(i) Human plasminogen (Pg, profibrinolysin)

The gene for human plasminogen (52 kb) is located on chromosome 6 and consists of 19 exons and 18 introns. Human plasminogen (92 kDa) is synthesized as a single polypeptide chain of 810 amino acids while the mature protein is 791 amino acids (2373 bp) having a leader sequence of 19 residues (57 bp) that is cleaved during secretion. Blood Derivatives And Thrombolytic Drugs Essay. The primary source of plasminogen is the liver, however, other sources like the adrenal glands, thymus, lung, brain, gut, uterus, kidney, spleen, testis and heart have also been identified. Its concentration in the plasma is 2.2 µM. The protein is glycosylated at Asn²⁸⁹, Thr³⁴⁶, Ser²⁴⁸, Thr³³⁹ and phosphorylated at Ser⁵⁷⁸.¹⁵ Plasminogen activators catalyze the activation of plasminogen to plasmin (Pm) by inducing a proteolytic cleavage at the Arg⁵⁶¹-Val⁵⁶²bond. Plasmin is a 2-chain molecule held together by 2 disulfide linkages. The N-terminal heavy chain of human plasmin (Glu¹-Arg⁵⁶¹) carries 5 kringle (K) domains that are triple-disulfide linked protein modules involved in interaction with lysine. Domains K1 and K4 exhibit the strongest affinity with lysine, while K2 has the weakest affinity.¹⁵Binding of plasmin to fibrin is mediated via the lysine-binding sites on these kringle domains. Kringle 1 is also involved in plasmin binding to the α2-antiplasmin inhibitor, one of the fastest protein-protein interactions (rate of the inhibition reaction is ∼10⁷ M⁻¹s⁻¹).¹⁶ Consequently, plasmin bound to fibrin impedes inactivation by this inhibitor. The C-terminal light chain carries the serine protease domain with His⁶⁰³, Asp⁶⁴⁶ and Ser⁷⁴¹as the catalytic triad.¹⁶ The heavy chain also accommodates a 77 residues N-terminal peptide region, which is cleaved during in vitro activation of plasminogen by autolysis. The Glu¹-Pm and Lys⁷⁸-Pm can catalyze the hydrolysis of the N-terminus 77 amino acids of Glu¹-Pg or Glu¹-Pm, converting Glu¹-Pg to Lys⁷⁸-Pg and Glu¹-Pm to Lys⁷⁸-Pm. Such a plasmin or plasminogen molecule having an N-terminal glutamic acid residue has a closed conformation while that with lysine as the N-terminal residue has a more open conformation making it the preferred substrate for activation by plasminogen activators.¹⁵ Plasmin degrades fibrin as well as fibrinogen, and other coagulation factors including factor V, VIII, IX, XI, XII, insulin and growth hormones.¹⁷ Various cell types such as monocytes, lymphocytes, granulocytes and endothelial cells express plasminogen receptors, which interact with plasminogen to accelerate its conversion to plasmin. The surface bound plasmin has an enhanced activity and is also protected from inactivation via inhibitors compared to plasmin in the blood plasma. Blood Derivatives And Thrombolytic Drugs Essay. Cell surface associated plasmin can also mediate the degradation of extracellular matrix and basement membrane molecules.¹⁸

(ii) Fibrinogen

It is the most abundant plasma protein (2–4 g/L) synthesized mainly by the hepatocytes and in smaller amounts by the epithelium of the intestine, cervix, and lungs with a half-life of approximately 4 d.¹⁹ The FGA, FGB and FGG genes, coding for 3 fibrinogen chains namely Aα, Bβ and γ, are located on chromosome 4 in a ∼50 kb region.²⁰ The first step in fibrinogen assembly is the formation of 2 chain complexes Aα-γ and Bβ-γ. In the next step, a third chain is added to form a 3 chain complex Aα-Bβ-γ. Finally, 2 of the 3 chain complexes join at the N-termini to form the dimeric hexamer (Aα-Bβ-γ)₂ of 340 kDa held together by 29 disulfide bonds.¹⁹ The central portion of this glycoprotein (domain E) comprises of the N-termini of the 6 chains. The domain D is a globular structure composed of the carboxy terminal of the Bβ and γ chains. The coiled coils of the α-helices of the 3 chains connect these domains. The spontaneous polymerization of the fibrinogen molecule is prevented due to the presence of negative charges at the N-terminus of the Aα and Bβ chains. Thrombin catalyzes the hydrolysis across the Arg¹⁶-Gly¹⁷ and Arg¹⁴-Gly¹⁵ peptide bonds in the N-terminal of the Aα and Bβ chains, respectively, due to which fibrinopeptides A and B are cleaved from fibrinogen. This partially degraded form of fibrin is called the fibrin monomer. Cleavage of fibrinopeptides A and B unmasks positively charged end groups which are the sites for polymerization termed as E_A (Gly-Pro-Arg-Val) and E_B (Gly-His-Arg-Pro). They interact non-covalently with negatively-charged complementary polymerization sites D_a and D_b in the C-terminal portion of the γ and β chains.²¹ The fibrin monomers interact with each other in a half-staggered manner to form protofibrils, a 2 stranded fibrin polymer, leading to the polymerization process. Alongside, thrombin catalyzes the conversion of factor XIII to active factor XIIIa, a transglutaminase that stabilizes the fibrin structure by cross-linking. The cross-linked fibrin is mechanically stronger and more resistant to proteolysis by plasmin. Blood Derivatives And Thrombolytic Drugs Essay.

During degradation, plasmin mediates cleavage in the C-terminal portion of the Aα chain and attacks the amino and carboxy terminal part of the Bβ chain. The γ chain hydrolysis is relatively slow.²² The first degradation product termed fragment X (∼260–300 kDa) is formed after the cleavage and removal of the C-terminal region of the Aα chain at Lys²⁰⁸-Met²⁰⁹, Lys²²¹-Ser²²² or Lys²³²-Ala²³³. This fragment is clottable by thrombin with an increased affinity toward plasmin and has a faster lysis rate compared to clots formed by intact fibrin.²² Fragment X is further hydrolyzed giving rise to the fragments Y (∼150 kDa) and D (∼94 kDa). Fragment Y is further degraded producing another fragment D and fragment E (∼50 kDa). Degradation of cross-linked fibrin produces D-dimers instead of D fragments.²³