The Proteome Content of Blood Clots Observed Under Different Conditions: Successful Role in Predicting Clot Amyloid(ogenicity)

Abstract

A recent analysis compared the proteome of (i) blood clots seen in two diseases-sepsis and long COVID-when blood was known to have clotted into an amyloid microclot form (as judged by staining with the fluorogenic amyloid stain thioflavin T) with (ii) that of those non-amyloid clots considered to have formed normally. Such fibrinaloid microclots are also relatively resistant to fibrinolysis. The proteins that the amyloid microclots contained differed markedly both from the soluble proteome of typical plasma and that of normal clots, and also between the diseases studied (an acute syndrome in the form of sepsis in an ITU and a chronic disease represented by Long COVID). Many proteins in the amyloid microclots were low in concentration in plasma and were effectively accumulated into the fibres, whereas many other abundant plasma proteins were excluded. The proteins found in the microclots associated with the diseases also tended to be themselves amyloidogenic. We here ask effectively the inverse question. This is: can the clot proteome tell us whether the clots associated with a particular disease contained proteins that are observed uniquely (or are highly over-represented) in known amyloid clots relative to normal clots, and thus were in fact amyloid in nature? The answer is in the affirmative in a variety of major coagulopathies, viz., venous thromboembolism, pulmonary embolism, deep vein thrombosis, various cardiac issues, and ischaemic stroke. Galectin-3-binding protein and thrombospondin-1 seem to be especially widely associated with amyloid-type clots, and the latter has indeed been shown to be incorporated into growing fibrin fibres. These may consequently provide useful biomarkers with a mechanistic basis.

Keywords: amyloid; clotting; cross-seeding; fibrils; fibrinaloid; proteomics.

Figure 1

Different classes or types of protein co-aggregation: Titration; Sequestration; Axial and Lateral. Reprinted from the open access preprint [29], which was itself adapted from [31].

Figure 2

The relationship between forward and inverse strategies (as in systems biology [35]), in which here we seek to assess the normal or amyloid nature of clots as judged by their proteome. In the forward strategy, we calibrate the system by asking which proteins differ in the two cases where the amyloid nature is known [36]. In the inverse case, developed here, we use the observed protein entrapments to infer or to suggest whether the clots are likely to be amyloid in nature.

Figure 3

Changes in the plasma proteome in individuals with deep vein thrombosis after trauma. Data are taken from Supplementary Table S15 of [72] and visualised using the Spotfire program (http://spotfire.com/, accessed on 30 January 2025). Those with a log2 change of <−2 or >2 are labelled.

Figure 4

Illustration of the pathways from inflammation-induced cellular damage to the formation of amyloid microclots, highlighting the roles of two notably amyloidogenic proteins (LG3BP and TSP-1) in abnormal fibrinogen interactions and the resulting fibrinolysis-resistant clot structures. (1) Inflammation and Cellular Stress: Triggers that initiate vascular damage, often due to infection or systemic inflammation. (2) Damage to Cells: Injury to endothelial cells and activation of immune cells like dendritic cells and macrophages. (3) Circulating Inflammatory Molecules: Release of inflammatory mediators that exacerbate vascular stress and promote clot formation. (4) Key Amyloidogenic Proteins: Production of Galectin-3-Binding Protein (LG3BP) and Thrombospondin-1 (TSP-1) by immune and endothelial cells. (5) Protein–Protein Interaction with Fibrinogen: Interaction of LG3BP and TSP-1 with fibrinogen, promoting amyloid-like fibrin formation. (6) Amyloid Microclotting: Formation of amyloid microclots that are resistant to fibrinolysis, contributing to disease pathology.