B. Schwippert

Platelet membrane activation markers are predictive for increased risk of acute ischemic events after PTCA.

BackgroundWe wished to investigate whether platelet activation is related to the clinical outcome during the 24 hours immediately after elective percutaneous transluminal coronary angioplasty(PTCA). Methods and ResultsIn 102 patients with high-grade coronary stenosis admitted for elective PTCA, preprocedural platelet activation was characterized by flow cytometric measurement of the proteins CD62, CD63, and thrombospondin expressed on the platelet surface membrane. The prevalence of acute ischemic events during the 24 hours immediately after the procedure was then related to the pre-PTCA platelet activation status. Fifty-six patients were classified as “nonactivated,” whereas 46 patients showed an increased percentage of activated platelets. Two patients developed acute occlusion (1.96 %) and four patients high-grade restenosis (3.92%), as confirmed by second-look coronary angiography. All events occurred in patients classified as “activated” (six of 46, or 13%). None of these patients received P-blocker medication, which was associated with lower expression of platelet membrane activation markers. In the nonactivated patient group, no clinical events were found (0 of 56, or 0%). This difference in prevalenceD is significant (P=0.007). ConclusionsWe conclude that analysis of platelet membrane activation markers may help to predict an increased risk of acute ischemic events after angioplasty.

Exposure of Adhesion Molecules on Activated Platelets in Patients with Newly Diagnosed IDDM Is Not Normalized by Near-Normoglycemia

It has been suggested that platelet hyperactivity contributes to the early evolution of diabetic vascular disease per se. This study directly evaluates the level of intravascular platelet activation in newly diagnosed IDDM patients before and after tight metabolic control. Platelet activation was determined by the Duesseldorf-III flow cytometry assay in 21 recent-onset hyperglycemic IDDM patients before insulin, after 3 days of treatment with intravenous insulin, and after 14 and 60 days of intensified conventional insulin therapy. The intravasal platelet activation status was quantified by the percentage of platelets exposing the activation-dependent molecules CD62 (P-selectdn), thrombospondin (TSP), and CD63 (GP53) as well as the activated fibrinogen receptor (GPIIB/IIIA). Fifty matched normal subjects served as control subjects. Fourteen patients completed the 60-day study design. After initial recompensation, near-normoglycemic control was achieved after 14 days (fasting blood glucose, 117.0 ± 19.0 mg/dl), and the HbA1 concentration was 7.6 ± 1.2% after 60 days. CD62+ (4.0 ± 4.5%), TSP+ (2.0 ± 1.8%), CD63+ (11.0 ± 7.0%), and activated-GPIIB/IIIA+ (7.6 ± 7.7%) platelet levels were initially 5, 3.3, 5.7, and 2.8 times higher than the mean level of normal. There was no correlation with any of the nearly normalized metabolic parameters. Thus, more activated platelets circulate in newly diagnosed IDDM patients, which supports the assumption of a prethrombotic condition even in disease stages without apparent vascular damage. Metabolic control does not appear to be successful in attenuating activated cellular hemostasis.

Large platelets continue to circulate in an activated state after myocardial infarction

Abstract This study was intended to investigate the actual platelet activation status after an acute coronary event. The activation status of circulating platelets was assayed directly by measuring the membrane activation markers CD62 and CD63 with the Düssel‐dorf III flow cytometry test in 22 patients with the diagnosis of acute myocardial infarction during the 48‐h observation period following the acute event. The number of activated, marker‐positive sample platelets was significantly increased in the post‐MI patients: CD62: 5·8%× 2·25±1 vs. 3·5%× 2·32±1, P≤ 0·05; CD63: 18·7%× 1·77±1 vs. 4·6%× 2·16±1, P≤ 0·00·1. The platelet volume and count were concomitantly increased (12·1 ± 2·4 fl/ 236 ± 90 times 103μl‐1 compared to 8·3 ± 1·6 fl/187 ± 42 times 103μl‐1) in the control group. Particularly large platelets were identified as being activated documented by the exponential increase in the difference in CD63‐binding sites per sample platelet above the 90%‐percentile and below the 10%‐percentile of the volume distribution: Δ+ 1341 ± 903 (MI patients) vs. Δ+ 276 ± 126 (controls), P≤ 0·00·1. Significant creatine kinase elevation and decrease in platelet count was found in the non‐survivor subset (n= 5). We conclude that predominantly large platelets continue to circulate in an activated state after MI. This study provides direct evidence that the assumption of an increased thrombotic potential becomes operative in vivo in MI patients. Besides CK elevation and decrease in platelet count this might possibly constitute a prognostic factor for the short‐term outcome of the patients.

Increased GPIIB/IIIA expression and altered DNA‐ploidy pattern in megakaryocytes of diabetic BB‐rats

Abstract. Hyperactive platelets contribute to angio‐pathic complications in diabetes mellitus. It is unclear whether the increased platelet function is a primary pathogenetic factor in diabetes or follows vascular injury. Increased platelet size and numbers of glyco‐protein receptors on diabetic platelets suggest that thrombopoiesis is altered in diabetes mellitus. For further support of this hypothesis we studied whether megakaryocytes are changed with regard to the DNA‐ploidy pattern and the GPIIB/IIIA expression in 10 acute diabetic (AD) and 24 insulin treated diabetic (ITD) BB rats in comparison with 22 diabetes resistant (ND) BB rats. In the AD group megakaryocyte size (P=0.035) and the modal DNA‐ploidy distribution dropped (P=0.0001) concomitant with increased TNF‐alpha activity (P=0.001). GPIIB/IIIA expression and the peripheral platelet status were unchanged. After 4 weeks of insulin substitution metabolic parameters (glucose, cholesterol, triglycer‐ides) were lowered, but remained still elevated. As compared to the AD group the modal DNA‐ploidy pattern reversed, but the relative percentage of 64n megakaryocytes increased 2.3‐fold and GPIIB/IIIA expression increased 1.6‐fold. Simultaneously, the peripheral platelet count and size increased. From these results we conclude that alterations of the megakaryocyte compartment occur at early onset of diabetes. These changes could reflect a response to increased systemic cytokine production during inflammatory islet cell destruction. The peripheral platelet thrombotic potency increased with insulin treatment. This was associated with an increase of 64n‐megakary‐ocytes with upregulated GPIIB/IIIA expression and could reflect a mitogenic effect of insulin upon the endomitotic cycle of the megakaryocytes.

Thrombospondin binds normally to glycoprotein IIIb deficient platelets

Glycoprotein IIIb (GPIV, CD36) has been proposed as the platelet receptor for thrombospondin (TSP). We found two healthy blood donors, whose platelets were shown to be GPIIIb deficient. These platelets expressed endogeneous TSP as control platelets and their binding capacity for exogeneous TSP was the same. These results indicate that GPIIIb is not the major TSP receptor on platelets. However, it is not yet possible to exclude that in GPIIIb-deficient platelets other proteins may substitute for GPIIIb in TSP binding.

Expression of Adhesion Molecules on the Surface of Activated Platelets is Diminished by PGI2-analogues and an NO (EDRF)-Donor: A Comparison Between Platelets of Healthy and Diabetic Subjects

Adhesion molecules such as P-selectin (CD 62), glycoprotein (CP) 53 (CD63) and thrombospondin play a decisive role in the thrombogenic transformation of platelets. Here we present evidence obtained using flow cytometric analysis that the PGI(2)-mimetics iloprost and taprostene, and an NO (EDRF)donor (SIN-1) are able to inhibit the expression of P-selectin, GP 53 and thrombospondin on human platelets activated by submaximal concentrations of thrombin. Since the half-maximal concentrations for inhibition of antigen expression (0.15 nM for iloprost, 3.0-5.3 nM for taprostene) are much lower than for activation of adenylate cyclase (1.4 nM for iloprost and 29.4 nM for taprostene) our data suggest that the occupation of a small number of PGI(2)-receptors is sufficient to inhibit the thrombogenic transformation and that spare PGI(2)-receptors are present on human platelets. In diabetes, the EC(50) for inhibition of expression of platelet antigens is shifted to higher concentrations suggesting that platelets from type 1 diabetic patients are partly resistant to PGI(2). Since the dose dependent increase in c-AMP by iloprost is not changed and intraplatelet c-AMP is elevated in platelets of diabetic patients, we assume that steps in the activation cascade subsequent to activation of adenylate cyclase are disturbed in diabetes.

Increased growth of human fibroblasts and arterial smooth muscle cells from diabetic patients related to diabetic serum factors and cell origin

Fibroblasts from 3 diabetic patients (DF) grew faster, resulting in higher cell counts in the stationary phase than fibroblasts from 3 age-matched healthy volunteers (NF). This difference was apparent when DF or NF were cultured in either diabetic (DS) or normal serum (NS). Diabetic serum increased growth of both DF and NF compared with normal serum. Total protein content per plate paralleled the increase of cell number per plate in relation to cell origin and serum type. DS increased growth and total protein per plate in the arterial smooth muscle cell line from a non-diabetic patient in a way similar to in DF and NF. It is concluded that increased growth of DF in vivo could result in an increased turnover of vascular cells with a shortened replicative lifespan, leading to an accumulation of basal lamina. This effect would be even further accentuated by exposure of DF to DS. Taken together with the increased protein synthesis the accelerated development of diabetic angiopathy could be the final consequence.

Increased growth stimulation of fibroblasts from diabetics by diabetic serum factors of low molecular weight

Serum factors from non-ketotic poorly controlled diabetic patients when compared to serum factors from normal subjects, stimulate growth and protein synthesis of cultured fibroblasts from diabetic patients by 25-50%. This increased growth stimulating effect of diabetic serum is mainly related to low molecular weight components (mol. wt. < 12,000 daltons), but not to insulin or glucose. These low molecular weight components of diabetic serum are effective only in combination with serum factors of a molecular weight > 12,000 daltons which are essential for initiation and continuous stimulation of cellular growth. As the growth stimulation by diabetic serum factors with a molecular weight < 12,000 daltons does not differ from comparable normal serum factors, the relevance of these serum factors (e.g. growth hormone, lipoproteins) for the increased growth stimulation of mesenchymal cells in diabetes mellitus seems to be only of limited importance. In as much as these in vitro results represent the in vivo situation, chronic exposure of vascular cells from diabetics to these serum factors could be related to the increased angiopathic risk in diabetes mellitus.

Adhesive proteins in platelet-endothelial interactions

Abstract. Adhesion molecules mediate the interaction between endothelium and platelets as well as other blood cells and the endothelium. The structure and function of some of these molecules will be reviewed and discussed. The expression of these molecules is largely affected by disease states such as hypertension, diabetes, and cardiac failure. Determination of adhesion molecules expressed on the surface of endothelial cells and platelets by cytoflowmetry enables a new approach to estimate the activity state of these cells and might be helpful to identify patients with an increased thrombotic risk.

Regulation of diabetic serum growth factors for human vascular cells by the metabolic control of diabetes mellitus

Serum factors from non-ketotic poorly controlled non-insulin-dependent diabetic patients stimulated growth and protein synthesis of human arterial smooth muscle cells and fibroblasts by 15-42%, compared to serum factors from well controlled diabetics. In contrast, the growth stimulating effect of pooled sera from well controlled diabetics did not differ from the effect of normal sera. Single sera from the same diabetics before and after improvement of the metabolic control stimulated cell growth to a similar degree as the respective pooled sera from different diabetic populations. As far as increased growth stimulation of vascular cells is related to increased angiopathic risk in diabetics, this metabolic regulation of growth factors supports the demand for a continuous optimal control of diabetic metabolism.