Decoration of Fibrin with Extracellular Chaperones

Abstract

Background Many proteins bind to fibrin during clot formation in plasma. We previously identified by mass spectrometry the most abundant proteins that noncovalently bind to fibrin clots. Several of these proteins (e.g., apolipoprotein J/clusterin, haptoglobin, α2-macroglobulin, α1-antitrypsin) can act as extracellular chaperones. Objective We hypothesize that clot-binding proteins may interact with fibrin as chaperones. The goal of this study is to test this hypothesis and to investigate the origin of the cross-β or amyloid structures in fibrin clots, which are associated with protein unfolding. Methods and Results A thioflavin T assay was used to detect cross-β structures. A steadily increasing amount was measured in the fibrinogen fraction of plasma during heat stress, a standard treatment to induce unfolding of proteins. Heat-stressed plasma was clotted and clot-bound proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results showed that the amounts of the clot-bound proteins were related to the duration of the heat stress. This indicates that cross-β structures in unfolded fibrin(ogen) are involved in clot binding of the proteins, which supports our chaperone hypothesis. A contributing role of fibrin formation itself was studied by clotting purified fibrinogen with thrombin in the presence of thioflavin T. The fluorescence intensity increased in time in the presence of thrombin, but did not increase in its absence. This provides evidence for the generation of cross-β structures during fibrin formation. Conclusion Fibrin clots generated in plasma are decorated with extracellular chaperones. The binding of these chaperones involves cross-β structures originating both from unfolded fibrinogen and from fibrin formation.

Glycoprotein VI is not a Functional Platelet Receptor for Fibrin Formed in Plasma or Blood

“…Numerous plasma proteins become associated with the fibrin fibers during coagulation of plasma as shown here and in previous studies. [31][32][33] Among the 18 noncovalently and 47 covalently fibrin bound proteins (through cross-linking via FXIIIa) 31,33,42 are albumin, fibronectin, vitronectin, tissue plasminogen activator, plasminogen, lipoprotein(a), α2-antiplasmin, plasminogen activator inhibitor-2 (PAI-2), factor Xa, factor V, tissue factor pathway inhibitor, thrombin-activatable fibrinolysis inhibitor, and VWF. 31,33 Certain plasma proteins such as albumin and fibronectin modulate fibrin formation, structure, and stability by directly interacting with fibrinogen or the fibrin network during fibrin assembly.…”

“…Fibrin generated in plasma by the coagulation cascade binds noncovalently and covalently numerous plasma proteins, [30][31][32][33] and thus differs from fibrin prepared with purified fibrinogen. We therefore compared the effects of anti-GPVI antibodies and inhibitors of GPVI signaling through Syk and Btk on flow-dependent platelet thrombus formation onto fibrins prepared from purified and recombinant fibrinogen, and endogenous fibrin generated by the coagulation cascade in plasma or blood.…”

Rupture of blood clots: Mechanics and pathophysiology

“…The current work on the rupture resistance of fibrin has been performed on blood plasma clots at a physiological fibrin volume fraction, following natural covalent cross-linking by plasma transglutaminase factor XIIIa, and in the presence of all plasma proteins that can potentially modulate fibrin properties (18,19). The rupture resistance of fibrin basically determines the mechanical stability of the entire blood clot because there is not a substantial difference in the mechanical response of human platelet-poor plasma clots, platelet-rich plasma clots, and whole-blood clots (5).…”

Could Some Nonhemostatic Plasma Proteins Serve as Refuse Collectors for Fibrin(ogen)?

“…The cross-β-structures in fibrinogen and fibrin were formed by prolonged heating of plasma (mimicking unfolding that occurs with aging) as well as upon enzymatic conversion of fibrinogen to fibrin, in which formation of β-structures can occur. 1,3 Also, the authors showed an inhibitory effect of caseins (known as milk chaperones) on fibrin formation.…”

“…In the October issue of Thrombosis and Haemostasis, Talens et al 1 proposed a general physiological role for certain plasma proteins previously shown to bind strongly but noncovalently to fibrinogen and/or fibrin. After excluding the proteins with known hemostatic activities, the authors recognized that some of the remaining fibrin-bound proteins (clusterin, haptoglobin, α2-macroglobulin, apolipoproteins E and AI, albumin, serum amyloid P, and α1-antitrypsin) belong to a family of "extracellular chaperones."…”