John D. Vickers

Elevated cholesterol in tissues of chicken embryos with hereditary myotonic muscular dystrophy.

Abstract The cholesterol content of muscle, liver, serum, and brain was examined both in embryos of chickens with hereditary muscular dystrophy and in normal control embryos between 9 and 16 days in ovo. The superficial pectoral muscles, which are severely affected by muscular dystrophy later in development, have a significantly higher cholesterol content than those of normal embryos at all ages examined. In contrast, the cholesterol content of thigh muscles that are not severely affected by the dystrophic process is no different from that of control embryos. The cholesterol contents of liver and serum from dystrophic embryos are also increased, but the cholesterol content of the brain is not. The increased cholesterol contents of liver and serum are due to proportionate increases of both free cholesterol and cholesteryl esters. In contrast, in superficial pectoral muscles the ratio of cholesteryl esters to free cholesterol is significantly increased. These data indicate that, in addition to the elevated cholesterol in the liver and serum of dystrophic embryos, the regulation of cholesteryl ester content in the dystrophic pectoral muscles is abnormal.

ADP-Induced changes in [32P]phosphate labeling of phosphatidylinositol-4,5-bisphosphate in washed rabbit platelets made refractory by prior ADP stimulation

Changes in 32P labeling of phosphatidylinositol-4,5-bisphosphate (PIP2) were examined during ADP-induced aggregation of washed rabbit platelets prelabeled with [32P]phosphate. ADP caused a significant decrease in the amount and 32P labeling of PIP2 at 10 and 60 sec. The decrease in labeling persisted at 2.5 min when the platelets were still aggregated, but disappeared by 10 min. Platelets refractory to ADP showed no further significant change in 32P in PIP2 when exposed to ADP; a decrease in PIP2 labeling could be induced, however, after platelets had recovered their disc shape and sensitivity to ADP. These data indicate that PIP2 may play a role in the response of platelets to ADP.

Differences between platelet phosphoinositide metabolism stimulated by thrombin or SFLLRN are not accounted for by interaction of thrombin with glycoprotein Ib

The formation of inositol phosphates was compared in aspirin‐treated, washed human platelets suspended in Tyrodes‐albumin solution containing 2 mM calcium and stimulated with SFLLRN (thrombin receptor‐activating peptide) or thrombin. SFLLRN (20 μM) and thrombin (1 U/ml) resulted in maximal irreversible aggregation and 80–90% secretion of dense granule contents. SFLLRN (50–100 μM) caused larger increases at 10 sec than 20 μM SFLLRN in the formation of inositol trisphosphate (IP3, measured as [3H]inositol label). These increases were not significantly less than those caused by thrombin (1 unit/ml). However, whereas the labeling of IP3 increased from 10–60 sec with thrombin, with SFLLRN it was much less at 60 sec than that at 10 sec. The decrease was not due to degradation of SFLLRN by ectopeptidases, since it was not prevented by amastatin, an inhibitor of ectopeptidases. Degradation of glycoprotein Ib (GPIb) with an O‐sialoglycoprotein endopeptidase did not affect the thrombin‐stimulated labeling of inositol phosphates, indicating that binding to GPIb is not involved in the sustained thrombin‐induced formation of inositol phosphates. The finding that the thrombin‐stimulated formation of IP3 was not dependent on Ca2+ in the medium (EGTA added) indicates that the transient SFLLRN‐induced formation of IP3 is not due to failure to cause Ca2+ influx. The finding that formation of IP3 was transient in SFLLRN‐stimulated platelets, whereas platelet aggregation and secretion were maximal, indicates that the sustained activation of phospholipase C caused by thrombin may have roles related to later processes in which platelets participate. Am. J. Hematol. 54:288–295, 1997.

Polyphosphoinositide Changes in Rabbit Platelets Stimulated with Platelet Activating Factor During the Formation of Platelet-fibrin Clots

Phosphoinositide metabolism in rabbit platelets prelabelled with [(32)P]phosphate and [(3)H]inositol was stimulated by platelet activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) with stirring at 200 rpm for 120 s in the presence of polymerising fibrin produced by the action of batroxobin (B. atrox) (also referred to by the proprietary name Reptilase) on fibrinogen. Under these conditions platelet-fibrin clots formed and retracted around the stirring bar. Phosphoinositides were extracted with chloroform: methanol: HC1. The role of the secretion of platelet granule contents in the phosphoinositide changes was examined by comparison of the effects of 1 nM PAF which did not cause secretion, with 50 nM PAF which caused extensive secretion. Stimulation of platelets with PAF in the presence of polymerising fibrin caused a greater decrease in the amount and labelling of extractable phosphatidylinositol 4,5-bisphosphate (PIP(2)) than was observed with platelets stimulated in the presence of fibrinogen. With 1 nM PAF, the decrease (1.26 ± 0.11 nmol/10(9) platelets) in amount of extractable PIP(2) when platelets were stimulated in the presence of polymerising fibrin compared with in the presence of fibrinogen was accounted for by an increase in the amount of phosphatidylinositol 4-phosphate (PIP). With 50 nM PAF, the decrease in amount of extractable PIP(2) (1.09±0.11 nmol/10(9) platelets) was not accounted for by an increase in the amount of PIP; the decrease in the amount of [(3)H]inositol label in PIP(2) in platelets stimulated in the presence of polymerising fibrin was accounted for by the sum of the increases in PIP labelling and the label associated with interfacial protein from the lipid extractions. When fibrin polymerisation was blocked with glycyl-L-prolyl-L-arginyl-L-proline (GPRP), the large decrease in extractable PIP(2) and the increase in the association of label with the interfacial protein did not occur. Thus, both the formation of a fibrin network, and the changes that accompany the secretion of granule contents, are necessary for the association of the (3)H-labelled material with interfacial protein. Blocking thromboxane A(2) formation had no effect on the changes in response to 50 nM PAF. Although PAF stimulated phospholipase C, resulting in increases in amount and (32)P-labelling of phosphatidic acid and (3)H-labelling of inositol bisphosphate and inositol phosphate, the increases were similar in the presence of polymerising fibrin or fibrinogen. Thus, further stimulation of phospholipase C does not occur in association with clot formation. The specific radioactivities of labelling with [(3)H]inositol of the phosphoinositides in unstimulated platelets differed (PIP(2)> phosphatidylinositol (PI) > PIP). Upon stimulation of the platelets with 1 nM PAF, the specific radioactivity of PIP rose above that of PI and toward that of PIP(2), indicating that the increase in PIP was due to degradation of PIP(2). Thus, the large decrease in extractable PIP(2) and increase in formation of PIP caused by the presence of polymerising fibrin appear to be due to increased degradation of PIP(2) to PIP.

Changes in Phosphoinositides in Rabbit Platelets during Clot Formation. Comparison of Platelets Stimulated by ADP or by Thrombin in the Presence of Polymerising Fibrin

Platelet phosphoinositide metabolism was examined during platelet-fibrin clot formation stimulated by ADP (10 μM) plus reptilase, or by thrombin (1 U/ml), for 120 s in the presence of fibrinogen, to determine which changes are specifically associated with this process. Stirring at 200 rpm was used to minimise the contribution of aggregation to the platelet changes. Under these conditions, thrombin caused extensive release of the contents of platelet granules; ADP plus reptilase did not. The presence of fibrinogen decreased the amount of extractable phosphatidylinositol 4,5-bisphosphate (PIP(2)) by 46.4±5.5% when thrombin was the stimulus, and by 47.4±5.5% when the platelets were stimulated by ADP plus reptilase. Fibrinogen did not decrease the extraction of other phospholipids. The amount of phosphatidylinositol 4-phosphate (PIP) increased when platelets were stimulated in either the presence or absence of fibrinogen. These increases were greater in the presence of fibrinogen and the thrombin-induced increase was smaller than the increase induced by ADP plus reptilase; with ADP plus reptilase, the increase in PIP more than accounted for the loss of extractable PIP(2). In platelets prelabelled with [(3)H]inositol, the decrease in PIP(2) labelling induced by fibrinogen with ADP plus reptilase as the stimulus was accounted for by the increase in PIP labelling; the decrease induced by fibrinogen with thrombin as the stimulus was not. With thrombin, 46.5% of the decrease in PIP(2) labelling, caused by fibrinogen, was accounted for by label that remained with the interfacial protein after lipid extraction; with ADP plus reptilase, the amount of label with this protein was the same with or without fibrinogen. Only thrombin increased the amount of label in inositol trisphosphate (IP(3)) and the amount of phosphatidic acid (PA); these changes were not increased by fibrinogen. Thus, the results with ADP plus reptilase indicate that clot formation is not dependent on release of granule contents, formation of detectable IP(3) or PA (and hence does not require activation of phospholipase C) or association of [(3)H]inositol-labelled compounds with protein. Clot formation is associated with a shift in the PIP(2)-PIP equilibrium toward PIP.

In contrast to fibrinogen or fibrin, peptide and peptide mimetic binding to alpha does not cause outside-in signalling as judged by measurements of phosphatidylinositol 4,5-bisphosphate beta (GPIIb-IIIa) IIb 3

Binding of ligands, including RGD-containing peptides, to the platelet fibrinogen receptor, integrin alpha beta has been reported to cause outside-in signals, which result in clustering of occupied receptors and changes in conformation of the receptor and its cytoplasmic tails. Thus, the peptides that are usually used as inhibitors may function as partial agonists. Binding of ligand, fibrinogen or polymerizing fibrin, to platelets with activated alpha beta causes decreases in phosphatidylinositol 4,5-bisphosphate (PIP ), which may affect actin organization. IIb 3 Whether or not binding to unactivated platelets of the peptide RGDS, the fibrinogen gamma -chain C-terminal dodecapeptide (H12), or a high affinity RGD mimetic SC-54701B affects phosphoinositide metabolism was tested. Although incubation of RGDS (230 mu M), dodecapeptide (400 mu M) or SC-54701B (10 mu M) with platelets for 2 min caused trends towards decreases in PIP , no significant decreases were found. As a positive control, 2 SC-54701B was sh...