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Clot retraction refers to the process whereby activated platelets transduce contractile forces onto the fibrin network of a thrombus, which over time increases clot density and decreases clot size. This process is considered important for promoting clot stability and maintaining blood vessel patency. Insights into the mechanisms regulating clot retraction at sites of vascular injury have been hampered by a paucity of in vivo experimental models. By pairing localized vascular injury with thrombin microinjection in the mesenteric circulation of mice, we have demonstrated that the fibrin network of thrombi progressively compacts over a 2-hour period. This was a genuine retraction process, as treating thrombi with blebbistatin to inhibit myosin IIa-mediated platelet contractility prevented shrinkage of the fibrin network. Real-time confocal analysis of fibrinolysis after recombinant tissue-type plasminogen activator (tPA) administration revealed that incomplete proteolysis of fibrin polymers markedly facilitated clot retraction. Similarly, inhibiting endogenous fibrinolysis with tranexamic acid reduced retraction of fibrin polymers in vivo. In vitro clot retraction experiments indicated that subthreshold doses of tPA facilitated clot retraction through a plasmin-dependent mechanism. These effects correlated with changes in the elastic modulus of fibrin clots. These findings define the endogenous fibrinolytic system as an important regulator of clot retraction, and show that promoting clot retraction is a novel and complementary means by which fibrinolytic enzymes can reduce thrombus size., (© 2017 by The American Society of Hematology.)

Unlabelled: Essentials C57BL/6J-tissue plasminogen activator (tPA)-deficient mice are widely used to study tPA function. Congenic C57BL/6J-tPA-deficient mice harbor large 129-derived chromosomal segments. The 129-derived chromosomal segments contain gene mutations that may confound data interpretation. Passenger mutation-free isogenic tPA-deficient mice were generated for study of tPA function., Summary: Background The ability to generate defined null mutations in mice revolutionized the analysis of gene function in mammals. However, gene-deficient mice generated by using 129-derived embryonic stem cells may carry large segments of 129 DNA, even when extensively backcrossed to reference strains, such as C57BL/6J, and this may confound interpretation of experiments performed in these mice. Tissue plasminogen activator (tPA), encoded by the PLAT gene, is a fibrinolytic serine protease that is widely expressed in the brain. A number of neurological abnormalities have been reported in tPA-deficient mice. Objectives To study genetic contamination of tPA-deficient mice. Materials and methods Whole genome expression array analysis, RNAseq expression profiling, low- and high-density single nucleotide polymorphism (SNP) analysis, bioinformatics and genome editing were used to analyze gene expression in tPA-deficient mouse brains. Results and conclusions Genes differentially expressed in the brain of Plat(-/-) mice from two independent colonies highly backcrossed onto the C57BL/6J strain clustered near Plat on chromosome 8. SNP analysis attributed this anomaly to about 20 Mbp of DNA flanking Plat being of 129 origin in both strains. Bioinformatic analysis of these 129-derived chromosomal segments identified a significant number of mutations in genes co-segregating with the targeted Plat allele, including several potential null mutations. Using zinc finger nuclease technology, we generated novel 'passenger mutation'-free isogenic C57BL/6J-Plat(-/-) and FVB/NJ-Plat(-/-) mouse strains by introducing an 11 bp deletion into the exon encoding the signal peptide. These novel mouse strains will be a useful community resource for further exploration of tPA function in physiological and pathological processes., (© 2016 International Society on Thrombosis and Haemostasis.)

Tissue-type plasminogen activator (tPA) is an extracellular protease that converts plasminogen into plasmin. For tPA to generate plasmin under biologic conditions, a cofactor must first bring tPA and plasminogen into physical proximity. Fibrin provides this cofactor for tPA-mediated plasmin generation in blood. Despite being naturally devoid of fibrin(ogen), tPA-mediated plasmin formation also occurs in the brain. The fibrin-like cofactor(s) that facilitates plasmin formation in the injured brain has remained unknown. Here we show that protein aggregates formed during neuronal injury provide a macromolecular, nonfibrin cofactor that promotes tPA-mediated plasmin formation and subsequent cell breakdown. The binding of plasminogen and tPA to these protein aggregates occurs via distinct mechanisms. Importantly, nonneuronal cell types also exhibit this cofactor effect upon injury, indicating a general phenomenon. This novel cofactor identified in nonviable cells has ramifications for ischemic stroke where tPA is used clinically and where plasmin activity within the injured brain is unwanted. A means of selectively inhibiting the binding of tPA to nonviable cells while preserving its association with fibrin may be of benefit for the treatment of ischemic stroke.