septicshock

The Pathophysiology and Treatment of Septic Shock:

The alpha-2-Macroglobulin Hypothesis

Mohammad M. Khan, MBBS., PhD., Alexander M. Scharko, MD. Children’s Hospital of Philadelphia, Philadelphia, PA, USA.

Reviewed by Nahid T. Iftekhar, MBBS, MRCP, MSc, MPhil, Norfolk and Norwich University Hospital, UK.

INTRODUCTION

Septic shock is defined as infection-induced hypotension that is resistant to fluid resuscitation (1).  Septic shock is an important medical problem with a mortality rate of 20% translating to greater than 200,000 deaths per year in the United States (2).  Its incidence appears to be rising.  It is frequently reported that the most common cause of septic shock is endotoxin from the cell walls of Gram-negative bacteria.  However, septic shock also occurs in the context of infection with Gram-positive bacteria, fungi, rickettsia, and viruses (3).

The pathophysiology of septic shock is not completely understood.  It is thought that the pathophysiology revolves around perturbations in inflammation, coagulation, and fibrinolysis (4-6).  Tumor necrosis factor alpha (TNF-a) along with other proinflammatory cytokines are frequently cited as mediating septic shock, but interventions aimed at these cytokines have not been successful.  We hypothesize that although the triad of inflammation, coagulation, and impaired fibrinolysis is part of the pathophysiology of septic shock, the primary factor that drives the septic shock process is the depletion of endogenous alpha-2-macroglobulin (a2M) early during the course of infection.  This then implies that simple replenishment of a2M may be the definitive treatment for septic shock.

THE ROLE OF a-2-MACROGLOBULIN

a2M is a plasma protein of hepatic origin that functions as a non-specific protease inhibitor via a trapping mechanism (7). In evolutional terms, a2M is a highly conserved component of innate immunity that is present in a number of animal phyla including humans(8).  Foreign proteases - bacterial, fungal, rickettsial, or viral - are effective virulence factors that enable microbiological invasion, growth, and subsequent disease in the host. a2M limits these foreign proteases by capturing them and eliminating them.  The protease/a2M complex binds to cell surface lipoprotein receptor (CD91), is internalized, and delivered to lysosomes (9).  It has been observed that a2M is consumed early in the course of septic shock with no trend to normalize (10).

PATHOPHYSIOLOGY OF SEPTIC SHOCK: a-2-MACROGLOBULIN HYPOTHESIS

Most, if not all, individuals who suffer septic shock have marked abnormalities of coagulation and fibrinolysis.  This creates a prothrombotic condition within those individuals with subsequent vascular damage and multi-system organ failure.  We posit that the unopposed action of foreign protease activates Hageman Factor (XII) driving the intrinsic cascade and in turn the kallikrein/kinin pathway(11).  This results in almost continuous clotting and continuous bradykinin formation.  As the plasma concentration of a2M decreases, the concentration of foreign protease increases.  By mass action the intrinsic cascade and the kallikrein/kinin systems simply follow Le Châtelier’s principle resulting in relentless clotting (causing vascular damage and multi-system organ failure) and vasodilatation (causing hypotension that is resistant to fluid resuscitation).  The consumption of a2M then explains much of the pathophysiology of septic shock (12, 13). 

TREATMENT OF SEPTIC SHOCK

A close parallel to our hypothesis is acetaminophen poisoning(14).  In this case the accumulation of the highly reactive metabolite N-acetyl-p-benzoquinoneimine depletes glutathione resulting in oxidative hepatocellular injury.  The definitive treatment is to administer N-acetylcysteine, which restores glutathione.  We suggest the same approach to the case of septic shock…to give the patient what he needs and that is a-2-macroglobulin.  Restoration and maintenance of a2M to physiological levels (2 to 4 mg per milliliter) would resolve the septic shock process by removal of foreign protease.  This in turn would allow homeostatic systems to reintroduce equilibrium in the intrinsic cascade and the kallikrein/kinin pathway thus moderating clotting and vasodilation back to more normal levels.  

SEVERAL LINES OF EVIDENCE SUPPORT THE IDEA THAT a-2-MACROGLOBULIN IS ABLE TO RESOLVE THE SEPTIC SHOCK PROCESS

I. Khan, MM et al. (1993): Pseudomonal Elastase Injection Causes Low Vascular Resistant Shock In Guinea Pigs (15).

In the guinea pig model an intravenous injection of culture supernatants derived from elastase (a serine protease) producing Pseudomonas aeruginosa (IFO-3455) strain caused an immediate fall of mean arterial blood pressure from 63.8 mmHg to 35.6 mmHg, increased heart rate from 249.6 beats/min to 272.6 beats/min, and increased respiratory rate from 44.8/min to 68.6/min - this is the picture of shock in the guinea pig model.  In contrast, culture supernatants derived from an non-elastase producing strain of Pseudomonas aeruginosa (PA-103) did not induce any cardiopulmonary changes even though the same concentration of endotoxin was contained in the supernatants used.

Intravenous or intracardiac injection of purified Pseudomonas aeruginosa elastase of 1.2 mg/kg immediately induced those cardiopulmonary changes indicative of guinea pig shock followed by death within 45 minutes.  Administration of endotoxin up to 2.0 mg/kg did not induce shock in the guinea pig.  Pretreatment of animals with anti-pseudomonal elastase rabbit antibody or with a synthetic inhibitor of pseudomonal elastase prevented the ability of pseudomonal elastase to induce shock.

Taken together, these data suggest a major role for microbial protease in the pathophysiology of septic shock.

II.  Khan, MM et al. (1993): Role of Hageman Factor/Kallikrein-Kinin System in Pseudomonal Elastase-Induced Shock Model (11).

Using a guinea pig model intrajugular injection of pseudomonal elastase of 1.2 mg/kg induced lethal shock characterized by hypotension, tachycardia, and respiratory distress.  Elastase induced lethal shock was prevented by pretreating animals with a bradykinin receptor antagonist. 

During elastase induced lethal shock process plasma concentrations of Hageman factor, prekallikrein, kininogen, and albumin were determined by single radial immunodiffusion and functional assays.  Hageman factor was decreased by 45.8%.  High molecular weight kininogen (HMWK) was reduced by 85.2%.  Prekallikrein became undetectable.  Albumin concentration was unchanged. This indicated that Hageman factor, prekallikrein, and kininogen were being consumed.

Hageman factor, prekallikrein, or HMWK each were removed from respective groups of animals by treatment with specific antibodies against each component. Albumin was used as the control as was normal rabbit antibody.  After removal of any one of Hageman factor, prekallikrein, or HMWK animals showed no response to a lethal dose of pseudomonal elastase.  Control animals all developed fatal shock.

Incubation of pseudomonal elastase in normal human plasma resulted in the generation of a large amount of bradykinin.  This bradykinin release was not detected when saline was used instead of pseudomonal elastase. 

Taken together, these data suggest that microbial protease precipitates shock through the activation of the Hageman factor/kallikrein-kinin pathway. 

III.  Khan, MM et al. (1994): Alpha-2-Macroglobulin as the Major Defense in Acute Pseudomonal Septic Shock in the Guinea Pig Model (13). 

The inhibitory capacity of guinea pig a2M and human a2M against pseudomonal elastase was determined by measuring the elastase hydrolytic activity after incubation in either normal guinea pig plasma or normal human plasma or in a2M depleted plasmas.  Four concentrations of elastase were used: 10 mg/ml, 20 mg/ml, 30 mg/ml, and 60 mg/ml.  In the case of human plasma the inhibition of elastase was essentially complete at the 10 mg/ml concentration with almost 100% loss of elastase activity.  At 60 mg/ml concentration there was 68.7% loss of elastase activity.  In marked contrast a2M depleted plasma yielded the same elastase activity as elastase treated with saline. 

Blood samples from guinea pigs taken before and during pseudomonal elastase induced shock showed that the concentration of a2M was reduced 69.4%.  Albumin levels were unchanged.  The loss of a2M was likely secondary to the formation of a2M/elastase complexes with subsequent removal. 

Treatment of animals with anti-a2M monoclonal antibody prior to administration of pseudomonal elastase resulted in inducing fatal shock when only 0.05 mg/kg of elastase was injected - some died with as little as 0.025 mg/kg of elastase.  Control animals showed no cardiovascular perturbations.  When pseudomonal elastase was pretreated with a synthetic inhibitor and then injected into a2M depleted animals, no cardiovascular perturbations were observed.

Reconstituting a2M depleted guinea pigs with purified human a2M and then challenging these animals with pseudomonal elastase 0.05 mg/kg resulted in no cardiovascular perturbations.  In contrast, reconstituted with heat-activated a2M resulted in lethal shock when challenged with elastase at 0.3 mg/kg.  It is noteworthy that when normal guinea pigs were given purified guinea pig a2M to raise total a2M concentration to 150%, an injection of a lethal dose of 1.2 mg/kg pseudomonal elastase caused only a mild hypotension that resolved.

Double depletion of Hageman factor and a2M in guinea pigs then injected with pseudomonal elastase up to 0.2 mg/kg did not develop shock.  In animal controls normal rabbit antibody was infused instead of anti-Hageman factor antibody.  These control animals were also depleted of a2M.  These guinea pigs developed lethal shock when only 0.025 mg/kg of pseudomonal elastase was injected.

Culture supernatants derived from elastase producing Pseudomonas aeruginosa (IFO-3455) caused lethal shock in all normal guinea pigs given intravenous injections of 2 ml supernatant (elastase dose 0.73 mg/kg).  Injection of 0.3 ml of supernatant (elastase dose 0.10 mg/kg) resulted in no cardiopulmonary changes.  In those guinea pigs depleted of a2M the animals immediately developed lethal shock at a dose of 0.3 ml of supernatant and a dose of 0.02 ml (elastase dose 0.0073 mg/kg) was enough to cause immediate hypotension in those a2M depleted guinea pigs and death within one hour.  Reconstitution of guinea pigs depleted of a2M with purified human a2M and then injected with 0.3 ml of supernatant resulted in no cardiopulmonary changes.  Further, culture supernatant pretreated with synthetic elastase inhibitor and then injected into a2M depleted guinea pigs did not cause any cardiopulmonary changes even with 2 ml of supernatant.  In contrast, when the culture supernatants of the non-elastase producing strain of Pseudomonas aeruginosa (PA-103) were injected into animals no cardiopulmonary changes were observed in either normal or a2M depleted guinea pigs.

Taken together, these data suggest that a2M is a major inhibitor of microbial protease and prevents the development of septic shock.

IV.  Khan, MM et al. (1995): Role of a-2-Macroglobulin and Bacterial Elastase in Guinea Pig Pseudomonal Septic Shock (12).

In this study an in vivo sepsis model was used in which bacterial peritonitis was induced by introduction of an elastase producing strain of Pseudomonas aeruginosa (IFO-3455) that in encased within a fibrin/thrombin clot.  With guinea pigs under pentobarbital anesthesia, laparotomies were done and the fibrin/thrombin clots contain 1x109  cfu/kg each were inoculated into the abdominal cavity.  The control was animal inoculation with sterile fibrin/thrombin clot.  All animals recovered from anesthesia in 1 to 2 hours with experimental and control groups indistinguishable in terms of physical condition.  In the case of the IFO-3455 inoculated group all animals became ill 3 to 4 hours post-inoculation and developed lethal shock about one hour before death. 

In those animals receiving IFO-3455 their plasma a2M levels decreased by 50% one hour post-inoculation and remained at that level until the development of lethal shock when plasma a2M decreased again to 30% of it initial level prior to inoculation [note: interestingly, a similar decrease in a2M with persistence has been observed in human patients with septic shock(16)].  At 4 hours post-inoculation a2M was depleted from circulation by injecting anti-a2M antibody in a subgroup of ill animals.  All of these animals immediately developed severe hypotension, tachycardia, and respiratory distress and died within one hour.  Injection of normal rabbit antibody did not induce immediate changes in cardiopulmonary status. 

At 4 hours post-inoculation a synthetic inhibitor of elastase was injected intravenously into another subgroup of ill animals.  After 5 minutes the circulating a2M was depleted by specific antibody.  Lethal shock did not immediately develop and these guinea pigs survived as long as the non-a2M-depleted animals.  Further, when the solvent of the synthetic elastase inhibitor was administered prior to depletion of a2M, the animals immediately developed shock and died within one hour.

To investigate the role of Hageman factor in the septic shock process the effect of depleting Hageman factor and a2M was examined.  At time 3 ½ hours post-inoculation circulating Hageman factor was removed by infusion of anti-guinea pig Hageman factor goat antibody.  At time 4 hours post-inoculation the circulating a2M was removed by specific antibody.  Only a mild and reversible hypotension developed in these double depleted animals.  These animals survived 1 to 2 hours longer than the non-depleted individuals.  As a control Fab of normal IgG was infused into another group of ill animals.  With subsequent depletion of a2M in those control animals resulted in immediate shock and death in 30 minutes. 

The intrajugular injection of purified human a2M into a2M depleted guinea pigs at the onset of severe hypotension (about 8 minutes after a2M depletion) resulted in full recovery from the hypotensive crisis.  These animals then survived as long as the non-a2M-depleted animals.  The use of heat-inactivated human a2M or saline had no beneficial effect and all these animals died within one hour.

Taken together, these data suggest that a2M is the major innate immune factor against foreign protease, that foreign protease activation of the Hageman factor/kallikrein-kinin cascade is the driver of septic shock process, and that exogenous a2M is the definitive treatment for septic shock.

V.  Khan, MM (unpublished data): Plasma a2M levels are decreased in adults with HIV infection.

There is evidence that the lack of a2M may facilitate entry of HIV into CD4 expressing cells. 

REFERENCES

1.             Marik P, Lipman J. The definition of septic shock: Implications for treatment. Critical Care and Resuscitation. 2007;9(1):101-3.

2.             Mitchell R. Hemodynamic disorders, thrombolic disease, and shock. In: Kumar V, Abbas A, Fausto N, Aster J, editors. Robbins and Cotren Pathological Basis of Disease. 8th ed. Philadelphia: Saunders/Elsevier; 2010. p. 111-34.

3.             Brower R, Rock P, Maughan W, Sylvester J. Shock. In: Harvey A, Johns R, McKusick V, Owens A, Ross R, editors. The Principles and Practice of Medicine. 22nd ed. Norwalk, CT: Appleton & Lange; 1988. p. 226-34.

4.             Bone R. The pathogenesis of sepsis. Annals of Internal Medicine. 1991;115(6):457-69.

5.             Kidokoro A, Iba T, Fukunaga M, Yagi Y. Alterations in coagulation and fibrinolysis during sepsis. Shock. 1996;5(3):223-8.

6.             Vervloet M, Thijs L, Hack C. Derangements of coagulation and fibrinolysis in critically ill patients with sepsis and septic shock. Seminars in Thrombosis and Hemostasis. 1998;24(1):33-44.

7.             Feldman S, Gonias S, Pizzo S. Model of alpha2-macroglobulin structure and function. Proceedings of the National Academy of Sciences of the USA. 1985;82:5700-4.

8.             Armstrong P. Proteases and protease inhibitors: A balance of activities in host-pathogen interaction. Immunobiology. 2006;211(4):263-81.

9.             Borth W. Alpha-2-macroglobulin, a multifactorial binding protein with targeting characteristics. FASEB Journal. 1992;6(15):3345-53.

10.          Witte J, Jochum M, Scherer R, Schramm W, Hochstrasser K, Frotz H. Disturbances of selected plasma proteins in hyperdynamic septic shock. Intensive Care Medicine. 1982;8(5):215-22.

11.          Khan M, Yamamoto T, Araki H, Shibuya Y, Kambara T. Role of hageman factor/kallikrein-kinin system in pseudomonal elastase-induced shock model. Biochimica et Biophysica Acta. 1993;1157:119-26.

12.          Khan M, Shibuya Y, Kambara T, Yamamoto T. Role of alpha-2-macroglobulin and bacterial elastase in guinea-pig pseudomonal septic shock. International Journal of Experimental Pathology. 1995;76:21-8.

13.          Khan M, Shibuya Y, Nakagaki T, Kambara T, Yamamoto T. Alpha-2-macroglobulin as the major defense in acute pseudomonal septic shock in the guinea-pig model. International Journal of Experimental Pathology. 1994;75:285-93.

14.          Marzullo L. An update of N-acetylcysteine treatment for acute acetaminophen toxicity in children. Current Opinion in Pediatrics. 2005;17(2):239-45.

15.          Khan M, Yamamoto T, Araki H, Ijiri Y, Shibuya Y, Okamoto M, et al. Pseudomonal elastase injection causes low vascular resistant shock in guinea pigs. Biochimica et Biophysica Acta. 1993;1182(1):83-93.

16.          Fritz H. Protease inhibitors in severe inflammatory processes (septic shock and experimental endotoxaemia): Biochemical, pathophysiological and therapeutic aspects. Ciba Foundation Symposium. 1979;75:351-79.

 Submitted to Rare Diseases India on August 31, 2012.

Book on this subject:

A2M-Miracle Protein, the lifesaver By Mohammad Khan MBBS PhD

Publication Date: January 15, 2017

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Pages: 51

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