A synthesis approach of mouse studies to identify genes and proteins in arterial thrombosis and bleeding.

Antithrombotic therapies reduce cardiovascular diseases by preventing arterial thrombosis and thromboembolism, but at expense of increased bleeding risks. Arterial thrombosis studies using genetically modified mice have been invaluable for identification of new molecular targets. Because of low sample sizes and heterogeneity in approaches or methodologies, a formal meta-analysis to compare studies of mice with single-gene defects encountered major limitations. To overcome these, we developed a novel synthesis approach to quantitatively scale 1514 published studies of arterial thrombus formation (in vivo and in vitro), thromboembolism, and tail-bleeding of genetically modified mice. Using a newly defined consistency parameter (CP), indicating the strength of published data, comparisons were made of 431 mouse genes, of which 17 consistently contributed to thrombus formation without affecting hemostasis. Ranking analysis indicated high correlations between collagen-dependent thrombosis models in vivo (FeCl3 injury or ligation/compression) and in vitro. Integration of scores and CP values resulted in a network of protein interactions in thrombosis and hemostasis (PITH), which was combined with databases of genetically linked human bleeding and thrombotic disorders. The network contained 2946 nodes linked to modifying genes of thrombus formation, mostly with expression in megakaryocytes. Reactome pathway analysis and network characteristics revealed multiple novel genes with potential contribution to thrombosis/hemostasis. Studies with additional knockout mice revealed that 4 of 8 (Apoe, Fpr2, Ifnar1, Vps13a) new genes were modifying in thrombus formation. The PITH network further: (i) revealed a high similarity of murine and human hemostatic and thrombotic processes and (ii) identified multiple new candidate proteins regulating these processes

A Novel Small Molecule 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose Mimics the Antiplatelet Actions of Insulin

BACKGROUND: We have shown that 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose (α-PGG), an orally effective hypoglycemic small molecule, binds to insulin receptors and activates insulin-mediated glucose transport. Insulin has been shown to bind to its receptors on platelets and inhibit platelet activation. In this study we tested our hypothesis that if insulin possesses anti-platelet properties then insulin mimetic small molecules should mimic antiplatelet actions of insulin. PRINCIPAL FINDINGS: Incubation of human platelets with insulin or α-PGG induced phosphorylation of insulin receptors and IRS-1 and blocked ADP or collagen induced aggregation. Pre-treatment of platelets with α-PGG inhibited thrombin-induced release of P-selectin, secretion of ATP and aggregation. Addition of ADP or thrombin to platelets significantly decreased the basal cyclic AMP levels. Pre-incubation of platelets with α-PGG blocked ADP or thrombin induced decrease in platelet cyclic AMP levels but did not alter the basal or PGE(1) induced increase in cAMP levels. Addition of α-PGG to platelets blocked agonist induced rise in platelet cytosolic calcium and phosphorylation of Akt. Administration of α-PGG (20 mg kg(-1)) to wild type mice blocked ex vivo platelet aggregation induced by ADP or collagen. CONCLUSIONS: These data suggest that α-PGG inhibits platelet activation, at least in part, by inducing phosphorylation of insulin receptors leading to inhibition of agonist induced: (a) decrease in cyclic AMP; (b) rise in cytosolic calcium; and (c) phosphorylation of Akt. These findings taken together with our earlier reports that α-PGG mimics insulin signaling suggest that inhibition of platelet activation by α-PGG mimics antiplatelet actions of insulin

Relation between energy production and adenine nucleotide metabolism in human blood platelets

The relation between ATP production and adenine nucleotide metabolism was investigated in human platelets which were starved by incubation in glucose-free, CN−-containing medium and subsequently incubated with different amounts of glucose. In the absence of mitochondrial energy production (blocked by CN−) and glycogen catabolism (glycogen almost completely consumed during starvation), lactate production increased proportionally with increasing amounts of glucose. The generated ATP was almost completely consumed in the various ATP-consuming processes in the cell except for a fixed portion (about 7%) that was reserved for restoration of the adenylate energy charge. During the first 10 min after glucose addition, the adenine nucleotide pool remained constant. Thereafter, when the glycolytic flux, measured as lactate formation, was more than 3.5 μmol · min−1 · 10−11 cells, the pool increased slightly by resynthesis from hypoxanthine-inosine and then stabilized; at a lower flux the pool decreased and metabolic ATP and energy charge declined to values found during starvation. Between moments of rising and falling adenylate energy charges, periods of about 10 min remained in which the charge was constant and ATP supply and demand had reached equilibrium. This enabled comparison between the adenylate energy charge and ATP regeneration velocity. A linear relation was obtained for charge values between 0.4 and 0.85 and ATP regeneration rates between 0.6 and 3.5 ATP equiv. · min−1 · 10−11 cells. These data indicate that in starved platelets ATP regeneration velocity and energy charge are independent and that each appears to be subject to the availability of extracellular substrate

Na+/H+ exchange and Ca2+ influx

AbstractCell stimulation raises the cytosolic free Ca2+ concentration, [Ca2+]i, and induces activation of Na+/H+ exchange which raises the cytosolic pH, pHj. Recent studies have addressed the question whether Na+/H+ exchange plays a role in Ca2+ influx and, specifically, whether a rise in pHi alone suffices to open Ca2+ channels in the plasma membrane. Artificial cytosolic alkalinization can induce Ca2+ uptake across the plasma membrane of endothelial cells, lymphocytes and smooth muscle cells. Furthermore, inhibition of Na+/H+ exchange reduces aganist-induced Ca2+ influx in endothelial cells and platelets which supports the concept that pHi may regulate the opening of Ca2+ channels in the plasma membrane. Although these findings argue in favour of a role of Na+/H+ exchange in Ca2+ influx, the onset of pHi and Ca2+ rises, measured with fluorescent indicators, suggests that the increase in [Ca2+]i distinctly precedes the increase in pHi. This challenges the concept that alkalinization per se is a sufficient signal for the opening of Ca2+ channels in the plasma membrane

Metabolic energy is required in human platelets at any stage during optical aggregation and secretion

The relationship between metabolic energy and platelet aggregation and secretion was investigated by sudden exhaustion of the cell energy content after these platelet responses had been initiated. In normal platelets, optical aggregation was at any stage susceptible to energy exhaustion, whereas single platelet disappearance and secretion were hardly affected. Prelowering the platelet energy content, while preserving the adenylate energy charge, made both optical aggregation and the secretion from dense, α- and acid hydrolase-containing granules susceptible to energy exhaustion, but single platelet disappearance was not affected. Complete arrest of secretion occurred when the energy content had fallen below 3–3.5 μmol ATP equivalents (ATPeq)/1011 platelets, while optical aggregation was interrupted below 2–2.5 μmol ATPeq/1011 platelets. At any stage of optical aggregation and the three secretion responses, the dependence on energy remained the same, indicating a tight coupling between these functions and metabolic energy, which held during the entire responses

Alterations in 32P-labelled intermediates during flux activation of human platelet glycolysis

Using a newly developed isotopic tracer technique for the measurement of 32P-labelled intermediates in glycolysis and nucleotide metabolism in platelets, we studied the variations in 32P-labelled intermediates during activation of the glycolytic flux by cyanide and platelet-activating agents. The major variations occured in [32P]Fru-1,6-P2, dihydroxy acetone phosphate, ATP and Pi. There was a quantitative covariance between the increase in lactate production and the rise in [32P]Fru-1,6-P2 induced by different platelet-activating agents. In contrast, cyanide induced weaker activation of the flux and greater accumulation of [32P]Fru-1,6-P2. Variations in 32P-labelled intermediates were apparent 5 s after flux activation, but the major changes in [32P]Fru-1,6-P2 occurred much later and fell in periods in which a constant lactate formation was maintained. The cyanide-induced changes in 32P-labelled intermediates depended on the extracellular level of glucose, showing a predominant ATP → Pi conversion in glucose-depleted medium that shifted to an ATP → Fru-1,6-P2 conversion at excess glucose. At about 40 μM glucose, flux activation occured without major changes in [32P]Fru-1,6-P2, dihydroxy acetone phosphate and Pi, with only a small fall in [32P]ATP. The data provide evidence for a role of the aldolase reaction in flux control and demonstrate rapid changes in Fru-1,6-P2 and ATP during flux activation with an additiona

Human platelet 6-phosphofructokinase . Relation between inhibition by Mg · ATP2 - and cooperativity towards fructose 6-phosphate and investigations on the formation of a ternary complex

Human platelet 6-phosphofructokinase (EC 2.7.1.11) shows cooperativity towards Fru-6-P and is allosterically inhibited by high Mg . ATP2− concentrations. No relation could be demonstrated between the cooperativity towards Fru-6-P and the inhibition by Mg . ATP2−. Increasing the concentrations of Mg . ATP2− only raised the apparent Km values for Fru-6-P, but did not change the Hill constants. a possible formation of a Mg . ATP2− : enzyme . Fru-6-P complex during catalysis was investigated. Our calculations suggest that such a ternary complex is indeed formed during the reaction

Influence of Mg2+, ITP4- and ATP4- on human platelet phosphofructokinase

In the reaction catalyzed by human platelet phosphofructokinase, Mg2+ is required for optimal activity. Maximal MG2− activation was obtained at [Mg2+] = [MgITP2−] or higher. At high MgATP2− concentrations there is an increase in the allosteric inhibition by ATP4−