The Fibrinolytic System in the Interstitial Space | 5

ABSTRACT

The fibrinolytic system contains a number of proteins, including proenzyme/enzymes, inhibitors, modulators, and one biologically active peptide, angiostatin, derived from plasminogen/plasmin (see Table 5.1). ¹ ^, ² The canonical function of the fibrinolytic system in the vascular space is the orderly dissolution of fibrin clots in the process of wound healing and the dissolution of intravascular fibrin in the prevention of thrombosis. It is clear, however, that the components of the fibrinolytic system have considerable function beyond the dissolution of fibrin, ³ and it is likely that the majority of these functions occur in the interstitial space in tissues. Indeed, the “off-label” activity of the various components of the fibrinolytic system may well be the predominant function in the interstitial space. The actual process of fibrinolysis as demonstrated by the digestion of fibrin and fibronectin is likely limited to the resolution of fibrotic lesions formed by fibroblastic proliferation into provisional fibrin matrices. As an example, transforming growth factor (TGF)-β1 is suggested to enhance the expression of plasminogen activator inhibitor (PAI)-1, which is considered to be a profibrotic agent based on its inhibition of urokinase-type plasminogen activator (uPA). ⁴ In the study by Samarakoon and coworkers ⁴ and in reviews of uPA and its receptor (uPAR), the emphasis is not on the dissolution of fibrin in the interstitial space but rather on the activation of matrix metalloproteinases (MMPs) and action on cell surface receptors. ⁵ ^, ⁶ The effect of TGF-β on the components of fibrinolysis in pleural mesoepithelial cells and the subsequent effect on fibrosis is shown in Figure 5.1. Briefly, TGF-β enhances the release of PAI-1, resulting in the inhibition of plasmin formation, which in turn decreases production of MMP-1 and the resolution of fibrosis. Some of the plasminogen inhibitors may have pleomorphic functions that remain to be decscribed ⁷ ^– ⁹ but could be of great importance in the interstitial space. Table 5.1 Components of the Fibrinolytic System in the Interstitial Space

Component	Description/Function	Presence in Interstitial Space
Plasminogen (Plg)	Plasminogen is a single-chain precursor of plasmin existing in several forms with molecular weights of approximately 90 kDa. ¹ ^– ⁴ Activation of the single chain yields plasmin consisting of two polypeptide chains joined by disulfide bonds. Proteolytic digestion releases angiostatin, which is involved in angiogenesis. Plasminogen binds to cell surface receptors, such as a-enolase, that possess a C-terminal carboxyl group and to pro-uPA bound to uPAR. Plasminogen can also bind to cell surface receptors on bacteria, promoting activation by bacterial plasminogen activators. ⁵ Kringle domains were first described in plasminogen as 80 peptide regions with three disulfide bonds, with structural similarity to a Danish pastry; the kringle domains bind lysine-containing ligands and macromolecules such as fibrinogen. Plasminogen exists in multiple glycoforms, ⁶ which may have functional consequences. ⁷	Presence in rat interstitial fluid at approximately 40% of plasma. ⁸ Presence in interstitial fluid is also inferred from analysis of peripheral lymph. ⁹ Plasminogen has been shown to be present in cerebrospinal fluid. ¹⁰ Degraded forms of plasminogen are found in synovial fluid. ¹¹ ^, ¹²
Plasmin (Pm)	Plasmin is a serine protease derived from plasminogen with a preference for cleavage at peptide bonds, where the carboxyl group is provided by lysine and, to lesser extent, arginine. The canonical function is digestion of fibrin, but plasmin is important in the interstitial space for the initiation of proteolytic cascades and digestion of fibronectin in the ECM. Thus, plasmin is an interesting protease that has both a digestive function in its action on fibrin and fibronectin and a regulatory function in the activation of pro-uPA and some MMPs.	It is likely that plasmin is generated from plasminogen in the interstitial space. ⁸ Plasmin does not appear to degrade fibrinogen or fibrin in the interstitial space as fibrinogen but not fibrin degradation products are present in lymph. ¹³ Plasmin that is generated in the interstitial space is probably inactivated rapidly by inhibitors such as α₂-antiplasmin.
Urokinase-type plasminogen activator (uPA, urokinase)	uPA is responsible for the conversion of plasminogen to plasmin. The conversion of plasminogen to plasmin by uPA is most efficient on the cell surface in the presence of plasminogen and uPAR. Urokinase was a therapeutic product (Abbokinase®) originally isolated from urine for the lysis of fibrin clots; Abbokinase is now obtained from cell culture technology. Urokinase is a serine protease that can be isolated in several forms that differ in molecular weight.	No direct measurement of uPA in lymph. However, the presence of uPA in the interstitial/extravascular space is inferred from in vitro studies. ¹⁴ ^, ¹⁵
Urokinase-type plasminogen activator receptor (uPAR)	uPAR is a highly glycosylated membrane protein with a molecular weight of approximately 60 kDa bound to the membrane by a glycosylphosphatidylinositol anchor. ¹⁶ As such, uPAR can initiate intracellular signaling but must rely on interaction with lateral partners such as integrins. It is best known for binding pro-uPA and uPA. uPAR also binds to vitronectin. There is considerable interest in the role of uPAR in oncology. ¹⁷	uPAR has a wide tissue distribution including monocytes and fibroblasts. Distribution to various organs is increased with tissue remodeling. ¹⁸ ^, ¹⁹ There is increased expression in tumors and during inflammation. A soluble form is released during inflammation.
Streptokinase	A 45 kDa protease secreted by several strains of Streptococcus that converts plasminogen to plasmin on the surface of the bacteria. ²⁰ It is an important factor in the virulence of the bacteria as it provides a mechanism for tissue destruction and concomitant bacterial invasion via either the direct action of plasmin or MMP. Streptokinase has use as a therapeutic for the lysis of fibrin clots.	The interstitial space is the area where bacterial invasion and ECM degradation takes place.
Staphylokinase	Somewhat less known is staphylokinase, a plasminogen activator secreted by lysogenic strains of S. aureus. ²¹ Staphylokinase is involved in the invasion of pathogenic bacteria by promotion of plasmin formation, which destroys the ECM. ²²	The interstitial space is the area where bacterial invasion and ECM degradation takes place.
Tissue plasminogen activator (tPA)	tPA is a serine protease expression on the luminal surface of endothelial cells and is responsible for initiation of intravascular fibrinolysis. ²³ Recombinant tPA is used for the treatment of intravascular thrombosis.	Little evidence to support the presence of sufficient tPA in the interstitial space to activate plasminogen. Secretion of tPA by endothelial synthesis is apical (luminal).
Plasminogen activator inhibitor type 1 (PAI-1, serpinE1)	PAI-1 is a serpin that is considered to be the most important inhibitor of uPA in the interstitial space. PAI-1, as with certain other serpins, can adopt a latent conformation that is not effective as a protease inhibitor ²⁴ but can be converted into an active form by treatment with chaotropic agents. ²⁴ ^, ²⁵ PAI-1 expressed in cell culture contains more than 90% latent material. In plasma and interstitial space, PAI-1 is stabilized by binding to vitronectin. ²⁶ ^, ²⁷ The complex of PAI-1 with target proteases such as uPA, tPA, and perhaps thrombin binds to lipoprotein receptor–related protein.	It was not possible to find information on the concentration of PAI-1 in interstitial fluid or lymph. The concentration of PAI-1 in serum has been determined in oncology patients. Much tissue measurement of PAI-1 is performed using immunohistochemistry, which measures PAI-1 associated with vitronectin.
Plasminogen activator inhibitor type 2 (PAI-2, serpinB2)	PAI-2 is a largely intracellular protein lacking a clearly defined function. ²⁸ The intracellular form has a molecular weight of approximately 44 kDA, while the extracellular form has a molecular weight of approximately 60 kDa, reflecting the glycosylation of the intracellular protein prior to secretion. ²⁹	Keratinocytes secrete PAI-2 to control uPA in the interstitial space of skin. ³⁰
Plasminogen activator inhibitor type 3 (PAI-3, serpinA5)	PAI-3 was found to be identical with protein C inhibitor. ³¹	Protein C inhibitor is present in bronchoalveolar lavage fluid and is associated with reduced fibrinolytic activity in interstitial lung disease. ³²
α₂-Antiplasmin (serpinF2)	α₂-Antiplasmin is a glycoprotein with a molecular weight of approximately 70 kDa and is considered to be the major inhibitor of plasmin in the vascular space. ³³ α₂-Antiplasmin is best known for the inhibition of plasmin; it can also inhibit human kallikrein 5 (KLK-5) with modest affinity, ³⁴ as well as trypsin and chymotrypsin. The reaction of α₂-plasmin inhibitor with trypsin is far more rapid than with either plasmin or chymotrypsin, which are in turn three orders of magnitude more rapid than the reaction with KLK-5. Oxidation of α₂-antiplasmin did not affect the rate of reaction with either trypsin, chymotrypsin, or plasmin. ³⁵ Plasmin bound to uPAR after formation from plasminogen was resistant to inactivation by α₂-antiplasmin. ³⁶ Plasminogen has an unusual structure consisting of three domains: the C-terminal region, which contains a cluster of basic amino acid residues and interacts with plasmin; the serpin domain, which contains the site that reacts with plasmin, resulting in inactivation; and an N-terminal extension that can be cross-linked to fibrin by factor XIIIa. ³⁷	Present in interstitial fluid at approximately 20% of the plasma concentration as shown in a rat model. ⁷
Vitronectin	A protein component of the ECM that binds PAI-1. The binding is associated with the stabilization of PAI-1 and enhances the inactivation of tPA and uPA. Upon binding to vitronectin, PAI-1 also becomes a slow-acting thrombin inhibitor. The role of vitronectin is complicated by the viability of a vitronectin knockout mouse. ³⁸	Soluble monomeric vitronectin is thought to be transported from the vascular space into the interstitial space and deposited into the ECM. ³⁹ ^, ⁴⁰ There has been some suggestion of extravascular sites of synthesis. ⁴⁰
Angiostatin	A fragment derived from plasminogen via the limited proteolysis and reduction of two disulfide bonds. A reduced form of plasminogen can serve as a precursor for angiostatin with the participation of phosphoglycerate kinase. ⁴¹ The size of angiostatin fragments depends on the protease responsible for the fragmentation of plasminogen. While angiostatin formation is closely associated with tumor cells, it has also been shown to be derived from macrophages during inflammation. Angiostatin can exist in multiple glycoforms derived from plasminogen glycoforms. ⁶	Though there is no evidence for the formation of angiostatins in the interstitial space, there is rapid transport of both angiostatin I and angiostatin II from the intravascular space to extravascular space. ⁴²

Figure 5.1 The role of the fibrinolytic system in TGF-β in the development of fibrosis. Activation of macrophages releases TGF-β in a latent form, which is activated by proteolysis. TGF-β enhances the release of PAI-1 from pleural mesothelial cells. (From Idell, S., et al., In vivo, Am. J. Respir. Cell Mol. Biol. 7, 414–426, 1992.) The net effect is the inhibition of plasmin, which prevents MMP activation and the subsequent resolution of fibrosis. (Adapted from Samarkoon, R., et al., Cell. Signal. 25, 264–268, 2013.) As can be observed, PAI-1 has additional profibrotic activities. https://s3-euw1-ap-pe-df-pch-content-public-p.s3.eu-west-1.amazonaws.com/9781315374307/eafc876c-1c18-4a14-bc4b-9d9665df6318/content/fig5_1_B.jpg" xmlns:xlink="https://www.w3.org/1999/xlink"/>