Platelet-monocyte Aggregation Research Articles

Human atheromatous plaques stimulate thrombus formation by activating platelet glycoprotein VI

Lipid-rich atherosclerotic plaques are vulnerable, and their rupture can cause the formation of a platelet- and fibrin-rich thrombus leading to myocardial infarction and ischemic stroke. Although the role of plaque-based tissue factor as stimulator of blood coagulation has been recognized, it is not known whether plaques can cause thrombus formation through direct activation of platelets. We isolated lipid-rich atheromatous plaques from 60 patients with carotid stenosis and identified morphologically diverse collagen type I- and type III-positive structures in the plaques that directly stimulated adhesion, dense granule secretion, and aggregation of platelets in buffer, plasma, and blood. This material also elicited platelet-monocyte aggregation and platelet-dependent blood coagulation. Plaques exposed to flowing blood at arterial wall shear rate induced platelets to adhere to and spread on the collagenous structures, triggering subsequent thrombus formation. Plaque-induced platelet thrombus formation was observed in fully anticoagulated blood (i.e., in the absence of tissue factor-mediated coagulation). Mice platelets lacking glycoprotein VI (GPVI) were unable to adhere to atheromatous plaque or form thrombi. Human platelet thrombus formation onto plaques in flowing blood was completely blocked by GPVI inhibition with the antibody 10B12 but not affected by integrin alpha2beta1 inhibition with 6F1 mAb. Moreover, the initial platelet response, shape change, induced by plaque was blocked by GPVI inhibition but not with alpha2beta1 antagonists (6F1 mAb or GFOGER-GPP peptide). Pretreatment of plaques with collagenase or anti-collagen type I and anti-collagen type III antibodies abolished plaque-induced platelet activation. Our results indicate that morphologically diverse collagen type I- and collagen type III-containing structures in lipid-rich atherosclerotic plaques stimulate thrombus formation by activating platelet GPVI. This platelet collagen receptor, essential for plaque-induced thrombus formation, presents a promising new anti-thrombotic target for the prevention of ischemic cardiovascular diseases.

W03-O-002 Dietary supplementation with oily fish reduces platelet-monocyte aggregation in man

W03-O-002 Dietary supplementation with oily fish reduces platelet-monocyte aggregation in man

Platelet-Monocyte Aggregates are Independently Associated with Occurrence of Carotid Plaques in Type 2 Diabetic Patients

Recent evidence suggests important roles for platelet activation in the progression of atherosclerosis. We have recently shown that P-selectin expression or the presence of platelet-monocyte aggregates, a well-characterized marker of platelet activation, is associated with carotid atherosclerosis in the general population. It is not clear, however, whether platelet activation is also associated with carotid atherosclerosis in patients with type 2 diabetes. In the present study, we measured circulating levels of platelet-monocyte aggregates in 120 patients with type 2 diabetes and 120 age- and gender-matched non-diabetic subjects, and examined their association with carotid atherosclerosis determined by arterial ultrasound. The percentage of platelet-monocyte aggregates was analyzed by CD41-positivity determined by whole-blood flow cytometry. Diabetic subjects (7.73 +/- 4.04%, mean +/- SD) showed significantly higher percentages of platelet-monocyte aggregates than non-diabetic subjects (6.03 +/- 4.38%). The percentage of these aggregates was significantly and positively correlated with HbA(1c) in both diabetic and non-diabetic subjects, with the association independent of other clinical factors. Logistic multiple regression analyses revealed that platelet-monocyte aggregates were significantly associated with the presence of carotid plaques independent of the status of glycemic control in diabetic subjects. Thus, an increase in platelet-monocyte aggregation in type 2 diabetic patients appears to be involved in the pathophysiology of carotid atherosclerosis.

Functional Consequences of Cellular Interactions with Heparin-PF4 Antibodies in Heparin-Induced Thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a potentially devastating complication of heparin therapy. The condition is due to formation of antibodies to the heparin-platelet factor 4 (HPF4) complex that activate platelets and/or endothelial cells by binding to FcγIIa (CD32) receptors. It is not clear why only a sub-population of patients that form HPF4 antibodies also develops clinical HIT, and why only a fraction of those develop thrombosis. We hypothesized that some functionally active HPF4 antibodies (i.e. that activate platelets) may also activate leukocytes and endothelial cells by upregulating procoagulant activity; a property not shared by non-functional HPF4 antibodies. Citrated plasmas were obtained from patients clinically suspected of HIT. A commercially available ELISA (GTI, Brookfield, WI) combined with a 14C-serotonin release assay (SRA) was used to stratify patient plasmas into functional (F) and non-functional (NF) HPF4 antibody groups. Pooled normal plasma (NP) served as negative control. Some (n=20) GTI-ELISA positive samples (both F and NF) were also tested by flow cytometry using HPF4 coated microspheres (DiaMed-Caribbean, Inc., Miami, FL) for immunological antibody detection, and fluorescent annexin V binding to detect functional activity. The ability of HPF4 antibodies to cause platelet-monocyte aggregate (conjugate) formation was evaluated by two-color flow cytometry. The functional consequences of HPF4 antibody interaction with EaHy926 hybridoma endothelial cells were studied in the presence/absence of washed platelets and therapeutic heparin levels. Cell surface procoagulant activity (PCA) was measured by a single-stage clotting assay. Approximately 25% of ELISA positive plasmas were also positive by SRA. Although most ELISA positive samples were strongly positive in the microsphere flow cytometry assay (5–20 fold increase in mean fluorescence intensity [MFI] compared to NP) there was no direct correlation between the ELISA OD values and MFI. In the functional flow cytometric assay, not all the SRA positive samples were able to cause platelet activation.Platelet-monocyte aggregate formation in the presence of functional HPF4 antibodies was increased by 2–6 fold (MFI increase 50–300%). This was significantly higher than seen with the NF (p<0.05, n=6) or NP (p<0.01, n=6) plasma pools. Exposure of confluent EaHy926 cells to functional HPF4 antibodies and washed platelets caused a significant and time-dependent increase (20–35%) in cellular PCA as evidenced by the shortening of plasma clot time (235±15 vs. 330±12 sec, p<0.05). The clot time was not significantly different in samples incubated with NP or with HPF4 positive samples incubated without platelets. These observations suggest that in addition to platelet activation, a subset of functional HPF4 antibodies can cause platelet-monocyte aggregation and platelet-dependent endothelial cell interaction. This may lead to upregulation of cellular PCA (tissue factor) and thereby contribute to the pathogenesis of HIT. Non-functional HPF4 antibodies, even at comparable titer, appear to be devoid of such cellular effects. A prospective study to relate these observations to clinical findings in HIT patients is currently underway.

Platelet–monocyte aggregates predict troponin rise after percutaneous coronary intervention and are inhibited by Abciximab

Background: Platelet–monocyte aggregates and other markers of platelet activation were investigated before and after percutaneous coronary intervention (PCI) with abciximab therapy. The study sought to assess the relationship between the level of platelet–monocyte aggregation and increases in cardiac troponin I post coronary intervention. Methods: Blood samples were collected from 40 patients before PCI and 10 min after abciximab administration. These were tested for platelet activation markers by flow cytometry. Cardiac troponin I levels were assayed at baseline and at 24 h post PCI. Results: Compared to healthy controls, patients with coronary artery disease had elevated markers of platelet activation including platelet–monocyte aggregates, P-selectin and PAC-1 (a marker specific for activated glycoprotein IIb/IIIa) prior to PCI. Increased levels of platelet–monocyte aggregates before PCI were associated with increased expression of P-selectin on the platelet surface. Abciximab therapy reduced platelet–monocyte aggregate levels but had no effect on P-selectin expression. The high levels of expression of activated glycoprotein IIb/IIIa (PAC-1) on platelets prior to PCI was reduced with abciximab therapy. Patients with higher levels of platelet–monocyte aggregates prior to PCI were more likely to develop an elevation of cardiac troponin I during the 24 h after PCI. Conclusions: Increased levels of platelet–monocyte aggregates may predict patients at risk for troponin elevation following PCI and identify those most likely to benefit from abciximab.

Increased CD40 ligand and platelet–monocyte aggregates in patients with type 1 diabetes mellitus

Background: Diabetes mellitus is a major risk factor for cardiovascular disease and is associated with a proinflammatory and prothrombotic state. We investigated whether CD40 ligand (L) expression and platelet–monocyte aggregation are increased in patients with type 1 diabetes. Methods: Serum C-reactive protein (CRP) and soluble (s) CD40L concentrations, platelet surface CD40L expression and platelet–monocyte aggregates were measured in 22 patients with uncomplicated type 1 diabetes and 22 age- and sex-matched non-diabetic control subjects. Results: In comparison to controls, patients with type 1 diabetes had higher serum CRP concentrations (3.29 ± 0.9 mg/L versus 0.99 ± 0.2 mg/L, P = 0.01), serum sCD40L concentrations (10.0 ± 1.4 ng/mL versus 4.6 ± 0.6 ng/mL, P = 0.006), and platelet surface expression of CD40L (13.8 ± 0.9% versus 8.5 ± 1.1%, P < 0.001). Platelet–monocyte aggregates were also significantly elevated in type 1 diabetes (35.9 ± 3.3% versus 26.4 ± 2.9%, P = 0.005; n = 10). We also observed a significant correlation between plasma glucose and serum CRP ( r = 0.53, P = 0.01) as well as platelet–monocyte aggregates ( r = 0.69, P = 0.03). Conclusions: Type 1 diabetes is associated with increased CD40L expression and platelet–monocyte aggregation, which may contribute to the proinflammatory and prothrombotic state as well as the accelerated atherogenesis associated with this disorder.

Upregulation of the CD40/CD40 ligand dyad and platelet-monocyte aggregation in cigarette smokers.

Smoking is a potent cardiovascular risk factor and is associated with proinflammatory and prothrombotic responses. The CD40/CD40 ligand (CD40L) dyad and platelet-monocyte aggregation mediate a range of proinflammatory and prothrombotic processes thought to be important in atherothrombosis. We investigated whether expression of the CD40/CD40L dyad and platelet-monocyte aggregation are altered in cigarette smokers. C-reactive protein (CRP), soluble (s) CD40L, and surface expression of CD40L on platelets and T cells and of CD40 on monocytes and platelet-monocyte aggregates were compared in 25 cigarette smokers and 25 age- and gender-matched nonsmokers. Cigarette smokers had increased serum CRP (2.47+/-2.60 versus 0.94+/-0.96 mg/L, P=0.008) and appeared to have elevated plasma sCD40L (0.8+/-1.09 versus 0.37+/-0.21 ng/mL, P=0.07) concentrations. Smokers also had increased surface expression of CD40 on monocytes (45.9+/-7.7% versus 39.9+/-6.5%, P=0.006), of CD40L on platelets (2.9+/-1.0% versus 2.3+/-0.6%, P=0.03), and of platelet-monocyte aggregates (26.6+/-10.9% versus 19.7+/-8.6%, P=0.02). Plasma cotinine concentrations correlated with monocyte CD40 expression, platelet CD40L expression, and platelet-monocyte aggregates. Cigarette smokers have upregulation of the CD40/CD40L dyad and platelet-monocyte aggregation that may account for the atherothrombotic consequences of this major cardiovascular risk factor.