Thrombin-stimulated Human Platelets Research Articles

Further studies of the secretory pathway in thrombin-stimulated human platelets

The pathway followed by secretory products stored in platelet alpha granules during the release reaction remains controversial. Tannic acid has been used in the present study as an electron-dense stain to follow the secretory process in thrombin-stimulated platelets. Preliminary experiments demonstrated that tannic acid precipitates fibrinogen, and binds osmium tetroxide to fibrinogen and fibrin strands. Examination of platelets fixed at short intervals after exposure to thrombin and incubated in solutions containing tannic acid revealed electron-dense deposits of osmium not apparent in resting platelets. Granules and lumina of channels making up the open canalicular system (OCS) were unstained in discoid cells. However, exposure to thrombin at concentrations of 1 to 5 U/mL for thirty seconds or more resulted in intense staining of alpha granules by osmium. Some granules communicated directly with dilated channels of the OCS, and several were frequently connected to the same canaliculus. The electron-dense substance in swollen granules and channels appeared to be in the process of extrusion through narrow or dilated openings of the OCS onto the platelet surface. Granule-to-granule fusion and formation of sealed vacuoles of fused granule products unstained by tannic acid-osmium were not observed. The findings support the concept that secretion by stimulated human platelets results from development of direct communications between granules and channels of the OCS and subsequent extrusion of products through channel pores to the surrounding medium.

Further Studies of the Secretory Pathway in Thrombin-Stimulated Human Platelets

The pathway followed by secretory products stored in platelet alpha granules during the release reaction remains controversial. Tannic acid has been used in the present study as an electron-dense stain to follow the secretory process in thrombin-stimulated platelets. Preliminary experiments demonstrated that tannic acid precipitates fibrinogen, and binds osmium tetroxide to fibrinogen and fibrin strands. Examination of platelets fixed at short intervals after exposure to thrombin and incubated in solutions containing tannic acid revealed electron-dense deposits of osmium not apparent in resting platelets. Granules and lumina of channels making up the open canalicular system (OCS) were unstained in discoid cells. However, exposure to thrombin at concentrations of 1 to 5 U/mL for thirty seconds or more resulted in intense staining of alpha granules by osmium. Some granules communicated directly with dilated channels of the OCS, and several were frequently connected to the same canaliculus. The electron-dense substance in swollen granules and channels appeared to be in the process of extrusion through narrow or dilated openings of the OCS onto the platelet surface. Granule-to-granule fusion and formation of sealed vacuoles of fused granule products unstained by tannic acid-osmium were not observed. The findings support the concept that secretion by stimulated human platelets results from development of direct communications between granules and channels of the OCS and subsequent extrusion of products through channel pores to the surrounding medium.

Thrombin-induced transfer of arachidonic acid in human platelets is not inhibited by trifluoperazine

Human platelets have been shown to contain a Ca++- and CoA-independent transacylase enzyme that catalyzes the transfer of arachidonic acid from phosphatidylcholine (PC) to lysoplasmenylethanolamine. It has been suggested that this route may represent a major source for released arachidonic acid in stimulated platelets. In this study, we have shown using arachidonic-labelled human platelets that the thrombin-induced activation of a transacylase reaction was not affected by concentrations of trifluoperazine (TFP) (15 micrograms/2 X 10(9) cells) which abolished the accumulation of free [3H]arachidonic acid in the presence of the cyclooxygenase/lipoxygenase inhibitor BW755C. TFP, at this concentration failed to block the hydrolysis of phosphatidylcholine (PC) completely and had no effect on the increased radioactivity seen in total phosphatidylethanolamine (PE) (160% of control after 4 min of incubation). These results suggest that the transacylase pathway activated in response to thrombin is not likely dependent on calcium. As TFP blocks effectively both the accumulation of free [3H]arachidonic acid and the mass of arachidonic acid without affecting the transfer of this fatty acid from PC to PE in thrombin-stimulated human platelets, it is very unlikely that the transacylation pathway represents a major source of release arachidonic acid. Based on these findings, we conclude that the above pathway may be primarily involved in the turnover of plasmenylethanolamine lipids in stimulated human platelets.

Signal transduction in normal and pathological thrombin-stimulated human platelets

Human blood platelets stimulated by thrombin undergo very rapid morphological changes, the most characteristic of which are pseudopod formation and granule centralization. These early changes in shape are accompanied by a transient decrease (30%) in phosphatidyl inositol 4,5-biphosphate (PIP 2) which occurs in the first 10 s after thrombin addition. Transient decreases in phosphatidyl inositol 4-phosphate (PIP) and phosphatidyl inositol (PI) occur later (20–30 s). These events lead to the formation of inositol 1,4,5-triphosphate (IP 3) and diacylglycerol (DG) and hence phosphatidate (PA). Two polypeptides are phosphorylated during the same time span: the myosin light chain (P20) and a 43 kDa protein (P43). Concomitant with these molecular changes, platelet ‘release reaction’ occurs, i.e., liberation of the different granule constituents into the external medium: the earliest concerns dense bodies which liberate adenine nucleotides, calcium and serotonin; α-granules then liberate adhesive and specific proteins and are followed by lysosomes which liberate hydrolases. Pathological platelets from patients with inherited disorders, presenting well-characterized and specific defects of either the platelet membrane (GT) or storage granules (GPS and HPS), have also been studied. The results obtained lead to the following conclusions: (1) the transducing system is normal in platelets unable to aggregate; (2) phosphorylation of P20 and P43 proteins can be complete with impaired release; and (3) when platelets lack α-granules the transducing system as well as the release of other granule populations are impaired. These results evidence the relationship between the absence of intraplatelet components and metabolic events.

Resolution of radiolabeled molecular species of phospholipid in human platelets: effect of thrombin.

Resolution of individual molecular species of human platelet 1,2-diradyl-sn-glycero-3-phosphocholines and 1,2-diradyl-sn-glycero-3-phosphoethanolamines by reverse phase high pressure liquid chromatography (HPLC) allowed a thorough analysis of those phospholipids labeled with [3H]arachidonic acid. Approximately 54% and 16% of the total incorporated radiolabel was found in choline glycerophospholipids and ethanolamine glycerophospholipids, respectively, with ca. 90% of this being found in the 1,2-diacyl molecular species. Eighty percent of [3H]-arachidonic acid incorporated into 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine in resting platelets was equally distributed between 1-palmitoyl-2-arachidonoyl and 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, while 70% of the radiolabel in 1-acyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine was found in 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine. Thrombin stimulation (5 U/ml for 5 min) resulted in deacylation of all 1-acyl-2-[3H]arachidonoyl molecular species of 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-acyl-2-arachidonoyl-sn-glycero-3-ethanolamine. There was also a slight increase in 1-O-alkyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine and a significant increase in 1-O-alk-1'-enyl-2-[3H]arachidonoyl-sn-glycero-3-phosphoethanolamine molecular species of over 300%. Thus, HPLC methodology indicates that arachidonoyl-containing molecular species of phosphatidylcholine and phosphatidylethanolamine are the major source of arachidonic acid in thrombin-stimulated human platelets, while certain ether phospholipid molecular species become enriched in arachidonate.

トロンビン刺激血小板でのイノシトール１，２‐サイクリック‐４，５‐三リン酸の生成と代謝

Detection and metabolism of inositol-1, 2-cyclic-4, 5-trisphosphate (cIP3) were investigated in the thrombin-stimulated human platelets. The reaction of 32P-labeled platelets with thrombin were stopped by addition of CHCl3/MeOH. The water soluble fraction was applied on the Whatman Partisil-10 column chromatogrphy. The unknown radioactive peak of 32P was detected in the fraction between inositol-1, 4-diphosphate (IP2) and inositol-1, 4, 5-trisphosphate (IP3) in the chromatography. The unknown radioactive peak moved to IP3 position with the internal standard of 3H-cIP3 by acidification of the sample. Furthermore, the unknwon radioactive peak of 32P was collected and was treated with IP3-5-phosphatase. The radioactive peak moved to the position of cIP2. It indicates that in the thrombin-stimulated platelets, the radioactive compound of 32P detected between IP2 and IP3 fraction in the chromatography is cIP3.Rabiolabeled inositolphosphates and inositol-cyclic-phosphates were incubated with the cytosol fraction of human pletelets. The 1, 2-ring of cIP3 was not cleaved immediatly after the production from platelets and the cIP3 did not convert to IP3 in the metabolic process. It indicates that cIP3 is metabolited as follow: cIP3→cIP2→cIP1→IP1.

Divalent Cation-Dependent and Independent Surface Expression of Thrombospondin on Thrombin-Stimulated Human Platelets

Thrombospondin (TSP), a platelet alpha-granule protein, becomes expressed on the surface of thrombin-stimulated platelets. The surface expression of this protein occurs through two distinct mechanisms. At low platelet concentrations (1 X 10(8)/mL), a divalent ion-dependent, low-capacity mechanism predominates. At higher cell concentrations, a divalent ion-dependent, higher capacity mechanism prevails that can account for greater than 90% of all the TSP surface expression measured. This mechanism requires the presence of both calcium and magnesium (Ca + Mg). The dependence of the divalent ion-dependent surface expression on platelet concentration suggests that release of the molecule from the cell followed by its binding to the cell surface mediates this component of the endogenous TSP-platelet interaction. These data are consistent with a two-receptor model for the platelet-surface expression of the endogenous TSP pool.

Divalent cation-dependent and independent surface expression of thrombospondin on thrombin-stimulated human platelets

Thrombospondin (TSP), a platelet alpha-granule protein, becomes expressed on the surface of thrombin-stimulated platelets. The surface expression of this protein occurs through two distinct mechanisms. At low platelet concentrations (1 X 10(8)/mL), a divalent ion-dependent, low-capacity mechanism predominates. At higher cell concentrations, a divalent ion-dependent, higher capacity mechanism prevails that can account for greater than 90% of all the TSP surface expression measured. This mechanism requires the presence of both calcium and magnesium (Ca + Mg). The dependence of the divalent ion-dependent surface expression on platelet concentration suggests that release of the molecule from the cell followed by its binding to the cell surface mediates this component of the endogenous TSP-platelet interaction. These data are consistent with a two-receptor model for the platelet- surface expression of the endogenous TSP pool.

The relative degradation of [14C]eicosapentaenoyl and [3H]arachidonoyl species of phosphatidylinositol and phosphatidylcholine in thrombin-stimulated human platelets.

The platelet-rich plasma from volunteers who had consumed a supplement containing eicosapentaenoate (EPA) was incubated with [3H]arachidonic acid (AA) and [14C]EPA so as to provide for the labelling of these fatty acids in the individual platelet phospholipids. Washed dual-labelled platelet suspensions were prepared and incubated with and without thrombin in the presence of BW755C and in the presence and absence of trifluoperazine (TFP) or indomethacin. The platelet lipids were extracted and the individual phospholipids, as well as diacylglycerol and phosphatidic acid, were separated by thin-layer chromatography and the radioactivity in each fraction was determined. The [3H]AA/[14C]EPA dpm ratio for the loss of radioactivity from phosphatidylcholine (PC) upon thrombin stimulation, as well as that in the residual PC remaining after stimulation, was similar to that in PC in the resting platelets. This suggests no marked selectivity in the degradation of EPA-versus AA-containing species of PC during platelet activation. The [3H]/[14C] ratios for the increased radioactivity appearing in diacylglycerol (DG) and phosphatidic acid (PA) upon thrombin stimulation were not significantly different from the corresponding ratio in phosphatidylinositol (PI) from resting platelets, suggesting little or no preference for 1-acyl-2-eicosapentaenoyl PI over 1-acyl-2-arachidonoyl PI in the pathway from PI to DG to PA. These results suggest that the relative formation of the 2- and 3-series prostaglandins, including thromboxane (Tx) A2 and A3, in stimulated platelets is not regulated by a preferential loss of one of the corresponding eicosanoid precursors over the other from membrane PC and PI.

Inositol cyclic triphosphate [inositol 1,2-(cyclic)-4,5-triphosphate] is formed upon thrombin stimulation of human platelets.

Cleavage of polyphosphoinositides in vitro by phospholipase C results in formation of both cyclic and noncyclic inositol phosphates. We have now isolated the cyclic product of phosphatidylinositol 4,5-bisphosphate cleavage, inositol 1,2(cyclic)-4,5-triphosphate [cIns(1:2,4,5)P3], from thrombin-treated platelets. We found 0.2-0.4 nmol of cIns-(1:2,4,5)P3 per 10(9) platelets at 10 sec after thrombin; none was found in unstimulated platelets or in platelets 10 min after thrombin addition. We conclude that cIns(1:2,4,5)P3 is a major product of polyphosphoinositide metabolism in thrombin-stimulated platelets.

Protein kinase C phosphorylates human platelet inositol trisphosphate 5′-phosphomonoesterase, increasing the phosphatase activity

Phosphoinositide breakdown in response to thrombin stimulation of human platelets results in the formation of the calcium-mobilizing messenger molecules inositol 1,4,5-trisphosphate and inositol 1,2-cyclic-4,5-trisphosphate and of diglyceride, which activates protein kinase C. We find that protein kinase C phosphorylates and thereby increases the activity of inositol 1,4,5-trisphosphate 5′-phosphomonoesterase, a phosphatase that hydrolyzes these molecules to inert compounds. The 5′-phosphomonoesterase phosphorylated using [γ- 32P]ATP comigrates on SDS-polyacrylamide gels with a protein (40 kd) phosphorylated rapidly in response to thrombin stimulation of 32PO 4-labeled platelets. Peptide maps of proteolytic digests of these two phosphorylated proteins indicate that they are the same. We propose that platelet Ca 2+ mobilization is regulated by protein kinase C phosphorylation of the inositol 1,4,5-trisphosphate 5′-phosphomonoesterase. These results explain the observation that phorbol ester treatment of intact human platelets results in decreased levels of inositol trisphosphate and decreased Ca 2+ mobilization upon subsequent thrombin addition.

Attenuation of sn-1,2-diacylglycerol second messengers by diacylglycerol kinase. Inhibition by diacylglycerol analogs in vitro and in human platelets.

Diacylglycerol kinase is though to play a central role in the metabolism of diacylglycerol second messengers in agonist-stimulated cells. A series of diacylglycerol analogs were tested for their ability to act as substrates or inhibitors of diacylglycerol kinase with the goal of determining the substrate specificity of the enzyme, and of discovering inhibitors. Screening of these compounds was performed using a partially purified diacylglycerol kinase from pig brain. Modified assays for this enzyme using co-sonicated mixtures of diacylglycerol and anionic phospholipids were developed. This enzyme was found to be quite specific for sn-1,2-diacylglycerol (KM 24 microM for dioctanoyl-glycerol). Among the analogs investigated, only 1,2-dioctanoyl-2-amino-1,3-propanediol was utilized at a significant rate. Two analogs, dioctanoylethylene glycol (KI 58 microM) and 1-monooleoylglycerol (KI 91 microM), were potent inhibitors in vitro. These compounds were tested for effects on diacylglycerol formation and metabolism in thrombin-stimulated human platelets. Dioctanoylethylene glycol inhibited diacylglycerol phosphorylation in platelets (70-100% at 100 microM) leading to a longer-lived diacylglycerol signal. This compound may be a useful tool for studies of diacylglycerol kinase in other cell types. 1-Monooleoylglycerol treatment elevated diacylglycerol levels up to 4-fold in unstimulated platelets and up to 10-fold in thrombin-stimulated platelets. The implications with regard to the pathways of diacylglycerol metabolism in human platelets are discussed.

Inhibitory action of cyclic GMP on secretion, polyphosphoinositide hydrolysis and calcium mobilization in thrombin-stimulated human platelets

The effect of cyclic GMP (cGMP) on human platelet activation was investigated, using its metabolically stable analogue, 8-bromo cGMP (8-bcGMP). Thrombin-induced serotonin secretion was inhibited by pretreatment with 8bcGMP in a dose-dependent manner. Production of inositol trisphosphate (IP 3), a Ca 2+ releaser was inhibited by 8bcGMP pretreatment of platelets. Preincubation of platelets with 8bcGMP was without effect on the basal level of cytosolic free Ca 2+, measured by fluorescent indicator quin2, but suppressed its thrombin-induced enhancement independently of extracellular Ca 2+. These results indicate that cGMP may be implicated in phospholipase C activation and Ca 2+ mobilization (both influx through the plasma membrane and efflux from internal stores) in thrombin-activated human platelets.

Diacylglycerol lipase pathway is a minor source of released arachidonic acid in thrombin-stimulated human platelets

It has been postulated that the diacylglycerol lipase pathway is a predominant source of the free arachidonic acid which is released from phospholipids upon the exposure of human platelets to thrombin. The amount of released arachidonic acid and other fatty acids in thrombin-stimulated platelets was determined in the presence of BW755C, the cyclooxygenase/lipoxygenase inhibitor, and in relation to phosphatidylinositol degradation and phosphatidic acid formation. A stearic acid:arachidonic acid molar ratio approaching unity would be expected in the free fatty acid fraction if the latter pathway were a major source of released arachidonic acid. Our results indicate that the diacylglycerol lipase pathway contributes a maximum of 3–4 nmol of arachidonic acid/2×10 9 platelets or 12–15% of the total arachidonic acid released (25.8 nmol/2×10 9 platelets) upon exposure to thrombin (2 units/ml) for 4 min. Trifluoperazine inhibited most of the thrombin-dependent free arachidonic acid release but only 15% of the absolute loss of arachidonic acid from phosphatidylinositol. Therefore, we conclude that the diacylglycerol lipase pathway represents only a minor source of the free arachidonic acid that is released upon thrombin stimulation of human platelets.

Degradation of plasma membrane phospholipids in thrombin-stimulated human platelets following pre-labeling with different lipid precursors

It has been established from pre-labeling experiments and compositional analyses that a rapid degradation of cellular phospholipid accompanies the activation of human platelets with thrombin, s'e's'jr'na3,24 This results in the hydrolysis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) via phospholipase A2 activity with the release of arachidonic acid and the loss of phosphatidylinositol (PI) via a combination of phospholipase C, phospholipase A2, and PI kinase activities, s,e,s,m°,16,n.23,24 The latter reaction is considered to be related to the enhanced degradation of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C t2° with the release of the second messengers, 1,2-diacylglycero121 and inositol trisphosphate. 4 Although an involvement of plasma membrane PI in cellular responses to external stimuli is generally assumed, 19 only recently has direct evidence for its participation been forthcoming from research involving the exposure of isolated rat hepatocytes or plasma membranes to vasopressin or norepinephrine. 9.~4~s~ The work in • stimulated platelets to date has been conducted using intact cells with no direct evidence for the breakdown of plasma membrane phospholipids having been reported. The present research was conducted to evaluate the possible participation of individual phospholipids residing in the plasma membrane in human platelet responses to an external agonist. For this purpose, platelet phospholipids were pre-labeled with [3I-l]glycerol, [m'C]arachidonic acid (AA), or [~%-']oleic acid (OA) prior to exposing intact platelets to thrombin and isolation of the plasma membranes.

Reversal by protein kinase C inhibitor of suppressive actions of phorbol-12-myristate-13-acetate on polyphosphoinositide metabolism and cytosolic Ca 2+ mobilization in thrombin-stimulated human platelets

In the presence of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinase C in vitro, phorbol-12-myristate-13-acetate (PMA) did not suppress the thrombin-induced increase of cytosolic Ca2+ concentration in human platelets. The H-7 reversal of the inhibitory action of PMA was also observed in thrombin-induced polyphosphoinositide breakdown by phospholipase C. These results provide additional support to the developing theory that the inhibition of PMA on Ca2+ mobilization and phosphoinositide turnover may be mediated by protein kinase C activation.

ヒト血小板のＰＭＡ（ｐｈｏｒｂｏｌ ｍｙｒｉｓｔａｔｅ ａｃｅｔａｔｅ）前処理による刺激応答の変化

The effects of tumor promoter (PMA, phorbol-12-myristate-13-acetate) on thrombin-stimulated human platelet responses and membrane phospholipid metabolism were investigated. Thrombin (0.1U/ml)-induced serotonin release was inhibited by pretreatment with PMA (50μg/ml) for 5min. This inhibition was associated with a suppression of polyphosphoinositide breakdown and increase of cytoplasmic free Ca2+ ([Ca2+] i). PMA was observed to increase the incorporation of [32P]phosphate into phosphatidylinositol 4, 5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP). This indicates that treatment with PMA suppresses the hydrolysis of PIP2 by phospholipase C, resulting in the insufficient supply of a Ca2+ releaser, IP3. A considerably high radioactivity in PIP would be due to the enhanced conversion of phosphatidylinositol (PI) to PIP via PI kinase rather than the inhibited PIP breakdown, since there was no indication of PIP hydrolysis during stimulation with thrombin alone. These observations provide evidence that protein kinase C subserves a negative feedback role in a receptor-mediated cell activation.

Related binding mechanisms for fibrinogen, fibronectin, von Willebrand factor, and thrombospondin on thrombin-stimulated human platelets

Fibrinogen, fibronectin, von Willebrand factor, and thrombospondin are four large glycoproteins that bind to thrombin-stimulated platelets and influence cellular adhesive functions. The effects of five monoclonal antibodies that react with platelet membrane glycoproteins (GP) IIb and/or IIIa on the binding of these four molecules to stimulated platelets were assessed. Tab and PMI-1, antibodies recognizing GPIIb, had no effect, whereas 10E5 and 2G12, antibodies that immunoprecipitate both GPIIb and IIIa in the presence of calcium, inhibited binding of all four ligands by greater than 85%. T10, an antibody specific for the GPIIb-IIIa complex, produced partial inhibition (60% to 80%) of the binding of each ligand. Inhibitory antibodies were effective in the same dose range for all four proteins and also inhibited binding of fibrinogen, fibronectin, and von Willebrand factor to receptors fixed in an induced state (thrombin-stimulated platelets fixed with paraformaldehyde). Thrombospondin did not bind to these fixed cell preparations. The results suggest that these four adhesive proteins have a related mechanism of binding to thrombin-stimulated platelets. This related mechanism may entail the sharing of some, but not necessarily all, binding sites for the four ligands or a proximal relationship between these binding sites.

Related Binding Mechanisms for Fibrinogen, Fibronectin, von Willebrand Factor, and Thrombospondin on Thrombin-Stimulated Human Platelets

AbstractFibrinogen, fibronectin, von Willebrand factor, and thrombospondin are four large glycoproteins that bind to thrombin-stimulated platelets and influence cellular adhesive functions. The effects of five monoclonal antibodies that react with platelet membrane glycoproteins (GP) IIb and/or IlIa on the binding of these four molecules to stimulated platelets were assessed. Tab and PMI-1, antibodies recognizing GPIIb, had no effect, whereas 10E5 and 2G12, antibodies that immunoprecipitate both GPIIb and IIIa in the presence of calcium, inhibited binding of all four ligands by >85%. T10, an antibody specific for the GPIIb—IIIa complex, produced partial inhibition (60% to 80%) of the binding of each ligand. Inhibitory antibodies were effective in the same dose range for all four proteins and also inhibited binding of fibrinogen, fibronectin, and von Willebrand factor to receptors fixed in an induced state (thrombin-stimulated platelets fixed with paraformaldehyde). Thrombospondin did not bind to these fixed cell preparations. The results suggest that these four adhesive proteins have a related mechanism of binding to thrombin-stimulated platelets. This related mechanism may entail the sharing of some, but not necessarily all, binding sites for the four ligands or a proximal relationship between these binding sites.

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

(1) 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. (2) 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° C. (3) 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°C in the presence of 100 μM 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.