Clinical Importance of Thrombomodulin Serum Levels

European Review for Medical and Pharmacological Sciences 2000; 4: 59-66 Clinical importance of thrombomodulin serum levels

F. CALIFANO, T. GIOVANNIELLO, P. PANTONE, E. CAMPANA, C. PARLAPIANO, F. ALEGIANI*, G.M. VINCENTELLI*, P. TURCHETTI

Department of Clinical Sciences, Policlinico Umberto I, “La Sapienza” University – Rome (Italy) *Servizio Pronto Soccorso, Reparto Breve Osservazione, “S. Giovanni Calibita” Hospital, Fatebenefratelli – Rome (Italy)

Abstract. – Thrombomodulin is a glyco- represents the soluble or plasmatic form3. protein that can bind to thrombin and activate The concentration of TM bound to the cell protein C, thus mitigating the effects of cytokines produced by inflammatory and immuno- surface is regulated by genetic factors. The logical processes. The molecule exerts a protec- presence of sequence mutations of the gene tive function on endothelial cells. or mutations of the regulatory sequences of Thrombomodulin is cleaved to its soluble TM transcription give place to the production form by neutrophil elastase and by other sub- of a molecule with impaired function or its stances produced during acute and chronic in- lower expression4. The concentration of flammatory responses, immunologic reactions serum TM levels varies accordingly. and complement activation. ELISA technique yields normal serum levels The proteinaceous component of the TM of 3,1 ± 1,3 ng/ml; in males these levels are high- molecule has multiple moieties, each formed er; TM levels also rise during menopause. Other by about forty amino acid residues, called do- circumstances associated with an increase of mains, that influence the tertiary structure serum TM levels are smoking, disseminated in- and are responsible for the specificity of ac- travascular coagulation (DIC), cardiac surgery, tion of the glycoprotein. Different domains atherosclerosis, ARDS, liver cirrhosis, diabetes mellitus, cerebral and myocardial infarction, and have different functions. These amino acid multiple sclerosis. sequences can act as cofactors in the activa- Serum levels of TM represent an useful prog- tion of protein C and have an anticoagulant nostic index, because they are associated with an effect by binding to thrombin. increase in mortality rate, or however a progres- Thrombomodulin can stimulate endothe- sion of the underlying pathological condition. lial cell growth. This characteristic of TM de- Key Words: pends from the molecular substrate of the se- Thrombomodulin, Thrombin, Protein C, Inflammation, quences, known as EGF-like (epidermal Cytokines. growth factor-like). The binding of thrombin with thrombomodulin activates protein C and the trombin- thrombomodulin complex functions as an anticoagulant and anti-inflammatory stimulus5. The reaction is regulated by the concentra- Introduction tion of surface TM and also by the function of protein C, and is conditioned by genetic mutations that can alter the anticoagulant ef- Thrombomodulin is a membrane glycopro- ficacy. Many factors such as cytokines (IL-1, tein expressed on endothelial cells1. TNF) and neutrophils can have a regulatory Thrombomodulin is present in the body in role on TM activity. Cytokines reduce surface two forms2; the first type has a higher molec- TM expression and cleave the molecule when ular weight, and it is bound to the cytoplas- activation of inflammatory processes takes matic membrane of endothelial cells; the sec- place6. Neutrophils, on the other hand, influ- ond form has a lower molecular weight and ence TM activity by the enzyme elastase.

59 F. Califano, T. Giovanniello, P. Pantone, E. Campana, C. Parlapiano, F. Alegiani, et al.

Molecular structure ture compressing the C-terminal ring) and the conjunction of the tail amino acids to the Thrombomodulin is a surface glycoprotein C-terminal ring region. of endothelial cells, whose gene, localized on The cofactor activity of the fourth and chromosome 207 does not contain introns8. sixth EGF-like domain depends on the pres- Thrombomodulin is found in the body in two ence of the molecule’s fourth EGF-like do- forms with different molecular weight. The main. The fact that the fourth domain does heavy form weighs 150 kDa and the light not bind well to the thrombin molecule sug- form 69 kDa. gests that, although necessary for protein C The domains with epidermal growth factor- activation, it is however unable to exert its like structure play an important role as cofac- action if not in conjunction with the fifth tors. These domains are formed by sequences EGF-like domain. This leads to the conclu- of about forty amino acids, with six cystein sion that the main region were the thrombo- residues that form three disulphuric bonds. modulin-thrombin bond is possible can be The molecular sequence of thrombomod- localized in the fifth EGF-like domain and ulin contains an N-terminal lectin-like ele- in the region connecting the fifth to the ment (1-154 residues), an hydrophobic region sixth EGF-like domain. The structural char- (155-222 residues ), six EGF-like modules acteristics of this region formed by the (223-462 residues), one dominium rich in amino acids C409-E•426 is the cyclic struc- Ser/Thr (463-497 residues), a trans-mem- ture formed by the disulphuric bond be- brane moiety composed by 23 amino acids tween C409 and C421 and by the bond be- (498-521 residues) and a tail of 35 cytoplas- tween the amino acid tail and this ring. The mic amino acids (522-557 residues). ring can interact with the tail so as to deter- Thrombin binds to the EGF-like modules mine specificity of the bond. Thrombo- and this bond is strengthened by the presence modulin cofactorial activity, evaluated by of the sulphuric glycosaminoglycans of the the quantity of protein C that can be acti- Ser/Thr rich region8. vated by thrombin, is measured by incuba- The structure of the EGF shows two main tion of thrombin and TM together with pro- rings of 9-15 amino acids. The N-terminal tein C, and than by dosage of the activated ring is formed by the amino acids located be- protein C. A directly proportional relation- tween the third and fourth cystein that make ship between activated protein C and con- up two disulphuric bonds between the first centration of TM has been thus demonstrat- and the third cystein and the second and ed. The sequence of the TM structure that fourth cystein, respectively. The C-terminal depends from the O region of the sugar, ring is formed by the amino acids between where the condroitin-sulphate bond takes the fifth and the sixth cystein and is a simple place, favours the inhibition of thrombin by ring with one disulphuric bond. The model anti-thrombin III and raises the affinity of considered shows that the third and the TM for thrombin5. fourth cystein, in the EGF sequences, are close enough to form a disulphuric bond. The thrombomodulin region that goes from the Genetic features fifth to the sixth domain inhibits the break- down of fibrinogen, but also plays a role as a The importance of the primary, secondary competitive inhibitor of protein C activation, and tertiary structure of TM is evident in pa- without however showing a cofactor-like ac- tients with gene mutation. These subjects tivity. The fourth EGF-like domain is the one present an elevated risk of cardiovascular necessary for the cofactor activity. A frag- accidents. The mutation of thrombomodulin ment containing the fourth, fifth and sixth structure from 127 G (guanine) to 127 A EGF-like moieties can solicit a structure vari- (adenine) leads to the mutation Ala (ala- ation at a distance of 15 A (Angstrom) from nine) 25 Thr (treonine) that raises the risk the active site of the thrombin molecule1. of myocardial infarction in men. This risk Thrombomodulin binds to thrombin by naturally increases also in association with two of its structural distinctive features: the other metabolic factors9. In a study conduct- disulphuric bond (that forms a cyclic struc- ed in patients surviving from a myocardial

60 Clinical importance of thrombomodulin serum levels infarction, the C/T (cytosine/timine) dimor- Metabolism phism of the thrombomodulin gene (nu- cleotide 1418) appeared to be an important Thrombomodulin serum levels also depend predictive index of early myocardial infarc- from the degradation of cellular TM present tion7. The C/T dimorphism (characterized by on endothelial cells detached by natural the substitution of the valine (Val) with ala- turnover. The elimination of TM is guaran- nine (Ala455) in the sixth EGF-like domain teed by renal and hepatic metabolism10. of the TM) when expressed by the allele in- Thrombomodulin levels are elevated in renal dicates a greater prevalence of myocardial failure and this increase shows a significant infarction4. The substitution of the Val with correlation with creatinine serum levels. Half Ala 455 generates a TM molecule with an al- life of TM is ten minutes11. tered function and spatial conformation in Serum TM may be expressed as the ratio the thrombin binding and protein C activat- between soluble TM/serum creatinine, to dis- ing region. The mutations of the molecular tinguish the habitual elevation of TM occur- structure can also influence the protein’s ring during renal failure from that due to en- susceptibility to proteolytic degradation and dothelial cell damage. Endothelial cell pro- influence the serum concentration of the duction of TM is up regulated mainly by c- fragments. The allelic combination C/T, C/C AMP, retinoic acid and IL-4 and IL-1310. and T/T in position 1418 determines a consequent reduction of the activation of protein C and a related increase of thrombin. In Physiology of TM patients with a previous myocardial infarction the difference in Ala 455 and Val allele Thrombomodulin production is regulated concentration differed significantly from by the sequences close to its gene4 and de- that of controls: 82% for Ala and 18% for pends from the vascular micro-environment12. Val versus 74% and 26% respectively. Many factors such as the presence of tPA (tis- However, C/T dimorphism is only one of the sue plasminogen activator) and PAI-1 predisposing factors toward myocardial in- (Plaminogen activator inhibitor)13, inflamma- farction, since this disease has a known mul- tory cytokines, immune reactions and coagu- tifactorial origin. Substitutions of single lation cascade activation influence TM lev- bases have been identified in others genes els14. An interesting observation is the shar- that codify for coagulation cascade proteins. ing of the receptor site of the thrombin mole- Two of the three mutations have been iden- cule with that of the TM15 molecule, through tified in the region corresponding to the a relationship based on the concentration of thrombomodulin gene. Moreover, the analy- TM. When TM reaches high levels it binds to sis of the reporter gene, using mutants with thrombin with a 1:1 ratio1, blocking the acti- promoter gene deletion, demonstrated that vation of the coagulation cascade and activat- the sequence from –33 to –70 is important ing protein C. Both fibrinogen and factor VII for the transcription of the regulatory se- participate in the formation of the clot. High quences for the transcription of the throm- factor VII levels favour the formation of bomodulin gene. One of the identified muta- thrombin (key enzyme for the development tions occurs at the end of this region close to of the arterial thrombus) that is also a mitosis the “TATA-box”. This mutation is more fre- promoting factor for smooth muscle cells and quent in the Asiatic population. The muta- fibroblasts. Thrombin is also a potent platelet tion in position –9/-10 from GG to AT has activator. The platelet activating factor acti- been identified only in patients with myocar- vates neutrophils and promotes the appear- dial infarction and in none of the controls. ance of leucocyte adhesion molecules on the This mutation as well occurs in the region endothelial cells. Through interaction with close to the “TATA-box”. thrombomodulin, thrombin can start an anti- Mutations and band shifts have been ob- coagulant cascade, by the activation of pro- served in the promoter region in 4.8% of pa- tein C. Thrombin thus seems to be able to tients with myocardial infarction. All these mediate vascular responses to events that mutations occurred in fragments tm2 and favour coagulation, anti-coagulation, inflam- tm34. mation and proliferation15.

61 F. Califano, T. Giovanniello, P. Pantone, E. Campana, C. Parlapiano, F. Alegiani, et al.

Two main mechanisms contribute to breaking up of the plaque. The low TM levels thrombotic disease in humans: the anticoagu- in patients with mutations of the 5’ region of lant pathway of protein C and the heparin- the TM gene can generate a protein C variant antithrombin mechanism. These two physio- that behaves less actively during transitory is- logical systems work together to regulate the chemic attacks and can consequently increase coagulation processes. The protein C path- the probability of cardiovascular injury. way inhibits the function of the regulatory Thrombomodulin also shows a growth factor proteins factor Va and factor VIIIa. The he- like function. It should be remembered that parin-antithrombin system inhibits the coagu- the repeated EGF like sequences of the solu- lation proteases. ble TM form, corresponding to the proteolyt- ically degraded TM, have a promoting action Protein C activation pathways and on mitosis of fibroblast and endothelial cell clinical events associated with defects of lines15. these pathways The protein C pathway starts with the interaction between thrombin and thrombo- Physiopathological characteristics modulin that transforms thrombin in an enzyme that starts an anti-coagulant response. In endothelial injury determinism, a great Due to the common polymorphism of protein importance must be attributed to the compo- C, it is possible to examine the relationship nents that modulate TM expression and con- between the resistance to its activated form sequently influence protein C, S5 and the and cardiovascular diseases. The altered processes associated to these proteins (in- function of protein C contributes to the inflammation, immune responses). Protein C creased risk of myocardial infarction. The ac- plays the fundamental role in endothelial in- tivation of protein C is almost certainly due jury physiopathology by blocking the inflam- to the formation of the thrombin-TM com- matory reactions and the injury secondary to plex. It can be assumed that low TM levels liberation of inflammatory peptides during weaken the anti-coagulant response when coagulation, fibrinolysis and complement ac- systemic thrombin levels rise, thus promoting tivation. Naturally, in this process trombo- the formation of the clot on sites of vascular modulin plays a fundamental role due to its damage. It has been demonstrated that the ability to activate protein C. Above protein presence of TM diminishes the thrombin’s C, we can find the regulation of cell surface ability of activating cells through thrombin TM expression that is influenced by other receptors. These studies have been conducted substances. Different peptides, such as brady- on cells that express thrombin receptors, but chinine, complement factor C3a, fibrinogen not TM receptors. These cells were transfect- degradation products (PDF)14 function as ed with TM, and the response to thrombin stimuli for inflammatory cell (neutrophils, could be attenuated according to the rise in monocytes, macrophages) production of cy- TM concentrations. The inhibition taking tokines. Cytokines such as IL-1 and TNF place can be at least in part explained by the down regulate the expression of TM, in par- fact that the thrombin and the thrombomod- ticular by acting on the membrane form. Also ulin receptors share some common binding TNF, endotoxins such as lipopolysaccharide sites. This demonstrates that thrombomod- (LPS), neutrophil elastase, oxygen free radi- ulin inhibits thrombin mediated cellular acti- cals, hydrogen peroxide and anaphilotoxin vation by acting on thrombin’s receptors. are capable of reducing TM activity by influ- Keeping in mind that the same regulation al- encing the “cleavage” of the extracytoplas- so takes place on the endothelium, the re- matic part6. duced expression of TM, conditioned by the The cytokines that down regulate TM con- mutation on the 5’ region of the gene, proba- centration on endothelial cells also influence bly can favour the activation of endothelial protein C. In this situation an increase of t- cells and the consequent expression of the PA and PAI.1 takes place13. adhesion molecules. Consequently, the loss of For what pertains to the interaction of en- function of TM can contribute locally to the dothelium with the cellular component of development of atherosclerosis and to the blood, it must be remembered that activated

62 Clinical importance of thrombomodulin serum levels neutophils can liberate TM from endothelial lymphatic vascular cells, TM is produced also cells. The oxidation of TM methionine by trophoblasts, leptomeninges, mesothelium residues by neutrophil activation products and mesangium10. can block the cofactor-like activity6. The acti- Moreover, a significant difference in sol- vated neutrophils desensitise TM anticoagu- uble TM levels has been demonstrated in lant activity by their products elastase, the different blood groups (AB0)20. catepsin G, H2O2, and exercise a proteolytic Thrombomodulin concentration is lower in activity that can cause detachment of en- group 0 and A subjects and higher in group dothelial cells. B subjects. In AB subjects TM levels are in- termediate between those of group A and group B. Trombomodulin serum levels and dosages TM levels during DIC During sepsis with DIC, the high TM levels Different pathological situations increase seem to participate in the development of soluble circulating TM. The endothelial cells MOF (multiple organ failure)21. Patients with more exposed to hemodynamic turbulence higher TM levels during DIC have a higher (such as those on the bifurcation of major ar- mortality rate compared to patients who sur- teries) liberate a great quantity of TM10. vived. Thrombomodulin levels range from 3 to 300 ng/ml. It is believed that normal levels are 3.1 TM levels during cardiac surgery ± 1.3 ng/ml, with slightly higher levels in Thrombomodulin levels rise in patients un- males2. It seems that in the women TM levels dergoing cardiac surgery with variations re- rise during menopause. Women with surgical lated to age, continuity of the disease and induced menopause have soluble TM levels complexity of the surgical procedure22. well above normal. After six weeks of hormone replacement therapy a significant re- TM levels during myocardial infarction duction of TM levels takes place16. Gene mutations of TM or alterations of the Thrombomodulin levels vary according to regulatory sequences for gene expression race and Blacks seem to have lower levels17. favour myocardial infarction4. The presence Thrombomodulin levels are usually mea- of these alterations causes the production of sured with ELISA method. During the study TM with impaired function that at the same of the different weight circulating fragments, serum levels, presents an inferior anticoagu- it was realized that the lighter fragments lant power23. However, soluble TM levels de- (M.W. 69 kDa) were the extracellular domain crease because of the cytokine mediated inhi- of the TM, while the heavier fragments bition of the mechanisms responsible for TM (M.W. about 150 kDa) are formed by TM production, and conversely, rise again after tied to the plasma membrane and its intracy- 24-48 hours from thrombolysis2. toplasmatic component3. Other studies conducted with the same immunoenzymatic TM levels during atherosclerosis method have shown a variation of these two Atherosclerosis is associated with high components in different diseases (disseminat- soluble TM levels24. Soluble TM levels rise ed intravascular coagulation (DIC), pul- in patients with localized atherosclerotic monary thromboembolism, adult respiratory disease (carotid artery disease, iliac or distress syndrome, acute renal and chronic femoral artery disease) with a significant renal failure)18. Thrombomodulin levels also further elevation in patients with multivas- vary in moderate alcohol drinkers, because cular disease (contemporary presence of alcohol concentration is inversely correlated carotid disease, coronary disease and iliac with the soluble form of TM17. A positive sig- or femoral disease). Soluble TM levels are nificant correlation has been found between thus correlated with the extension of the TM levels and the number of years of tobac- vascular pathology25. It has also been co smoking19. Vascular smooth muscle cells demonstrated that TM mRNA present in and some tumor cells produce TM both in vi- monocytic cells positively correlates to low vo and in vitro. Besides endothelium and density lipoproteins (LDL) concentration26.

63 F. Califano, T. Giovanniello, P. Pantone, E. Campana, C. Parlapiano, F. Alegiani, et al.

TM levels in ARDS tion of factor 1 + 2, proportional to thrombin Only a significant TM elevation (> 100 concentration. With the increasing number of ng/ml) is able to predict an increased inci- lacunae, we assist to a parallel rise of the ma- dence of ARDS in patients at risk of this jor parameters and risk factors of coagulation complication27. Moreover, TM levels have activation, among those soluble TM levels. been demonstrated to be very high in bron- Moreover, it should be remembered that choalveolar lavage fluid in ARDS patients28. Lp(a) levels, considered to be a reliable marker of atherosclerosis progression, show a TM levels in liver cirrhosis significant positive relation with TM levels (p Patients with alcoholic cirrhosis present < 0.05). the highest TM levels, comparable to those in patients with chronic active hepatitis B o TM levels in multiple sclerosis C29. In hepatic cirrhosis the high weight Thrombomodulin levels are significantly forms are significantly reduced, with an in- elevated in patients with clinical recurrences crease of the low weight molecules. It is in- of multiple sclerosis and it has been shown teresting to note that in acute hepatic fail- that steroid therapy can reduce them36. ure the different TM forms rise in a similar In conclusion, soluble thrombomodulin fashion18. serum levels are a valid prognostic index during many diseases. Thrombomodulin levels TM levels in diabetes mellitus can be interpreted as a marker of disease pro- Elevated serum glucose levels can cause gression both in acute and chronic pathologi- endothelial cell damage. Thrombomodulin cal conditions21. Higher serum TM levels are rises in adult diabetic patients and also in pa- associated with greater disease severity and tients with juvenile diabetes, also in absence progression probability indicating a progres- of vascular disease30. sive evolution of inflammatory and immuno- Thrombomodulin levels are in direct rela- logical mechanisms. tion with proteinuria. In patients with NID- DM TM levels are higher in those with macroalbuminuria compared to those with microalbuminura31. Serum levels of TM correlate with the du- References ration of the disease both in patients with type I and type II diabetes mellitus. 1) LOUGHEED JC, BOWMAN CL, MEININGER DP, KOMIVES EA. Thrombin inhibition by cyclic peptides from Serum TM levels are increased in patients thrombomodulin. Protein Sci 1995; 4: 773-780. with a growing number of complications s s (nephropathy, retinopathy, arterial occlusive 2) HLIN A, MORSER J, HLIN H. Soluble thrombomod- 32 ulin antigen in plasma is increased in patients disease, neuropathy, hypertension) . with acute myocardial infarction treated with Moreover, progression of diabetic retinopa- thrombolytic therapy. Thromb Res 1996; 82: 313- thy can be predicted by monitoring of serum 322. TM levels that rise with the progression of 3) TOMURA S, DEGUCHI F, M ARUMO F, A OKI N. Enhanced the disease33. presence of thrombomodulin in the glomeruli of Elevated TM serum and urinary levels in lupus glomerulonephritis. Clin Nephrol 1994; 41: diabetic patients are predictive of an early 205-210. evolution towards diabetic nephropathy34. 4) IRELAND H, KUNZ G, KONSTANTINOS K, STUBBS PJ, LANE DA. Thrombomodulin gene mutations associated TM levels in cerebral vascular disesease with myocardial infarction. Circulation 1997; 96: 15-18. The presence of a silent lacunar infarction represents a risk factor for successive evolu- 5) ESMON CT. Inflammation and thrombosis: the im- pact of inflammation on the protein C anticoagu- tion towards a clinically evident ischemic lant pathway. Haematologica 1995; 80 (Suppl 2): 35 cerebral infarction . In the patients with 49-56. symptomatic and silent lacunar infarction to- 6) MACGREGOR IR, PERRIE AM, DONNELLY SC, HASLETT C. gether with an elevation of fibrinogen levels, Modulation of human endothelial thromomodulin a major activation of factor VII and thrombin by neutrophils and their release products. Am J is also present, as measured by the concentra- Respir Crit Care Med 1997; 155: 47-52.

64 Clinical importance of thrombomodulin serum levels

7) NORLUND L, HOLM J, ZÖLLER B, sHLIN AK. A com- lial cell markers and adhesion molecules. Br J mon thrombomodulin amino acid dimorphism is Hematol 1996; 92: 498-500. associated with myocardial infarction. Thromb 21) ENDO S, INADA K, NAKAE H, et al. Blood levels of Haemost 1997; 77: 248-251. endothelin-1 and thrombomodulin in patients with 8) IRELAND H, KYRIAKOULIS K, KUNZ G, LANE DA. Direct disseminated intravascular coagulation and sep- search for thrombomodulin gene mutations. sis. Res Comm Molec Pathol Pharmacol 1995; Haemostasis 1996; 26 (Suppl 4): 227-232. 90: 277-288.

9) DOGGEN JMC, KUNZ G, ROSENDAAL FR, et al. A mu- 22) BOLDT J, SCHINDLER E, KNOTHE CH, WELTERS I, tation in the thrombomodulin gene, 127G to A STERMANN WA, HEMPELMANN G. Endothelial-related coding for Ala25Thr, and the risk of myocardial in- coagulation in cardiac surgery. Br J Anaesth farction in men. Thromb Haemost 1998; 80: 743- 1995; 75: 174-179. 748. 23) NEUMANN FJ, OTT I, GAWAZ M, et al. Cardiac re- 10) BOFFA MC. Considering cellular thrombomodulin lease of cytochines and inflammatory responses distribution and its modulating factors can facil- in acute myocardial infarction. Circulation 1995; tate the use of plasma thrombomodulin as a reli- 92: 748-755. able endothelial marker? Haemostasis 1996; 26 24) TOHDA G, OIDA K, OKADA Y, et al. Espression of (Suppl 4): 233-243. thrombomodulin in atherosclerotic lesions and mi- 11) KUWASAKO K, KIDA O, KITAMURA K, KATO J, ETO T. togenic activity of recombinant thrombomodulin in Plasma adrenomedullin in cerebrovascular dis- vascular smooth muscle cells. Arterioscler ease: a possible indicator of endotelial injury. Int Thromb Vasc Biol 1998; 18: 1861-1869. Angiol 1997; 16: 272-279. 25) BLANN AD, SEIGNEUR M, STEINER M, BOISSEAU MR, C OLLUM . Circulating endothelial cell mark- 12) AIRD WC, EDELBERG JM, WEILER-GUETTLER H, SIMMONS M C CN ers in peripheral vascular disease: relationship to WW, SMITH TW, ROSEMBERG RD. Vascular bed-specific expression of an endothelial cell gene is pro- the location and extent of atherosclerotic disease. grammed by the tissue microenviroment. J Cell Eur J Clin Invest 1997; 27: 916-921. Biol 1997; 138: 1117-1124. 26) OIDA K, TOHDA G, ISHII H, et al. Effect of oxidized low density lipoprotein on thrombomodulin ex- 13) WADA H, MORI Y, S HIMURA M, et al. Poor outcome in disseminated intravascular coagulation or throm- pression by THP-1 cells. Thromb Haemost 1997; botic thrombocytopenic purpura patients with se- 78: 1228-1233. vere vascular endotelial cell injuries. Am J 27) BAJAJ MS, TRICOMI SM. Plasma levels of the three Hematol 1998; 58: 189-194. endothelial-specific proteins von Willebrand factor, tissue factor pathway inhibitor, and thrombo- 14) INFIAMMAZIONE E PROCESSI RIPARATIVI. In Robbins SL, modulin do not predict the development of acute Cotran RS. Le basi patologiche delle malattie. respiratory distress syndrome. Intensive Care Padova: Piccin, 1984: 56-91. Med 1999; 25: 1259-1266. 15) ESMON CT. Defect in natural anticoagulant path- 28) TANIGUCHI H, KONDOH Y, I KUTA N, TAKAGI K. ways as potential risk factors for myocardial in- Diagnosis of acute respiratory distress syndrome: farction. Circulation 1997; 96: 9-11. analysis of bronchoalveolar lavage fluid. [Article 16) LIP GY, BLANN AD, JONES AF, BEEVERS DG. Effects of in Japanese]. Nihon Kyobu Shikkan Gakkai hormone-replacement therapy on hemostatic fac- Zasshi 1995; 33 (Suppl): 251-256. tors, lipid factors, and endothelial function in 29) SIDO B, DATSIS K, MEHRABI A, et al. Soluble throm- women undergoing surgical menopause: implica- bomodulin Ð a marker of reperfusion injury after tions for prevention of atherosclerosis. Am Heart orthotopic liver transplantation. Transplantation J 1997; 134: 764-771. 1995; 60: 462-466. 17) SALOMAA V, M ATEI C, ALEKSIC N, et al. Soluble throm- 30) MCLAREN M, ELHADD TA, GREENE SA, BELCH JJ. bomodulin as a predictor of incident coronary Elevated plasma vascular endothelial cell growth heart disease and symptomless carotid artery factor and thrombomodulin in juvenile diabetic atherosclerosis in the Atherosclerosis Risk in patients. Clin Appl Thromb Hemost 1999; 5: 21- Communities (ARIC) Study: a case-cohort study. 24. Lancet 1999; 353: 1729-1734. 31) SUMIDA Y, W ADA H, FUJII M, et al. Increased soluble 18) SHOGO T, K IMURA S, OHDAMA S, AOKI N. Plasma fibrin monomer and soluble thrombomodulin level thrombomodulin in health and disease. Blood in non-insulin dependent diabetes mellitus. Blood 1990; 76: 2024-2029. Coagul Fibrinolysis 1997; 8: 303-307.

19) MARKULJAK I, IVANKOVA J, KUBISZ P. Thrombomodulin 32) GABAT S, KELLER C, KEMPE HP, et al. Plasma throm- and von Willebrand Factor in smokers and during bomodulin: a marker for microvascular complica- smoking. Nouv Rev Fr Hematol 1995; 37: 137- tions in diabetes mellitus. Vasa 1996; 25: 233- 139. 241. 20) BLANN AD, DALY RJ, AMIRAL J. The influence of age, 33) IKEDA T, S ATO K, IWASAKI T, K UME C, HIRAISHI S, ISHII H. gender and AB0 blood group on soluble endothe- Thrombomodulin in diabetic retinopathy [Article in

65 F. Califano, T. Giovanniello, P. Pantone, E. Campana, C. Parlapiano, F. Alegiani, et al.

Japanese]. Nippon Ganka Gakkai Zasshi 1994; Arterioscler Thromb Vasc Biol 1996; 16: 734- 98: 283-286. 741.

34) SHIMANO H, TAKAHASHI K, KAWAKAMI M, et al. 36) TSUKADA N, MATSUDA M, MIYAGI K, YANAGISAWA N. Elevated serum and urinary thrombomodulin Thrombomodulin in the sera of the patients with levels in patients with non-insulin-dependent di- multiple sclerosis and human lymphotropic virus abetes mellitus. Clin Chim Acta 1994; 225: 89- type-1-associated myelopathy. J Neuroimmunolo- 96. gy 1995; 56: 113-116.

35) KARIO K, MATSUO T, K OBAYASHI H, ASADA R, MATSUO 37) HAYNES BF, FAUCI AS. Introduzione al sistema im- M. Silent cerebral infarction is associated with munitario. In: Fauci AS, Braunwald E, Isselbacher hypercoagulability, endothelial cell damage, KJ, et al. Principi di Medicina Interna. Milano: and high Lp(a) levels in elderly Japanese. Mac Graw-Hill Libri Italia srl, 1999: 1999-2025.