Chemical Adjustment of Fibrinolysis.

Fibrinolysis is the process of the fibrin-platelet clot dissolution initiated after bleeding has been stopped. It is regulated by a cascade of proteolytic enzymes with plasmin at its core. In pathological cases, the balance of normal clot formation and dissolution is replaced by a too rapid lysis, leading to bleeding, or an insufficient one, leading to an increased thrombotic risk. The only approved therapy for emergency thrombus lysis in ischemic stroke is recombinant tissue plasminogen activator, though streptokinase or urokinase-type plasminogen activators could be used for other conditions. Low molecular weight compounds are of great interest for long-term correction of fibrinolysis dysfunctions. Their areas of application might go beyond the hematology field because the regulation of fibrinolysis could be important in many conditions, such as fibrosis. They enhance or weaken fibrinolysis without significant effects on other components of hemostasis. Here we will describe and discuss the main classes of these substances and their mechanisms of action. We will also explore avenues of research for the development of new drugs, with a focus on the use of computational models in this field.

Design, Synthesis, and Evaluation of New Hybrid Derivatives of 5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-2(1H)-one as Potential Dual Inhibitors of Blood Coagulation Factors Xa and XIa.

Cardiovascular diseases caused by blood coagulation system disorders are one of the leading causes of morbidity and mortality in the world. Research shows that blood clotting factors are involved in these thrombotic processes. Among them, factor Xa occupies a key position in the blood coagulation cascade. Another coagulation factor, XIa, is also a promising target because its inhibition can suppress thrombosis with a limited contribution to normal hemostasis. In this regard, the development of dual inhibitors as new generation anticoagulants is an urgent problem. Here we report the synthesis and evaluation of novel potential dual inhibitors of coagulation factors Xa and XIa. Based on the principles of molecular design, we selected a series of compounds that combine in their structure fragments of pyrrolo[3,2,1-ij]quinolin-2-one and thiazole, connected through a hydrazine linker. The production of new hybrid molecules was carried out using a two-stage method. The reaction of 5,6-dihydropyrrolo[3,2,1-ij]quinoline-1,2-diones with thiosemicarbazide gave the corresponding hydrazinocarbothioamides. The reaction of the latter with DMAD led to the target methyl 2-(4-oxo-2-(2-(2-oxo-5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1(2H)-ylidene)hydrazineyl)thiazol-5(4H)-ylidene)acetates in high yields. In vitro testing of the synthesized molecules revealed that ten of them showed high inhibition values for both the coagulation factors Xa and XIa, and the IC50 value for some compounds was also assessed. The resulting structures were also tested for their ability to inhibit thrombin.

Platelet functional abnormalities in pediatric patients with kaposiform hemangioendothelioma/Kasabach-Merritt phenomenon.

Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor of infancy that is commonly associated with a life-threatening thrombocytopenic condition, Kasabach-Merritt phenomenon (KMP). Platelet CLEC-2, tumor podoplanin interaction is considered the key mechanism of platelet clearance in these patients. Here, we aimed to assess platelet functionality in such patients. Three groups of 6 to 9 children were enrolled: group A with KHE/KMP without hematologic response (HR) to therapy; group B with KHE/KMP with HR; and group C with healthy children. Platelet functionality was assessed by continuous and end point flow cytometry, low-angle light scattering analysis (LaSca), fluorescent microscopy of blood smears, and ex vivo thrombi formation. Platelet integrin activation in response to a combination of CRP (GPVI agonist) and TRAP-6 (PAR1 agonist), as well as calcium mobilization and integrin activation in response to CRP or rhodocytin (CLEC-2 agonist) alone, were significantly diminished in groups A and B. At the same time, platelet responses to ADP with or without TRAP-6 were unaltered. Thrombi formation from collagen in parallel plate flow chambers was also noticeably decreased in groups A and B. In silico analysis of these results predicted diminished amounts of CLEC-2 on the platelet surface of patients, which was further confirmed by immunofluorescence microscopy and flow cytometry. In addition, we also noted a decrease in GPVI levels on platelets from group A. In KHE/KMP, platelet responses induced by CLEC-2 or GPVI activation are impaired because of the diminished number of receptors on the platelet surface. This impairment correlates with the severity of the disease and resolves as the patient recovers.

New Hybrid Tetrahydropyrrolo[3,2,1-ij]quinolin-1-ylidene-2-thioxothiazolidin-4-ones as New Inhibitors of Factor Xa and Factor XIa: Design, Synthesis, and In Silico and Experimental Evaluation.

Despite extensive research in the field of thrombotic diseases, the prevention of blood clots remains an important area of study. Therefore, the development of new anticoagulant drugs with better therapeutic profiles and fewer side effects to combat thrombus formation is still needed. Herein, we report the synthesis and evaluation of novel pyrroloquinolinedione-based rhodanine derivatives, which were chosen from 24 developed derivatives by docking as potential molecules to inhibit the clotting factors Xa and XIa. For the synthesis of new hybrid derivatives of pyrrolo[3,2,1-ij]quinoline-2-one, we used a convenient structural modification of the tetrahydroquinoline fragment by varying the substituents in positions 2, 4, and 6. In addition, the design of target molecules was achieved by alkylating the amino group of the rhodanine fragment with propargyl bromide or by replacing the rhodanine fragment with 2-thioxoimidazolidin-4-one. The in vitro testing showed that eight derivatives are capable of inhibiting both coagulation factors, two compounds are selective inhibitors of factor Xa, and two compounds are selective inhibitors of factor XIa. Overall, these data indicate the potential anticoagulant activity of these molecules through the inhibition of the coagulation factors Xa and XIa.

Hypochlorite-induced oxidation of fibrinogen: Effects on its thermal denaturation and fibrin structure.

BACKGROUND: Human fibrinogen, which plays a key role in plasma haemostasis, is a highly vulnerable target for oxidants. Fibrinogen undergoes posttranslational modifications that can potentially disrupt protein structure and function. METHODS: For the first time, by differential scanning calorimetry, dynamic and elastic light scattering and confocal laser scanning microscopy, the consequences of HOCl/-OCl-induced oxidation of fibrinogen on its thermal denaturation, molecular size distribution and fibrin clot network have been explored. RESULTS: Within a wide range of HOCl/-OCl concentrations (50-300 µM), the molecular size distribution remained unimodal; however, the average size of the hydrated molecules decreased. HOCl/-OCl-induced oxidation of fibrinogen resulted in the diminished thermal stability of regions D and E. As evidenced by elastic light scattering and confocal laser scanning microscopy, HOCl/-OCl caused the formation of abnormal fibrin with a decreased diameter of individual fibres. CONCLUSIONS: The current results along with data from previous studies enable one to conclude that the effect of HOCl/-OCl-mediated oxidation on the thermal stability of region D is influenced directly by oxidative damage to the D region structure. Since the E region is not subjected to oxidative modification, its structural damage is likely to be mediated by the oxidation of other protein structures, in particular α-helical coiled-coils. GENERAL SIGNIFICANCE: The experimental findings acquired in the current study could help to elucidate the consequences of oxidative stress in vivo on damage to the structure of fibrinogen/fibrin under the action of different ROS species.

Asymmetrical Forces Dictate the Distribution and Morphology of Platelets in Blood Clots.

Primary hemostasis consists in the activation of platelets, which spread on the exposed extracellular matrix at the injured vessel surface. Secondary hemostasis, the coagulation cascade, generates a fibrin clot in which activated platelets and other blood cells get trapped. Active platelet-dependent clot retraction reduces the clot volume by extruding the serum. Thus, the clot architecture changes with time of contraction, which may have an important impact on the healing process and the dissolution of the clot, but the precise physiological role of clot retraction is still not completely understood. Since platelets are the only actors to develop force for the retraction of the clot, their distribution within the clot should influence the final clot architecture. We analyzed platelet distributions in intracoronary thrombi and observed that platelets and fibrin co-accumulate in the periphery of retracting clots in vivo. A computational mechanical model suggests that asymmetric forces are responsible for a different contractile behavior of platelets in the periphery versus the clot center, which in turn leads to an uneven distribution of platelets and fibrin fibers within the clot. We developed an in vitro clot retraction assay that reproduces the in vivo observations and follows the prediction of the computational model. Our findings suggest a new active role of platelet contraction in forming a tight fibrin- and platelet-rich boundary layer on the free surface of fibrin clots.

Binding of Coagulation Factor XIII Zymogen to Activated Platelet Subpopulations: Roles of Integrin αIIbß3 and Fibrinogen.

Factor XIIIa (fXIIIa) is a transglutaminase that plays a crucial role in fibrin clot stabilization and regulation of fibrinolysis. It is known to bind to procoagulant platelets. In contrast, the zymogen fXIII interaction with platelets is not well characterized. We investigated the interaction of zymogen fXIII with activated platelet subpopulations. Confocal microscopy and flow cytometry using fluorescently labelled factors and antibodies. Phosphatidylserine (PS)-positive activated platelets bound 700 to 800 molecules/cell of fXIII at 100 nM, while both PS-negative activated platelets and resting platelets bound 200 to 400 molecules/cell. The binding was reversible, calcium-independent and linear within the fXIII concentration range of up to 1,000 nM. fXIII predominantly bound to the caps of procoagulant platelets and co-localized with fibrinogen. Exogenous fibrinogen promoted fXIII binding by activated PS-negative platelets; this effect was abolished by the integrin αIIbß3 antagonist monafram. The fXIII binding was 1.5- to 3-fold decreased for platelets from four patients with grey platelet syndrome, and was variable for platelets from six patients with Glanzmann's thrombasthenia. Strong platelet stimulation, fibrinogen and αIIbß3 play essential roles in fXIII binding, without any of them fXIII does not bind to platelets. The preferential binding in the cap-like structures might be important for increasing local fXIII concentration in platelet thrombi.

Coagulation factors bound to procoagulant platelets concentrate in cap structures to promote clotting.

Binding of coagulation factors to phosphatidylserine (PS)-exposing procoagulant-activated platelets followed by formation of the membrane-dependent enzyme complexes is critical for blood coagulation. Procoagulant platelets formed upon strong platelet stimulation, usually with thrombin plus collagen, are large "balloons" with a small (∼1 µm radius) "cap"-like convex region that is enriched with adhesive proteins. Spatial distribution of blood coagulation factors on the surface of procoagulant platelets was investigated using confocal microscopy. All of them, including factors IXa (FIXa), FXa/FX, FVa, FVIII, prothrombin, and PS-sensitive marker Annexin V were distributed nonhomogeneously: they were primarily localized in the "cap," where their mean concentration was by at least an order of magnitude, higher than on the "balloon." Assembly of intrinsic tenase on liposomes with various PS densities while keeping the PS content constant demonstrated that such enrichment can accelerate this reaction by 2 orders of magnitude. The mechanisms of such acceleration were investigated using a 3-dimensional computer simulation model of intrinsic tenase based on these data. Transmission electron microscopy and focal ion beam-scanning electron microscopy with Annexin V immunogold-labeling revealed a complex organization of the "caps." In platelet thrombi formed in whole blood on collagen under arterial shear conditions, ubiquitous "caps" with increased Annexin V, FX, and FXa binding were observed, indicating relevance of this mechanism for surface-attached platelets under physiological flow. These results reveal an essential heterogeneity in the surface distribution of major coagulation factors on the surface of procoagulant platelets and suggest its importance in promoting membrane-dependent coagulation reactions.

Hysteresis-like binding of coagulation factors X/Xa to procoagulant activated platelets and phospholipids results from multistep association and membrane-dependent multimerization.

Binding of coagulation factors X (fX) and Xa (fXa) to activated platelets is required for the formation of membrane-dependent enzymatic complexes of intrinsic tenase and prothrombinase. We carried out an in-depth characterization of fX/fXa binding to phospholipids and gel-filtered, thrombin-activated platelets. Flow cytometry, surface plasmon resonance, and computational modeling were used to investigate interactions of fX/fXa with the membranes. Confocal microscopy was employed to study fXa binding to platelet thrombi formed in flowing whole blood under arterial conditions. Binding of fX/fXa to either vesicles or procoagulant platelets did not follow a traditional one-step reversible binding model. Their dissociation was a two-step process resulting in a plateau that was up to 10-fold greater than the saturation value observed in the association experiments. Computational modeling and experimental evidence suggested that this was caused by a combination of two-step association (mainly for fX) and multimerization on the membrane (mainly for fXa). Importantly, fX formed multimers with fXa, thereby improving its retention. The same binding/dissociation hysteresis was observed for annexin V known to form trimers on the membranes. Experiments with platelets from gray syndrome patients showed that alpha-granular factor Va provided an additional high-affinity binding site for fXa that did not affect the hysteresis. Confocal microscopy observation of fXa binding to platelet thrombi in a flow chamber and its wash-out confirmed that this phenomenon persisted under physiologically relevant conditions. This suggests its possible role of "locking" coagulation factors on the membrane and preventing their inhibition in plasma and removal from thrombi by flow.

Platelet surface-associated activation and secretion-mediated inhibition of coagulation factor XII.

Coagulation factor XII (fXII) is important for arterial thrombosis, but its physiological activation mechanisms are unclear. In this study, we elucidated the role of platelets and platelet-derived material in fXII activation. FXII activation was only observed upon potent platelet stimulation (with thrombin, collagen-related peptide, or calcium ionophore, but not ADP) accompanied by phosphatidylserine exposure and was localised to the platelet surface. Platelets from three patients with grey platelet syndrome did not activate fXII, which suggests that platelet-associated fXII-activating material might be released from α-granules. FXII was preferentially bound by phosphotidylserine-positive platelets and annexin V abrogated platelet-dependent fXII activation; however, artificial phosphotidylserine/phosphatidylcholine microvesicles did not support fXII activation under the conditions herein. Confocal microscopy using DAPI as a poly-phosphate marker did not reveal poly-phosphates associated with an activated platelet surface. Experimental data for fXII activation indicates an auto-inhibition mechanism (ki/ka = 180 molecules/platelet). Unlike surface-associated fXII activation, platelet secretion inhibited activated fXII (fXIIa), particularly due to a released C1-inhibitor. Platelet surface-associated fXIIa formation triggered contact pathway-dependent clotting in recalcified plasma. Computer modelling suggests that fXIIa inactivation was greatly decreased in thrombi under high blood flow due to inhibitor washout. Combined, the surface-associated fXII activation and its inhibition in solution herein may be regarded as a flow-sensitive regulator that can shift the balance between surface-associated clotting and plasma-dependent inhibition, which may explain the role of fXII at high shear and why fXII is important for thrombosis but negligible in haemostasis.

Procoagulant platelets form an α-granule protein-covered "cap" on their surface that promotes their attachment to aggregates.

Strongly activated "coated" platelets are characterized by increased phosphatidylserine (PS) surface expression, α-granule protein retention, and lack of active integrin αIIbß3. To study how they are incorporated into thrombi despite a lack of free activated integrin, we investigated the structure, function, and formation of the α-granule protein "coat." Confocal microscopy revealed that fibrin(ogen) and thrombospondin colocalized as "cap," a single patch on the PS-positive platelet surface. In aggregates, the cap was located at the point of attachment of the PS-positive platelets. Without fibrin(ogen) retention, their ability to be incorporated in aggregates was drastically reduced. The surface fibrin(ogen) was strongly decreased in the presence of a fibrin polymerization inhibitor GPRP and also in platelets from a patient with dysfibrinogenemia and a fibrinogen polymerization defect. In contrast, a fibrinogen-clotting protease ancistron increased the amount of fibrin(ogen) and thrombospondin on the surface of the PS-positive platelets stimulated with collagen-related peptide. Transglutaminases are also involved in fibrin(ogen) retention. However, platelets from patients with factor XIII deficiency had normal retention, and a pan-transglutaminase inhibitor T101 had only a modest inhibitory effect. Fibrin(ogen) retention was normal in Bernard-Soulier syndrome and kindlin-3 deficiency, but not in Glanzmann thrombasthenia lacking the platelet pool of fibrinogen and αIIbß3. These data show that the fibrin(ogen)-covered cap, predominantly formed as a result of fibrin polymerization, is a critical mechanism that allows coated (or rather "capped") platelets to become incorporated into thrombi despite their lack of active integrins.