plan of anti-coagulation management pre-operatively, intra-operatively, during follow up and also during redo surgeries. There was dearth of cases in our study, due to COVID 19 pandemic and there is need for a larger study involving a large number of patients to further study the incidence of AT III deficiency.

1. Shore-Lesserson L, Baker RA, Ferraris VA, Greilich PE, Fitzgerald D, Roman P et al. The Society of Thoracic Surgeons, The Society of Cardiovascular Anesthesiologists, and The American Society of Extra Corporeal Technology: Clinical practice guidelines—Anticoagulation during cardiopulmonary bypass. The Annals of thoracic surgery. 2018;105(2):650-62.
2. O’gara PT, Kushner FG, Ascheim DD, Casey Jr DE, Chung MK, De Lemos JA et al. 2013 ACCF/AHA guideline for the management of ST-elevation myocardial infarction: executive summary: A report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Circulation. 2013;127(4):529-55.
3. Hirsh J, Anand SS, Halperin JL, Fuster V. Mechanism of action and pharmacology of unfractionated heparin. Arteriosclerosis, thrombosis, and vascular biology. 2001;21(7):1094-6.
4. Ersdal-Badju E, Lu A, Zuo Y, Picard V, Bock SC. Identification of the antithrombin III heparin binding site. J Biol Chem. 1997;272(31):19393-400.
5. Seegers WH, Johnson JF, Fell C. "An antithrombin reaction to prothrombin activation". Am. J. Physiol. 1954;176 (1): 97–103.
6. Pagana KD, Pagana TJ, Pagana TN. Mosby's Diagnostic & Laboratory Test Reference. 14th ed. St. Louis, MO: Elsevier;2019.
7. Pol-Fachin L, Franco Becker C, Almeida Guimarães J, Verli H. Effects of glycosylation on heparin binding and antithrombin activation by heparin. Proteins. 2011;79(9):2735-45.
8. Amiral J, Seghatchian J. Revisiting antithrombin in health and disease, congenital deficiencies and genetic variants, and laboratory studies on α and β forms. Transfus Apher Sci. 2018;57(2):291-297.
9. Langdown J, Johnson DJ, Baglin TP, Huntington JA. Allosteric activation of antithrombin critically depends upon hinge region extension. J. Biol. Chem. 2004;279(45):47288–97.
10. Delavenne X, Ollier E, Chollet S, Sandri F, Lanoiselée J, Hodin S et al. Pharmacokinetic/pharmacodynamic model for unfractionated heparin dosing during cardiopulmonary bypass. British Journal of Anaesthesia. 2017;118(5):705-12.
11. Hattersley PG. Activated coagulation time of whole blood. Jama. 1966;196(5):436-40.
12. Anderson JA, Saenko EL. Heparin resistance. British Journal of Anaesthesia. 2002;88(4):467-9.
13. Spiess BD. Treating heparin resistance with antithrombin or fresh frozen plasma. Ann Thorac Surg. 2008;85(6):2153-60.
14. Maclean PS, Tait RC. Hereditary and acquired antithrombin deficiency: epidemiology, pathogenesis and treatment options. Drugs. 2007;67(10):1429–40.
15. Egeberg O. Inherited antithrombin deficiency causing thrombophilia. Thrombosis and Haemostasis.1965;13(02):516-30.
16. Levy JH, Despotis GJ, Szlam F, Olson P, Meeker D, Weisinger A. Recombinant human transgenic antithrombin in cardiac surgery: a dose-finding study. The Journal of the American Society of Anesthesiologists. 2002;96(5):1095-102.
17. Carlos J, Toledo Y, Modolo R, Moreno H. Smoking and the Endothelium. In: Protásio L. Da Luz, Peter Libby, Antonio C.P. Chagas, Francisco R.M. Laurindo, editors. Endothelium and Cardiovascular Diseases: Academic Press;2018; pp 537-554.
18. Fernández JA, Gruber A, Heeb MJ, Griffin JH. Protein C pathway impairment in non-symptomatic cigarette smokers. Blood Cells Mol Dis. 2002;29(1):73-82.
19. Wang H, Cao J, Su JB, Wang XQ, Zhang DM, Wang XH. The relationship between insulin sensitivity and serum antithrombin 3 activities in patients with type 2 diabetes. Endocr Connect. 2021;10(7):667-675.
20. RH Kauffmann, JJ Veltkamp, N van Tilburg. Acquired antithrombin III deficiency and thrombosis in the nephrotic syndrome. Am J Med. 1978; 65:607-10.
21. Joshi D, Manohar S, Goel G, Saigal S, Pakhare AP, Goyal A. Adequate Antithrombin III Level Predicts Survival in Severe COVID-19 Pneumonia. Cureus. 2021;13(10):18538.
22. Linden MD, Schneider M, Baker S, Erber WN. Decreased concentration of antithrombin after preoperative therapeutic heparin does not cause heparin resistance during cardiopulmonary bypass. J Cardiothorac Vascular Anaesthesia. 2004;18(2):131-5.