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A cross-sectional study to estimate the prevalence of protease activity in the plasma of chronically ill patients in Bangladesh and identify its predictive relationship with protease inhibitor, alpha 2-macroglobulin (A2M).
Mohammad M Khan1*, Mohammad A Muqueet2, Intekhab Hossain, Munir E Khan, Ishmam Mustavi, Mohammad H Shibli, Meherab Hossain, Mohammad E Hossain.
1Biomark Bangladesh Foundation and , 2Assistant Professor, Department of Nephrology, Mymensingh Medical College, Mymensingh.
Reviewed by Abul Sadeque, MBBS, MA, MPH, DrPH, University of Texas, TX, USA & North South University, Dhaka, Bangladesh.
SUMMARY
This study is a continuation of our previous two publications in this online journal (www.rarediseasesindia.org/septicshock, 2012 and www.rarediseasesindia.org septic shock/protease inhibition, 2015). In our current study, 1.6 ml of human blood was collected from 30 chronically ill patients with a variety of diseases and 30 healthy volunteers. We measured alpha- 2-macroglobulin (A2M) and protease activity in their plasma. We found that in all chronically ill patients with a variety of diseases, A2M levels went down significantly (p<0.001), (n=30) compared to the level of normal healthy individuals (n=30) while also observing a significant increase in protease activity (p<0.001), (n=20) compared with the level of normal healthy individuals (n=20). These results indicated an inverse relationship between A2M and protease activity levels in chronically ill patients in Bangladesh.
INTRODUCTION
Microorganism’s invasion, metabolism, and virulence depend on protease(s) which are secreted by prokaryotic and eukaryotic microorganism1. Studies have found evidence of evolutionarily conserved and functional remnants of the innate immune system in human cells that date back to theoretically ancestral unicellular models2, 3. A2M is a plasma protein that traps and acts as a major component of the innate immune system by inhibiting a broad range of proteases2, 4. A2M provides immediate defense against infection and is an evolutionarily conserved defense mechanism found in plants, fungi, insects, bacteria, and primitive multi-cellular organisms1, 2. Low levels of A2M in protease-induced diseases are well documented in animal research5-8. These findings show an inverse relationship between protease(s) and protease inhibitor, indicative of a potential function of A2M in the patho-physiology of protease related disease processes. Results from animal models have shown that administration of A2M during advanced stages of septic shock drastically improved both conditions and recovery rates in all subjects7, 8. Micro-organisms secrete enzymes, known as proteases (one of the virulent factors), that facilitate the penetration and degradation of target proteins and cell membranes4, 9. As previously discussed, an extensive variety of proteases are involved in a spectrum of common diseases in humans4. There are over 500 human proteases that account for 2% of human exome. Similar proportions of genomic proteases are found in plants, insects, marine organisms, and all infectious organisms that cause disease10. Proteases play regulatory constituents in conception, birth, digestion, growth, maturation, senescence, and mortality. Furthermore, they have contributing roles in physiological processes, such as controlling the activation, synthesis, and turnover of proteins. Proteases are also essential for viruses, bacteria, and parasites during replication and spread of infectious diseases in insects, organisms, and animals resulting in effective transmission of disease in addition to the mediation and sustenance of diseases in animal and human hosts11. It is now known that non- synonymous mutations found in over 50 human proteases result in hereditary/genetic diseases. Other genetic or environmental conditions can result in an over/under-abundance of particular crucial protease(s) and/or abnormal levels of natural inhibitors/activators of proteases, leading to abnormal physiology and disease11.
A2M is a large glycoprotein which is present in the body fluids of both invertebrates and vertebrates12. It has many diversified and complex functions, but it is primarily known by its ability to inhibit a broad spectrum of proteases without the direct blockage of the protease active site. A2M is also known to be involved in the regulation, transport, and a host of other functions. For example, apart from inhibiting proteases, it regulates binding of transferrin to its surface receptor13, binds defensin and myelin basic protein14, etc., binds several important cytokines, including basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), interleukin-1β (IL-1β), and interleukin-6 (IL-6), and modify their biological activity12. A2M also binds a number of hormones and regulates their activity. A2M is said to protect the body against various infections7-8, and hence, can be used as a biomarker for the diagnosis and prognosis of a number of diseases. However, this multipurpose anti-protease is not "fail safe" and could be damaged by reactive species generated endogenously or exogenously, leading to various pathophysiological conditions12.
Given this understanding of A2M function and the relationship with protease activity found in animal models of septic shock, we posit that low levels of A2M and high levels of protease activity exist in patients with severe chronic illnesses and administration of A2M will reduce protease activity, increase A2M levels and improve patient recovery.
MATERIALS & METHODS:
Blood collection and preparation of plasma: After obtaining approval from the Institutional ethical committee of Mymensingh Medical College, Mymensingh, Bangladesh and / Bangladesh Medical Research Council (BMRC), voluntary consent was obtained from patients with chronic diseases and healthy volunteers. An amount of 1.6 ml of venous blood was obtained in 3.8% sodium citrate containing vacutainer blood collection tubes in room temperature from patients of different category (n=30) and healthy individuals (n=30). Plasma was prepared by conventional method15. We collected data on the plasma levels of A2M and protease activity from treatment-naïve randomly selected severely and chronically ill patients who were admitted at different departments in the Mymenshing Medical College Hospital, Mymensingh, Bangladesh. We measured A2M by sandwich ELISA (Enzyme–Linked Immunosorbent Assay) assay kit from abcam, ab 108888-alpha 2 Macroglobulin Human ELISA Kit, USA and protease (trypsin as a representative of proteases) assay by Protease assay kit from Creative BioMart, USA. Both assays were performed according to the manufacturer’s instruction.
Study Design: A pilot study with cross-sectional design conducted over a short period with the following objectives:
General Objectives: The study was conducted to assess the risk factor of protease activity in chronic patients, and develop understanding of common disease aetiology, generate hypothesis for future study.
Specific Objectives: To investigate the association between protease activity and plasma level of Alpha 2-macroglobulin among patients of chronic disease in Bangladesh. To estimate the prevalence of the protease activity of interest for the same population.
Rationale: In Bangladesh and many other developed and developing countries the role of proteases from microorganisms in disease processes have not been extensively studied yet. We would like to study the involvement of proteases and its inhibitor, A2M in different diseases.
a) Inclusion criteria: Inclusion criteria includes (1) Males or females age 10 to 80 years of any race or ethnicity (2) Chronically ill patients suffering from any diseases for at least 6 months (3) Informed consent/assent as applicable (4) For control subjects: healthy individuals without any known disease(s).
b) Exclusion criteria: Exclusion criteria includes (1) Substance abuse or dependence within 12 months prior to enrollment, (2) use of medication known to alter immune function within four weeks prior to enrollment, such as steroid or antiretroviral medications, (3) pregnancy, lactation, or the use of oral contraceptives (4) hemoglobin <9 g/dl.
Sampling method: drawing blood from peripheral vein with all aseptic precaution and sterile techniques15.
Statistical basis of sample size: Total patients: 30 and Normal subjects: Control: 30 are very nice sample size for following statistical analysis procedures for this study. Statistical analysis was performed by parametric techniques such as t-test, analysis of variance (ANOVA) and an analysis of covariance (ANCOVA) if the data are approximately normal or can be normalized using a suitable transformation.
Statistical Analysis: Student's t test was used for comparisons between control and patient’s protein levels (A2M) in ELISA assay and enzymatic activity for the analysis of protease activity (trypsin) in plasma.
Table 1 shows the age, sex and diagnosis diagnosis of patients’ enrolled in the study who were suffering for at least 6 months.
RESULTS
Our results show protease activity in patients’ plasma was 5021.22 ± 61.63 mg/ml compared to healthy individuals (control) was 516.16 ± 17.17 mg/ml (p<0.0001) (Figure 1 A). In contrast, A2M levels in patient’s plasma was 487.64 ± 9.70 g/ml compared to healthy individuals (control) was 2013.30 ± 52.00 g/ml (p<0.0001) (Figure 1 B). All severely chronic ill patients showed a significant increase in protease activity (Figure 1 A). In contrast, all severely chronic ill patients showed a significant decrease in A2M level (Figure 1 B).
Figure. 1 A A2M level in plasma of patients of different diseases (n=30) compared with normal healthy individuals (control) (n=30).
Figure 1B Protease activity in plasma of patients of different diseases (n=20) compared with normal healthy individuals (control) (n=20).
DISCUSSION
Our results further support evidence in human diseases (in chronic ill patients), that plasma levels of A2M goes down and oppositely protease activity levels in plasma goes up, suggesting an inverse relationship taken on by protease and protease inhibitors in severe to terminal ill patients, which we have also seen in animal research models7-8. Previously we have observed in animal models that when A2M was depleted out for two hours, only one hundredth of the lethal dose of a protease (Pseudomonas aeruginosa elastase) for guinea pigs is enough to kill them7-8. In another experimental group from the same study, we observed that injecting purified A2M at the crucial stage of protease induced septic shock model drastically improved conditions of all subjects, resulting in full recoveries7-8. Again, if we increased the A2M concentration in plasma by injecting purified human A2M to 150%, all of the subjects were protected from their lethal doses 7-8. Our results from these animal models had given us the interest to investigate blood plasma A2M levels and protease activity levels in chronically ill patients with hopes of seeking a comparisonal relationship (if any) between proteases and A2M – a method of investigational thought still novel in the field of medicine.
Corresponding to previously studied animal models and proposed hypothesis, we observed that A2M levels in plasma dropped significantly in all chronically suffering ill patients while also observing an inversed increase in their protease activity. This phenomenon suggests that under normal conditions, A2M actively functions to remove proteases. It can be further inferred that maintained A2M activity confers protective immunity from various protease induced pathological symptoms by limiting protease(s) capacity to destroy different protective or defense systems (coagulation / complement / immunoglobulin etc.).
In animal models we also observed that during the crisis-stage of septic shock pathogenicity, A2M levels came down to 30% of its original level 7-8. Therefore under chronic and severely ill conditions, A2M, which is supplied from liver, continuously attempts to maintain normal immune conditions via protease inhibition. However, increased severity and chronicity of infection progressively limits A2M conferred immunity after an interim of prolonged and continuous secretion of proteases by micro-organisms. This inevitably depletes the ability for endogenous A2M based immunity via protease inhibition, either due to deteriorating production or exhaustion from liver, indicatively leading to the key event for the beginning of disease process 4 (Figure 2).
Contrasting levels of A2M and protease(s) activity must be further investigated in order to understand the pathophysiology of many disease processes where the necessary amount of A2M needed to maintain normal immunity is jeopardized due to production depletion/exhaustion in chronic illness.
Proteins of the A2M family are present in a variety of animal phyla, including the nematodes, arthropods, mollusks, echinoderms, urochordates, and vertebrates and invertebrates or even plant kingdom1. A shared suite of unique functional characteristics have been documented for the A2Ms of vertebrates, arthropods, and mollusks. The A2Ms of nematodes, arthropods, mollusks show significant sequence identity in key functional domains. Thus, the A2Ms comprise an evolutionarily conserved arm of the innate immune system with similar structure and function in animal phyla separated by 0.6 billion years of evolution1. A2M prevents protease induced activation of coagulation by inhibiting thrombin and inhibitor of fibrinolysis by inhibiting plasmin and kallikrein. Numerous growth factors, cytokines and hormones bind to A2M. A2M also binds soluble beta-amyloid, of which it mediates degradation as seen in the brain of Alzheimer's disease (AD) patients. A2M is synthesized mainly in liver, but also locally by macrophages, fibroblasts, and adrenocortical cells. A2M is also produced in the brain where it binds multiple extracellular ligands and is internalized by neurons and astrocytes16-22.
In protease induced septic shock (animal models) 23-24, the activation of contact system followed by generation of bradykinin within few seconds by protease, Pseudomonas aeruginosa elastase, was shown23. Bradykinin was solely responsible for severe hypotension in that animal model. The beneficial effects of A2M in that model7-8 was observed because, probably in addition to inhibition of proteases, A2M also inhibits contact25-26, coagulation27 or complement systems27 and clears their by-products of different bioactive toxic products generated during disease processes12,16-22. Inhibition of contact system had further shown beneficial effect in animal model of artritis28. Recent evidence has also shown the beneficial effects of treatment with autologous A2M in post-traumatic osteoarthritis patients29.
It is well known that until now natural defense mechanisms or the innate immune system also known as the in-born immunity system, always deals with the description about a pathogen/micro-organism when they enter the body. The innate immune system, is an important subsystem of the overall immune system that comprises of the cells and mechanisms that defend the host from infection by other organisms. The cells of the innate system recognize and respond to pathogens in a generic way, but, unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host. Innate immune systems provide immediate defense against infection, and are found in all classes of plant and animal life30. After entry, a number of orchestrated defense systems start playing a role to opsonize them and eliminate them from the body. However, the dynamic roles shared by protease(s) and the protease inhibitor, A2M, have never been so keenly studied in regards to its application to human diseases, especially for severe and chronically ill patients.
We suggest to measure protease activity and levels of broad spectrum protease inhibitor (A2M) in plasma in chronically ill patients. An external supply of A2M to counterbalance insufficient endogenous levels of A2M in plasma could provide a promising new medical treatment in chronic ill patients after measuring their deficiency in more diseases.
Author Contributions
M. M. K. contributed to the major overall conception of the hypothesis and setup for all experiments. M. M. K., I. H and M. E. K contributed data analysis, and writing of the manuscript. M. M. K. performed experimental work with I. H. All patients’ samples were arranged by M. A. M. All control samples were arranged by I. H. Statistical data analysis and figures were made by M. E. K. All authors contributed to the project.
Acknowledgments
We thank Mr. Sariful Islam of Ibn Sina laboratory at Dhaka, Bangladesh for technical help regarding blood sample collections.
*Corresponding author:
Mohammad M Khan, MBBS, PhD
Professor and Founding Member,
Biomark Bangladesh Foundation, 5th Floor,
Hosna Center, 106 Gulshan Avenue, Gulshan, Dhaka, Bangladesh
drkhan@gmail.com
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Submitted to Rare Diseases India on February 22, 2016.
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A2M-Miracle Protein, the lifesaver By Mohammad Khan MBBS PhD
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