Fibrinogen Cleavage by Lectin Pathway Proteases: Roles and Implications

Special Article: Fibrinogen

J Blood Disord. 2023; 10(2): 1075.

Fibrinogen Cleavage by Lectin Pathway Proteases: Roles and Implications

Madhuri M¹; Kumari P¹; Ali A¹; Roy D¹; Hajela S²; Kumari B¹; Hajela K³*; Sharma S¹*

1All India Institute of Medical Sciences, Patna, India

2Mahakaushal University, Jabalpur, Madhya Pradesh, India

3School of Life Sciences, DAVV, Indore, India

*Corresponding author: Sadhana Sharma Department of Biochemistry, All India Institute of Medical Sciences, Patna - 801507, Bihar, India Tel: 9631280481 E-mail: [email protected], [email protected]

Received: June 07, 2023 Accepted: July 06, 2023 Published: July 13, 2023


The lectin pathway proteases (MASPs) are diverse in regard to their substrate selectivity and also have substrates outside the complement pathway. These proteases have been reported to cleave proteins of the coagulation cascade. MASP-1 has thrombin-like activity and therefore cleaves fibrinogen, fibrin and prothrombin. MASP-2 also participates in coagulation by cleaving prothrombin. Thus, these proteases are key players in the process of thrombosis and thrombolysis, therefore have implications in thrombotic vascular diseases. The cleaved fragments of fibrinogen and fibrin, consequently produced during coagulation and fibrinolysis, are important in the repair process and hence may have involvement in inflammatory and fibro-proliferative diseases. Also, altered levels of MBL and its associated serine proteases reported to be involved in different diseases such as cardiovascular diseases, diabetes, cancer, sepsis, stroke, COVID-19 etc. may have a role in thrombosis and thrombolysis. Upregulated lectin pathway-associated proteins predispose towards autoimmune disease susceptibility and may also exhibit off-target activities resulting in clot formation. Thus, the lectin pathway and its associated proteases are of great importance in normal circumstances where it plays a role in the maintenance of hemostasis and homeostasis, playing a preventive role in infections. Modulation in the level of MASPs proteases could be developed as a promising approach to treat a variety of infections and diseases.

Keywords: Fibrinogen; MBL; MASPs; Thrombin; Coagulation; Fibrinolysis

Abbreviations: MASPs: MBL Associated Serine Protease; rMASP- Recombinant MASP; FPA and FPB: Fibrinopeptide A and B, Respectively; TAFI: Thrombin-Activated Fibrinolysis Inhibitor; DIC: Disseminated Intravascular Coagulation; CVD: Cardiovascular Diseases; LP: Lectin Pathway.


Fibrinogen is a serum glycoprotein which is synthesized by the liver. It is a large molecular weight (340kDa) protein with three pairs of non-identical polypeptide chains (Hexameric homodimer) [1]. Such a structure of fibrinogen supports its complex roles in hemostasis and homeostasis [2]. During an inflammatory response, this protein is upregulated and expressed more. Different variants of fibrinogen are also formed as a result of alternative splicing. These have unique properties to contribute to the coagulation process following any vascular injury [3]. During coagulation, fibrinogen conversion to fibrin occurs via thrombin-mediated proteolytic cleavage. It produces intermediate protofibrils followed by mature fibers which eventually provide stable hemostatic clots and prevent blood loss at sites of tissue damage [4]. The processes of hemostasis and thrombosis involve interactions between fibrinogen and/or fibrin and plasma proteins and receptors on platelets, leukocytes, endothelial cells, and other cells [4]. Disorders in fibrinogen concentration and/or function increase the risk of bleeding, thrombosis, and infection [5]. Also, recent studies suggest that abnormal thrombin generation patterns produce abnormally structured clots that are associated with an increased risk of bleeding or thrombosis [6,4].

Further, a network of circulating blood cells and soluble proteins constitutes the host’s immune system which not only provides protection from various diseases but maintains homeostasis too. The complement system is one component of the immune system, which is a set of soluble proteins, which get activated in response to pathogens and help in clearing infection. There are three types of complement pathways, namely Classical, Alternative and Lectin. The lectin pathway [Mannan-Binding Lectin (MBL)], of complement activation, plays a key role in all types of infections and diseases. MBL, a pathogen recognition molecule of the innate immune system, remains associated with three serine proteases as MASP-1, MASP-2 and MASP-3 (MASP: MBL associated serine protease) [7].

The purple notches on top are CRD (carbohydrate recognition domain) through which MBL binds PAMPs on pathogen surface and associated serine proteases MASP-1, MASP-2 and MASP-3 are activated leading to lysis of pathogen [8].

Their role in the lectin pathway has been reported extensively; however, these serine proteases are pretty diverse in regard to their substrate selectivity and have substrates outside the complement pathway also. MASPs have been reported to share similar substrate specificity as that of thrombin which is also a serine protease. Hence, proteins of the coagulation pathway like fibrinogen, fibrin and prothrombin are cleaved by MASPs, however, the sites may be different [9].

During coagulation and fibrinolysis, the other cleaved fragments of fibrinogen and fibrin are released to perform various other roles in the repair process such as cell adhesion and spreading, vasoconstriction and chemotaxis [10]. These cleaved fragments have also been reported to act as mitogens for several cell types including fibroblasts, endothelial and smooth muscle cells. Such roles of these fragments open newer strategies to develop molecules for tissue repair or some preventive strategy for fibrosis in inflammatory and fibro-proliferative diseases where endothelial cell damage or chronic leakage of blood proteins is a feature [11].

This review deals with the unconventional cleavage of fibrinogen by MBL-associated serine proteases and its implications.

The Basic Mechanism of Fibrinogen Cleavage by Thrombin

Thrombin, a serine protease, plays a pivotal role in the blood coagulation pathway. It acts to convert various factors, including fibrinogen, into their active forms [6]. A significant part of this process is the conversion of soluble fibrinogen into insoluble strands of fibrin, which is critical for blood clot formation [12]. Thrombin accomplishes this by catalyzing the cleavage of fibrinopeptides A and B from the respective Aa and Bβ chains of fibrinogen, thus forming fibrin monomers [1]. This conversion process is one among a cascade of reactions that thrombin catalyzes in the coagulation pathway.

Fibrinogen, the substrate for this reaction, is a complex molecule composed of three pairs of different polypeptide chains: Aa, Bβ, and γ, held together by disulfide bonds [13]. The specific sites at which thrombin cleaves fibrinogen are located at the N-terminus of the Aa and Bβ chains. Following the cleavage, small peptides known as fibrinopeptides A and B are released. The specific cleavage sites are situated after residues Arg16-Gly17 on the Aa chain (releasing fibrinopeptide A) and after Arg14-Gly15 on the Bβ chain (releasing fibrinopeptide) [2].

Upon the cleavage of fibrinopeptides by thrombin, fibrinogen undergoes a transformation to fibrin monomers. This process exposes new polymerization sites on the fibrinogen molecule, allowing it to polymerize into fibrin, the main protein of blood clots [2]. It is this transformation that changes the soluble fibrinogen into insoluble fibrin strands. The fibrin strands then interact via "knobs" exposed by fibrinopeptide removal in the central region, with "holes" always exposed at the ends of the molecules. The resulting half-staggered, double-stranded oligomers lengthen into protofibrils, which aggregate laterally to make fibers [12]. These fibers then branch, yielding a three-dimensional network that forms the basis of a blood clot [2]. This entire process is crucial for hemostasis, the cessation of bleeding following vascular injury [13].

MASP-1 in Coagulation

The activation of serine proteases namely MASP-1, 2, and 3 of the lectin pathway of the complement system is an important event for its functioning to achieve pathogen clearance. MASP-1 forms an association with MBL/Ficolins in response to foreign moieties and auto-activates itself, downstream of that it also activates other associated serine proteases such as MASP-2 [14]. Further, the complement system, through the mediation of the coagulation components, builds a greater response to invading pathogens [15,16], probably causing aggregation of pathogens [17], thus limiting the dissemination of infection. Such studies have clearly demonstrated that the complement system and the coagulation cascade are intricately interlinked. Furthermore, in vitro and in vivo studies have also supported that MBL/Ficolins-MASP-1/2 have a pivotal role in the coagulation system [9,18].

In the thrombotic pathway, the involvement of MASP-1 in the lectin pathway is crucial [19] as it plays a predominant role in the coagulation system [20]. MASP-1 interacts with a broad range of proteins involved in the coagulation system such as coagulation Factor XIII (FXIII), fibrinogen, prothrombin etc. [21,17,22] (discussed in Figure 3). In coagulation pathway, prothrombin gets activated to thrombin in a conventional way by activated Factor X (FXa), however, recombinant MASP-1(rMASP-1) and rMASP-2 also have shown to catalyse the same reaction [21,17]. In this catalytic cleavage, the underlined mechanism of prothrombin cleavage by rMASPs may be different from FXa. Functional studies by [21] have shown that MASP-1 may potentially activate the prothrombin by cleaving the arginine (R) at either 271 or 393 position however, in the conventional pathway of thrombin generation, prothrombin cleavage occurs at R155 and R284, which are crucial sites, however, it is not true for MASP-1 [21]. Apart from this, prothrombin cleavage by MASP-1 at R320 can also activate it to thrombin. We were the first to demonstrate that MASP-1 also possesses thrombin-like activity [23] and shares similar substrate specificity as thrombin but with reduced catalytic potential [17]. Biochemical evidence suggests that both MASP-1 and thrombin act on fibrinogen. These share an identical cleavage site for the β-chain but a differential cleavage site for the a-chain of the fibrinogen during its polymerization. During its cleavage, MASP-1 leads to the generation of fragmented γ2-chain along with a few oligomers of a-chain. Upon cleavage of the fibrinogen, MASP-1 mainly releases fibrinopeptide B in contrast to thrombin, which releases both fibrinopeptide A and B. In terms of target specificity (a-chain), MASP-1 seems to be less specific than thrombin [17].

Cleavage of fibrinogen by thrombin results in the polymerization of the alpha chain (alpha n) with the complete disappearance of the alpha chain band. The γ chain dimmers are formed by factor XIII present as a contaminant present in fibrinogen. Factor XIII is converted to Factor XIIIa by the action of thrombin in the presence of calcium. Fibrinogen cleavage by purified MASP-1 essentially required Ca++ as in the absence of Ca++ fibrinogen cleavage is not observed. (Figure 02 lane 4, lane 6). However, no difference is observed in fibrinogen cleavage by recombinant MASP-1 in the presence and absence of Ca++ (data not shown). In both cases, only the alpha chain is cleaved. It is to be noted that in our studies recombinant MASP-1 lacks the Ca++ binding EGF domain. The fibrinogen cleavage by MASP-1 was found to be completely inhibited in the absence of Ca++ or the presence of 5 mM EDTA (lane 6) or 5 mM EGTA (lane 8). In our study presence of Mg in place of Ca++ could not restore the fibrinogen-cleaving activity of MASP-1(Lane 10). The fibrinogen cleavage by serum-purified MASP-1 is stopped completely by a C-1 inhibitor [23].