Plasma Counterpart Research Articles

Characterization and Origin of Fibrinogen in Blood Platelets

SummaryThe discovery of thrombin-clottable protein in platelet homogenates initiated studies on the location, properties, and origin of such material, the ultimate aim of which has been to define its physiologic role. It was established that fibrinogen is present both on the platelet membrane as well as in the cytoplasmic α-granules. The membrane-bound material is apparently fibrinogen adsorbed from plasma, while the intracellular fibrinogen appears to have unique biochemical and functional properties. Differences in properties observed between platelet fibrinogen and plasma fibrinogen are not due to in vitro modifications during handling. Whether the intracellular fibrinogen is synthesized in the platelet (or megakaryocyte) or whether it is derived through uptake and limited modification of plasma fibrinogen in vivo has remained an open question. Thus, to investigate the aspect of origin of platelet fibrinogen, radioactively-labelled fibrinogen was injected into rats, blood was collected at time intervals and the radioactivity in the subcellular fractions of platelets was examined. A part of the injected fibrinogen became associated with the platelets, but very little was found in the granule fraction. Previous findings on human platelet fibrinogen, together with the present data obtained using rats, suggest that platelet fibrinogen may not be derived from plasma fibrinogen in vivo. It thus is apparent that the intracellular fibrinogen is synthesized by a unique genetic mechanism. Molecular properties of fibrinogen derived from rat platelet granules were shown analogous to the properties of fibrinogen from human platelet granules. Fibrinogen of intracellular origin is less stable than its plasma counterpart. The results obtained by others in which identity of platelet and plasma fibrinogens was reported may be explained on the basis of recovery of only the fibrinogen component bound to the platelet membrane, which is adsorbed plasma fibrinogen. It is suggested that the term platelet fibrinogen may be used to denote that present in the α-granules, with the membrane-bound component being referred to as platelet-associated fibrinogen.

Washout kinetics of red cells and plasma from the spleen.

Radioiodinated (125I) serum albumin was injected inttravenously in cats and allowed to equilibrate in the circulation. Red cell and plasma washout from the isolated spleens were enrom the isolated spleens were then compared during perfusion with oxygenated Ringer solution, the respective concentrations in the outflow being measured by celloscope and scintillation counters. Washout kinetics yielded three exponential components for cells (perfusate volumes for 50% washout (V1/2) being 0.067, 4.70, and 97 ml/g spleen) but only two for plasm (V1/2, 0.14 and 2.40 ml/g). There is no plasma counterpart to the slowly released cells, i.e., they do not represent a separate vascular space. This is an accord with a previous view that these are immature cells, delayed through adherence to fine structures of the red pulp. Compartment analysis indicates that the plasma and two remaining cell components represent washout from two separate vascular spaces, containing 0.09 ml/g blood at arterial hematocrit 37% and 0.42 ml/g blood at hematocrit 75%, perfused by 0.9 and 0.1 of the arterial inflow respectively. Evidence suggests these spaces are i) blood vessels, and ii) red pulp.

Platelet alpha2-macroglobulin and alpha1-antitrypsin.

Subcellular membrane and granule fractions derived from human platelets contain immunologically identifiable alpha2-macroglobulin and alpha1-antitrypsin. These platelet-derived inhibitors show a reaction of immunologic identity when compared to alpha2-macroglobulin and alpha1-antitrypsin purified from human plasma. Further, the platelet protease inhibitors possessed a similar subunit polypeptide chain structure to their plasma counterparts as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. Studies of the binding of radiolabeled trypsin to the various solubilized platelet subcellular fractions suggest that the granule-associated alpha2-macroglobulin and alpha1-antitrypsin, as well as membrane-associated alpha2-macroglobulin were functionally active. Quantitatively, circulating platelets contain relatively small concentrations of these inhibitors as compared to platelet-associated fibrinogen and factor VIIIAGN. Platelet protease inhibitors may modulate the protease-mediated events involved in the formation of hemostatic plugs and thrombi.

In vivo plasma and urine folate binding after ingestation of 3H-folic acid and 14C-methyl-folate.

After simultaneous ingestion of equivalent amounts of [3H]folic acid (3H-PteGlu) and [14C]N5-methyl-tetrahydrofolic acid (14C-CH3-H4PteGlu) we were able to demonstrate progressive macromolecular binding of radiofolate in plasma, which appeared to be near maximal at 6 h. Bound radiofolate was predominantly of 14C-CH3H4PteGlu origin, and only at 24 h could 3H incorporation be demonstrated. The binder eluted with albumin from Sephadex DEAE-A50 columns. In urine a smaller bound radiofolate fraction, with approximately equal amounts of 3H and 14C, appeared after 5.5 h. Plasma chromatography showed radio-PteGlu (peak 1) to be rapidly converted to CH3-H4PteGlu (peak 2), with subsequent appearance of two further radiofolate peaks (peaks 3 and 4) the nature of which is as yet unclear. Urine showed similarly placed fractions but their magnitude differed, and urinary peak 3 in particular was much more prominent than its plasma counterpart.

Apoprotein profile of plasma and chylous ascites lipoproteins

Plasma and chylous ascites lipoproteins were compared in a rare case of exudative enteropathy associated with stenosis of the thoracic duct. All ascites lipoproteins separated by ultracentrifugation showed a much higher triglyceride/ protein ratio and a lower cholesterol ester content than their plasma counterparts. Polyacrylamide gel electrophoresis of apoproteins before and after fractionation on Sephadex G-200 essentially showed, compared to normal plasma, a reduction of apoprotein C in VLDL and HDL particularly obvious for apoCIII 1 and CIII 2. Ascites lipoproteins were characterized by an increased apoA content in chylomicrons, VLDL and LDL and a reduced percentage of apoCII and CIII 1, mostly in chylomicrons, VLDL and HDL. The differences in composition between plasma and ascites might be explained by different origins for the various apoprotein subfractions. The transsudation of chyle into the abdominal cavity might divert some peptides of intestinal origin from their normal entry into the circulation.

Cylindrical solitons in a warm, multi-ion plasma

The soliton problem for ion acoustic waves propagating in a warm plasma consisting of a mixture of two ion species is solved for cylindrically symmetric ingoing waves. The small amplitude approximation is made and a modified Korteweg–de Vries equation is obtained. The early time solution show that the solitons for the two ion species plasma are narrower in spatial width and propagate faster than their counterpart in the single ion plasma. Also, a given initial disturbance will break up into more solitons in the multi-ion, warm plasma than in the cold single-ion plasma.

A sufficient condition for instability of BGK-type waves

By investigating three dimensional perturbations, a sufficient condition for instability of BGK waves is found. New perturbed modes, having no counterpart in a linear plasma, are also found. Known results are recovered in various limits.

Factor X Friuli: An immunological study in plasma and in serum using several methods.

An immunological study of factor X Friuli was carried out using an anti-human-factor X antiserum. The results obtained with different methods were compared. Only an ill-defined factor X band or precipitate was evident on standard immuno-electrophoresis and on immunodiffusion. Using a non-absorbed antiserum a precipitate was evident on eletroimmunoassay, in normal, factor X Friuli and in factor X deficient plasma. On the contary, using an antiserum which had been previously absorbed with factor X deficient plasma, a precipitate was evident only with factor X Friuli plasma or normal plasma. Excellent results were obtained with the cross-over electrophoresis. Using this method a major or factor X band was evident in normal plasma, factor II,P FACTOR VII, factor IX deficient plasmas and in the factor X FRIULI PLASMA. No factor X band was evident in factor X deficient plasmas and in absorbed normal plasma. Lighter, more cathodic bands were seen in all plasmas. These were due to secondary activities of the antiserum and could be disregarded. Friuli serum factor X and normal serum factor X showed a slightly more anodic migration as compared to their plasma counterparts. In coumarin plasma two factor X bands were evident; one normal in position and one slightly more catholidic. In the bidimensional immunoelectrophoresis two precipitates are visible in normal plasma and in factor X Friuli plasma. In factor X deficient plasma only one precipitate is evident. This latter precipitate is due to the secondary activity of the antiserum used. On the basis of these data it is concluded that factor X Friuli behaves, immunologically, as normal factor X.

Platelet fibrinogen. Identity and initial observations on the mode of its degradation by plasmin

Fibrinogen has been purified from human platelets. Platelet fibrinogen exhibits a characteristic pattern in agar gel immunoelectrophoresis different from that of plasma fibrinogen. Stepwise plasmin degradation has been used in further elucidation of the molecular properties of the platelet protein. Examination of comparative digests by immunologic and gel electrophoretic methods has revealed that (1) the platelet protein is more resistant to plasmin degradation, (2) the plasmin-produced fragments of platelet fibrinogen differ consistently from those of its plasma counterpart, and (3) platelet fibrinogen is different from fragment X of plasma fibrinogen. It is suggested that platelet fibrinogen may contribute to the stability of the thrombus.

Comparison of Human Plasma Fibrinogen Subfractions and Early Plasmic Fibrinogen Derivatives

Abstract Human plasma fibrinogen Subfractions I-8 and I-9 have previously been shown to consist of catabolic intermediates of fibrinogen characterized by the lack of COOH-terminal portions of Aα chains. The present study was undertaken to determine the structural relationships between these catabolites and core derivatives formed in vitro by the action of plasmin. First, these fractions were compared with plasma Subfraction I-4, which is representative of native fibrinogen, by polyacrylamide gel electrophoresis of unreduced samples in sodium dodecyl sulfate. Fraction I-4 resolved into two major bands, designated I and II (the higher the number the greater the anodal migration and the smaller the molecular size), and a trace component (Band IIIa). Fractions I-8 and I-9 exhibited four bands (IIIa, IV, V, and VI) of which Band IIIa was the major component and VI, the least abundant. Fractions I-8 and I-9 were also compared with subfractions isolated from stage 1 plasmic digests of Fraction I-4 (termed I-9Dn, where n = clottability of the digest from which the subfraction was obtained). An early plasmic subfraction, I-9D88, contained Bands IIIa (major component), V, and VI plus a minor amount of Band VII; a subfraction from a more advanced stage 1 digest (I-9D50) contained a faster migrating Band III (IIIb) and relatively increased amounts of Bands VI and VIII. Despite the fact that Fraction I-9D88 closely resembled its plasma counterpart (I-9) in clottability (96%), electrophoretic, chromatographic, and solubility behavior, several differences were apparent. Band IV was detected only in the plasma fractions; Band VII was present only in the plasmic derivatives. Each of the 10 core Aα chain remnants (viz., Aα/2, Aα/4, Aα/6 to Aα/13) identifiable by gel electrophoresis of reduced plasma fibrinogen subfractions can apparently be generated in vitro by plasmin digestion. However, whereas Aα/8 and Aα/9 were the most abundant of the small Aα/ remnants (mol wt g40,000) in Fractions I-8 and I-9, Aα/11 predominated in I-9D88. Moreover, Bβ chain degradation was somewhat more advanced in I-9D88 than in its plasma counterpart. The failure to demonstrate complete identity between I-9 and I-9D88 underscores the present uncertainty as to whether plasmin alone catalyzes the formation of circulating catabolites in vivo. Experiments involving gel slicing, reduction of the eluted protein, and re-examination by gel electrophoresis, plus related electrophoretic procedures and chromatographic analyses, yielded the following results. Formation of species migrating within Bands II, IIIa, IV, and V could be accounted for by the loss of COOH-terminal portions of the Aα chains; species migrating in Band IIIa or more anodally exhibited no intact Aα chains. Formation of Band IIIb (from species like those in Band IIIa) occurs by the loss of the NH2-terminal region of one Bβ chain; material within Bands VI and VII is associated not only with the removal of this region from the other Bβ chain but also with internal cleavages such as those eventually leading to the separation of Fragments D and E.

Studies on platelet proteins: IX. The identity of fibrinogen

The molecular differences between human platelet fibrinogen and plasma fibrinogen observed previously were further investigated to determine whether the platelet protein might be a proteolytic derivative of its plasma counterpart. Purified plasma fibrinogen labeled with 125I was added to washed, intact platelets, and the total fibrinogen was isolated in the presence of proteolytic inhibitors by the same procedure used for platelet fibrinogen alone. Comparison of control 125I-labeled plasma fibrinogen, total fibrinogen, and platelet fibrinogen by sodium dodecylsulfate gel electrophoretic staining patterns, as well as the radioactivity distribution for control and total fibrinogen, showed no conversion of the added plasma fibrinogen to platelet fibrinogen. Furthermore, the electrophoretic pattern of platelet fibrinogen was different from the slowly clottable plasmin derivative of plasma fibrinogen, termed Fragment X. It is concluded that platelet fibrinogen is not derived, during the course of its isolation, from plasma fibrinogen.

The uptake and metabolism of plasma lysophosphatidylcholine in vivo by the brain of squirrel monkeys.

1. Adult squirrel monkeys were injected intravenously with doubly labelled lysophosphatidylcholine (a mixture of 1-[1-(14)C]palmitoyl-sn-glycero-3-phosphorylcholine and 1-acyl-sn-glycero-3-phosphoryl[Me-(3)H]choline; (3)H:(14)Cratio 3.75) complexed to albumin, and the incorporation into the brain was studied at times up to 3h. 2. After 20min, 1% of the radioactivity injected as lysophosphatidylcholine had been taken up by the brain. 3. Approx. 70% of the doubly labelled lysophosphatidylcholine taken up by both grey and white matter was converted into phosphatidylcholine, whereas about 30% was hydrolysed. 4. The absence of significant radioactivity in the phosphatidylcholine, free fatty acid and water-soluble fractions of plasma up to 30min after injection of doubly labelled lysophosphatidylcholine rules out the possibility that the rapid labelling of these compounds in brain could be due to uptake from or exchange with their counterparts in plasma. 5. The similarity between the (3)H:(14)C ratios of brain phosphatidylcholine and injected lysophosphatidylcholine demonstrates that formation of the former occurred predominantly via direct acylation. 6. Analysis of the water-soluble products from lysophosphatidylcholine catabolism revealed that appreciable glycerophosphoryl-[Me-(3)H]choline did not accumulate in the brain and that radioactivity was incorporated into choline, acetylcholine, phosphorylcholine and betaine. 7. The role of plasma lysophosphatidylcholine as both a precursor of brain phosphatidylcholine and a source of free choline for the brain is discussed.

Human Fibrinogen Heterogeneities

The heterogeneity of human plasma fibrinogen manifested as differences in solubility reflects the presence of early catabolic intermediates, which are more soluble, have a longer thrombin-clotting time, and are of a lower molecular weight than the parent material from which they are formed. In this study the S-sulfo subunits from fibrinogen fractions of low (I-4) and high (I-8 and I-9) solubility were compared. Separation of Aα, Bβ, and γ chains was achieved by gradient elution chromatography on CM-cellulose. The tryptic peptide maps were characteristic for each type of chain. The only observable differences were in the maps of Aα chains; in those of I-8 and I-9 a considerable number of spots were absent or reduced relative to I-4. NH2-terminal analyses of isolated chains before and after thrombin treatment showed the characteristic residues of the Aα, Bβ, and γ chains, respectively. Thus the NH2-terminal portions of all chains of I-4, I-8, and I-9 were intact. Molecular weight estimation of subunit chains was made by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate under conditions in which differences in mobility due to the presence or absence of A or B peptides were distinguishable. Electrophoresis in this medium before and after reptilase or thrombin treatment served for identification of the chains. The γ chains moved as a single band in this system (molecular weight, 50,700) and were indistinguishable from one another; the same was true of Bβ chains (molecular weight, 60,400). However, electrophoresis of Aα chains revealed the presence of 13 more-or-less distinct bands (numbered in order of increasing anodal mobility), of which all but two (Band 3, a Bβ contaminant and Band 5, a γ chain contaminant) were shown to be intact Aα chain or COOH-terminally degraded Aα remnants. The molecular weights ranged from that of intact Aα chain (70,900) to 15,400 (Band 13). The highest proportion of intact Aα chains was in I-4; the lowest was in I-9, which had virtually no detectable intact Aα chain. Conversely, I-9 contained the highest proportion of Aα remnant chains; I-4, the lowest. These data suggested that cleavage at any or all of at least 10 sites along the Aα chain resulted in the formation of early catabolic intermediates. Comparisons with highly clottable derivative fractions I-8D and I-9D produced by plasmin treatment of I-4 in vitro showed that they were quite similar to their respective plasma counterparts (I-8 and I-9) in that the γ and Bβ chains appeared to be intact, but few or no intact Aα chains were present. These observations thus strengthen the notion that it is plasmin which catalyzes the formation of early intermediates in vivo. Examination of Fragment ‘X', another fibrinogen derivative produced by plasmin digestion, revealed considerable depletion of intact Bβ as well as of Aα chains, indicating that Fragment ‘X' was more extensively degraded than I-8D or I-9D.

In Vivo Studies on Glycosphingolipid Metabolism in Porcine Blood

Abstract Porcine plasma and erythrocytes were found to contain the same glycosphingolipids as humans, namely glucosyl ceramide, lactosyl ceramide, galactosylgalactosylglucosyl ceramide, and globoside, and the actual concentrations were very similar. The in vivo studies were carried out by using uniformly labeled 14C-d-glucose as the isotopic precursor and isolating the glycolipids from 30-ml samples of blood removed at frequent intervals over the 3-month study period. The results suggested that erythrocyte lactosyl ceramide, galactosylgalactosylglucosyl ceramide and globoside were synthesized in the bone marrow, did not exchange with their plasma counterparts, and were released into the plasma at the time of general erythrocyte catabolism. Erythrocyte glucosyl ceramide did not appear to be synthesized in the marrow and was in dynamic equilibrium with plasma glucosyl ceramide. The label was rapidly and randomly incorporated into all the plasma glycosphingolipids but after 9 days no labeling could be detected until the erythrocytes began to be catabolized (50 to 70 days). The relative amounts of label in the glycolipids during the 1st week and the latter period from 40 to 60 days confirmed that the rapid source of plasma glycosphingolipid biosynthesis is a minor pathway and showed that the release or catabolism of erythrocyte glycosphingolipids (especially globoside, the major component) is the major source of all four plasma glycosphingolipids.

Nonlinear Hydromagnetic Solitary Waves in a Collision-Free Plasma with Isothermal Electron Pressure

Nonlinear hydromagnetic solitary waves propagating in a collision-free plasma of cold ions and isothermal electrons are investigated on the basis of hydrodynamical transport equations. Both cases of wave propagation along and across a magnetic field are studied in detail. In the parallel case, two types of solitary wave are found. The first type is an ordinary one which reduces to the zero-pressure wave in the cold limit and the other is a particular one which has no counterpart in the cold plasma. In the transverse case, only one type of solitary wave similar to that in the cold plasma is found. The effect of electron pressure shortens the relative widths of the waves in the parallel case (for the first type) while widens those in the transverse case. Magneto-ion-dynamics is formulated for the plasma under consideration, where the relation between the two-fluid model and the one-fluid magneto-hydrodynamics is established.

Plasma and urinary metabolites of progesterone-4-C 14 administered orally or subcutaneously to rabbits

The fate of orally versus subcutaneously administered progesterone-4-C14 was studied in rabbits. During the first 6 hours after subcutaneous injection of labeled hormone, plasma radioactivity rose smoothly with free steroids accounting for 8–30% of the total. On the other hand, ingested radioprogesterone was rapidly absorbed and converted to conjugated metabolites which constituted more than 99% of the total plasma radioactivity. Fractionation and thin layer chromatography of hydrolyzed plasma extracts revealed at least 6 metabolites, the preponderant one corresponding to 5β-pregnane-3α,20α-diol (pregnanediol). More pregnanediol was present in plasma following oral administration rather than subcutaneous injection of progesterone. In contrast to these findings, route of administration of the radiohormone had no observable influence on the pattern of excretion products in the urine. Urinary metabolites were in general qualitatively similar to those found in plasma. However, urine also contained considerable alkali-extractable radioactivity which had no counterpart in plasma.