Fibers Generated by Plasma Des-AA Fibrin Monomers and Protofibril/Fibrinogen Clusters Bind Platelets: Clinical and Nonclinical Implications

Abstract

Objective Soluble fibrin (SF) is a substantial component of plasma fibrinogen (fg), but its composition, functions, and clinical relevance remain unclear. The study aimed to evaluate the molecular composition and procoagulant function(s) of SF. Materials and Methods Cryoprecipitable, SF-rich (FR) and cryosoluble, SF-depleted (FD) fg isolates were prepared and adsorbed on one hydrophilic and two hydrophobic surfaces and scanned by atomic force microscopy (AFM). Standard procedures were used for fibrin polymerization, crosslinking by factor XIII, electrophoresis, and platelet adhesion. Results Relative to FD fg, thrombin-induced polymerization of FR fg was accelerated and that induced by reptilase was markedly delayed, attributable to its decreased (fibrinopeptide A) FpA. FR fg adsorption to each surface yielded polymeric clusters and co-cryoprecipitable solitary monomers. Cluster components were crosslinked by factor XIII and comprised ≤21% of FR fg. In contrast to FD fg, FR fg adsorption on hydrophobic surfaces resulted in fiber generation enabled by both clusters and solitary monomers. This began with numerous short protofibrils, which following prolonged adsorption increased in number and length and culminated in surface-linked three-dimensional fiber networks that bound platelets. Conclusion The abundance of adsorbed protofibrils resulted from (1) protofibril/fg clusters whose fg was dissociated during adsorption, and (2) adsorbed des-AA monomers that attracted solution counterparts initiating protofibril assembly and elongation by their continued incorporation. The substantial presence of both components in transfused plasma and cryoprecipitate augments hemostasis by accelerating thrombin-induced fibrin polymerization and by tightly anchoring the resulting clot to the underlying wound or to other abnormal vascular surfaces.

Novel characteristics of soluble fibrin: hypercoagulability and acceleration of blood sedimentation rate mediated by its generation of erythrocyte-linked fibers

“…Similar heterogeneity (8 S and 24 S) reported later in studies of rabbit fg was attributed to dissociable fibrin/fg complexes, reviewed in Shainoff and DiBello (2001). In hindsight, this heterogeneity likely reflected the recently discovered multimeric SF/fg clusters (Galanakis et al 2021).…”

“…Reagents, supplies, and their sources were as detailed previously (Galanakis et al 2021). Buffers used included the following: 0.01 M Tris-HCl, pH 7.4, in 0.15 NaCl (TBS); 0.01 M Na 2 PO 4 , pH 6.4 or 7, in 0.15 M NaCl (PBS); and 0.05 M or 0.02 M Na 2 PO 4 , pH 6.4.…”

“…For example, when fg was isolated (Ware et al 1947), SF most likely co-isolated with it and was later prepared and further investigated (Shainoff and Page 1962). A key attribute of SF is that at sub-physiologic temperatures and decreased ionic strength SF tends to form precipitates and gels that redissolve on rewarming to 37 °C (Shainoff and Page 1962;Shainoff and DiBello 2001;Galanakis et al 2021).…”

“…Plasma cryoprecipitate is still used today during massive transfusions and when the relative factor concentrates are not available. Using the cold washing observation (vide supra), we isolated SF by washing plasma cryoprecipitate with cold buffer (Galanakis et al 2021). Another group (Morrison et al 1948) purified fg by an ethanol and glycine precipitation method and their otherwise highly pure fg was heterogeneous by ultracentrifugation yielding an S 20 value of 8.5 S with minor components showing 15 S and both higher and lower values.…”

“…Meanwhile, the substantial SF levels shown in blood bank plasma (Galanakis 1995) raised the possibility of an SF role in the thrombotic complications of fg replacement therapy, particularly with use of the inherently SF-rich plasma cryoprecipitate, and multiple perioperative transfusions (Goel et al 2018). Also, thrombotic complications within and around intravascular devices (Cost and Journeycake 2011) argue for a possible SF role, since many devices possess hydrophobic surface on which plasma SF readily adsorbs and self-assembles (Galanakis et al 2021). Our present investigations of SF focus on its molecular imprint profile, stiffness and lysis of its thrombininduced clots, and its possible role in ESR acceleration suggested by fg binding to RBCs (Maeda et al 1984(Maeda et al , 1987De Olivera et al 2012;Lominadze and Dean 2002).…”