Heart Vessels (2008) 23:301–307 DOI 10.1007/s00380-008-1053-x

© Springer 2008

ORIGINAL ARTICLE Manabu Horii · Shiro Uemura · Masahito Uemura Masanori Matsumoto · Hiromichi Ishizashi Keiichi Imagawa · Hajime Iwama · Yukiji Takeda Hiroyuki Kawata · Tamio Nakajima · Yoshihiro Fujimura Yoshihiko Saito

Acute myocardial infarction as a systemic prothrombotic condition evidenced by increased von Willebrand factor protein over ADAMTS13 activity in coronary and systemic circulation Received: November 27, 2007 / Accepted: March 7, 2008

Abstract The aim of the present study is to clarify the roles of circulating ADAMTS13 and von Willebrand factor (VWF) in the formation of coronary artery thrombi in acute myocardial infarction (AMI). Twenty-six AMI patients, 37 age-matched healthy controls, and 20 young controls were studied. Plasma ADAMTS13 activity and levels of VWF antigen (VWF :Ag) and unusually large VWF multimer (UL-VWFM) were measured in the femoral vein (FV), aortic root (Ao), and coronary sinus (Cs) immediately before percutaneous coronary intervention (PCI) during the acute phase of AMI, as well as 6 months later. During the acute phase of AMI, plasma levels of VWF :Ag were similar in FV, Ao, and Cs, and were higher than those of age-matched control. In contrast, ADAMTS13 activity in three sampling points in AMI patients was similar to that of age-matched controls. Thus, the ratio of VWF :Ag to ADAMTS13 activity in the acute phase of AMI was significantly higher in all three sampled sites than that of agematched controls. In the chronic phase, plasma levels of VWF :Ag, ADAMTS13 activity, and the ratio of VWF :Ag to ADAMTS13 activity were similar to those of age-matched controls. UL-VWFM was detected in the acute phase of AMI but not in the chronic phase. The present study showed that the plasma VWF :Ag levels are increased and ADAMTS13 activity is relatively decreased in both systemic and coronary circulation during the acute phase of AMI, suggesting that an imbalance between the enzyme and its substrate may play a role in the formation of occlusive thrombi in a coronary artery. M. Horii · S. Uemura · K. Imagawa · H. Iwama · Y. Takeda · H. Kawata · T. Nakajima · Y. Saito (*) First Department of Internal Medicine, Nara Medical University, 840 Shijo-cho, Kashihara 634-8521, Japan Tel. +81-744-22-3051; Fax +81-744-22-9826 e-mail: [email protected] M. Uemura Third Department of Internal Medicine, Nara Medical University, Kashihara, Japan M. Matsumoto · H. Ishizashi · Y. Fujimura Department of Blood Transfusion Medicine, Nara Medical University, Kashihara, Japan

Key words Acute coronary syndromes · Blood coagulation · Coronary circulation · Platelets · Thrombosis

Introduction The rapid closure of the coronary artery by acutely formed arterial thrombi, which are composed of platelets, fibrin, and inflammatory cells, is the major cause of acute myocardial infarction (AMI).1,2 Although the exact mechanism of coronary thrombus formation is not fully understood, the binding of von Willebrand factor (VWF) to glycoproteins Ibα and IIb/IIIa on the surface of platelets is known to lead to platelet activation and subsequent aggregation, which is an initial step toward formation of coronary thrombi.3,4 Earlier reports have shown that circulating levels of VWF antigen (VWF :Ag) is elevated in patients during the acute phase of AMI,5,6 and increased levels of plasma VWF :Ag can predict primary and secondary coronary events.7–9 Thus, VWF appears to be involved in the formation of coronary thrombi as a cause of AMI, although blocking of VWF function has not yet been clinically proven to prevent the onset of AMI. It is not clear, however where and how VWF is produced during AMI. Von Willebrand factor is synthesized in vascular endothelial cells and then released into the plasma as unusually large VWF multimer (UL-VWFM),4 which has most potent biological activities interacted with platelet, and is rapidly degraded into smaller VWF multimers by ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs 13),4,9 a metalloproteinase that specifically cleaves multimeric VWF between Tyr1605 and Met1606 within the VWF A2 domain.9 Loss-of-function mutation of ADAMTS13 leads to Upshaw–Schulman syndrome, a form of congenital thrombotic thrombocytopenic purpura. Reduction of ADAMTS13 activity keeps circulating ULVWFM levels high, which leads to platelet clumping and formation of platelet-rich thrombi. Recently, Sakai et al.6 reported that UL-VWFM was detected in plasma drawn from peripheral veins in patients with AMI. To understand

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the mechanism for the formation of coronary arterial thrombi in AMI, we measured plasma ADAMTS13 activity together with circulating levels of its substrate, VWF :Ag, in three sites: the aorta (Ao) near the ostium of the infarction-related coronary artery, the coronary sinus (Cs), and the femoral vein (FV). Samples were taken immediately before the percutaneous transluminal coronary intervention (PCI) during the acute phase of AMI and compared with those taken during the chronic phase.

Materials and methods Patients We studied 26 Japanese patients with AMI (5 women and 21 men; mean age 67.8 ± 11.6 years; range 38–89 years) admitted to the Nara Medical University Hospital between August 2004 and February 2005. The diagnosis of AMI was based on sustained chest pain of typical character and location, electrocardiographic ST-T elevation in two or more leads, disrupted regional wall motion on echocardiograms, and plasma levels of cardiac enzymes, including creatine phosphokinase (CK) and its MB fraction, that were greater than twice the normal upper limit. Of the 26 patients, 18 had hypertension, 21 had dyslipidemia, 13 had diabetes mellitus, 5 were obese, and 19 smoked. All of the patients received emergency coronary angiography and PCI within 24 h from the onset of AMI (the first symptoms). Clinical characteristics and drugs used are summarized in Table 1. The culprit lesions were in the right coronary artery in 6 patients, the left anterior descending coronary artery in 18, and the left circumflex coronary artery in 2. The peak CK level in AMI patients averaged 2 960 IU/l and ranged from

344 to 12 930 IU/l. All of the patients received intracoronary stents, implanted at the culprit lesions, and were subsequently given aspirin (81 mg/day, per os) and ticlopidine (200 mg/day, per os) or cilostazol (200 mg/day, per os) as antiplatelet therapy. An angiotensin-converting enzyme inhibitor and/or angiotensin-II receptor blocker were also administered to all patients. In addition, 10 patients received a β-blocker, 6 a calcium channel blocker, 7 a diuretic, and 15 a statin. Six months after the first onset of AMI, coronary angiography was again carried out in all of the patients. Written informed consent was obtained from all patients and control subjects participating in the study. The protocol was approved by the institutional review board of Nara Medical University (#2002-009). Young and age-matched healthy control subjects Study participants included both young and age-matched healthy control subjects. Young healthy subjects consisted of 30 volunteers (15 women and 15 men) aged from 20 to 39 years with a mean age of 30 ± 12.0 years, and agematched healthy subjects consisted of 37 healthy volunteers (19 women and 18 men) aged from 39 to 93 years with a mean age of 64.2 ± 14.0 years. Both groups had no history of angina, myocardial infarction, coronary artery bypass graft surgery, PCI, or any electrocardiographic abnormalities. Blood samples were collected from the antecubital vein early in the morning, before breakfast. Nine of the agematched controls (4 women and 5 men, mean age 48.1 ± 4.8 years, range 41–52 years) were also studied to evaluate the circadian variation of VWF :Ag and ADAMTS13 activity in plasma. In those subjects, blood samples were collected from the antecubital vein in the morning (09:30) and in the evening (20:00).

Table 1. Characteristics of patients with acute myocardial infarction

Age (years) Sex (female/male) Coronary risk factor (yes/no) Hypertension Dyslipidemia Diabetes mellitus Obesity Smoking Peak CK (IU/l) (mean) Location of AMI RCA/LAD/LCx Medication (yes/no) Aspirin Ticlopidine or Cilostazol ACE-I or ARB β-Blocker Calcium-antagonist Diurea Statin

Patient

Age-matched control subjects

P value

67.8 (38–89) 5/21

64.2 (39–93) 19/18

0.29 <0.01

8/18 21/5 13/13 5/21 19/7 2 960 (344–12 930)

0/37 2/35 3/34 7/30 7/30

<0.01 <0.01 <0.01 0.41 <0.01

6/18/2 26/0 26/0 26/0 10/16 6/20 7/19 15/11

Values in parentheses indicate range CK, creatine phosphokinase; AMI, acute myocardial infarction; RCA, right coronary artery; LAD, left anterior descending artery; LCx, left circumflex artery; ACE-I, angiotensin-converting enzyme inhibitor; ARB, angiotensin-II receptor blocker

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Assays of ADAMTS13 activity, VWF :Ag, and UL-VWFM

Blood sampling In the AMI patients, emergency cardiac catheterization was performed within 90 min of their arrival in our hospital. Blood samples were collected using a 7-F sheath inserted into the patient’s femoral vein (FV), a 6-F Cs catheter placed in the Cs through an FV sheath, and a 4-F Judkins catheter placed at the Ao. Unfractionated heparin and contrast medium were not used before pre-PCI blood sampling. Blood was sampled at the femoral vein (FV), the aortic root near the ostium of the infarction-related coronary artery (Ao), and the coronary sinus vein (Cs) immediately before and after emergency PCI. Six months after the onset of AMI, all 26 patients underwent a second round of coronary angiography, at which time blood was again collected from the same three areas. In young healthy and age-matched control subjects, blood samples were drawn from the antecubital vein. Preliminary experiments showed that there was no difference in plasma levels of VWF :Ag and ADAMTS13 activity among the antecubital vein, the FV, and the right atrium. Blood was collected into plastic tubes with 1/10th volume of 3.8% sodium citrate. Platelet-poor plasma was prepared by centrifugation at 3 000× g at 4°C for 15 min and stored in aliquots at −80°C until analysis.

Plasma ADAMTS13 activity was determined using a highly sensitive enzyme-linked immunosorbent assay (ELISA) recently developed by our laboratory.10 The assay system includes a recombinant GST-VWF73-His polypeptide as a substrate and a murine monoclonal antibody that specifically recognizes the Tyr1605 residue in the VWF-A2 domain exposed by ADAMTS13 cleavage; it does not recognize the uncleaved form of the peptide. Plasma VWF :Ag was measured by a sandwich enzyme immunoassay using rabbit antihuman VWF polyclonal antibody (Dako, Kyoto, Japan). Plasma ADAMTS13 activity and VWF :Ag levels were expressed as percentages of those of reference peripheral plasma obtained from 20 healthy volunteers aged 20–40 years. The lower detection limit of the ELISA for ADAMTS13 activity was 0.5% of the reference peripheral plasma activity. Plasma UL-VWFM was analyzed by sodium dodecyl sulfate – 0.9% agarose gel electrophoresis using 1 μl samples, after which VWF multimers were visualized by Western blotting and luminography, as described previously.11

Statistical analysis

150 A AM ADA MT TS1 13 aacttivity y (% %)

250 # 200 VW WF F:A Ag (% %)

T e rratiio of VW Th VWF:A Ag g to o ADA A AM MT TS113 activvityy

The data are expressed as mean ± SD. Comparison between acute and chronic data was performed using the paired

150 100

100 00

# 50

50 0 0 a

0 Ageg matched controls

Youngg healthyy controls

b

5 # 4 3 2 1 0

Ageg matched controls

Fig. 1a–c. Comparison of plasma von Willebrand factor antigen (VWF : Ag) levels and ADAMTS13 activity between healthy young subjects and age-matched controls. a Plasma VWF :Ag levels. b Plasma

Young healthyy controls

c

Ageg matched controls

Young healthyy controls

ADAMTS13 activity. c Ratios of VWF :Ag to ADAMTS13 activity. Shown are mean ± SD; #P < 0.001 vs young subjects

304

300

*

Student’s t-test or Wilcoxon signed-rank test, when appropriate. Comparison among the three groups of subjects was performed by analysis of variance. The analyses were carried out using the statistical software Statview (version 5.0; SAS Institute, Cary, NC, USA). A P value of less than 0.05 was considered statistically significant.

*

*

VWF:Ag (%)

250 200 150

Results

100

Differences between healthy young and age-matched controls

50 0

FV

a

Ao

CS

Acute p phase

FV

Ao

CS

Chronic p phase

Agege matched controls

ADAMTS13 activity (%)

100 80 60

VWF :Ag levels

40 20 0

FV

The ratio of VWF:Ag to ADAMTS13 activity

b

c

Plasma levels of VWF :Ag were significantly higher in healthy age-matched controls than in the young subjects (151% ± 58% vs 102% ± 33%, P < 0.001) (Fig. 1). Conversely, the plasma ADAMTS13 activity was lower in the age-matched controls than in the young subjects (51% ± 15% vs 104% ± 22%, P < 0.001), resulting in a three-fold higher ratio of VWF :Ag to ADAMTS13 activity in the age-matched controls than in young healthy controls (3.3 ± 1.4 vs 1.0 ± 0.3, P < 0.001) (Fig. 1).

Ao

CS

Acute p phase

*

*

FV

Ao

CS

Chronic phase p

Agege matched controls

*

6

During the acute phase of AMI before PCI, plasma VWF :Ag levels were significantly higher (P < 0.01) at the FV (211% ± 75%), Ao (204% ± 78%), and Cs (205% ± 90%) than in peripheral blood samples from the agematched controls (151% ± 58%) (Fig. 2a). During the chronic phase, these values (P < 0.05) fell to levels similar to those seen in the age-matched controls (FV, 149% ± 69%; Ao, 148% ± 73%; and Cs, 133% ± 52%). There also were no differences in VWF :Ag levels among sampling sites (Fig. 2a).

5

ADAMTS13 activity

4

Plasma ADAMTS13 activity did not differ among blood samples collected from the FV, Ao, and Cs before PCI during the acute phase of AMI (FV, 55% ± 22%; Ao, 57% ± 22%; Cs, 54% ± 19%), or during the chronic phase of AMI (FV, 51% ± 19%; Ao, 52% ± 17%; Cs, 51% ± 22%). In fact, all of these values were similar to ADAMTS13 activity in peripheral blood from the age-matched controls (51% ± 15%) (Fig. 2b). Moreover, ADAMTS13 activity in the acute phase was similar to that in the chronic phase at each sampling point. There was no significant inverse correlation between ADAMTS13 activity and plasma level of VWF :Ag in the acute phase of AMI.

3 2 1 0

FV

Ao

Acute p phase

CS

FV

Ao

CS

phase Chronic p

Agege matched controls

Fig. 2a–c. Plasma von Willebrand factor antigen (VWF : Ag) levels and ADAMTS13 activity and the ratio of VWF :Ag to plasma ADAMTS13 activity during the acute and chronic phases of AMI. a VWF :Ag levels, b ADAMTS13 activity, and c ratios of VWF :Ag to ADAMTS13 activity before percutaneous coronary intervention (PCI) during the acute phase and chronic phase of acute myocardial infarction (AMI). Measurements were made using plasma samples collected from the femoral vein (FV), aortic root (Ao), and coronary sinus (Cs) of the AMI patients and peripheral blood samples collected from control subjects. Shown are means ± SD; *P < 0.05 vs age-matched controls

The ratio of VWF :Ag to ADAMTS13 activity During the acute phase of AMI, the ratio of VWF :Ag to ADAMTS13 activity before PCI was significantly higher (P < 0.05) in the FV (4.5 ± 2.4), Ao (4.2 ± 2.5), and Cs (4.2 ± 2.4) than in the peripheral blood samples from age-matched

305 Fig. 3a,b. von Willebrand factor (VWF) multimeric analysis in two representative patients and its densitometric analysis. 1, patient 1 before PCI in acute phase of AMI; 2, after PCI of patient 1; 3, chronic phase of patients 1; 4 and 8, normal plasma; 5, before PCI of patient 2; 6, after PCI of patient 2; 7, chronic phase of patient 2. a von Willebrand factor multimeric analysis was performed using 1% agarose gel electrophoresis. b Densitometric analyses of unusually large VWF multimer (UL-VWFM) using NIH image J (developed by the National Institute of Health, http://rsb. Info.nih.gov/nih-image/). ULVWF multimer was detected in samples obtained from the femoral vein before and after PCI in all AMI patients tested, though it was not detected in samples collected during the chronic phase

UL-VWFM area (p (pixel))

1

a

5

2

6

3

7

4

8

controls (3.3 ± 1.4), though there was no significant difference in the ratio among sampling points (Fig. 2c). In the chronic phase, those ratios decreased to levels similar to those seen in age-matched controls (FV, 3.2 ± 1.9; Ao, 3.0 ± 1.6; Cs, 3.0 ± 1.5) (Fig. 2c). Detection of UL-VWFM We analyzed UL-VWFM in 6 out of 26 AMI patients. ULVWFM was detected in samples obtained from the FV before and after PCI in all patients (Fig 3a). It was not detected in samples collected during the chronic phase (Fig. 3a). To more easily quantify UL-VWFM levels, we performed densitometric analysis (Fig. 3b).

Circadian variation in plasma VWF :Ag and ADAMTS13 activity Von Willebrand factor antigen was significantly higher in the morning (100% ± 42%) than in the evening (89% ± 41%) (Fig. 4a); however, for ADAMTS13 activity there was no significant difference between the morning (55% ± 13%) and the evening (61% ± 18%) (Fig. 4b). The ratio of VWF :Ag to ADAMTS13 activity was significantly higher at 09:30 (1.9 ± 0.8) than at 20:00 (1.5 ± 0.8) (P < 0.05, Fig. 4c).

1

4 162 4,

2

3 714 3,

3

1 239 1,

4

569

5

4 872 4,

6

7 555 7,

7

699

8

544

b

Discussion In the present study we simultaneously measured the activity of ADAMTS13 and the levels of its substrates (VWF :Ag and UL-VWFM) in plasma samples obtained from the FV, Ao, and Cs during the acute and chronic phases of AMI. We demonstrate for the first time that during the acute phase, the ratio of VWF :Ag to ADAMTS13 activity in AMI patients was significantly higher at all three sampling sites than in the peripheral blood of age-matched controls and that during the chronic phase of AMI, these ratios had returned to levels similar to those seen in age-matched controls. Moreover, UL-VWFM was detected during the acute phase but not in the chronic phase of AMI, in agreement with recent reports by Sakai et al.6 and Goto et al.12 In agreement with our findings, Kaikita et al.13 also reported that the ratio of VWF :Ag to ADAMTS13 activity in peripheral venous plasma is higher in AMI patients than in those with stable exertional angina and chest pain syndrome. During the last decade, evidence has accumulated that release of VWF :Ag from endothelial cells and platelets is a key early step toward occlusive thrombus formation in the coronary circulation. With that in mind, before the beginning of the present study we hypothesized that VWF :Ag would be much higher in the Cs than in the Ao or FV, and that ADAMTS13 activity might be lower in the Cs than in the FV or Ao. However, our study showed that there was

306

160

Fig. 4a–c. Comparison with von Willebrand factor antigen (VWF : Ag), ADAMTS13, and the ratio of VWF :Ag to ADAMTS13 activity between in the morning and in the evening. a VWF :Ag was significantly higher in the morning (100% ± 42%) than in the evening (89% ± 41%). b ADAMTS13 activity was no significant difference between in the morning (55% ± 13%) and the evening (61% ± 18%). c The ratio of VWF :Ag to ADAMTS13 activity was significantly higher at 09:30 than at 20:00. Shown are mean ± SD, *P < 0.05 vs 20:00

* 140 VWF::Ag VW g (%))

120 100 80 60 40 20 a

0 9:30

20:00

9:30

20:00

ADA AD AM MTS S13 3 actiivitty (% %)

80 70 60 50 40 30 20 10

Ratio of VWF:Ag to ADAMTS13 activity

b

c

0

3 * 22.55 2 1.5 1 .55 0 9:30

20:00

no significant difference in the plasma level of VWF :Ag, ADAMTS activity, and the ratio of VWF :Ag to ADAMTS13 activity among three sampling points, clearly indicating that AMI is not a local but rather a systemic prothrombotic condition. In other words, the present findings support the recent concept that occlusive coronary thrombi develop in vulnerable blood (prone to thrombosis) or in vulnerable patients.14,15 Further studies are necessary to clarify whether increased VWF may lead to the formation of coronary thrombi or whether coronary thrombi itself may cause elevation of VWF levels in AMI. It was previously reported that AMI frequently occurs in the morning, between 06:00 and 12:00, a vulnerable period for cardiovascular events,16 and also frequently occurs after physical exercise, especially in the sun during summer. In that regard, we observed that the ratio of VWF :Ag to ADAMTS13 activity was higher in the morning than in the evening. Acute myocardial infarction is also more frequently observed in aged men than in young men. We previously reported that plasma ADAMTS13 activity declines and plasma VWF :Ag levels increase with increasing age, and that detectable levels of UL-VWFM were circulating in some older people.11 Here, we confirmed that plasma VWF :Ag is 50% higher while plasma ADAMTS13 activity is nearly 50% lower in healthy age-matched control subjects as compared to young healthy subjects, resulting in a three-fold higher ratio of VWF :Ag to ADAMTS13 activity in the former than in the latter. Interestingly, we have also observed that physical exercise increases the ratio of VWF :Ag to ADAMTS13 activity in healthy men.17 Thus the simultaneous measurement of ADAMTS13 activity and its substrate, VWF :Ag level, is useful in understanding the pathology of thrombotic diseases. Although it is not possible to identify the precise site of production of VWF and ADAMTS13, our findings (together with those of others5,6) suggest that, during the acute phase of AMI, production of VWF is increased not only in coronary arterial endothelial cells and/or locally activated platelets, but also in systemic vascular beds and/ or circulating endothelial cells. ADAMTS13 is mainly produced in hepatic stellate cells,18 and is also synthesized in both human endothelial cells19 and platelets,20 suggesting that this enzyme is produced in the systemic circulation as well as in the liver. In summary, we observed increased plasma VWF :Ag levels and relatively decreased ADAMTS13 activity in both systemic and coronary circulation during the acute phase of AMI, suggesting that an imbalance between the enzyme and its substrate may play an important predictive role in the formation of occlusive thrombi in a coronary

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artery, ultimately leading to AMI. Further analysis of the production–consumption relation between VWF and ADAMTS13 in the coronary and systemic circulations will be necessary to understand the pathophysiological significance of VWF and ADAMTS13 in the formation of occlusive thrombi. Acknowledgments This work was supported in part by research grants from the Ministry of Education, Culture, Sports, Science and Technology (to S.U., M.M., M.U., and Y.S.) and the Ministry of Health, Labour and Welfare (to M.M., Y.F., and Y.S.) of Japan.

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