THE FIBRINOLYTIC ENZYME SYSTEM

BLOOD STASIS AND THROMBOSIS

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THE FIBRINOLYTIC ENZYME SYSTEM Basic Principles and Links to Venous and Arterial Thrombosis Bjorn Wiman, MD

The fibrinolytic enzyme system is involved in many physiological and pathophysiologic processes. Removal of fibrin deposits from blood vessels and prevention of formation of fibrin clots in the circulatory system are perhaps the best-known functions. These processes have given the name fibrinolytic to this proteolytic cascade system that operates through plasminogen activation and plasmin proteolysis. Today it is known that degradation of fibrin is only one of several important functions for this system. Another is activation of metalloproteinases, which in turn have the capacity to degrade extracellular matrices. This mechanism is important in remodeling tissue as needed (e.g., in wound healing) and for invasive growth. This system also seems to have an important role in the mechanisms involved in cell migration. If the activity in the fibrolytic system is increased, these disturbances may cause an increased bleeding tendency. This increased bleeding tendency is observed, for example, in individuals with antiplasmin deficiency4 or plasminogen activator inhibitor-1 (PAI-1) defi~iency.~~ Both these components are inhibitors of the system. More commonly, however, the activity of the system is decreased. The decrease in activity may be caused by several different disturbances, such as plasminogen deficiency, deficient activator production or storage, or increased fibrinolysis inhibition, usually resulting from elevated plasma levels of PAI-

From the Department of Clinical Chemistry and Blood Coagulation, Karolinska Hospital, Karolinska Institute, Stockholm, Sweden

HEMATOLOGY/ ONCOLOGY CLINICS OF NORTH AMERICA VOLUME 14 * NUMBER 2 * APRIL 2000

325

1. Early clinical studies using nonspecific assays (e.g., clot lysis time) indicated that an impaired fibrinolytic function was often found in individuals suffering from venous thromboembolic di~ease.2~ Unfortunately the studies were retrospective. Because it is now well known that deep-vein thrombosis (DVT) is associated with a long-lasting inflammation that, in turn, affects fibrinolytic inhibitors, it is not possible to draw conclusions regarding cause or consequence from such studies. A number of prospective studies of fibrinolytic parameters associated with myocardial infarction (MI) have appeared. Most indicate a clear connection between an impaired fibrinolytic function and MI. This association is, however, complicated by the strong correlation that seems to exist between raised plasma PAI-1 levels and insulin resistance, which is also correlated to MI. Therefore it is still difficult to assign a precise causative role for impaired fibrinolytic function in MI; impaired fibrinolytic function and MI may be parallel phenomena. Evidence that an impaired fibrinolytic function is indeed involved in the development of thrombotic disease has also come from studies of mice with inactivated tissue plasminogen activator ( P A ) and urokinase plasminogen activator (uPA) genes. Mice with an extra P ! - 1 gene that leads to increased PAI-1 concentrations also suffer from an increased thrombotic tendency from birth. This article briefly describes some important aspects of the fibrinolytic system, its regulation, and possible disturbances of this system in connection with DVT and MI.

THE FlBRlNOLYTlC ENZYME SYSTEM

Compounds of the System Plasminogen

Table 1 summarizes the relevant properties of the factors in the fibrinolytic system. The central compound of the fibrinolytic enzyme system is plasminogen, the proenzyme of plasmin, which is responsible for the proteolytic degradation of fibrin. The activation of the singlechain 92-kd plasminogen molecule to plasmin occurs by the cleavage of a single peptide bond (Arg560-Val) by plasminogen activators, thus forming the two-chain plasmin molecule? The chain derived from the NH, terminus of the proenzyme (A-chain) consists of five homologous structures, known as kringles. These structures are lysine-binding sites and are responsible for the affinity of plasminogen and plasmin for fibrk1.6~These structures are thus of great importance for regulatory mechanisms within the fibrinolytic system.68The chain derived from the COOH terminus @-chain)carries the center for serine proteinase activity and is highly homologous with other serine proteinases, such as trypsin and chymotrypsin.

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Table 1. BASIC FACTS FOR SOME OF THE COMPOUNDS FORMING THE FIBRINOLYTIC SYSTEM

Compound

Plasminogen tPA, active scuPA Antiplasmin PAI-1 PAI-2 Vitronectin uPAR Mannose receptor LRP

Molecular Mass (kd)

92 65 54 70 50 4&70

67 45-65 175 600

Plasma Concentration

1-1.5 pmol/L -10 pmol/L ?

-1 pmol/L -0.3 nmol/L not applicable 3-4 pmol/L ?

not applicable not applicable

Synthesis Site

Liver Vascular endothelium Kidneys, macrophages Liver Multiple origins Placenta Liver Multiple origins Liver Liver

tPA = tissue plasminogen activator; scuPA = single-chain urokinase plasminogen activator (prourokinase); PAI-1 = plasminogen activator inhibitor-1; PAL2 = plasminogen activator inhibitor-2; uPAR = urokinase plasminogen receptor; LRP = lipoproteinlike receptor protein; ? = unknown

Activators The activators are either tPA or uPA. In the circulatory system, tPA seems to have a predominant role, whereas uPA seems to be relatively more important in exocrine glands, including the kidneys. Also, uPA seems to be relatively more important in cellular and pgicellular proteolysis. The tPA molecule is unique among serine proteinases in that it is fully active towards its natural substrate plasminogen in its single-chain form, especially in the presence of fibrin.56The COOH-terminal portion again contains the serine proteinase active center, whereas the NH,terminal portion contains two kringle structures, one a so-called ”finger structure” and one an epidermal growth factor (EGF) domain. The tPA molecule has an affinity for fibrin, for which kringle number 2 and, to some extent, the finger structure are resp~nsible.~~ The EGF structure is also responsible for the affinity of tPA to the low-density lipoproteinlike receptor protein (LRP) at the surface of the liver cells and therefore is involved in the clearance of tPA by the liver.& Activation of tPA by cleavage of the Arg,,,-Ile peptide bond (e.g., by plasmin) causes an increased activity towards some low-molecular-weight synthetic sub57 Only strates and also towards plasminogen in the absence of fibrin.28* small effects are observed in the presence of fibrin. The affinity of tPA to fibrin and its action directed towards this thrombus-stabilizing protein have caused tPA or variants of tPA to become the preferred drugs for thrombolytic therapy. The single-chain uPA (scuPA, i.e., prourokinase) contains a serine proteinase domain, one kringle domain and one EGF domain.22In its single-chain form, uPA seems not to have any proteolytic or activator activity; it needs to be cleaved (e.g., by plasmin), forming a two-chain structure which possesses activator activity.” Both uPA and SCUPAare also used as thrombolytic agents.

In addition to these physiologically important plasminogen activators in humans, different bacterial species produce efficient plasminogen activators of their own, most of which have great importance for those bacterial species. For example, the strains of streptococci that produce streptokinase typically have efficient invasive capacity. The specific plasminogen activator produced by Yersfniiu pestis also seems to be an important factor affecting virulence and to be involved in invasive ability.62 Streptokinase is well known as a plasminogen activator because of its use in thrombolytic treatment. Initially it was used in the treatment of venous thromboembolism (mostly pulmonary embolism and severe DVT), but later thrombolytic treatment of myocardial infarction became much more common. Streptokinase acts by forming a tight 1:l complex with plasminogen or plasmin, taking advantage of the potential plasmin active center, but its specificity changes dramatically.44The general proteolytic activity is decreased, and the plasminogen-activating activity becomes enormously increased. Streptokinase has no preferential action towards plasminogen bound to fibrin but acts equally well on all plasminogen molecules in the circulation, resulting in a generalized activity. This activity typically causes an extensive plasmin formation that consumes most of the antiplasmin. Therefore, free plasmin occurs in the circulating blood, leading to a massive fibrinogen degradation, which in turn affects general hemostasis. The action of staphylokinase, produced by certain strains of Stuphyloccus uureus, is similar to that of streptokinase, but with the important difference that staphylokinase needs to form a complex with plasmin (not plasminogen) to induce plasminogen activator activity. The action of staphylokinase is, therefore, directed to some extent towards fibrin. The mechanism responsible for this activity is probably that small amounts of plasmin are always formed in the vicinity of a fibrin clot or a thrombus, and, as mentioned before, active plasmin is needed for staphylokinase to form an active activator complex. Therefore, activation of plasminogen with staphylokinase occurs preferably in the vicinity of a thrombus. Interesting clinical results have recently been obtained using staphylokinase mutants as thrombolytic agents.12 Inhibitors

Several serine proteinase inhibitors (serpins), such as antiplasmin, PAI-I, and PAI-2, are involved in regulating the activity of the system. Antiplasmin, a glycoprotein with a molecular mass of about 70 kd, seems to inhibit activity at the plasmin level exclusively. Its extremely rapid reaction with plasmin depends on both a free active site in the plasmin molecule and free lysine-binding sites.7oThe inhibitor is produced in the liver, and its concentration in plasma is about 70 mg/L (- 1 Fmol/L). A small but important portion of antiplasmin becomes cross-linked to fibrin by factor XIIIa during clotting, thus increasing the initial resistance of fibrin to the action of p l a ~ r n i n .This ~ ~ resistance allows repair processes to start before clot lysis.

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Plasminogen activator inhibitor-1 operates at the activator level.l1* It also belongs to the serpin superfamily of proteins, and it reacts equally well with tPA and uPA.'O, 37 Plasminogen activity inhibitor-1 has a molecular mass of about 50 kd, and its concentration in plasma is very low, less than about 0.3 nmol/L in healthy individuals. In plasma, PAI1is completely bound to vitronedin, which increases the stability of this molecule, which is labile under physiologic conditions.25By a spontaneous conformational change, PAI-1 converts to a so-called "latent" form that has a half-life of a few hours.4O Although the concentration of PAI1 in plasma is very low, its reaction rate with the plasminogen activators is very high, so PAI-1 is still an important regulator of in vivo fibrinolysis.l0 The origin of PAI-1 in plasma is not fully understood. Several cells may be involved in its formation, for example, vascular endothelial cells, hepatocytes, smooth muscle cells, and adipocytes.2l. 38, 41, 59 In plasma, PAI-1 acts as an acute-phase reactant and behaves in a similar fashion as C-reactive protein.13 The concentration of PAI-1 also follows a typical diurnal pattern, with peak levels in the early morning and lower levels during the day and evening? Plasminogen activity inhibitor-1 is also found in platelets, although a large portion of the PAI-1 in platelets seems to be latent.% Nevertheless, it has been demonstrated that the PAL1 present in platelets is important for the thrombolytic dissolution of platelet-rich thrombi." 66 Several polymorphisms have been described within the PN-2 gene. The 4G / 5G polymorphism in the promoter region of the PAI-1 gene is of special interest, because individuals who are homozygous for the 4G allele have sigruficantly higher plasma PAI-1 c~ncentrationsl~ and also have higher PAI-1 content in their platelets." Plasminogen activity inhibitor-2 (PAI-2) is also a serpin and preferentially inhibits uPA. It is typically produced in placental tissue and is therefore almost exclusively found in plasma during ~regnanCy.7~ It is not known to be involved either in bleeding problems or in thrombotic complications.

35, 41

Receptors and Other Compounds

At least two receptor proteins at the surface of liver cells are important for the rapid clearance of tPA or uPA from plasma, namely LRP and the mannose receptor.%Each seems to be responsible for about half of the rapid tPA clearance. Lipoproteinlike receptor protein interacts with the EGF domains in the NH,-terminal portions of both tPA and uPA, whereas the mannose receptor interacts with carbohydrate structures in tPA. Removal of these sites by molecular engineering has resulted in tPA molecules with extensively prolonged half-lives in the circulation. Weitz et aF7have published a recent review. A specific receptor for uPA and uPA, urokinase plasminogen activity receptor (uPAR), has been found on many normal and tumor ce11s.l Binding of scuPA to uPAR seems to facilitate activation of uPA, giving a surface-bound plasminogen activator that may participate in such

processes as matrix degradation, wound healing, and invasion, and also in migration of cells. A few years ago, a carboxypeptidase (CPU) was found in plasma that removed COOH-terminal lysine residues from proteins. Because free lysine residues (e.g., in fibrin) are important for activating plasminogen, this carboxypeptidase works as a fibrinolytic inhibitor. It is now recognized as thrombin-activatable fibrinolytic inhibitor (TAFI)."" Its pathophysiologic sigruficance remains to be clarified.

Regulatory Aspects The central compound in the fibrinolytic system is the highly aggressive proteolytic enzyme plasmin, which has the potential capacity to degrade and destroy most proteins. Because the formation and inadivation of plasmin is highly regulated in vivo, however, this enzyme is normally active only temporarily and locally. Activation occurs much more efficiently at the surface of fibrin, because both tPA and plasminogen have affinity for fibrin and form a ternary complex with this protein. Formation of this complex causes a decrease of about 500-fold in Michaelis constant (K,) for the tPA-dependent plasminogen activation at the fibrin surface; hence, this process becomes localized.28,57 The release of tPA from the vascular endothelium, mediated by compounds such as vasopressin, bradykinin, or catecholamines, occurs easily during physical exercise or during ischemia.17 If, however, no fibrin is present in the blood vessels, the tPA is rapidly cleared (half-life 5 minutes) from the circulation, after just one passage through the liver by the action of LRP and the mannose receptor. Because of the high K, for the tPA-catalyzed plasminogen activation, this reaction is typically insigruficant in the absence of fibrin. If the plasma concentrations of PAI-1 are elevated, as they are in many individuals with thrombotic disease, the half-life of tPA is decreased to less than 60 seconds because of the rapid formation of the inactive tPA/ PAI-1 complex.'O, 48 Thus, increased plasma PAI-1 levels have a direct effect on the fibrinolytic activity. It has, in fact, been demonstrated that the plasma concentration of PAI-1 correlates negatively with the plasma concentration of plasmin/antiplasmin complex, a good marker of ongoing or recent fibrinolytic activity? 71 The plasmin formed at the fibrin surface by the localized activation of plasminogen is only poorly inactivated by antiplasmin as long as its active site and lysine-binding sites are occupied by the interaction with fibrina, 70 Once the fibrin is degraded and free plasmin leaks into the circulation, however, these plasmin molecules are rapidly and irreversibly inactivated by antiplasmin. The mechanisms described thus localize the activity of these potentially dangerous proteolytic enzymes to selected areas.

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IMPAIRED FlBRlNOLYSlS IN DEEP-VEIN THROMBOSIS

Early studies of fibrinolytic function in patients with thromboembolic disease consistently demonstrated that 30% to 40% of these patients had an impaired fibrinolytic function.2gBecause the data were retrospective and nonspecific assays were used, the value of the investigations is limited. The finding of a few families with plasminogen deficiency whose members frequently suffered from thromboembolic disease in early life clearly suggested that an impaired fibrinolytic function indeed might be associated with thrombotic disease? Recent experiments with mice with inactivated plasminogen or fPAand uPA genes further support this hypothesis.8Another interesting finding also indicating that PAI-1 may be involved in the thrombotic process is that transgenic mice with an extra human PAI-2 gene, resulting in increased PAI-1 concentrations, suffer from venous thrombosis shortly after birth.Is In epidemiologic studies using specific assays to measure the compounds within the fibrinolytic system, it became evident that increased plasma PAI-1 level is the most common reason for an impaired fibrinolytic function. Quite frequently however, this increase is combined with a decreased capacity to release tPA on venous occlusion. Many patients with DVT suffer from a long-lasting inflammatory response, which certainly contributes to an impaired fibrinolytic function by elevating the PAI-1 levels in plasma. As mentioned previously it is well known that PAI-1 acts as an acute-phase reactant. Therefore, retrospective studies give only limited information regarding cause or consequence. A recent longitudinal study of patients with idiopathic DVT, however, demonstrated that increased PAI-1 levels in plasma predict the development of a new thrombotic event? The difference in plasma PAI-1 concentration between the patients who experienced a subsequent event, as compared with the event-free patients, was quite small in comparison to the standard deviation. In other words, there is a large overlap between the groups. Therefore the data seem to be of limited prognostic value for an individual patient, even though a statistically significant difference in PAI-1 concentration was obtained between the groups. It is possible that plasma PAI-1 elevation by itself is of limited importance in the pathogenesis of DVT. In combination with other mild prethrombotic conditions, however, a decreased fibrinolytic potential caused by plasma PAI-1 elevation might be more aggravating. Indeed, increased levels of plasma PAI-1 in combination with increased plasma levels of histidinerich glycoprotein have been described in several families with recurrent DVT.3 Some studies of genetic markers for fibrinolytic compounds associated with venous thromboembolism have recently appeared. Several small studies have indicated that, in regard to the 4G / 5G polymorphism in the PAI-1 promoter region, the 4G allele seems to be overrepresented in individuals with thromboembolic disease.60As already mentioned, the 4G allele is associated with a higher plasma PAI-1 c~ncentration.'~

Postoperative Deep-Vein Thrombosis

Venous thromboembolism is still a sigruficant problem in patients who undergo major surgery. The fibrinolytic function has been extensively studied in a variety of surgical settings. In particular, these studies have shown a transient postoperative fibrinolytic shutdown caused by a temporary increase in plasma PAI-1 activity, probably as part of an acute-phase reaction.@In two studies of patients who had total hip replacement, preoperative PAI-1 levels in plasma or the perioperative or immediately ostoperative increase in plasma PAI-1 correlated sigrufrStudies of cantly with tEe risk of developing postoperative DVT.19,51 fibrinolytic function in connection with major abdominal surgery failed to confirm such a relationship.42Unlike patients subjected to total hip replacement, however, patients undergoing major abdominal surgery are a very heterogeneous group. More studies are therefore needed to evaluate correctly the importance of impaired fibrinolytic function for the development of postoperative DVT. Thus, the clinical studies available today indicate that a poor fibrinolytic function may be a causative mechanism in idiopathic or postoperative DVT. It is not yet clear, however, if measurements of these compounds have value as predictive markers for the individual patient. IMPAIRED FlBRlNOLYSlS IN MYOCARDIAL INFARCTION

In several longitudinal cohort studies of patients with manifest coronary heart disease (CHD), elevated plasma PAI-1 or tPA antigen concentrations have been linked to future cardiovascular events such as myocardial infarction. Our research group first reported the possibility of using elevated plasma PAI-1 concentrations as a predictor of MI from a follow-up study of men who had survived an MI occurring before the age of 45 ~ears.2~ This study suggested that high plasma concentrations of PAI-1 activity independently correlated with reinfarction within 3 years of a primary event.u Several additional longitudinal studies of patients with manifest coronary heart disease and also of initially healthy individuals have provided confirmatory information on the role of impaired fibrinolysis as a marker for an increased risk of MI.14, An elevated basal tPA antigen level has also been found to be associated with an increased risk of cardiovascular events during a Pyear followup period in a larger cohort of patients with severe angina pectoris and angiographically documented coronary artery disease.30A predictive value for plasma tPA antigen concentration for long-term mortality was subsequently established in this Furthermore, in the prospective Physicians Health Study, higher tPA antigen concentrations were found among participants who later suffered MI.% Cortellaro et all4 studied hemostatic function in a case-controlled study of 953 patients with atherosclerotic disease. During a 1-year follow-up period, 60 patients @

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suffered a thrombotic event. In most cases, these were MIS, but some patients with transient ischemic attacks and a few cases with peripheral arterial thrombosis were also included in the group of patients experiencing a thrombotic event. These patients were compared with 94 patients who were event-free during the same time period. A highly statistically sigruficant difference in plasma PAI-1 activity was observed between the two groups, those with events having higher plasma PAI-1 activity levels. Elevated levels of the fibrin degradation product Ddimer were associated with thrombotic events. Data from the European Concerted Action on Thrombosis and Disabilities Group Angina Pectoris Study regarding an investigation of hemostatic factors and the risk of MI in 3043 atients with angina pectoris were published a few years ago.=, 65 In e 106 coronary events recorded during a follow-up period of 2 years, tPA antigen was the hemostatic factor that independently correlated most strongly with a future cardiovascular event. Also, PAI-1 activity and PAI-1 antigen correlated with the development of MI but were not independent, because the signhcance disappeared after adjustments were made for confounding factors. Data from the Angina Pectoris Study in Stockholm (APSIS), consisting of 631 patients with angina pectoris followed during 3 years for MI,% also demonstrated that increased plasma PAI-1 levels predicted a forthcoming MI?7 Increased plasma levels of tPA antigen and a decreased release of tPA activity after a specified work load also predicted MI. This is the first study to demonstrate that, besides fibrinolytic inhibition, an impaired release of tPA during exercise might also be linked to the development of MI. Recent data from the Stockholm Heart Epidemiological Study (SHEEP) have also indicated that the specific measurement of tPA/PAI-l complex is more accurate than measuring the PAI-1 activity and tPA antigen in predicting an MI.71aThe data have been obtained from 86 patients with reinfarction, from a group of 1212 patients (893 men and 374 women) with a first MI," who were subjected to blood sampling in a metabolically stable period (about 3 months after the primary event) and compared with 134 age- and sex-matched patients without reinfarction (Table 2), and 261 matched healthy control subjects (Table 3). Similar results were obtained for men and for women. The odds-risk ratio for individuals with a tPA/PAI-1 complex concentration above the seventy-fifth percentile (control subjects)were calculated as 1.8 (95% confidence inter-

tK

Table 2. CONCENTRATION" OF SOME FlBRlNOLYTlC PARAMETERS IN PATIENTS WITH MYOCARDIAL INFARCTION AND HEALTHY CONTROL SUBJECTS Component

PAI-1 (arb.U/mL) P A antigen (pg/L) tPA/PAI-l (pg/L)

Patlents

19.9

?

17.7

11.3

& &

3.6 3.3

7.0

Number

Controls

Number

24 220 220

15.5 & 11.4 10.0 & 3.6 5.6 & 3.0

264 258 257

*Mean standard deviation PAI-1 = plwmhogen adivator inhibitor-1; tPA

= tissue plasmhogen activator

P Value

2.4 x 1.7 x 3.0 x

lo-'

Table 3. CONCENTRATION* OF SOME FlBRlNOLYTlC PARAMETERS IN MYOCARDIAL INFARCTION PATIENTS WITH OR WITHOUT REINFARCTION ________~

Component

Reinfarction

Number

No Reinfarction

Number

P Value

PAI-1 (arb. U/mL) P A antigen (kg/L) tl"f'AI-1 (kg/L)

21.2 t 17.7 12.0 k 4.0 7.8 t 3.6

89 87 87

18.1 k 15.3 10.8 f 3.4 6.5 -t 3.0

134 133 133

0.17 0.026 0.0047

*Mean f standard deviation PAI-1 = plasminogen activator inhibitor-1; tPA = tissue plasmhogen activator

Val 1.1-3.1). A close correlation between tPA/PAI-1 complex concentration and PAI-1 activity was observed, suggesting that increased levels of this complex reflect an impaired fibrinolytic function. Recently, the results from a prospective, nested case-controlled study of 78 individuals with MI and 156 matched control subjects selected from a cohort of initially healthy individuals also demonstrated that PAI-1 and tPA antigen predict a MI.@In conclusion, these data all support the hypothesis that an impaired fibrinolytic function, caused either by increased PAI-1 levels or by an impaired ability to release tPA activity from the vessel wall, may be linked with the development of MI. Besides the association between elevated plasma PAI-1 levels and MI, it has been demonstrated that individuals with this laboratory abnormality also seem to have a more pronounced progression of the atherosclerotic process7 The reasons for this finding and the mechanisms are not yet known. Connection Between Plasma PAI-1 Levels and Other Metabolic Factors

Data available today have clearly pointed out a link between an impaired fibrinolytic function caused by plasma PAL1 elevation and th& so-called "insulin resistance" syndrome. (Juhan-Vague and Alessi have published a recent review?2) Thus, it has been found that PAI-1 in plasma is correlated with serum triglycerides, blood glucose, and insulin levels, body mass index (BMI), waist-to-hip circumference ratio, and hypertension. The mechanisms involved are not yet known. PAI-1 Genotypes and Myocardial Infarction

Studies of polymorphisms within the PAI-1 gene in individuals with CHD have not been consistent. A few small initial studies of the 4G/5G polymorphism in the PAI-1 gene indeed demonstrated a higher frequency of the 4G allele among MI patients than in healthy 49 In several larger studies, however, no correlation was observed between

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the different genotypes for the 4G/5G polymorphism in the PAI-1 promoter region and risk of MI.16,w 54, 73 In the SHEEP study, the authors have investigated this polymorphism in 1300 MI patients and in 1700 healthy controls. No difference in allele frequency or genotype frequency have been found between these groups (Wiman, Andersson, Falk, et al, unpublished data). Treatment of MI in the acute stage with thrombolysis, using infusion of a plasminogen activator to activate the fibrinolytic enzyme system, is occasionally not successful because of the resistance of the thrombus to treatment. It has been demonstrated that PAI-1 within the platelets plays a role in this behavior by certain thrombi, particularly platelet-rich thrombi.63In fact, the pool within the platelets constitutes the major source of PAI-1 antigen in the circulating blood, although there are somewhat conflicting opinions about how active this PAI-1 really is.6,66 Most data obtained have suggested a low activity of the PAI-1 released from platelets. The authors’ own data have suggested that there may be a large variation among individuals and that occasionally more than 50% of this PAI-1 is active.47Nevertheless, because of the relatively high concentration of PAI-1 antigen in the platelets, enough PAI-1 activity is present, at least occasionally, to delay thrombolysis during thrombolytic treatment. In conclusion, a reduced fibrinolytic potential cannot yet be considered as a fully established risk factor for MI in the conventional epidemiologic sense, because prospective studies of initially healthy individuals that include measurement of PAI-1, tPA antigen, or tPA/PAI-l complex are still very sparse. The data produced so far, however, strongly suggest that the compounds of the fibrinolytic enzyme system must be taken into consideration in forthcoming prospective studies. Even though much of the available data suggest a connection between an impaired fibrinolytic function and MI, the authors still cannot be certain about an causative role. References 1. Andreasen PA, Kjoller L, Christensen L, et al: The urokinase-type plasminogen activator system in cancer metastasis: A review. Int J Cancer 721, 1997 2. Andreotti F, Davies GJ, Hackett DR, et al: Major circadian fluctuations in fibrinolytic factors and possible relevance to time of onset of myocardial infarction, sudden cardiac death and stroke. Am J Cardiol 62635, 1988 3. Angles Can0 E, Gris JC, Loyau S, et al: Familial association of high levels of histidinerich glycoprotein and plasminogen activator inhibitor-1 with venous thromboembolism. J Lab Clin Med 121:646, 1993 4. Aoki N, Sakata Y, Matsuda M, et al: Fibrinolytic states in a patient with congenital deficiency of alpha 2-plasmin inhibitor. Blood 55:48, 1980 5. Aoki N: Physiological endogenous fibrinolysis: Its congenital abnormalities. Progress in Fibrinolysis 6:3, 1983 6. Booth NA, Croll A, Bennet B: The activity of plasminogen activator inhibitor-1 (PAI1)of human platelets. Fibrinolysis 4:138, 1990 7. Blvenholm P, deFaire U, Landou C, et al: Progression of coronary artery disease in young male post-infarction patients is linked to disturbances of carbohydrate and lipid metabolism and to impaired fibrinolytic function. Eur Heart J 19:402, 1998

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