Fibrinolysis of Contracted Blood Clots Depends on Whether Plasminogen Activator Acts from inside or Outside

Abstract Fibrinolysis involves the dissolution of polymeric fibrin networks that is required to restore blood flow through vessels obstructed by clots and thrombi. The efficiency of lysis depends on the susceptibility of fibrin to enzymatic digestion, which is governed by the structure and spatial organization of fibrin fibers as well as porosity and composition of the clot. Platelet-driven clot contraction results in compaction of the erythrocytes into the core of the clot, effectively reducing the permeability of the clot, and influences fibrin network structure. We have shown that clot contraction is reduced in blood from patients with thrombotic conditions such ischemic stroke and deep vein thrombosis, which points to the clinical importance of understanding the influence of clot contraction on efficacy of fibrinolysis. Here, we examined the effects of clot contraction on the rate of internal fibrinolysis emanating from within the clot to simulate (patho)physiological conditions, and external fibrinolysis initiated from the clot exterior to simulate therapeutic thrombolysis. Fibrinolysis was induced and the kinetics of lysis was measured in parallel in contracted versus uncontracted clots from the same citrated human blood samples. Clot formation and platelet activation were initiated with 1 U/ml thrombin and 2 mM CaCl2. Clot contraction was either unaffected or impaired by inhibiting platelet non-muscle myosin IIa (blebbistatin), actin polymerization (latrunculin A), and platelet-fibin(ogen) binding (abciximab). To examine internal fibrinolysis, 75 ng/ml of human recombinant tissue plasminogen activator (tPA) was added prior to initiation of clotting, allowing for tPA to be uniformly distributed through the clot volume and for fibrinolysis occur after the clot has formed. We used optical tracking to follow clot size in a time dependent manner. Contracted clots were completely lysed at a rate that was at least 2 times faster than clots with impaired contraction. Specifically, the average time to complete lysis was 33±4 minutes for contracted clots versus 59±3, 84±4, 75±3 minutes when contraction was impaired by blebbistatin, latrunculin A, and abciximab, respectively (p This difference in the susceptibility of contracted and uncontracted clots to internal versus external lysis suggests that the lysis rate is dominated by the interplay of clot permeability to fibrinolytic enzymes and the spatial proximity of the fibrin fibers themselves. Despite limitations of in vitro experimental models, numerous studies on fibrinolysis have demonstrated the relevance of experimental findings to pathophysiological fibrinolysis and therapeutic thrombolysis. Enhancement of fibrinolysis in contracted blood clots is consistent with the need to dissolve mature clots once they have performed their hemostatic function in a vessel on in a wound. The reduced rates of dissolution of contracted clots in our model of externally applied tPA could account for the inefficacy of therapeutic thrombolysis of old thrombi that likely underwent more compaction compared to newer thrombi. Our studies point to the clinical importance of understanding how mechanical remodeling of clots and thrombi may influence their fibrinolytic resolution and could inform the development of improved thrombolytic therapies. This work, in part, was supported by the Program for Competitive Growth at KFU. Disclosures No relevant conflicts of interest to declare.