Dense and dangerous: The tissue plasminogen activator-resistant fibrinolysis shutdown phenotype is due to abnormal fibrin polymerization

Abstract

BACKGROUND Both hyperfibrinolysis and fibrinolysis shutdown can occur after severe trauma. The subgroup of trauma patients with fibrinolysis shutdown resistant to tissue plasminogen activator (t-PA)-mediated fibrinolysis have increased mortality. Fibrin polymerization and structure may influence fibrinolysis subgroups in trauma, but fibrin architecture has not been characterized in acutely injured subjects. We hypothesized that fibrin polymerization measured in situ will correlate with fibrinolysis subgroups. METHODS Blood samples were collected from trauma patients and noninjured controls. We selected samples across a range of fibrinolysis phenotypes (shutdown, physiologic, hyperfibrinolysis) and t-PA sensitivities (sensitive, physiologic, resistant) determined by thrombelastography. Plasma clots were created in situ with fluorescent fibrinogen and imaged using confocal microscopy for analysis of clot architecture in three dimensions. For each clot, we quantified the fiber resolvability, a metric of fiber distinctness or clarity, by mapping the variance of fluorescence intensity relative to background fluorescence. We also determined clot porosity by measuring the size and distribution of the gaps between fibrin fibers in three-dimensional space. We compared these measures across fibrinolysis subgroups. RESULTS Fiber resolvability was significantly lower in all trauma subgroups compared with controls (n = 35 and 5, respectively; p < 0.05). We observed markedly different patterns of fibrin architecture among trauma patients stratified by fibrinolysis subgroup. Subjects with t-PA–resistant fibrinolysis shutdown exhibited abnormal, densely packed fibrin clots nearly devoid of pores. Individuals with t-PA–hypersensitive fibrinolysis shutdown had highly irregular clots with pores as large as 2500 μm3 to 20,000 μm3, versus 78 μm3 to 1250 μm3 in noninjured controls. CONCLUSION Fiber resolvability was significantly lower in trauma patients than controls, and subgroups of fibrinolysis differ in the porosity of the fibrin clot structure. The dense fibrin network in the t-PA–resistant group may prevent access to plasmin, suggesting a mechanism for thrombotic morbidity after injury.

COVID-19 patient plasma demonstrates resistance to tPA-induced fibrinolysis as measured by thromboelastography

“…The mechanism is thought to be due to the downregulation of angiotensin converting enzyme by the coronavirus (SARS-CoV-2), leading to increased levels of angiotensin II, an inducer of PAI-1 in endothelial cells [ 18 ]. Another possibility may simply be the high fibrinogen levels lead to clot formation with a dense fibrin structure, making it less susceptible to tPA-induced fibrinolysis [ 19 ]. Increased fibrinogen in COVID-19 patients has been shown to correlate with levels of the inflammatory cytokine IL-6, which is also associated with more severe disease [ 20 , 21 ].…”

Increased Histone-DNA Complexes and Endothelial-Dependent Thrombin Generation in Severe COVID-19

“…Patients with thromboembolic diseases also form structurally abnormal clots that are resistant to fibrinolysis [26]. Similar observations have been made in patients with severe trauma [17], which is also characterized by a disseminated intravascular coagulation-like phenotype [27]. As patients with acute thromboembolic disease [20] and trauma [13] also have elevated circulating histone levels, these structurally-and functionally-altered clots may be the result of covalent (via factor XIIIa crosslinking) and noncovalent interactions of histones with fibrin [28] in addition to dysregulated thrombin formation.…”

“…Fibrin clot formation was assessed as previously described [17]. Plasma was diluted 1:1 with HBS and incubated on glass chamber slides with AlexaFluor488-labeled fibrinogen (220 nM) (ThermoFisher Scientific, Waltham, MA), tissue factor (8.7 pM) and CaCl 2 with HistoChoice (MilliporeSigma, Burlington, MA), and an anti-photobleaching agent (Agilent Technologies, Santa Clara, California).…”

Estradiol provokes hypercoagulability and affects fibrin biology: A mechanistic exploration of sex dimorphisms in coagulation

“…Using this methodology, we evaluated macroscopic fibrin structure using custom in-house software to provide a quantifiable measurement of fiber resolvability that is relevant to hemostatic potential, as previously described 16 . In brief, “fiber resolvability index” is a measurement of the distinctness or clarity of fibrin polymerization.…”