Critical analysis of a computer-assisted tutorial on ECG interpretation and its ability to determine competency.

BACKGROUND: We developed a computer-based tutorial and a posttest on ECG interpretation for training residents and determining competency. METHODS: Forty residents, 6 cardiology fellows, and 4 experienced physicians participated. The tutorial emphasized recognition and understanding of abnormal ECG features. Active learning was promoted by asking questions prior to the discussion of ECGs. Interactivity was facilitated by providing rapid and in-depth rationale for correct answers. Responses to questions were recorded and extensively analyzed to determine the quality of questions, baseline knowledge at different levels of training and improvement of grades in posttest. Posttest grades were used to assess improvement and to determine competency. RESULTS: The questions were found to be challenging, fair, appropriate and discriminative. This was important since the quality of Socratic questions is critical for the success of interactive programs. The information on strengths and weakness in baseline knowledge at different levels of training were used to adapt our training program to the needs of residents. The posttest revealed that the tutorial contributed to marked improvement in feature recognition. Competency testing distinguished between residents with outstanding grades and those who needed remediation. CONCLUSIONS: The strategy for critical evaluation of our computer program could be applied to any computer-based educational program, regardless of topic.

Integrins involved in the adhesion of megakaryocytes to fibronectin and fibrinogen.

We studied integrins involved in the adhesion of resting and activated megakaryocytes (MK) to fibronectin (FN) and fibrinogen (FGN). Guinea pig MK were isolated and in some experiments were activated by thrombin. MK adhering to FN or FGN coated on coverslips were quantitated by a computerized image analysis program. The binding of soluble human FN to MK was detected by Western blotting. Anti-integrin antibodies, disintegrins, and cyclic RGD peptides were used to identify integrins involved in the adhesion of MK to FN or FGN. Resting MK adhered to coverslips with immobilized FN. The adhesion of MK to FN was primarily inhibited by an anti-alpha5 antibody and EMF-10, a distintegrin highly specific for alpha5 beta1. However, the adhesion of MK to FN was not blocked by agents that inhibit alphaIIb beta3, alphav beta3 or alpha4 beta1. A beta1 activating antibody increased the number of MK bound to FN due to the activation of alpha5 beta1. The binding of soluble FN was also primarily inhibited by agents that block alpha5 beta1. Resting MK did not adhere to FGN. However, MK activated by thrombin did adhere to FGN. This binding was mediated by alphaIIb beta3, because binding was inhibited by bitistatin, a disintegrin, and a cyclic RGD peptide that are known to block this integrin. The binding of thrombin-activated MK to FN was mediated by both alpha5 beta1 and alphaIIb beta3 based on the additive effect of agents that inhibit these integrins. The study indicates that resting MK bind to FN but not to FGN and that alpha5 beta1 is the major integrin involved in the binding of MK to FN. Activated MK bind to FGN primarily by alphaIIb beta3. However, the binding of activated MK to FN is due to both alpha5 beta1 and alphaIIb beta3. The demonstration that alpha5 beta1 and that alphaIIb beta3 are involved in MK adhesion indicates that these integrins may have a role in MK maturation and platelet production.

Synthesis and secretion of von Willebrand factor and fibronectin in megakaryocytes at different phases of maturation.

Our goals have been to define the biochemical characteristics of megakaryocytes during maturation that are critical for platelet assembly and release into the circulation and to introduce biochemical markers for megakaryocytes. To achieve these goals, we have studied fibronectin (FN) and von Willebrand factor (vWF), which are large adhesive proteins that are synthesized by megakaryocytes, stored in alpha granules, and thought to have a fundamental role in hemostasis. The study demonstrated that vWF is primarily synthesized in mature megakaryocytes, which synthesized 7.5 times more vWF than immature megakaryocytes. Brefeldin A, which blocks the exit of proteins from the rough endoplasmic reticulum (RER), inhibited the formation of vWF multimers but did not affect the synthesis of monomers and dimers in mature megakaryocytes. These data are consistent with the formation of vWF dimers in the RER and the assembly of vWF multimers in the trans- and post-golgi. The synthesis of both the 260-kD and 275-kD pro-vWF was detected. However, the synthesis of 275-kD pro-vWF and 220-kD mature vWF was only evident after 2 hours, suggesting that the transit time of nascent vWF through the RER is about 2 hours. Constitutive secretion of vWF was demonstrated in megakaryocytes. About 14.5% and 4.6% of synthesized vWF was secreted by mature and immature megakaryocytes, respectively. In contrast, the synthesis of FN monomers and dimers was established in immature megakaryocytes, and their synthesis in mature megakaryocytes was very similar. Constitutive secretion of FN was not seen in megakaryocytes. Brefeldin A did not inhibit the synthesis of FN dimers; thus, formation of FN dimers occurs in the RER. The demonstration that vWF and FN are synthesized at different phases of megakaryocyte maturation and that only vWF is constitutively secreted by megakaryocytes provides new information relevant to alpha granule formation and possibly bone marrow matrix assembly.

The synthesis and localization of alternatively spliced fibronectin EIIIB in resting and thrombin-treated megakaryocytes.

There are several species of alternatively spliced fibronectin (FN). One of these, FN EIIIB, is primarily present in embryonic and in proliferating and migrating cells and is believed to be important for cell maturation. We have studied the synthesis, localization, and secretion of this FN isoform in isolated guinea pig megakaryocytes, nonmegakaryocytic bone marrow cells, and platelets. There was 7.5 times more general FN in megakaryocytes than in nonmegakaryocytic cells based on the analysis of equivalent amounts of protein. FN EIIIB was detected by Western blotting in megakaryocytes but not in nonmegakaryocytic cells present in bone marrow. Neither megakaryocytes nor platelets secreted FN EIIIB, while megakaryocytes secreted 25.3% +/- 4.6% general FN and platelets secreted about 61% general FN in response to thrombin. Analysis of immunostained cells by confocal microscopy revealed that FN EIIIB had been redistributed to the surface of megakaryocytes in response to thrombin. Synthesis was studied by metabolic labeling, and megakaryocytes were shown to synthesize FN and FN EIIIB. Thus, megakaryocytes and platelets are among a small number of adult cells and tissues that synthesize and contain FN EIIIB. The expression of FN EIIIB on the megakaryocyte surface may influence migration and maturation.

The acylation of megakaryocyte proteins: glycoprotein IX is primarily myristoylated while glycoprotein Ib is palmitoylated.

The acylation of megakaryocyte proteins was studied with special emphasis on the myristoylation and palmitoylation of the glycoprotein (GP) Ib complex. Guinea pig megakaryocytes were purified and separated into subpopulations at different phases of maturation. Cells were incubated with [3H]myristate, [3H]palmitate, or [3H]acetate to study endogenous protein acylation. Cycloheximide was used to distinguish between cotranslational and posttranslational acylation and hydroxylamine to distinguish between thioester and amide linkages. After incubations, delipidated proteins or GPIb complex subunits, immunoprecipitated with PG-1, AN-51 or FMC-25 monoclonal antibody, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and assessed by fluorography. Radiolabeled fatty acids bound to GPIX and GPIb were also analyzed by high pressure liquid chromatography (HPLC) and scintillation spectrometry. With [3H]myristic acid and [3H]acetate, GPIX was found to be a major myristoylated protein in megakaryocytes and CHRF-288 cells. Myristic acid was linked to GPIX by an amide bond, and this process occurred cotranslationally. With [3H]acetate, GPIb was primarily palmitoylated, but with [3H]myristate, GPIb was acylated with about equal mounts of myristic acid and palmitic acids. Both fatty acids were linked to GPIb by thioester bonds, and acylation was posttranslational. The myristoylation of GPIX while the palmitoylation of GPIb occurred throughout megakaryocyte maturation. Myristoylation and palmitoylation may have different functions relevant to the assembly of the GPIb complex in megakaryocytes.

Fibronectin isoforms in megakaryocytes.

Our studies have shown that megakaryocytes (MK) can synthesize fibronectin (FN) and alternatively spliced fibronectin, FN EIIIB. FN EIIIB is primarily present in embryonic, proliferating and migrating cells, and thought to be important for cell maturation. MK, but not nonmegakaryocytic bone marrow cells, contain FN EIIIB and thus, MK and platelets are among a small number of adult cells and tissues that synthesize and contain FN EIIIB. Thrombin can induce the secretion of general FN, but does not cause the secretion of FN EIIIB into the medium. Analysis of immunostained cells by confocal microscopy revealed that both general FN and FN EIIIB accumulated on the MK surface following thrombin treatment. Thus, FN EIIIB can be released only to be bound to the MK surface. The expression of FN EIIIB on the MK surface may have a unique role in MK migration and maturation.

The expression of acetyl coenzyme A carboxylase is related to megakaryocyte maturation.

We have previously demonstrated that de novo fatty acid synthesis predominantly occurs in later phases of megakaryocyte maturation. Therefore we have investigated the expression of fatty acid synthase (FAS) and acetyl coenzyme A carboxylase (ACC), key enzymes for fatty acid synthesis in megakaryocytes at different phases of maturation. Immature and mature megakaryocytes were isolated. Guinea pig-specific FAS and ACC cDNA probes were prepared by reverse transcriptase reaction-polymerase chain reaction (RT-PCR). The probes were used to assess the expression of mRNA for ACC and FAS by Northern blotting. The hybrids were quantitated by densitometry. Endogenous megakaryocyte ACC was quantitated by virtue of its biotin content by Western blotting with streptavidin and enhanced chemiluminescence (ECL). The ratio of ACC mRNA between mature and immature megakaryocytes was 2.43 +/- 0.86, and the ratio of FAS mRNA was 0.50 +/- 0.13 (mean +/- SD, n = 4). The ratio of endogenous ACC in mature and immature megakaryocytes was 1.96 +/- 0.62 (n = 6). The study showed that the FAS mRNA was expressed in all phases of megakaryocyte maturation. However, both mRNA for ACC and endogenous ACC were demonstrated primarily in mature megakaryocytes. Thus de novo fatty acid synthesis in megakaryocytes may depend on the expression of ACC in mature cells. The expression of ACC occurs during the terminal phases of megakaryocyte maturation and may be a marker of megakaryocyte maturity.

P-selectin mRNA is expressed at a later phase of megakaryocyte maturation than mRNAs for von Willebrand factor and glycoprotein Ib-alpha.

The assembly of alpha-granules occurs exclusively in megakaryocytes because platelets have limited capacity for the synthesis of macromolecules. Thus far, alpha-granule development in megakaryocytes has been primarily evaluated by ultrastructural studies. The aim of the study was to obtain molecular and biochemical evidence for the expression of selected alpha-granule proteins in megakaryocytes. Guinea pig megakaryocytes were purified and separated into subgroups at different phases of maturation by the Celsep procedure (Schick et al. Blood 1989;73:1801-8). Guinea pig-specific probes for P-selectin, von Willebrand factor (vWF), glycoprotein Ib-alpha (GpIb-alpha), and phosphoglycerate kinase were prepared by using the polymerase chain reaction. By Northern blot analysis, P-selectin messenger ribonucleic acid (mRNA) was primarily expressed in the mature megakaryocyte Celsep subgroup, whereas vWF and GpIb-alpha mRNA were expressed at all phases of megakaryocyte maturation. In situ hybridization confirmed that P-selectin mRNA was primarily expressed at later stages of cytoplasmic maturation: 14% +/- 6.2% of stage I, 35.5% +/- 6.1% of stage II, 72% +/- 5.2% of stage III, and 47.0% +/- 3.3% of stage IV megakaryocytes expressed P-selectin mRNA. Thus, the expression of mRNA for P-selectin appeared to peak in stage III cells. In contrast vWF mRNA was expressed in immature megakaryocytes and persisted throughout megakaryocyte maturation. In situ hybridization did not demonstrate a relationship between the expression of mRNA for P-selectin or vWF with megakaryocyte ploidy.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasminogen activator inhibitor-1 mRNA is expressed in platelets and megakaryocytes and the megakaryoblastic cell line CHRF-288.

Plasminogen activator inhibitor-1 (PAI-1) is present in the platelet alpha-granule and is released on platelet activation. Platelet PAI-1 could either be synthesized by the megakaryocyte or taken up from the plasma. In this report we confirm the presence of PAI-1 protein in human megakaryocytes by Western blot analysis and show its synthesis in guinea pig megakaryocytes by metabolic labeling. We document the presence of PAI-1 mRNA in human platelets and show a 3-kb mRNA species on Northern blot analysis of guinea pig megakaryocytes. Neither untreated CHRF-288 cells, a megakaryoblastic cell line, nor human erythroleukemia (HEL) cells expressed PAI-1 mRNA. Phorbol ester (phorbol 12-myristate 13-acetate, 160 nM) treatment of CHRF-288 and HEL cells for 4 days induced PAI-1 mRNA expression in CHRF-288 cells but not in HEL cells. These studies show that PAI-1 is synthesized by megakaryocytes. Megakaryocytes most likely determine the PAI-1 content of platelets and thereby establish the antifibrinolytic potential of the platelet.

Development of storage granules during megakaryocyte maturation: accumulation of adenine nucleotides and the capacity for serotonin sequestration.

The specific release of platelet-dense body constituents such as adenine nucleotides and serotonin has a fundamental role in hemostasis. The content of adenine nucleotides in platelet dense bodies is probably established in megakaryocytes, but very little is known about this process. To gain a better understanding of platelet development, we studied the storage and metabolic pools of adenine nucleotides and the capacity for serotonin sequestration in storage granules during megakaryocyte maturation. Megakaryocytes were isolated from guinea pig bone marrow and separated in subgroups at different phases of maturation. The sequestration of adenine nucleotides and serotonin in storage granules was assessed by using calcium ionophore to induce secretion under nonlytic conditions, and metabolic pool adenine nucleotides were evaluated by using digitonin under controlled lysis conditions. The study showed that there were similar amounts of cytoplasmic adenosine triphosphate (ATP) in the mature and immature fractions based on digitonin-induced controlled lysis (1.26 +/- 0.37 nmol/microgram phosphorus vs 1.23 +/- 0.44 nmol/microgram phosphorus). However, only the mature cells contained a significant amount of storage pool ATP (0.41 +/- 0.19 nmol/microgram phosphorus vs 0.05 +/- 0.05 nmol/microgram phosphorus) released in response to A23187. The subgroups of megakaryocytes contained equal amounts of total adenosine diphosphate (ADP) extractable with ethanol (0.49 +/- 0.14 nmol/microgram phosphorus vs 0.55 +/- 0.50 nmol/microgram phosphorus) but only mature cells contained ADP in the storage granules (0.25 +/- 0.13 nmol/microgram phosphorus vs 0.07 +/- 0.05 nmol/microgram phosphorus).(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of olive oil, hydrogenated palm oil, and omega-3 fatty acid-enriched diets on megakaryocytes and platelets.

Unsaturated fatty acids are thought to prevent thrombotic and arteriosclerotic disease, whereas saturated fatty acids are thought to increase the incidence of these disorders. However, the effects of these diets on megakaryocytes and platelets are not well understood. We compared the effects of diets enriched with 8.4% olive oil, 8.4% hydrogenated palm oil, or 10.2% omega-3 fatty acid ethyl esters on guinea pig megakaryocytes and platelets. In plasma, changes in fatty acid composition reflected the composition of each diet. However, in platelets and megakaryocytes, hydrogenated palm oil induced a decrease in 16:0 and an increase in 18:2 while the olive oil diet caused a marked increase in 18:1 and a decrease in most other fatty acids. The differences in the effects of the diets on cellular versus plasma fatty acids suggest that megakaryocytes and platelets have an extensive capacity to regulate their fatty acid composition. Thrombocytosis occurred with the omega-3 fatty acid-enriched diet: 12.9 +/- 1.78 x 10(5) compared with 7.45 +/- 1.08 x 10(5) platelets per microliter of platelet-rich plasma in control animals. There was an increase in megakaryocyte size, ploidy, and morphological stage (cytoplasmic maturation) with the omega-3 fatty acid-enriched diet but not with the other diets. The omega-3 fatty acid-enriched diet decreased platelet thromboxane production while the other diets had no effect. Platelet hypersensitivity was suggested in collagen aggregation studies with olive oil but not with the hydrogenated palm oil diet. Although saturated fatty acid diets are thought to be atherogenic, this diet had no affect on platelet function.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in thromboxane A2 synthesis by megakaryocytes and platelets.

The capacity for thromboxane A2 synthesis in response to exogenous arachidonic acid, calcium ionophore A23187, thrombin, and collagen was studied during megakaryocyte maturation. Studies were performed in (1) isolated megakaryocytes not separated, (2) isolated megakaryocytes separated into subgroups at different stages of maturation, and (3) washed platelets. When comparisons were based on equal amounts of cell protein (10(5) megakaryocytes vs 10(8) platelets), isolated megakaryocytes, not separated into subgroups, responded to exogenous arachidonic acid with synthesis of thromboxane A2 equal to that of platelets from the same animals at their respective times of maximum synthesis (30 minutes vs 10 minutes). In similar fashion, megakaryocytes and platelets synthesized thromboxane A2 from endogenous arachidonic acid at the same minimum concentration of A23187, 0.1 mumol/L, and showed equal maximum synthesis at 1 mumol/L (167 +/- 9 pmol and 150 +/- 18 pmol, respectively). In contrast, maximum thromboxane A2 synthesis in response to thrombin (10 U/ml) was three times higher in platelets than in megakaryocytes (230 +/- 15 pmol and 74 +/- 5 pmol, respectively), and synthesis in response to collagen (20 micrograms/ml) was 20 times higher in platelets (130 +/- 20 pmol vs 7 +/- 1.2 pmol). When synthesis was studied in isolated megakaryocytes at different stages of maturation, the capacity for thromboxane A2 synthesis was established in immature megakaryocytes but was not fully developed in the most immature megakaryocytes. Synthesis in response to thrombin was not significantly enhanced by megakaryocyte maturation. Thus the ability to metabolize arachidonic acid occurs early during megakaryocyte maturation, but the ability to respond to thrombin and collagen is only fully established in platelets.

Fatty acylation of heparan sulfate proteoglycan from human colon carcinoma cells.

A number of transmembrane proteins have been recently reported to be modified by the covalent addition of saturated fatty acids which may contribute to membrane targeting and specific protein-lipid interactions. Such modifications have not been reported in cell-associated heparan sulfate proteoglycans, although these macromolecules are known to be hydrophobic. Here, we report that a cell surface heparan sulfate proteoglycan is acylated with both myristate and palmitate, two long-chain saturated fatty acids. When colon carcinoma cells were labeled with [3H]myristic acid, a significant proportion of the label was shown to be specifically incorporated into the protein core of the proteoglycan. Characterization of fatty acyl moiety in the purified proteoglycan by reverse-phase high pressure liquid chromatography revealed that approximately 60% of the covalently bound fatty acids was myristate. We further show that this relatively rare 14-carbon fatty acid was bound to the protein core via a hydroxylamine- and alkali-resistant amide bond. The remaining 40% was the more common 16-carbon palmitate, which was bound via a hydroxylamine- and alkali-sensitive thioester bond. Palmitate appeared to be added post-translationally and derived in part from intracellular elongation of myristate, a process that occurred within the first two hours and was insensitive to inhibition of protein synthesis. Acylation of heparan sulfate proteoglycan represents a novel modification of this gene product and could play a role in a number of biological functions including specific interactions with membrane receptors and ligand stabilization.

Composition and synthesis of glycolipids in megakaryocytes and platelets: differences in synthesis in megakaryocytes at different stages of maturation.

The composition and synthesis of megakaryocyte and platelet glycolipids were compared since these lipids are thought to be important for biologic activities such as adhesion and maturation. Highly purified guinea pig megakaryocytes at different stages of maturation and platelets were studied. Glycolipids and gangliosides were extracted, separated by thin-layer chromatography, and the carbohydrate content was analyzed by gas-liquid chromatography (GLC). Synthesis of ceramides and glycolipids was determined by the incubation of megakaryocytes with [14C]acetate, [3H]palmitic acid, and [3H]galactose. A major neutral glycolipid present in guinea pig megakaryocytes and platelets was identified as asialoGM2 by selective enzymatic hydrolysis with beta-N-acetylhexosaminidase, alpha-galactosidase and endo-beta-galactosidase, and carbohydrate analysis by GLC. Trace amounts of asialoGM1 were detected immunologically. The cells also contained glucosyl ceramide and lactosyl ceramide. Several ganglosides were detected of which one was identified as GM1 by its reaction with the beta-subunit of cholera toxin and by the identification of an asialoGM1 core with anti-asialoGM1 antibody after desialylation. The synthesis of ceramides from palmitic acid and acetate was 5 and 10 times greater, respectively, in megakaryocytes than in platelets. Ceramide and glycolipid synthesis from palmitic acid occurred primarily in immature megakaryocytes while synthesis from acetate occurred primarily in more mature megakaryocytes. The glycosylation of ceramides from galactose was 42 times greater in megakaryocytes than in platelets. Thus, ceramides and glycolipids are primarily synthesized in megakaryocytes, but platelets retain the capacity to synthesize significant amounts of free ceramides. The glycosylation of free ceramides occurs almost exclusively in megakaryocytes and only in trace amounts in platelets. These data indicate that megakaryocytes determine the composition of glycolipids in platelets and that there is considerable compartmentalization of glycolipid synthesis and membrane assembly at various stages of megakaryocytes development.

Lower motor neuron disease in a patient with autoantibodies against Gal(beta 1-3)GalNAc in gangliosides GM1 and GD1b: improvement following immunotherapy.

We followed a patient with a lower motor neuron form of motor neuron disease whose neurologic disorder improved following immunotherapy. The patient did not have an M protein but did have IgM antibodies to ganglioside GM1 detectable at serum titers of 1:2,000 by ELISA. These antibodies were found only in the IgM fraction with lambda light chains and immunoreacted with GD1b and Gal (beta 1-3) GalNAc.

Effects of marine oil-enriched diets on guinea pig megakaryocyte and platelet lipids: effects on thromboxane synthesis and platelet function.

The effects of marine oil-enriched diets on the fatty acid composition of lipids in guinea pig megakaryocytes (MK) and platelets were studied to obtain a better understanding of the mechanisms for changes in platelet fatty acid composition and platelet function. Animals were fed 2%, 5% and 10% menhaden oil-enriched diets for up to 35 days. Platelets and MK were isolated and MK subpopulations at various stages of development were prepared. The diets did not cause a change in the cholesterol/phospholipid ratio in MK or platelets. The diets induced a dose related incorporation of eicosapentaenoic (20:5) and docosahexaenoic acid (22:6) and an associated decrease in linoleic acid (18:2) in both MK and platelets. However, there was a considerable greater depression of 20:4 in platelets than in MK. These changes were evident with 2% marine oil diets and maximal with 10% diets. Half maximal changes in fatty acid composition occurred after 3 days and maximal changes at 10 days after the initiation of the diets and no further changes occurred up to 35 days. Based on percent of total fatty acids in individual phospholipids, 20:5 had been primarily incorporated into phosphatidylethanolamine (PE) and phosphatidylinositol (PI) and 22:6 into PE and phosphatidylserine (PS) in both MK and platelets. 18:2 was decreased in all phospholipids. 20:4 was decreased only in PI in MK while 20:4 was decreased in PE, PI and PS in platelets. In animals on the 10% marine oil diet, more 20:5 and 22:6 were incorporated into mature than immature MK but the greatest amount of 20:5 and 22:6 had accumulated in platelets. Ingestion of marine oil-enriched diets did not cause thrombocytopenia or affect MK maturation based on the analysis of morphologic stage, ploidy or size. Marine oil-enriched diets caused a decrease in thromboxane synthesis in response to thrombin and calcium ionophore in platelets and MK at all stages of maturation. In platelet-rich plasma, collagen induced platelet aggregation, ATP secretion and thromboxane synthesis were decreased to a greater degree at 35 days than 10 days. Thus, the study indicates that the ingestion of marine oil-enriched diets resulted in the compartmentalization of 20:5 and 22:6 in acidic phospholipids in mature MK and platelets. The observation that marine oil-enriched diet induced maximal changes in lipid composition in MK and platelets within 10 days but caused progressive inhibition of platelet function for up to 35 days indicates that as yet undefined membrane and cellular changes may occur at later time points.

Lipid composition and metabolism in megakaryocytes at different stages of maturation.

The lipid composition and metabolism of isolated guinea pig megakaryocyte subgroups at various stages of maturation were investigated. Three groups were studied: 1) 67% of megakaryocytes in Group A were immature; 2) Group B was heterogeneous and contained both immature and mature subgroups of megakaryocytes; 3) 92% of megakaryocytes in Group C were mature. Lipid composition was determined by thin-layer chromatography, lipid-phosphorus, and gas-liquid chromatography. Cholesterol, ceramide, and de novo fatty acid synthesis were evaluated with [14C]acetate. [14C]Glycerol was used to assess de novo phospholipid synthesis. 14C-Labeled fatty acids were used to evaluate fatty acid uptake. The phospholipid and cholesterol content was found to be four times greater in mature megakaryocytes than that in immature megakaryocytes, which paralleled the protein content and volume of mature and immature cells. The cholesterol-phospholipid ratio was similar and there were no differences in the phospholipid species in the three groups. Phospholipid and cholesterol synthesis were established in immature megakaryocytes and persisted at about the same level in mature megakaryocytes. The uptake of arachidonic and palmitic acids also occurred primarily in immature cells, while the de novo synthesis of palmitic acid occurs predominantly in mature megakaryocytes. There was an inverse relationship between the uptake of exogenous palmitic acid and fatty acid synthesis, but the uptake of palmitic acid primarily inhibited fatty acid synthesis in mature megakaryocytes. There were differences in the acylation of phospholipid species with arachidonic acid in megakaryocytes at different stages of maturation since the acylation of phosphatidylcholine occurred primarily in immature megakaryocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of guinea pig megakaryocyte subpopulations at different phases of maturation prepared with a Celsep separation system.

We introduce a new method for preparing subpopulations of guinea pig megakaryocytes (MK). MK, partially purified by a density gradient, were separated according to size by sedimentation, starting as a monolayer, in an albumin gradient at unit gravity. Twenty-two fractions were collected. Cells were cytocentrifuged, ploidy was assessed by microdensitometry, and small MK were identified with anti-von Willebrand factor (vWF) immunoglobulin. Immaturity was assessed by uptake of 3H thymidine and synthesis of proteoglycans from 35S sulfate. About 88% of cells in fractions 2 through 18 were MK, of which 90% were viable. Fractions containing the largest cells were composed of 98% stage III and IV MK; fractions with the smallest cells contained up to 80% stage I and II MK. Six MK classes were isolated: immature cells, both stage I and II cells, at either the 8N, 16N or 32N ploidy class; mature cells, both stage III and IV cells, at either the 8N, 16N or 32N ploidy class. The fractions were pooled into three groups: (a) 8% of MK in group 1, fractions 2 through 11, were immature, and group 1 was composed of 92% of 16N and 32N mature classes; (b) 29% of MK in group 2, fractions 12 through 15, were immature, and group 2 was composed of 52% 16N mature, 24% 16N immature, and 13% 8N mature classes; 67% of MK in group 3, fractions 16 through 18, were immature, and group 3 contained 51% 8N immature, 14% 16N immature, and 18% mature 16N classes. The mean protein content of the three groups was 1.251, 0.624, and 0.284 mg/10(6) MK, respectively. Nine percent of cells in group 3 but no cells in group 1 took up large amounts of 3H thymidine. The synthesis of high-molecular-weight (high-mol-wt) proteoglycans in group 3 and synthesis of lower mol wt proteoglycans in groups 1 and 2 provided further evidence for differences in MK maturity. Thus, the method can isolate MK subpopulations that are viable and can be used to investigate the biochemical characteristics of MK at different phases of maturation.

Arachidonic acid is preferentially incorporated by immature megakaryocytes.

Megakaryocytes are bone marrow cells responsible for the synthesis of circulating platelets. Subgroups of megakaryocytes can be recognized on the bases of morphologic stage, DNA content (ploidy), and size. However, the physiologic roles and biochemical characteristics of these subgroups have not been defined. We have investigated arachidonic acid uptake in megakaryocyte subgroups. Purified guinea pig megakaryocytes were incubated with tritiated arachidonic acid, cytocentrifuged, and subjected to autoradiography. Eight hundred megakaryocytes were analyzed in four experiments by four parameters: (1) grains per cell (arachidonic acid uptake), (2) ploidy, (3) morphologic stage, and (4) size. Lipids were extracted from aliquots of megakaryocytes subjected to autoradiography and were assessed for arachidonic acid metabolism. Arachidonic acid had not been overtly metabolized nor oxygenated, and thus, radioactivity represented arachidonic acid. The capacity for arachidonic acid uptake was dependent primarily on megakaryocyte cytoplasmic maturation as judged by morphologic stage. Stage I and II megakaryocytes, immature cells, took up three times more arachidonic acid than stage III megakaryocytes and four times more than stage IV megakaryocytes, mature megakaryocytes, when calculated as uptake per cell. When uptake was calculated per megakaryocyte area, an approximation of megakaryocyte volume, arachidonic acid uptake was six times greater in stage I and II than in stage III and IV megakaryocytes. Arachidonic acid uptake was related to ploidy to a lesser extent, because 8N megakaryocytes took up two times more arachidonic acid than 32N megakaryocytes when calculated as uptake per cell area. Arachidonic acid uptake was independent of megakaryocyte size when assessed within each morphologic stage group and ploidy class.(ABSTRACT TRUNCATED AT 250 WORDS)

Covalent crosslinking of human coagulation factor V by activated factor XIII from guinea pig megakaryocytes and human plasma.

Coagulation factor V (FV) has been shown to be synthesized in both the liver and megakaryocytes. We now present evidence that FV can be covalently crosslinked by an enzyme originating from megakaryocytes to form polymeric multimers of factor V. The guinea pig megakaryocyte enzyme appears to be factor XIIIa since the FV-crosslinking activity (1) had an absolute requirement for Ca++, (2) was completely inhibited by iodoacetamide, 5,5'-dithiobis- (2-nitrobenzoic acid), p-chloromercuribenzene sulfonic acid, and N-ethylmaleimide, all known alkylators of the thiol group at the active site of the factor XIIIa, (3) was blocked by known pseudoamine donor substrates of factor XIIIa including dansylcadaverine and putrescine, and (4) could be directly demonstrated in the guinea pig megakaryocyte lysate by a specific activity staining procedure. No tranglutaminase was detected in guinea pig megakaryocytes in contrast to red cells and liver. A similar pattern of covalent crosslinking of human FV by purified activated human plasma factor XIII was also demonstrated. Analysis of the crosslinked products of FV formed by the guinea pig enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicates the formation of intermediate as well as higher molecular weight polymers, suggesting that the crosslinking is a stepwise polymerization process.