**2. A new approach to PE**

#### **2.1 The role of matrix metalloproteinases**

Experimental evidence indicated that the pathophysiology of PE implies the activation of matrix metalloproteinases (MMPs) (Uzuelli et al., 2008; Dias-Junior et al., 2009; Souza-Costa et al., 2005; Souza-Costa et al., 2007; Palei et al., 2005; Fortuna et al., 2007). Indeed, hemodynamic derangements associated with this condition improved with the inhibition of MMPs. Neutrophil activation (Eagleton et al., 2002) and rapid release of granules containing large amounts of MMP-9 in inflammation (Van den Steen et al., 2002) and during PE explains how MMPs, especially MMP-9, are involved in pathophysiology of PE. The increased activity and levels of MMP-9 found in ischemic stroke, or the upregulation of the enzyme after cerebral ischemia are interestingly similar to PE (Asahi et al., 2000). The degradation of type IV collagen, laminin, and fibronectin by MMP-9, may contribute to hemorrhagic transformation after cardioembolic stroke as these components are the main structure of the vascular matrix (Rosell et al., 2008; Montaner et al., 2001). Also, tissue plasminogen activator (or alteplase) can amplify MMP-9 levels by upregulation, thus increasing ischemic brain damage (Wang et al., 2004; Burggraf et al., 2007; Ning et al., 2006; Tsuji et al., 2005). There is evidence, that increased plasmin concentration may activate MMPs. Previous experimental work by our group aimed to assess the levels of MMPs following fibrinolysis for acute PE. Circulating levels of MMPs were measured serially (MMP-9 and MMP-2). Their endogenous inhibitors, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were also measured in alteplase and in ultra-high dose streptokinase-treated patients with acute PE (Mühl et al., 2010).

#### **2.2 Measurements and discussion of TIMP/MMP changes in PE**

In our study MMP levels were assessed by sodium-dodecil-sulphate polyacrylamide gel electrophoresis, TIMP levels were measured with a commercially available ELISA kit (Mühl et al., 2010). Significant increases in pro-MMP-9 concentrations were found after TL therapy in both groups, but these were not associated with significant alterations in TIMP-1 levels. Pro-MMP-9/TIMP-1 ratio increased significantly. Interestingly, earlier increases in pro-MMP-9 levels and in pro-MMP-9/TIMP-1 ratio were found in subjects treated with streptokinase. From the 3rd day pro-MMP-9 levels and pro-MMP-9/TIMP-1 ratio returned to normal. No significant changes in pro-MMP-2 concentrations were measured after TL. Moreover, we found no significant changes in TIMP-2 concentrations or in pro-MMP-2/TIMP-2 ratio.

Although there is a lack of firm evidence, the possible explanation for increased MMP-9 levels during treatment with alteplase is the promotion of MMP-9 release by neutrophils (Cuadrado et al., 2008). According to our knowledge, no previous study has reported that streptokinase induces the release of MMP-9.

A slower increase of pro-MMP-9 was found in alteplase treated patients, but the precise explanation for this difference between fibrinolytic agents is not yet elucidated. There is significant interindividual variability in neutrophil degranulation (Cuadrado et al., 2008), therefore a multi-central study may draw firm evidence on this question.

No definitive conclusion can be drawn yet, but it is widely acknowledged that intracerebral hemorrhage is the most feared bleeding complication of TL (Arcasoy & Kreit, 1999). The use of alteplase enhanced MMP-9 levels, which has already been widely associated with hemorrhagic transformation after cardioembolic stroke (Rosell et al., 2008; Montaner et al., 2001). This observation offers an explanation for the hemorrhagic transformation during stroke.

It is possible that the MMP inhibitors may decrease the risk of intracerebral hemorrhage or other bleeding complication of TL for acute PE (Murata et al., 2008; Sumii & Lo, 2002; Machado et al., 2009) and may have beneficial hemodynamic effects (Fortuna et al., 2007; Palei et al., 2005).
