Thrombomodulin is found on endothelium of arteries, veins, capillaries, and lymphatics, and on syncytiotrophoblast of human placenta.

We have used antibodies to human thrombomodulin isolated from placenta to investigate the distribution of this cofactor for protein C activation in human tissues. Thrombomodulin was found on endothelial cells of arteries, veins, capillaries, and lymphatics by immunocytochemical staining using an avidin-biotin peroxidase method. Thrombomodulin was not detected on sinusoidal lining cells of liver or on postcapillary high-endothelial venules of lymph node, although the latter contained another endothelial antigen, von Willebrand factor. Other cells noted to contain thrombomodulin antigen are those of the syncytiotrophoblast in placenta. The thrombomodulin in syncytiotrophoblast was primarily on the plasma membrane surface that forms the maternal blood sinus. Syncytiotrophoblast also stained with antibodies to von Willebrand factor, which implies that these cells have multiple endothelial functions. Thrombomodulin antigen was found in all organs studied, with the notable exception of brain.

The effects of thrombin on phytohemagglutinin receptor sites in human platelets.

We have previously demonstrated that lentil phytohemagglutinin (lentil-PHA) binds to human platelet membranes without causing either aggregation or the release reaction. When platelets are treated with thrombin, there is an increase in lentil-PHA binding suggesting the appearance of new receptor sites on the cell surface. We prepared a lentil-PHA-ferritin conjugate using affinity chromatography which was used to saturate cell surface receptor sites. Studies using this conjugate suggest that thrombin causes a complex change in the platelet surface involving a decrease in the number of lentil-PHA receptor sites on the external platelet surface with a marked increase in sites within the center of the canalicular system. These increased sites may result from fusion of granule membranes with the canalicular membranes during the secretion process. There is no obvious relationship between lentil-PHA receptor sites and intramembranous particles.

Uptake and subcellular distribution of [3H]arachidonic acid in murine fibrosarcoma cells measured by electron microscope autoradiography.

We have used quantitative electron microscope autoradiography to study uptake and distribution of arachidonate in HSDM1C1 murine fibrosarcoma cells and in EPU-1B, a mutant HSDM1C1 line defective in high affinity arachidonate uptake. Cells were labeled with [3H]arachidonate for 15 min, 40 min, 2 h, or 24 h. Label was found almost exclusively in cellular phospholipids; 92-96% of incorporated radioactivity was retained in cells during fixation and tissue processing. All incorporated radioactivity was found to be associated with cellular membranes. Endoplasmic reticulum (ER) contained the bulk of [3H]arachidonate at all time points in both cell types, while mitochondria, which contain a large portion of cellular membrane, were labeled slowly and to substantially lower specific activity. Plasma membrane (PM) also labeled slowly, achieving a specific activity only one-sixth that of ER at 15 min in HSDM1C1 cells (6% of total label) and one-third of ER in EPU-1B (10% of total label). Nuclear membrane (NM) exhibited the highest specific activity of labeling at 15 min in HSDM1C1 cells (twice that of ER) but was not preferentially labeled in the mutant. Over 24 h, PM label intensity increased to that of ER in both cell lines. However, NM activity diminished in HSDM1C1 cells by 24 h to a small fraction of that in ER. In response to agonists, HSDM1C1 cells release labeled arachidonate for eicosanoid synthesis most readily when they have been labeled for short times. Our results therefore suggest that NM and ER, sites of cyclooxygenase in murine fibroblasts, are probably sources for release of [3H]arachidonate, whereas PM and mitochondria are unlikely to be major sources of eicosanoid precursors.

Acyl carrier protein: effects of acetylation and tryptic hydrolysis on function in fatty acid synthesis.

Acetylation of the four lysine residues and the amino group of the terminal serine residue of Escherichia coli acyl carrier protein has no effect on the ability of this protein to function in fatty acid synthesis. Subsequent trypsin hydrolysis resulting in complete inactivation cleaves a single arginyl peptide bond, releasing the amino terminal hexapeptide from the molecule.

Identity of inositol 1,2-cyclic phosphate 2-phosphohydrolase with lipocortin III.

The amino acid sequences of three fragments of cyanogen bromide-digested human placental inositol 1,2-cyclic phosphate 2-phosphohydrolase, an enzyme of the phosphatidylinositol signaling pathway, are identical to sequences within lipocortin III, a member of a family of homologous calcium- and phospholipid-binding proteins that do not have defined physiological functions. Lipocortin III has also been previously identified as placental anticoagulant protein III (PAP III) and calcimedin 35 alpha. Antibodies to PAP III detected PAP III and inositol 1,2-cyclic phosphate 2-phosphohydrolase with identical reactivity on immunoblotting. In addition, inositol 1,2-cyclic phosphate 2-phosphohydrolase was stimulated by the same acidic phospholipids that bind lipocortins.

The metabolism of phosphoinositide-derived messenger molecules.

The phosphoinositides are minor phospholipids present in all eukaryotic cells. They are storage forms for messenger molecules that transmit signals across the cell membrane and evoke responses to extracellular agonists. The phosphoinositides break down to liberate messenger molecules or precursors of messenger molecules. Many different compounds are formed, although the functions of only a few are understood. Recent studies elaborating the pathways for formation of products from phosphoinositides and the factors controlling their metabolism are summarized here.

Prostaglandins: critical roles in pregnancy and colon cancer.

Cyclooxygenase catalyses a key step in prostaglandin biosynthesis, and recent work suggests that one isoenzyme, COX-2, has important roles in early stages of pregnancy; it also appears to be involved in the somewhat analogous process of colon tumor formation and spread.

The protein tyrosine phosphatase SHP-1 regulates integrin-mediated adhesion of macrophages.

The Src homology 2 domain phosphatase-1 (SHP-1) is a tyrosine phosphatase containing two amino-terminal SH2 domains and is expressed primarily by hematopoietic-derived cells [1]. The viable motheaten (Hcphme-v) mutant mice (mev) suffer from progressive inflammation due to a deficiency of SHP-1 enzyme activity [2,3] and die at 3-4 months of age from macrophage and neutrophil accumulation in the lung [4]. The mechanism by which SHP-1 deficiency leads to inflammation is unknown. We found that macrophages from mev mice adhered and spread to a greater extent than normal macrophages through alpha m beta 2 integrin-mediated contacts. Whereas macrophages deficient in the transmembrane tyrosine phosphatase CD45 (CD45-/-) spontaneously detached from alpha m beta 2 integrin contacts [5], cells deficient in both CD45 and SHP-1 did not. In SHP-1 deficient macrophages there was a 10-15-fold increase in D-3 phospholipid products of phosphatidylinositol (PI) 3-kinase. Concomitantly, there was a 2-5-fold increase in membrane-associated PI 3-kinase activity in mev macrophages relative to normal macrophages. Treatment of macrophages with the PI 3-kinase inhibitors wortmannin or LY294002 resulted in a dramatic detachment of cells, indicating that PI 3-kinase activity is required for adhesion. These data demonstrate that SHP-1 is necessary for detachment from alpha m beta 2 integrin-mediated contacts in primary macrophages and suggest that a defect in this pathway may contribute to inflammatory disease.

Multiple forms of an inositol polyphosphate 5-phosphatase form signaling complexes with Shc and Grb2.

BACKGROUND: Shc and Grb2 form a complex in cells in response to growth factor stimulation and link tyrosine kinases to Ras during the resulting signaling process. Shc and Grb2 each contain domains that mediate interactions with other unidentified intracellular proteins. For example, the Shc PTB domain binds to 130 kDa and 145 kDa tyrosine-phosphorylated proteins in response to stimulation of cells by growth factors, cytokines and crosslinking of antigen receptors. The Grb2 SH3 domains bind to an unidentified 116 kDa protein in T cells. We have identified three proteins, of 110 kDa, 130 kDa and 145 kDa, as a new family of molecules encoded by the same gene. In vivo studies show that these proteins form signal transduction complexes with Shc and with Grb2. RESULTS: The 130 kDa and 145 kDa tyrosine-phosphorylated proteins that associate with the Shc PTB domain were purified by conventional chromatographic methods. Partial peptide and cDNA sequences corresponding to these proteins, termed SIP-145 and SIP-130 (SIP for signaling inositol polyphosphate 5-phosphatase), identified them as SH2 domain-containing products of a single gene and as members of the inositol polyphosphate 5-phosphatase family. The SIP-130 and SIP-145 proteins and inositol polyphosphate 5-phosphatase activity associated with Shc in vivo in response to B-cell activation. By using an independent approach, expression cloning, we found that the Grb2 SH3 domains bind specifically to SIP-110, a 110 kDa splice variant of SIP-145 and SIP-130, which lacks the SH2 domain. The SIP proteins hydrolyzed phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5)-P3) and Ins (1,3,4,5)-P4, but not PtdIns (4,5)-P2 or Ins (1,4,5)-P3. CONCLUSIONS: These findings strongly implicate the inositol polyphosphate 5-phosphatases in Shc- and Grb2-mediated signal transduction. Furthermore, SIP-110, SIP-130 and SIP-145 prefer 3-phosphorylated substrates, suggesting a link to the phosphatidylinositol 3-kinase signaling pathway.

Fatty acid biosynthesis in human leukocytes.

Extracts from human leukocytes have been examined for the enzymes of de novo fatty acid biosynthesis. These extracts do not catalyze the synthesis of long-chain fatty acids because they lack acetyl CoA carboxylase, the first enzyme unique to the fatty acid synthesis pathway. Since these cells cannot form malonyl CoA, they are unable to synthesize long-chain fatty acids. This inability can be corrected by addition of either purified acetyl CoA carboxylase from rat liver or malonyl CoA to leukocyte extracts. The incorporation of acetate-1-(14)C into fatty acids by intact leukocytes is shown to represent chain elongation of preformed fatty acids rather than de novo synthesis by the fact that 60-100% of the label incorporated resides in the carboxyl carbon of the fatty acids formed.Both mature leukocytes and erythrocytes are unable to synthesize fatty acids because of a lack of acetyl CoA carboxylase even though both contain the other enzymes of fatty acid synthesis. It is possible that a precursor hematopoietic cell may have the capacity to synthesize fatty acids de novo. This hypothesis is supported by the finding of acetyl CoA carboxylase activity in extracts from human leukemic blast cells.The leukocyte fatty acid synthetase activity from malonyl CoA of a number of normal volunteers and of patients with a variety of hematologic diseases is reported.

Lipid metabolism in human platelets. II. De novo phospholipid synthesis and the effect of thrombin on the pattern of synthesis.

Washed human platelets were incubated with radioactive glycerol; the platelets were able to synthesize de novo the major phosphoglycerides including phosphatidic acid, phosphatidylinositol, phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl serine. The specific activities of the phosphoglycerides obtained after glycerol incorporation indicate that phosphatidic acid, phosphatidylinositol, and phosphatidyl choline are metabolically active relative to phosphatidyl ethanolamine and that formation of phosphatidyl serine occurs to a much more limited extent. When platelets were incubated with bovine thrombin, 1 U/ml, the pattern of glycerol incorporation into phospholipid was changed. There was a 3-fold decrease in the total incorporation into lipid in 30 min with a relative 5-fold decreased incorporation into phosphatidyl choline and phosphatidyl ethanolamine and a 5-fold increased incorporation into phosphatidyl serine. The increased incorporation into phosphatidyl serine. The increased incorporation into phosphatidyl serine was maximal within the first 2 min but was transient, since within 20 minutes, the rate returned to that seen in platelets incubated with glycerol alone. Purified human thrombin also produced this same effect on phospholipid synthesis in platelets. Trypsin produced effects on phosphoglyceride formation similar to those seen with thrombin, and the trypsin-induced effect was inhibited by prior incubation of trypsin with soybean trypsin inhibitor, suggesting that proteolysis may be required for the observed effects on phospholipid synthesis.

The measurement of thrombin in clotting blood by radioimmunoassay.

We have developed a radioimmunoassay for human thrombin using rabbit anti-human thrombin IgG. The assay can measure 2 ng thrombin/ml plasma, 500-fold more sensitive than clotting assays. Human prothrombin is less reactive in the assay than thrombin by at least four orders of magnitude, and there is no demonstrable cross-reactivity with human factor Xa, the clotting factor structurally most similar to thrombin. The assay does not detect thrombin bound to anthithrombin III. Using the assay, we have demonstrated that plasma from 20 normal subjects does not contain detectable thrombin. We measured thrombin generation in clotting blood in polypropylene tubes and observed that thrombin appears (approximately equal to 3 ng/ml) within 45 S-5 min after venipuncture. This material is thrombin, not intermediates of prothrombin activation, since it disappears after addition of heparin, which promotes thrombin antithrombin III complex formation. After a plateau of 2-10 min, there is further thrombin generation, which results in clotting after 15-27 min at a level of 40-50 ng thrombin/ml. The thrombin generated 9-25 min before clotting may activate factors V and VIII and stimulate platelet aggregation and release. In contrast, the cascade hypothesis assigns a role for thrombin only late in blood clotting. Radioimmunoassay of thrombin and other clotting factors will be useful for clinical and physiological studies of blood clotting especially since the assay seems specific for thrombin and is independent of other activities that affect bioassays.

The mechanism of the effect of aspirin on human platelets. I. Acetylation of a particulate fraction protein.

Aspirin (acetylsalicylic acid) inhibits platelet prostaglandin synthesis and the ADP- and collagen-induced platelet release reaction. The mechanism of the inhibitory effect is unknown but may involve protein acetylation, since aspirin acetylates a variety of substrates, including platelet protein. We have examined the relationship between protein acetylation and aspirin's physiologic effect on platelets. Suspensions of washed human platelets were incubated at 37 degrees C with (3H)aspirin, and incorporation of radioactivity into protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Exposure to (acetyl-3H)aspirin but not (aromatic ring-3H)aspirin resulted in radioactive labeling of three platelet proteins, suggesting that the drug acetylates these three proteins. The acetylation of two of the proteins (located in the supernatant fraction) was not saturable, implying that these reactions may not be physiologically significant. Acetylation of the third protein, approximate mol wt 85,000 (located in the particulate fraction), saturated at an aspirin concentration of 30 muM and was complete within 20 min. Platelets prepared from aspirin-treated donors did not incorporate any (acetyl-3H)aspirin radioactivity into the particulate protein for 2 days after drug treatment and did not show full pretreatment uptake of radioactivity for 12 days thereafter. The course of increasing incorporation of (acetyl-3H)aspirin radioactivity parralleled that of platelet turnover. Therefore, in addition to its saturability, acetylation of the particulate fraction protein by aspirin was permanent. In two respects, the inhibition of platelet function by aspirin correlates well with the aspirin-mediated acetylation of the particulate fraction protein. Both persist for the life-span of the aspirin-treated platelet, and both occur at a similar saturating aspirin concentration. The evidence suggests that the physiologic effect of aspirin on human platelets is produced by acetylation of a single protein located in the particulate fraction. The acetylated protein may be related to cyclo-oxygenase, the prostaglandin G2 biosynthetic enzyme.

The perturbation of thrombin binding to human platelets by anions.

Thrombin binds with high affinity to specific cell-surface receptors on washed human platelets. We present experiments indicating that thrombin binding correlates withe the release reaction when binding is perturbed by anions. Marked differences in the affinity of human 125I-thrombin for platelets wer observed in various isotonic buffers at pH 7.4. At low concentrations of thrombin (0.001-0.01 U/ml), binding was 5-fold greater in Tris-sodium acetate and 12-fold greater in Tris-sodium cacodylate than in Tris-sodium chloride. These anion-induced changes in 125I-thrombin binding paralleled changes in [14C] serotonin release when both parameters were measured in the same platelets. Thus, equivalent release occurred for equal amounts of thrombin bound in all buffers, even though the thrombin concentration varied by up to 30-fold. After approximately 100 molecules of thrombin bound per platelet, complete release occurred in all buffers in 2 min. The effect of anions was specific for the thrombin-receptor interaction as there was no corresponding effect on the binding of erythroagglutinating phytohemagglutinin (E-PHA) to platelets nor on E-PHA or collagen-induced serotonin release. The various anions did not alter platelet morphology as judged by electron microscopy. The anions had no effect on thrombin esterase catalytic activity. In addition, the total number of thrombin receptors per platelet was approximately the same in all buffers. Thus anions alter the affinity between platelet thrombin receptors and a site on thrombin distinct from the catalytic site. We conclude that the thrombin receptor is essential for thrombin-induced platelet reactions.

Inhibition of human platelet aggregation by monovalent antifibrinogen antibody fragments.

Monovalent goat antibody fragments (Fab) that were monospecific for human fibrinogen were isolated by affinity chromatography on fibrinogen-Sepharose and used as a direct probe for the involvement of fibrinogen in platelet aggregation and the release reaction. The antifibrinogen Fab inhibited aggregation of washed human platelets induced by thrombin (0.1-10 U/ml) by 50-95%, but had no effect on (14-C)-serotinin release and only a slight inhibitory effect on 125-I-thrombin binding to platelets. Inhibition of aggregation was not observed with nonimmune goat Fab or rabbit antihuman albumie bound tightly at saturation to surface fibrinogen molecules. After washing the platelets once to remove unbound Fab, aggregation by subsequently added thrombin was no longer inhibited. The antifibrinogen Fab inhibited the clotting of fibrinogen by thrombin but did not effect the rate of fibrinopeptide A release, indicating that the Fab inhibits clotting by interfering with the polymerization of fibrin monomers. Our experiments suggest that fibrinogen released from platelets is directly involved in thrombin-induced aggregation of washed platelets, perhaps through polymerization of fibrin monomers generated by proteolytic cleavage of released fibrinogen.

Thrombomodulin is present in human plasma and urine.

Thrombomodulin is an endothelial cell membrane protein that is a cofactor required for the rapid activation of plasma protein C. We now report that plasma and urine of normal subjects contains a modified form of thrombomodulin that is soluble. The levels measured by radioimmunoassay were 292 +/- 60 ng thrombomodulin/ml plasma and 102 +/- 38 ng thrombomodulin/ml urine. Thrombomodulin was isolated from both plasma and urine by immunoaffinity chromatography using a polyclonal anti-human thrombomodulin IgG column. The apparent molecular weight of soluble thrombomodulin was estimated by immunoblot analysis using 125I-monoclonal anti-thrombomodulin IgG. When run without 2-mercaptoethanol, soluble thrombomodulin appeared as two polypeptides, Mr = 63,000 and 54,000, while samples run with 2-mercaptoethanol migrated mainly at Mr = 85,000. These results imply that the soluble form of thrombomodulin is smaller than the cellular form, presumably because of a lack of the membrane-binding domain. Soluble thrombomodulin is similar to cellular thrombomodulin in its intrinsic protein C-activating cofactor activity as measured by antibody neutralization. The apparent Km for protein C was the same for cellular and soluble thrombomodulin, while the soluble form requires a higher concentration of thrombin (three- to fivefold) for one-half maximal activity than the cellular form. Thrombomodulin functional activity cannot be directly measured in plasma because of some inhibitory substance(s). The physiological significance of circulating and urinary thrombomodulin is presently obscure.

Patients with congenital factor V deficiency have decreased factor Xa binding sites on their platelets.

Human platelets have binding sites for plasma coagulation Factor X(a) that are available only after the platelet release reaction. Platelets from 15 normal donors bound 216+/-52 (SD) molecules of Factor X(a) per platelet. The association of Factor X(a) with its platelet surface receptor results in a 300,000-fold increase in the catalytic activity of Factor X(a) in forming thrombin from prothrombin. The turnover number for platelet-bound Factor X(a) was 1,850+/-460 mol thrombin/ml per min per mol Factor X(a) in experiments with platelets from 15 normal donors. Platelets from five patients with varying degrees of Factor V deficiency were investigated to determine whether or not coagulation Factor V participates in either aspect of the Factor X(a)-platelet interaction. The binding of Factor X(a) to platelets and the accompanying increase in rate of thrombin formation were either reduced in parallel or absent in each case with values ranging from 0 to 45% of control values. The apparent affinity of Factor X(a) from Factor V-deficient patients was normal when platelet binding was detected. The supernate from thrombin-treated control platelets, which contains Factor V activity, corrected the Factor X(a) binding deficiency of the platelets from three patients tested. Immunoreactive Factor V determined with an homologous antibody corresponded to the functional Factor V activity of platelets from one patient with Factor V deficiency, suggesting that the patient's platelets have a decreased amount of normal Factor V. The ability of platelets from the patients to bind Factor X(a) and increase the rate of thrombin formation correlated with the severity of each patient's bleeding disorder better than the plasma level of Factor V. The results indicate that Factor V is required for the Factor X(a)-platelet interaction and that thrombin formation at the platelet surface is important in normal hemostasis.

Isolation and quantitation of the platelet membrane glycoprotein deficient in thrombasthenia using a monoclonal hybridoma antibody.

We used the hybridoma technique to characterize further the platelet glycoprotein abnormality in Glanzmann's thrombasthenia. Spleen cells from Balb/c mice immunized with human platelets were fused to mouse myeloma cell line Sp2/0-Ag14. Hybridoma lines producing a variety of antiplatelet antibodies were isolated by hypoxanthine-aminopterin-thymidine selection and cloned, and purified monoclonal IgG from six lines was prepared. One of these lines, 8aB5-9, produced an antibody, Tab, that binds to a protein on normal but not thrombasthenic platelets. We isolated this protein from Triton X-100 solubilized normal platelet membranes by affinity chromatography on Tab-Sepharose. As determined by SDS polyacrylamide gel electrophoresis, the isolated protein is a complex of glycoproteins IIb and IIIa, because the two subunits comigrate with glycoproteins IIb and IIIa of whole platelets and show identical changes in mobility after disulfide bond reduction. We prepared (125)I-Tab to determine the number of glycoprotein IIb-IIIa complexes on normal and thrombasthenic platelets by a direct binding assay. Platelets from 17 normal donors bound 39,000+/-4,600 (SD) Tab molecules/platelet. Platelets from four patients with thrombasthenia lacked Tab binding sites (<5%). Five obligate and four presumed heterozygotes for thrombasthenia bound 24,500+/-5,800 Tab molecules/platelet. The platelet alloantigen, Pl(Al), is not that recognized by Tab, because platelets from three Pl(Al)-negative subjects bound Tab normally. Studies with the Tab antibody have (a) enabled quantitation of the number of glycoprotein IIb-IIIa complexes on normal platelet membranes, (b) demonstrated that thrombasthenic homozygotes lack and heterozygotes have a partial deficiency of this complex, and (c) made possible the isolation of this membrane protein which may be required for normal platelet aggregation and clot retraction.

Inhibition of platelet prostaglandin synthetase by oral aspirin.

Aspirin inhibits platelet function by permanently acetylating the cyclooxygenase that forms prostaglandins. We determined the sensitivity of platelets to aspirin in normal subjects by measuring [3H-acetyl]aspirin-susceptible cyclooxygenase in washed platelets obtained at various times after aspirin ingestion. A single 325-mg aspirin dose inactivated 89% of platelet cyclooxygenase. The inhibition persisted for 2 days suggesting that oral aspirin also inactivated megakaryocyte cyclooxygenase. Thereafter, active enzyme returned with a time-course reflecting platelet turnover (life-span 8.2+/-2 days). Single doses of 20-650 mg aspirin resulted in 34- greater than 95% inhibition after 24 h. Daily doses of 20-325 mg aspirin for brief periods produced 61- greater than 95% inactivation when measured 24 h after cessation of the drug. Platelet cyclooxygenase is more sensitive to inactivation by aspirin than enzyme in sheep seminal vesicles.

Lipid metabolism in human platelets. I. Evidence for a complete fatty acid synthesizing system.

Extracts from human platelets contain the enzymes of de novo fatty acid biosynthesis. The pattern of incorporation of acetate-1-(14)C into fatty acids by intact platelets indicates that these enzymes function in platelets. The level of acetyl-coenzyme A (CoA) carboxylase activity in extracts of platelets from normal subjects is 0.036 +/-0.01 mmumole of malonyl-CoA formed per min per mg of protein and that of fatty acid synthetase is 0.075 +/-0.016 mmumole of malonyl-CoA utilized per min per mg of protein. Thus, platelets are the only formed elements of the blood capable of de novo fatty acid synthesis. The capacity of platelets to synthesize fatty acids is similar to human liver based on enzyme activity per milligram of soluble protein.Acetyl-CoA carboxylase was purified 16-fold from platelet extracts, and this partially purified enzyme was compared to enzyme from rat liver. The two enzymes were similar with respect to requirements, substrate affinities, pH profile of activity, inhibition by malonyl-CoA, and aggregation in the presence of citrate. Thus, while fatty acid synthesis may serve a different function in platelets than in liver, the properties of acetyl-CoA carboxylase from these tissues are alike. The levels of the enzymes of fatty acid synthesis were significantly higher in platelets from splenectomized subjects than in controls. Acetyl-CoA carboxylase levels were 0.086 +/-0.027 mmumole of malonyl-CoA formed per min per mg of protein, and fatty acid synthetase levels were 0.151 +/-0.039 mmumole of malonyl-CoA utilized per min per mg of protein. These changes in the enzymes of fatty acid synthesis occurred promptly after splenectomy with peak values being reached within 7-10 days.