Low Molecular Weight Heparin Treatment Decreases MMP-9 Plasma Activity in Patients with Abdominal Aortic Aneurysm

Article Outline

• Abstract
• Introduction
• Report
• Discussion
• Acknowledgements
• References
• Copyright

Large abdominal aortic aneurysms (AAAs) are associated with coagulation abnormalities, which are significantly reduced by low molecular weight heparin (LMWH). Considering anti-inflammatory properties of heparin we verified, whether LMWH influences MMP-2/-9 in AAA patients. The study involved 26 AAA individuals, 10 patients with coagulation abnormalities received LMWH and 16 were a control group. The plasma activity of MMP-2/-9 was measured using zymography. We found that, in addition to the reduction of coagulation abnormalities, LMWH treatment was associated with the decreased MMP-9 but not MMP-2 activity. Therefore, LMWH use could be considered as a valuable pretreatment before an elective aneurysm repair.

Keywords: AAA, abdominal aortic aneurysm, LMWH, low molecular weight heparin, MMP-9, matrix metalloproteinase

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Introduction 

The abdominal aortic aneurysm (AAA) development results from chronic destruction of the aortic wall, mediated by extracellular matrix metalloproteinases (MMPs), mainly MMP-9.¹ The increased MMPs activity in the damaged aortic wall correlates with an increased risk of aneurysm rupture. Therefore, it is possible that introduction of MMPs inhibitors to the clinical practice would decrease the aneurysm rupture frequency. Indeed, such a therapeutic approach is currently under extensive investigation.² It seems to be especially useful in individuals with small AAA, to decrease the aneurysm dilatation rate. This approach would also be beneficial for patients with large AAA to prevent its rupture until an elective repair. Numerous agents have been analyzed establishing their capability of MMPs inhibition. However, their effectiveness, especially in context of possible adverse events, remains controversial.³

Recently, we have demonstrated that large aneurysms are usually associated with subclinical coagulation abnormalities, that could be successfully treated with low doses of low molecular weight heparin (LMWH).⁴ Since heparin, in addition to anti-coagulant activity, displays some anti-inflammatory properties,⁵ we attempted to verify whether short-term LMWH treatment could influence MMP-2 and/or MMP-9 plasma activity in AAA patients.

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Report 

The study involved 26 AAA individuals qualified for an elective aneurysm repair, after exclusion of any concomitant diseases, which could independently provoke haemostatic abnormalities (malignancy, diabetes, hepatic cirrhosis, rheumatoid arthritis, renal insufficiency). On the basis of an extended laboratory screening for subclinical blood coagulation abnormalities, patients were divided into two subgroups, as described previously.⁴ The analyzed parameters included D-dimer (with threshold level >3000ng/ml) and prothrombin fragment PF₁₊₂ (>2nM) concentration and activation of fibrinolysis (>20%) (Table 1).

Table 1. Clinical data

Patient' parameters	Control AAA patients (n=16)	LMWH-treated group (n=10)
Age	65.5±7.9	70.1±6.5
Sex (male/female)	15/1	9/1
AAA diameter (mm)	53.8±8.2	69.7±12.0*
Laboratory test	No LMWH	Before LMWH	After LMWH
Blood platelets (× 103/μl)	225±38	192±38	233±42
Activated partial tromboplastin time (s)	36.4±2.5	36.2±2.3	37.2±3.9
Prothrombin time (s)	15.3±1.1	15.8±0.8	15.2±0.9
Fibrinogen (mg/dl)	403±76	384±42	454±75**
D-dimer (ng/ml)	776±330	5260±2120*	3018±1044**
Prothrombin fragment 1+2 (nM)	1.15±0.32	4.13±1.55*	1.91±0.75**
Fibrinolysis activation in the plasma (%)	4±5	58±36*	23±31**

The first group (n=10) received LMWH (2850IU of nadroparinum calcium, s.c., twice daily) injection for 5–7 days,⁴ whereas the second group (n=16) was a control group. All patients gave their written informed consent to participate in the study that was approved by the local ethics committee.

Blood morphology and selected haemostatic parameters were measured as described previously.⁴ The changes in coagulation tests observed in LMWH-treated AAA patients were shown in Table 1.

Plasma activity of MMP-2 and MMP-9 was measured using standard zymography. Quantitative analysis of zymograms was performed using digital gel documentation system GDS (UVP, Cambridge, UK). The conversion of sample activity to the specific MMP concentration (ng/ml) was done by a comparison of sample in-gel gelatinolytic activity to the activity curve of respective recombinant MMP, used in serial dilutions, at known concentrations.

We found that individuals with subclinical coagulation abnormalities displayed mean MMP-9 plasma activity significantly higher, than AAA individuals with normal coagulation parameters (p<0.01, by Mann-Whitney U-test) (Fig. 1). Interestingly, after 5–7 days of LMWH administration we observed a statistically significant (p<0.003, by matched pair T-test) decrease in MMP-9 plasma activity (Fig. 1). The mean MMP-9 activity in LMWH-treated patients reached the level of that of the control group. In contrast to MMP-9, no difference in MMP-2 plasma activity between both groups, or any influence of LMWH administration on the MMP-2 activity has been observed (not shown).

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• Fig. 1 

  MMP-9 zymograms of AAA control group (panel A) and LMWH-treated AAA patients (panel B). Each band represents one individual's plasma sample, displaying MMP-9-specific activity, with the band thickness corresponding to the MMP-9 amount/activity. Each rectangle in panel B contains a pair of MMP-9 bands from one individual' plasma samples, before and after LMWH treatment, respectively. The calculated MMP-9 concentration (ng/ml) was shown in panel C. Squares represent patients from AAA control group, whereas circles represent LMWH-treated patients before (open circles) and after treatment (filled circles). In each group mean values have been shown as solid lines and median values are represented by dotted lines. NS – non significant.

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Discussion 

It has been demonstrated that large aneurysms usually correlate with subclinical or clinically overt coagulation abnormalities and require preoperative LMWH treatment.⁴ On the other hand, the aneurysm diameter and rupture risk directly correlate with MMP-9 level.¹ However, the relationship between AAA-related coagulation abnormalities and high levels of MMP-9 remains uncertain. Moreover, it is unclear, what is a chronology of events. Two hypotheses are to be taken under consideration. The first possible mechanism is the primary hyperactivity of MMP-9 leading to the development of abdominal aneurysm and consequently intravascular coagulation in the aneurysmal sack. This hypothesis could be supported by the observation of high MMP-9 levels in individuals with small AAA without coagulation abnormalities. The second proposed hypothesis is somehow the evolution of the first and includes two steps. Primary mild increase of MMP-9 activity results in AAA development and intraaneurysmal clotting formation. However, intravascular coagulation in the aneurysmal sack could lead to the further increase of MMP-9. This theory could be supported by the fact that LMWH treatment reduced coagulation abnormalities and was associated with the decrease of MMP-9 activity. The observed LMWH-dependent decrease of MMP-9 activity is probably indirect, since, in our in vitro studies LMWH had an effect on neither MMP-9 activity, nor on MMP-9 production by leukocytes (data not shown).

Thus, the use of LMWH, especially in large aneurysms, would offer an attractive approach with double benefit. Indeed, in our observation the LMWH treatment resulted in advantageous effect on the coagulation abnormalities ⁴ and attenuation of MMP-9 hyperactivity. Therefore, LMWH use could be considered as a valuable pretreatment before an elective aneurysm repair. However, the effect of LMWH on MMP-9 activity in individuals with small AAA remains to be analyzed.

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Acknowledgements 

The study was supported by individual Warsaw Medical University grants. No competing interest declared.

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References 

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