Meinrad Gawaz

A Critical Role of Platelet Adhesion in the Initiation of Atherosclerotic Lesion Formation

The contribution of platelets to the process of atherosclerosis remains unclear. Here, we show in vivo that platelets adhere to the vascular endothelium of the carotid artery in ApoE − / − mice before the development of manifest atherosclerotic lesions. Platelet–endothelial cell interaction involved both platelet glycoprotein (GP)Ibα and GPIIb-IIIa. Platelet adhesion to the endothelium coincides with inflammatory gene expression and preceded atherosclerotic plaque invasion by leukocytes. Prolonged blockade of platelet adhesion in ApoE − / − mice profoundly reduced leukocyte accumulation in the arterial intima and attenuated atherosclerotic lesion formation in the carotid artery bifurcation, the aortic sinus, and the coronary arteries. These findings establish the platelet as a major player in initiation of the atherogenetic process.

A Crucial Role of Glycoprotein VI for Platelet Recruitment to the Injured Arterial Wall In Vivo

Platelet adhesion and aggregation at sites of vascular injury is crucial for hemostasis but may lead to arterial occlusion in the setting of atherosclerosis and precipitate diseases such as myocardial infarction. A current hypothesis suggests that platelet glycoprotein (GP) Ib interaction with von Willebrand factor recruits flowing platelets to the injured vessel wall, where subendothelial fibrillar collagens support their firm adhesion and activation. However, so far this hypothesis has not been tested in vivo. Here, we demonstrate by intravital fluorescence microscopy of the mouse carotid artery that inhibition or absence of the major platelet collagen receptor, GPVI, abolishes platelet–vessel wall interactions after endothelial denudation. Unexpectedly, inhibition of GPVI by the monoclonal antibody JAQ1 reduced platelet tethering to the subendothelium by ∼89%. In addition, stable arrest and aggregation of platelets was virtually abolished under these conditions. Using different models of arterial injury, the strict requirement for GPVI in these processes was confirmed in GPVI-deficient mice, where platelets also failed to adhere and aggregate on the damaged vessel wall. These findings reveal an unexpected role of GPVI in the initiation of platelet attachment at sites of vascular injury and unequivocally identify platelet–collagen interactions (via GPVI) as the major determinant of arterial thrombus formation.

Platelets induce alterations of chemotactic and adhesive properties of endothelial cells mediated through an interleukin-1-dependent mechanism. Implications for atherogenesis

Platelets and alterations of chemotactic and adhesive properties of endothelium play an important role in the pathophysiology of atherosclerosis. We investigated the effect of platelets on secretion of monocyte chemotactic protein-1 (MCP-1) and on surface expression of intercellular adhesion molecule-1 (ICAM-1) of cultured endothelium. Pretreatment of cultured monolayers of endothelial cells with alpha-thrombin-activated platelets significantly enhanced secretion of MCP-1 and ICAM-1 surface expression (P<0.01) that could be inhibited by interleukin-1 (IL-1) antagonists by approximately 40%. Activation of transcription factor nuclear factor-kappaB (NF-kappaB) which regulates transcription of early inflammatory response genes such as MCP-1, was significantly increased in endothelial cells treated with activated platelets via an IL-1 mediated mechanism as determined by electrophoretic mobility shift assay (EMSA) and kappaB-dependent transcriptional activity. In trans-well experiments, alpha-thrombin-activated platelets enhanced IL-1-dependent surface expression of vitronectin receptor (alpha(v)beta(3)) on the luminal aspect of endothelial monolayers and promoted alpha(v)beta(3)-mediated platelet/endothelium adhesion that could be inhibited by the antiadhesive peptides GRGDSP and c(RGDfV). We conclude that activated platelets induce significant changes in chemotactic (secretion of MCP-1) and adhesive (surface expression of ICAM-1 and alpha(v)beta(3)) properties of cultured endothelium. These findings imply a potential pathophysiological mechanism of platelets in an early stage of atherogenesis.

Engagement of Glycoprotein IIb/IIIa (αIIbβ3) on Platelets Upregulates CD40L and Triggers CD40L-Dependent Matrix Degradation by Endothelial Cells

Background—CD40L-CD40 interactions induce inflammatory signals in cells of the vascular wall. We evaluated the effects of glycoprotein (GP) IIb/IIIa (&agr;IIb&bgr;3) engagement that occurs during platelet-endothelium interactions on CD40L surface exposure on platelets and initiation of proteolytic activity in human umbilical vein endothelial cells (HUVECs). Methods and Results—Transient (60-minute) adhesion of thrombin-prestimulated platelets enhanced HUVEC expression of urokinase-type plasminogen activator receptor and membrane type-1 matrix metalloproteinase (MT1-MMP) (reverse transcriptase–polymerase chain reaction, flow cytometry) and secretion of urokinase-type plasminogen activator, tissue-type plasminogen activator, and MMP-1 (ELISA) and induced proteolytic activity via MMP-2 and MMP-9 (gelatin zymography). These effects were abrogated by hindrance of physical platelet-endothelial contacts using transwell systems or inhibited by GRGDSP, mAbs anti–GP IIb/IIIa (7E3), anti-&agr;v&bgr;3 (LM609), or anti-CD40L (TRAP1). In addition, MMP-2 and MMP-9 were inhibited by specific GP IIb/IIIa antagonists tirofiban, lamifiban, or integrelin. On endothelial cells, induction of proteolytic activity by activated platelets was mimicked by CD40 engagement using soluble CD40L but not affected by antibody clustering of &agr;v&bgr;3. On platelets, CD40L and CD62P exposure was enhanced on adhesion to HUVECs or immobilized fibrinogen and was abrogated by GRGDSP or LM609. In suspension, cross-linking of GP IIb/IIIa by fibrinogen plus secondary mAb upregulated CD40L surface exposure. Consistently, bivalent mAb 7E3 upregulated CD40L, whereas ligation of GP IIb/IIIa by soluble fibrinogen alone or monovalent Fab-fragment c7E3 had no effect. Conclusions—Platelet adhesion via GP IIb/IIIa upregulates CD40L and CD62P surface exposure. Proteolytic activity of HUVEC is induced by the concerted action of &bgr;3-integrin–mediated platelet adhesion and subsequent CD40L-induced signals in HUVECs. Effective anti–GP IIb/IIIa or anti-CD40L strategies might, therefore, contribute to plaque stabilization.

G13 is an essential mediator of platelet activation in hemostasis and thrombosis

Platelet activation at sites of vascular injury is essential for primary hemostasis, but also underlies arterial thrombosis leading to myocardial infarction or stroke. Platelet activators such as adenosine diphosphate, thrombin or thromboxane A2 (TXA2) activate receptors that are coupled to heterotrimeric G proteins. Activation of platelets through these receptors involves signaling through Gq, Gi and Gz (refs. 4–6). However, the role and relative importance of G12 and G13, which are activated by various platelet stimuli, are unclear. Here we show that lack of Gα13, but not Gα12, severely reduced the potency of thrombin, TXA2 and collagen to induce platelet shape changes and aggregation in vitro. These defects were accompanied by reduced activation of RhoA and inability to form stable platelet thrombi under high shear stress ex vivo. Gα13 deficiency in platelets resulted in a severe defect in primary hemostasis and complete protection against arterial thrombosis in vivo. We conclude that G13-mediated signaling processes are required for normal hemostasis and thrombosis and may serve as a new target for antiplatelet drugs.

Neutrophil and platelet activation at balloon-injured coronary artery plaque in patients undergoing angioplasty☆

OBJECTIVESnThis study sought to investigate changes in the expression of activation-dependent adhesion receptors on neutrophils and platelets after exposure to the balloon-injured coronary artery plaque.nnnBACKGROUNDnActivation of blood cells at the balloon-injured coronary artery plaque may contribute to abrupt vessel closure and late restenosis after percutaneous transluminal coronary angioplasty.nnnMETHODSnIn 30 patients undergoing elective coronary angioplasty, blood specimens were obtained through the balloon catheter proximal to the plaque before dilation and distal to the plaque after dilation. Simultaneous blood samples obtained through the guiding catheter served as control samples. Total surface expression of the inducible fibrinogen receptor (CD41) and surface expression of the activated fibrinogen receptor (LIBS1) on platelets as well as Mac-1 (CD11b) and L-selectin (CD62L) surface expression on neutrophils were assessed by flow cytometry.nnnRESULTSnAfter exposure to the dilated coronary artery plaque, surface expression of LIBS1 on platelets increased by 40.5 +/- 11.0 mean (+/-SE) fluorescence (p=0.001) and that of CD11b on neutrophils increased by 20.1 +/- 4.4 mean fluorescence (p=0.018). Concomitantly, anti-CD62L binding on neutrophils decreased by 6.6 +/- 2.4 mean fluorescence (p=0.022). In contrast, surface expression of the adhesion receptors did not change significantly between the coronary ostium and the prestenotic coronary segment.nnnCONCLUSIONSnThe results of this study demonstrate neutrophil and platelet activation at the balloon-injured coronary artery plaque. This cellular activation may serve as a target for pharmacologic interventions to improve the outcome of coronary angioplasty.

Effects of Statins on Platelet Inhibition by a High Loading Dose of Clopidogrel

Background—Recent studies suggested that some HMG-CoA reductase blockers might inhibit the antiplatelet activity of clopidogrel. Therefore, we analyzed how various statins together with a high loading dose of clopidogrel (600 mg) affect platelet aggregation. Methods and Results—Seventy-seven patients with stable angina scheduled for elective coronary stenting were studied. Patients were randomized to receive atorvastatin, fluvastatin, lovastatin, pravastatin, simvastatin (each 20 mg), cerivastatin (0.3 mg), or placebo, plus a high loading dose of 600 mg of clopidogrel. ADP-induced platelet aggregation (5 and 20 &mgr;mol/L) was determined before and 2 and 4 hours after first clopidogrel administration. All patients were taking aspirin (100 mg/d) regularly. We found that none of the statins significantly influenced inhibition of platelet aggregation by clopidogrel. Conclusions—Concomitant use of statins with clopidogrel does not significantly inhibit antiplatelet activity, at least when clopidogrel is administered at a high loading dose of 600 mg.

Induction of Cytokine Expression in Leukocytes in Acute Myocardial Infarction

OBJECTIVESnThis study sought to investigate whether cytokine expression in leukocytes may be induced by plasma from the reperfused heart of patients with an acute myocardial infarction (MI).nnnBACKGROUNDnReperfusion in acute MI is associated with deleterious local and systemic inflammatory responses that are regulated by cytokines. Induction of cytokine expression in resident leukocytes could contribute to inflammatory responses of the ischemic and reperfused heart.nnnMETHODSnBlood samples of 10 patients with an acute MI were obtained simultaneously from the coronary sinus and the aorta before and 5 min after recanalization of the coronary occlusion. Ten patients with elective percutaneous transluminal coronary angioplasty served as a control group. We incubated leukocytes from healthy donors with plasma samples and analyzed mRNA expression of interleukin (IL)-1 beta, IL-6, IL-8 and tumor necrosis factor-alpha (TNF-alpha) by Northern blot analysis.nnnRESULTSnIn patients with an acute MI, plasma obtained from the coronary sinus after recanalization increased the mRNA expression of IL-1 beta and IL-8 compared with that of plasma before recanalization (median [quartiles] difference before vs. after recanalization: 34.5 [4, 137], p = 0.017, for IL-1 beta; 18.5 [4, 35], p = 0.032, for IL-8) and simultaneously obtained aortic plasma (median [quartiles] coronary sinus-aortic differences after recanalization: 45.5 [-3, 115], p = 0.021, for IL-1 beta; 16 [4, 52], p = 0.005, for IL-8). No induction of IL-6 and TNF-alpha expression could be observed. No changes found in the study patients were detectable in the control group.nnnCONCLUSIONSnPlasma from the ischemic and reperfused heart stimulates the expression of IL-1 beta and IL-8 in leukocytes. Therefore, leukocyte-derived cytokines may contribute to the regulation of cardiac inflammatory responses in patients with an acute MI.

Radionuclide Imaging: A Molecular Key to the Atherosclerotic Plaque

Despite primary and secondary prevention, serious cardiovascular events such as unstable angina or myocardial infarction still account for one-third of all deaths worldwide. Therefore, identifying individual patients with vulnerable plaques at high risk for plaque rupture is a central challenge in cardiovascular medicine. Several noninvasive techniques, such as magnetic resonance imaging, multislice computed tomography, and electron beam tomography are currently being tested for their ability to identify such patients by morphological criteria. In contrast, molecular imaging techniques use radiolabeled molecules to detect functional aspects in atherosclerotic plaques by visualizing their biological activity. Based upon the knowledge about the pathophysiology of atherosclerosis, various studies in vitro and in vivo and the first clinical trials have used different tracers for plaque imaging studies, including radioactive-labeled lipoproteins, components of the coagulation system, cytokines, mediators of the metalloproteinase system, cell adhesion receptors, and even whole cells. This review gives an update on the relevant noninvasive plaque imaging approaches using nuclear imaging techniques to detect atherosclerotic vascular lesions.

Prospective evaluation of hemostatic predictors of subacute stent thrombosis after coronary Palmaz-Schatz stenting.

OBJECTIVESnThis study sought to investigate hemostatic predictors of subacute occlusive coronary stent thrombosis.nnnBACKGROUNDnBetter hemostatic monitoring may improve antithrombotic therapy after stenting.nnnMETHODSnIn 140 consecutive patients undergoing Palmaz-Schatz stent implantation for suboptimal angioplasty results, we obtained serial blood samples immediately before and daily for 12 days after stenting. We prospectively tested the hypothesis that subacute stent thrombosis was more frequent if the surface expression of the inducible fibrinogen receptor on platelets (flow cytometry) or the concentration of plasma fibrinogen or that of the prothrombin fragments F1 + 2 before stent implantation exceeded the 75th percentile of the entire study cohort.nnnRESULTSnAll five stent occlusions encountered during the study occurred in patients with platelet fibrinogen receptor expression above the 75th percentile. Thus, the rate of stent occlusion differed significantly between the groups defined by platelet fibrinogen receptor expression (14.3% vs. 0%, p = 0.0008). In both the group with fibrinogen concentration and that with F1 + 2 concentration above the 75th percentile, three stent occlusions occurred. Between the groups defined by these variables, the rate of stent occlusion did not differ significantly (8.6% vs. 1.9%, p = 0.10). Logistic regression analysis, including angiographic and hemostatic variables, confirmed platelet fibrinogen receptor expression as an independent predictor of stent occlusion (p = 0.020). Stent occlusion could not be predicted by the time course of any of the hemostatic variables.nnnCONCLUSIONSnPlatelet fibrinogen receptor expression is an independent predictor of subacute stent occlusion. However, fibrinogen and F1 + 2 concentrations do not show a strong relation to the risk of stent occlusion.