Effects of hyperglycemia and hyperinsulinemia on the tissue factor pathway of blood coagulation

Tissue factor (TF) is the primary initiator of blood coagulation. Circulating TF procoagulant activity (TF-PCA) is associated with blood cells and microparticles and is elevated in patients with type 2 diabetes mellitus. Combined hyperinsulinemia and hyperglycemia and to a lesser degree selective hyperinsulinemia for 24 hours in healthy volunteers increased circulating TF-PCA, monocyte TF surface expression and mRNA, plasma thrombin generation, and coagulation factors VII and VIII activities, suggesting that the coagulation system had been activated. In addition, platelet CD40L and platelet-monocyte aggregates increased, indicating platelet activation. Somatostatin abolished these changes. We conclude that hyperinsulinemia, but particularly the combination of hyperinsulinemia and hyperglycemia, creates a prothrombotic state and may, in addition, be proinflammatory and proatherogenic by virtue of the actions of CD40L and TF.     

Increased platelet activation in subjects chronically exposed to cadmium: A pilot study

Cadmium exposure has been reported to be associated with the risk of vascular disorders. Here, we investigated platelet activity in subjects with chronic cadmium exposure. Eighteen and 15 women participated in this study as chronically cadmium-exposed and control non-exposed subjects, respectively. Plasma P-selectin and CD40 ligand (CD40L), soluble markers of platelet activation, were measured. Platelet aggregation in whole blood, P-selectin and activated glycoprotein (aGP) IIb/IIIa expression on platelets and platelet–leukocyte aggregates were determined. The levels of plasma P-selectin and CD40L increased in subjects with chronic cadmium exposure compared with control subjects. Platelet aggregation induced by adenosine diphosphate (ADP) was higher in cadmium-exposed subjects than control subjects. Cadmium-exposed subjects had higher baseline and ADP-induced aGPIIb/IIIa expression on platelets than control subjects. Platelet–neutrophil aggregates also increased in cadmium-exposed subjects. Blood cadmium correlated with ADP-induced aggregation, aGPIIb/IIIa expression and platelet–neutrophil aggregates, while urinary cadmium correlated with soluble P-selectin. However, cadmium only at high concentration (15?µM) could potentiate ADP-induced platelet activation in vitro. In conclusion, our pilot data show that cadmium-exposed subjects have increased baseline platelet activation and reactivity.     

Exercise-induced platelet and leucocyte activation is not enhanced in well-controlled Type 1 diabetes,despite increased activity at rest

Aims/hypothesis Stress can evoke a prothrombotic state with activated platelets and leucocytes, and increased platelet activation at rest has been reported for diabetic subjects. Thus, prothrombotic responses to stress may be enhanced in diabetes mellitus. We therefore evaluated platelet and leucocyte responses to exercise in Type 1 diabetic patients.Methods Type 1 diabetes mellitus patients with good metabolic control and healthy subjects matched for age and body mass index (n=15 for both) performed a maximal exercise test. Platelet and leucocyte activation and their heterotypic aggregation were monitored by whole blood flow cytometry.Results Diabetic platelets did not show higher basal levels of P-selectin expression, but were more reactive to ADP and thrombin stimulation. Diabetic patients had increased lymphocyte and monocyte CD11b expression, and increased circulating platelet–monocyte aggregates. Exercise evoked thrombocytosis, increased circulating platelet P-selectin expression, enhanced platelet sensitivity to ADP and thrombin, and elevated plasma levels of soluble P-selectin to a similar degree in diabetic patients and healthy subjects. Exercise induced marked leucocytosis and elevated plasma elastase, but only slightly increased leucocyte CD11b expression and leucocyte reactivity to stimulation by N-formyl-methionyl-leucyl-phenylalanine. In all of these there was no difference between diabetic patients and healthy subjects. The numbers, but not percentages of circulating platelet–leucocyte aggregates were markedly increased by exercise, but the increase was less prominent among diabetic patients.Conclusions/interpretation Strenuous exercise evokes platelet and leucocyte activation in Type 1 diabetic patients and healthy subjects. Platelet and monocyte hyperactivity were found at rest, but responses to stress were not augmented in metabolically well-controlled Type 1 diabetes mellitus patients.Abbreviations FITC fluorescein isothiocyanate - fMLP N-formyl-methionyl-leucyl-phenylalanine - MAb monoclonal antibody - PLA platelet–leucocyte aggregate - P-Lym platelet–lymphocyte aggregate - P-Mon platelet–monocyte aggregate - P-Neu platelet–neutrophil aggregate - RPE R-phycoerythrin     

Circulating tissue factor procoagulant activity and thrombin generation in patients with type 2 diabetes: effects of insulin and glucose

CONTEXT: Type 2 diabetes mellitus (T2DM) is a hypercoagulable state. Tissue factor (TF) is the principal initiator of blood coagulation. OBJECTIVE: Our objective was to examine the effects of hyperglycemia and hyperinsulinemia on the TF pathway of blood coagulation in T2DM. DESIGN: Three study protocols were used: 1) acute correction of hyperglycemia (with iv insulin) followed by 24 h of euglycemia, 2) 24 h of selective hyperinsulinemia, and 3) 24 h of combined hyperinsulinemia and hyperglycemia. SETTING: The study took place at a clinical research center. STUDY PARTICIPANTS: Participants included 18 T2DM patients and 22 nondiabetic controls. RESULTS: Basal TF-procoagulant activity (TF-PCA), monocyte TF mRNA, plasma coagulation factor VII (FVIIc), and thrombin-anti-thrombin complexes were higher in T2DM than in nondiabetic controls, indicating a chronic procoagulant state. Acutely normalizing hyperglycemia over 2-4 h resulted in a small ( approximately 7%) but significant decline in TF-PCA with no further decline over 24 h. Raising insulin levels alone raised TF-PCA by 30%, whereas raising insulin and glucose levels together increased TF-PCA (by 80%), thrombin-anti-thrombin complexes, and prothrombin fragment 1.2. Plasma FVIIa and FVIIc declined with increases in TF-PCA. CONCLUSION: We conclude that the combination of hyperglycemia and hyperinsulinemia, common in poorly controlled patients with T2DM, contributes to a procoagulant state that may predispose these patients to acute cardiovascular events.     

Nephropathy in Type 1 diabetes is associated with increased circulating activated platelets and platelet hyperreactivity

Patients with diabetes mellitus (DM) have increased platelet activation compared to non-diabetic controls. Platelet hyperreactivity has been associated with adverse cardiovascular outcomes in Type 2 DM, and with diabetic nephropathy. We investigated the relationship between platelet activation and nephropathy in Type 1 DM. Patients with Type 1 DM and diabetic nephropathy (n = 35), age- and sex-matched Type 1 DM patients with persistent normoalbuminuria (n = 51), and healthy age- and sex-matched controls (n = 30) were studied. Platelet surface P-selectin, platelet surface activated GPIIb/IIIa, monocyte-platelet aggregates (MPAs) and neutrophil-platelet aggregates (NPAs) were measured by whole blood flow cytometry as markers of platelet activation. Platelet reactivity was assessed in response to exogenously added ADP and thrombin receptor activating peptide (TRAP). Platelet surface P-selectin (basal and in response to 0.5 or 20 µM ADP) was higher in nephropathy patients compared with normoalbuminuric patients (P = 0.027), and non-diabetic controls (P = 0.0057). NPAs were higher in nephropathy patients compared to normoalbuminuric patients (P = 0.0088). MPAs were higher in nephropathy patients compared to non-diabetic controls (P = 0.0075). There were no differences between groups in activated GPIIb/IIIa or in response to TRAP at any end-point. More patients with nephropathy received aspirin (71.4%) compared to normoalbuminuric patients (27.4%) (P < 0.0001). Type 1 diabetic nephropathy, as compared with normoalbuminuria, is associated with circulating activated platelets and platelet hyperreactivity to ADP, despite the confounding variable of more nephropathy patients receiving aspirin. This platelet activation is likely to contribute to the known increased risk of cardiovascular events in patients with diabetic nephropathy.     

Increased platelet,leukocyte, and coagulation activation in primary myelofibrosis

Platelet and leukocyte activation has been demonstrated in polycythemia vera (PV) and essential thrombocythemia (ET), but such information is limited in primary myelofibrosis (PMF). Platelet, leukocyte, endothelial, and coagulation activation status was assessed in 26 PMF patients and compared with data from 22 age- and sex-matched healthy individuals. Study included flow cytometry assessment of platelet P-selectin expression [at baseline and after adenosine diphosphate (ADP), thrombin and arachidonic acid stimulation], platelet–neutrophil and platelet–monocyte complexes, and CD11b expression in neutrophils and monocytes. Additionally, soluble P-selectin, sCD40L, tissue factor, thrombomodulin, prothrombin fragment 1 + 2 (F1 + 2), and D-dimer were measured by enzyme-linked immunosorbent assays. The above parameters were correlated with the patients’ clinical data and presence of the JAK2 V617F mutation. Compared with controls, PMF patients had increased baseline platelet activation, as shown by significantly higher levels of soluble and platelet P-selectin expression, and also higher percentages of platelet–monocyte complexes. Neutrophil and monocyte CD11b expression was significantly higher in patients with the JAK2 mutation than in those with wild-type allele or the controls. Endothelial and coagulation activation, as demonstrated by increased plasma levels of thrombomodulin and F1 + 2, was also found in PMF, with patients with the JAK2 mutation showing significantly higher values of F1 + 2 than those with wild-type allele. In conclusion, PMF patients have platelet, leukocyte, endothelial, and coagulation activation similar to that in PV and ET. CD11b overexpression and F1 + 2 are correlated with the presence of the JAK2 mutation.     

Increased Platelet Binding to Circulating Monocytes in Idiopathic Pulmonary Fibrosis

Purpose

Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial pneumonia and its prognosis is poor. Epidemiological evidence suggests an association of IPF with vascular disease and thrombotic tendency, which may be related to platelet activation.

Methods

Platelet–monocyte adhesion in peripheral blood was examined by flow cytometry in patients with IPF (n = 19), interstitial lung disease (ILD) other than IPF (n = 9), and control subjects without pulmonary fibrosis (n = 14). Expression of platelet activation markers P-selectin (CD62P), PSGL-1 (CD162), and CD40 ligand (CD40L) on leukocytes and platelets were studied. Plasma concentrations of soluble P-selectin and CD40L were measured by ELISA.

Results

Significantly elevated levels of platelet–monocyte binding were found in patients with IPF (35.6 ± 4.34 % [mean ± SEM]) compared with patients with non-IPF ILD (23.5 ± 3.68 %) and non-ILD control subjects (16.5 ± 2.26 %; P < 0.01). There was a trend towards increased divalent cation-independent platelet–monocyte binding in IPF (6.0 ± 0.77 % [mean ± SEM]) compared with non-IPF ILD (4.3 ± 1.38 %) and control subjects without ILD (3.1 ± 1.75 %; P = 0.058). There was no differential surface expression of platelet activation markers on subsets of leukocytes or platelets. Plasma concentrations of CD40L and soluble P-selectin did not differ between IPF and control subjects. Platelet–monocyte binding had no significant correlation with percent predicted TLco or FVC.

Conclusions

Platelet–monocyte binding is increased in IPF, suggesting increased platelet activation. This conjugation is predominantly calcium-dependent, but there may be more calcium-independent adhesion in IPF. These findings support further research into the role of platelet activation in IPF.     

Increase in expression of monocytic tissue factor (CD142) with monocytes and blood platelet activation in liver cirrhosis.

Tissue factor (TF) is one of the proteins that participate in hemostatic and inflammatory processes. Activated monocytes present in the liver increase expression of TF, and while accumulating in the organ they can intensify inflammation. The aim of the present study was to evaluate the expression of TF on monocytes in advanced liver cirrhosis with regard to other activation markers. The flow cytometric analysis of TF (CD142), CD14, adhesive molecules CD11b and CD11c, costimulatory molecules CD40, CD80 and CD86, and HLA-DR on monocytes was carried out in 45 patients with postalcoholic liver cirrhosis (Child Pugh B, 20 patients; Child Pugh C, 25 patients) and in 25 healthy persons. The positive correlation between monocytic TF expression and monocyte [soluble CD14 (sCD14), CD11b, monocyte aggregates] activation, the expression of costimulatory molecules on monocytes (CD40, CD80), blood platelet (soluble P-selectin, microplatelets) activation, the level of tumor necrosis factor-alpha, biochemical parameters of liver damage (alanine aminotransferase, aspartate aminotransferase, alkaline phosphate, gamma-glutamyltransferase, and bilirubin) as well as coagulation disorders were observed in the study. In conclusion, the study revealed that the activation of monocytes and blood platelets is accompanied by the elevation of monocytic TF expression in advanced liver cirrhosis. The monocytic TF is a significant link connecting clotting processes and inflammatory and immunological phenomena in liver cirrhosis.     

Role of platelet P-selectin and CD40 ligand in the induction of monocytic tissue factor expression

Activated platelets can express CD40 ligand (CD40L) and trigger inflammatory response and tissue factor (TF) expression in endothelial cells through interaction with CD40. This pathway is also important for T cell-induced monocyte and endothelial cell procoagulant activity. We have studied the potential role of the CD40-CD40L pathway in platelet-induced TF expression in a monocytic cell line and in whole-blood monocytes. In vitamin D(3)-differentiated U-937 cells, thrombin-stimulated platelets increased TF expression as measured by mRNA quantification, flow cytometry, and procoagulant activity. Maximum antigen expression occurred after 2 hours. Neutralizing anti-P-selectin antibody yielded a 50% suppression of procoagulant activity, whereas antibody to CD40L had no effect. In thrombin receptor activator-stimulated citrated blood, monocytes were up to 77% TF-positive, with peak expression after only 15 minutes. However, no TF mRNA was detectable at that time. Anti-P-selectin antibody reduced TF by 50%, whereas antibody to CD40L gave a 17% reduction. Thus, we conclude that P-selectin exposed on activated platelets induces the expression of TF in both U-937 cells and whole-blood monocytes but by different mechanisms. Platelet CD40L does not display any significant effect on U-937 cells but may be of some importance on whole-blood monocytes. This suggests a possible functional difference between U-937 and monocyte CD40. Another important finding in this study is the rapid appearance of surface TF on monocytes without detectable mRNA formation. This indicates that TF may be stored intracellularly in these cells and can be exposed on the surface independent of de novo protein synthesis.     

Increased platelet CD63 and P-selectin expression persist in atherosclerotic ischemic stroke

Platelet activation is an important process in the pathogenesis of atherothrombosis. However, the serial changes of platelet activation in atherosclerotic ischemic stroke have not been determined. In this study, we measured serially platelet expression of CD63 and P-selectin and platelet aggregability to ADP and collagen. Measurements were made 24 and 72 h and 7 and 90 days after the ischemic event in 29 patients with atherosclerotic ischemic stroke. Platelet aggregability was significantly decreased after 72 h compared to that at 24 h of stroke onset. However, platelet CD63 and P-selectin expression remained high even 90 days after the events. These findings suggest that platelet hyperactivation in atherosclerotic ischemic stroke may be sustained for a considerable period.     

Response to antiplatelet therapy and platelet reactivity to thrombin receptor activating peptide-6 in cardiovascular interventions: Differences between peripheral and coronary angioplasty

Background

The long-term prognosis of patients with peripheral arterial disease (PAD) is significantly worse than the prognosis of coronary artery disease (CAD) patients. Detrimental platelet activation could contribute to the increased rate of adverse cardiovascular events in PAD. We therefore investigated whether response to antiplatelet therapy and thrombin inducible platelet activation differ between patients with best medical therapy undergoing angioplasty and stenting for symptomatic PAD (n = 166) or CAD (n = 104).

Methods

Adenosine diphosphate (ADP), arachidonic acid (AA) and thrombin receptor activating peptide (TRAP)-6 inducible platelet reactivity was measured by multiple electrode aggregometry (MEA). Platelet surface expression of P-selectin and activated glycoprotein IIb/IIIa (GPIIb/IIIa) in response to ADP, AA, and TRAP-6, and the formation of monocyte-platelet aggregates (MPA) in response to ADP and TRAP-6 were assessed by flow cytometry.

Results

Patients with PAD had significantly higher platelet reactivity in response to ADP and AA by MEA compared to CAD patients. Likewise, the expression of P-selectin and GPIIb/IIIa following stimulation with ADP and AA, and MPA formation in response to ADP were significantly higher in PAD patients than in CAD patients. In response to TRAP-6, patients with PAD showed a significantly increased platelet aggregation by MEA, higher expression of activated GPIIb/IIIa, and more pronounced formation of MPA than CAD patients.

Conclusion

Following angioplasty and stenting, PAD patients exhibit a significantly diminished response to dual antiplatelet therapy and an increased susceptibility to TRAP-6 inducible platelet activation compared to CAD patients.     

High glucose levels enhance platelet activation: involvement of multiple mechanisms

Diabetes mellitus (DM) and hyperglycaemia are associated with platelet activation. The present study was designed to investigate how high glucose levels influence platelet function. Fasting human blood was incubated with different concentrations of D-glucose (5, 15 and 30 mmol/l) and other sugars without or with in vitro stimuli. Platelet activation was monitored by whole blood flow cytometry. High glucose levels enhanced adenosine diphosphate (ADP)- and thrombin receptor-activating peptide (TRAP)-induced platelet P-selectin expression, and TRAP-induced platelet fibrinogen binding. Similar effects were seen with 30 mmol/l L-glucose, sucrose and galactose. Hyperglycaemia also increased TRAP-induced platelet-leucocyte aggregation. Protein kinase C (PKC) blockade did not counteract the enhancement of platelet P-selectin expression, but abolished the enhancement of TRAP-induced platelet fibrinogen binding by hyperglycaemia. Superoxide anion scavenging by superoxide dismutase (SOD) attenuated the hyperglycaemic enhancement of platelet P-selectin expression, but did not counteract the enhancement of TRAP-induced platelet fibrinogen binding. Hyperglycaemia did not alter platelet intracellular calcium responses to agonist stimulation. Blockade of cyclo-oxygenase (COX), phosphotidylinositol-3 (PI3) kinase, or nitric oxide synthase, or the addition of insulin did not influence the effect of hyperglycaemia. In conclusion, high glucose levels enhanced platelet reactivity to agonist stimulation through elevated osmolality. This occurred via superoxide anion production, which enhanced platelet P-selectin expression (secretion), and PKC signalling, which enhanced TRAP-induced fibrinogen binding (aggregablity).     

Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules

Platelet activation leads to secretion of granule contents and to the formation of microvesicles by shedding of membranes from the cell surface. Recently, we have described small internal vesicles in multivesicular bodies (MVBs) and alpha-granules, and suggested that these vesicles are secreted during platelet activation, analogous to the secretion of vesicles termed exosomes by other cell types. In the present study we report that two different types of membrane vesicles are released after stimulation of platelets with thrombin receptor agonist peptide SFLLRN (TRAP) or alpha-thrombin: microvesicles of 100 nm to 1 microm, and exosomes measuring 40 to 100 nm in diameter, similar in size as the internal vesicles in MVBs and alpha-granules. Microvesicles could be detected by flow cytometry but not the exosomes, probably because of the small size of the latter. Western blot analysis showed that isolated exosomes were selectively enriched in the tetraspan protein CD63. Whole-mount immuno-electron microscopy (IEM) confirmed this observation. Membrane proteins such as the integrin chains alpha(IIb)-beta(3) and beta(1), GPIbalpha, and P-selectin were predominantly present on the microvesicles. IEM of platelet aggregates showed CD63(+) internal vesicles in fusion profiles of MVBs, and in the extracellular space between platelet extensions. Annexin-V binding was mainly restricted to the microvesicles and to a low extent to exosomes. Binding of factor X and prothrombin was observed to the microvesicles but not to exosomes. These observations and the selective presence of CD63 suggest that released platelet exosomes may have an extracellular function other than the procoagulant activity, attributed to platelet microvesicles.     

Platelet-CD40 ligand interaction with melanoma cell and monocyte CD40 enhances cellular procoagulant activity.

Platelet-tumor cell interactions are believed to be important in tumor metastasis. Tumor cell tissue factor (TF) expression enhances metastasis and angiogenesis, and is primarily responsible for tumor-induced thrombin generation and the formation of tumor cell-platelet aggregates. Activated platelets express and release CD40 ligand (CD40L), which induces endothelial TF expression by ligation to CD40. We investigated the effect of platelet-derived CD40L on the TF activity of human CD40-positive melanoma cells and monocytes by incubating supernatants from activated or resting platelets with tumor cells or monocytes, and by bringing resting or activated platelets into close apposition with tumor cell monolayers. CD40L was present on the surface of activated (but not resting) platelets and was also released following platelet activation. Both recombinant soluble CD40L (rsCD40L) and activated platelet supernatants increased procoagulant activity (PCA) and TF antigen in tumor cells and monocytes. The increase in TF activity induced by both rsCD40L and activated platelet supernatants was inhibited by anti-CD40L antibody. Furthermore, contact of activated platelets with tumor cells increased cellular PCA, and this effect was also inhibited by anti-CD40L. In malignancy, the increase in cellular TF activity via CD40 (tumor cell)-CD40L (platelet) interaction may possibly enhance intravascular coagulation and hematogenous metastasis.     

Blood platelet and monocyte activations and relation to stages of liver cirrhosis

AIM: Blood platelets (pIt) and monocytes are the cells that play a crucial role in the pathogenesis of liver damage and liver cirrhosis (LC). In this paper, the analysis of mutual relationship between platelets and monocytes activation in LC was conducted. METHODS: Immunofluorescent flow cytometry was used to measure the percentage of activated platelet populations (CD62P, CD63), the percentage of plt-monocyte aggregates (pma) (CD41/CD45), and activated monocytes (CD11b, CD14, CD16) in the blood of 20 volunteers and 40 patients with LC. Platelet activation markers: sP-selectin, platelet factor 4 (PF4), beta-thromboglobulin (PTG) and monocyte chemotactic peptide-1 (MCP-1) were measured and compared in different stages of LC. RESULTS: Platelet activation with the increase in both βTG serum concentration and elevation of pIt population (CD62P and CD63 as well as MIF CD62P and CD63) is elevated as LC develops and thrombocytopenia rises. There is a positive correlation between medial intensity of fluorescence (MIF) CD62P and MIF CD63 in LC. We did not show any relationship between monocyte activation and pma level. SP-selectin concentration correlates positively with pIt count and pma, and negatively with stage of pIt activation and MIF CD62P and MIF CD63. There was no correlation between MCP-1 concentration and pIt, monocyte activation as well as pma level in LC. CD16 monocytes and MIF CD16 populations are significantly higher in the end stage of LC. A positive correlation occurs between the value of CDllb monocyte population and MIF CD14 and MIF CD16 on monocytes in LC. CONCLUSION: Platelet and monocyte activation plays an important role in LC. Platelet activation stage does not influence monocyte activation and production of pIt aggregates with monocytes in LC. With LC development, thrombocytopenia may be the result of pIt consumption in platelet-monocyte aggregates.     

Platelet activation and function during eltrombopag treatment in immune thrombocytopenia

We monitored platelet activation by means of P-selectin and platelet monocyte aggregates (PMA) and platelet function by whole blood multiple electrode aggregometry and platelet adhesion under high shear in chronic immune thrombocytopenia patients to define changes in platelet activation during treatment with eltrombopag. Overall, platelet activation and function normalized with increasing platelet counts. However, P-selectin, which was already elevated before treatment, and PMA increased further transiently during the first weeks. The increases in P-selectin and in PMA indicate ongoing platelet activation during the early period of treatment.     

Enhanced P-selectin expression and increased soluble CD40 Ligand in patients with Type 1 diabetes mellitus and microangiopathy: evidence for platelet hyperactivity and chronic inflammation

Aims/hypothesis Platelet activation, endothelial dysfunction and inflammation may be involved in early stages of diabetic microangiopathy. We therefore investigated patients with Type 1 diabetes mellitus, without (n=19) and with (n=20) microangiopathy, matched for glycaemic control and duration of disease, and matched with healthy control subjects (n=27).Methods Platelet activation was measured as platelet P-selectin expression using whole blood flow cytometry and as soluble P-selectin by immunoassay. Von Willebrand factor antigen in plasma, serum soluble E-selectin, CD40 ligand (sCD40L) and C-reactive protein (CRP) served as markers for endothelial function and inflammation.Results Thrombin-induced platelet P-selectin expression was enhanced, and soluble P-selectin and sCD40L concentrations were increased in patients with microangiopathy compared with the control subjects (p<0.01 for both) and with patients without microangiopathy (p<0.05 for P-selectin expression and sP-selectin), whereas all three parameters were similar in patients without microangiopathy and in the control subjects. CRP and soluble E-selectin were increased in patients with microangiopathy, compared with the control subjects (p<0.01 and p<0.05), whereas von Willebrand factor did not differ between the groups.Conclusions/interpretation Microangiopathy in Type 1 diabetes is associated with platelet hyperactivity, endothelial dysfunction and low-grade inflammation, indicating an increased risk for cardiovascular disease.Abbreviations sP-selectin Soluble P-selectin - sCD40L soluble CD40 Ligand - CRP C-reactive protein     

Increased Platelet Reactivity and Circulating Monocyte-Platelet Aggregates in Patients With Stable Coronary Artery Disease

Objectives. We sought to examine whether patients with stable coronary artery disease (CAD) have increased platelet reactivity and an enhanced propensity to form monocyte-platelet aggregates.Background. Platelet-dependent thrombosis and leukocyte infiltration into the vessel wall are characteristic cellular events seen in atherosclerosis.Methods. Anticoagulated peripheral venous blood from 19 patients with stable CAD and 19 normal control subjects was incubated with or without various platelet agonists and analyzed by whole blood flow cytometry.Results. Circulating degranulated platelets were increased in patients with CAD compared with control subjects (mean [±SEM] percent P-selectin–positive platelets: 2.1 ± 0.2 vs. 1.5 ± 0.2, p < 0.01) and were more reactive to stimulation with 1 μmol/liter of adenosine diphosphate (ADP) (28.7 ± 3.9 vs. 16.1 ± 2.2, p < 0.01), 1 μmol/liter of ADP/epinephrine (51.4 ± 4.6 vs. 37.5 ± 3.8, p < 0.05) or 5 μmol/liter of thrombin receptor agonist peptide (TRAP) (65.7 ± 6.8 vs. 20.2 ± 5.1, p < 0.01). Patients with stable CAD also had increased circulating monocyte-platelet aggregates compared with control subjects (percent platelet-positive monocytes: 15.3 ± 3.0 vs. 6.3 ± 0.9, p < 0.01). Furthermore, patients with stable CAD formed more monocyte-platelet aggregates than did control subjects when their whole blood was stimulated with 1 μmol/liter of ADP (50.4 ± 4.5 vs. 28.1 ± 5.3, p < 0.01), 1 μmol/liter of ADP/epinephrine (60.7 ± 4.3 vs. 48.0 ± 4.8, p < 0.05) or 5 μmol/liter of TRAP (67.6 ± 5.7 vs. 34.3 ± 7.0, p < 0.01).Conclusions. Patients with stable CAD have circulating activated platelets, circulating monocyte-platelet aggregates, increased platelet reactivity and an increased propensity to form monocyte-platelet aggregates.     

Platelet-induced expression of tissue factor procoagulant activity in freshly isolated human mononuclear cells: implications for experimental use

Monocytes express tissue factor (TF) as a result of cytokine stimulation or endothelial adherence. We evaluated monocyte-platelet interaction in vitro as another trigger for monocyte TF enhancement in human mononuclear cells isolated by density gradient centrifugation from peripheral blood. Cell TF procoagulant activity (TF-PCA) was quantitated by a one-stage recalcification clotting time assay. Platelets were counted and identified by whole blood flow cytometry as CD61 positive particles, activated platelets were characterized by P-Selectin (CD62) expression, and monocytes by surface CD14 expression. A significant correlation between normalized TF-PCA of isolated mononuclear cells and platelet count was shown (r = 0.43, P < 0.001). Percentage of activated platelets in baseline samples was 4.2 +/- 3.5 while adenosine diphosphate (ADP) increased platelet positivity to 34 +/- 17% (P < 0.001). After isolation, 52 +/- 12% of platelets within suspensions were activated (P < 0.0001). Percentage of CD62-positive monocytes (CD14+ particles) increased from baseline 5% to 13 +/- 6% in ADP-stimulated samples to 53 +/- 17% after isolation (P < 0.001). These findings suggest that density gradient centrifugation activates platelets and that an adhesive interaction between monocytes and platelets may promote TF-PCA expression in isolated mononuclear suspensions. Enhanced monocyte TF expression as a result of an activated platelet-monocyte interaction seems to be an important laboratory effect requiring consideration when utilizing this technique in an experimental setup.     

Venous hypertension induces increased platelet reactivity and accumulation in patients with chronic venous insufficiency

The objective of this study was to determine whether there are changes in platelet activation and rheology in patients with chronic venous insufficiency (CVI) and what their impact is on this disease. Anticoagulated peripheral venous blood collected from 21 patients with CVI and 13 normal control subjects in different bodily positions was incubated either with 0.5 mumol/L adenosine diphosphate (ADP) or without agonist and analyzed by whole blood flow cytometry. Soluble P-selectin was analyzed in obtained sera by enzyme-linked immunosorbent assay. Platelet count was determined by a whole blood analyzer. Circulating platelets were more reactive to stimulation with 0.5 mumol/L ADP in patients with CVI compared with control subjects. There was no statistically significant change in platelet activation without ADP and the level of soluble P-selectin as a function of posture. Under simulated venous hypertension, platelet accumulation was observed in patients with CVI. Patients with CVI had increased platelet reactivity and accumulation during orthostasis, suggesting this might be a contributory factor to CVI pathogenesis.