scholarly journals Recombinant Destabilase from Hirudo medicinalis Is Able to Dissolve Human Blood Clots In Vitro

2021 ◽  
Vol 43 (3) ◽  
pp. 2068-2081
Author(s):  
Pavel Bobrovsky ◽  
Valentin Manuvera ◽  
Izolda Baskova ◽  
Svetlana Nemirova ◽  
Alexandr Medvedev ◽  
...  
Keyword(s):  
Response Curve ◽  
Blood Clot ◽  
Morphological Characteristics ◽  
Fibrin Clot ◽  
Blood Clots ◽  
Isopeptide Bonds ◽  
Isopeptidase Activity ◽  
Stabilized Fibrin ◽  
Poisonous Animals

Leeches are amazing animals that can be classified as conditionally poisonous animals since the salivary cocktail they produce is injected directly into the victim, and its components have strictly defined biological purposes, such as preventing blood clot formation. Thrombolytic drugs are mainly aimed at treating newly formed blood clots. Aged clots are stabilized by a large number of isopeptide bonds that prevent the action of thrombolytics. These bonds are destroyed by destabilase, an enzyme of the leech’s salivary glands. Here, we conducted a pilot study to evaluate the feasibility and effectiveness of the use of destabilase in relation to blood clots formed during real pathological processes. We evaluated the isopeptidase activity of destabilase during the formation of a stabilized fibrin clot. We showed that destabilase does not affect the internal and external coagulation cascades. We calculated the dose–response curve and tested the ability of destabilase to destroy isopeptide bonds in natural blood clots. The effect of aged and fresh clots dissolving ability after treatment with destabilase coincided with the morphological characteristics of clots during surgery. Thus, recombinant destabilase can be considered as a potential drug for the treatment of aged clots, which are difficult to treat with known thrombolytics.

scholarly journals TITANIUM NANOPARTICLES CONJUGATED WITH STREPTOKINASE AS A MODIFIED THROMBOLYTIC AGENT

2020 ◽  
pp. 18-25
Author(s):  
HAYDER H. ABED ◽  
ESTABRAQ AR. ALWASITI ◽  
AMIR T. TAWFEEQ
Keyword(s):  
Thrombolytic Agent ◽  
Blood Clot ◽  
Control Group ◽  
Thrombolytic Activity ◽  
Protein Assay ◽  
Blood Clots ◽  
Ft Ir ◽  
Titanium Nanoparticles ◽  
D Dimer

Objective: Blood clots are the main cause of death worldwide by stroke and myocardial infarction. Streptokinase a thrombolytic agent that is used in the treatment of circulatory disorders. Methods: Titanium Nanoparticles was supplied from Changsha Santech Co. Its characterized were studied using (FT-IR, XRD, AFM, FE-SEM). Streptokinase at concentration 0.1 mg/ml was conjugated with Titanium nanoparticles using PH equal to 5.2 with continuous stirring. Formation of Streptokinase loading Titanium nanoparticles confirmed using FT-IR, Ninhydrine’s test and Bradford protein assay. Physicochemical Properties were studied in vitro. Thrombolytic activity in vitro was determined using d–dimer indicator and weight of blood clot after treatment as indicators of thrombolytic activity. Results: Titanium nanoparticles show particle size at range 31 nm. The thrombolytic activity of streptokinase loading Titanium nanoparticles shows significant value in d-dimer and weight of blood clot compared with the control group and non-significant compared with an equivalent amount of streptokinase alone. Conclusion: Titanium nanoparticles conjugated with streptokinase show high thrombolytic activity against blood clots in vitro.

Effect of Progesterone and Synthetic Progestins on Whole Blood Clot Formation and Erythrocyte Structure

2017 ◽  
Vol 23 (3) ◽  
pp. 607-617 ◽  
Author(s):  
Albe C. Swanepoel ◽  
Odette Emmerson ◽  
Etheresia Pretorius
Keyword(s):  
Whole Blood ◽  
Blood Clot ◽  
Thrombotic Event ◽  
Clot Formation ◽  
Erythrocyte Aggregation ◽  
Erythrocyte Morphology ◽  
Increased Risk ◽  
Blood Clots ◽  
Membrane Ultrastructure

AbstractCombined oral contraceptive (COC) use is a risk factor for venous thrombosis (VT) and related to the specific type of progestin used. VT is accompanied by inflammation and pathophysiological clot formation, that includes aberrant erythrocytes and fibrin(ogen) interactions. In this paper, we aim to determine the influence of progesterone and different synthetic progestins found in COCs on the viscoelasticity of whole blood clots, as well as erythrocyte morphology and membrane ultrastructure, in an in vitro laboratory study. Thromboelastography (TEG), light microscopy, and scanning electron microscopy were our chosen methods. Our results point out that progestins influence the rate of whole blood clot formation. Alterations to erythrocyte morphology and membrane ultrastructure suggest the presence of eryptosis. We also note increased rouleaux formation, erythrocyte aggregation, and spontaneous fibrin formation in whole blood which may explain the increased risk of VT associated with COC use. Although not all COC users will experience a thrombotic event, individuals with a thrombotic predisposition, due to inflammatory or hematological illness, should be closely monitored to prevent pathological thrombosis.

scholarly journals Comparison of Autologous Blood Clots with Fibrin Sealant as Scaffolds for Promoting Human Muscle-Derived Stem Cell-Mediated Bone Regeneration

Biomedicines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 983
Author(s):  
Xueqin Gao ◽  
Haizi Cheng ◽  
Xuying Sun ◽  
Aiping Lu ◽  
Joseph J. Ruzbarsky ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Regeneration ◽  
Osteogenic Differentiation ◽  
Fibrin Sealant ◽  
Blood Clot ◽  
Autologous Blood ◽  
Human Muscle ◽  
Blood Clots ◽  
Autologous Blood Clot

Background. Fibrin sealant has been used as a scaffold to deliver genetically modified human muscle-derived stem cells (hMDSCs) for bone regeneration. Alternatively, autologous blood clots are safe, economic scaffolds. This study compared autologous blood clot (BC) with fibrin sealant (FS) as a scaffold to deliver lenti-BMP2/GFP-transduced hMDSCs for bone regeneration. Methods. In vitro osteogenic differentiation was performed using 3D pellet culture and evaluated using microCT and Von Kossa staining. The lenti-GFP transduced cells were then mixed with human blood for evaluation of osteogenic differentiation. Furthermore, a murine critical- sized calvarial defect model was utilized to compare BC and FS scaffolds for lenti-BMP2/GFP-transduced hMDSCs mediated bone regeneration and evaluated with micro-CT and histology. Results. Lenti-BMP2/GFP transduced hMDSCs formed significantly larger mineralized pellets than non-transduced hMDSCs. hMDSCs within the human blood clot migrated out and differentiated into ALP+ osteoblasts. In vivo, BC resulted in significantly less new bone formation within a critical-sized calvarial bone defect than FS scaffold, despite no difference observed for GFP+ donor cells, osteoclasts, and osteoblasts in the newly formed bone. Conclusions. Human lenti-BMP2/GFP-transduced hMDSCs can efficiently undergo osteogenic differentiation in vitro. Unexpectedly, the newly regenerated bone in BC group was significantly less than the FS group. The autologous blood clot scaffold is less efficacious for delivering stem cells for bone regeneration than fibrin sealant.

Clot Accumulation Characteristics of Plasminogen-bearing Liposomes in a Flow-system

1998 ◽  
Vol 79 (01) ◽  
pp. 144-149 ◽  
Author(s):  
H. Feitsma ◽  
C. Kluft ◽  
J. L. M. Heeremans ◽  
R. Prevost ◽  
D. J. A. Crommelin
Keyword(s):  
Closed System ◽  
Whole Blood ◽  
Surface Density ◽  
Closed Loop ◽  
Flow System ◽  
Blood Clot ◽  
Fibrin Clot ◽  
Partial Degradation ◽  
Accumulation Characteristics

SummaryIn this study, the clot accumulation properties of liposome-coupled plasminogen were compared to those of free (non-liposomal) plasminogen in an in vitro, closed-loop, flow-system. After introduction of a clot into the closed system, double-radiolabelled plasminogen-liposomes were administered and the accumulation of radiolabel on the entire clot was measured.Liposomal plasminogen showed improved accumulation over free plasminogen, on both a fibrin clot and a whole blood clot. Moreover, once liposomal plasminogen was fibrin associated, it could not be washed away with buffer, in contrast to free plasminogen. Liposomal plasminogen was able to compete successfully with an excess of free plasminogen. The plateau levels for the accumulated amount of plasminogen depended on the incubated amount of plasminogen and were influenced by partial degradation of the clot. Furthermore, it was shown that a threshold liposomal plasminogen surface-density was needed for optimum clot accumulation.

Clot properties and cardiovascular disease

2014 ◽  
Vol 112 (11) ◽  
pp. 901-908 ◽  
Author(s):  
Katherine Bridge ◽  
Helen Philippou ◽  
Robert Ariëns
Keyword(s):  
Cardiovascular Disease ◽  
Blood Clot ◽  
Fibrin Clot ◽  
Disease States ◽  
Inflammatory Conditions ◽  
Number Of Factors ◽  
Venous Diseases ◽  
Cardiovascular Medications ◽  
Clot Structure

SummaryFibrinogen is cleaved by thrombin to fibrin, which provides the blood clot with its essential structural backbone. As an acute phase protein, the plasma levels of fibrinogen are increased in response to inflammatory conditions. In addition to fibrinogen levels, fibrin clot structure is altered by a number of factors. These include thrombin levels, treatment with common cardiovascular medications, such as aspirin, anticoagulants, statins and fibrates, as well as metabolic disease states such as diabetes mellitus and hyperhomocysteinaemia. In vitro studies of fibrin clot structure can provide information regarding fibre density, clot porosity, the mechanical strength of fibres and fibrinolysis. A change in fibrin clot structure, to a denser clot with smaller pores which is more resistant to lysis, is strongly associated with cardiovascular disease. This pathological change is present in patients with arterial as well as venous diseases, and is also found in a moderate form in relatives of patients with cardiovascular disease. Pharmacological therapies, aimed at both the treatment and prophylaxis of cardiovascular disease, appear to result in positive changes to the fibrin clot structure. As such, therapies aimed at ‘normalising’ fibrin clot structure may be of benefit in the prevention and treatment of cardiovascular disease.

scholarly journals Effects of Hyperhomocysteinemia on the Platelet-Driven Contraction of Blood Clots

Metabolites ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 354
Author(s):  
Rustem I. Litvinov ◽  
Alina D. Peshkova ◽  
Giang Le Minh ◽  
Nail N. Khaertdinov ◽  
Natalia G. Evtugina ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Rat Model ◽  
Binding Capacity ◽  
Blood Clot ◽  
Fibrinogen Binding ◽  
Blood Clots ◽  
Clot Contraction ◽  
Dose Dependent

Hyperhomocysteinemia (HHcy) is associated with thrombosis, but the mechanistic links between them are not understood. We studied effects of homocysteine (Hcy) on clot contraction in vitro and in a rat model of HHcy. Incubation of blood with exogenous Hcy for 1 min enhanced clot contraction, while 15-min incubation led to a dose-dependent suppression of contraction. These effects were likely due to direct Hcy-induced platelet activation followed by exhaustion, as revealed by an increase in fibrinogen-binding capacity and P-selectin expression determined by flow cytometry. In the blood of rats with HHcy, clot contraction was enhanced at moderately elevated Hcy levels (10–50 μM), while at higher Hcy levels (>50 μM), the onset of clot contraction was delayed. HHcy was associated with thrombocytosis combined with a reduced erythrocyte count and hypofibrinogenemia. These data suggest that in HHcy, platelets get activated directly and indirectly, leading to enhanced clot contraction that is facilitated by the reduced content and resilience of fibrin and erythrocytes in the clot. The excessive platelet activation can lead to exhaustion and impaired contractility, which makes clots larger and more obstructive. In conclusion, HHcy modulates blood clot contraction, which may comprise an underappreciated pro- or antithrombotic mechanism.

scholarly journals In Vitro Clot Dissolving Activity of Carbomer Based Gel Containing Ethanolic Extract of Calotropis Gigantea Leaves

2018 ◽  
Vol 8 (6-s) ◽  
pp. 101-104
Author(s):  
Prerna Alawa ◽  
Mayank Tenguria ◽  
Vijayshree Nilofey
Keyword(s):  
Blood Clot ◽  
Circulatory System ◽  
Ethanolic Extract ◽  
Thrombolytic Activity ◽  
Calotropis Gigantea ◽  
Blood Clots ◽  
The Family ◽  
Carbopol 940 ◽  
Ethanolic Leaf Extract

Blood clotting is an important and vital process that takes places in humans, animals and birds. At the same time blood clots sometimes considered an issue when developed in the circulatory system due to failure of hemostasis causes vascular blockage and leads to serious consequences in thrombolytic diseases such as acute myocardial or cerebral infarction which may cause death. Certain substances like Alteplase, anistreplase, streptokinase, urokinase, tissue plasminogen activator, Heparin and Aspirin are used as clot dissolving agents. Plants have also been proved to perform such activity. Calotropis gigantea L. is a traditional medicinal plant with several pharmacological properties belongs to the family Apocyanaceae of Asclepiadaceous was investigated for its efficacy for blood clot dissolving activity under in vitro conditions when incorporated into carbomer based gels. The ethanolic extract of the leaves of C. gigantea was prepare by soxhlation which upon analysis reported to contain alkaloids, glycosides, flavonoids, tannins, terpenoids and saponins as usual components as were reported in earlier investigations. There were 3 gels with ethanolic leaf extract C. gigantea with 1 mg, 0.5 mg and 0.25 mg per gram of 1% Carbopol-940 gel containing methyl paraben, glycerol and water. The two gels containing sterile distilled water and 0.1 mg per gram gel streptokinase enzyme served as –ve and positive controls respectively. The in vitro thrombolytic of clot dissolving activity war performed on goat blood collected from slaughter houses. From the results the percentage clot dissolving activity of gels containing 1 mg, 0.5 mg and 0.25 mg C. gigantea extract per gram were reported as 34%, 27% and 14% respectively considered significant when compared to the activity of standard streptokinase in the gel. The activity of these experimental gels describes the possibility of development of new safe, and non reactive topical anti-clot gel products using herbal or plant components like C. gigantea extracts. Keywords: Calotropis gigantea L, Streptokinase, Thrombolytic activity, Carbopol gel, Biopharmaceutical.

scholarly journals Acoustic and Elastic Properties of a Blood Clot during Microbubble-Enhanced Sonothrombolysis: Hardening of the Clot with Inertial Cavitation

Pharmaceutics ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1566
Author(s):  
Laurent Auboire ◽  
Damien Fouan ◽  
Jean-Marc Grégoire ◽  
Fréderic Ossant ◽  
Camille Plag ◽  
...  
Keyword(s):  
Elastic Properties ◽  
Blood Clot ◽  
Tissue Type ◽  
High Frequency Ultrasound ◽  
Shear Wave Speed ◽  
Ultrafast Imaging ◽  
Type Plasminogen Activator ◽  
Blood Clots ◽  
Therapeutic Alternatives

Stroke is the second leading cause of death worldwide. Existing therapies present limitations, and other therapeutic alternatives are sought, such as sonothrombolysis with microbubbles (STL). The aim of this study was to evaluate the change induced by STL with or without recombinant tissue-type plasminogen activator (rtPA) on the acoustic and elastic properties of the blood clot by measuring its sound speed (SoS) and shear wave speed (SWS) with high frequency ultrasound and ultrafast imaging, respectively. An in-vitro setup was used and human blood clots were submitted to a combination of microbubbles and rtPA. The results demonstrate that STL induces a raise of SoS in the blood clot, specifically when combined with rtPA (p < 0.05). Moreover, the combination of rtPA and STL induces a hardening of the clot in comparison to rtPA alone (p < 0.05). This is the first assessment of acoustoelastic properties of blood clots during STL. The combination of rtPA and STL induce SoS and hardening of the clot, which is known to impair the penetration of thrombolytic drugs and their efficacy.

scholarly journals Force-Independent Cleavage of Talin by Calpain Promotes Platelet-Mediated Fibrin Clot Contraction

2021 ◽  
Author(s):  
Karen P. Fong ◽  
Kathleen S. Molnar ◽  
Nicholas J. Agard ◽  
Rustem I Litvinov ◽  
Oleg V. Kim ◽  
...  
Keyword(s):  
Binding Sites ◽  
Blood Clot ◽  
Tensile Force ◽  
Structural Rearrangement ◽  
Fibrin Clot ◽  
Clot Retraction ◽  
Clot Contraction ◽  
Α Helix ◽  
Unstructured Regions

Blood clot contraction is driven by traction forces generated by the platelet cytoskeleton that are transmitted to fibrin fibers via the integrin αIIbβ3. Here we show that clot contraction is impaired by inhibitors of the platelet cytosolic protease calpain. We used subtiligase-mediated labeling of amino-termini and mass spectrometry to identify proteolytically-cleaved platelet proteins involved in clot contraction. Of 32 calpain-cleaved proteins after TRAP stimulation, fourteen were cytoskeletal, most prominently talin and vinculin. A complex of talin and vinculin constitutes a "mechanosensitive clutch" connecting integrins bound to the extracellular matrix to the actin cytoskeleton. Accordingly, we focused on talin and vinculin. Talin is composed of an N-terminal head domain and a C-terminal rod domain organized into a series of four- and five-helix bundles. The bundles contain 11 vinculin binding sites (VBS), each of which is an α-helix packed into a bundle interior and requiring a structural rearrangement to initiate vinculin binding. We detected 8 calpain-mediated cleavages in talin, 2 previously identified in unstructured regions and 6 in α-helical regions in proximity to a VBS. There is evidence in vitro that applying mechanical force across talin enables vinculin binding to the talin rod. However, we found that inhibiting platelet cytoskeletal contraction had no effect on talin cleavage, indicating that talin cleavage by calpain in platelets does not require cytoskeleton-generated tensile force. Thus, it is likely that calpain acts in the later stages of clot retraction through focal adhesion disassembly.

Abstract 3284: Influence of Blood Clot Age, Density and Thrombin Content on In Vitro Sonothrombolysis Rate and Efficacy

Stroke ◽  
2012 ◽  
Vol 43 (suppl_1) ◽  
Author(s):  
Michael J Borrelli ◽  
Eric Hamilton ◽  
Leah Hennings ◽  
Laura J Bernonck ◽  
William C Culp
Keyword(s):  
Myocardial Infarct ◽  
Mass Density ◽  
Blood Clot ◽  
Vascular Occlusion ◽  
Rabbit Serum ◽  
Blood Clots ◽  
Glass Tubes ◽  
Different Characteristics ◽  
Physical And Chemical

Sonothrombolysis (STBL) uses ultrasound to lyse thrombi that cause stroke, myocardial infarct and other vascular occlusion diseases; and efficacy is improved markedly by combining tPA and/or microbubbles (MBs) with ultrasound. The efficacy of in vitro STBL varies considerably depending upon how blood clots are formed. However, the relationship between the physical and chemical nature of blood clots and STBL efficacy has not been quantified. This study measured the effects that clot age, density and thrombin content had on STBL efficacy and the ability of tPA and MBs to complement STBL. Blood clots with different characteristics were produced by mixing pooled rabbit plasma with rabbit blood cells, adding varied amounts of thrombin and calcium, followed by incubation at 37 o C in glass tubes for 3 h to 72 h. Blood clots were then cured at 5 o C for 24 h to 72 h. The volume and mass density of the clots were measured, 9-11 mg pieces were cut from the main clot, weighed and then insonated with 1 MHz, pulsed (20% duty factor) ultrasound in a Mylar chamber through which fresh rabbit serum containing tPA and or MBs was flowed continuously past the clot. STBL efficacy was measured as % of clot mass lysed in 15 min. For clots incubated 3 h to 24 h at 37 o C with 5 mM Ca + 2 and 5 U of thrombin, maximal STBL with ultrasound plus tPA was observed with 0.1 mg/ml tPA for all ultrasonic intensities tested. Using MBs (1.0-2.5 x10 8 /ml) produced a comparable level of STBL. More effective STBL was produced by combining tPA with MBs, even with reduced tPA concentrations (0.006-0.01 mg/ml). However, increasing clot thrombin, up to 60 U, proportionally reduced STBL efficacy with tPA or MBs, and combining tPA with MBs no longer produced an additive STBL effect. This was also true for clots incubated with 25 mM Ca + 2 that did not contract and thus had a fourfold lower mass density. Increasing thrombin levels produced clots whose STBL efficacy was markedly lower with either tPA or MBs, and for which combining tPA with MBs produced no additive STBL effect. Hence, the level of thrombin used during clot formation (and putatively the degree of fibrin networks) was the major factor in determined STBL efficacy and whether or not tPA combined with MBs were additive in potentiating STBL. Surprisingly, the mass density of the clot alone had no effect on STBL efficacy.

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