Marie-Claude Guillin

Plasma Matrix Metalloproteinase-9 as a Marker of Blood Stasis in Varicose Veins

Background—Possible intermediate circulating markers linking blood stasis to vein remodeling were explored in patients with varicose veins in the lower limbs. Methods and Results—Blood was sampled at rest (supine position) and after a stasis of 30 minutes both in the varicose vein (limbs hanging down) and in the brachial vein (arm hanging down) as a paired control. Several endothelial and leukocyte markers were measured in plasma with the use of ELISA kits. Angiotensin-converting enzyme activity was determined by use of a specific substrate. Matrix metalloproteinases (MMPs) 9 and 2 were evaluated with the use of gelatin zymography. No markers were significantly modified after 30 minutes of blood stasis in the brachial vein. After 30 minutes of blood stasis in the varicose vein, oxygen partial pressure decreased (P <0.01). Although thrombomodulin, von Willebrand factor, vascular endothelial growth factor, and MMP-2 were not modified in these conditions, the proteins released by proteolysis from the endothelial membrane intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and angiotensin-converting enzyme were increased (P <0.01). After blood stasis in varicose veins, the leukocyte markers lactoferrin, myeloperoxidase, and interleukin-8 were not modified, whereas L-selectin shed from leukocytes increased (P <0.05), and a major increase in pro-MMP-9, which is released from tertiary granules during polymorphonuclear activation, was observed (P =0.0001). Conclusions—The marked increase in plasma pro-MMP-9 activity provides evidence of polymorphonuclear activation and granule release in the varicose vein in response to postural blood stasis. Similarly, detection in the plasma of membrane proteins shed from the endothelium or leukocytes provides evidence of pericellular proteolysis.

The Serpin Protease-Nexin 1 Is Present in Rat Aortic Smooth Muscle Cells and Is Upregulated in l-NAME Hypertensive Rats

Objective—Protease-nexin 1 (PN-1) belongs to the serpin superfamily and behaves as a specific thrombin inhibitor in the pericellular environment. Little is known about PN-1 expression and its regulation in the vascular system. In this study, we examined the expression of functionally active PN-1 in vitro in rat aortic smooth muscle cells and in vivo in rat arterial media and its regulation in hypertensive rats. Methods and Results—The vascular PN-1 formed specific covalent complexes with thrombin involving the catalytic site of the protease, and heparin increased the formation of these complexes. We also demonstrated PN-1 in rat arterial media by immunohistochemical staining. Moreover, we examined in vivo vascular expression of PN-1 in a model of chronic hypertension induced by long-term administration of NG-nitro-l-arginine methyl ester (l-NAME). Marked increases in PN-1 mRNA (3-fold) and protein (2-fold) were observed after 2 months of hypertension. Increased expression of PN-1 in the vascular wall was associated with an increase in the formation of complexes between radiolabeled-thrombin and PN-1, indicating that PN-1 was functional. Conclusions—PN-1 may thus participate in the mechanisms that regulate thrombin activity in the vessel wall.

Protease Nexin-1: A Cellular Serpin Down-Regulated by Thrombin in Rat Aortic Smooth Muscle Cells

Protease nexin‐1 (PN‐1), a potent inhibitor of serine proteases, is present in vascular cells and forms complexes with thrombin, plasminogen activators, and plasmin. We examined the effect of thrombin on PN‐1 expression by rat aortic smooth muscle cells (RASMCs). PN‐1 expression was determined by measuring protein and mRNA levels, using respectively immunoblotting and semi‐quantitative reverse transcriptase polymerase chain reaction (PCR). Thrombin down‐regulated PN‐1 expression in a dose‐ and time‐dependent manner. This effect was mediated via the interaction of thrombin with its receptor protease activated receptor (PAR‐1) since the peptide thrombin receptor activating peptide (TRAP) reduced PN‐1 expression. PN‐1 secreted by smooth muscle cells remained essentially associated to cell‐surface glycosaminoglycans and was released from the cell surface by heparin. A lower amount of PN‐1 was released by heparin from TRAP‐stimulated versus unstimulated cells and correlated with a decreased capacity to inhibit thrombin. In addition, the ability to generate peri‐cellular plasmin was increased in cells with a low PN‐1 expression. Pre‐treatment of smooth muscle cells with cycloheximide abolished the reduction of PN‐1 expression by thrombin. Furthermore, conditioned media from thrombin‐treated cells reproduced the effect of thrombin, suggesting that thrombin acted via the induction of auto/paracrine mediator(s). We observed that fibroblast growth factor‐2 (FGF‐2)‐neutralizing antibodies abolished thrombin effect whereas FGF‐2 reproduced it, indicating that FGF‐2 is one of the involved mediator. Together, these results indicate that (i) PN‐1 modulates the activity of endogenous and exogenous serine proteases in RASMCs, (ii) thrombin down‐regulates PN‐1 expression and thus may increase its own activity on cells. J. Cell. Physiol. 201: 138–145, 2004.

Thrombin-induced platelet PAR4 activation: role of glycoprotein Ib and ADP.

Summary.  Thrombin activates human platelets via the cleavage of two protease‐activated G‐protein coupled receptors (PARs), PAR1 and PAR4 that respond to low and high concentrations of thrombin, respectively. The aim of the present study was to examine the relative contributions of GPIbα and ADP receptors in response to thrombin‐induced PAR1 and PAR4 stimulation. Platelet responses (aggregation, secretion and calcium mobilization) elicited by low thrombin concentrations were impaired when thrombin interaction with GPIbα was blocked. In contrast, blockade of thrombin interaction with GPIbα had no effect when PAR4‐coupled responses were specifically elicited by high thrombin concentrations in the presence of PAR1 antagonists or after PAR1 desensitization. These results confirmed that unlike PAR1, PAR4 does not require GPIbα as a cofactor for thrombin‐mediated activation. Both apyrase and selective antagonists of P2Y1 and P2Y12 inhibited PAR1‐coupled responses but did not modify PAR4‐coupled responses, indicating that in contrast to PAR1, PAR4 signals are not reinforced by ADP secretion and binding to the platelets. These results provide the direct evidence that, in human platelets, GPIbα and ADP act in synergy to amplify PAR1 coupled responses while PAR4 is activated independently of GPIbα and ADP.

Association of the −92C/G and 807C/T Polymorphisms of the α2 Subunit Gene With Human Platelets α2β1 Receptor Density

Objective—Platelet adhesion to the subendothelial tissue via the collagen receptor α2β1 is a crucial event in vascular biology. Although evidence has been provided that the number of platelets α2β1 copies is genetically determined, the molecular change primary responsible has not been yet elucidated. The aim of our present study was to investigate the effect of combined polymorphisms within both regulatory (−52C/T and −92C/G) and coding regions (807C/T and 1648A/G) of the α2 subunit gene on human platelets α2β1 receptor density and/or susceptibility to coronary artery disease (CAD). Methods and Results—Among 254 cardiac surgery patients, no evidence was found for an association between the α2 subunit gene polymorphisms and CAD. In contrast, in a subgroup of 113 patients, we observed a significant association between all polymorphisms except −52C/T and α2β1 receptor level. Furthermore, when 3 groups of patients were defined according to the tertiles of platelets α2β1 copies, the −92C/807T haplotype was more frequent in the group of patients with high α2β1 receptor level. Conclusion—These results suggest that an individual effect of each polymorphism located either in the coding or promoter sequence of the α2 gene may act in combination to modulate variations in platelets α2β1 receptor density.