Carol Dangelmaier

Molecular basis for ADP-induced platelet activation, I : Evidence for three distinct ADP receptors on human platelets

Acting through cell surface receptors, ADP activates platelets resulting in shape change, aggregation, thromboxane A2 production, and release of granule contents. ADP also causes a number of intracellular events including inhibition of adenylyl cyclase, mobilization of calcium from intracellular stores, and rapid calcium influx in platelets. However, the receptors that transduce these events remain unidentified and their molecular mechanisms of action have not been elucidated. The receptor responsible for the actions of ADP on platelets has been designated the P2T receptor. In this study we have used ARL 66096, a potent antagonist of ADP-induced platelet aggregation, and a P2X ionotropic receptor agonist, α,β-methylene adenosine 5′-triphosphate, to distinguish the ADP-induced intracellular events. ARL 66096 blocked ADP-induced inhibition of adenylyl cyclase, but did not affect ADP-mediated intracellular calcium increases or shape change. Both ADP and 2-methylthio-ADP caused a 3-fold increase in the level of inositol 1,4,5-trisphosphate over control levels which peaked in a similar fashion to the Ca2+ transient. The increase in inositol 1,3,4-trisphosphate was of similar magnitude to that of inositol 1,4,5-trisphosphate. α,β-Methylene adenosine 5′-triphosphate did not cause an increase in either of the inositol trisphosphates. These results clearly demonstrate the presence of two distinct platelet ADP receptors in addition to the P2X receptor: one coupled to adenylyl cyclase and the other coupled to mobilization of calcium from intracellular stores through inositol trisphosphates.

Tec Kinases Mediate Sustained Calcium Influx via Site-specific Tyrosine Phosphorylation of the Phospholipase Cγ Src Homology 2-Src Homology 3 Linker

Tyrosine phosphorylation of phospholipase Cγ2 (PLCγ2) is a crucial activation switch that initiates and maintains intracellular calcium mobilization in response to B cell antigen receptor (BCR) engagement. Although members from three distinct families of non-receptor tyrosine kinases can phosphorylate PLCγ in vitro, the specific kinase(s) controlling BCR-dependent PLCγ activation in vivo remains unknown. Brutons tyrosine kinase (Btk)-deficient human B cells exhibit diminished inositol 1,4,5-trisphosphate production and calcium signaling despite a normal inducible level of total PLCγ2 tyrosine phosphorylation. This suggested that Btk might modify a critical subset of residues essential for PLCγ2 activity. To evaluate this hypothesis, we generated site-specific phosphotyrosine antibodies recognizing four putative regulatory residues within PLCγ2. Whereas all four sites were rapidly modified in response to BCR engagement in normal B cells, Btk-deficient B cells exhibited a marked reduction in phosphorylation of the Src homology 2 (SH2)-SH3 linker region sites, Tyr753 and Tyr759. Phosphorylation of both sites was restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation of the PLCγ2 carboxyl-terminal sites, Tyr1197 and Tyr1217, was unaffected by the absence of functional Btk. Together, these data support a model whereby Btk/Tec kinases control sustained calcium signaling via site-specific phosphorylation of key residues within the PLCγ2 SH2-SH3 linker.

Role of Phosphoinositide 3-Kinase β in Glycoprotein VI-mediated Akt Activation in Platelets

Glycoprotein (GP) VI is a critical platelet collagen receptor. Phosphoinositide 3-kinase (PI3K) plays an important role in GPVI-mediated platelet activation, yet the major PI3K isoforms involved in this process have not been identified. In addition, stimulation of GPVI results in the activation of Akt, a downstream effector of PI3K. Thus, we investigated the contribution of PI3K isoforms to GPVI-mediated platelet activation and Akt activation. A protein kinase C inhibitor GF 109203X or a P2Y12 receptor antagonist AR-C69931MX partly reduced GPVI-induced Akt phosphorylation. Platelets from mice dosed with clopidogrel also showed partial Akt phosphorylation, indicating that GPVI-mediated Akt phosphorylation is regulated by both secretion-dependent and -independent pathways. In addition, GPVI-induced Akt phosphorylation in the presence of ADP antagonists was completely inhibited by PI3K inhibitor LY294002 and PI3Kβ inhibitor TGX-221 indicating an essential role of PI3Kβ in Akt activation directly downstream of GPVI. Moreover, GPVI-mediated platelet aggregation, secretion, and intracellular Ca2+ mobilization were significantly inhibited by TGX-221, and less strongly inhibited by PI3Kα inhibitor PIK75, but were not affected by PI3Kγ inhibitor AS252424 and PI3Kδ inhibitor IC87114. Consistently, GPVI-induced integrin αIIbβ3 activation of PI3Kγ−/− and PI3Kδ−/− platelets also showed no significant difference compared with wild-type platelets. These results demonstrate that GPVI-induced Akt activation in platelets is dependent in part on Gi stimulation through P2Y12 receptor activation by secreted ADP. In addition, a significant portion of GPVI-dependent, ADP-independent Akt activation also exists, and PI3Kβ plays an essential role in GPVI-mediated platelet aggregation and Akt activation.

Measurement of arachidonic acid liberation in thrombin-stimulated human platelets. Use of agents that inhibit both the cyclooxygenase and lipoxygenase enzymes.

The formation of radiolabelled oxygenated products of arachidonic acid in thrombin-stimulated, [3H]arachidonic acid-prelabelled human platelets is inhibited in a concentration-dependent manner by BW 755C (3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline) or propyl gallate, both of which are combined inhibitors of lipoxygenase and cyclooxygenase. These compounds do not inhibit the thrombin-induced decrease in the radioactivity of platelet phospholipids but, instead, allow the accumulation of free radiolabelled arachidonic acid. Thrombin causes an increase in the levels of free, endogenous palmitic, stearic, oleic, linoleic and arachidonic acids of up to 10 nmol/10(9) platelets. In the presence of BW 755C or propyl gallate, further increases in the level of free arachidonic acid, of 20-50 nmol/10(9) platelets, occur. The enzyme inhibitors do not affect the accumulation of the other free fatty acids. The increase in arachidonic acid is optimal at 1 U/ml thrombin and 60% complete by 1 min at 37 degrees C. In the platelets from eight donors, the average increases in free fatty acids (in nmol/10(9) platelets) induced by 5 U/ml thrombin in 5 min at 37 degrees C in the presence of 100 microM BW 755C were 1 for linoleic acid, 3.6 for oleic acid, 4.5 for palmitic acid, 7.6 for stearic acid and 32.0 for arachidonic acid.

Determination of acid hydrolases in human platelets

Abstract Most acid hydrolases in platelets are determined by methods developed for other tissues. Therefore, we have established the optimal conditions for the measurements of 13 acid glycosidases, aryl phosphatase, aryl sulfatase, and β-glycerophosphatase in crude and dialyzed lysates of human platelets. The substrates used were the glycosides, phosphates and sulfates of p-nitrophenol and 4-methylumbelliferone, phenolphthalein-β-glucuronide, p-nitrocatechol sulfate and β-glycerophosphate. The pH optimum for each enzyme activity did not depend on the type of substrate, whereas Km and V could vary as much as 100-fold. For most of the acid hydrolases, V was higher than the enzyme level determined in the usual way, with the highest substrate concentration obtainable, because this concentration was close to Km. The possible existence of low molecular weight enzyme inhibitors in the lysates was tested by comparing Km at two different lysate concentrations and by comparing the enzyme level in crude lysate with that obtained after dialysis. Only inhibitors for α- and β-galactosidase and aryl sulfatase were found. α-Glucosidase had two pH optima, one at pH 4.1 and the other at 6.0. p-Nitrocatechol sulfatase had a nonlinear time course, similar to the enzyme present in liver. Where possible, Km and V were determined in both citrate-phosphate and acetate buffers, and differences were found for β-fucosidase, α-galactosidase, β-glucosidase, β-N-acetylglucosaminidase, and α-mannosidase. The sensitivity and accuracy of the fluorophor-based assays with 4-methylumbelliferone-containing substrates were markedly better than the chromophore-based assays. In contrast, the determination of β-glycerophosphatase was particularly inaccurate due to formation of large amounts of phosphate from endogenous sources in the lysates. The following acid hydrolases activities were found in human platelet lysates (listed in descending order of total levels): Aryl phosphatase, β-N-acetylglucosaminidase, β-glucuronidase, β-galactosidase, α-mannosidase, aryl sulfatase (p-nitrocatechol substrate), α-arabinosidase, β-N-acetylgalactosaminidase, α-galactosidase, α-fucosidase, β-fucosidase, β-glucosidase, and α-glucosidase (pH 4.1). β-Cellubiosidase and α-xylosidase were not present in the lysates.

[17] Measurement of secretion of serotonin

Publisher Summary The principle of the measurement of secretion of serotonin states that the radiolabeled serotonin that has been taken up equilibrates rapidly and totally with the endogenous serotonin and, therefore, behaves functionally as endogenous serotonin. This allows the quantitation of serotonin by a simple measurement of the total radioactivity. However, this ability also poses difficulties for secretion experiments because the secreted serotonin will rapidly re-enter the same platelets that secreted it. In the procedures outlined in this chapter, imipramine is added to a platelet suspension before secretion is induced. Alternatively, serotonin forms a fluorophore with O-phthalaldehyde (OPT), which can be easily detected down to 22 pmol under optimal fluorimetric conditions. The content of serotonin varies in platelets from different species, from 300 μ mol/10 11 human platelets to 6,930 μ mol/10 11 rabbit platelets. The OPT method described in the chapter is, therefore, suitable for human platelet suspensions of 1–3 × 10 8 cells/mL.

ADP stimulates IP3 formation in human platelets

Aspirinated human platelets labeled with 32PO4 showed a 1.7‐fold increase in [32P]IP3 when stimulated with ADP. ADP‐stimulated mobilization of internal Ca2+ and phosphorylation of myosin were enhanced in the presence of extracellular Ca2+ but the increase in IP3 was not significantly affected by external Ca2+. The Ca2+ ionophore, ionomycin, elevated internal Ca2+ and induced myosin phosphorylation without a detectable change in IP3. These results indicate that the mechanism of ADP stimulation of human platelets is similar to that of other platelet agonists and supports the theory that IP3 functions to liberate internal Ca2+.

A novel histidine tyrosine phosphatase, TULA-2, associates with Syk and negatively regulates GPVI signaling in platelets

T-cell ubiquitin ligand-2 (TULA-2) is a recently discovered histidine tyrosine phosphatase thought to be ubiquitously expressed. In this work, we have investigated whether TULA-2 has a key role in platelet glycoprotein VI (GPVI) signaling. This study indicates that TULA-2 is expressed in human and murine platelets and is able to associate with Syk and dephosphorylate it. Ablation of TULA-2 resulted in hyperphosphorylation of Syk and its downstream effector phospholipase C-γ2 as well as enhanced GPVI-mediated platelet functional responses. In addition, shorter bleeding times and a prothrombotic phenotype were observed in mice lacking TULA-2. We therefore propose that TULA-2 is the primary tyrosine phosphatase mediating the dephosphorylation of Syk and thus functions as a negative regulator of GPVI signaling in platelets.

Adenine nucleotide metabolism of blood platelets: X. Formaldehyde stops centrifugation-induced secretion after A23187-stimulation and causes breakdown of metabolic ATP

A23187 induced shape change, aggregation and secretion of platelets in plasma. When rapid cooling was used to stop secretion and centrifugation to separate the cells from the medium, maximal amounts of storage ATP plus ADP and preadsorbed [14C]serotonin were found in the supernatant immediately (less than 5 s) after A23187 addition. These results suggested that A23187 could cause shape change and aggregation through secreted ADP and not directly. When secretion was stopped with chilling and formaldehyde treatment before centrifugation, the secreted substances appeared after a lag of 60-120 s, i.e. after shape change was terminated and aggregation was well on its way. These two platelet responses thus seemed to be independent of secretion and induced directly by A23187. The absence of a lag period when secretion was stopped by chilling alone was thought to be due to centrifugation-induced secretion of platelets conditioned by A23187. Formaldehyde completely inhibited centrifugation-induced secretion. At 37 degrees C, formaldehyde caused rapid breakdown of metabolic ATP in platelets with a pattern dependent on the formaldehyde concentration: Below 50 mM, ATP was converted to inosine plus hypoxanthine via ADP, AMP and IMP and the adenylate energy charge was preserved. Above 100 mM, AMP was the end product with a drastic reduction in the adenylate energy charge. These changes were not due to lysis of the platelets, but were apparently caused by an formaldehyde-induced increase in cellular ATP consumption. Platelet secretion is usually associated with a conversion of metabolic ATP to hypoxanthine. Formaldehyde had to be used to stop secretion and since it caused breakdown of ATP, additional smaples were taken out for nucleotide determination during stirring of platelet-rich plasma with A23187. It was found that metabolic ATP was converted to inosine plus hypoxanthine only during the secretion step.

Differential phosphorylation of myosin light chain (Thr)18 and (Ser)19 and functional implications in platelets

Summary. Background:  Myosin IIA is an essential platelet contractile protein that is regulated by phosphorylation of its regulatory light chain (MLC) on residues (Thr)18 and (Ser)19 via the myosin light chain kinase (MLCK).