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

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<0.001). To examine external fibrinolysis, blood spiked with purified human 125I-fibrinogen was allowed to clot and contract (unless contraction was inhibited) prior to the addition of 75 ng/ml tPA. Clots with impaired contraction released 2-4-fold more radiolabeled soluble degradation products during the first 30 minutes and continued to lyse at a rate 4-fold faster than contracted clots over the initial 4 hours following addition of tPA. This reduction of the fibrinolysis rate in contracted clots was not due to the expulsion of serum-soluble anti-fibrinolytic compounds during the contraction process because serum replacement with a buffer did not affect the lysis rate. 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.

Post-reperfusion syndrome in patients with ischemic heart disease after coronary stenting.

Post-reperfusion syndrome is a chronic multi-factorial syndrome, caused by restoration of coronary blood flow of de novo in patients after coronary stenting. Pathophysiological processes occurring in stenting segment (incomplete endothelization of vessel, excessive regeneration of neointima, formation of neoatherosclerosis) the risk of partial or total stent thrombosis due to the low level of compliance of the patient, as well as local changes in blood flow to the myocardium and modification of hemodynamics in stented arteries have a significant impact on the clinical course and prognosis of the disease. In contrast to the syndrome of reperfusion occurring after restoration of blood flow in the infarct-dependent coronary artery syndrome and postischemic reperfusion syndrome in patients with acute myocardial infarction after spontaneous or therapeutic thrombolysis, post-reperfusion syndrome is characterized by prolonged course (months and years). Clinical symptoms depend of the localization of pathological process (in stented arteries or in other non-stented arteries of the heart), as well as the presence of comorbidity and the factors of risk of the coronary heart disease.

Clot Lysis in a Perfusion System

SummaryMost information on clot lysis is derived from in vitro methods whereby various components of the clotting and fibrinolytic systems are mixed before an actual clot is formed. This situation bears little relationship to thrombolysis in vivo. Therefore several techniques have been recently proposed, in which pre-formed clots are exposed to the effects of active agents by contact and diffusion rather than by intimate mixing prior to clotting. We describe an apparatus whereby a perfusion is delivered at controlled rates to clots of standard size and volume formed in calibrated tubes. The composition of the clots can be varied as well as the rate of perfusion and the content of perfusate. The surface of contact between the fluid and the fibrin gel is kept constant throughout and the clot-perfusate relationship is as close as possible to the in vivo situation during thrombolytic therapy. Under these conditions clot lysis by Streptokinase appears as a linear function of time, and the rate of lysis is directly related to kinase concentration. Since the clot intrinsic plasminogen-proactivator content is sufficient to ensure lysis, the lysis time finally depends upon the rate of diffusion of the kinase into the gel. Inhibition obtained with various amounts of E-aminocaproic acid incorporated to the clots or added to the perfusion fluid also suggests that diffusion problems are of major importance in physiological and therapeutic thrombolysis.