Inherited single amino acid substitutions are an important source of potential phenotypic variation between individuals that can lead to disease risk (1, 2) and can contribute to complex multifactorial disorders (3). About one-half of known genetic conditions are caused by nonsynonymous single nucleotide polymorphisms (nsSNPs) (3, 4). Single amino acid mutational studies are limited but naturally-occurring missense variants with associated phenotypes can provide very valuable information for analysis of structure-function relationships of proteins. Techniques such as targeted exome sequencing play key roles in discovering alleles associated with Mendelian and complex disorders.

A comprehensive review of disease-associated PLG missense variants in world populations is highly relevant to more fully comprehend their overall causative influences on coagulopathies and inflammatory diseases and the involvement of the fibrinolytic system in these processes. In this review, we explore the ramifications of naturally occurring variants on the abundant multi-functional soluble plasma protein zymogen, PLG, and its activated product, the serine protease, plasmin. We begin this review with a summary of the background on PLG/plasmin structure-function which is necessary to better understand the mechanisms of the effects of missense variants on the properties of PLG and plasmin.

Human plasminogen is encoded by the PLG gene, which is located on human chromosome 6q26. The PLG DNA contains 19 exons separated by 18 introns and is 51,861 bp in length (http://genome.ucsc.edu/) (5). PLG is translated primarily in the liver (6), along with minor production in extrahepatic cells. The translated protein is a single-chain 810 amino acid protein without enzymatic activity. Upon maturation, the 19-amino acid signal peptide is removed, and two carbohydrate chains are placed on PLG side-chains, Asn²⁸⁹ and Thr³⁴⁶ (Figure 1) (7–9), as well as a phosphorylation site of unknown significance located at Ser⁵⁷⁸ (10).

In numbering PLG residues, the fully translated protein, which includes the 19-residue signal peptide, is frequently used in the literature when referring to genomic data and clinical case reports. For example, the fully translated protein numbering for the codominant allelic variant of PLG is written as p.D⁴⁷²N, while the corresponding mature protein number is PLG/D⁴⁵³N (lacks the signal peptide). In this review, we used the mature protein numbering for all PLG variants in the text. To enhance comparison with data from the literature, the fully translated and the mature protein numbering for PLG variants are stated side-by-side in the Tables.

The mature PLG protein (Glu¹-PLG) is multi-modular, containing consecutively from the amino terminus (Figure 1): a 77-residue activation peptide (AP), followed by five ∼80 residue triply disulfide-linked kringle (K) domains separated by variable length inter-kringle residues; an activation cleavage site (R⁵⁶¹-V⁵⁶²) susceptible to the catalytic cleavage activity of plasminogen activators (PAs), and a light chain homologous to serine proteases, such as trypsin and chymotrypsin. After direct hydrolysis of the R⁵⁶¹-V⁵⁶² peptide bond, as catalyzed by PAs, such as urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), or indirect activation by bacterial activators, e.g., streptokinase (SK) and staphylokinase (Sak), the final protease, plasmin (EC 3.4.21.7), is formed. Plasmin consists of the plasmin/[K⁷⁸-R⁵⁶¹] heavy chain (HC), containing all five kringles, doubly disulfide-linked to the PLG/[V⁵⁶²-N⁷⁹¹] light chain, or serine protease (SP) domain, containing the serine protease catalytic triad, His⁶⁰³-Asp⁶⁴⁶-Ser⁷⁴¹ (11–13). After activation, the resulting plasmin lacks the AP, the removal of which is autocatalyzed by plasmin (14). The HC and the LC are latent in the zymogen (PLG). Also provided in Figure 1 are other derivatives of Glu¹-PLG, which have occasionally been described in the literature, e.g., mini-PLG and micro-PLG (μPLG), but these are proteolytic products of native PLG, or cloned fragments of this protein, and are not further discussed herein. The post-translational product, Lys⁷⁸-PLG, is an important activation intermediate of Glu¹-PLG and will be referred to in this review.

Of essential importance to PLG/plasmin function, are the five kringle domains of the PLG-HC, four of which, viz., K1, K2, K4, and K5, bind to lysine with varying affinities. Figure 2A shows the x-ray crystal structure of the binding of a lysine analog, ε-aminocaproic acid (EACA), to isolated PLG-K1 and the figure highlights the critical lysine binding residues (15). Figure 2B represents a generic 79-residue lysine binding kringle, based on the numbering in PLG-K1. The location of the important lysine binding residues for each of the kringle modules of PLG is summarized in Table 1. Of course, other residues can assist in the stabilization of the ligand, but the residues shown are important for binding in each of the kringle domains.

There are three major centers within the LBS that are essential for the lysine-binding event (Figure 2A). Firstly, an anionic center, formed by two aspartates, Asp⁵⁴ and Asp⁵⁶ (numbering beginning at C¹ of the generic kringle), that coordinate with the amino group side chain of lysine, lysine isosteres, and lysine analogs, such as EACA. Notably, one of these aspartates is replaced by glutamate in PLG-K2 and by lysine in PLG-K3. Secondly, a hydrophobic core center, in which two aromatic amino acids, in this case, Trp⁶¹ and Tyr⁷¹_, form a cluster that stabilizes the central methylene groups of EACA, and lastly, a cationic center, composed of basic residue(s), which interact with the carboxylate group of the ligand. As shown in Figure 2A, Arg⁷⁰ interacts with the COOH group of EACA, while Tyr⁶³ not only supports the hydrophobic core but also forms a hydrogen bond with the COOH group of EACA. Multiple studies indicate that residue-to-residue variations among the kringle domains, highlighted in Table 1, affect their lysine binding affinities.

Additionally, at least for the binding of EACA to K1-PLG, an Arg at position 34 further stabilizes the carboxyl group of the ligand. Phe³⁵ contributes to the hydrophobic cluster that surrounds the backbone of EACA, while the side chains of Tyr⁷¹ and Tyr⁷³ support the anionic center by having interatomic distances that suggest that it can serve as a hydrogen binding partner for EACA and for Asp⁵⁶, respectively, thus stabilizing this latter residue in the lysine binding pocket (Figure 2A). In studies with the isolated kringle domains, PLG-K1 has the highest lysine binding affinity, followed by PLG-K4, PLG-K5 and PLG-K2, while PLG-K3 poorly binds to EACA (16), due to the presence of a lysine (Lys⁵⁶) instead of the acidic amino acid side chain, Asp⁵⁶ (Figure 2B), in its anionic center. Further, PLG-K5 contains Leu⁷⁰, rather than Arg⁷⁰ in its cationic center, a factor that likely governs its weaker binding to EACA (17, 18).

The lysine binding sites (LBS) of kringle domains are critical for the functional properties of PLG and allow PLG and plasmin to bind to cellular receptors utilizing C-terminal lysine residues (19) or internal through-space isosteric lysines formed from proper spacing of amino acid side chains (20). This binding activity stimulates the activation to plasmin and places the potent protease, plasmin, on cell surfaces where it is also resistant to inactivation by natural inhibitors, e.g., α2-antiplasmin (α2AP) and α2-macroglobulin (α2M) (21).

The PLG/plasmin system is primarily involved in the degradation of fibrin but also is a key participant in other proteolytic migratory cellular functions, including tissue repair, extracellular matrix degradation, angiogenesis, tumor invasion, inflammatory cell migration, complement protein interactions, and in maintaining healthy body mucosal surfaces by removing fibrin and misfolded proteins from extravascular tissues (22–27). The PLG activation system is tightly regulated by serpin inhibitors of PAs, such as plasminogen activator inhibitors-1 (PAI-1) and -2 (PAI-2).

Since any free plasmin generated in plasma would be rapidly inactivated by circulating protease inhibitors, most of the pathophysiological cell migratory functions of plasmin, e.g., wound healing, employ cell-bound plasmin. Thus, specific PLG/plasmin cellular receptors are needed. In mammalian cells, glycolytic moonlighting proteins, such as enolase, play important roles in this regard (19, 28–32), whereas in microbial cells, surface proteins, such as M-protein, and even enolase, which migrates from the cytoplasm to the cell surface by an unknown mechanism, are important PLG receptors used by bacteria for migration and dissemination (33).

Not only do the lysine binding sites (LBS) of kringle domains mediate PLG interactions with other proteins, but also basic amino acid side-chains within the AP interact intramolecularly with LBS' of kringle domains, esp., K2-PLG, K4-PLG, and K5-PLG, to place PLG in a tight (T) poorly activatable conformation (34–36). Biochemical and biophysical studies, in addition to the x-ray crystal structure of PLG (37), indicate that the LBS residues of intact PLG, viz., Asp⁴¹¹ and Asp⁴¹³ (equivalent to Asp⁵⁴ and Asp⁵⁶ of the isolated PLG-K4), make several interactions with Arg⁶⁸ and Arg⁷⁰ in the AP domain. Additionally, Asp⁵¹⁸ in the anionic center of the PLG-K5 interacts with Lys⁵⁰ of the AP domain. Likewise, in the LBS of PLG-K2, Asp²¹⁹, Glu²²¹, and Arg²³⁴, interact with other residues located in the SP domain of PLG. These interactions serve to place PLG in a tightly folded and closed activation-resistant T-conformation (32, 38–40), thus maintaining PLG in plasma, which otherwise would be activated, with the resulting plasmin rapidly inactivated by circulating inhibitors. Upon binding to cellular receptors via the LBS, the intramolecular interactions between the LBS' and the AP and SP residues are displaced, inducing a change that relaxes the conformation of the bound PLG (R) rendering it highly activatable (41). This step results in an increased susceptibility of PLG (R) to convert to Lys-PLG by the cleavage of the exposed Arg⁵⁶⁰-Val⁵⁶¹ peptide bond by plasminogen activators. Most recently, systematic inactivation of critical LBS residues in the various kringle domains of PLG was used to determine their effects on PLG activation by tPA, uPA, or SK. The results indicated that the LBS of PLG-K2 has the highest influence on relaxing the PLG conformation and enhancing its activation potential, followed by PLG-K4 and PLG-K5, with PLG-K1 having the smallest influence (32).

Several posttranslational variants of PLG are found in plasma samples and also in purified preparations of the protein. Without inclusion of protease inhibitors in the purification media, a portion of the native Glu¹-PLG can be converted to Lys⁷⁸-PLG by proteolytic removal of the 77-residue N-terminal AP (Figure 1). The product, Lys⁷⁸-PLG, is far more activatable to Lys⁷⁸-plasmin than is Glu¹-PLG, but both forms of PLG are converted to the same plasmin, viz., Lys⁷⁸-plasmin (14, 42). Another source of variation in PLG is the two glycoforms separable by specific affinity chromatography on Lysine-Sepharose. This is a general feature of plasminogens from plasmas of different mammalian species (43). These glycoforms have been characterized as a population of PLG not N-glycosylated at Asn²⁸⁹ and another form of PLG which is glycosylated with N-linked biantennary complex carbohydrate at Asn²⁸⁹. Thr³⁴⁶ is fully O-glycosylated in the entire PLG population (7, 8, 44). The properties of these glycoforms have been studied extensively since their discovery and differences between them have been found in lysine binding, PLG activation rates, fibrin binding, and catabolic rates (43, 45–47). In addition, isoelectric focusing (IEF) reveals a number of PLG subspecies ranging in pI from 6.4–8.5 that are primarily derived from differences in sialic acid content on the carbohydrate. Treatment with neuraminidase reduces the number of these bands (45, 48).

Since these variations of PLG are not allelic variants, but are post-translational modification subforms, they will not be further considered in this review.

Mutations found in the PLG gene include nonsense, missense, frameshift, splice site, deletion and insertion variants that can affect the structure and function of the PLG protein zymogen and its activated product, plasmin. In this review, we focus on missense variants and, to the extent possible, discuss the mechanisms by which these variants can affect the structure and function of PLG and plasmin.

PLG contains several relatively abundant alleles with non-synonymous single nucleotide polymorphisms (nsSNP) that result in missense variants. Some of these alleles appear globally, while others are restricted to different populations. Most of the common PLG missense variants are not thought of being directly deleterious. However, they may contribute via a cumulative effect to increase disease risk when in combination with other PLG variants, or with other protein pathogenic variants and with environmental factors (49, 50). Because PLG plays a critical role in inflammation and disease, it is important to be aware of major PLG variants in the population and their potential effects of PLG/plasmin dysfunction.

The fibrinolytic potential and plasmin generation capacity in individuals can vary significantly and this fact requires attention as to which fibrinolytic drugs should be used in different patients (27). An earlier study reported that the ability to generate plasmin can vary 8-fold in healthy individuals in addition to differences attributed to gender, age, and the use of contraceptives (51). It is not clear whether the existence of polymorphic PLG contributes to some of this variation. The ability to activate PLG to plasmin using different PAs needs to be considered before administering therapeutic treatments to patients carrying certain PLG variants. Understanding the relative world abundance and potential phenotypical consequences of relevant PLG variants is therefore of interest to medicine and population biology, as well as forensics.

In population genetics, the most common allele for a given SNP is referred to as the major allele, while less common alleles are termed minor alleles. The frequency of occurrence of the less common allele (aka, the second-most common allele) is presented as the Minor Allele Frequency (MAF). The MAFs are useful as they provide information about how common a particular SNP is within a given population. The MAF often varies geographically, and both global and regional numbers are important and useful when focusing on populations or resulting protein variants encoded by the allele. Rare alleles are prone to appear locally while common alleles are shared over a wider population range (52).

In this review, MAFs are classified into four groups based on relative abundance ranges: (1)

Polymorphisms: those variants with MAF% ≥5%, corresponding to a MAF ≥0.05.

While many rare Mendelian diseases are caused by rare (and ultra-rare) variants with large effects, it is believed that both rare and common variants with smaller effects play roles in both complex diseases, but how they work together is unclear (49). Low frequency variants have an important impact in the phenotypic variation at a population scale (53). Genome-wide association studies (GWAS) cannot fully explain the heritability of complex traits (54). This missing heritability effect can be explained by common variants having a weak effect in combination with low-frequency rare variants, which together can lead to complex diseases (55).

The term PLG polymorphisms was first used in the 1970s when researchers started to discover PLG protein variants. During this period, there was no information about MAFs, and PLG polymorphisms were only defined by PLG protein variants carried by the population. Moreover, phenotyping of PLG became of great interest when an abnormal PLG with an unusual electrophoretic mobility pattern was reported in a patient with recurrent thrombosis (56).

Other PLG polymorphisms have since been described using isoelectric focusing (IEF) gel electrophoresis (57, 58). Usually, the procedure to detect PLG polymorphisms by IEF involves treatment of patient plasmas with neuraminidase to remove negatively charged sialic acid from glycan structures and reduce the complexity of the isoforms. The treated plasma is usually next submitted for IEF gel electrophoresis at a pH range 3–10 (or 5–8). PLG is then functionally assayed by activation with uPA or SK with a chromogenic substrate-containing assay kit, and/or by following the lysis of casein in an agar overlay (59). PLG patterns are often obtained by immuno-detection and Western blots.

The interest in PLG polymorphisms increased upon observations that ethnically different populations presented with dissimilar frequencies for certain PLG variants as detected by IEF (60). Accounts of PLG variants in individuals started to accumulate mostly between 1970 and 2000 (56, 61, 62). Some polymorphisms were initially confirmed by amino acid sequencing (63). The PLG/D⁴⁵³N polymorphism was identified when the PLG gene was first characterized (5). The phenotypic distribution for the PLG/Asp⁴⁵³ and PLG/Asn⁴⁵³ alleles was found to fit the Hardy-Weinberg equilibrium, with an autosomal codominant inheritance matching a Mendelian inheritance mode (64).

To identify the many different PLG phenotypes discovered from individual plasmas, an alpha numeric nomenclature system was proposed (65). This nomenclature is based on using as a reference the IEF mobility of the two most common PLG polymorphic codominant alleles. They were initially labeled, PLGA, with A for acidic, and PLGB, with B for basic. Other alleles were compared to A and B mobilities in terms of being more acidic or more basic than these major forms. The identification therefore included A-like and B-like designations. The letter M was used to refer to an intermediate variant or medium (between A and B) and C was used for common (65). It was soon realized that PLG-based allelic signatures could help generally identify an individual. This gave rise to the use of PLG polymorphisms in forensic hemogenetics, which included paternity examinations (59, 66, 67). It was later found that the polymorphic IEF phenotypes, PLGA and PLGB, were generated by a single amino acid substitution of the more acidic PLG/Asp⁴⁵³ for the relatively more basic PLG/Asn⁴⁵³, respectively (68). These polymorphisms were included in many PLG deficiency (PD) case reports and became a reference for the IEF phenotype nomenclature (68).

Prior to standardizing this nomenclature, PLG polymorphisms were difficult to refer to and to compare. Different designations were initially given, including the city of origin of the patient. For example, PLG-Tochigi (69), a mutant with reduced plasmin activity after normal activation, was identified as IEF-M5 and later associated to PLG/A⁶⁰¹T. PLG-Osaka also produced a PLG variant that led to a form of plasmin with reduced activity (70). This was classified as IEF-M and later identified as PLG/D⁶⁷⁶N. The most frequent IEF patterns often included combinations of one or two wild-type (WT) PLG alleles with a combination of one or two common PLG alleles. Overall, about eighteen phenotypic PLG polymorphisms were initially identified using IEF (71). Other names for variants included PLG-Nagoya (72), PLG-Chicago (73), PLG-Frankfurt (74), and Plasminogen Paris (75). On occasion, phenotypes were identified with designations such as PLG-1 which was later associated with the A-phenotype. Case reports of novel PLG polymorphisms after the year 2000 occasionally use the city of origin of the proband. The PLG-Kanagawa-I polymorphism was reported in 2002 and corresponds to a dysfunctional PLG activity caused by the PLG/G⁷³²R variant (76).

PLG phenotyping based on the IEF protocol has several advantages, viz.: (1) PLG is readily available from patient plasmas for further characterization; (2) the PLG protein band pattern corresponding to the translated alleles from the blood of an individual can be readily visualized; (3) the electrophoretic mobility provides information about the overall charge of the protein as compared to wild-type (WT)-PLG and differences can be an indication of amino acid changes and different alleles; (4) many times an allele is expressed in a lower amount and the relative abundance of alleles could provide phenotypic information; and (5) the isoelectric point for a protein with a known amino acid sequence can be calculated. Since IEF changes may reveal alterations of the PLG structure, the IEF pattern adds valuable information for a phenotypic characterization of a PLG variant in a patient and a first step towards a diagnosis of a PLG deficiency.

The IEF protocol helped to visualize the existence of various PLG phenotypes in plasma and PLG variants sometimes associated with disease. PLG genetic analysis was later introduced, especially when young individuals presented unusual symptoms, e.g., thrombosis, which made the search for abnormalities in this gene a valuable approach. The need to purify PLG variants for further analysis was also suggested when subjects were found to carry different IEF patterns (77).

Whereas IEF analysis is still often used as a characterization step, this is usually followed by genomic DNA analysis of PLG, including the use of the polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP) analysis, and/or direct DNA sequencing (68, 78). A summary of several IEF phenotypes with corresponding PLG molecular variations has been reported (79).

IEF from case reports of patients and families, combined with DNA sequence information, has contributed to the discovery of many amino acid substitutions in PLG deficiencies. IEF, followed by DNA sequence analysis, was most recently used in the discovery of the PLG/K³¹¹E missense variant that leads to a rare disease known as hereditary angioedema (HAE) with normal C1 inhibitor. This variants has been cataloged as a clinical variant (80).

Most PLG missense variants of interest lack functional studies and their clinical significance are missing or uncertain. Amino acid variants can range from benign to pathogenic. Predictive in silico computational methods can provide highly likely scenarios of amino acid substitutions in proteins, especially when using different approaches (81–83). To facilitate a more comprehensive discussion of the pathogenic variants that will be discussed later in this review, we consider it essential to conduct an in silico analysis that predicts potential structural and functional perturbations resulting from various amino acid substitutions in PLG variants. This analysis will enable us to better understand the molecular implications of these variants and provide valuable insights into their pathogenic potential. Herein, we used the following in silico prediction tools. SIFT (Sorting Intolerant From Tolerant), which is based on sequence conservation (84); Polyphen-2, which assesses the impact of amino acid substitutions on protein structure/function (85); mCSM, which predicts the effect of variants in proteins using graph-based signatures (86); MUpro, which predicts protein stability changes based on protein sequence and structure and uses Support Vector Machine (SVM) (87); and DynaMut2, which combines Normal Mode Analysis (NMA) methods to capture protein motion and graph-based signatures (88). PLG structural data used for mCSM, MUpro, and DynaMut2 was based on the x-ray structure of Glu¹-PLG (PDB ID, 4DUR) (36) and the cryo-EM structure of PLG (PDB ID, 8UQ6).

For the amino acid substitution effects using Polyphen-2 and SIFT, the score for substitution of each residue in each prediction tool was first recorded. A red-green heat map was then created from those values by assigning bright red for the most damaging score and bright green for the most tolerated score for each prediction tool. Specifically, Polyphen-2 score 0, benign (green); score 0.5 (mid-range), possibly damaging; score 1, probably damaging (red). SIFT score < 0.05 is predicted to be deleterious (red); score 0, variants can affect protein function (red); and score 1, tolerated (green). We excluded nonsense variants since stop codons cannot be modelled with the prediction software. Clinical variant classifications were based on the ClinVar (NHLBI) algorithm (https://www.ncbi.nlm.nih.gov/clinvar/). Accession numbers for PLG missense clinical variants are provided in the text as appropriate.

The high-resolution structures of PLG have contributed greatly to the understanding of its structure/function relationships and facilitates making credible functional predictions. Studying the effects of single amino acid substitutions in PLG that lead to clinical outcomes, as found in congenital PLG deficiencies, also presents a convenient informational source that can provide critical insights into its role in vivo. Animal models, such as PLG gene-altered mice (89, 90), in combination with various other related transgenic murine models, continue to be instrumental in understanding the mechanisms of PLG function.

In general, population data of a variant is important when evaluating its pathogenicity. Usually, the most abundant variants are not directly pathogenic but may contribute in a minor way to complex diseases, especially if the variant is predicted as pathogenic and if it occurs in a protein like PLG which is involved in many disease mechanisms (50). The chances of a pathogenic condition increase in homozygous or compound heterozygous states where the additive effect increases the penetrance (50).

The gnomAD browser v4.0.0 currently lists ∼1,000 missense PLG variants detected from a wide variety of large-scale sequencing projects (https://gnomad.broadinstitute.org/). Most of the PLG nsSNPs are rare or ultra-rare (MAF≤ 0.1%), while less than 2% of the variants (Table 2) are relatively abundant (MAF ≥0.1%) in various genetic ancestries in the world. From the 2% group, most major PLG missense variants are assumed to be benign, but in fact, not much is known about them at the molecular level and, therefore, are also of great interest in this review.

In addition to the data from gnomAD browser v4.0.0, the data obtained from the PAGE population study (Table 3) are included in this review because they provide access to genomic data from various American populations involving various races and ethnicities that have not been sufficiently represented in a world in which diversity is progressively increasing (91). The multiscale nature of the MAF% distribution of major PLG variants in different ethnic backgrounds is evident in both Tables 2, 3. The PAGE population study compiles allelic data from various populations, including Native Hawaiians and Native Americans, not readily available in the past (91), and studies on a genetic propensity for stroke in such populations can now benefit from these data. As an example, it has been recently reported that these populations have a higher-than-normal propensity to stroke at younger ages with significantly higher stroke mortality in comparison to other regional ancestries in local populations (92).

The overall relative distribution trend found with the gnomAD browser for the second minor allele for PLG missense variants was consistent with two other populations studies, the ALFA project (https://ncbiinsights.ncbi.nlm.nih.gov/2020/03/26/alfa/), and with the 1000 Genomes project (https://www.ncbi.nlm.nih.gov/bioproject/28889).

All the PLG missense variants discussed in the present review are illustrated in Figure 3 showing their location along the primary structure of the PLG protein.

PD-associated PLG missense variants have attracted much attention, and they are among the most described in the literature. Except for the relatively abundant PLG/K¹⁹E and PLG/A⁶⁰¹T variants, most PD-associated nsSNPs are rare or ultra-rare and are not necessarily always detected in population studies. In fact, most are described in case reports from diseases running within families.

Two types of PD, which include type I (PDI) and type II (PDII), have been described. The PDI and PDII missense variants are represented in red and green font, respectively, in Figure 3. Notably, PLG/K¹⁹E and PLG/A⁶⁰¹T are both abundant and related to PD and those have an added asterisk to represent this duality. Single gene Mendelian diseases like PD offer a unique opportunity to study protein structure/function in relationship to disease phenotype.

PDI, also known as true PLG deficiency or hypoplasminogenemia, is a genetic disease characterized by low or undetectable PLG antigen. Thus, reduced plasmin activity in plasma is found. This condition results in compromised fibrin clearance (93). Congenital PDI is mostly inherited as an autosomal recessive trait and it is cataloged as a rare disease by the National Organization of Rare Disorders (NORD) (https://rarediseases.org/).

To date, about 45 single amino acid substitutions in PLG have been discovered in probands with PDI. Table 4 summarizes the amino acid substitutions reported for PDI, their domain mapping, and the result of our in silico predictions. The current clinical significance for the majority of these substitutions is either missing or has conflicting interpretations.

The reduced PLG antigen concentration and/or activity characteristic of PDI leads to extravascular accumulation of undigested fibrin and impairs wound healing in mucosal surfaces. This debris causes thick white-yellowish pseudomembranous lesions with a wood-like (ligneous) appearance. Histologically, pseudo-membranes show accumulation of hyaline-like substances, impaired epithelial and fibrin debris with inflammatory cells, fibroblasts, and eosinophilic infiltration (25). The dominant presentation of PDI occurs in the eye-lid surface or conjunctiva, known as ligneous conjunctivitis (LigC), which accounts for up to 80% of the clinical presentations. Similar systemic lesions may occur in additional mucosal tissues, including the gingiva (known as ligneous gingivitis or LigG), the middle ear, the larynx, and the female genital tract. Periodontal disease can be the first clinical manifestation of LigC and PDI (94). Approximately 60% of cases of LigC also develop LigG in PDI with∼a 2:1 ratio of female to male presentation (95). The PLG/K¹⁹E mutant has been reported in 34% of the LigG cases (95).

Table 5 (Cases A to D) summarizes data from four previously reported case studies involving PLG nsSNPs associated with PDI. The data were adapted from case reports and reviews of patients and family members with PDI with no other known health conditions. Notably, data from case reports are often missing critical information. Where available, %PLG activity, %PLG antigen, zygosity, gender, reporting age, and phenotypes are presented. From these available data, PDI symptoms become evident when the %PLG activity is <40%, regardless of the variant. Also, in most cases, homozygotes or compound heterozygotes for certain PLG nsSNPs were needed for clinical manifestations of PDI. This is true for PLG/K¹⁹E, PLG/R²¹⁶H, PLG/W⁵⁹⁷C, and PLG/L¹²⁸P, suggesting an additive effect and different penetrance. PLG/K¹⁹E may have lower penetrance since at least one homozygous relative did not show clinical manifestations. Variable penetrance and expressivity of variants is a recognized limitation that affects our understanding of the effect of a variant when comparing case reports with the population at large (99). Occasionally, a variant may be sufficiently pathogenic in a given individual to be able to cause clinical symptoms in a heterozygous state, as is the case for PLG/A⁵⁰⁵V variant in Case A, patient 2, in Table 5.

An important finding resulting from case studies of heterozygous relatives of congenital PLG deficiency patients is that PLG activity and PLG antigen concentration can be significantly lower than that considered to be normal, with individuals appearing healthy. As an example, the PLG antigen concentration could go as low as 2 mg/dL, with no disease phenotype reported, when having 66% PLG activity (Table 5, Case C, Patient 1, mother). An estimate of how low the PLG activity can be without disease phenotypes based on these cases is about 50%. When the PLG activity and antigen concentration of PLG are too low, clinical manifestations of quantitative PLG deficiency become evident. Beyond a threshold level, symptomatic patients present extravascular fibrin deposition and systemic inflammation.

Overall, PDI can result in severe consequences, including blindness, tooth loss, and infertility in both males and females (25, 100). This debilitating illness significantly impacts quality of life (101). Furthermore, as a rare disease, PDI poses diagnostic challenges worldwide, resulting in delayed access to limited but potentially life-changing interventions (102).

The overall prevalence of congenital PD as homozygous or compound heterozygous is reported to be 1/625,000 (orphanet.net). These numbers are expected to be high within regions where consanguineous unions are common. Notably, about 0.13%–0.42% of the world population can be asymptomatic heterozygous carriers of PD alleles (103). This is an important issue since most PDI-associated variants are predicted to be pathogenic and, as such, they may contribute to disease even in heterozygous individuals. Such pathogenic variants may add to the variation in PLG levels and activities observed in the population and possibly contribute to complex, non-Mendelian diseases.

A significant number of patients with PDI are of Turkish origin (25, 93, 104). Turkey has a 19% consanguineous union frequency, with 58% of those being between first-cousins (105). One report showed that 21 of 50 studied patients were of Turkish origin with consanguineous union between parents in 19 members of that study group (96). The Middle East also presents some of the highest rates of consanguinity in the world, with Arabian first cousin consorts reaching 25%–30% of all marriages (106). Likewise, there is a high rate of common ancestral unions in inner Asia (107) and North African countries (108). Unfortunately, allele databases from these regions are not readily available. There are ongoing efforts to improve the current limited access to genomic data from Lebanon and Africa (109, 110). India is also known to have a high burden of rare recessive genetic diseases (111). Importantly, the most recent version of the gnomAD browser (v4.0.0) now includes Middle Eastern ancestry data, which is known for having high consanguinity.

It is not clear how the different PLG missense variants lead to PD and the nature of the molecular mechanisms are equally uncertain. Most of the PLG variants associated with PDI have not been fully characterized beyond IEF. Herein, we utilized prediction software to assess the impact of the resulting substitutions on PLG structure and function. It is believed that the structural changes that occur in PDI variants may result in an impaired secretion and/or a reduction in half-life. Unfortunately, the half-lives of most known PDI variants have not been reported.

As indicated by the red-green heat map in Table 4, most PDI substitutions were predicted to be damaging (bright red) for the sequence-based predictions Polyphen-2 and SIFT. The ΔΔG values from the structure-based predictions are consistent with most PDI substitutions being destabilizing when using either protein data base (PDB) structure files. It is seen from this Table that the majority of the PDI variants are located in the very conserved kringle domains of PLG (Column 4, Table 4 and Figures 4A,B) which would destabilize the protein. Interestingly, several PDI variants were consistently predicted to be highly destabilizing. Those variants include PLG/R²¹⁶H in K2-PLG, PLG/R⁵¹³H in K5-PLG, and three variants (PLG/W⁵⁹⁷C, PLG/P⁷⁴⁴S, and PLG/R⁷⁷⁶H) in the SP domain of PLG/plasmin (Table 4). These predictions are consistent with recent findings that show that a functional LBS in K2-PLG is critical to maintain the PLG closed conformation by interactions with the SP domain, and that the LBS’ in K4-PLG and K5-PLG are also critical to maintaining the activation resistant form (32). PDI variants in the SP domain probably mostly destabilize the closed conformation leading to PLG short half-lives. About 20% of PDI substitutions occur in the SP domain (Figure 4B). Herein, we discuss the potential pathogenic mechanisms of some of the variants.

Studying isolated PLG from patient plasmas would be of great interest in order to determine the impacts of critical variants that result in PDI. Activation with SK, uPA, and tPA would also provide important information. Most case reports are limited to overall PLG activation assays using SK and a chromogenic commercial kit, as well as an antigen assay. The method by which PLG activity is measured in patients can affect the conclusions regarding the fibrinolytic potential of PLG variant carriers. The ability to bind fibrin and the activation kinetics with various PAs require clarification. Analysis of PLG variants obtained from homozygous probands should include PLG activation assays with various PAs (123). A functional assay would facilitate defining the molecular mechanism of the disease (114). Measuring fibrinolytic and thrombin generation simultaneously is also a potential strategy.

The manner of measuring fibrinolysis needs reevaluation when PLG deficiencies are reported (124). An important point is that alternative functional PLG/fibrinolysis assays should be considered when evaluating PLG-deficient plasma. A simultaneous test of thrombin and plasmin generation within defined populations may facilitate obtaining a more comprehensive sense of hemostasis regulation among individuals and this strategy has been proposed for a more comprehensive assessment of PD patients and their clinical outcomes (125). This approach was recently used to compare hemostasis among different species, and it was found that using a tPA activation assay of PLG is a much more sensitive and reliable method for plasmin generation than measurement of clot lysis times (126).

PDII, also known as dysplasminogenemia is characterized by normal or slightly lower PLG antigen level with a diminished or abnormal activity. The East Asian abundant PLG/A⁶⁰¹T variant and six other rare and ultra-rare PLG missense variants have been associated with dysplasminogenemia (Table 6). The PLG/A⁶⁰¹T variant is inherited as an autosomal codominant trait. It circulates in a heterozygous form in otherwise healthy subjects and can lead to a reduced plasmin activity of 14% as a homozygous variant and 57% in heterozygous individuals. It has been proposed that carrying this allele could increase the risk of thrombosis when combined with other factors. Approximately 86% of the reported missense variants associated with PDII are clustered along the serine protease domain, possibly causing disruptions to the catalytic site (Figures 3, 4C).

Dysplasminogenemias are generally considered to be disorders caused by PLG variants but may not necessarily lead to disease or thrombotic risk. Others believe that this condition can be a risk factor when individuals are challenged by trauma, infection, and environmental influences. An interesting and unexplained observation is that the typical mucosal lesions observed with PDI have never been reported for PDII in either homozygous or compound heterozygous PDII patients (98). The most surprising observation is the apparent lack of deep vein thrombosis (DVT) in most PDII patients. The reason for this may be due to the fact that most PDII associated missense variants have intact kringle domain residues which allow PLG to bind to extravascular receptors. It is hypothesized that mild dysfunction of plasmin activity may suffice to support fibrin degradation. Another hypothesis, supported by previous findings, is that upon binding to receptors, a more functional PLG conformation is favored that may compensate for a dysfunctional activity (41). In a clinical case report, a PLG activity as low as 7.7% was reported for a combined heterozygous Japanese individual carrying PLG/A⁶⁰¹T and PLG/G⁷³²R, viz., the Kanagawa-I PLG phenotype (76). The patient had a healthy lifespan, but senile dementia developed at age ≥70, possibly due to unconfirmed multiple cerebral strokes (76). It is hypothesized that low or dysfunctional plasmin activity represents an increased risk for small vessel circulatory diseases. PDI and PDII are both reportedly associated with circulatory abnormalities, including small optic thrombotic retinopathy, ischemic optic neuropathy, and occlusion of the central retinal artery or vein (127). The PLG/A⁶⁰¹T missense variant was reported in three individuals with retinochoroidal vascular disorder, which presented with low antigen and low PLG activity, but a more severe macular choroidal occlusion occurred when the homozygous disorder was present (127). Recently, a 34-year-old Korean patient heterozygous for PLG/A⁶⁰¹T presented with a history of recurrent thrombotic arterial embolism and cardiac myxoma. The proband's mother, homozygous for PLG/A⁶⁰¹T, had a 75% decrease in plasmin activity, but surprisingly had no history of DVT. This lack of correlation of the homozygous variants with thrombotic disease is unclear (128). The PLG/A⁶⁰¹T variant is, therefore, still classified as a variant of conflicting pathogenicity (VCV000013574.7).

PLG/A⁶⁰¹T is the most reported PLG missense variant associated with dysplasminogenemia and is present in up to 4% of East Asian individuals (129). It is associated with the PLG phenotypes of Tochigi I and II, Kagoshima, and Nagoya. This variant is abundant in the Chinese Han population (130), in Korea (131), and in Japan (132). It is also found to be a low-frequency variant of concern in Native Hawaiians. The high frequency of PLG/A⁶⁰¹T in East Asians is believed to have occurred by a founder effect (131).

The mechanisms behind PDII variants may be mostly related to a reduced ability to activate PLG to an effective serine protease. PLG residue Ala⁶⁰¹ is strongly conserved through multiple species. The molecular mechanism behind the Ala⁶⁰¹Thr variants has been proposed to involve the nearby His⁶⁰³ of the serine protease catalytic triad, which becomes unable to serve as an effective proton acceptor (133). The mouse PLG/A⁶⁰³T model, equivalent to the human PLG/A⁶⁰¹T Tochigi phenotype, showed significantly reduced Pm plasmin activity (8%) after activation by uPA, but did not show a significant difference in a brain ischemia model (129), strongly suggesting that other factors are needed to induce thrombosis. For the PDII-associated PLG missense variant PLG/D⁶⁷⁶N, it was proposed that this variant, which is associated with the PLG-Osaka (IEF M), may render PLG inactive because it produces a new N-glycosylation site at the tripeptide, Asn⁶⁷⁶-Arg⁶⁷⁷-Thr⁶⁷⁸, which would disrupt the protease active site (70).

It has been proposed that dysplasminogenemias should be characterized using highly purified PLG from the plasmas of patients (77). It was argued that the unaccounted existence of SK antibodies, PA inhibitors, and plasmin inhibitors in plasma affect plasmin generation in patients. It was also recognized that patients may be homozygous or heterozygous for PLG variants, which would add extra complexity to phenotypes. It was further proposed to study the binding of SK to the purified PLG, the equimolar PLG-SK complex formation rate, and the active site generation in the complex. For example, PLG may activate with uPA but not with SK. Even purified PLG may contain inactive and active proteins. In those cases, it is important to either confirm with a homozygous protein or to model the variants in vitro. This challenge was recognized early and a classification of PLG variants was suggested using twelve different PLG deficiencies that were sorted by classifying active site-deficient PLG. Overall, a further understanding of how the different variants perform will require isolating the variants or expressing them in vitro.

Although elevated PAI-1 and thrombin-activatable fibrinolysis inhibitor (TAFI) are major risk factors for venous thrombosis, the PLG level continues be a potential underlying risk (134). The role of PD-associated variants in contributing to thrombosis is unclear. There are no currently recommended routine genetic determinations for PLG polymorphisms to help determine risk for venous or arterial thrombosis. While thrombosis has been observed in PLG-deficient individuals (135), it has mostly been reported in isolated cases within families. Often, an individual nsSNPs is directly paired to a phenotype by clinicians attempting to make a connection between single variants and the in vivo PLG mechanism. Such reports also offer a glimpse of their variable penetrance. However, case reports, while providing valuable first analyses, need to be considered with caution because they may be compounded with unknown genetic and environmental factors (136). Such reports need to be supported with other data including those derived from predictive tools, population studies, and functional analysis.

A study in a Japanese population with low PLG activity did not show a significant relationship with thrombosis risk as compared to those with normal PLG activity (137). However, the deficiency was not uniquely characterized in each person, and they were assumed to all have the PLG/A⁶⁰¹T variant. In a single case report, an individual heterozygous for PLG/A⁶⁰¹T presented with pulmonary embolism (138). This heterozygous patient only had a 59% decreased PLG activity with no other obvious risk factors, yet this individual required lifelong treatment. In another study, PD could not be excluded as a risk for thromboembolism since no other known or testable factors were present (123). A further report describes a 21-year-old individual with a combined homozygous protein C (PC) deficiency (although it seems unreasonable that this patient would have survived the neonatal period with a total PC deficiency), along with a heterozygous PLG variants, presented with recurrent DVT (139). Most recently, two young males (ages 14 and 16) and a young female (age 19) presenting with cerebral infarction were all found to have a significantly reduced PLG activity (∼50%–60%), likely caused by an inherited PLG/A⁶⁰¹T heterozygous variant. These recent findings continue to support an association of PD with an increased risk of thrombosis. Thrombotic disease is a complex multi-factorial disorder (140) and the role of age, gender, and ethnic background on venous thromboembolism has been recognized and discussed elsewhere (141).

Important contributing factors for the uncertainties of thrombotic disease and PLG deficiencies include the limited number of patients, the diversity of the PLG variants, the variable penetrance, the involvement of different tissues, and the lack of systematic guidelines for reporting the condition. These issues are being addressed by the HISTORY project (102). An important issue is the possibility that PD patients carrying abnormal PLG may be at higher risk of not responding well to classic therapeutic fibrinolytic agents, such as tPA, if a pro-coagulant state is present (27). Heterozygous carriers of abnormal PLG(s) may be more susceptible to incorrect therapeutics due to lack of risk awareness.

Studies investigating the correlation between PD and thrombosis risk are hampered by limited sample sizes, primarily due to the rarity of PD as a recessive disease, making it challenging to get enough participants. A study of 9,611 blood donors in Scotland (142) and a study in Japan (137) indicate a wide variation in PLG levels and activities within these populations. Thus, it is difficult to correlate PLG levels/activity with thrombosis. Moreover, these findings imply that defining a universal normal PLG concentration/activity may be challenging, necessitating a reevaluation of the criteria used to define PLG concentration/activity in healthy patients. For example, there could be a spectrum of functional PLG activation potential in different individuals and ethnicities, PLG may be in excess in many individuals, and/or there may be evolutionary conserved compensatory mechanisms for low PLG concentration/activity. Regardless, the physiological consequences of very low PLG antigen and/or plasmin activity in the vasculature or extravascular tissue nevertheless represent a potential risk factor for disease.

Systematic studies reporting plasma PLG concentrations and activities in different human populations are very limited. It is pertinent to reexamine the normal PLG plasma concentration and activity range. Most reports have included a small number of healthy blood donors from a few geographical regions. A PLG antigen concentration average of 16.0 mg/dl (range: 12.3–19.7 mg/dl) was obtained from 100 donors in a study in England (143). While a PLG antigen concentration average of 12.2 mg/dl (range: 7.7–16.8 mg/dl) and a PLG activity of 96.3% (range: 65.9%–126.8%) was reported from 43 blood donors in Hamburg, Germany (144). A review that same year on PLG proposes 20 mg/dl as the normal plasma concentration for Glu¹-PLG (145). In the Scotland study earlier mentioned, the 9,611 participants age were between 15 and 65, and the PLG level ranged from 9.0–15.0 mg/dl (average, 12 mg/dl), consistent with the∼2-fold range found previously, but the PLG activity showed a surprising 8-fold range variation (20%–200%) in otherwise healthy individuals (51). Another study reported a normal PLG activity ranging between 75 and 120% (78). Moreover, an investigation of PLG activity from 4,517 normal donors from Japan (ages 32–89), prompted by the fact that the Japanese population have an increased tendency to carry the PLG/A⁶⁰¹T variant, reported a∼4-fold variation in %PLG activity (ranging from 42%–160%) (61, 137).

The reasons for the wide variations in PLG concentration/activity are not fully understood and studies on the factors that determine plasma levels of PLG are also limited (134). The heritability of PLG levels and activity in plasma is not fully understood and has not been addressed systematically in different populations. More recently, GWAS with a cohort of 2,304 young healthy individuals in Ireland (Trinity Student Study) and a group of 507 siblings at the University of Michigan, indicated a heritability factor of up to ∼50% for plasma PLG levels (146). In that study, a relatively abundant PLG missense variant, PLG/R⁵⁰⁴W, was found to be strongly associated with reduced PLG levels (∼13% reduction per allele). Nevertheless, the molecular nature of this PLG mutant has not been addressed. On the other hand, factors found to increase plasma PLG levels in normal individuals included tobacco smoking, female gender, and the use of oral contraceptives (146). The potential phenotypic impact of PLG/R⁵⁰⁴W, and other abundant PLG missense variants in the population, is uncertain. Such PLG variants may further contribute to variations in the fibrinolytic potential among individuals and influence multifactorial disorders.

Additional evidence suggests that nsSNPs in PLG gene generate variants that contribute to a range of disorders beyond PD. Moreover, various PD associated variants have also been linked to other diseases. Pathogenic missense variants associated with a specific disease can help identify causal genes (147).

PLG is often a target in GWAS due to its multiple roles in hemostasis. The use of targeted gene panels, or segregation with disease, has allowed the discovery of the association of PLG with several traits and disorders. These include, but are not limited to, bleeding, thrombosis, platelet conditions (148, 149), susceptibility to infection (150), coronary artery disease, periodontitis (151), quantitative trait loci relevant for absorption, distribution metabolism, excretion of drugs in human liver (152), giant cell arteritis (153) and plasma Lp(a) levels (152).

PLG bound to a variety of cell surface receptors is involved in various cellular responses, such as fibrinolysis, cell migration, wound healing, inflammation, and angiogenesis. Therefore, it is not surprising that PD associated variants and other PLG pathogenic missense variants may contribute to other diseases.

In this section, we will discuss specific PLG variants and their contributions to diseases in addition to PD. The first group (A to F) below includes rare or ultra-rare PLG missense variants (Tables 4, 7) and the second group (G to K) below includes relatively abundant PLG missense variants in the population (Tables 2, 3, 8).

To date, no PLG germline genetic variants have been reported to directly lead to cancer. However, a potential role of PLG missense somatic variants in cancer (and other diseases) should not be ruled out. The PLG/plasmin system plays a fundamental role in the migration of malignant cells and metastasis in solid tumors, and it is directly involved in the activation of matrix metalloproteases (184, 185). Pericellular plasmin facilitates the invasion process and PLG receptors are found on the surface of most tumors. The expression of PLG receptors can be used for cancer prognosis and survival (186). Regulation of the PLG/plasmin system can result in the stimulation or suppression of cancer (187). Whole genome/exome database and computational and experimental analyses can facilitate identification of driver genes and to determine the role of missense variants in cancer (2). PLG is a cancer-related gene based on experiments involving insertional mutagenesis in mice, but it is not considered to be a cancer-driver gene. Somatic mutations accumulate during malignant transformation (188) and other complex diseases (189). Mutations that directly lead to a tumor proliferative advantage are considered driver mutations but those account for a very low (3%) proportion of observed genetic aberrations in cancer. PLG somatic genetic mutations found in tumors are curated by the catalogue of somatic mutation in cancer (COSMIC) among other databases. Hundreds of somatic variants of PLG have been reported and catalogued from diverse tumors. The most common type of PLG variants found in tumor samples curated by COSMIC are variants in the protein coding sequence, with missense variants representing 45% of the total.

Whereas missense mutations are frequently found in malignancy, their role is not easy to predict (190). To date, no PLG somatic mutations have been identified as a driver cancer mutations Many of the PLG missense variants discussed in this review have been also detected as somatic variants in diverse tumors but they are predicted as passenger mutations using the FATHM cancer algorithm prediction tool (https://fathmm.biocompute.org.uk) (191). The role of passenger mutations in cancer however is currently poorly understood but the concept of such genes playing important roles in malignancy evolution is increasingly supported (192). Passenger mutations constitute most (>97%) of the somatic mutations present in tumors. Some passenger mutations can become established and become part of the clonal progression of a tumor and may affect the tumor phenotype, e.g., drug susceptibility and antigenicity. Passenger mutations can also affect tumor growth properties or even lead to tumor regression. These mutations can also be used to classify tumor type and help determine the origin and history of metastatic lesions by serving as a molecular clock on cancer evolution (193). The collective burden caused by passenger mutations can help to explain the progression of cancer not explainable by driver genes alone (194). The tumor type, and its evolution and prognosis, can be influenced by the accumulation of somatic mutations (195).

A systematic study of the progression of missense somatic mutations of PLG and their potential role in cancer evolution is lacking. It is possible that particularly pathogenic PLG missense variants, like those that cause PDI and PDII, will impair tumor progression and will not be selected in the clonal expansion of a tumor. But those that facilitate PLG binding to cellular receptors or enhance PLG activation would possibly promote malignancy and may represent important therapeutic targets. Two critical parameters, sometimes missing from tumor databases, include confirmation of somatic vs. germline origin of mutations and zygosity. These gaps can hinder a comprehensive understanding of the role of missense variants in tumor evolution.

PLG passenger somatic missense variants may be involved in the cancer mutational progression landscape and represents a potentially important point to investigate further.

Missense variants in PLG may also play an indirect role in cancer by affecting the type of posttranslational modifications that occur. Phosphorylation is a reported post-translational modification that can lead to many types of cancer (196, 197). Recently residue PLG/Tyr⁹² present in PLG-K1 was flagged by a novel bioinformatic proteomics and cancer co-clustering tool as a potentially relevant cancer-associated phosphorylation site in PLG (196).

The pandemic of COVID-19 emphasized how diverse host genetic differences at the individual level can affect the outcome of the disease (198). For instance, COVID-19 presenting with an inflammatory response can become a systemic thrombotic disease in susceptible individuals and, as such, the circulating PLG concentration is a current new key parameter obtained from patients on hospital admission (199). PLG and plasmin are key participants in homeostasis and other pathological states (200). These proteins play critical and complex roles in COVID-19 pathogenesis (201) and their dysregulation can influence the outcome of COVID-19 patients. Recent studies revealed that low PLG levels were the most significant prognosticators of death in COVID-19 patients (199, 202), being also associated with higher inflammation parameters. Potentially, patients with reduced PLG may be more susceptible to poorer outcomes in COVID-19 and other inflammatory diseases. In these cases, treating patients with PLG during the acute phase of the disease has been found to be beneficial (201).

The heterogeneity of susceptibility and outcomes to COVID-19 can be affected by genetic variants in the population (198). Ethnic genetic variants of PLG as potential determinants of heterogeneity in response to COVID-19 has been recently suggested (203). The importance of PLG in COVID-19 accentuates a potential clinical significance of polymorphic PLG carried in different populations toward this and other diseases.

Atypically low concentrations of PLG may be contributing risk factors for this and other diseases. These PD states can be genetic in combination with single or combined PLG polymorphisms or acquired during the disease state. In any event, studies relating to the PLG genetic complexities with diseases, such as COVID-19, are lacking.

While not the focus of this review, the generation of PLG^−/− mice allowed unprecedented studies of the role of PLG in vivo at multiple levels. Mice with a total deficiency of PLG (mouse PLG^−/−) have severe lifelong challenges, including deficiencies in vascular wound healing (204) and vascular remodeling after arterial injury (205), as well as venous and arterial thrombosis (134), despite the fact that PLG is not the only fibrin degrading enzyme in the vasculature (89, 90). A study assessing the development of LigC in PLG^−/− mice demonstrated an equivalent phenotype to that observed in PLG-deficient humans. However, mice deficient for both PLG and fibrinogen did not develop ligneous conjunctivitis thereby linking PLG/plasmin-mediated clearance of fibrin as a regulatory mechanism for this disease (135). Endothelial cells from mice deficient in PAs or mouse PLG can penetrate fibrin barriers with metalloproteinases acting as fibrinolysins (206). Moreover, endothelial cells ensure and contribute to vascular system patency by producing fibrinolytic activity through MMPs in the absence of PLG (207).

A mouse PLG knock-in carrying the homozygous mouse PLG/A⁶⁰³T allele did not show an increased susceptibility to thrombosis, as compared to WT mice when challenged in experimental thrombotic models (129). Unlike the reported PLG deficiencies in humans, the PLG^−/− mouse model, wherein PLG is totally absent, shows a fundamental need for PLG for a healthy life (90). Thus, it is reasonable to extrapolate that the complete absence of plasmin in humans will be damaging and having low PLG activity could increase susceptibility to thrombosis after a challenge (137, 208, 209).

The most well-known PLG variants are a group of rare pathogenic missense variants that lead to PDI and PDII and are described in family case reports. The true prevalence of these variants is unclear, and they may constitute a disease risk in heterozygous carriers. In addition to the codominant PLG/D⁴⁵³N, approximately ten other PLG missense variants are rather abundant in various world genetic ancestries. Some of them have disease association and predicted pathogenicity, including PLG/K¹⁹E and PLG/A⁶⁰¹T, which associate with PDI and PDII, respectively. The abundant PLG/R⁵⁰⁴W variant that lowers Pg levels, and several other prevalent and predicted pathogenic variants, such as PLG/R²⁴²H, PLG/R⁴⁷¹Q, PLG/A⁴⁷⁵V, and PLG/T¹⁸¹A, most likely contribute to complex disorders and deserve further attention. These findings are consistent with PLG/plasmin having important involvement not only in fibrinolysis, but also in wound healing, inflammation, immune response, and pathogen invasion. The PLG concentration and activity vary considerably among the global population which could in part be a consequence of carrying some of those PLG alleles. PLG/K¹⁹E and PLG/A⁶⁰¹T, initially described more than 20 years ago, are still relevant today when individual heterogeneity and differential susceptibility to disease has become increasingly evident. The different susceptibilities to the progression of COVID-19 require clearer understanding of unique genetic background of critical parameters, such as the PLG levels and the PLG activation potentials. We herein review how ethnic backgrounds influence the nature of the PLG variants carried, and what regions in the world are more susceptible to these genetic diseases. This knowledge is relevant when designing global therapeutic and prophylactic interventions. Several other rare PLG missense variants have been associated with disease by GWAS. It is thought that many complex diseases can result from additive effects of even moderate pathogenicity from individual missense variants. The important role of PLG in inflammation and allergy is confirmed by the direct connection of the PLG/K³¹¹E variant with HAE with normal C1 inhibitor. The present review highlights how PLG activity and concentration can be much lower than originally expected. Variations in the PLG level and activity in plasma and extravascular tissues can have severe consequences in combination with other factors. This comprehensive view of PLG missense variants and disease association in a global context is relevant to epidemiology of diseases. The information discussed herein can impact personalized medicine, e.g., a knowledge of specific variants and associated pathology can help in diagnosis (development of targeted diagnostic kits) and tailored treatment strategies (development of novel therapeutics), optimizing outcome of PLG associated disorders and minimizing adverse reactions. Moreover, it is useful for the development of prophylactic strategies in different world populations carrying certain variants. The use of IEF for detecting PLG alleles can still be of use for paternity tests where no molecular biology methods are feasible. Also, the information provided in this review is relevant for genetic counselling and risk assessment. For instance, identifying high-risk populations can lead to early interventions and monitoring, potentially preventing disease progression. Deciphering molecular mechanisms of PLG-related genetic diseases will continue to reveal the in vivo significance of the PLG/plasmin system. Our intention is to bring a global perspective and awareness of PLG heterogeneity to the population and their susceptibilities to disease beyond fibrinolysis. Awareness of the clinical significance and disease risk of PLG polymorphisms will provide useful information that will assist development of new therapies for a number of diseases in which PLG plays a role.