Identification of Fibrin Clot-Bound Plasma Proteins

Abstract

Several proteins are known to bind to a fibrin network and to change clot properties or function. In this study we aimed to get an overview of fibrin clot-bound plasma proteins. A plasma clot was formed by adding thrombin, CaCl2 and aprotinin to citrated platelet-poor plasma and unbound proteins were washed away with Tris-buffered saline. Non-covalently bound proteins were extracted, separated with 2D gel electrophoresis and visualized with Sypro Ruby. Excised protein spots were analyzed with mass spectrometry. The identity of the proteins was verified by checking the mass of the protein, and, if necessary, by Western blot analysis. Next to established fibrin-binding proteins we identified several novel fibrin clot-bound plasma proteins, including α2-macroglobulin, carboxypeptidase N, α1-antitrypsin, haptoglobin, serum amyloid P, and the apolipoproteins A-I, E, J, and A-IV. The latter six proteins are associated with high-density lipoprotein particles. In addition we showed that high-density lipoprotein associated proteins were also present in fibrinogen preparations purified from plasma. Most plasma proteins in a fibrin clot can be classified into three groups according to either blood coagulation, protease inhibition or high-density lipoprotein metabolism. The presence of high-density lipoprotein in clots might point to a role in hemostasis.

Comment on Ågren et al. Increased Incorporation of Antiplasmin Into the Fibrin Network in Patients With Type 1 Diabetes. Diabetes Care 2014;37:2007–2014

“…It is anticipated that only noncovalently fibrin-bound antiplasmin is extracted by urea. Although noncovalently bound antiplasmin might theoretically be present in plasma clots (3), in a recent study we did not find evidence for the presence of this form of antiplasmin in plasma clots prepared and extracted in a comparable manner as in the study byÅgren et al (4).…”

Factors intrinsic and extrinsic to blood hamper the development of a routine blood test for human prion diseases

“…(2) The non-specific binding of several plasma proteins, including PrP Sc and the infectious agent, to fibrin clots which can often be made from plasma samples in vitro (Talens et al, 2012).…”

“…(3) The entrapment of two important protease inhibitors, a2-macroglobulin and a1-antitrypsin, into fibrin clots (Talens et al, 2012) may increase the spontaneous digestion of PK-sensitive PrP Sc , reducing its effective concentration in plasma samples.…”

Decreased fibrinolytic potential and morphological changes of fibrin structure in dermatitis herpetiformis

“…The median lysis time of plasma clots from untreated DH patients was more than 3-fold longer in this assay compared to When a plasma clot is formed, a broad range of proteins associated with fibrinogen is entrapped in its structure [25]. The prevalent cryofibrinogenemia in DH [9] raises the possibility that the altered structural and lytic profile of fibrin in DH is due to a factor associated with circulating fibrinogen.…”