Mechanisms of Thrombosis and Thrombolysis

Key Points

The fundamental processes involved in thrombus formation, thrombus dissolution, and thrombus stability and their relevance to the central nervous system (CNS) are described.
The role(s) of endogenous plasminogen activators (PAs, including tissue-type plasminogen activator, urokinase-type plasminogen activator) in thrombus dissolution are presented, together with considerations of their regulation in vivo. Their relevance to derived therapeutics is emphasized.
Fibrinolytic agents tested or used as pharmaceuticals including recombinant and purified endogenous PAs and exogenous PAs (including streptokinase, staphylokinase, PAs derived from Desmodus species, and novel plasminogen activators) are presented.
The molecular basis for PA inhibition and modulation of vascular fibrinolysis is made.
These considerations form a basis for exploration of current information about the impact of PAs and of plasmin generation on CNS vessel and microvessel integrity.
Exploration of the role(s) of endogenous PAs in CNS development, CNS integrity, and on neuronal function in the CNS is presented, and the potential effects of therapeutic PAs on the CNS.
The pioneering use of therapeutic plasminogen activation in the acute setting in ischemic/thrombotic stroke, acute cerebral arterial recanalization, and its consequences are described.
The use of PAs in experimental cerebral ischemia, recanalization and tissue injury reduction, and their limitations and relevance to the clinical setting are discussed.
The risks of PAs in the acute intervention in ischemic stroke and the quantitative effects on intracerebral hemorrhage are presented. Limitations to the clinical use of fibrinolytic agents in ischemic stroke are considered.

Thrombosis, and thrombus growth, dissolution, and migration are inextricably connected. Thrombus formation involves activation of platelets, activation of the coagulation system, and the processes of fibrin dissolution. The central feature of each of these processes is the generation of thrombin from prothrombin. Thrombin, in turn, generates the thrombus fibrin network by the cleavage of circulating fibrinogen with formation of the fibrin network. Excess local vascular fibrin deposition can contribute to thrombus growth, while vascular injury and excess degradation of fibrin in “hemostatic plugs” at sites of vascular injury can lead to hemorrhage. Plasmin can degrade fibrin and fibrinogen. Plasminogen activators (PAs), which convert plasminogen to plasmin, have been exploited to dissolve clinically significant vascular thrombi acutely. Notably, all substances that promote plasmin formation have the potential to increase the risk of hemorrhage.

The acute use of PAs has been associated with detectable clinical improvement in selected patients with symptoms of focal cerebral ischemia. Acute thrombolysis has thus attained pride of place in the treatment of ischemic stroke so far. Currently, recombinant tissue plasminogen activator (rt-PA) is licensed in the United States, Japan, Europe, and many other countries for the treatment of ischemic stroke within 3 hours of symptom onset, and up to 4.5 hours in some jurisdictions. ^, Early studies, a phase III prospective trial, and more recent experience suggest that extension of the treatment window is possible with strict limitations to patient selection. ^, Early on, few studies of acute rt-PA delivery correlated improvement in patient outcome with imaging evidence of recanalization of an occluded brain-supplying artery, however.

The development of agents that promote fibrin degradation in the clinical setting stems from observations in the 19th century of the spontaneous liquefaction of clotted blood and the dissolution of fibrin thrombi. A growing understanding of plasma proteolytic digestion of fibrin paralleled enquiry into the mechanisms of streptococcal fibrinolysis. Streptokinase (SK) was the first PA employed to dissolve closed space (intrapleural) fibrin clots, but purified preparations were required for lysis of intravascular thrombi. The development of PAs for therapeutic lysis of vascular thrombi has progressed along with insights into the mechanisms of thrombus formation and degradation. It should be remembered that the concentrations of PAs used to degrade fibrin thrombi clinically far exceed those required to perform the same task endogenously.

Thrombus Formation

The relative platelet-fibrin composition of a specific thrombus depends on the vascular bed, the local development of fibrin, platelet activation, and regional blood flow or shear stress. Even in the same arterial territory there may be considerable variability and local heterogeneity in thrombus composition as evidenced by thrombi removed in situ. Pharmacologic inhibition of the platelet activation/aggregation and coagulation processes can also alter thrombus composition and volume. At arterial flow rates thrombi are predominantly platelet rich, whereas at lower shear rates characteristic of venous flow, activation of coagulation seems to predominate. It has been suggested that the efficacy of pharmacologic thrombus lysis depends on (i) the relative fibrin content and (ii) the extent of fibrin cross-linking of the thrombus that may reflect thrombus age and thrombus remodeling. The latter may vary with location within a vascular bed (e.g., arterial, capillary, or venular).

Thrombin (factor IIa) is the central player in clot formation ( Fig. 2.1 ). Thrombin, a serine protease, cleaves fibrinogen to generate fibrin, which forms the scaffolding for the growing thrombus. Inter-fibrin strand cross-linking requires active factor XIII, a transglutaminase bound to fibrinogen that is itself activated by thrombin. Factor XIIIa stabilizes the fibrin network ( Fig. 2.2 ). ^, Thrombin-mediated fibrin polymerization leads to the generation of fibrin I and fibrin II monomers and to the release of fibrinopeptide A (FPA) and fibrinopeptide B (FPB).

Fig. 2.1, Intrinsic and extrinsic coagulation pathways (see text). Phospholipid-containing membranes (e.g., platelets) provide the scaffold for accelerating coagulation pathway activation. Both intrinsic and extrinsic pathways lead to prothrombin (factor II) activation, with fibrin generation from circulating fibrinogen. The extrinsic pathway initiates coagulation through the interaction of factor VII with tissue factor (TF) in the vascular adventitia, brain perivascular parenchyma, and activated monocytes. The TF:VIIa complex catalyzes activation of factor X and acceleration of thrombin generation. The intrinsic system involves activation of components within the vascular lumen. Initiation of coagulation through this pathway involves pre-kallikrein, kallikrein, high-molecular-weight kininogen (HMWK) , and factors XI and XII. (A) Thrombin generation . The intrinsic system activates factor X through the “tenase” complex (factors VIIIa and IXa, and Ca 2+ on phospholipid). Both intrinsic and extrinsic pathways activate prothrombin through the common “prothrombinase” complex (factors Xa and Va, and Ca 2+ ). The platelet surface has receptors for factors Va and VIIIa. Cleavage of prothrombin generates the prothrombin fragment 1.2 (PF 1.2) and thrombin (factor IIa). (B) Thrombin has multiple stimulatory positive feedback effects. It catalyzes activation of factors XI and VIII as well as the activities of the tenase and prothrombinase complexes. Thrombin also stimulates activation of platelets and granule secretion via specific thrombin receptors on their surface. (C) Coagulation activation is regulated by interleaving inhibitor pathways. The effects of factors Va, Xa, and VIIIa are modulated by the protein C pathway. Activated protein C (APC) , generated by the action of the endothelial cell receptor thrombomodulin on protein C, with its cofactor protein S, inhibits the action of factor V. AT , Antithrombin; HC-III , heparin cofactor-III.

Fig. 2.2, Generation of cross-linked fibrin. Fibrinogen is cleaved successively to form fibrin I and fibrin II by thrombin (factor IIa) with the release of fibrinopeptides A and B (FPA and FPB) . Thrombin activates factor XIII to the active transglutaminase, which promotes cross-linking of fibrin and stabilization of the growing thrombus.

Platelet activation is required for thrombus formation under arterial flow conditions and accompanies thrombin-mediated fibrin formation. Platelet membrane receptors and phospholipids form a workbench for the generation of thrombin through both the intrinsic and extrinsic coagulation pathways. Platelets promote activation of the early stages of intrinsic coagulation by a process that involves the factor XI receptor and high-molecular-weight kininogen (HMWK) (see Fig. 2.1 ). Also, factors V and VIII interact with specific platelet membrane phospholipids (receptors) to facilitate the activation of factor X to Xa (the “tenase complex”) and the conversion of prothrombin to thrombin (the “prothrombinase complex”) on the platelet surface. Platelet-bound thrombin-modified factor V (factor Va) serves as a high-affinity platelet receptor for factor Xa. These mechanisms accelerate the rate of thrombin generation, further catalyzing fibrin formation and the fibrin network.

This process also leads to the conversion of plasminogen to plasmin and to the activation of endogenous fibrinolysis. Thrombin provides one direct connection between thrombus formation and plasmin generation, through the localized release of tissue plasminogen activator (t-PA) and single chain urokinase (scu-PA) from endothelial cells. Thrombin has been shown in vitro and in vivo to markedly stimulate t-PA release from endothelial stores. ^, In one experiment, infusion of factor Xa and phospholipid into non-human primates resulted in a pronounced increase in circulating t-PA activity, suggesting that significant vascular stores of this PA can be released by active components of coagulation. Other vascular and cellular stimuli also augment PA release, thereby pushing the hemostatic balance toward thrombolysis (see below).

The development of arterial or venous thrombi requires loss of the constitutive antithrombotic characteristics of endothelial cells. In addition to both the antithrombotic properties of endothelial cells and the circulating anticoagulants and their cofactors (i.e., activated protein C [APC], protein S), thrombus growth is limited by the endogenous thrombolytic system. Thrombus dissolution or remodeling results from the preferential conversion of plasminogen to plasmin on the thrombus surface. There, fibrin binds t-PA in proximity to its substrate (fibrin-bound) plasminogen, thereby accelerating local plasmin formation, in concert with local shear stress. The parallel role of scu-PA is discussed below.

These processes may also promote embolization into the downstream cerebral vasculature. However, little is known about the endogenous generation and secretion of PAs within cerebral vessels. Exogenous application of pharmacologic doses of PAs can accelerate conversion of plasminogen to plasmin and thereby prevent thrombus formation and promote thrombus dissolution, as discussed later.

Fibrinolysis

Plasmin formation is central to the lysis of vascular thrombi. The endogenous fibrinolytic system comprises plasminogen, scu-PA, urokinase (u-PA), and t-PA, and their inhibitors. Hence, plasmin degrades fibrin (and fibrinogen). Plasminogen, its activators, and their inhibitors contribute to the balance between vascular thrombosis and hemorrhage ( Fig. 2.3 ; Tables 2.1 and 2.2 ).

TABLE 2.1

Plasminogen Activators.

Plasminogen Activators	Molecular Weight (kDa)	Chains	Plasma Concentration (mg/dL)	Plasma Concentration Half-Life (t _1/2 )	Substrates
Endogenous
Plasminogen	92	2	20	2.2 days	(Fibrin)
Tissue PA (t-PA)	68 (59)	1→2	5 × 10 ⁻⁴	5–8 min	Fibrin/plasminogen
Single-chain urokinase PA (scu-PA)	54 (46)	1→2	2–20 × 10 ⁻⁴	8 min	Fibrin/plasmin(ogen)
Urokinase PA (u-PA)	54 (46)	2	8 × 10 ⁻⁴	9–12 min	Plasminogen
Exogenous
Streptokinase	47	1	0	41 and 30 min	Plasminogen, fibrin(ogen)
Anisoylated plasminogen-streptokinase activator complex (APSAC)	131	Complex	0	70–90 min	Fibrin(ogen)
Staphylokinase	16.5		0		Plasminogen
Desmoteplase	52	1	0	138 min	Plasminogen

Table 2.2

Plasminogen Activator Inhibitors.

Inhibitor	Molecular Weight (kDa)	Chains	Plasma Concentration (mg/dL ⁻¹ )	Plasma Concentration Half-Life (t _1/2 )	Inhibitor Substrates
Plasmin Inhibitors
α ₂ -antiplasmin	65	1	7	3.3 min	Plasmin
α ₂ -macroglobulin	740	4	250		Plasmin (excess)
Plasminogen Activator Inhibitors
PAI-1	48–52	1	5 × 10 ⁻²	7 min	t-PA, u-PA
PAI-2	47, 70	1	<5 × 10 ⁻⁴	24 h	t-PA, u-PA
PAI-3	50				u-PA, t-PA

PAI , Plasminogen activator inhibitor; t _1/2 , half-life; t-PA , tissue plasminogen activator; u-PA , urokinase plasminogen activator.

Plasmin formation occurs (i) in the plasma, where it can cleave circulating fibrinogen and fibrin into soluble products, and (ii) on reactive surfaces (e.g., thrombi or cells). The fibrin network provides the scaffold for plasminogen activation, whereas various cells, including polymorphonuclear (PMN) leukocytes, platelets, and endothelial cells, express receptors for plasminogen to bind to. Specific cellular receptors concentrate plasminogen and specific activators (e.g., urokinase plasminogen activator [u-PA]) on the cell surface, thereby enhancing local plasmin production. Similar receptors on tumor cells (e.g., the urokinase plasminogen activator receptor [u-PAR], which concentrates u-PA) also facilitate dissolution of basement membranes and matrix, promoting metastases. u-PA and u-PAR are both expressed by microvessels and neurons in the ischemic bed. ^, Plasmin can also cleave various extracellular matrix (ECM) glycoprotein components (e.g., laminins, collagen IV, perlecan) found in the basal lamina of microvessels of the central nervous system, and in other organs.

Plasminogen

The naturally circulating PAs, single-chain t-PA and single-chain u-PA (scu-PA or pro-UK), catalyze plasmin formation. ^, Plasmin derives from the zymogen plasminogen, a glycosylated single-chain 92-kDa serine protease. ^, Structurally, plasminogen contains five kringles and a protease domain, two of which (K1 and K5) mediate the binding of plasminogen to fibrin through characteristic lysine-binding sites ( Fig. 2.4 ). ^, ^, Glu-plasminogen has an NH ₂ -terminal glutamic acid, and lys-plasminogen, which lacks an 8-kDa peptide, has an NH ₂ -terminal lysine. Plasmin cleavage of the NH ₂ -terminal fragment of glu-plasminogen generates lys-plasminogen. Glu-plasminogen has a plasma clearance half-life (t _1/2 ) of ∼2.2 days, whereas the t _1/2 of lys-plasminogen is 0.8 days. Both t-PA and u-PA catalyze the conversion of glu-plasminogen to lys-plasmin through either of two intermediates, glu-plasmin or lys-plasminogen. The lysine-binding sites of plasminogen mediate the binding of plasminogen to α ₂ -antiplasmin, thrombospondin, components of the vascular ECM, and histidine-rich glycoprotein (HRG). α ₂ -Antiplasmin prevents binding of plasminogen to fibrin by this mechanism. Partial degradation of the fibrin network enhances the binding of glu-plasminogen to fibrin, promoting further local fibrinolysis.

Plasminogen Activation

Plasminogen activation is tied to activation of the coagulation system and can involve secretion of physiologic PAs (“extrinsic activation”). It has been suggested that kallikrein, factor XIa, and factor XIIa, in the presence of HMWK, can directly activate plasminogen. ^, Several lines of evidence suggest that scu-PA activates plasminogen under physiologic conditions. Tissue-type PA, which is secreted from the endothelium and other cellular sources, appears to be the primary PA in the vasculature. Thrombin, generated by either intrinsic or extrinsic coagulation, stimulates secretion of t-PA from endothelial stores. ^,

Several serine proteases can convert plasminogen to plasmin by cleaving the arg ⁵⁶⁰ -val ⁵⁶¹ bond. Serine proteases have common structural features, including an NH ₂ -terminal “A” chain with substrate-binding affinity, a COOH-terminal “B” chain with the active site, and intra-chain disulfide bridges. Plasminogen-cleaving serine proteases include the coagulation proteins factor IX, factor X, and prothrombin (factor II), protein C, chymotrypsin and trypsin, various leukocyte elastases, the plasminogen activators u-PA and t-PA, and plasmin itself.

Activation of plasminogen by t-PA is accelerated by a ternary complex with fibrin. In the circulation, plasmin binds rapidly to the inhibitor α ₂ -antiplasmin and is thereby inactivated. Activation of thrombus-bound plasminogen also protects plasmin from the inhibitors α ₂ -antiplasmin and α ₂ -macroglobulin. Here, the lysine-binding sites and the catalytic site of plasmin are occupied by fibrin, thereby blocking its interaction with α ₂ -antiplasmin. Furthermore, fibrin and fibrin-bound plasminogen render t-PA relatively inaccessible to inhibition by other circulating plasma inhibitors.

Thrombus Dissolution

Fibrinolysis occurs predominantly at the surface, and so may be augmented by increased local blood flow, but also by flow within the thrombus. ^, During thrombus consolidation, plasminogen bound to fibrin and to platelets allows local release of plasmin. In the circulation , plasmin cleaves the fibrinogen Aα chain appendage, generating fragment X (DED), Aα fragments, and Bβ. Further cleavage of fragment X leads to the generation of fragments DE, D, and E. By contrast, degradation of the fibrin network generates YY/DXD, YD/DY, and the unique DD/E (fragment X = DED and fragment Y = DE). Cross-linkage of DD with fragment E is vulnerable to further cleavage, producing D-dimer fragments. The measurement of D-dimer levels can have clinical utility, in that the absence of circulating D dimer correlates with the absence of massive thrombosis. Ordinarily, in the setting of focal cerebral ischemia, the thrombus load is small and the meaning of any D-dimer elevation is uncertain. The generation of the degradation products has two consequences: (i) incorporation of some of these products into the forming thrombus destabilizes the fibrin network of the thrombus and (ii) reduced circulating fibrinogen and the generation of breakdown products of fibrin(ogen) limits the protection from hemorrhage by hemostatic thrombi.

Plasminogen Activators

All fibrinolytic agents are obligate PAs (see Table 2.1 ). Tissue PA, scu-PA, and u-PA are endogenous PAs involved in physiologic fibrinolysis. Recombinant t-PA, scu-PA, and u-PA, as well as SK, acylated plasminogen streptokinase activator complex (APSAC), staphylokinase (STK), PAs from Desmodus species, and other newer novel agents in clinical use (e.g. reteplase [r-PA], and tenecteplase [TNK]), are termed exogenous PAs. ^, t-PA, scu-PA, and a number of novel agents have relative fibrin and thrombus specificity.

Endogenous Plasminogen Activators

Tissue Plasminogen Activator

Tissue PA is a 70-kDa, single-chain glycosylated serine protease that has four distinct domains—a finger (F-) domain, an epidermal growth factor (EGF) domain (residues 50–87), two kringle regions (K1 and K2), and a serine protease domain ( Fig. 2.5 ). The COOH-terminal serine protease domain contains the active site for plasminogen cleavage, and the finger and K2 domains are responsible for fibrin affinity. ^, The two kringle domains are homologous to the kringle regions of plasminogen.

The single-chain form of t-PA is converted to the two-chain form by plasmin cleavage of the arg ²⁷⁵ -isoleu ²⁷⁶ bond. Both single-chain and two-chain species are enzymatically active and have relatively fibrin-selective properties. Infusion studies in humans indicate that both single-chain and two-chain t-PA have circulating plasma t _1/2 values of 3–8 minutes, although the biologic t _1/2 s are longer. Tissue PA is considered to be fibrin-selective because of its favorable binding constant for fibrin-bound plasminogen and its activation of plasminogen in association with fibrin. Significant inactivation of circulating factors V and VIII does not occur with infused rt-PA, and an anticoagulant state is generally not produced. However, if sufficiently high rt-PA dose-rates are employed, clinically measurable fibrinogenolysis and plasminogen consumption can be produced.

Physiologically, secretion of t-PA from cultured endothelial cells is stimulated by thrombin, ^, APC, histamine, phorbol myristate esterase, and other mediators. Physical exercise and certain vasoactive substances produce measurable increases in circulating t-PA levels, and 1-deamino(8- d -arginine) vasopressin (DDAVP) may produce a 3-4-fold increase in t-PA antigen levels within 60 minutes of parenteral infusion in some patients. Both t-PA and u-PA have been reported to be secreted by endothelial cells, neurons, astrocytes, and microglia in vivo or in vitro . ^, The reasons for this broad cell expression are not known, however.

Urokinase-Type Plasminogen Activator

Single-chain u-PA is a 54-kDa glycoprotein synthesized by endothelial and renal cells as well as by certain malignant cells ( Fig. 2.6 ). This single-chain proenzyme of u-PA is unusual in that it has fibrin-selective plasmin-generating activity and also has been synthesized by recombinant techniques.

Fig. 2.6, The secondary structure of single-chain urokinase plasminogen activator (scu-PA; 54 kDa). Activation by plasmin takes place at the 158–159 bond (arrow) . The zigzag line represents the glycosylation site.

The relationship of scu-PA to u-PA is complex: cleavage or removal of lys ¹⁵⁸ from scu-PA by plasmin produces 54-kDa, two-chain u-PA. This PA consists of an A-chain (157 residues) and a glycosylated B-chain (253 residues), which are linked by the disulfide bridge between cys ¹⁴⁸ and cys ²⁷⁹ . Further cleavages at lys ¹³⁵ and arg ¹⁵⁶ produce low-molecular-weight (31-kDa) u-PA. Both high- and low-molecular-weight species are enzymatically active.

The 54-kDa urokinase (u-PA) activates plasminogen by first-order kinetics. ^, The two forms of u-PA exhibit measurable fibrinolytic and fibrinogenolytic activities in vitro and in vivo , and have plasma t _1/2 values of 9–12 minutes. ^, When infused as a therapeutic agent, pharmacologic doses of u-PA lead to plasminogen consumption and inactivation of factors II (prothrombin), V, and VIII. The latter changes constitute the systemic lytic state.

It has been postulated that t-PA is primarily involved in the maintenance of hemostasis through the dissolution of fibrin, whereas u-PA is involved in generating pericellular proteolytic activity by cells expressing the u-PA receptor, which is needed for degradation of the ECM for migration. The roles of these two PAs in central nervous system cell function are not fully understood. However, recent work has provided further insight between the interactions of t-PA and the u-PA precursor.

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