The biologic role of components of the plasminogen-plasmin system

Progress

Cardiovascular VOL XXXIV,

in

Diseases

NO 5

The Biologic

MARCH/APRIL

1992

Role of Components of the Plasminogen-Plasmin Sys tern Hau C. Kwaan

T

HE PLASMINOGEN-PLASMIN system is composed of the proteolytic enzyme plasmin, with its precursor plasminogen and its activators of the urokinase-type (uPA) and the tissue-type (tPA).’ Their catalytic actions are modulated by inhibitors of plasmin and of plasminogen activators. Plasmin inhibitors include a,-antiplasmin and g-macroglobulin, and plasminogen activator inhibitors (PAI) include the type 1 (PAI-1) and type 2 (PAI-2). The actions of the plasminogen activators are facilitated by the presence of receptors of plasminogen, uPA and tPA, on cell surfaces as well as in the circulation. In recent years, much knowiedge has been gained on the interactions of all these components at the cellular level. This has increased our understanding of the complex pathogenesis of thrombosis and the mechanism of thrombolysis as well as of atherogenesis. An attempt to review some of these observations will be made in this article as befitting this symposium on thrombolysis. CELLULAR EXPRESSION OF PLASMINOGEN ACTIVATORS AND THEIR INHIBITORS

Expression of the various components of the plasminogen-plasmin system has been observed in many cells in culture and in tissues. These components are now believed to be actively involved in many biologic functions at the cellular level in such processes as embryogenesis, ovulation, neuron growth, muscle regeneration, wound healing, angiogenesis, and tumor growth and invasion (Table 1). One notable example of their importance is the expression of the plasminogen activators, especially uPA, in many tumor cells in culture and in human tumor

Progress

in CardiovascularDiseases,

Vol XXXIV,

No 5 (March/April),

tissues.“’ In carcinoma of the breast,‘-” co1on,11.12 prostate,7*‘3-16lung,” and malignant melanoma,18 the expression of uPA was correlated with an unfavorable clinical prognosis. Though most of these studies have been performed on cultured tumor tissues and cultured tumor cells, the findings are applicable to the other biologic processes mentioned above. In particular, the hypothesis on the mechanism of tumor cell invasion as shown in Fig 1 applies equally to that on cell migration in other biologic processes. The expression of uPA, tPA, and the PAIs in endothelial cells and in smooth muscle cells (SMC) is relevant to their role in thrombosis and in atherogenesis, especially during the reparative process following vascular injury. Their expression is modulated by the transcriptional regulation of a variety of cytokines,19-u including interleukin-1, interleukin-4, interferon gamma, and tumor necrosis factor,24 by hormones, such as corticosteroids25.26 and gonadotrophins,27,28 and by growth factors,3zu including epidermal growth factor,29.3Dbasic fibroblast growth factor,31-35transforming growth factors alpha and beta,3,23 platelet-derived growth factors,36-38platelet-derived endothelial cell growth factor,39 and colony stimulating factors.40

From the Department of Medicine, Northwestern University Medical School, and the HematologylOncologv Division, VA Lakeside Medical Center, Chicago, IL. Address reprint requests to Hau C. Kwaan, MD, Chief; HematologvlOncology, VA Lakeside Hospital, 333 E Huron St, Chicago, IL 60611. Copyright 0 I992 by W B. Saunders Company 0033-0620/92/3405-0001$5.00/O

1992:

pp 309-316

309

310

HAU

Table 1. Physiological and Pathological Conditions in Which Plasminogen Activator-Mediated Extracellular Proteolysis Has Been

Described Cells

Physiological conditions Embryogenesis

Trophoblastic tion””

Ovulation

Leading

growth

edge

of neuronsoa’

Keratinocytes2.”

Connective tissue Capillaries Skeletal muscle Vascular injury

MODE

implanta-

Oocytez8 Thecal and granulosa cellsz7 (gonadotrophin regulated)

Neuron growth Pathological conditions Wound healing Epidermis

Tumor sion

Involved

Fibroblasts Endothelial Myotubular Endothelial

cells” formation cells, smooth

muscle Endothelial

and inva.

sisF Tumor

cells’3 cells cells

In many biologic processes, such as tissue repair and remodeling, cell movement is an essential step; the migrating cell has to penetrate its surrounding extracellular matrix. This is accomplished by the focal proteolysis of the extracellular matrix proteins consisting of collagen, laminin, fibronectin, elastin, vitronectin, and fibrinogen.4’-43 A group of three major metalloproteinases, namely collagenase, stromel-

TGF

e

ysin, and gelatinase, are synthesized and secreted by many cells, enabling them to degrade the extracellular matrix.44-47 These metalloproteinases are regulated by tissue inhibitors.46V47 When secreted, the metalloproteinases are in a latent form with their activation in vivo accomplished primarily by plasmin.5@q49 Thus, the availability of plasmin, generated by uPA or tPA, is an important step in cell migration in both physiological and in pathological conditions. Recent observations that uPA can activate the latent metalloproteinases in the absence of plasminogen focuses attention to the role of uPA rather than that of tPA.5,49 ROLE OF tPA

(angiogene-

OF ACTION OF UPA-FACILITATING CELL MIGRATION

TRANSCRIPTIONAL REGULATION: (STEROIDS, TNF, INTERLEUKINS, OTHERS)

C. KWAAN

tPA has long been considered to be the physiological plasminogen activator generated by the endothelial cell and has a major role in the regulation and control of fibrin deposition on the intimal surface. However, such may not be the case with its cellular function. In the case of tumor growth and invasion, the respective effects of uPA and tPA are sometimes quite opposite. For example, in carcinoma of the breast and of the colon, uPA, but not tPA, was found to be an independent and strong prognostic marker for relapse and overall survival.“” Whether or not the expression of tPA is important for the regulation of endothelial cell and smooth muscle function is not presently known.

......x..r .,:.:. .,:>, ‘:: .:y :.;:.:.,I,1..,:,:,.,gcy :? .: .:, “=,‘j,yt,x~ DNA::..”..... : .A.: :.... i.. ..I.._ :: : : :,.:::.j,. ... ::.:.‘:‘:‘:‘.:.: E?:;::: ;: .:.:.:.:... ..... .:I ‘:::, ,:p:,:..:.::.::::.: m:.::.:.:‘:.:::: .:

$’

\

y& +j$ . ..:..\

EXTRACELLULAR> ‘ii? ,CO,“~~~~“LA”,k-+=~ FlBROlVECT;N. OTHER)

ELASTIII, *“e;; k ‘,A+?!!*,.;, VZ’ /y-x:.;:

flETALLOPROTEASES ( COLLACEWASES. ELASTASE. LAIlllASE . OTHERS )

Fig 1. Hypothesis cell invasion. (Reprinted mission.‘)

of tumor with per-

BIOLOGY

OF UROKINASE

311

Although both tPA and uPA are plasminogen activators, their discordance in cellular function may be explained by many recent observations relative to their respective inhibition by the PAIs uPA is secreted by cells in the singlechained form, which is resistant to inhibition by PAIs. The single-chained form is bound to its receptor on the cell surface, where it can then be converted to the two-chained form. In contrast, tPA is not protected from inhibition by the PAIs. Membrane-bound uPA is phosphorylated, resulting in a 60% decrease in its reactivity with the PAIs, while at the same time altering its catalytic efficiency with a fivefold increase in its Kcat.” A similar picture has not yet been observed with tPA. MODE

OF ACTION OF uPA-ROLE PROLIFERATION

IN CELL

In addition to its role in proteolysis of the extracellular matrix protein, uPA was found to be a growth stimulant for the human epidermal tumor cell line” and to be mitogenic for malignant renal cells5’ and for an osteoblast-like cell line.53 The aminoterminal fragment (Ser[l]Glu[143]) (ATF) may be the domain in the uPA molecule responsible for this mitogenic effect. A specific uPA-cleaving enzyme catalyzing the proteolysis of the Glu[143]-Leu[144] bond has been isolated from cultures of renal cells. This fragment may also be produced by the proteolytic action of plasmin at the Lys[134]-Ljrs[135] bond. The ATF contains the epidermal growth factor-like domain and the kringle structure in the A-chain but not the catalytic domain. Therefore, the mitogenic element is distinct from that of the proteolytic function of uPA. These findings may have implications in the proliferation of endothelial cells and smooth muscle cells during vascular repair and atherogenesis, because vascular injury induces increased expression of uPA in these cells. ROLE OF UPA RECEPTOR l

Biologic events taking place on the cell surface involving uPA are greatly enhanced by the presence of a specific receptor for this enzyme.5”59 This receptor has been identified as a 55 to 60 kd single-chained protein molecule anchored to the cell membrane by a glycolipid. The ligand binding site is located at the growth

factor domain in the A chain of uPA. Receptorbound uPA has a twofold lower Km and a sixfold higher Kcat than the solution-phase uPA, giving it greatly increased catalytic efficiency (Kcat/Km). The presence of this receptor allows uPA to be localized on the cell surface in high concentrations at focal points where activation of plasminogen takes place during cell migration. uPA produced by cells is mostly in the single-chained form. The receptor is able to bind both the single- and two-chained uPA. Receptor-bound single-chained uPA is rapidly converted to the two-chained form by various proteases on the cell surface, such as plasmin, leading to further amplification of its proteolytic action. ROLE OF PAI

Both PAI- and PAIare present on cell surfaces. In contrast to uPA, which is localized on the cell surface at focal areas of cell adhesion sites, PAI- is uniformly distributed in the cell substratum.@‘s6’ Such differences in the distribution of uPA and its inhibitor allow proteolysis to occur at the focal points where the activator is located. It would appear that the inhibitor in the cell substratum provides a foothold for the migrating cell, whereas the uPA-induced proteolysis of the extracellular matrix at the focal cell adhesion sites allows cell movement. The inhibition of the activator is also attenuated on the cell surface in several ways. Single-chained uPA and phosphorylated uPA found in the cell membrane is resistant to inhibition by both PAI- and PAI-2.50 As such, uPA produced by the cell can be secreted and attached to its receptor without much interference by its inhibitor on its proteolytic function until after it is converted to the two-chained form. ENDOTHELIAL

CELL

Although much of the previous picture applies to endothelial cells in culture, few data are presently available on what occurs in vivo during vascular injury. From the available published information, one can summarize the various interactions between the components of the plasminogen-plasmin system and the events taking place in endothelial cells (Fig 2).62 Both the activators, uPA and tPA, are produced by

312

HAU

PLASMIN

P.C. -

t

A.P.C.

k

Fig 2. Interaction of the various components of the plarminogan-plasmin system on endothelial cell surface. Arrows denote activation, arrowheads denote secretion, and interrupted arrows denote inhibition of the respective reactions. P.C., protein C; A.P.C., activated protein C; TM, thrombomodulin; UPA-R, UPA receptor; tPA-Ft. tPA receptor; PG. plasminogen; PG-l?, plasminogen receptor; Lp (a), lipoprotein(a).

the endothelial cells. Ligand binding proteins are also present on the cell surface, specificly for their respective plasminogen activators, uPA and tPA, and for plasminogen. Receptors for plasminogen are present in high density on the cell surface, thereby providing plentiful zymogen available for the activation by UPA or tPA. In addition, the endothelial cells convert circulating native Glu-plasminogen to Lysplasminogen, an intermediate form of zymogen that has a higher affinity for fibrin and can be more readily activated to plasmin by either UPA or tPA.63 The fibrinolytic system can also be modulated on the endothelial surface through other pathways. (1) A glycoprotein, thrombomodulin, generated by the endothelial cells binds thrombin in the event of clotting on the endothelial surface.@ A thrombin/thrombomodulin complex is formed and is able-to activate protein C in the circulating blood. Activated protein C enhances fibrinolytic activity by inhibiting PAI-1. The importance of protein C is reflected in the high thromboembolic risk noted in patients with congenital deficiency of this protein. (2) Recent epidemiologic studies showed that high plasma levels of lipoprotein(a) is associated with an increased risk of atherosclerotic cardiovascular disease.65 A subunit marker of this lipoprotein, ape(a) has a structure with extensive homology to that of plasminogen and, as such, lipoprotein(a) can compete with plasminogen in impairing several fibrinolytic functions, including binding with fibrin, with plasminogen receptors on the endothelial surface, and with heparinbound tPA.66*67 On the other hand, lipopro-

C. KWAAN

tein(a) may also compete with PAI- for fibrinogen bound tPA, exerting the opposite effect on fibrinolysis. Further studies of this interesting lipoprotein will undoubtedly cast new light on these interactions. (3) Endothelial cell production of tPA, uPA, and the uPA receptor may also be modulated through the cyclic adenosine monophosphate (CAMP) and the protein kinase C pathways.68,69For example, it can be regulated by histamine and by thrombin which stimulates the protein kinase C pathway. (4) A major adhesive glycoprotein, vitronectin, is present in the circulating blood as well as in the subendothelial matrix and has multiple functions with the fibrinolytic system on the vascular surface.” It stabilizes PAI- by forming a binary complex. Similar binding also takes place with the other inhibitors of plasminogen activators, namely PAI- and protease nexin I. SMOOTH

MUSCLE

CELLS

During the process of healing following all forms of vascular injury, proliferation of SMCs takes place. Excessive growth of the SMC can lead to intimal thickening, which is frequently the major cause of restenosis following arterial reconstructive surgery, such as endarterectomy and bypass grafts, and following angioplasty.‘l Intimal thickening from SMC hyperplasia is also an essential component in atherogenesis. The regulation of SMC proliferation has been extensively studied, and the important role of various growth factors, such as the plateletderived growth factor and basic fibroblast growth factor, is well recognized and reviewed elsewhere.37’72 Recently, the components of the plasminogen-plasmin system have been found to be involved in the regulation of SMC growth.73 Following intimal injury, several reparative steps occur (Fig 3). Platelet deposition takes place on the injured surface. Growth factors from these platelets may contribute to stimulating the proliferation of the reparative cells (endothelial cells and SMCs). The injury also leads to monocyte infiltration bringing with them growth factors that will upregulate uPA production of both endothelial and SMCs. Monocytes themselves may also express uPA. Endothelial cells regenerate by ingrowth from adjacent uninjured intima, but this is frequently limited and may

BIOLOGY

OF UROKINASE

313

VASCULAR

MONOCYTE DERIVED

-

(MECHANICAL, INFLAMMATORY, IMhlUNOLOGIC,

INJURY

ENDOTOXfN,GI‘C)

J RELEASE

QROWI-H FACTORS (LFGF, PDGF, PDRGF, TGFb)

OF

(TNF, &

I

CYTOKINES

IL-l,

IL-4,

IFN-‘6

,)

OF HORMONES ( CORTICOSTEROIDS,

TRANSCRIIl’IONAL OF uPA, tPA,

)

1

INDUCITON

PAi-1,

PAI-

1 EXPRESSION

OF uPA,

tPA,

PAI-1,

PAI-2

1 ENDOTHELIAL (ANQIOQENESIS)

SMOOTH

CELL

MUSCLE

PROLIFERATION

CELL

MIGRATION

AND

PROLIFERATION

I MTlMAL

Fig 3. Events intimal hyperplasia.

following

HYPERPLASIA

vascular

injury

that

may

lead

to

not completely cover the damaged intimal surface. At the same time, there is migration of proliferating SMCs from the media to the intima, leading to a myointimal hyperplasia. The resultant thickening of the intima is undesirable as it causes narrowing of the vascular lumen. The initial SMC hyperplasia is stimulated by endogenous mitogens, such as basic fibroblast growth factor, released by the injury, and is also brought to the site of injury by monocytes. Subsequent migration of the SMC is probably regulated by platelet-derived growth factor. Concomitant with these changes, the participating cells exhibited the mRNA of both tPA and uPA, as well as the protein expression and secretion of these proteins. It is believed that both the plasminogen activators enable the proliferating SMCs to migrate from the media to the intima. At present, there are no data available on expression of PAIs by these cells. THERAPEUTIC

IMPLICATIONS

Much of the work on the plasminogenplasmin system in the past has been devoted to thrombolysis. Attention has now been shifted to the cellular level in many biologic processes. The significance lies in the regulation of two important steps in tissue repair and remodeling, namely cell migration and cell proliferation. In

vascular disease, lumen narrowing and thrombosis are the ultimate complications stemming from pathological reparative processes leading to intimal thickening following injury. Recent findings described previously suggest that factors stimulating the expression of the components of the plasminogen-plasmin system are released following injury. A better understanding of the regulation and modulation of these proteins may lead to a new approach in the development of therapeutic inhibitors of intima1 hyperplasia. Identification of the growth factors that upregulate the expression of uPA and tPA in cells may allow the development of mutant growth factors that compete, thus blocking the action of these factors. An example is observed in mutants of acidic fibroblast growth factors74 and of platelet-derived growth factor receptor.75 Synthetic peptides can also be designed based on the structure of the binding ligand.76 For instance, the epidermal growth factor domain of UPA is the binding element of uPA to its receptor. Such binding can be intercepted by a competing peptide with a structure similar to this portion of the uPA molecule. Finally, specifically designed drugs may be developed. For example, heparin in large doses can prevent SMC proliferation and intimal hyperplasia following arterial injury, but at the same time stimulate endothelial cell hyperplasia.“~” Of great interest is the finding that this heparin effect is not related to its anticoagulant property. Consequently, a non-anticoagulant fraction of heparin may be the future drug that can be given in sufficiently large doses to prevent clinical restenosis following arterial reconstruction. In conclusion, the study of the plasminogenplasmin system has, in the past, contributed to the understanding of thrombolysis. With wider and more successful use of thrombolytic therapy, a number of new problems in vascular disease are now being focused on, including restenosis following thrombolytic therapy, angioplasty, arterial reconstruction, or injury. Thus far, drug prevention of this complication has been unsuccessful. With a better understanding of the cellular and molecular mechanisms of vascular repair, a new approach may be made for the development of drug intervention.

314

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C. KWAAN

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l

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