Serum protein synthesis in mutant mice with abnormal hepatic endoplasmic reticulum.

Severe ultrastructural abnormalities of liver endoplasmic reticulum have been described in newborn mice homozygous for radiation-induced deletion alleles at the colour locus. The ultrastructural defects were accompanied by deficiencies of several enzymes and lowered serum protein levels. Studies on serum protein synthesis were undertaken to see if decreased rates of synthesis, especially of constituents thought to be synthesized on membrane-bound ribosomes, were the cause of the deficiencies. Although decreases or absence of several serum proteins were shown, radiopulse-immunoprecipitation studies of albumin and fibrinogen synthesis suggested that the decreased synthesis rates were a secondary defect. Serum glycoproteins were not altered more than other constituents in the mutant material.     

Factor XIII-induced crosslinking in solutions of fibrinogen and fibronectin

In solutions containing fibrinogen and fibronectin, factor XIIIa catalyzes the formation of two types of crosslinked polymers: hybrid oligomers consisting of equimolar amounts of fibrinogen and fibronectin, and fibrinogen oligomers. The two types of oligomers are produced in amounts proportional to the starting concentration of fibronectin and fibrinogen in the reaction mixture. Increasing the fibronectin concentration relative to the fibrinogen concentration results in the production of more hybrid and less fibrinogen type oligomers. The lowest molecular weight hybrid oligomer, a dimer, is formed by ligation of one molecule of fibrinogen and fibronectin. The A alpha-chain of fibrinogen and one fibronectin subunit participate in the crosslinking. Larger size hybrid oligomers form by the joining of two hybrid dimers to each other via gamma-chain dimerization in the fibronectin moiety of the dimers. In fibrinogen oligomer formation, fibrinogen molecules are ligated by gamma-chain dimerization in a step-wise fashion producing fibrinogen dimers, trimers, tetramers, etc. without A alpha-chain crosslinking. The hybrid type and the fibrinogen type of oligomer grow in size and eventually become crosslinked to each other yielding large molecular weight complexes that interact to form a gel network.     

Purification and characterization of Atlantic salmon (Salmo salar) fibrinogen

This study describes a purification protocol of salmon fibrinogen that gives a consumable and highly clottable fibrinogen. Some characteristics of salmon and human fibrinogen are compared. Fibrinogen was purified from barium sulphate adsorbed plasma of Atlantic salmon, using two steps of 25% ammonium sulphate precipitation followed by ultrafiltration. The clottability of the purified salmon fibrinogen was 91%. The Aalpha chains of salmon fibrinogen were heterogeneous with a molecular mass of 90-110 kDa, compared to approximately 67 kDa of human fibrinogen Aalpha chains. The Bbeta and gamma chains of salmon and human fibrinogen had molecular masses of approximately 55 and 50 kDa, respectively. Western blotting revealed that polyclonal rabbit anti-human fibrinogen antibodies had affinity for the gamma chains of salmon fibrinogen, making it possible to study factor XIII activity in purified salmon fibrinogen. Cross-linking of either gamma-gamma or gamma-alpha chains was not detected upon incubation of the purified fibrinogen with thrombin and calcium alone, but was detected when clotting was performed in plasma indicating absence of factor XIII activity in the purified product.     

Copper ion binding and heparin interactions of human fibrinogen

The binding of copper ions to fibrinogen was studied by the equilibrium dialysis technique in neutral Tris buffer. The presence of copper causes precipitation of fibrinogen-copper complexes, the amount of which varies with the copper ion concentration. Solutions of 96% clottable fibrinogen (whole fibrinogen) displayed a maximum binding capacity about four times greater than that of fibrinogen solutions from which the cold-insoluble precipitate had been removed (cold-soluble fibrinogen). Binding in both systems apparently involves two classes of sites in fibrinogen, and the class of lower affinity is associated with cooperative interactions with copper. The copper concentration at which this cooperative uptake occurs is identical to the concentration at which the amount of precipitated material increases sharply and also to the concentration at which a sharp decrease is observed in the sedimentation coefficient of soluble fibrinogen, suggesting some relationship between copper binding, solubility, and solution properties. The presence of heparin markedly affects the sedimentation coefficient of fibrinogen in the presence of copper ions, although showing a lesser effect in the absence of the metal. The sedimentation coefficient of fibrinogen is increased in the presence of heparin and copper ion, compared to the value of fibrinogen-copper systems without heparin, and this effect is enhanced by changing the fibrinogen:heparin molar ratio to larger values. The precipitation of fibrinogen from solution, apparently without a coincident removal of heparin, also increases with increasing copper ion concentration and fibrinogen:heparin molar ratio. The possible significance of these effects in terms of heparin anti-coagulant activity is discussed.     

Atomic force microscopy of liposomes bearing fibrinogen

Extruded liposomes formed from dipalmitoylphosphatidylcholine and cholesterol, with and without fibrinogen, were examined by atomic force microscopy (AFM). The sequence of events involved in the transition from attached liposomes to bilayer patches on mica supports was viewed by tapping mode in liquid. After adhesion to the mica surface, both liposomes without fibrinogen and liposomes with attached fibrinogen collapsed into patches. The fibrinogen layer attached to the liposomes was 2.6 nm thick. This implied that the protein was spread over the entire liposome and the protein characteristic trinodular structure disappeared. To check the type of bond between fibrinogen and liposome, sequential images were taken after the incubation of fibrinogen with liposomes with and without a chemical group for attaching the protein. The results clearly confirmed that fibrinogen bound covalently to liposomes.     

Influence of fibrinogen on fibrin polymerization. Ultracentrifugation studies

During the transformation of fibrinogen to fibrin, excess fibrinogen suppresses further polymerization of fibrin, thereby enabling the nascent fibrin to be transported in a soluble form in blood. The question of possible complex formation between fibrin and fibrinogen was addressed by analyzing fibrin/fibrinogen (1:20, mol/mol) mixtures in the presence of calcium ions in stable linear sucrose density gradients by ultracentrifugation at 37 degrees C. During the period of ultracentrifugation in independent experiments, 40% of desAA-fibrin and 30% of desAABB-fibrin, respectively, precipitated without the participation of fibrinogen. The desAABB-fibrin, recovered in the gradient fractions, appeared as high-molecular-weight polymers (22 S), whereas the recovered desAA-fibrin exhibited only a slight increase in molecular weight (9 S) compared to fibrinogen (8 S). In contrast to this finding, both types of fibrin were totally recovered in gradient fractions provided that fibrinogen was present in the gradient at a uniform concentration of 2 mg/ml. In addition, the presence of fibrinogen but not human serum albumin reduced the size of desAABB-fibrin polymers (17 S). However, stable fibrin-fibrinogen complexes could not be demonstrated, since cosedimentation of differently labelled desAABB-fibrin and fibrinogen was not detectable. These studies suggest a specific but weak interaction of the solubilizing fibrinogen with the soluble fibrin polymers as demonstrated by a rapid exchange of both macromolecules.     

Fibrin but not adsorbed fibrinogen supports fibronectin assembly by spread platelets. Effects of the interaction of alphaIIb beta3 with the C terminus of the fibrinogen gamma-chain

We investigated the assembly of soluble fibronectin by lysophosphatidic acid-activated platelets adherent to fibrinogen or fibrin. More fibronectin was assembled by activated platelets spread on fibrin matrices than by platelets spread on adsorbed fibrinogen. The difference between platelets adherent to fibrinogen and fibrin occurred under both static and flow conditions. Similar differences were seen in binding of the 70-kDa N-terminal fragment of fibronectin that recognizes fibronectin assembly sites on adherent cells. Antibody and peptide blocking studies demonstrated that alphaIIb beta3 integrin mediates platelet adhesion to fibrinogen, whereas both alphav beta3 and alphaIIb beta3 mediate platelet adhesion to fibrin. The hypothesis that engagement of the C-terminal QAGDV sequence of the fibrinogen gamma-chain by alphaIIb beta3 inhibits the ability of the platelet to assemble fibronectin was tested by several experiments. Activated platelets adherent to adsorbed mutant fibrinogen lacking the QAGDV sequence (gammadelta5FG) were assembly-competent, as were platelets adherent to adsorbed normal fibrinogen that had been pretreated with the 7E9 antibody to the C terminus of the gamma-chain. Moreover, adsorbed normal fibrinogen but not gammadelta5FG suppressed the ability of co-adsorbed fibronectin to direct assembly of soluble fibronectin by spread platelets. The suppressive effect was lost when a surface of co-adsorbed fibronectin and fibrinogen was pretreated with 7E9. These results support a model in which the engagement of alphaIIb beta3 by the C-terminal sequence of the fibrinogen gamma-chain initiates signals that suppress subsequent fibronectin assembly by spread platelets. This interaction is less dominant when platelets adhere to fibrin, resulting in enhanced fibronectin assembly.     

Babesia argentina: Studies on the nature of an antigen associated with infection

A crude soluble haemagglutination antigen obtained from a mixture of Babesia argentina parasites and infected erythrocyte stromata contained fibrinogen. The fibrinogen was removed by precipitation in an initial attempt to purify the antigen. However, most if not all of the antigenic activity was located in the fibrinogen precipitate. After consideration of the evidence available, it was concluded that the antigen was either a babesial moiety complexed with fibrinogen or a fibrinogen molecule altered by the metabolic activity of the parasite.     

The colour of flowers in spectrally variable illumination and insect pollinator vision

The spectral reflectance of differently coloured Australian native plant flowers and foliage was measured and plotted in a colour triangle to represent the colour space of the honeybee. Spectral variations in illumination are shown to significantly change plant colours for bee vision without colour constancy. A model of chromatic adaptation based upon the von Kries coefficient law shows a reduction in plant colour shift, with the degree of correction depending upon position in colour space. A set of artificial reflectances is used to map relative colour shift caused by spectrally variable illumination for the entire colour space of the honeybee. The rarity of some flower colours in nature shows a correlation to a larger colour shift for these colours when illuminated by spectrally variable radiation. The model of chromatic adaptation is applied to illuminations used in a behavioural study on honeybee colour constancy by Neumeyer 1981. Surface colours used by Neumeyer are plotted in colour space for the various illuminations. The results show that an illumination-dependent colour shift correlates to a decrease in the frequency of bees correctly choosing a colour to which it was trained. Accepted: 23 February 1998     

Localization of fibrinogen during ADP- or thrombin-induced aggregation of washed rabbit platelets

In contrast to human platelets, which aggregate poorly in response to ADP unless fibrinogen is present in the external medium, washed rabbit platelets form large aggregates in response to ADP without fibrinogen in the suspending medium. Addition of fibrinogen to the suspending medium of rabbit platelets frequently has little or no effect on the extent of ADP-induced platelet aggregation. We examined washed rabbit platelets by immunocytochemistry during ADP-induced aggregation and deaggregation and during thrombin-induced aggregation when the external medium did not contain added fibrinogen to determine if (a) fibrinogen was expressed on the surface of rabbit platelets that could support aggregation when the platelets were stimulated, or (b) fibrinogen secreted from the alpha granules supports platelet aggregation. Glutaraldehyde-fixed samples were prepared at different times after addition of ADP or thrombin, embedded in Lowicryl K4M, sectioned, incubated with sheep anti-rabbit fibrinogen, washed, reacted with gold-labeled anti-sheep IgG, and prepared for electron microscopy. The alpha granules of rabbit platelets were heavily labeled with immunogold; the platelet membrane was not labeled. During platelet aggregation and deaggregation in response to ADP, fibrinogen was not detectable on the platelet surface. In response to thrombin, large aggregates formed before fibrinogen was secreted from the alpha granules; fibrinogen was detectable focally at sites of granule discharge by 30-60 sec and fibrin formed by 3 min. Therefore, stimulated washed rabbit platelets can adhere to each other without large amounts of fibrinogen taking part in the close platelet-to-platelet contact, since aggregation occurs before detectable secretion, and large areas where the platelets are in contact are devoid of fibrinogen between the adherent membranes. Adhesion mechanisms not involving fibrinogen may support the aggregation of washed rabbit platelets.     

Fibrinogen Manchester. Detection of a heterozygous phenotype in the intraplatelet pool.

Family members heterozygous for the congenitally abnormal fibrinogen designated fibrinogen Manchester, A alpha 16Arg----His, have previously been shown by h.p.l.c. and amino acid analysis to release a variant fibrinopeptide, [His16]fibrinopeptide A, from plasma fibrinogen after the addition of thrombin. The present study was designed to determine if the same abnormal phenotype was also present in the intraplatelet fibrinogen pool. Fresh platelets were washed in buffers containing EDTA until it could be shown that all washable plasma fibrinogen was removed. Normal platelets were then lysed by freezing and thawing to release their intracellular proteins, which were then treated with thrombin. The fibrinopeptides, cleaved from the intraplatelet fibrinogen, could be detected by an optimized h.p.l.c. technique. Quantification of the intraplatelet fibrinogen gave a result (means +/- S.D., n = 5) of 110 +/- 30 and 90 +/- 30 micrograms/10(9) platelets, when determined by h.p.l.c. quantification of fibrinopeptide B content and fibrinogen fragment E radioimmunoassay respectively. Examination of fibrinopeptides released from the platelet fibrinogen from the family with fibrinogen Manchester with the same techniques showed elution peaks in the same positions as both [His16]fibrinopeptide A and normal fibrinopeptide A. The identity of these peaks was further substantiated by analysis of the h.p.l.c. peaks by using specific radioimmunoassay to fibrinopeptide A. Our results therefore demonstrate that platelet fibrinogen expresses the heterozygous A alpha 16His phenotype. This supports the view that the A alpha chains of platelet and plasma fibrinogen are produced from a single genetic locus.     

Affinity labeling of a human platelet membrane protein with 5'-p-fluorosulfonylbenzoyl adenosine. Concomitant inhibition of ADP-induced platelet aggregation and fibrinogen receptor exposure

Incubation of washed human blood platelets with 5'-p-fluorosulfonylbenzoyl [3H]adenosine (FSBA) covalently labels a single polypeptide of Mr = 100,000. Protection by ADP has suggested that an ADP receptor on the platelet surface membrane was modified. The modified cells, unlike native platelets, failed to aggregate in response to ADP (100 microM) and fibrinogen (1 mg/ml). The extent of binding of 125I-fibrinogen and aggregation was inhibited to a degree related to the incorporation of 5'-p-sulfonylbenzoyl adenosine (SBA) into platelets, indicating FSBA could inhibit the exposure of fibrinogen receptors by ADP necessary for aggregation. Incubation of SBA platelets with alpha-chymotrypsin cleaved the covalently labeled polypeptide and concomitantly reversed the inhibition of aggregation and fibrinogen binding. Platelets proteolytically digested by chymotrypsin prior to exposure to FSBA did not require ADP for aggregation and fibrinogen binding. Moreover, subsequent exposure to FSBA did not inhibit aggregation or fibrinogen binding. The affinity reagent FSBA can displace fibrinogen bound to platelets in the presence of ADP, as well as promote the rapid disaggregation of the platelets. The apparent initial pseudo-first order rate constant of dissociation of fibrinogen was linearly proportional to FSBA concentrations. These studies suggest that a single polypeptide can be altered either by ADP-induced conformational changes or proteolysis by chymotrypsin to reveal latent fibrinogen receptors and promote aggregation of platelets after fibrinogen binding.     

Histaminylation of fibrinogen by tissue transglutaminase-2 (TGM-2): potential role in modulating inflammation

Plasma fibrinogen plays an important role in hemostasis and inflammation. Fibrinogen is converted to fibrin to impede blood loss and serves as the provisional matrix that aids wound healing. Fibrinogen also binds to cytokine activated endothelial cells and promotes the binding and migration of leukocytes into tissues during inflammation. Tissue transglutaminase (TGM-2) released from injured cells could cross-link fibrinogen to form multivalent complexes that could promote adhesion of platelets and vascular cells to endothelium. Histamine released by mast cells is a potent biogenic amine that promotes inflammation. The covalent attachment of histamine to proteins (histaminylation) by TGM-2 could modify local inflammatory reactions. We investigated TGM-2 crosslinking of several biogenic amines (serotonin, histamine, dopamine and noradrenaline) to fibrinogen. We identified histaminylation of fibrinogen by TGM-2 as a preferred reaction in solid and solution phase transglutaminase assays. Histamine caused a concentration-dependent inhibition of fibrinogen cross-linking by TGM-2. Fibrinogen that was not TGM-2 crosslinked bound to un-activated endothelial cells with low affinity. However, the binding was increased by sevenfold when fibrinogen was cross-linked by TGM-2. Histaminylation of fibrinogen also inhibited TGM-2 crosslinking of fibrinogen and the binding to un-activated HUVEC cells by 75–90 %. In summary, the histaminylation of fibrinogen by TGM-2 could play a role in modifying inflammation by sequestering free histamine and by inhibiting TGM-2 crosslinking of fibrinogen.     

Fibrinogen distribution on surfaces and in organelles of ADP stimulated human blood platelets

The fibrinogen distribution in platelet organelles after ADP-stimulation was investigated with anti-human fibrinogen using protein A-gold applied to serial sections. Fibrinogen was detected in the so-called alpha-granules of platelets and also in granule protrusions which were observed after ADP-stimulation. The ends of these protrusions were formed as coated membranes and the tips were often in apposition to the surface connected membranes or the plasmalemma. At such places fusion events and hence signs of an exocytosis could be demonstrated by means of cryofixation and cryosubstitution. Examination of serial sections revealed fibrinogen on all these granule profiles. Surface connected membranes, free surfaces and the characteristic structure of the contact zones of aggregated platelets were also labelled by gold particles but less than anticipated. On the platelet surfaces and surface connected membranes fibrinogen was rarely demonstrable with ferritin-labelled anti-human fibrinogen on washed or thrombin-stimulated, almost fibrinogen free platelets. After addition of human fibrinogen to the thrombin stimulated and disaggregated platelets a part of the platelets aggregated spontaneously and formed characteristic contact zones. Anti-human fibrinogen was observed on the free surfaces, in filamentous bridges between the contact spaces and in a tubular surface connected membrane system with involvement of coated membranes at the central ends of these structures. The results indicate the following: all alpha-granules contain fibrinogen; after ADP-stimulation secretion takes place with involvement of coated membranes; during aggregation fibrinogen binds to platelet surfaces and forms contact spaces; fibrinogen is taken up by the surface connected system with involvement of coated membranes.     

ON THE NATURE OF FORCES OPERATING IN BLOOD CLOTTING : II. THE CLOTTING OF FIBRINOGEN AS A TWO-STEP REACTION

It is found that clotting of fibrinogen by thrombin does not occur on the acid side of the isoelectric point of the fibrinogen. At such pH values, however, a primary reaction between thrombin and fibrinogen takes place, leading to the formation of profibrin, a compound of thrombin and fibrinogen. At pH values at which clotting is possible, fibrinogen is negatively, thrombin positively charged, whereas profibrin has a pattern of positive and negative charges. The primary reaction, the formation of profibrin by combination of thrombin and fibrinogen, is inhibited by urea but not by neutral salts. The combination of thrombin with fibrinogen most probably takes place by hydrogen bonds. The second reaction, the polymerisation of profibrin to fibrin, is inhibited by neutral salts in the same way as complex or autocomplex coacervates. It is caused therefore by electrostatic attraction between the positive and the negative charges of the profibrin.     

Peroxynitrite-mediated oxidation of fibrinogen inhibits clot formation

The clotting activity of human fibrinogen was fully inhibited in vitro by peroxynitrite. The decrease of activity followed an exponential function and the concentration of peroxynitrite needed to inhibit 50% of fibrinogen clotting was 22 microM at 25 degrees C. The oxidative modification(s) induced by the peroxynitrite system (i.e. ONOO-, ONOOH and ONOOH*) appeared specifically to affect fibrin clot formation (through the inhibition of fibrinogen polymerization) since the interaction of peroxynitrite-modified fibrinogen with thrombin appeared to be unaffected. The addition of NaHCO3 decreased the peroxynitrite effect on fibrinogen clotting, suggesting that the reactive species formed by the reaction of CO2 with peroxynitrite are less efficient oxidants of peroxynitrite itself. Similar effects were observed after addition of bilirubin, which also exerted a significant protection against peroxynitrite-mediated modification of fibrinogen.     

Differential degradation of the three fibrinogen chains by proteasomes: involvement of Sec61p and cytosolic Hsp70

HepG2 cells, which synthesize and secrete fibrinogen, accumulate surplus Aalpha and gamma chains. The nonsecreted fibrinogen chains are degraded both by proteasomes and lysosomes, with unassembled chains primarily degraded by proteasomes and an Aalpha-gamma complex by lysosomes. To further determine the mechanisms by which unassembled fibrinogen chains are degraded, and to explain the pools of Aalpha and gamma chains that occur in HepG2 cells, the association of fibrinogen chains with Sec61beta, a component of the translocon, and with a cytosol chaperone, Hsp70, was studied in both HepG2 cells and COS cells expressing single fibrinogen chains. Retrotranslocation from the lumen of the endoplasmic reticulum was shown by treatment with MG132, a proteasome inhibitor. MG132 caused glycosylated Bbeta to accumulate on Sec61beta in COS cells expressing Bbeta and acted similarly with all three fibrinogen chains in HepG2 cells. In HepG2 cells, Bbeta was associated with Sec61beta ahead of Aalpha and gamma chains, suggesting that pools of Aalpha and gamma chains may be caused by unequal rates of retrotranslocation. In COS cells, retrotranslocation into the cytoplasm was demonstrated by the ATP-sensitive association of ubiquitinylated Aalpha, Bbeta, and gamma chains bound to Hsp70. More Aalpha and gamma than Bbeta accumulated on Hsp70 of HepG2 cells, consistent with more rapid degradation of Bbeta. Overexpression of Hsp70 in HepG2 cells resulted in decreased secretion, but not synthesis, of fibrinogen. Decreased secretion may be due to enhanced degradation of unassembled fibrinogen chains, indicating that proteolysis by proteasomes might regulate both the intracellular pools of fibrinogen chains and fibrinogen secretion.     

Congenital afibrinogenemia: intracellular retention of fibrinogen due to a novel W437G mutation in the fibrinogen Bbeta-chain gene

Congenital afibrinogenemia is a rare autosomal recessive coagulation disorder characterised by hemorrhagic manifestations of variable entity and by severe plasma fibrinogen deficiency. Among the 31 afibrinogenemia-causing mutations so far reported, only 2 are missense mutations and both are located in the fibrinogen Bbeta-chain gene. Direct sequencing of the fibrinogen gene cluster in two afibrinogenemic Iranian siblings revealed a novel homozygous T>G transversion in exon 8 (nucleotide position 8025) of the fibrinogen Bbeta-chain gene. The resulting W437G missense mutation involves a highly conserved amino acid residue, located in the C-terminal globular D domain. The role of the W437G amino acid substitution on fibrinogen synthesis, folding, and secretion was assessed by in vitro expression experiments in COS-1 cells, followed by qualitative and quantitative analyses of intracellular and secreted mutant fibrinogen. Results of both pulse-chase experiments and enzyme-linked immunosorbent assays demonstrated intracellular retention of the mutant W437G fibrinogen and marked reduction of its secretion. These data, besides elucidating the pathogenetic role of the W437G mutation in afibrinogenemia, underline the importance of the Bbeta-chain D domain in fibrinogen folding and secretion.     

Stimulation of fibrinogen synthesis in cultured rat hepatocytes by fibrinogen fragment E

Because of the inherent difficulties of experimentation in intact animals, we used primary monolayer cultures of non-proliferating adult rat hepatocytes to study the effects of fibrinogen degradation products on fibrinogen biosynthesis. The freshly isolated hepatocytes obtained by collagenase perfusion of the liver in situ were cultured in a chemically defined serum-free medium. The rate of fibrinogen synthesis in control cultures was $\text{40–50}\text{pmol}\text{2.5·10}{}^6$ cells per 24 h. Additions of 20, 60 or 100 μg of homologous stage I fibrinogen degradation products had no effect on fibrinogen synthesis. In contrast, addition of the same amounts of homologous or heterologous (human) stage III fibrinogen degradation products resulted in a concentration-dependent increase in fibrinogen biosynthesis without affecting the rate of synthesis of albumin. When purified stage III fibrinogen degradation products D and E (human) were tested in 10, 30 or 50 μg/3 ml medium only fragment E showed a significant increase in fibrinogen biosynthesis (1.9-, 2.8- and 5.6-fold, respectively, over the control cultures). The presence of excess fibrinogen had no effect. These results suggest that fibrinogen fragment E may be a specific stimulator of fibrinogen biosynthesis which may play an important role in maintaining normal levels of plasma fibrinogen.     

Synthesis and secretion of fibrinogen and albumin by isolated rat hepatocytes

In order to investigate the regulation of synthesis of some of the plasma proteins, especially fibrinogen, at the cellular level, we have chosen to work with suspensions of hepatocytes isolated by the perfusion of rat liver with crude bacterial collagenase. By adding soybean trypsin inhibitor to the collagenase and by avoiding mechanical damage, we have prepared cell suspensions that synthesize and secrete fibrinogen and albumin and that survive for longer than twenty hours. The fibrinogen secreted is clottable and shows the same pattern in acrylamide gel electrophoresis as fibrinogen purified from rat plasma. After a three hour lag, the rate of synthesis of fibrinogen, as measured by a solid-phase radioimmunoassay, continually accelerates, so that rates several fold greater than the $\text{in vivo}$ rate have been observed after twenty hours incubation. Cycloheximide (0.1 mM) completely abolished the appearance of fibrinogen in the medium; whereas colchicine (0.3 mM) reduced the rate by 85%. Insulin and cortisol succinate enhanced fibrinogen synthesis and secretion. The albumin secretion profile differs in several respects from that of fibrinogen, reflecting differences in intracellular pool levels and probably distinct regulatory mechanisms.