Contraction (retraction) of the blood clot is a part of the clotting process driven by activated platelets attached to fibrin that can potentially modulate the obstructiveness and integrity of thrombi. The aim of this work was to reveal the pathogenic importance of contraction of clots and thrombi in venous thromboembolism (VTE). We investigated the kinetics of clot contraction in the blood of 55 patients with VTE. In addition, we studied the ultrastructure of ex vivo venous thrombi as well as the morphology and functionality of isolated platelets. Thrombi from VTE patients contained compressed polyhedral erythrocytes, a marker for clot contraction in vivo. The extent and rate of contraction were reduced by twofold in clots from the blood of VTE patients compared with healthy controls. The contraction of clots from the blood of patients with pulmonary embolism was significantly impaired compared with that of those with isolated venous thrombosis, suggesting that less compacted thrombi are prone to embolization. The reduced ability of clots to contract correlated with continuous platelet activation followed by their partial refractoriness. Morphologically, 75% of platelets from VTE patients were spontaneously activated (with filopodia) compared with only 21% from healthy controls. At the same time, platelets from VTE patients showed a 1.4-fold reduction in activation markers expressed in response to chemical activation when compared with healthy individuals. The results obtained suggest that the impaired contraction of thrombi is an underappreciated pathogenic mechanism in VTE that may regulate the obstructiveness and embologenicity of venous thrombi.

Clot contraction influences the rate of fibrinolysis in vitro. Internal fibrinolysis is enhanced ∼2‐fold in contracted vs. uncontracted blood clots. External fibrinolysis is ∼4‐fold slower in contracted vs. uncontracted blood clots. Contraction can modulate lytic resistance and potentially the clinical outcome of thrombosis. Summary BackgroundFibrinolysis involves dissolution of polymeric fibrin networks that is required to restore blood flow through vessels obstructed by thrombi. The efficiency of lysis depends in part on the susceptibility of fibrin to enzymatic digestion, which is governed by the structure and spatial organization of fibrin fibers. How platelet‐driven clot contraction affects the efficacy of fibrinolysis has received relatively little study. ObjectiveHere, 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. MethodsClot contraction was prevented by inhibiting platelet myosin IIa activity, actin polymerization or platelet‐fibrin(ogen) binding. Internal fibrinolysis was measured by optical tracking of clot size. External fibrinolysis was determined by the release of radioactive fibrin degradation products. Results and ConclusionsClot contraction enhanced the rate of internal fibrinolysis ∼2‐fold. In contrast, external fibrinolysis was ~4‐fold slower in contracted clots. This dichotomy in the susceptibility of contracted and uncontracted clots to internal vs. external lysis suggests that the rate of lysis is dependent upon the interplay between accessibility of fibrin fibers to fibrinolytic agents, including clot permeability, and the spatial proximity of the fibrin fibers that modulate the effects of the fibrinolytic enzymes. Understanding how compaction of blood clots influences clot lysis might have important implications for prevention and treatment of thrombotic disorders.

Polyhedral erythrocytes, named polyhedrocytes, are formed in contracted blood clots and thrombi, as a result of compression by activated contractile platelets pulling on fibrin. This deformation was shown to be mechanical in nature and polyhedrocytes were characterized using light and electron microscopy. Through three-dimensional reconstruction, we quantified the geometry of biconcave, intermediate, and polyhedral erythrocytes within contracting blood clots. During compression, erythrocytes became less oblate and more prolate than the biconcave cells and largely corresponded to convex, irregular polyhedra with a total number of faces ranging from 10 to 16. Faces were polygons with 3 to 6 sides. The majority of the faces were quadrilaterals, though not all sides were straight and not all faces were flat. There were no changes in the surface area or volume. These results describe the gradual natural deformation of erythrocytes as a part of compaction into a tightly packed array that is an important but understudied component of mature blood clots and thrombi.

Breast tumors are heterogeneous and composed of different subpopulation of cells, each with dynamic roles that can change with stage, site, and microenvironment. Cellular heterogeneity is, in part, due to cancer stem-like cells (CSC) that share properties with stem cells and are associated with treatment resistance. CSCs rewire metabolism to meet energy demands of increased growth and biosynthesis. O-GlcNAc transferase enzyme (OGT) uses UDP-GlcNAc as a substrate for adding O-GlcNAc moieties to nuclear and cytoplasmic proteins. OGT/O-GlcNAc levels are elevated in multiple cancers and reducing OGT in cancer cells blocks tumor growth. Here, we report that breast CSCs enriched in mammosphere cultures contain elevated OGT/O-GlcNAcylation. Inhibition of OGT genetically or pharmacologically reduced mammosphere forming efficiency, the CD44 H /CD24 L , NANOGþ, and ALDHþ CSC population in breast cancer cells. Conversely, breast cancer cells overexpressing OGT increased mammosphere formation, CSC populations in vitro, and also increased tumor initiation and CSC frequency in vivo. Furthermore, OGT regulates expression of a number of epithelial-to-mesenchymal transition and CSC markers including CD44, NANOG, and c-Myc. In addition, we identify Kr€ uppel-like factor 8 (KLF8) as a novel regulator of breast cancer mammosphere formation and a critical target of OGT in regulating CSCs.Implications: These findings demonstrate that OGT plays a key role in the regulation of breast CSCs in vitro and tumor initiation in vivo, in part, via regulation of KLF8, and thus inhibition of OGT may serve as a therapeutic strategy to regulate tumor-initiating activity.

Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy sometimes associated with thrombosis. The hallmark of HIT is antibodies to the heparin/platelet factor 4 (PF4) complex that cause thrombocytopenia and thrombosis through platelet activation. Despite the clinical importance, the molecular mechanisms and late consequences of immune platelet activation are not fully understood. Here, we studied immediate and delayed effects of the complexes formed by human PF4 and HIT-like monoclonal mouse anti-human-PF4/heparin IgG antibodies (named KKO) on isolated human platelets in vitro. Direct platelet-activating effect of the KKO/PF4 complexes was corroborated by the overexpression of phosphatidylserine (PS) and P-selectin on the platelet surface. The immune platelet activation was accompanied by a decrease of the mitochondrial transmembrane potential (ΔΨm), concurrent with a significant gradual reduction of the ATP content in platelets, indicating disruption of energy metabolism. A combination of PS expression and mitochondrial depolarization induced by the PF4-containing immune complexes observed in a substantial fraction of platelets was considered as a sign of ongoing platelet death, as opposed to a subpopulation of activated live platelets with PS on the plasma membrane but normal ΔΨm. Both activated and dying platelets treated with KKO/PF4 formed procoagulant extracellular microvesicles bearing PS on their surface. Scanning and transmission electron microscopy revealed dramatic morphological changes of KKO/PF4-treated platelets, including their fragmentation, another indicator of cell death. Most of the effects of KKO/PF4 were prevented by an anti-FcγRII monoclonal antibody IV.3. The adverse functional and structural changes in platelets induced by the KKO/PF4 complexes were associated with strong time-dependent activation of calpain, but only trace cleavage of caspase 3. The results indicate that the pathogenic PF4-containing HIT-like immune complexes induce direct prothrombotic platelet activation via FcγRIIA receptors followed by non-apoptotic calpain-dependent death of platelets, which can be an important mechanism of thrombocytopenia during HIT development.