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.     

Correlation between immature platelet fraction and reticulated platelets. Usefulness in the etiology diagnosis of thrombocytopenia

Background: Reticulated platelets (RP) are a surrogate marker for megakaryocytic activity, but the limitation of this determination is the lack of standardization of methodology. The determination of the immature platelet fraction (IPF) is performed in a simple, automated, and reproducible way between laboratories. We analyzed the correlation between IPF and RP, and usefulness of IPF in patients with thrombocytopenia. Methods: RP were determined by flow cytometry using double staining with thiazole orange and CD61 PerCP^®. IPF was performed with Sysmex XE2100 analyzer. We used a control group with normal platelets, and thrombocytopenic patients were classified into three groups: Group 1. Central thrombocytopenia, Group 2. Thrombocytopenia as a result of enhanced peripheral platelet destruction, and Group 3. Peripheral non‐immune thrombocytopenia by abnormal distribution. Results: Fourteen controls and 66 patients were analyzed. Group 1: 25 patients, they had mean and confidence interval 95% (95% CI) for IPF 8.67% (6.49–10.46%) and RP 4.08% (2.86–5.30%). Group 2: 20 patients, they had mean and 95%CI for IPF 16.80% (12.20–21.39%) and RP 16.14% (9.89–22.40%). Group 3: 21 patients, they had mean and 95% CI for IPF 9.04% (6.95–11.14%) and RP 5.23% (3.41–7.05%). The overall Pearson linear correlation between IPF and RP was r: 0.65. There were statistically significant differences in values of IPF and RP between Group 2 and the other two groups (P < 0.01). Conclusion: There is a good correlation between IPF and RP mainly in thrombocytopenia by peripheral destruction. Determination of IPF is an easy technique in their implementation, standardized and reproducible, so it could be a useful screening technique in patients with thrombocytopenia.     

Atrial fibrillation is associated with increased mean platelet volume in patients with type 2 diabetes mellitus

Platelet abnormalities in diabetes mellitus (DM) and atrial fibrillation (AF) may underline the etiology of a prothrombotic state in these conditions. Increased mean platelet volume (MPV) is a marker of abnormal platelet function and activation. We aimed to investigate the possible association of chronic AF with MPV in patients who have type 2 DM. Patients who had type 2 DM with either chronic (≥6 months) AF or normal sinus rhythm (NSR) were included in the study. A total of 162 patients (aged 38–89 years) were divided into 2 groups according to the presence of either AF or NSR. Group 1 consisted of 81 diabetic patients with AF, and group 2 consisted of 81 diabetic patients with NSR. The two groups were not significantly different in terms of age, and gender, as well as in hypertension, smoking, history of coronary artery disease, previous cerebrovascular accidents, microalbuminuria, retinopathy, duration of DM, body mass index, hemoglobin A_1c, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglyceride (p?>?0.05 for all variables). Although no significant difference was present between groups concerning platelet count; for patients with AF, MPV was higher compared with patients with NSR (9.0?±?0.2?fl vs. 8.4?±?0.2?fl; p?=?0.001). Furthermore, no significant difference was noted between groups regarding routine medications received by patients. In multivariate logistic regression analysis, MPV was the only variable independently related to AF (OR?=?2.659; 95% CI, 1.286–5.498; p?=?0.008). Consequently, it is concluded that AF is associated with increased MPV in patients with type 2 DM, suggesting the presence of tentatively related processes leading to reciprocal interaction.     

高血压患者循环内皮细胞微颗粒水平的变化

目的研究高血压患者内皮细胞微颗粒水平的变化及其与血压的关系。方法采用流式细胞术检测26例高血压患者(高血压组)和30例健康志愿者(健康对照组)血浆中CD31+/CD42-内皮细胞微颗粒的水平,并比较其与血压的关系。结果与健康对照组比较,高血压组患者血浆中内皮细胞微颗粒水平明显增高[(1 900.7±226.4)个/μlvs(944.9±124.5)个/μl,P<0.001]。内皮细胞微颗粒水平与收缩压和脉压呈正相关(r=0.38,P=0.004;r=0.42,P=0.001)。结论高血压患者内皮细胞微颗粒水平增高,内皮细胞微颗粒水平与血压呈正相关。     

A diagnostic test for heparin-induced thrombocytopenia: detection of platelet microparticles using flow cytometry

Based on our previous observation that heparin-induced thrombocytopenia (HIT) sera can generate platelet microparticles from washed platelets in a heparin-dependent fashion, we developed a test for HIT using flow-cytometry to measure heparin-dependent platelet microparticle formation. During the developmental phase of the assay the optimal physical conditions for microparticle generation were defined. 133 sera were then evaluated using the microparticle assay and the serotonin release assay to determine the threshold for defining a positive result that gave optimal sensitivity and specificity. The microparticle assay was then prospectively evaluated against the serotonin release assay in 202 sera referred to our laboratory for HIT testing. Overall agreement between the two assays was 96% (Cohen's kappa = 0.91). When the clinical data were reviewed on patients whose sera gave discrepant results between the two assays, no case of HIT was detected by one assay and missed by the other. The platelet microparticle assay is as accurate as the serotonin release assay and may be a useful non-radioactive test for HIT.     

Platelet-derived microparticle levels are significantly elevated in patients treated by elective stenting compared to subjects with diagnostic catheterization alone

Significant platelet reactivity with endothelial damage may occur during percutaneous coronary intervention such as balloon angioplasty and elective stenting in patients with stenotic or occluded arteries in coronary artery disease. Activated platelets express several surface epitopes to aggregate and form heterotypic interactions with leukocytes and endothelial cells, secrete biomarkers to enhance their prothrombotic properties and also generate increased level of microparticles (PMPs). These events may contribute to subacute stent thrombosis induced by the procedure-mediated trauma. Our aim was to study the level of PMPs and other platelet activation markers at an early time point after stenting with bare metal stent in patients on aspirin antiplatelet pre-treatment, and these results were compared to data obtained from subjects with diagnostic catheterization alone. We found that at 15 minutes after the completion of stenting, the levels of PMPs (557 ± 83/µl versus 325 ± 42/µl), the platelet P-selectin expression (2.7 ± 0.3% versus 1.99 ± 0.1%) and the ratio of platelet-monocyte heterotypic aggregates (48 ± 4% versus 38 ± 3%) were significantly (p < 0.05) elevated in patients compared to unstented subjects, but no difference was found in soluble P-selectin values. We suggest that the observed cellular changes are early and sensitive markers to detect the platelet-activating effect of stent implantation.     

Raised platelet levels in diabetes mellitus complicated with nephropathy

Abstract . Sterner G, Carlson J, Ekberg G (Malmö University Hospital, Malmö, Sweden). Raised platelet levels in diabetes mellitus complicated with nephropathy. J Intern Med 1998; 244 : 437–41.
Objectives To study if elevated platelet levels are found in early and late diabetic nephropathy.
Design A retrospective study was performed of platelet count in different subgroups of patients with insulin-dependent diabetes mellitus, including patients with long and short duration of diabetes and with early signs of nephropathy. Patients with severe renal failure subjected to renal transplantation were also included.
Results The results suggest that raised platelet levels were associated with early signs of diabetic nephropathy and with female gender. Amongst diabetics progressing to uraemia leading to renal transplant, the platelet level was 50% higher than in non-diabetic uraemic patients.
Conclusion It is concluded that raised platelet levels are a common finding in patients with diabetes mellitus complicated by nephropathy.     

Flow cytometric analysis of platelet cyclooxygenase-1 and -2 and surface glycoproteins in patients with immune thrombocytopenia and healthy individuals

Immature platelets may contain more platelet enzymes such as cyclooxygenase (COX)-1 and COX-2 than mature platelets. Patients with immune thrombocytopenia (ITP) have a higher fraction of immature platelets and can therefore be utilized as a biological model for investigating COX-1 and COX-2 platelet expression. The aims were to develop flow cytometric assays for platelet COX-1 and COX-2 and to investigate the COX-1 and COX-2 platelet expression, platelet turnover, and platelet glycoproteins in ITP patients (n = 10) compared with healthy individuals (n = 30). Platelet count and platelet turnover parameters (mean platelet volume (MPV), immature platelet fraction (IPF), and immature platelet count (IPC)) were measured by flow cytometry (Sysmex XE-5000). Platelet COX-1, COX-2, and the glycoproteins (GP)IIb, IX, Ib, Ia, and IIIa were all analyzed by flow cytometry (Navios) and expressed as median fluorescence intensity. COX analyses were performed in both whole blood and platelet rich plasma (PRP), whereas platelet glycoproteins were analyzed in whole blood only. ITP patients had significantly lower platelet count (55 × 10⁹/L) than healthy individuals (240 × 10⁹/L, p < 0.01), but a higher MPV (p = 0.03) and IPF (p < 0.01). IPC was similar for the two groups (p = 0.74). PRP had significantly lower MPV (p < 0.01) and significantly higher platelet count and IPC (both p-values <0.03) when compared with whole blood. IPF was similar for PRP and whole blood (p = 0.18). COX-1 expression was 10 times higher and COX-2 expression was 50% higher in PRP than in whole blood (p_COX-1 < 0.01, p_COX-2 < 0.01). Platelet COX-1 expression was higher in ITP patients than healthy individuals using whole blood (p_COX-1 < 0.01) and PRP, though this was nonsignificant in PRP (p_COX-1 = 0.17). In ITP patients, positive correlations were found between platelet turnover and COX-1 expression (all p-values <0.01, rho = 0.80–0.94), whereas healthy individuals showed significant though weaker correlations between platelet turnover and COX-1 and COX-2 expressions (all p-values <0.03, rho = 0.44–0.71). GPIIb, IX, and Ib expression was increased in ITP patients compared with healthy individuals (all p-values < 0.03). GPIIb, IX, Ib, and IIIa showed positive correlations with platelet turnover in ITP patients (all p-values <0.02, rho = 0.71–0.94), but weak and nonsignificant correlations in healthy individuals (all p-values >0.14, rho = 0.11–0.28). In conclusion, ITP patients expressed higher COX-1 and platelet glycoprotein levels than healthy individuals. COX-1 and platelet glycoproteins demonstrated positive correlations with platelet turnover in ITP patients. In healthy individuals, COX-1 and COX-2 expression correlated positively with platelet turnover. PRP was more sensitive compared with whole blood as regards determination of COX. Therefore, PRP is the recommended matrix for investigating COX-1 and COX-2 in platelets.     

Coronary heart disease is associated with mean platelet volume in type 2 diabetic patients

Background: Mean platelet volume (MPV) is an indicator of platelet activation which is a central process in the pathophysiology of coronary heart disease (CHD). The aim of the study was twofold; first to determine whether MPV values is increased in patients with DM, and secondly to evaluate the relation between diabetic complications and MPV. Methods: The study population included 258 patients divided into two groups. Group A composed of 158 type 2 diabetic patients with coexistent coronary artery disease (stenotic lesions of 50%) (78 women, 80 men; mean age 53.9_10.8; mean diabetes duration 13.1_6.0). One hundred subjects (48 women, 52 men; mean age 53.9_11) without type 2 diabetes with normal coronary angiographies were taken as the control group (group B). To evaluate the extension of CHD, Gensini scoring system was used. Results: The MPV was significantly different in the patient group compared to the controls (9.79 ± 1.5 fl vs 8.3 ± 0.9 fl, P<0.001). The existence of CHD was associated with MPV with odds ratio (95% CI) of 2.31 (1.55–4.42, p50.001). Conclusion: We have found that diabetic patients with coronary heart disease have significantly higher MPV values compared to control subjects without diabetes and with angiographically normal coronary arteries.     

Reticulated platelet levels in patients with ulcerative colitis

Background and aims In this study, we investigated whether reticulated platelets (RP) would be useful markers in the evaluation of ulcerative colitis (UC) activity and also aimed to gain indirect information about the platelet kinetics. Materials and methods Complete blood count, C-reactive protein, erythrocyte sedimentation rate, and proportion of RP were measured in 16 active, 21 inactive UC patients, and 20 healthy blood donors. UC activity was assessed by Truelove–Witts criteria. Results Mean platelet count was increased in patients with active compared to inactive UC (p = 0.008) or healthy donors (p = 0.000). Mean platelet volume (MPV) was significantly decreased in patients with active compared to inactive (p = 0.015) and healthy donors (p = 0.001). RP values was significantly decreased in active and inactive UC groups compared to healthy donors (p = 0.000, p = 0.000, respectively), while there was no significant difference between active and inactive UC patients (p = 0.980). Significant negative correlation between platelet count and MPV in patients with active UC (r = −0.542, p = 0.030) was observed. Conclusions RP values is reduced in active and inactive UC patients compared to healthy donors. To our knowledge, this is the first study about proportion of RP with UC in literature. However, the role of low RP values have not been determined clinically. Further studies are needed to evaluate the role of platelet abnormalities and changes in megakaryopoiesis caused by inflammatory state on low MPV and RP values during the course of UC.     

Flow cytometric evaluation of platelet activation in blood collected into EDTA vs. Diatube-H,a sodium citrate solution supplemented with theophylline,adenosine, and dipyridamole

With platelet activation, there is modulation of platelet surface molecule expression. In flow cytometric analyses of in vivo platelet activation, results are often confounded by activation induced in vitro by the preparative procedures. It is particularly important therefore to prevent or retard platelet activation as soon as possible after withdrawal of the blood sample. Taking blood into paraformaldehyde, or fixing the cells with paraformaldehyde as soon as possible after withdrawal, has been employed to prevent platelet activation in vitro, but paraformaldehyde-fixed platelets cannot be further used in functional studies. We investigated the efficacy of Diatube-H, a commercially available combination of platelet antagonists (theophylline, adenosine, and dipyridamole), in preventing or retarding platelet activation in vitro, along with its effects on modulation of platelet membrane glycoproteins (GP) and adhesion molecules. In contrast to blood taken into EDTA, blood taken into Diatube-H vacutainer tubes could be stored at room temperature for up to 4 hr prior to paraformaldehyde fixation without significant in vitro platelet activation, as measured by CD62P, CD63 and modulation of GPIb and GPIIbIIIa surface expression. Hence, paraformaldehyde fixation could be deferred for several hours, permitting transport of samples from distant sites. Studies of thrombin-induced platelet activation indicated that platelets taken into Diatube-H remained functional i.e. were able to be activated. Expression of the CD29, CD49b and CD31 adhesion molecules on the platelet surface was unaffected by storage in Diatube-H. The results suggest that Diatube-H may be a useful reagent for flow cytometric studies of platelets when the samples cannot be processed immediately.     

Elevated levels of remnant lipoproteins are associated with plasma platelet microparticles in patients with type-2 diabetes mellitus without obstructive coronary artery disease.

AIMS: Platelets participate in the pathogenesis of arterial thrombosis and it has been demonstrated that enhanced platelet activation occurs in patients with diabetes mellitus (DM). Dyslipidaemia is a common feature of diabetes. We investigated the association between certain lipid fractions and plasma platelet-derived microparticle (PMP) levels in patients with type-2 DM. METHODS AND RESULTS: We measured fasting serum levels of remnant-like lipoprotein particles-cholesterol (RLP-cholesterol) and assessed in vivo platelet activation by quantifying the number of PMP in the plasma detected as CD42b-positive microparticles by flow cytometry in Japanese type-2 DM patients without obstructive coronary artery disease who were more slender when compared with Western diabetic patients. The levels of total cholesterol, triglycerides, RLP-cholesterol, and plasma glucose were significantly higher in patients with type-2 DM (n = 105) than in non-diabetic patients (n = 92). The plasma levels of PMP were elevated significantly in type-2 DM patients when compared with non-diabetic control subjects [7.41(5.39-10.50) x 10(6) vs. 3.44(2.43-4.41)x10(6), P < 0.001]. We found that RLP-cholesterol levels were the best predictor of PMP in multivariable linear regression analyses (beta = 0.375, P < 0.001). Lipid-lowering medication with bezafibrate successfully reduced levels of both RLP-cholesterol and PMP in patients with type-2 DM (P < 0.05). CONCLUSIONS: RLP-cholesterol and platelet microparticles are both elevated in type-2 DM patients when compared with controls. RLP-cholesterol is the primary and only predictor of platelet microparticles in the multivariable analysis, which include several standard atherosclerosis risk factors. This suggested that reducing elevated RLP-cholesterol with lipid-lowering therapy may be an effective strategy to prevent thrombogenic vascular complications in type-2 DM.     

冠心病患者血小板功能在精神应激后的变化

目的观察冠心病（CAD）患者血小板功能在精神应激前后的变化。方法用流式细胞术测定冠状动脉造影证实的22例CAD患者精神应激（心算10min）前、结束后即刻、休息10min后外周血血小板膜糖蛋白ⅡbⅢa复合物的α亚单位（CD41）、P选择素（CD62p）的变化。结果精神应激后CAD患者外周血中CD41阳性率由（75±20）%增加到（89±10）%（P<0.05）;CD62p阳性百分率由（2.5±0.9）%增加到（3.5±0.8）%（P<0.05）;休息10min后则降至应激前水平。结论精神应激可以激活CAD患者血小板功能。     

流式细胞术研究特发性血小板减少性紫癜患者血小板相关抗体

目的：探讨特发性血小板减少性紫癜（ITP）患者血小板表面相关抗体（PAIg）在诊断及预后的价值。方法：应用流式细胞术（FCM）检测84例ITP患者及20例正常人PAIgG、PAIgM、PAIgA。结果：初发ITP和复发ITP患者组与正常对照组比较，PAIgG、PAIgA差异有统计学意义（P〈0．01），PAIgM差异无统计学意义（P〉0．05）。PAIgM与PAIgA之间有显著相关性，r=0．451（P〈0．01）。结论：①PAIg增高可作为诊断初发ITP的重要指标之一；②FCM检测ITP患者血小板表面PAIg敏感性好、特异性高，适用于临床，对ITP的诊断及预后评价有较好的实用价值；③初发型ITP患者以PAIgG和PAIgA增高为主，预后较好；复发型以PAIgM增高为主，预后较差，易复发。     

Storage of platelets: effects associated with high platelet content in platelet storage containers

Background

A major problem associated with platelet storage containers is that some platelet units show a dramatic fall in pH, especially above certain platelet contents. The aim of this study was a detailed investigation of the different in vitro effects occurring when the maximum storage capacity of a platelet container is exceeded as compared to normal storage.

Materials and methods

Buffy coats were combined in large-volume containers to create primary pools to be split into two equal aliquots for the preparation of platelets (450–520×10⁹ platelets/unit) in SSP+ for 7-day storage in two containers (test and reference) with different platelet storage capacity (n=8).

Results

Exceeding the maximum storage capacity of the test platelet storage container resulted in immediate negative effects on platelet metabolism and energy supply, but also delayed effects on platelet function, activation and disintegration.

Conclusion

Our study gives a very clear indication of the effects in different phases associated with exceeding the maximum storage capacity of platelet containers but throw little additional light on the mechanism initiating those negative effects. The problem appears to be complex and further studies in different media using different storage containers will be needed to understand the mechanisms involved.     

Inverse relationships between coronary blood flow obstruction and platelet reactivity in stable angina pectoris

This study investigates relationships between platelet reactivity and coronary blood flow obstruction in stable angina pectoris. Consented were 36 patients with single-vessel disease. The subjects were divided into two groups. One group (n?=?14) had less severe (<?=?80%) and the second group (n?=?22) had severe coronary flow impairment (90%). Before elective coronary angiography platelet in vitro reactivity in venous whole blood was determined using a flow cytometry technique. A thrombin-receptor activating peptide (TRAP-6) (0.77 and 0.06?g/l) and ADP (8.5 and 1.7?µmol/l) were used to activate platelets. The number of fibrinogen positive cells (%) i.e., activated platelets after stimulation was employed as experimental parameter. Less severe flow obstruction was associated with more reactive platelets. When stimulating with 0.77?g/l TRAP-6 the number of activated platelets was 64?±?15 (SD)%. The corresponding value for the group with severe flow obstruction was 40?±?17(SD)%. The difference is significant (P?<?0.001). 0.06?g/l TRAP-6 yielded similar results (P?<?0.01). Also when using 8.5?µmol/l ADP to challenge platelets less severe flow obstruction was associated with enhanced reactivity (P?<?0.01). 1.7?µmol/l ADP generated comparable results (P?<?0.05). Thus, in stable angina pectoris coronary flow obstruction is inversely related to platelet reactivity.     

Mean platelet volume is associated with myocardial perfusion defect in diabetic patients

Diabetes mellitus (DM) is considered a coronary artery risk equivalent.1 DM is associated with an increased risk of cardiovascular morbidity and mortality.2,3 DM may cause myocardial perfusion defects involving the main coronary artery and myocardial microvascular circulation. Myocardial perfusion imaging (MPI) is a useful non-invasive tool to determine whether there is a myocardial perfusion defect.4Platelet volume is a marker of platelet activation and function and is measured as mean platelet volume (MPV).5 MPV has become a prognostic factor in coronary heart disease and may eventually be accepted as a parameter of platelet activity.6 MPV is emerging as a new risk factor for vascular complications of DM of which atherothrombosis plays a crucial role.7However, to the best of our knowledge, there have been no reports in the literature to evaluate the relationship between MPV and myocardial perfusion defect using MPI in patients with diabetes. Our aim was to evaluate whether there was a relationship between myocardial perfusion defect using myocardial perfusion scintigraphy and MPV in selected diabetic patients.