Herbert K. Lau

Percutaneous coronary intervention results in acute increases in oxidized phospholipids and lipoprotein(a): short-term and long-term immunologic responses to oxidized low-density lipoprotein.

Background— This study was performed to assess whether oxidized low-density lipoprotein (OxLDL) levels are elevated after percutaneous coronary intervention (PCI). Methods and Results— Patients (n= 141) with stable angina pectoris undergoing PCI had serial venous blood samples drawn before PCI, after PCI, and at 6 and 24 hours, 3 days, 1 week, and 1, 3, and 6 months. Plasma levels of OxLDL-E06, a measure of oxidized phospholipid (OxPL) content on apolipoprotein B-100 detected by antibody E06, lipoprotein(a) [Lp(a)], autoantibodies to malondialdehyde (MDA)-LDL and copper-oxidized LDL (Cu-OxLDL), and apolipoprotein B-100–immune complexes (apoB-IC) were measured. OxLDL-E06 and Lp(a) levels significantly increased immediately after PCI by 36% (P < 0.0001) and 64% (P < 0.0001), respectively, and returned to baseline by 6 hours. In vitro immunoprecipitation of Lp(a) from selected plasma samples showed that almost all of the OxPL detected by E06 was bound to Lp(a) at all time points, except in the post-PCI sample, suggesting independent release and subsequent reassociation of OxPL with Lp(a) by 6 hours. Strong correlations were noted between OxLDL-E06 and Lp(a) (r = 0.68, P < 0.0001). MDA-LDL and Cu-OxLDL autoantibodies decreased, whereas apoB-IC levels increased after PCI, but both returned to baseline by 6 hours. Subsequently, IgM autoantibodies increased and peaked at 1 month and then returned to baseline, whereas IgG autoantibodies increased steadily over 6 months. Conclusions— PCI results in acute plasma increases of Lp(a) and OxPL and results in short-term and long-term immunologic responses to OxLDL. OxPL that are released or generated during PCI are transferred to Lp(a), suggesting that Lp(a) may contribute acutely to a protective innate immune response. In settings of enhanced oxidative stress and chronically elevated Lp(a) levels, the atherogenicity of Lp(a) may stem from its capacity as a carrier of proinflammatory oxidation byproducts.

Plasma Urokinase Antigen and Plasminogen Activator Inhibitor-1 Antigen Levels Predict Angiographic Coronary Restenosis

BACKGROUND The fibrinolytic system is intimately involved in several processes that contribute to restenosis, including clot dissolution, cell migration, and tissue remodeling. However, the role of the individual activators (urokinase [uPA] and tissue plasminogen [tPA] activators) and inhibitors (plasminogen activator inhibitor [PAI-1]) of the fibrinolytic system in maintaining patency after coronary artery angioplasty and stenting is unclear. METHODS AND RESULTS We prospectively studied 159 patients with stable angina who underwent successful elective angioplasty (n=110) or stenting (n=49) of de novo native coronary artery lesions. Plasma samples were drawn at baseline (before angioplasty) and serially after angioplasty (immediately afterward and 6 hours, 24 hours, 3 days, 7 days, 1 month, 3 months, and 6 months afterward). Antigen and activity assays were performed for uPA, tPA, and PAI-1. Follow-up quantitative coronary angiography was performed in 92% of eligible patients. The overall angiographic restenosis rate (diameter stenosis >50%) was 31% (37% in PTCA patients, 17% in stented patients). At all time periods, including baseline, uPA antigen levels were significantly higher and PAI-1 antigen levels were significantly lower in patients with restenosis. Restenosis rates for patients in the upper tertile of baseline uPA antigen levels were 2-fold higher than for those in the lower 2 tertiles (46% versus 24% and 22%, respectively; P<0.004). In a stepwise regression multivariate analysis, obstruction diameter after the procedure and uPA antigen were significant predictors of follow-up diameter stenosis. CONCLUSIONS Plasma uPA antigen levels and PAI-1 antigen levels identify patients at increased risk for restenosis after percutaneous coronary revascularization.

Intravenous immunoglobulin inhibits anti-glycoprotein IIb-induced platelet apoptosis in a murine model of immune thrombocytopenia.

We have previously shown that injection of anti‐glycoprotein (GP) IIb induces murine immune thrombocytopenia (ITP) and that intravenous immunoglobulin (IVIg) ameliorates ITP. We hypothesise that murine ITP may be associated with platelet apoptosis, which is upregulated by anti‐GPIIb and downregulated by IVIg. The current study demonstrated that anti‐GPIIb injection induced three critical apoptosis manifestations in platelets: (i) mitochondrial inner transmembrane potential (ΔΨm) depolarisation; (ii) caspase‐3 activation; and (iii) phosphatidylserine (PS) exposure. IVIg administration inhibited caspase‐3 activation and PS exposure, but not ΔΨm‐depolarisation, in anti‐GPIIb‐treated platelets, demonstrating that IVIg ameliorates thrombocytopenia concomitantly with inhibiting late, but not early mechanisms of platelet apoptosis.

Thrombin-activatable fibrinolysis inhibitor (TAFI): a novel predictor of angiographic coronary restenosis

The fibrinolytic system is closely related to several processes that are involved in restenosis. We previously showed that low PAI-1 plasma levels predicted restenosis. Recently, a different fibrinolytic inhibitor, TAFI, has been described. The aims of this study were to evaluate the relationship between pre-procedural plasma levels of TAFI and late angiographic restenosis and the interaction between TAFI and PAI-1.We prospectively studied 159 patients with stable angina who underwent successful elective angioplasty or stenting of de novo native coronary artery lesions. TAFI and PAI-1 antigen levels were measured in plasma samples drawn before the procedure. Follow-up coronary angiography was performed in 92% of patients. There was a significant correlation between pre-procedural TAFI levels and 6-month % diameter stenosis (DS) (r = 0.21; p = 0.013). The overall angiographic restenosis rate (DS>50%) was 31%. Pre-procedural TAFI levels were significantly higher in patients with restenosis (108 +/- 33% versus 94+/-30%, p = 0.011). Restenosis rates for patients in the upper tertile of TAFI levels were 2-fold higher than for those in the lowest tertile (45% versus 22%; p = 0.016). A combination of high TAFI and low PAI-1 levels identified patients at the highest risk of restenosis (53%) compared to 14% in patients with low TAFI and high PAI-1 levels; p = 0.027. In conclusion, pre-procedural plasma TAFI antigen levels identify patients at increased risk for restenosis after PCI.

The interaction between platelets and factor VII/VIIa

Blood coagulation normally occurs when factor VII interacts with its specific cellular receptor, tissue factor, which is exposed when a blood vessel is severed. The factor VII/tissue factor complex then initiates a cascade of proteolytic reactions involving factors IX, X, prothrombin and fibrinogen, culminating in the formation of a fibrin clot. The role of platelets in the initiation phase of blood coagulation is still unclear. It has been postulated that platelets bind activated factor VIIa independently of tissue factor, and that this interaction forms the basis of the usefulness of high-dose recombinant factor VIIa in treating hemophiliacs with inhibitory antibodies, and other thrombocytopenia-like syndromes. In this review, we will examine the evidence for and against such an hypothesis, as well as discuss an alternative mechanism for the efficacy of high-dose factor VIIa in treating hemophilic patients with inhibitors.

Modulation of the plasminogen activation system in murine macrophages.

We have dissected the state of fibrinolytic balance in the C57/BL mouse macrophages, by means of immunotrap assays and zymography. We have monitored the individual changes of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) activities of cellular lysates and secretions of these macrophages, after they were stimulated by various exogenous agents. The resident peritoneal macrophages were found to have very little PA but high level of PAI, and are therefore highly anti-fibrinolytic in nature. Upon stimulation by thioglycollate, PA activity increased and PAI activity decreased, thus raising the fibrinolytic balance in these macrophages. Upon incubation of resident or thioglycollate-activated macrophages by lipopolysaccharide (LPS), the PA level was depressed while the PAI level was increased, resulting in a large drop in the total fibrinolytic balance of the activated cells. When resident or thioglycollate-activated macrophages were incubated with the anti-inflammatory agent dexamethasone, the drug depressed both the expressions of PA and PAI, in the lysate and conditioned medium of both cell types. Thus cell-bound or secreted forms of macrophage PA and PAI activities were either increased or decreased in response to thioglycollate, LPS or dexamethasone challenge. The changes in PA and PAI resulted in different state of fibrinolytic balance in macrophages, and could be related to the different functions of these macrophages at different stages of their development.

Mental Stress-Induced Platelet Activation Among Patients With Coronary Artery Disease

Objective: To study patients with coronary artery disease (CAD) scheduled for coronary angioplasty and to examine platelet activation in response to mental stress as a potential mechanism involved in the association between psychosocial factors and cardiac outcomes. Psychosocial factors have been identified as risk factors for CAD and adverse cardiac outcomes, although the underlying mechanisms are poorly understood. Methods: Markers of platelet activation and platelet reactivity in response to experimentally induced mental stress (mental arithmetic and anger recall) were examined, using flow cytometry analysis and &bgr;-thromboglobulin (BTG) assays among 249 CAD patients (age = 60.3 ± 9.0 years, 15% women) who were scheduled to undergo elective percutaneous coronary intervention. Results: Mental stress-induced increases in platelet activation (CD41 (GP IIb/IIIa), p = .002; percent of mononuclear cells positive for CD41, p = .01; CD62P (P-selectin) expression, p = .005; and percent platelets positive for CD62P, p < .001). The degree of platelet reactivity was not related to demographic, clinical, or psychological variables, or cardiovascular hemodynamic changes. Conclusions: Experimentally induced mental stress induced platelet activation in patients with CAD. This mechanism may partially explain the link between psychosocial variables and the development of adverse cardiac outcomes in patients with CAD. ACE = angiotensin-converting enzyme; ASA = acetylsalicylic acid; CAD = coronary artery disease; BDI = Beck Depression Inventory; BP = blood pressure; bpm = beats per minute; BTG = &bgr;-thromboglobulin; DBP = diastolic blood pressure; FDR = false discovery rate; GP = glycoprotein; HR = heart rate; MLD = minimum lumen diameter; MI = myocardial infarction; MNC = mononuclear cells; NE = norepinephrine; PCI = percutaneous coronary interventions; SBP = systolic blood pressure; STAEI = State-Trait Anger Expression Inventor.

Regulation of plasminogen activator inhibitor-1 secretion by urokinase and tissue plasminogen activator in rat epithelioid-type smooth muscle cells.

Summary. Tissue plasminogen activator (tPA) and urokinase (uPA) are targets of plasminogen activator inhibitor‐1 (PAI‐1) inhibition. We have previously shown that both proteases can also induce PAI‐1 secretion in rat smooth muscle cells (SMCs). We now report that both proteases appear to use very similar cellular mechanisms for signal transduction. They induced PAI‐1 secretion using a pathway(s) involving protein kinase C (PKC). They also activated the Raf/Mek/mitogen‐activated protein kinase (MAPK) pathway, which lies downstream of PKC activation. Activation of protein kinase A (PKA), however, lowered PAI‐1 secretion induced by uPA and tPA, as a result of an inhibition of the PKC pathway and inhibition of Raf, Mek and MAPK phosphorylations. Src and syk family non‐receptor tyrosine kinases (TK) were also involved in PAI‐1 induction. The mechanisms of interaction of these tyrosine kinases with other pathways appeared to be quite different: src appeared to act within the PKC and PKA pathways, while syk operated independently of these pathways. Furthermore, whereas src inhibition resulted in inhibition of Raf/Mek/Erk phosphorylations, syk inhibition could only inhibit Mek and Erk phosphorylations but not the phosphorylation of Raf. These multiple pathways utilized by uPA and tPA to modulate PAI‐1 secretion might be involved in determining the proteolytic or antiproteolytic potential of the SMCs under different pathophysiological conditions.

C-reactive protein modulates vagal heart rate control in patients with coronary artery disease

Systemic inflammation is associated with sympathetic cardiac activation and decreased HRV (heart rate variability) in subjects at high risk of CAD (coronary artery disease). In the present study, we examined the influence of systemic inflammation, measured by CRP (C-reactive protein), on vagal HR (heart rate) control during behavioural relaxation in patients with CAD. It was hypothesized that CRP would be associated with decreased vagal HR modulation. Consecutive patients were screened 2 weeks prior to elective PTCA (percutaneous transluminal coronary angioplasty). The study was comprised of 29 subjects who represented the first and fourth quartiles of the CRP distribution: Low (0.47+/-0.07 microg/ml)- and High (8.19+/-1.95 microg/ml)-CRP groups respectively. Vagal HR control was quantified as RR high-frequency spectral power (0.15 to 0.40 Hz), and was assessed in log-transformed absolute units (logHF power). Near-IR particle immunoassay was used to determine high-sensitivity CRP concentration. Assessment entailed 5 min of silent reading and self-guided behavioural relaxation. RR logHF power was decreased in the High-CRP group across both assessment procedures (P=0.032). Behavioural relaxation increased RR logHF power for both the Low- and High-CRP groups (P=0.033). Hierarchical linear regression determined that CRP accounted for 18.9% of the variance in RR logHF power during behavioural relaxation (P=0.03), independent of baseline RR interval, cardiac medication, respiratory logHF power and body mass index. In conclusion, patients with CAD had augmented vagal HR control with behavioural relaxation, but this effect was moderated by the severity of CRP. Therefore it may be advisable to assess systemic inflammation in interventions aimed at improving neurocardiac regulation in patients with CAD.

Characterization of immunotrap assays for urokinase plasminogen activator and its inhibitors and measurements of these molecules in human plasma and mouse macrophage in culture

1. Immunotrap assays that can measure the activities of urokinase-type plasminogen activator (uPA) and its inhibitors (PAIs) were characterized. 2. Both human plasma and mouse macrophages in culture were found to contain much higher inhibitor activity than uPA-like activity. 3. The balance between pro- and anti-fibrinolytic activities was quantitatively changed in the murine macrophages after the injection of thioglycollate. uPA-like materials were synthesized by the macrophages and secreted to the conditioned medium continuously, while PAI activity was unchanged during the same time period.