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Fibrinogen synthesis was investigated in guinea pig megakaryocytes. Purified megakaryocytes were incubated with 35S-methionine in methionine-free incubation medium for 18 hours. Newly synthesized fibrinogen in megakaryocyte lysates enriched with purified carrier guinea pig fibrinogen was immunoprecipitated with a specific anti-guinea pig fibrinogen antiserum produced in rabbits. Proteins in the immunoprecipitates were analyzed with a 3.5% to 10.0% gradient polyacrylamide slab gel electrophoresis and autoradiography. Radioactivity was detected in a protein band of 340,000 daltons. In order to verify fibrinogen synthesis, immunoprecipitate was analyzed by two-dimensional slab gel electrophoresis: (1) the first dimension separated unreduced fibrinogen using a 3.5% to 10.0% gradient gel; (2) following reduction by 2-β-mercaptoethanol, fibrinogen chains were separated in the second dimension using a 10% gel. Alpha, beta, and gamma fibrinogen chains, which represented carrier guinea pig plasma fibrinogen, were visualized by Coomassie brilliant blue. Autoradiography identified the incorporation of radioactivity into the three fibrinogen chains. In control experiments, immunoprecipitates, produced by exposing megakaryocyte lysates to preimmune rabbit serum and goat anti-rabbit IgG, were also analyzed by the two-dimensional gel system. Radioactivity was not detected in sites corresponding to the migration of fibrinogen subunits. The study demonstrates that isolated guinea pig megakaryocytes can synthesize fibrinogen. The electrophoretic mobility of newly synthesized fibrinogen and subunits is similar to that of guinea pig plasma fibrinogen. © 1985 by Grune & Stratton, Inc.

The characteristics of the surface of guinea pig megakaryocytes were investigated with wheat germ agglutinin (WGA). Purified guinea pig megakaryocytes and platelets were incubated with WGA conjugated with rhodamine, cytocentrifuged, and then exposed to Chromomycin A3 for the assessment of ploidy. The fluorescence emission of the DNA-Chromomycin complex was similar to that of fluorescein, and thus both rhodamine-WGA and Chromomycin A3 fluorescence could be analyzed in the same cell. Ploidy was assessed by microdensitometry of Chromomycin A3 fluorescence. Eight hundred megakaryocytes were analyzed by four parameters: (1) labeling by WGA, (2) ploidy, (3) morphological stage, and (4) size. The results were analyzed by a computer-assisted program. Although all platelets had reacted with WGA, only about half of the isolated megakaryocytes had been labeled by the lectin. The analysis of the megakaryocytes that had reacted revealed that 72% of stage III and 77% of stage IV megakaryocytes as compared with 35% of stage I and 29% of stage II cells had been labeled by the lectin. WGA reacted with 44% of 8N megakaryocytes and 60% and 59% of 16N and 32N cells. However, WGA labeling was independent of megakaryocyte size. The digestion of 15% and 48% of megakaryocyte sialic acid with neuraminidase from Vibrio cholera resulted in a 67% and 89% decrease in the binding of rhodamine-WGA, respectively, as determined by microdensitometry. The study indicated that sialic acid serves as a receptor for WGA and that sialoglycoproteins and possibly gangliosides become exposed on the surface of mature megakaryocytes. WGA can recognize mature megakaryocytes by biochemical criteria and the assessment of lectin binding could complement the morphological staging of megakaryocytes.

There are several species of alternatively spliced fibronec-tion (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.

Because thrombus formation at the site of endothelial injury has been thought to be a critical step in the pathogenesis of bacterial endocarditis, the effect of aspirin on experimental valvular thrombosis and bacterial endocarditis in rabbits was evaluated. Aortic valvular injury and thrombosis were induced in aspirin-treated and control rabbits with intracardiac catheters. A subsequent inoculation of Streptococcus viridansresulted in the development of infective endocarditis. Rabbits were sacrificed as early as 6 hr, and the effectiveness of aspirin was determined by the weight of the sterile vegetations and the quantitation of bacteria in the thrombotic vegetation. Aspirin, in levels in excess of 50 mg/dl did not attenuate the evolution of infective endocarditis, since the formation of sterile thrombotic vegetation and bacterial endocarditis in aspirin-treated rabbits was similar to those in controls.

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 Xllla 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 Xllla, (3) was blocked by known pseudoamine donor substrates of factor Xllla including dansylcadaverine and putrescine, and (4) could be directly demonstrated in the guinea pig megakaryocyte lysate by a specific activity staining procedure. No tranglu-taminase 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.

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/106 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.© 1989 by Grune & Stratton, Inc. 0006-4971/89/7307-0012$3.00/0

The activation of prekallikrein by factor XII fragments (XIIf), during incubation in plastic tubes was previously noted to be increased by high molecular weight (HMW) kininogen as well as other plasma proteins. In this report, we investigated the mechanism responsible for this increase. Although we confirmed that HMW kininogen, bovine serum albumin, fibrinogen, cold insoluble globulin, and mixed phospholipids apparently increased prekallikrein activation, we found that the product of prekallikrein activation (kallikrein) lost substantial activity in less than 0.5 min after exposure to a variety of fresh surfaces. This loss was partially prevented by the presence of various proteins and phospholipids. Similar protection against inactivation of XIIf, the enzyme in this reaction, was also found. In contrast, no loss of the substrate, prekallikrein, was observed during incubation. The loss of kallikrein activity was found to be proportional to the surface area of the incubation vessel as well as the concentration of kallikrein. Further loss of kallikrein activity could also be prevented by pretreating the vessel with kallikrein. We therefore conclude that various substances apparently affect prekallikrein activation in a purified system by preventing the enzyme and product in the reaction mixture from losing activity due to adsorption to a surface.

The lipid composition of platelet cytoskeletons was analyzed. Triton X-100 (0.5%) was used to prepare cytoskeletons from thrombin-treated platelets. The lipid/protein ratio of platelet cytoskeletons was 0.260 and the phospholipid/protein ratio was 0.177, which were comparable to the ratios present in platelets. However, there was a selective enrichment of platelet lipids in platelet cytoskeletons. Only 2 of the 5 major platelet phospholipids were detected. About 14% platelet sphingomyelin and 2% platelet phosphatidylcholine were present in platelet cytoskeletons. Only 1 of the 4 platelet neutral glycolipids, trihexosyl ceramide, was detected and was about 7% of that in intact platelets. Two percent of platelet hematoside, the predominant ganglioside in platelets, was found in cytoskeletons. Six percent of platelet cholesterol was present in platelet cytoskeletons, while no other neutral lipid could be detected. The study demonstrates that the lipid/protein ratio of platelet cytoskeletons is similar to that in platelets, but the composition of cytoskeleton lipids is specific and distinctly different from that in platelets. The selective glycolipid and phospholipid composition of cytoskeletons may be important for cytoskeleton and platelet function.