G. Pennings

The role of the fibrocyte in intimal hyperplasia

Summary.  Background: Experimental animal studies have shown that the intimal hyperplasia (IH) responsible for occlusion after successful revascularization procedures may be partially caused by a bone marrow‐derived cell that migrates to the site of vascular injury. Concurrent studies have demonstrated an extensive role in wound healing for the circulating fibrocyte. Objectives: We aimed to trace the path of the circulating cell that contributes to IH and determine if it is the fibrocyte. Methods and results: We established an in vitro model whereby purified monocytes from six healthy human volunteers were cultured into fibrocytes. These cells were morphometrically similar to the vascular smooth muscle cell (VSMC) found in IH and expressed alpha‐smooth muscle actin (α‐SMA) as well as CD34, CD45 and Collagen I (Col I), markers indicative of the fibrocyte. In an in vivo ovine carotid artery synthetic patch graft model, carboxyfluorescein diacetate, succinimidyl ester (CFSE) labeled circulating leukocytes were observed throughout the graft as well as in the neointima in 18 sheep. These cells were shown to produce collagen and α‐SMA at 1, 2 and 4 weeks. These cells then underwent immunohistochemical analysis and were found to express a set of markers unique to the fibrocyte (CD34, CD45, Vimentin and α‐SMA) and also to double stain for CD34 and α‐SMA. Conclusions: IH in an ovine carotid artery patch graft model is partially derived from a hematopoietic circulating progenitor cell that acquires mesenchymal features as it matures at the site of injury.

Pathologic shear triggers shedding of vascular receptors: a novel mechanism for down-regulation of platelet glycoprotein VI in stenosed coronary vessels.

Ligand-induced ectodomain shedding of glycoprotein VI (GPVI) is a metalloproteinase-dependent event. We examined whether shear force, in the absence of GPVI ligand, was sufficient to induce shedding of GPVI. Human-citrated platelet-rich plasma or washed platelets were subjected to increasing shear rates in a cone-plate viscometer, and levels of intact and cleaved GPVI were examined by Western blot and ELISA. Pathophysiologic shear rates (3000-10 000 seconds(-1)) induced platelet aggregation and metalloproteinase-dependent appearance of soluble GPVI ectodomain, and GPVI platelet remnant. Shedding of GPVI continued after transient exposure to shear. Blockade of α(IIb)β(3), GPIbα, or intracellular signaling inhibited shear-induced platelet aggregation but minimally affected shear-induced shedding of GPVI. Shear-induced GPVI shedding also occurred in platelet-rich plasma or washed platelets isolated from a von Willebrand disease type 3 patient with no detectable VWF, implying that shear-induced activation of platelet metalloproteinases can occur in the absence of GPVI and GPIbα ligands. Significantly elevated levels of sGPVI were observed in 10 patients with stable angina pectoris, with well-defined single vessel coronary artery disease and mean intracoronary shear estimates at 2935 seconds(-1) (peak shear, 19 224 seconds(-1)). Loss of GPVI in platelets exposed to shear has potential implications for the stability of a forming thrombus at arterial shear rates.

Intracoronary shear-related up-regulation of platelet P-selectin and platelet-monocyte aggregation despite the use of aspirin and clopidogrel

Recent in vitro studies have shown that shear stress can cause platelet activation by agonist-independent pathways. However, no studies have assessed the extent of shear-induced platelet activation within human coronary arteries. We sampled blood from the coronary arteries proximal and distal to coronary lesions and from the coronary sinus in humans with stable coronary disease who were taking both aspirin and clopidogrel. A novel, computationally based technique for estimating shear stress from 3-dimensional coronary angiographic images of these arteries was developed, and the effect of stenosis severity and calculated shear stress on in vivo platelet and related leukocyte activation pathways were determined. We provide evidence of intracoronary up-regulation of platelet P-selectin, platelet-monocyte aggregation, and monocyte CD11b without platelet glycoprotein IIb-IIIa activation or soluble P-selectin up-regulation. This correlates with intracoronary stenosis severity and calculated shear stress and occurs despite the concurrent use of aspirin and clopidogrel. Our results show for the first time shear-related platelet and monocyte activation in human coronary arteries and suggest this as a potential therapeutic target that is resistant to conventional antiplatelet agents.

Expression of EMMPRIN (CD147) on circulating platelets in vivo

Summary.  Background and Objectives: EMMPRIN (CD147) is a matrix metalloproteinase inducer present on leukocytes and recently identified on platelets in vitro. We examined platelet CD147 expression in vivo and in correlation with markers of platelet activation and coronary artery disease (CAD). Patients/Methods: This prospective observational study involved 70 subjects (55 patients with CAD and 15 controls). Platelet CD62P expression, PAC‐1 expression, platelet–leukocyte aggregates and CD147 (both platelet and leukocyte) expression were assessed by flow cytometry, and soluble CD62P expression was assessed by enzyme‐linked immunosorbent assay. A full blood count and high‐sensitivity C‐reactive protein test were performed. Results: CD147 was expressed on 20.45% ± 1.63% (mean ± standard error of the mean) of circulating platelets, whereas CD62P and PAC‐1 were expressed on 0.87% ± 0.12% and 0.90% ± 0.27% of platelets, respectively. Platelet CD147 expression correlated with CD62P expression (r = 0.359, P = 0.002), PAC‐1 expression (r = 0.428, P < 0.001), leukocyte CD147 expression (monocyte, r = 0.416, P = 0.001; granulocyte, r = 0.434, P < 0.001), C‐reactive protein level and neutrophil/lymphocyte ratio (NLR). CAD patients had significantly higher CD147 mean fluorescence intensity than controls on circulating platelets (2.41 ± 0.14 vs. 2.87 ± 0.09, P = 0.014), monocytes (8.57 ± 1.20 vs. 12.3 ± 0.57, P = 0.006) and granulocytes (4.30 ± 0.65 vs. 6.50 ± 0.34, P = 0.005). Age adjustment eliminated the association between platelet CD147 expression and CAD, but the association between leukocyte CD147 expression and CAD persisted. According to multivariate analysis, the independent predictors of platelet CD147 expression were monocyte CD147 expression, NLR and age. Conclusions: Platelet CD147 expression is evident in vivo and correlates moderately with traditional platelet activation markers and leukocyte CD147 expression. Platelet CD147 expression shows a stronger association with age, and leukocyte CD147 expression a stronger association with clinical CAD, suggesting differences in the regulation of platelet and leukocyte CD147 expression in vivo.

Detection of hypofibrinolysis in stable coronary artery disease using the overall haemostatic potential assay

INTRODUCTION Patients with stable coronary artery disease (CAD) are at risk of arterial thrombosis causing myocardial infarction. Detection of global haemostatic markers of hypercoagulability and hypofibrinolysis may be important for risk stratification and individualised treatment. We examined overall haemostatic potential (OHP) and thrombin generation in a group of stable CAD patients. We also sought to investigate associations between fibrinolytic inhibitors and abnormal global fibrinolysis in these patients. MATERIALS AND METHODS Blood samples were collected from 56 patients defined by coronary anatomy as symptomatically stable CAD. Medications were recorded. Samples were analysed using the global coagulation assays OHP and thrombin generation (calibrated automated thrombogram, CAT), platelet aggregometry measured by Multiplate®, and levels of plasminogen activator inhibitor-1 (PAI-1) antigen measured by ELISA. Results were compared with a reference group of healthy controls. RESULTS Stable CAD patients displayed increased fibrin and thrombin generation and impaired fibrinolysis (decreased overall fibrinolytic potential, OFP, and increased clot lysis time) compared with healthy controls. No effect of antiplatelet agents or other medications on these parameters was observed using platelet-poor plasma. After multivariate adjustment, OFP of healthy individuals was significantly associated with fibrinogen, but in CAD patients PAI-1 became an important determinant. CONCLUSIONS Hypercoagulability of plasma is observed in stable CAD, with both increased thrombin generation and reduced fibrinolytic potential making a significant contribution. The OHP assay may provide a simple method of identifying hypercoagulability in individual patients.

Global hypercoagulability in patients with schizophrenia receiving long-term antipsychotic therapy

BACKGROUND Patients with schizophrenia are at increased risk of venous thromboembolism. The mechanisms underlying this association are poorly understood. AIMS We investigated whether there is a global hypercoagulable state in patients with schizophrenia utilising the overall haemostatic potential (OHP) assay which assesses overall coagulation potential (OCP), haemostatic potential (OHP) and fibrinolytic potential (OFP). METHOD Citrated plasma was collected for OHP assays from patients with schizophrenia on long-term antipsychotic treatment and compared with healthy age- and sex-matched controls. Time courses of fibrin formation and degradation were measured by spectrophotometry (absorption of 405nm) after the addition of tissue factor and tissue plasminogen activator to plasma. RESULTS Ninety patients with schizophrenia (antipsychotic treatment-15.9±9.7years) and 30 controls were recruited. Patients with schizophrenia had higher rates of smoking and levels of inflammatory markers (high-sensitivity C-reactive protein and neutrophil-to-lymphocyte ratio) than controls. Whilst D-dimer, fibrinogen and platelet count did not differ between patients with schizophrenia and controls, the OCP (54.0±12.6 vs 45.9±9.1, p=0.002) and OHP (12.6±5.8 vs 7.2±3.7, p<0.001) were higher, and OFP was lower (76.6±9.8% vs 84.9±6.4%, p<0.001) in patients with schizophrenia, implying both a hypercoagulable and hypofibrinolytic state in these patients. Importantly, abnormalities in overall coagulation were independently predicted by levels of plasminogen-activator-inhibitor-1, fibrinogen, platelet count, inflammatory markers and plasma triglycerides, suggesting a multifactorial aetiology. CONCLUSION Patients with schizophrenia have evidence of a global hypercoagulable and hypofibrinolytic state which may contribute to their increased risk of venous thromboembolism.

Circulating levels of soluble EMMPRIN (CD147) correlate with levels of soluble glycoprotein VI in human plasma.

Abstract Extracellular matrix metalloproteinase inducer (EMMPRIN; CD147), which binds to the platelet-specific collagen receptor glycoprotein (GP) VI, is expressed in a range of cell types including platelets and leukocytes, and has been implicated in neoplastic disease and atherosclerotic coronary disease. Both CD147 and GPVI can be shed from cell membranes and detected in plasma. However, while the relationship between soluble CD147 (sCD147), soluble GPVI (sGPVI) and standard markers of platelet activation has received little attention, such analysis may help reveal pathways mediating release of sCD147. We investigated the relationship between sCD147 and platelet markers including sGPVI, soluble and platelet-bound CD62P (P-selectin), active αIIbβ3 (assessed by PAC-1 binding) and platelet CD147 in 25 patients with stable angina pectoris (SAP), 13 patients with no coronary artery disease (CAD) and 10 healthy donors. Plasma levels of sCD147 significantly correlated with sGPVI (r = 0.46, p = .004), but did not correlate with any other platelet markers examined. Linear regression analysis identified that sCD147 levels could be predicted by sGPVI levels (β = .445, p = 0.003) and age (β = 0.304, p = 0.038), but were independent of potential clinical confounders such as CAD, diabetes and medication usage. As sCD147 strongly correlates with platelet-specific sGPVI, a common platelet source and/or mechanism of release may contribute to sCD147 levels in vivo.

Persistent global hypercoagulability in long-term survivors of acute pulmonary embolism.

Hypercoagulable and/or hypofibrinolytic states are risk factors for venous thromboembolism (VTE) including acute pulmonary embolism. Current screening for thrombophilia is targeted towards identifying a specific defect and guidelines recommend a population-based rather than individualized strategy for anticoagulation treatment. We investigated whether there is a global hypercoagulable state in long-term survivors of pulmonary embolism no longer receiving therapeutic anticoagulation utilizing the overall haemostatic potential (OHP) assay, which assesses overall coagulation potential (OCP), OHP and overall fibrinolytic potential (OFP). Long-term survivors of acute pulmonary embolism were identified from a local registry and OHP assays were performed and compared with age and sex-matched controls without pulmonary embolism. Time courses of fibrin formation and degradation were measured by spectrophotometry (absorption 405 nm) after addition of tissue factor and tissue plasminogen activator to plasma. OHP assays were performed in 67 long-term survivors of single pulmonary embolism (7.9 ± 1.4 years after pulmonary embolism) and 20 age (61.7 ± 11.2 vs 56.6 ± 6.4 years, P = 0.06) and sex (P = 0.45)-matched controls. Survivors of pulmonary embolism were more hypercoagulable as reflected by significantly higher OCP (56.4 ± 13.0 vs 49.9 ± 6.9, P = 0.03) and had impaired fibrinolysis with higher OHP (12.6 ± 7.0 vs 5.9 ± 2.0, P < 0.001) and lower OFP (78.1 ± 9.4 vs 88.2 ± 2.9, P < 0.001) compared with controls. Importantly, these abnormalities in overall coagulation were independently predicted by levels of fibrinogen, platelet count, shortened activated partial thromboplastin time and inflammatory markers suggesting a multifactorial cause. Long-term survivors of pulmonary embolism demonstrate enhanced global coagulation and reduced fibrinolytic potential. Assessment of global coagulation may provide new insights into the aggregate effects of multiple prothombotic factors and long-term risk of VTE recurrence.

CD147 in Cardiovascular Disease and Thrombosis

Thrombotic and inflammatory pathways play a key role in coronary artery disease (CAD) development. Extracellular matrix metalloproteinase (aka CD147) is a member of the immunoglobulin superfamily that is expressed on many cell types including hematopoietic, endothelial cells, leukocytes, keratinocytes, platelets, and others. The binding partners of CD147 are numerous and diverse, and give some indication to the various roles that CD147 can play; these include homophilic interactions, integrins, cyclophilins, glycoprotein VI (GPVI), caveolin 1, and monocarboxylate transporters. Recent evidence suggests a role for CD147 in both thrombosis and inflammation, as well as involvement in CAD and cancer. In this review, we summarize the role of CD147 and its binding partners in platelets, thrombosis, and arterial disease and assess mechanistic aspects of CD147 biology.

Intracoronary upregulation of platelet extracellular matrix metalloproteinase inducer (CD147) in coronary disease.

BACKGROUND CD147, also known as extracellular matrix metalloproteinase inducer, is present on circulating platelets and leukocytes in patients with coronary disease and is implicated in atherogenesis, and plaque rupture. We investigated whether CD147 (platelet, leukocyte and soluble) is upregulated within the coronary circulation in patients with stable coronary disease, and whether CD147 levels are associated with coronary shear stress levels. METHODS A total of 25 patients undergoing intervention of a single coronary lesion had blood sampled within the coronary (n=15) and peripheral circulation (n=10). Platelet and leukocyte CD147 expression was measured by flow cytometry. Soluble CD147 was measured by enzyme-linked immunosorbent assays. Shear stress was calculated using computational fluid dynamics analysis. The effect of shear on platelet CD147 expression in vitro was investigated using a cone-plate viscometer. RESULTS Platelet CD147 was higher in the coronary sinus (CS) compared to femoral vein (mean ± SD fluorescence intensity: 3.1 ± 0.7 vs. 2.2 ± 0.5, p=0.01). There was a significant linear trend for increased platelet CD147 expression from the proximal artery to the distal artery, and subsequently to the CS (p=0.01), indicating trans-lesion and transmyocardial upregulation. There were no differences between the various sites for monocyte, granulocyte or soluble CD147 levels (all p=ns). Trans-lesion gradients of CD147 did not correlate with shear stress (all p=ns). Blood subjected to shear in vitro had higher levels of platelet P-selectin (p=0.04) but similar levels of platelet CD147 (p=0.46) compared to rested blood. CONCLUSION Platelet CD147 expression is upregulated in the coronary circulation in patients with stable coronary disease, but its upregulation is independent of shear stress.