Gertie Gorter

Platelet Inhibition by Insulin Is Absent in Type 2 Diabetes Mellitus

Objective—ADP-induced P2y12 signaling is crucial for formation and stabilization of an arterial thrombus. We demonstrated recently in platelets from healthy subjects that insulin interferes with Ca2+ increases induced by ADP-P2y1 contact through blockade of the G-protein Gi, and thereby with P2y12-mediated suppression of cAMP. Methods and Results—Here we show in patients with type 2 diabetes mellitus (DM2) that platelets have lost responsiveness to insulin leading to increased adhesion, aggregation, and procoagulant activity on contact with collagen. Using Ser473 phosphorylation of protein kinase B as output for insulin signaling, a 2-fold increase is found in insulin-stimulated normal platelets, but in DM platelets there is no significant response. In addition, DM2 platelets show increased P2y12-mediated suppression of cAMP and decreased P2y12 inhibition by the receptor antagonist AR-C69931MX. Conclusion—The loss of responsiveness to insulin together with increased signaling through P2y12 might explain the hyperactivity of platelets in patients with DM2.

The phospholipid composition and cholesterol content of platelet‐derived microparticles: a comparison with platelet membrane fractions

Summary.  Background: The processes that govern the distribution of molecules between platelets and the microparticles (MP) they release are unknown. Certain proteins are sorted selectively into MP, but lipid sorting has not been studied. Objectives: To compare the phospholipid composition and cholesterol content of platelet‐derived MP obtained with various stimuli with that of isolated platelet membrane fractions. Methods: Washed platelets from venous blood of healthy individuals (n = 6) were stimulated with collagen, thrombin, collagen plus thrombin, or A23187. Platelet activation, MP release and antigen exposure were assessed by flow cytometry. MPs were isolated by differential centrifugation. Platelet plasma‐, granule‐ and intracellular membranes were isolated from platelet concentrates (n = 3; 10 donors each) by pressure homogenization and Percoll density gradient fractionation. The phospholipid composition and cholesterol content of MPs and membrane fractions were analyzed by high performance thin layer chromatography. Results: The phospholipid composition of MPs was intermediate compared with that of platelet plasma‐ and granule membranes, and differed significantly from that of intracellular membranes. There were small but significant differences in phospholipid composition between the MPs produced by the various agonists, which paralleled differences in P‐selectin exposure in case of the physiological agonists collagen, thrombin, or collagen plus thrombin. The cholesterol content of MPs tended to be higher than that of the three‐platelet membrane fractions. Conclusions: Regarding its phospholipid content, the MP membrane is a composite of the platelet plasma‐ and granule membranes, showing subtle differences depending on the platelet agonist. The higher cholesterol content of MPs suggests their enrichment in lipid rafts.

LDLs increase the exposure of fibrinogen binding sites on platelets and secretion of dense granules.

Because previous studies show that lipoproteins affect platelet aggregation, we studied the effect of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) on the binding of fibrinogen, which mediates platelet-platelet contact. Neither LDL nor HDL induced 125I-fibrinogen binding at concentrations up to 2 g protein/L. In contrast, platelets stimulated with 10 mumol/L ADP bound 63 734 +/- 2453 molecules of fibrinogen per platelet. A 5-minute preincubation with LDL (0.5 to 2 g/L protein) induced a dose-dependent increase to 91 307 +/- 2164 molecules of fibrinogen per platelet at 1.5 g/L, which is in the range found after optimal stimulation with alpha-thrombin. The increased fibrinogen binding in the presence of LDL resulted in faster aggregation with a 16% increase in single platelet disappearance and a faster optical aggregation at 5 mumol/L ADP and 1.5 g protein/L LDL. Inhibition of prostaglandin G2/H2-thromboxane A2 formation with indomethacin (30 mumol/L) did not change the stimulation by LDL. In contrast, modification of lysine residues of LDL, which is known to prevent specific binding to platelets, completely abolished the effect of LDL. Under the same conditions HDL did not change fibrinogen binding or aggregation. LDL also enhanced alpha-thrombin-induced [14C]serotonin secretion, but this property was not affected by lysine modification of LDL. These data indicate that LDL enhances platelet aggregation by stimulating the mechanisms that control exposure of fibrinogen binding sites on the glycoprotein IIB/IIIA complex via a mechanism that differs from the effect of LDL on secretion.

Enhancement of platelet functions by low density lipoproteins.

Platelet suspensions, that secreted about 50% of their dense granule contents upon stimulation with alpha-thrombin, showed a dose-dependent increase in secretion after 30 min preincubation with 0.5-3.0 g low density lipoprotein (LDL) protein/1. A 1-5 min preincubation had no effect. The enhancement by LDL only occurred at about 20% secretion or more, indicating that a minimal degree of activation was required for LDL to become effective. Lysine-modified LDL was equally effective as native LDL. The effect of LDL on secretion was accompanied by enhanced thromboxane B2 formation caused by stimulation of the liberation of arachidonate from phosphatidylcholine and/or phosphatidylinositol. However, when thromboxane formation was inhibited or the prostaglandin H2-thromboxane A2-receptor was blocked, LDL remained a potent stimulator of the secretion response. Thus, LDL enhances platelet secretion by a thromboxane A2-dependent and a thromboxane A2-independent mechanism via an effect that is independent of specific binding sites on the platelet.

Effect of Oxidation on the Platelet-Activating Properties of Low-Density Lipoprotein

Objective—Because of the large variation in oxidizing procedures and susceptibility to oxidation of low-density lipoprotein (LDL) and the lack in quantification of LDL oxidation, the role of oxidation in LDL–platelet contact has remained elusive. This study aims to compare platelet activation by native LDL (nLDL) and oxidized LDL (oxLDL). Methods and Results—After isolation, nLDL was dialyzed against FeSO4 to obtain LDL oxidized to well-defined extents varying between 0% and >60%. The oxLDL preparations were characterized with respect to their platelet-activating properties. An increase in LDL oxidation enhances platelet activation via 2 independent pathways, 1 signaling via p38MAPK phosphorylation and 1 via Ca2+ mobilization. Between 0% and 15% oxidation, the p38MAPK route enhances fibrinogen binding induced by thrombin receptor (PAR-1)-activating peptide (TRAP), and signaling via Ca2+ is absent. At >30% oxidation, p38MAPK signaling increases further and is accompanied by Ca2+ mobilization and platelet aggregation in the absence of a second agonist. Despite the increase in p38MAPK signaling, synergism with TRAP disappears and oxLDL becomes an inhibitor of fibrinogen binding. Inhibition is accompanied by binding of oxLDL to the scavenger receptor CD36, which is associated with the fibrinogen receptor, &agr;IIb&bgr;3. Conclusion—At >30% oxidation, LDL interferes with ligand binding to integrin &agr;IIb&bgr;3, thereby attenuating platelet functions.

Development of Platelet Inhibition by cAMP during Megakaryocytopoiesis

Prostacyclin is a potent inhibitor of agonist-induced Ca2+ increases in platelets, but in the megakaryocytic cell line MEG-01 this inhibition is absent. Using human megakaryocytic cell lines representing different stages in megakaryocyte (Mk) maturation as well as stem cells and immature and mature megakaryocytes, we show that the inhibition by prostacyclin develops at a late maturation stage shortly before platelets are formed. This late appearance is not caused by insufficient cAMP formation or absent protein kinase A (PKA) activity in immature cells. Instead, the appearance of Ca2+ inhibition by prostacyclin is accompanied by a sharp increase in the expression of the catalytic subunit of PKA (PKA-C) but not by changes in the expression of the PKA-regulatory subunits Iα/β, IIα, and IIβ. Overexpression of PKA-C in the megakaryocytic cell line CHRF-288-11 potentiates the Ca2+ inhibition by prostacyclin. Thus, up-regulation of PKA-C appears to be a key step in the development of Ca2+ inhibition by prostacyclin in platelets.

Kinetic analysis of α-granule secretion by platelets. A methodological report

Abstract A method is described for the kinetic measurement of α-granule secretion by platelets. The method uses formaldehyde as a secretion-blocking reagent. This treatment alters the antigenecity of β-thromboglobulin but not of Platelet Factor 4, both measured with commercially available reagents. Evidence is shown that this formaldehyde effect does not alter the secretion kinetics when the data are expressed as a percentage of a similarly treated reference sample. The method shows that following stimulation with thrombin or A 23187 α-granule secretion is much slower than dense granule secretion.

ATP‐ADP compartmentation in storage pool deficient platelets: correlation between granule‐bound ADP and the bleeding time

Platelets contain two major compartments of ATP and ADP, the cytosol and the dense granules. We separated the two compartments by controlled digitonin‐induced cell lysis and measured both fractions directly in the platelets of 16 patients with storage pool deficiency. The total contents of ATP and ADP in the platelets of these patients was significantly lower than in normal controls. This was primarily caused by decreased amounts of ATP and ADP in the granule compartment, but also cytosolic ADP was low in these patients. In contrast, cytosolic ATP was not significantly decreased. The lower the amount of granule‐bound ATP and ADP, the longer was the bleeding time in these patients. The best correlation was found with granule ADP. These data may indicate that ADP stored in platelet dense granules plays a role in the arrest of bleeding.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) inhibits low density lipoprotein-induced signaling in platelets

At physiological concentrations, low density lipoprotein (LDL) increases the sensitivity of platelets to aggregation- and secretion-inducing agents without acting as an independent activator of platelet functions. LDL sensitizes platelets by inducing a transient activation of p38MAPK, a Ser/Thr kinase that is activated by the simultaneous phosphorylation of Thr180 and Tyr182 and is an upstream regulator of cytosolic phospholipase A2 (cPLA2). A similar transient phosphorylation of p38MAPK is induced by a peptide mimicking amino acids 3359–3369 in apoB100 called the B-site. Here we report that the transient nature of p38MAPK activation is caused by platelet endothelial cell adhesion molecule 1 (PECAM-1), a receptor with an immunoreceptor tyrosine-based inhibitory motif. PECAM-1 activation by cross-linking induces tyrosine phosphorylation of PECAM-1 and a fall in phosphorylated p38MAPK and cPLA2. Interestingly, LDL and the B-site peptide also induce tyrosine phosphorylation of PECAM-1, and studies with immunoprecipitates indicate the involvement of c-Src. Inhibition of the Ser/Thr phosphatases PP1/PP2A (okadaic acid) makes the transient p38MAPK activation by LDL and the B-site peptide persistent. Inhibition of Tyr-phosphatases (vanadate) increases Tyr-phosphorylated PECAM-1 and blocks the activation of p38MAPK. Together, these findings suggest that, following a first phase in which LDL, through its B-site, phosphorylates and thereby activates p38MAPK, a second phase is initiated in which LDL activates PECAM-1 and induces dephosphorylation of p38MAPK via activation of the Ser/Thr phosphatases PP1/PP2A.

Regulation of glycolytic flux in human platelets relation between energy production by glyco(geno)lysis and energy consumption

Abstract 1. 1. The relation between energy production and energy consumption was studied in resting human platelets. Platelets were gel filtered in a medium with 1 mM CN − and without glucose and then incubated at 37°C for 60 min. Platelet glycogen was converted into lactate at a rate of 0.79 ± 0.23 (mean ± S.D., n = 6) μmol glycosyl residues/min per 10 11 cells. This conversion was linear with time for 40 min and then ceased abruptly. During a subsequent 20 min of incubation, glycogen and lactate levels remained constant, indicating a halt in energy production; the cells remained intact during this period. 2. 2. During the first 40 min, 14 C-labeled ATP and adenylate energy charge also decreased linearly with time. Thereafter, constant levels were maintained for another 20 min. 3. 3. Addition of glucose stimulated lactate production which originated primarily from endogenous glycogen at 0–50 μM glucose but which at higher glucose concentrations, originated predominantly from glucose. 4. 4. At lactate production rates of 3 μmol/min per 10 11 cells, a stable ATPm concentration and adenylate energy charge were maintained. At a lower flux, both parameters decreased and a linear relationship was observed between the decrease in ATPm level and adenylate energy charge versus lactate formation. When the lactate production was 1 μmol lactate formed/min per 10 11 cells, the adenylate energy charge fell 0.008 unit/min and ATPm level fell 1.1% of total radioactivity/min. 5. 5. From these data, the balance between production and consumption of ATPm was calculated. A decrease in production was almost completely matched by a decrease in consumption. A slight discrepancy remained which increased as the rate of ATPm formation fell. When ATPm formation was 2 μmol/min per 10 11 cells consumption exceeded production by 15 nmol ATPm/ min per 10 11 cells. The slight excess of ATPm consumption over production at suboptimal rates of ATPm production provides a tool for feedback control of ATPm-consuming processes on glycolysis and glycogenolysis.