Influence of temperature on spatial fibrin clot formation process in thrombodynamics

In this study we have investigated the process of spatial fibrin clot formation in non-steered platelet-free plasma at the temperatures from 20°C to 43°C using thrombodynamics – the novel in vitro hemostasis assay, which imitates the process of hemostatic clot growth in vivo. During data processing the following parameters were calculated: initial (V i ) and stationary (V st ) rates of clot growth which characterize initiation and propagation phases of clotting process, and clot size on the 30 th minute. The temperature dependence of extrinsic and intrinsic tenase activities, which determine values of the initial and stationary clot growth rates, respectively, have been also measured. It was established that the temperature lowering from 37°C to 24°C extends mainly on the initiation phase of clot growth, while the stationary rate of clot growth changes insignificantly. Meanwhile none of the thrombodynamics parameters shows the dramatic change of plasma coagulation system condition at the temperature of 24°C (acute hypothermia). Using the thrombodynamics assay an assumption, that the temperature lowering does not change the state of plasma hemostasis system significantly has been confirmed.

Download Full-text

Development of MDL 28,050, a Small Stable Antithrombin Agent Based on a Functional Domain of the Leech Protein, Hirudin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645196 ◽

1990 ◽

Vol 63 (02) ◽

pp. 208-214 ◽

Cited By ~ 46

Author(s):

John L Krstenansky ◽

Robert J Broersma ◽

Thomas J Owen ◽

Marguerite H Payne ◽

Mark T Yates ◽

...

Keyword(s):

Structural Optimization ◽

Systematic Study ◽

Fibrin Clot ◽

Clot Formation ◽

Functional Domain ◽

In Vivo Models ◽

Agent Based ◽

Structure Activity

SummaryMDL 28,050 is a decapeptide antithrombin agent that inhibits a-thrombin-induced fibrin clot formation by binding to a non-catalytic site on α-thromhin. It is the result of chemical and structural optimization of a functional domain of the leech anticoagulant, hirudin. In contrast to the contention that the polyanionic nature of this C-terminal functional domain governs its interaction with α-thrombin, systematic study of this region has shown the importance of the lipophilic residues for providing the functionality necessary foi potent binding to a-thrombin. The development of MDL 28,050 and other effective antithrombin agents are outlined through the description of the structure-activity relationships (SAR) for these peptides. These peptides are effective in a variety of in vitro and in vivo models of thrombosis.

Download Full-text

Slounase, a Batroxobin Containing Activated Factor X Effectively Enhances Hemostatic Clot Formation and Reducing Bleeding in Hypocoagulant Conditions in Mice

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/10760296211018510 ◽

2021 ◽

Vol 27 ◽

pp. 107602962110185

Author(s):

Reheman Adili ◽

Madeline Jackson ◽

Livia Stanger ◽

Xiangrong Dai ◽

Mandy Li ◽

...

Keyword(s):

Bleeding Time ◽

Fibrin Clot ◽

Biochemical Properties ◽

Clot Formation ◽

Factor X ◽

Wild Type ◽

Fibrin Formation ◽

Uncontrolled Bleeding

Uncontrolled bleeding associated with trauma and surgery is the leading cause of preventable death. Batroxobin, a snake venom-derived thrombin-like serine protease, has been shown to clot fibrinogen by cleaving fibrinopeptide A in a manner distinctly different from thrombin, even in the presence of heparin. The biochemical properties of batroxobin and its effect on coagulation have been well characterized in vitro. However, the efficacy of batroxobin on hemostatic clot formation in vivo is not well studied due to the lack of reliable in vivo hemostasis models. Here, we studied the efficacy of batroxobin and slounase, a batroxobin containing activated factor X, on hemostatic clot composition and bleeding using intravital microcopy laser ablation hemostasis models in micro and macro vessels and liver puncture hemostasis models in normal and heparin-induced hypocoagulant mice. We found that prophylactic treatment in wild-type mice with batroxobin, slounase and activated factor X significantly enhanced platelet-rich fibrin clot formation following vascular injury. In heparin-treated mice, batroxobin treatment resulted in detectable fibrin formation and a modest increase in hemostatic clot size, while activated factor X had no effect. In contrast, slounase treatment significantly enhanced both platelet recruitment and fibrin formation, forming a stable clot and shortening bleeding time and blood loss in wild-type and heparin-treated hypocoagulant mice. Our data demonstrate that, while batroxobin enhances fibrin formation, slounase was able to enhance hemostasis in normal mice and restore hemostasis in hypocoagulant conditions via the enhancement of fibrin formation and platelet activation, indicating that slounase is more effective in controlling hemorrhage.

Download Full-text

Formation of Clot Analogs Between Co-Flow Fluid Streams in a Microchannel Device

Volume 4: Fluid Measurement and Instrumentation; Micro and Nano Fluid Dynamics ◽

10.1115/ajkfluids2019-5513 ◽

2019 ◽

Author(s):

Sue-Mae Saw ◽

Anand K. Ramasubramanian ◽

Melinda Simon ◽

Sang-Joon John Lee

Keyword(s):

Soft Lithography ◽

Dominant Role ◽

Fibrin Clot ◽

Fresh Frozen Plasma ◽

Clot Formation ◽

Flow Rates ◽

Fresh Frozen ◽

Hydrodynamic Stresses

Abstract Hemodynamics plays an important role in the formation of blood clots, for which changes in hydrodynamic stresses and transport phenomena can initiate or inhibit the clotting process. Fibrin, which is converted from fibrinogen in blood plasma, plays a dominant role in structural mechanics of a clot. Clot analogs are conventionally fabricated in a static in vitro environment whereas clot formation in vivo occurs in the presence of dynamic blood flow. In this paper we demonstrate an ability to produce clot analogs at the boundary between active co-flow fluid streams. The time evolution of clot formation in microchannel flow was investigated using fluorescence imaging of fibrin clots at one-minute intervals. Time-tracking of skewness and kurtosis of fluorescence intensity data was conducted to monitor shape and density distribution changes in the clot. Soft lithography and casting techniques were used to fabricate a polydimethylsiloxane (PDMS) microfluidic device which consisted of a Y-shaped microchannel 300 μm wide × 12 μm deep × 10 mm long with two inlets and a single outlet. The first inlet introduced fresh frozen plasma (FFP), which contains fibrinogen and plasma proteins. The second inlet introduced thrombin, which initiated the conversion of fibrinogen to fibrin. Clot analogs were formed at the interface between these two parallel streams. Flow was driven by withdrawal of a syringe pump at flow rates of 50 nL/min and 100 nL/min. Clots that are formed in such an engineered device provide opportunities to recapitulate the flow rates and concentrations of reagents, to mimic in vivo scenarios in which clot density and composition gradients depend on flow conditions.

Download Full-text

Additive roles of platelets and fibrinogen in whole-blood fibrin clot formation upon dilution as assessed by thromboelastometry

Thrombosis and Haemostasis ◽

10.1160/th13-06-0493 ◽

2014 ◽

Vol 111 (03) ◽

pp. 447-457 ◽

Cited By ~ 21

Author(s):

Marisa Ninivaggi ◽

Gerhardus Kuiper ◽

Marco Marcus ◽

Hugo ten Cate ◽

Marcus Lancé ◽

...

Keyword(s):

Red Blood Cells ◽

Whole Blood ◽

Blood Cells ◽

Thrombin Generation ◽

Prothrombin Complex Concentrate ◽

Coagulation Factors ◽

Fibrin Clot ◽

Clot Formation

SummaryBlood dilution after transfusion fluids leads to diminished coagulant activity monitored by rotational thromboelastometry, assessing elastic fibrin clot formation, or by thrombin generation testing. We aimed to determine the contributions of blood cells (platelets, red blood cells) and plasma factors (fibrinogen, prothrombin complex concentrate) to fibrin clot formation under conditions of haemodilution in vitro or in vivo. Whole blood or plasma diluted in vitro was supplemented with platelets, red cells, fibrinogen or prothrombin complex concentrate (PCC). Thromboelastometry was measured in whole blood as well as plasma; thrombin generation was determined in parallel. Similar tests were performed with blood from 48 patients, obtained before and after massive fluid infusion during cardiothoracic surgery. Addition of platelets or fibrinogen, in additive and independent ways, reversed the impaired fibrin clot formation (thromboelastometry) in diluted whole blood. In contrast, supplementation of red blood cells or prothrombin complex concentrate was ineffective. Platelets and fibrinogen independently restored clot formation in diluted plasma, resulting in thromboelastometry curves approaching those in whole blood. In whole blood from patients undergoing dilution during surgery, elastic clot formation was determined by both the platelet count and the fibrinogen level. Thrombin generation in diluted (patient) plasma was not changed by fibrinogen, but improved markedly by prothrombin complex concentrate. In conclusion, in dilutional coagulopathy, platelets and fibrinogen, but not red blood cells or vitamin K-dependent coagulation factors, independently determine thromboelastometry parameters measured in whole blood and plasma. Clinical decisions for transfusion based on thromboelastometry should take into account the platelet concentration.

Download Full-text

A Comparison of the in Vitro and in Vivo Thrombogenic Activity of Factor IX Concentrates Using Stasis (Wessler) and Non-Stasis Rabbit Models

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650089 ◽

1980 ◽

Vol 44 (02) ◽

pp. 081-086 ◽

Cited By ~ 8

Author(s):

C V Prowse ◽

A E Williams

Keyword(s):

Platelet Rich Plasma ◽

Thrombus Formation ◽

Factor Ix ◽

Coagulation System ◽

In Vitro Tests ◽

Clinical Use ◽

Low Activity

SummaryThe thrombogenic effects of selected factor IX concentrates were evaluated in two rabbit models; the Wessler stasis model and a novel non-stasis model. Concentrates active in either the NAPTT or TGt50 in vitro tests of potential thrombogenicity, or both, caused thrombus formation in the Wessler technique and activation of the coagulation system in the non-stasis model. A concentrate with low activity in both in vitro tests did not have thrombogenic effects in vivo, at the chosen dose. Results in the non-stasis model suggested that the thrombogenic effects of factor IX concentrates may occur by at least two mechanisms. A concentrate prepared from platelet-rich plasma and a pyrogenic concentrate were also tested and found to have no thrombogenic effect in vivo.These studies justify the use of the NAPTT and TGt50 in vitro tests for the screening of factor IX concentrates prior to clinical use.

Download Full-text

The Roles of α2-Antiplasmin and Plasminogen Activator Inhibitor 1 (PAI-1) in the Inhibition of Clot Lysis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649570 ◽

1993 ◽

Vol 70 (02) ◽

pp. 301-306 ◽

Cited By ~ 51

Author(s):

Linda A Robbie ◽

Nuala A Booth ◽

Alison M Croll ◽

Bruce Bennett

Keyword(s):

Plasminogen Activator ◽

Plasminogen Activator Inhibitor ◽

Fibrin Clot ◽

Clot Lysis ◽

Plasminogen Activator Inhibitor 1 ◽

Dependent Inhibition ◽

Activator Inhibitor ◽

Pai 1

SummaryThe relative importance of the two major inhibitors of fibrinolysis, α2-antiplasmin (α2-AP) and plasminogen activator inhibitor (PAI-1), were investigated using a simple microtitre plate system to study fibrin clot lysis in vitro. Cross-linked fibrin clots contained plasminogen and tissue plasminogen activator (t-PA) at concentrations close to physiological. Purified α2-AP and PAI-1 caused dose-dependent inhibition. All the inhibition due to normal plasma, either platelet-rich or poor, was neutralised only by antibodies to α2-AP. Isolated platelets, at a final concentration similar to that in blood, 2.5 × 108/ml, markedly inhibited clot lysis. This inhibition was neutralised only by antibodies to PAI-1. At the normal circulating ratio of plasma to platelets, α2-AP was the dominant inhibitor. When the platelet:plasma ratio was raised some 20-fold, platelet PAI-1 provided a significant contribution. High local concentrations of PAI-1 do occur in thrombi in vivo, indicating a role for PAI-1, complementary to that of α2-AP, in such situations.

Download Full-text

Glioblastoma cell populations with distinct oncogenic programs release podoplanin as procoagulant extracellular vesicles

Blood Advances ◽

10.1182/bloodadvances.2020002998 ◽

2021 ◽

Vol 5 (6) ◽

pp. 1682-1694

Author(s):

Nadim Tawil ◽

Rayhaan Bassawon ◽

Brian Meehan ◽

Ali Nehme ◽

Laura Montermini ◽

...

Keyword(s):

Cancer Cells ◽

Tissue Factor ◽

Extracellular Vesicles ◽

Coagulation System ◽

Platelet Factor ◽

Increased Risk ◽

Cell Subpopulations ◽

Cancer Associated Thrombosis

Abstract Vascular anomalies, including local and peripheral thrombosis, are a hallmark of glioblastoma (GBM) and an aftermath of deregulation of the cancer cell genome and epigenome. Although the molecular effectors of these changes are poorly understood, the upregulation of podoplanin (PDPN) by cancer cells has recently been linked to an increased risk for venous thromboembolism (VTE) in GBM patients. Therefore, regulation of this platelet-activating protein by transforming events in cancer cells is of considerable interest. We used single-cell and bulk transcriptome data mining, as well as cellular and xenograft models in mice, to analyze the nature of cells expressing PDPN, as well as their impact on the activation of the coagulation system and platelets. We report that PDPN is expressed by distinct (mesenchymal) GBM cell subpopulations and downregulated by oncogenic mutations of EGFR and IDH1 genes, along with changes in chromatin modifications (enhancer of zeste homolog 2) and DNA methylation. Glioma cells exteriorize their PDPN and/or tissue factor (TF) as cargo of exosome-like extracellular vesicles (EVs) shed from cells in vitro and in vivo. Injection of glioma-derived podoplanin carrying extracelluar vesicles (PDPN-EVs) activates platelets, whereas tissue factor carrying extracellular vesicles (TF-EVs) activate the clotting cascade. Similarly, an increase in platelet activation (platelet factor 4) or coagulation (D-dimer) markers occurs in mice harboring the corresponding glioma xenografts expressing PDPN or TF, respectively. Coexpression of PDPN and TF by GBM cells cooperatively affects tumor microthrombosis. Thus, in GBM, distinct cellular subsets drive multiple facets of cancer-associated thrombosis and may represent targets for phenotype- and cell type–based diagnosis and antithrombotic intervention.

Download Full-text

Effect ofToona microcarpaHarms Leaf Extract on the Coagulation System

BioMed Research International ◽

10.1155/2014/615363 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Hao Chen ◽

Min Jin ◽

Yi-Fen Wang ◽

Yong-Qing Wang ◽

Ling Meng ◽

...

Keyword(s):

Leaf Extract ◽

Factor Xa ◽

Adenosine Diphosphate ◽

Coagulation System ◽

Dependent Manner ◽

Thrombin Time ◽

Antithrombotic Agent ◽

Thrombin Activity

Toona microcarpaHarms is a tonic, antiperiodic, antirheumatic, and antithrombotic agent in China and India and an astringent and tonic for treating diarrhea, dysentery, and other intestinal infections in Indonesia. In this study, we prepared ethyl-acetate extract from the air-dried leaves ofToona microcarpaHarms and investigated the anticoagulant activitiesin vitroby performing activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) assays. Antiplatelet aggregation activity of the extract was examined using adenosine diphosphate (ADP), collagen, and thrombin as agonists, and the inhibitions of factor Xa and thrombin were also investigated. Bleeding and clotting times in mice were used to determine its anticoagulant activitiesin vivo. It is found thatToona microcarpaHarms leaf extract (TMHE) prolonged APTT, PT, and TT clotting times in a dose-dependent manner and significantly inhibited platelet aggregation induced by thrombin, but not ADP or collagen. Clotting time and bleeding time assays showed that TMHE significantly prolonged clotting and bleeding timesin vivo. In addition, at the concentration of 1 mg/mL, TMHE inhibited human thrombin activity by 73.98 ± 2.78%. This is the first report to demonstrate that THME exhibits potent anticoagulant effects, possibly via inhibition of thrombin activity.

Download Full-text

Abstract 356: Adhesion of Staphylococcus Epidermidis to Fibrinogen Alters Clot Formation Kinetics and Ultimate Stiffness

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.356 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Carolyn Vitale ◽

Tianhui Ma ◽

Michael J Solomon ◽

J. Scott VanEpps

Keyword(s):

Stationary Phase ◽

Deposition Rate ◽

Staphylococcus Epidermidis ◽

Biofilm Formation ◽

Fibrin Clot ◽

Exponential Phase ◽

Automated Image Analysis ◽

Clot Formation ◽

Coagulation Proteins

Bacterial infection is known to increase the risk for thromboembolism. The mechanism underlying this correlation remains largely unknown. We recently showed that the common pathogen Staphylococcus epidermidis retards clot formation, increases clot elasticity and generates a heterogeneous clot structure that remodels over time. Here, we elucidate the mechanism of this process by evaluating the capacity for S. epidermidis to bind to fibrinogen as a function of its growth phase. We hypothesized that the effect of S. epidermidis on a fibrin clot is related to its propensity toward biofilm formation. Therefore, stationary phase (biofilm-like) S. epidermidis will have a more robust effect on clot kinetics and elasticity than exponential phase (planktonic). Furthermore, this difference is mediated by increased adhesion to fibrinogen. Rheometry was used to evaluate the formation and resultant elasticity of fibrin clots with exponential or stationary phase S. epidermidis . A functional in vitro model was developed to evaluate adhesion of S. epidermidis to a fibrinogen coated surface in a continuously flowing environment. Fluorescent labeled exponential and stationary phase S. epidermidis were visualized flowing through a parallel plate microfluidic chamber past immobilized fibrinogen. Images were obtained every 3 seconds for 30 min. Bacterial deposition rate and mean adhesion time were quantified by automated image analysis. A paired Student’s t-test was used for statistical analysis. Stationary phase S. epidermidis retards clot formation and increases resultant elasticity while exponential phase only slightly reduces elasticity. The bacterial deposition rate onto fibrinogen was significantly (p=0.03) greater for stationary phase (1741 ± 1513 cells/cm 2 · sec -1 ) vs exponential phase (676 ± 270 cells/cm 2 · sec -1 ). The average adhesion time however was similar for exponential and stationary phase cells. Coagulation proteins can provide a framework for bacterial adhesion, biofilm formation and infection. In turn infected thrombi with (biofilm-like) bacteria are stiffer which correlates to more frequent bacterial binding to fibrinogen. This provides a potential molecular mechanism for infection mediated thromboembolic events.

Download Full-text