Platelet phospholipids

The intrinsic pathway (Figure 51-1) involves factors XII, XI, IX, VIII, and X as well as prekallikrein, high-molecular-weight (HMW) kininogen, Ca, and platelet phospholipids. It results in the production of factor Xa (by convention, activated clotting factors are referred to by use of the suffix a). 

An excellent example of PLC applications in the indirect coupling version is provided by the works of Miwa et al. [12]. These researchers separated eight phospholipid standards and platelet phospholipids from the other lipids on a silica gel plate. The mobile phase was composed of methylacetate-propanol-chloro-form-methanol-0.2% (w/v) potassium chloride (25 30 20 10 10, v/v). After detection with iodine vapor (Figure 9.2), each phospholipid class was scraped off and extracted with 5 ml of methanol. The solvent was removed under a stream of nitrogen, and the fatty acids of each phospholipid class were analyzed (as their hydrazides) by HPLC. The aim of this study was to establish a standardized... 

Fig. 2. Generation of tenase and prothrombin complexes. PPL represents the anionic phospholipid surface provided by the platelets (platelet phospholipid). Cleavage of prothrombin by the prothrombinase complex results in the formation of thrombin and the release of a small fragment called prothrombin fragment 1.2 (PFI.2).

Fig. S. Schematic representation of coagulation pathway as currently understood. TF, tissue factor PPL, platelet phospholipid.

Hageman factor (XII), antihemophilic globulin (VIII), Christmas factor (IX), plasma thromboplastin antecedent (XI), calcium (IV), and platelet phospholipids... 

Ferrier, L., Caston, L., Leeson, S., Squires, J., Weaver, B., and Holub, B. 1995. a-Linolenic acid- and docosahexaenoic acid-enriched eggs from hens fed flaxseed Influence on blood lipids and platelet phospholipid fatty acids in humans. Am J. Clin Nutr. 62, 81-86. 

A typical separation of platelet phospholipids can be achieved as follows A sample containing 40-50 p,g of phospholipid (by weight) is applied to a p, Porasil (polar-type) column (Waters, Milford, CT) measuring 7 x 30 cm, and a solvent system of acetonitrile-methanol-45% phosphoric acid (130 5 1.5, v/v) is applied, as suggested by Chen and Kou (1982). The flow rate was 1 ml/min, and the elution was monitored by an ultraviolet detector sensitive at 203 nm. The total elution time in this case was 40 min, and the profile is illustrated in Figure 3-4. 

Arachidonic acid content of platelet phospholipids Decreased 124, 125, 127, 128... 

Platelet phospholipid and fatty acid composition was first reported in some detail by two groups (11, 12). Subsequently, our laboratory fully characterized individual fatty acid specific molecular species in human platelet phospholipids (13), in addition to documenting the molar concentration of individual phospholipids (Table 1). This work... 

Swann PG, Parent CA, Croset M, Fonlupt P, Lagarde M, Venton DL, and Le Breton GC. (1990) Enrichment of platelet phospholipids with eicosapentaenoic acid and docosahexaenoic acid inhibits thromboxane A2/prostaglandin H2 receptor binding and function. J. Biot. Cheia 263,21692-21697. 

AA (b>-6)] fixHn the sn-2 positicm of platelet phospholipids (22). The majm source of DAG appears to be predcxninantly c ved Wn pho hoinositides, whidi are rich in 1-stearoyl 2-arachiyl species (18, 22). Althmigh 1-stearoyl 2-arachid(H]oyl DAG species m also come fimn a PLD-mediated hydrolysis of phospholipids, it is less clear as to the extmt of amtributirm from this pathway fix die release of AA. FurthomcHe, the DAG lipase pathway appears to be an unlikely route fw the release... 

Alternatively, the release of platelet phospholipid AA is mediated by the activation of phospholipase Aj (PIA) coupled to PLC, intracellular Ca rise and protein kinase C (PKC) and tyrosine kinase-mediated protein phosphorylations (23,24) and/or by the activation of PLA that is not coupled to these events in platelets (25-29). Furthermore, the hydrolysis of phosphatidic acid (PA) by a PA-specific PLAj may also contribute to eicosanoid synthesis (30,31). It is evident that the differential sensitivity of platelets to multiple agonists and their signals, have contributed to the complexity in understanding the regulation of PLAj. 

Warmer, Tsai WJ, Chang SM, ShiaoYJ, and Yang CC. (1987). Different susceptibilities of platelet phospholipids to various phoqiholipases and modifications induced by thrombin. Possible evidence ofrearrangement of lipid domains. Biochimi. Biophys. Acta. 899,205-212. 

Huzoor-Akbar, Anwer, K., Wince, L. and Kundu, N. Platelet phospholipid metabolism and fatty acid... 

GLA at a dose of 5.23 g provided as borage oil for 42 d in male volunteers increased platelet phospholipid DGLA (Barre and Holub, 1992), but borage oil at doses of 3 g/d has been shown not to affect human platelet aggregation (Bard et al., 1997). 

Phospholipase A Inhibition - The liberation of arachidonic acid in stimulated platelets is inhibited by cyclic-AMP. 14,24 This action of cAMP does not seem to be a direct action on platelet phospholipase A2. Cyclic-AMP decreases the levels of free cytoplasmic Ca2+ and reduces in this way the availability of Ca2+ for phospholipase A2 activity. Trifluoperazine, which is a calmodulin antagonist, also inhibits the thrombin- or platelet activating factor-induced mobilization of arachidonic acid from platelet phospholipids.20,48... 

Kalofoutis, A., Lekakis, J., Koutselinis A. (1980) Effects of d9-THC on human platelet phospholipids. Pharmacol. Biochem. Behav. 12, 697-699. 

Srivastava et al. showed that curcumin inhibited platelet aggregation induced by arachidonate, adrenaline, and collagen. This compound inhibited thromboxane B2 production from exogenous [ " C] arachidonate (AA) in washed platelets and concomitantly increased the formation of 12 lipoxygenase products. Moreover, curcumin inhibited the incorporation of [ " C] AA into platelet phospholipids and inhibited the deacylation of AA-labeled phospholipids (liberation of free AA) on stimulation with... 

Approximately 30% of position sn-2 in human platelet phospholipids consists of arachidonate. Of course, the percentage of arachidonate varies within the different phospholipids species and the amounts of the different species of phospholipid vary in the platelet. This is illustrated in Table 1.1. which shows that sphingomyelin contains essentially t-o arachidonic acid while ethanolamine-phospholipids contain the most arachidonic acid. Although phosphatidylinositol is the phospholipid species that is most enriched in arachidonic acid, its contribution to the total content of arachidonate in blood platelets is smalP. ... 

Table 1.1 Composition and arachidonate content of human platelet phospholipids...

The release of 32 nmol of arachidonic acid per 10 platelets obtained using 5U/ml thrombin represents the hydrolysis of approximately 25% of the total arachidonate present in platelet phospholipids (Table 1.1) and is approximately twice the mass of phosphatidylinositol. Thus, the total hydrolysis of phosphatidylinositol by either a phospholipase pathway acting on this phospholipid or a phospholipase C-diglyceride lipase pathway could at the... 

A group of volunteers were put on a mackerel diet corresponding to a daily intake of 7-11 g of eicosapentaenoic acid. Analysis of their platelet lipids after six days on the diet showed a five-fold increase in eicosapentaenoic acid, while the arachidonic acid fraction decreased considerably. A decrease in collagen induced platelet aggregation was found that correlated with diminished TXBj formation and with an increase in the eicosapentaenoic acid to arachidonic acid ratio in platelet phospholipids [175]. 

Phospholipids The laborious combinations of thin-layer or liquid chromatography (LC) with gas chromatography (GC) for the analysis of phospholipids in biological fluids have been replaced by combinations of HPLC with MS (LC-MS). In many instances, the convenience of MS/MS mode of mass spectrometric identification of the erythrocyte and platelet phospholipids has been preferred to the LC-MS combination. A collision-induced dissociation of specific parent ions allows the deconvolution of the composition of the common mixtures of molecular species. For more complex phospholipid mixtures, a combined LC-MS approach is recommended. 

Han, X., Gubitosi-Klug, R. A., Collins, B. J. and Gross, R. W. (1996) Alterations in individual molecular species of human platelet phospholipids during thrombin stimulation electrospray ionisation mass spectrometry-fadlitated identification of the boundary conditions for the magnitude and selectivity of thrombin-induced platelet phospholipid hydrolysis. Biochemistry, 35, 5822-32. 

Both reversed-phase HPLC-ES-MS and flow injection ES-MS were used by Han et al (1996) for the investigation of the alterations in individual molecular species of human platelet phospholipids during thrombin stimula-... 

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