Enoxaparin augments alpha-1-antitrypsin inhibition of TMPRSS2, a promising drug combination against COVID-19

We found that enoxaparin, a low-molecular weight heparin, augments the ability of alpha-1-antitrypsin (a serine protease inhibitor = serpin) to inhibit TMPRSS2, the serine protease that is required to process the spike protein of coronaviruses, a necessary step for viral entry into cells.

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Pneumonia with refractory hypoxemia and septic shock is the most common cause of death in severe COVID-19 1. A key injury mechanism in those with severe COVID-19 is a delayed hyperinflammatory immune response – in which the pro-inflammatory cytokine interleukin-6 (IL-6) plays a prominent role. A number of investigational and repurposed agents have been researched against severe COVID-19 2. Only a few of these efforts have achieved success and most are modest at best. The glucocorticoid dexamethasone has shown mortality benefit for severe COVID-19 – with an absolute reduction in mortality of 3.5% to 11.7% in those with severe COVID-19 3. However, dexamethasone use in mild cases is associated with a trend toward worse outcome, perhaps due to the likelihood that an early pro-inflammatory response is necessary for viral clearance 3,4. Indeed, others do not recommend glucocorticoid use in COVID-19 5. Antagonists to IL-6 and the IL-6 receptor have shown either negative results or benefit in some patients recalcitrant to dexamethasone 6. The FDA has also issued a Black Box warning for JAK inhibitors due to risks of serious cardiac events and cancer. Monoclonal antibodies and anti-viral agents are used in those with mild disease to prevent progression but have no current indication for those with severe COVID-19. Furthermore, two leading monoclonal antibodies are now powerless against the Omicron SARS-CoV-2 variant. Thus, finding a better remedy for patients with severe COVID-19 is an unmet need.


Epidemiologic studies indirectly support the paradigm that alpha-1-antitrypsin (AAT), a prominent serum protein with activity against infection and inflammation 7, antagonizes SARS-CoV-2 infection. COVID-19 cases are increased in areas of Italy with an increased prevalence of AAT deficiency 8. AAT deficient subjects were 8.8-fold more likely to have symptomatic COVID-19 than the general Italian population 9. Shapira and colleagues 10 found a significant direct correlation between the frequency of the mutant AAT alleles, protease inhibitor (Pi)Z and PiS, with COVID-19 death rates in 67 countries. Yoshikura 11 reported a robust correlation between the Pi*Z variant and the number of COVID-19 cases (correlation coefficient (CC)=0.8584) and deaths (CC=0.8713) in 68 countries. McElvaney and co-workers 12 found that the IL-6:AAT ratio is markedly elevated in critically ill patients with COVID-19 compared with healthy volunteers or stable hospitalized COVID-19 patients; this ratio also directly correlated with prolonged hospital stay and mortality.


The cell surface serine protease Transmembrane Protease 2 (TMPRSS2) is required to cleave the spike protein of SARS-CoV-2 for viral entry into cells. Negatively-charged polysaccharides like heparin augment serpin activity by introducing a more favorable electrostatic interaction between the serpin and its protease, creating a more stable ternary complex of polysaccharide–protease–serpin to allow greater interaction beween the protease and its cognate serpin, resulting in greater inhibition of the former by the latter. Thus, we determined whether negatively-charged heparins, unfractionated heparin (UFH), enoxaparin, and nadroparin – the latter two being low molecular fractions of UFH – augment AAT inhibition of TMPRSS2. Enoxaparin significantly enhanced AAT inhibition of both TMPRSS2 activity and infection of human airway epithelial cells (hAEc) with the human coronavirus 229E (HCoV-229E). These biochemical and biological synergies are supported by detailed molecular modeling. These structural analyses revealed that: (i) the reactive center loop of AAT adopts an inhibitory-competent conformation compared with the crystal structure of TMPRSS2 bound to an exogenous (nafamostat) or endogenous (HAI-2) TMPRSS2 inhibitor and (ii) negatively-charged heparin bridges adjacent electropositive patches at the TMPRSS2–AAT interface, neutralizing otherwise repulsive forces and thus enhancing their interaction and inhibition of viral infection.


Confirmation and translation of our work will provide the foundation for future clinical studies of AAT + enoxaparin treatment in individuals severely ill with COVD-19. Moreover, since AAT: (i) levels could be low (due to inherited AAT deficiency); (ii) levels may be inadequately increased in response to an infection (in those with heterozygous AAT alleles); or (iii) may be oxidized leading to the loss of its serpin activity (due to high oxidative stress seen with severe COVID-19), we posit that this intervention will be beneficial, regardless of the basal AAT levels, in those with recalcitrant severe COVID-19.


In conclusion, based on our biochemical, virological, and Artificial Intelligence-based molecular modeling findings, enoxaparin synergizes with AAT to inhibit TMPRSS2 and reduce coronavirus burden in primary human airway epithelial cells. This combined viral-inhibitory activity of these two agents hold promise to improve the outcomes of those with severe COVID-19. Such host-directed therapy is less likely to be affected by SARS-CoV-2 mutations since neither agents are directly targeting the spike protein of the virus. Furthermore, given the known anti-inflammatory activities of both AAT and heparin, this form of treatment may target both the virus and the excessive inflammatory consequences of severe COVID-19.



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Ed Chan

Physician, National Jewish Health