The Effect In Adherence Platelets With HUVECs And In Patients With AMI After PCI By Tirofiban

Acute coronary syndromes is a kind of coronary atherosclerotic heart disease, including unstable angina and acute myocardial infarction(AMI). Mortality of AMI is 30% approximately, which is very harmful of public health.The rupture of the atherosclerotic unstable plague induced the formation of thrombotic occlusions,which is the cause of many cardiovascular maces. Enhanced turnover of the extracellular matrix (ECM) is thought to be involved in a variety of cardiovascular pathologies that underlie acute coronary syndromes. So many scientists were interested in matrix metalloproteinase.In the 1990s, use of antiplatelet agents proved that platelet aggregation caused thrombotic ischemic events resulting from the rupture of plaques in advanced lesions and from the vascular injuries inflicted by percutaneous interventions (PCI). GP IIb/IIIa antagonist tirofiban is the best antiplatelet agents,which inhibit cross-linking of fibrinogen as a bridging molecule within platelets.The object of my study is about to effects on MMPS and CD40L in coincobation platelets with HUVECs and in patients with AMI After PCI by tirofeiban.HUVEC monolayers were coincubated with platelets for 60 minutes, platelets were removed, and HUVECs were additionally incubated with medium. After 6 hours, HUVEC supernatant was stored .HUVEC incubation with activated platelets resulted in a increase in HUVEC surface expression of MT1-MMP by immunofluorescence microscopy . Lindemann et al have shown this effect that was comparably achieved by HUVEC stimulation with interleukin-1(IL-1 ). Notably, nonstimulated platelets did not alter HUVEC expression of MT1-MMP. SDS-PAGE zymography using the endothelial supernatants evealed constitutive expression of 72-kD gelatinase (MMP-2) and very low expression of 92-kD gelatinase (MMP-9), which were only slightly induced by resting platelets . In addition, a second, lower band at 66 kDa revealed MMP-2 activation on adhesion of activated platelets. MMP activity was exclusively of endothelial origin, because all platelets had been removed by repeated washing steps after the initial 60-minute coincubation period, as verified by light microscopy and flow cytometry .To discriminate between effects mediated by physical platelet-endothelial contact (eg, CD40L) versus effects of platelet-released soluble factors (eg, platelet factor 4), HUVECs were coincubated with activated platelets in a transwell system that prevents physical cell to cell contact. Platelet-induced HUVEC surface expression of MT1-MMP were almost completely absent when direct platelet-endothelium interactions were hindered by transwells . SDSPAGE zymographies reveal that the expression of MMP-2 and MMP-9 was deacreased in HUVEC supernatants by tirofiban,compaired with the act plt group.The destabilization of the vulnerable fibrous cap of the atherosclerotic plaque seems to result from an imbalance of the plasminogen and matrix metalloproteinase (MMP) activation systems. In a complex cascade, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) cleave plasminogen to plasmin, a serine protease of broad specificity, capable of degrading specific components of the ECM. In addition, plasmin activates MMPs, such as interstitial collagenase (MMP-1) and gelatinase (MMP-2).Endothelial cells secrete uPA, tPA, MMP-1, MMP-2, and MMP-9 in an activation-dependent manner1,6 and restrict proteolytic activity to the cell surface via membrane-anchored protease receptors such as urokinase receptor (uPAR) and membrane type-1 MMP (MT1-MMP), which bind and activate uPA and MMP-2, respectively. MT1-MMP not only serves as a receptor and catalyst of soluble MMPs but also directly degrades several ECM components.Clinical study state that the level of MMP-9 is relative to the extent of coronary stenosis. The level of MMP-9 in serum of coronary atherosclerotic heart disease is higher than that of health people. It suggested that MMP-9 is associated with the rupture of the atherosclerotic unstable plague and myocardial lesion.In addition, MMP-9 could promote proliferation and migration of smooth muscle cells. IL-1,TNF-A released by lesione vascular enhanced the expression of activated MMP-9 on VSMC in vitro.It may caused imbanlance of MMPs,which promote migration of smooth muscle cells and degradation of ECM.More over,increase of inflammatory cells may have important roles in the inflammatory aspects of atherosclerotic lesion progression. Recent study indicate MMPs may triggered inflammatory activity.Another important factor that Triggers metalloproteinase activation systems is CD40 ligend. CD40L-mediated signaling pathway play an impotant role in regulation unimmune system cells that involved in atherosclerotic lesions. CD40L is a transmembrane protein of the tumor necrosis factor family that was originally identified on cells of the immune system cells( mast cells, basophils, eosinophils, and natural killer cells). However, it was shown that CD40L and CD40 are also present on several cells of the vasculature, including endothelial cells, smooth muscle cells, monocytes, and macrophage. CD40L may induced the expression of MMP-9 on SMCs and endothelial cells.In addition ,this two kinds cells secret based level of MMP-2, CD40L could trigger the expression of activated MMP-2. Notably, this effect that was sifinicantly achieved by HUVEC stimulation with interleukin-1B .CD40L not only is activator of matrix metalloproteinase,but also is a key inflammatory factor.Because many of the proteins identified in the gene-targeting experiments outlined above can be induced by CD40L, it was particularly exciting when the linkage of this protein to atherosclerotic lesion progression was shown. Mach and coworkers7 found that disruption of CD40L function in the (LDLR)-/- mouse by administering a blocking CD40L antibody prevented the progression of atherosclerotic disease. CD40–CD40L interaction is also involved in plaque stability, most likely because of the release of matrix metalloproteinases.Platelets, however, are also the primary source of circulating CD40L, begging the question about its role in the progression of atherosclerotic disease, including the formation of thrombotic occlusions. Recent studies demonstrate that platelet CD40L becomes mobilized in acute coronary thrombotic indications. For example, increased levels of sCD40L are a consequence of various procedures known to have thrombotic and inflammatory components, including PCI and cardiac surgery requiring cardiopulmonary bypass.Increased levels of sCD40L are also found in patients with acute coronary syndromes,and peripheral arterial occlusive disease. An indication of the inflammatory activity of sCD40L comes from transfusion medicine. Storage of platelet concentrates for clinical transfusion is known to release≤50% of the platelet CD40L: Transfusion of concentrates into patients results in CD40L-dependent febrile responses.The production of sCD40L from platelets and its thrombotic activity appear to be intimately linked to the platelet integrin GP IIB/IIIA. GP IIB/IIIA is known to be involved in sCD40L production because GP IIB/IIIA antagonists attenuate the release of sCD40L from activated platelets in vitro. These antagonists block release from stimulated platelets even in the absence of aggregation, demonstrating a direct role for GP IIB/IIIA in the cleavage mechanism. Secondly, direct binding of sCD40L to GP IIB/IIIA indicates that the ability of sCD40L to promote and stabilize platelet thrombosis under high shear rates is a result of direct interactions between these 2 proteins.Receptor-specific platelet adhesion was performed in my study. Platelets, submaximally activated , were allowed to attach to the specific ligand of GP IIB/IIIA, ibrinogen . After 60 minutes, nonadherent platelets were removed. Adherent cells were stained with fluorescence-conjugated mAbs, cautiously detached, and evaluated by flow cytometry. CD40L expression were significantly enhanced on platelets that were allowed to adhere to fibrinogen . In addition ,flow cytometry reveal that the expression of CD40L was deacreased on platelets by tirofiban,compaired with the act plt group. In fact, Andreas E may et al have shown a significant increase in CD40L surface expression was only achieved when GP IIB/IIIA was clustered by combined fibrinogen/anti-fibrinogen treatment or by bivalent mAb 7E3.In contrast, ligation of GP IIB/IIIA by fibrinogen alone or monovalent c7E3 (Fab) had no effect. Thus,clustering of GP IIB/IIIA as it occurs during platelet-endothelial interactions seems to effectively trigger CD40L expression on platelets. Cross-linking of GP IIB/IIIA was found to be effective on platelets regardless of whether they were preactivated with thrombin, TRAP, or ADP, all of which are agonists of the fibrinogen receptor GP IIB/IIIA. Thus, GP IIB/IIIA–mediated CD40L upregulation seems to require prestimulation of GP IIB/IIIA affinity independent of the respective stimulus.This study describes a novel activation pathway on platelets that upregulate CD40L in direct response to GP IIB/IIIA engagement and thereby allow CD40L-mediated signaling. This pathway promotes proteolytic activity on endothelial cells on transient interactions with activated platelets. Andreas E may et al have shown compared with nonadherent thrombin-activated platelets in suspension. uPAR and MT1-MMP and to secrete uPA, tPA, and MMP-1 as well as MMP-2 and MMP-9.CD40L blockade inhibit this activation pathway. Platelet adhesion via GP IIB/IIIA to the endothelium or immobilized fibrinogen upregulates CD40L surface expression. however, the underlying mechanism remained unclear.The present data may have important pathophysiological and therapeutic implications for atherosclerotic diseases.CD40L-CD40 signaling substantially contributes to atherogenesis, because inhibition of CD40L retarded the progression of atherosclerosis in miceand led to a collagen-rich stable plaque phenotype very likely attributable to decreased MMP-1 and MMP-2 activity. Consistent with our data, CD40L-expressing T-cell membranes have been shown to enhance endothelial secretion of both pro-MMP-9 and activated MMP-2 in vitro.Our data present evidence that platelets can induce matrix-degrading activity in HUVEC via platelet-associated CD40L and that this mechanism is regulated by GP IIB/IIIA, a receptor involved substantially in platelet-endothelial interaction.The present data show that irrespective of the nature of GP IIB/IIIA antagonists, platelet-induced endothelial matrix degradation is substantially inhibited by all tested inhibitors, although maximal effects were found for inhibitors that block both GP IIB/IIIA andαvβ3.Whether these differential effects might be of clinical significance has to be answered in future studies. In addition ,my data show tirofiban may improve TIMI blood flow and EF after PCI of patients with AMI.The role of GP IIB/IIIA antagonist tirofiban not only bloke platlats aggregation ,but also may involved in cell molecular chemism.In conclusion, our data indicate that platelets can focus matrix-degrading activity to the particular sites of platelet adhesion at the vessel wall. GP IIB/IIIA blockade may not only inhibit platelet aggregation at the vulnerable plaque and thereby prevent physical vessel occlusion but also may prevent platelet-CD40L–mediated inflammatory cascades,leading to matrix degradation and plaque rupture. This mechanism may contribute to the understanding of beneficial effects of GP IIB/IIIA antagonists on ischemic complications in patients with acute coronary syndromes.In brief, the conclusion of my studies show that GP IIB/IIIA antagonist tirofiban could inhibit inflammatory reaction in coincobation platelets with HUVECs ,and may improve cronary blood flow during PCI of patients with AMI,and decrease MACE.