Higher levels of plasmin enhances the virulence and infectivity of SARS-CoV-2 by cleaving its spike protein

A new review suggests that higher-than-normal levels of an enzyme involved in blood clot prevention may be a common risk factor for developing COVID-19 – a respiratory disease caused by the novel coronavirus SARS-CoV-2 – in some populations.

People with diabetes, high blood pressure and heart, lung or kidney disease have a higher risk of developing COVID-19.

In addition, people with preexisting medical conditions typically become sicker when infected with SARS-CoV-2 than those in otherwise good health.

Research has found that one of the leading causes of death from COVID-19 is hemorrhage or bleeding disorders and that one of the characteristics of the disease is overactivity of the system responsible for removing blood clots (hyperfibrinolysis).

Elevated levels of plasminogen and plasmin have been found to be a common factor in people with diabetes and preexisting heart, lung and kidney conditions.

Plasminogen is an inactive substance in the blood. When substances in the cells of the blood vessels activate plasminogen, it generates plasmin, an enzyme that removes blood clots from the blood.

Higher-than-normal levels of both of these chemicals can lead to severe bleeding.

Studies report that more than 97% of people hospitalized with COVID-19 have increased levels of D-dimer, a protein in the blood that is produced when a blood clot dissolves.

D-dimer levels are associated with the amount of virus detected in the body and continue to rise as the severity of COVID-19 increases.

This is particularly true in people who develop the often-fatal complication of acute respiratory distress syndrome (ARDS). “In contrast, D-dimer levels decreased to control levels in [COVID-19] survivors or non-ARDS patients,” the review’s authors wrote.

“The time [period] for the elevated D-dimer [to go] down in mild [cases] or survivors is dependent.

Generally, it takes at least one week for mild [cases] but longer for severe patients,” explained Hong-Long Ji, MD, PhD, corresponding author of the review.

“Measurements of plasmin(ogen) levels and its enzymatic activity may be important biomarkers of disease severity” in people with COVID-19, the authors wrote.

In addition, treating hyperfibrinolysis “may prove to be a promising strategy for improving the clinical outcomes of patients with [additional medical] conditions,” they added.

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Plasminogen improves lung lesions of clinically moderate COVID-19 patients
To study the effects of plasminogen on lung lesions, high-resolution CT scanning of chest was performed on COVID-19 patients. Before administration of plasminogen, the chest CT images of 5 moderate COVID-19 patients showed multiple patchy/punctate ‘ground-glass’ opacities in bilateral lungs with unclear margins and heterogeneous density, and also scattered punctate density in mediastinum (Fig. 1 and Table 2).

Despite the 5 patients received the daily common supportive treatments with antibiotics and traditional Chinese medicine, the density and range of ‘ground glass’ opacities in lung increased over time, suggesting a rapid deleterious progress of the disease (CT scanning examination record, not shown here).

Surprisingly, just after 2-3 times of plasminogen inhalation, the CT results showed that the numbers, the range and the density of lung lesion-focal in all of the 5 patients treated have diminished or even partially disappeared.

Patchy or punctate ‘ground glass’ opacities have significantly decreased or absorbed. This indicated that atomization inhalation of plasminogen rapidly improves lung lesions caused by COVID-19 infection.

Plasminogen improves oxygen saturation of clinically severe and critical COVID-19 patients We further investigated the effects of plasminogen on oxygen saturation. Throughout the entire plasminogen administration period, the oxygen saturation levels were detected by patient monitors on clinically severe or critical COVID-19 patients with oxygen supplementation by a nasal cannula that delivered oxygen at constant concentrations of at least 80% or 100%, respectively.

As shown in Table 3, although generally having good oxygen saturation levels, the oxygen saturation values of 5 out of 6 severe COVID-19 patients still increased 1-4% after plasminogen administration.

Only in one patients, perhaps due to administration of plasminogen for just once, the oxygen saturation value decreased from 91% to 89%. In these 2 patients with critical conditions, the oxygen levels have significantly increased from 79-82% to 91% just about 1 hour after the first inhalation and stabilized at around 91% thereafter (Detailed data not shown here). However, due to the critical conditions of these two patients, they were transferred to intensive care unit, so that the follow-up treatment of plasminogen was interrupted.

Nevertheless, these data indicate that plasminogen generally improves the oxygen saturation levels of severe and critically severe COVID-19 patients, especially in these patients that have particularly low oxygen levels.

Plasminogen improves heart rates of COVID-19 patients All of the COVID-19 patients also underwent heart rate examination during the study. The results showed that whereas before inhalation of plasminogen, these patients generally showed higher heart rates.

After treatment, in 8 out of 13 COVID-19 patients the heart rates had slowed down as much as 26 beats per min, 2 increased and 3 no changes. Statistical analysis showed that despite patient samples are sparse, the average heart rates after treatment in general have decreased in all of the three groups and for the moderate group, the statistics reached significance (P<0.05) (in Table 4 and Fig 2).

These results indicate that plasminogen can generally slow down the high beating rates and alleviate the burden of hearts of COVID-19 patients.

Tables

Table 1: Treatment profiles of plasminogen for COVID-19 patients

Patient ID	Gender	Age (years old)	Severity of Disease	Inhalation times of plasminogen
1	male	58	moderate	2
2	male	30	moderate	2
3	female	49	moderate	2
4	female	48	moderate	3
5	male	48	moderate	2
6	male	46	severe	6
7	male	78	severe	6
8	male	47	severe	2
9	male	65	severe	2
10	female	56	severe	4
11	male	78	severe	1
12	male	48	critical	5
13	male	47	critical	2

Table 2 High-resolution chest CT scanning record in moderate COVID-19 patients

Patient ID	CT results before the first inhalation of plasminogen	CT results after the last inhalation of plasminogen
1	Multiple patchy/punctate ‘ground-glass’ opacities in bilateral lungs characterized with unclear margin and heterogeneous density, especially consolidation at lower lobe of right lung	Number and range of lesion-focal decreased and partially disappeared
2	Multiple ‘ground-glass’ appearance in bilateral lungs characterized with unclear margin and heterogeneous density, left consolidation at lower lobe	‘Ground-glass’ appearance in lower lobes of both lungs, flaky denser area in left lower lobe decreased and partially absorbed
3	Multiple patchy/punctate ‘ground-glass’ opacities in bilateral lungs characterized with unclear margin and heterogeneous density, mild consolidation at right lower lobe	‘Cloud dense’ area in bilateral lungs, number and area of lesion-focal decreased and almost all absorbed
4	Multiple ‘ground-glass’ appearance in bilateral lungs characterized with unclear margin and heterogeneous density, consolidation at lower lobes of bilateral lungs	Smaller Patchy/punctate ‘ground-glass’ opacities in lower lobes of both lung, mostly absorbed
5	Multiple ‘ground-glass’ appearance in bilateral lungs characterized with unclear margin and heterogeneous density, consolidation at right middle and lower lobes	Patchy/punctate ‘ground-glass’ opacities significantly decreased in right lower lobes

Table 3 Oxygen saturation values of severe and critical COVID-19 patients

Patient ID	Severity of Disease	Oxygen saturation values before the   first inhalation of plasminogen (%)	Oxygen saturation values after the   last inhalation of plasminogen (%)
6	severe	93	97
7	severe	95	96
8	severe	93	95
9	severe	93	96
10	severe	91	95
11	severe	91	89
12	critical	79	91
13	critical	82	91

Table 4 Heart rate of COVID-19 patients

Patient ID	Severity of Disease	Heart rate before the first inhalation of   plasminogen (beats per min)	Heart rate after the last inhalation of   plasminogen (beats per min)
1	moderate	92	76
2	moderate	84-104	80
3	moderate	82	82
4	moderate	82	82
5	moderate	84	76
6	severe	98	72
7	severe	81	70
8	severe	73	75
9	severe	73	73-75
10	severe	79	78
11	severe	71	72
12	critical	92	70
13	critical	110	89

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ELEVATED PLASMIN(OGEN) LEVELS IN COMORBID DISEASES OF COVID-19
Plasmin(ogen) in Hypertensive Patients

Plasmin is generated from the cleavage of plasminogen by either urokinase (uPA) or tissue-like plasminogen activator (tPA). In general, uPA is responsible for the plasmin in body fluid (BAL, urine, tear, pleural effusion, etc.), while circulating tPA proteolytically cleaves plasminogen in the plasma.

The most frequent comorbidity observed in COVID-19 patients is hypertension followed by diabetes, chronic cardiovascular conditions, cerebrovascular diseases, COPD, and chronic kidney illnesses (80, 101).

The plasminogen system is a druggable target in renal hypertension (76). Plasmin, a potent protease, cleaves up to 16 sites, including the cleavage sites for trypsin, chymotrypsin, prostasin, and elastases, of the human γ ENaC subunit (99).

Elevated renal plasmin results in hypertension by cleaving ENaC in the collecting tubule, which increases salt retention, causing expanded circulating volume. Urinary excretion of plasmin(ogen) and urokinase directly correlates with urine albumin in hypertensive subjects (2). On the other hand, amiloride, an inhibitor of ENaC, lowers blood pressure and urine plasminogen excretion (61).

ENaC proteins are located in the apical membranes of tight epithelia, and they are the major pathways for the entry of Na+. As such, they play an important role in maintaining the proper depth of airway and alveolar lining fluids, the reabsorption of edema fluid in injured lungs, and the regulation of salt retention in the collecting tubules (6, 14, 18, 28, 38, 39, 50, 64).

Proteolysis is an important regulatory mechanism of ENaC function (36, 63, 65, 67, 70, 99). Decreased ENaC function will result in increased fluid in body cavities (e.g., lung edema in the airspaces and increased blood volume), while increased function will cause dehydration of luminal fluid, as it occurs in cystic fibrosis (CF) and most likely dry eye syndrome.

Plasmin(ogen) in Cardiovascular Diseases

Significantly higher levels of urinary plasminogen and plasmin are reported in rats (71) and patients with chronic heart failure (76, 100). In addition, plasmin activity in patients with coronary artery disease is 1.7-fold greater compared with healthy subjects (16).

Plasmin(ogen) in Diabetes

Both types I and II diabetes are associated with higher plasmin(ogen) levels in plasma. A 25-year prospective study of type I diabetes documented an association with increased urinary plasmin(ogen), particularly in hypertensive subjects (69).

Concentrations of plasmin(ogen) in urine are correlated with the development of preeclampsia late in pregnancy (58). Aberrant plasmin in preurine may inappropriately activate ENaC in patients with type II diabetes and microalbuminuria (7).

Individuals with high plasma furin concentration have a pronounced dysmetabolic phenotype and elevated risk of diabetes mellitus and premature mortality (19).

Plasmin(ogen) in Other Comorbid Diseases

Higher levels of plasmin(ogen) are detected in the urine of various cancer patients as compared with healthy individuals (9). Elevated urine plasmin(ogen) levels, accompanied by increased exosomal α ENaC fragments, have been detected in pregnant women (59), a population susceptible to influenza (22).

Endogenous channel-activating proteases, as well as proteases released by inflammatory cells (trypsin, elastase), activate ENaC either by cleaving critical amino acids in α and γ ENaC subunits, or by activating signaling pathways (66, 72).

Aprotinin, a potent and reversible Kunitz-type inhibitor of several serine proteases, including trypsin, plasmin, and kallikreins, has been reported to inhibit sodium transport among a variety of epithelial cells (66). Other Kunitz-type serine protease inhibitors, such as hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2 (placental bikunin), have also been demonstrated to inhibit prostasin and ENaC activity (77).

Finally, a1-antitrypsin, an acute-phase glycoprotein and a member of the serine protease inhibitor (SERPIN) superfamily, inhibits ENaC in vitro and in vivo by decreasing protease activity (41). Of note, SARS S protein inhibits ENaC via the protein kinase C signaling pathway (35). It is worth noting that high plasmin levels may contribute to the development of comorbid bacteremia and sepsis (26, 74).

Interestingly, azithromycin, a common antibiotic for suppressing infection in the CF airways when combined with hydroxychloroquine, turns COVID-19 to SARS-CoV-2 negative in 5 days (21).

PLASMIN(OGEN) IN ARDS

Plasmin(ogen) Is Increased in ARDS

ARDS is a life-threatening disorder associated with respiratory and systemic infections, trauma, burns, inhalation of toxic gases, and aspiration of gastric contents injury (52). In addition to lung injury, patients with ARDS may develop MOF with a hallmark of excess D-dimer and other FDPs presenting in both BAL and blood biopsies.

Soluble D-dimer and D-monomer are predominately produced from the proteolytic cleavage of cross-linked fibrin and fibrinogen/non-cross-linked fibrin, respectively, by plasmin (fibrinolysis). Plasmin activity in BAL is detected in healthy subjects (71). Both D-dimer and D-monomer levels are significantly increased up to 17-fold in undiluted edema fluid in patients with ARDS (68).

Significant increase in both plasminogen and cleaved plasmin protein in the BAL of ARDS patients (32) and an animal model of DAD (33) have been reported. Augmented plasmin activity contributes to elevated D-dimer in the BAL of infected lungs in a time-dependent manner during the development of ARDS (20, 87).

This is further validated by the observation that plasmin-mediated fibrinolytic activity could be inhibited by 50% with α2-antiplasmin antibody (32). Kallikrein and neutrophil elastase may contribute to the residual proteolytic activity in BAL of ARDS (53). Also, radiation-induced lung injury in mesothelioma patients is accompanied by a significant elevation in BAL plasminogen and plasmin-associated fibrinolytic activity (49).

Fibrinolysis in COVID-19

In comparison with patients with mild COVID-19 (such as those who did not require ICU stays, did not develop ARDS or pneumonia, and who survived), patients with severe COVID-19 have higher comorbidities, including 56% for hypertension, 21% for heart diseases, 18% for diabetes, 12% for cerebrovascular diseases, and 7% for cancer (TABLE 1) (79, 97).

Some patients have more than one, even up to five preexisting conditions. Multivariate regression further links hypertension with increased incidence and fatality (85, 89, 101). Hyperfibrinolysis, reflected by elevated serum D-dimer levels, was present in 97% of COVID-19 patients at admission and increased further in all patients before death (TABLE 2) (97).

FDPs were significantly increased as well (79). This is accompanied by a prolonged prothrombin time particularly in non-survivors (31, 79, 97, 101). Platelet counts were decreased significantly in severe and dead patients (79, 97, 101). 71.4% of non-survivors meet the criteria of the International Society on Thrombosis and Hemostasis (ISTH) for DIC, suggesting the coexistence of coagulation activation and hyperfibrinolysis in patients with severe COVID-19 infection (78, 85). In contrast, D-dimer levels decreased to control levels in survivors or non-ARDS patients.

Table 1. Comorbidities of COVID-19 patients

	Epidemiology (death%) (n = 72,314)	Severe/non-severe Pneumonia (n = 38/72)	Severe/non-severe (n = 173/926)	ICU/non-ICU (n = 13/28)	ARDS/non-ARDS (n = 53/56)	Non-survivor/survivor (n = 54/137)	Non-survivor/survivor (n = 32/20)	Non-survivor (n = 82)
Hypertension	12.8/6	39.47/29.17c	23.7/13.4	15/14	39.6/28.6	48/23%c		56.1
Diabetes	5.3/7.3	21.05/9.72	16.2/5.7	8/25	20.8/1.8b	31/14b	22/10	18.3
Coronary heart diseases	4.2/10.5		5.8/1.8	23/11	5.7/7.1	24/1d	9/10	20.7
Cerebrovascular diseases		7.89/5.56	2.3/1.2		11.3/0b		22/0	12.2
COPD	2.4/6.3	10.53/2.78a	3.5/0.6	8/0	3.8/3.6	7/1a	6/10	14.6
Kidney diseases			1.7/0.5		15.1/3.6a	4/0a		4.9
Liver diseases			0.6/2.4	0/1		—		2.4
Cancer	0.5/5.6		1.7/0.8	0/1		0/1	3/5	7.3
Secondary infection			0.6/2.4					6.1
Immunodeficiency			0/0.2					17.1
Others						20/8a		3.7 (surgery)
Total	26/−		38.7/21	38/29		67/40c		76.8
Reference no.	80	85	24	31	45	101	95	97

ARDS, acute respiratory distress syndrome; COPD, chronic obstructive pulmonary disease; ICU, intensive care unit.

^aP < 0.05, – ^bP < 0.01, – ^cP < 0.001, – ^dP < 0.0001.

Table 2. Coagulation and fibrinolysis in patients with COVID-19

	Severe/non-severe (n = 173/926)	ARDS/non-ARDS (n = 53/56)	ICU/non-ICU (n = 13/28)	Severe/non-severe Pneumonia (n = 38/72)	Non-survivor/survivor (n = 54/137)	Non-survivor/survivor (n = 21/162)	Non-survivor/survivor (n = 32/20)	Non-survivor (n = 82)
D-dimer, >1 mg/L	59.6/43.2 (≥0.5 mg/L)	940/370c	2.4/0.5b	1.11/0.37c	5.2/0.6d	2.12/0.61c, 100/		97.5–100 (>0.55 mg/L)
FDP, mg/L						7.6/4.0c
Fibrinogen, g/L		3.4/2.9c				5.16/4.51, 28.6 (<1 g/L)		—
Platelets, <100 × 109/L	57.7/31.6 (<150,000/mm3)		8/4	144.5/179.5c (109/L)	20%/1%d	57.1/	191/164	63.2
Prothrombin time, ≥16 s			12.2/10.7a		13/3a	15.5/13.6c		100 (>12.1)
Antithrombin activity						84/91	12.9/10.9
APTT, s			26.2/27.7			44.8/41.2
ISTH DIC criteria						71.4/-
Reference no.	24	45	31	85	101	79	95	97

APTT, activated partial thromboplastin time; ARDS, acute respiratory distress syndrome; DIC, disseminated intravascular coagulation; FDP, fibrin degradation products; ICU, intensive care unit; ISTH, International Society on Thrombosis and Hemostasis.

^aP < 0.05, – ^bP < 0.01, – ^cP < 0.001,

The mortality rate of patients with COVID-19 who did not develop ARDS is 9 versus 49% for those who did develop ARDS (45). Of note, ARDS/respiratory failure remains the leading cause of death (70%), followed by sepsis/MOF (28%), heart failure (15%), hemorrhage (6%), and renal failure (4%) (TABLE 3).

Coagulation/hemorrhage ranks among the top three leading causes of death (97). Furthermore, multivariate regression analysis identifies D-dimer and age as independent risk factors for mortality (TABLE 4) (85, 89, 101). These findings suggest that the normalization of hyperactive fibrinolysis may be a therapeutic target.

Table 3. Outcomes or complications (%) in patients with COVID-19

Severe/non-severe (n = 173/926)	ICU/non-ICU (n = 13/28)	Non-survivor/survivor (n = 54/137)	Non-survivor/survivor (n = 32/20)	Injured Organs (n = 82)	Death Cause (n = 82)
Sepsis/MOF			100/42d	3/0	28.0e
Respiratory failure			98/36d			100
ARDS	16.5/1.1	85/4d	93/7d	81/45	69.5e
Septic shock	6.4/0.1	23/0a	70/0d
Acute cardiac injury		31/4a	59/1d	28/15		89.0
Heart failure			52/12d		14.6e
Coagulopathy/hemorrhage			50/7d		6.1e	80.5
Acute kidney injury	2.9/0.1	23/0a	50/1d	37.5/15		31.7
Secondary infection		31/0a	50/1d	9/20
Hypoproteinemia			37/1d
Acidosis			30/1d		2.4e
Renal failure	0.6/0				3.7e
Liver failure					1.2e	78.0
GI failure					2.4e	6.1
Reference no.	24	31	101	77	97	97

ARDS, acute respiratory distress syndrome; GI, gastrointestinal; ICU, intensive care unit; MOF, multiple organ failure.

^aP < 0.05, – ^bP < 0.01, – ^cP < 0.001, – ^dP < 0.0001. – ^ePercent of contribution to death.

Table 4. Risk factors of COVID-19 associated with mortality computed with multivariate logistic regression

	Non-survivor/survivor (OR, n = 54/137)	Severe/non-severe Pneumonia (OR, n = 38/72)	ARDS (HR, n = 201)
Age	1.10 (1.03, 1.17)b	25.314 (1.628, 92.664)c >60 yr	6.17 (3.26, 11.67)c
Lymphocyte	0.19 (0.01, 1.62)	0.322 (0.137, 0.756)b	0.51 (0.22, 1.17)
D-dimer	18.42 (2.64, 128.55)b	17.054 (2.547, 114.171)b	1.02 (1.01, 1.04)b
Reference no.	101	85	89

ARDS, acute respiratory distress syndrome; HR, hazard ratio; OR, odd ratio.

^aP < 0.05, – ^bP < 0.01, – ^cP < 0.001.

Uncoordinated Coexistence of Hypercoagulation and Hyperproteolysis
The specific plasmin inhibitor α2-antiplasmin is elevated by approximately one order of magnitude in patients with ARDS, while fibrinolytic activity is reduced approximately by half, and D-dimer is elevated 50-fold in BAL (25).

The nonproportional change between the expression and activity level of plasmin(ogen) and anti-plasmin indicates the stoichiometry of the plasmin-antiplasmin complexes may not be in a ratio of 1:1.

Increased levels of α2-antiplasmin and other antiproteases may not completely shield the proteolytic triad of plasmin in the complexes, suggesting that either their efficacy is inadequate or that plasmin is still able to cut fibrin to produce D-dimers and FDPs in ARDS patients.

The soluble complexes of the plasmin-antiplasmin in BAL may facilitate physical interactions with the vast deposition of fibrin at the luminal surface of alveoli. Based on the pathology and laboratory results, dynamic hypercoagulation occurs as evidenced by microthrombi throughout the blood vessels of multiple organs, accompanied with extremely reduced platelets in COVID-19 patients (TABLE 2).

On the other hand, hemorrhage and markedly elevated degraded fibrin products result from plasmin-associated hyperproteolysis. Whether patchy hemorrhage coexists with areas infected by SARS-CoV-2 is not known. Administration of anti-proteases may prove beneficial.

CLINICAL RELEVANCE AND PERSPECTIVE
The cleavage of the new furin sites in the S protein of SARS-CoV-2 virus by plasmin and other proteases may enhance its infectivity by expediting entry, fusion, duplication, and release in respiratory cells. Elevated plasmin(ogen) levels are a common feature in COVID-19 patients with underlying medical conditions.

The elevated plasmin(ogen) could be an independent factor for risk stratification of patients with COVID-19. Measurements of plasmin(ogen) levels and its enzymatic activity may be important biomarkers of disease severity in addition to resultant D-dimer. The administration of antiproteases to suppress plasmin activity in the respiratory system may prevent, or at least decrease, SARS-CoV-2 entry into respiratory cells and improve the clinical outcome of patients with COVID-19.

As demonstrated in vitro, a serine protease inhibitor for TMPRSS2 blocks SARS-CoV-2 S protein-driven entry into cells (30). Clinical trials conducted in China are testing various protease inhibitors (29). Currently there are no proper animal models of COVID-19 with underlying medical conditions to test new therapeutic agents. Healthy mice and monkeys infected with SARS-CoV-2 develop either mild lung injury or show no symptoms of disease (8).

It remains to be seen whether mice and monkeys with preexisting comorbid conditions and higher plasmin levels develop COVID-19 when infected with SARS-CoV-2. Targeting hyperfibrinolysis with a broad spectrum or specific anti-plasmin compounds may prove to be a promising strategy for improving the clinical outcome of patients with comorbid conditions.

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Source:
American Physiological Society