Fibrinogen fractionation

The clot is made by adding 2 U/mL of human thrombin (Sigma Chemical Co., St. Louis, MO) to 1% bovine fibrinogen (fraction I, Type IV, Sigma) in sterile 0.9% NaCl. 

Peerschke EIB, Galanakis DK. Binding of fibrinogen to ADP-treated platelets Comparism of plasma fibrinogen fractions and early plasmic fibrinogen derivatives. JLab Clin Med 1983 101 453-4. 

They experimented with four other proteins as carriers rabbit serum albumin, bovine serum albumin, bovine fibrinogen fraction I, and bovine )8-globulin fraction III. The structurally related derivatives of DDT and malathion, DDA, and 0,0-dimethyl S-carboxy-carboxyethyl phosphoro-dithioate (malathion half ester), respectively, were used as the specific haptens attached to these carrier proteins. These compounds contain free carboxyl groups, which when they reacted with thionylchloride, provide a means of coupling of the hapten to the amino groups of the protein carrier. 

A method for the fractionation of plasma, allowing albumin, y-globulin, and fibrinogen to become available for clinical use, was developed during World War II (see also Fractionation, blood-plasma fractionation). A stainless steel blood cell separation bowl, developed in the early 1950s, was the earhest blood cell separator. A disposable polycarbonate version of the separation device, now known as the Haemonetics Latham bowl for its inventor, was first used to collect platelets from a blood donor in 1971. Another cell separation rotor was developed to faciUtate white cell collections. This donut-shaped rotor has evolved to the advanced separation chamber of the COBE Spectra apheresis machine. 

Primary blood components iaclude plasma, red blood cells (erythrocytes), white blood cells (leukocytes), platelets (thrombocytes), and stem cells. Plasma consists of water dissolved proteias, ie, fibrinogen, albumins, and globulins coagulation factors and nutrients. The principal plasma-derived blood products are siagle-donor plasma (SDP), produced by sedimentation from whole blood donations fresh frozen plasma (FFP), collected both by apheresis and from whole blood collections cryoprecipitate, produced by cryoprecipitation of FFP albumin, collected through apheresis and coagulation factors, produced by fractionation from FFP and by apheresis (see Fractionation, blood-plasma fractionation). 

Arvin [9046-56-4] is a purified fraction from the cmde venom of Agkistrodon rhodostoma (48). The action of this venom fraction is selectively specific for fibrinogen and can rapidly deplete fibrinogen in vivo safely from the ckculating blood. Blood without fibrinogen cannot undergo clot formation. 

The clear serum of this example is an amber liquid free from prothrombin, thrombin, fibrinogen and fibrin. It contains profibrinolysin and is excellently suited to further purification by salt precipitation fractionation, as given below. 

Cry precipitate is a plasma protein fraction obtainable from whole blood. It is used to treat deficiencies or qualitative abnormalities of fibrinogen, such as that which occurs with disseminated intravascular coagulation and liver disease. A single unit of cryoprecipitate contains 300 mg of fibrinogen. 

For clinical purposes serum is preferable to plasma because the fibrinogen band interferes with the reading of globulin fractions. Moreover, it is easier to get serum free from traces of hemoglobin liberated by hemolysis. 

A commonly employed first separation step is ammonium sulfate precipitation. This technique exploits the fact that the solubility of most proteins is lowered at high salt concentrations. As the salt concentration is increased, a point is reached where the protein comes out of solution and precipitates. The concentration of salt required for this salting out effect varies from protein to protein, and thus this procedure can be used to fractionate a mixture of proteins. For example, 0.8 M ammonium sulfate precipitates out the clotting protein fibrinogen from blood serum, whereas 2.4 M ammonium sulfate is required to precipitate albumin. Salting out is also sometimes used at later stages in a purification procedure to concentrate a dilute solution of the protein since the protein precipitates and can then be redissolved in a smaller volume of buffer. 

From a strict biochemical point of view a clear-cut definition of the role of the liver in the biosynthesis of any particular plasma protein can be made only when the particular protein has been clearly and cleanly isolated, as in the case of fibrinogen. The practical difficulties of effecting such isolations on a small scale from isotopic labeling studies of the plasma proteins, such as we have described, seriously militate against such a detailed demonstration at present. The use of fractionation techniques with greater resolving power such as acrylamide gel electrophoresis already show some promise in our laboratory toward affording a more definitive picture of the biosynthetic role of the liver and the nonhepatic tissues in plasma protein production. 

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