Spatial Proteomics Analysis of Soft and Stiff Regions in Human Acute Arterial Thrombus

Abstract

Background: The soft regions of a thrombus tend to be more susceptible to r-tPA (recombinant tissue-type plasminogen activator)-mediated thrombolysis and are more easily removed by mechanical thrombectomy than the stiff counterpart. This study aimed to understand the molecular pathological differences between the soft and stiff regions of human arterial thrombus. Methods: We developed a spatial proteomic workflow combining proteomics with laser-captured microdissection to analyze human arterial thrombi with Masson trichrome staining to identify stiff and soft regions from 2 independent cohorts of patients with acute myocardial or cerebral infarction. Dysregulated proteins in a C57BL6/J male mouse model of arterial thrombosis were identified by pathway enrichment and pairwise analyses from the common gene ontology enrichment and dysregulated proteins between carotid and coronary arterial thrombi, and validated by immunohistochemistry. Results: Spatial proteomics of the coronary arterial thrombi collected from 7 patients with myocardial infarct revealed 7 common dysregulated proteins in 2 cohorts of patients, and upregulation of TGF-β1 (transforming growth factor β1) was the most prominent fibrosis-related protein. Inhibition of TGF-β1 resulted in delayed arterial thrombosis and accelerated blood flow restoration in mouse model. We further expanded the spatial proteomic workflow to the carotid artery thrombi collected from 11 patients with cerebral infarction. Pairwise proteomic analysis of stiff and soft regions between carotid and coronary arterial thrombi further revealed 5 common gene ontology clusters including features of platelet activation, and a common dysregulated protein COL1A1 (collagen type 1 alpha 1) that was reported to be influenced by TGF-β1. We also verified the expression in human and mice carotid arterial thrombi. Conclusions: This study demonstrates the spatially distinct composition of proteins in the stiff and soft regions of human arterial thrombi, and suggests that TGF-β1 is a key therapeutic target for promoting arterial thrombolysis.

How Clot Composition Influences Fibrinolysis in the Acute Phase of Stroke: A Proteomic Study of Cerebral Thrombi

“…14 In clinical studies, FA was assessed indirectly by its expected clinical-radiological benefits. [5][6][7][8]12,24 In an original way, we studied FA in vivo both at the bloodthrombus interface and inside the thrombus. As expected, FA in and FA out were significantly associated with r-tPA administration, which supports the validity of the methods used.…”

“…This inverse correlation between fibrin(ogen) level and erythrocyte content is consistent with the literature, which classically opposes fibrin-rich thrombi to erythrocyte-rich thrombi. 5 In fact, several reports suggest that dichotomization into fibrin-or erythrocyte-rich thrombi is linked to recanalization rate, thrombus volume variations, or clinical outcomes after IVT, [5][6][7][8]12,24 thereby indirectly suggesting different FA. In our study, only high FA at the blood-thrombus interface led to better clinico-radiological outcomes for stroke patients, although both FA out and FA in were driven by IVT.…”

“…5,6 However, studies of histological thrombi composition led to contradictory results and had major limitations. Indeed, thrombus composition varies along the thrombus structure, 7,8 but the classical histological approach can only provide a partial analysis of a thrombus fragment.…”