scholarly journals Inhibition of platelet adhesion, thrombus formation, and fibrin formation by a potent αIIbβ3 integrin inhibitor from ticks

2020 ◽  
Author(s):  
Danique L. Kerkhof ◽  
Magdolna Nagy ◽  
Kanin Wichapong ◽  
Sanne L.N. Brouns ◽  
Johan W. M. Heemskerk ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Fibrin Formation ◽  
Αiibβ3 Integrin ◽  
Integrin Inhibitor

Contribution of Intrinsic and Extrinsic Coagulation Pathways to Thrombus Formation in the Mouse Carotid Artery.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1900-1900
Author(s):  
Antonella Zampolli ◽  
Patrizia Marchese ◽  
Wolfram Ruf ◽  
Nigel Mackman ◽  
Zaverio M. Ruggeri
Keyword(s):  
Carotid Artery ◽  
Platelet Adhesion ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Artery Occlusion ◽  
Carotid Artery Occlusion ◽  
Flow Index ◽  
Fibrin Deposition ◽  
Fibrin Formation ◽  
Coagulation Pathway

Abstract We studied how the intrinsic and extrinsic coagulation pathways contribute to thrombus formation. Ex vivo experiments were performed with citrated blood re-calcified before perfusion over different thrombogenic substrates. This allowed evaluation of fibrin deposition along with platelet adhesion/aggregation in real time and under relevant shear rates. Under these conditions, human melanoma cells expressing human tissue factor (TF) supported platelet adhesion/aggregation and fibrin formation, but only under relatively low shear rate (200–800 s-1). Anti-TF antibodies markedly inhibited both platelet and fibrin deposition, and no reactivity was observed using the parental cell line not transfected with TF. Blood perfusion over fibrillar collagen type I resulted in platelet adhesion/aggregation and subsequent fibrin deposition even under relatively high shear rate (1500 s-1). Corn trypsin inhibitor (CTI), a specific inhibitor of coagulation factor XIIa, had no effect on platelet adhesion/aggregation but inhibited fibrin deposition, while anti-TF antibodies had no significant effect. Preincubation of blood with prostaglandin E1 or a monoclonal antibody against integrin αIIbβ3 blocked not only thrombus growth on the surface but also fibrin formation. Such a finding indicates that thrombin generation leading to fibrin deposition occurs after platelet activation. Blood perfusion over dermal fibroblast extracellular matrix (F-ECM) from mice expressing human but lacking murine TF resulted in the rapid formation of platelet- and fibrin-rich thrombi at all shear rates tested (up to 1500 s-1). Using this thrombogenic substrate containing both collagen and TF, we found that blockade of the intrinsic coagulation pathway with CTI had a minimal effect on platelet adhesion/aggregation and fibrin formation, while inhibition of the extrinsic coagulation pathway with anti-human TF antibodies reduced thrombus growth and stabilization and abolished fibrin formation. The results of these ex vivo studies demonstrate that the relative contribution of the intrinsic and extrinsic coagulation pathways to fibrin and platelets deposition depends on the composition of the thrombogenic surface exposed to blood flow, and indicate also that CTI and anti-TF antibodies can be used as specific inhibitors of thrombin generation through the intrinsic and extrinsic coagulation pathways, respectively. We then established a model of carotid artery injury induced by ferric chloride to evaluate whether the information obtained under controlled experimental conditions ex vivo can help interpret the mechanisms underlying arterial thrombus formation in vivo. In this model, all control C57BL6 mice exhibited a stable carotid artery occlusion within a predictable time frame. To obtain a quantitative parameter of the progression of thrombosis we calculated a flow index. This represents the ratio between the volume of blood that flew through the injured artery from the induction of the lesion to occlusion (or to the end of the predetermined 30 min observation period) and the volume of blood that during the same period of time should have flown through the artery if the initial (pre-injury) flow rate had been maintained. We found that blocking the intrinsic coagulation pathway with CTI injected through the jugular vein significantly delayed, and in most cases prevented, the formation of a stable occlusion, resulting in a significantly altered flow index. The injection of antibodies against human TF, on the other hand, resulted in a trend towards thrombus destabilization, but in most cases the artery still occluded and the flow index was less significantly altered that with CTI. Of note, CTI at a dose that inhibited carotid artery occlusion had no effect on the tail bleeding time. Co-administration of CTI and anti-TF antibodies showed a cooperative effect across the tested concentration range. In conclusion, extrinsic and intrinsic coagulation pathways have complementary roles in thrombus formation and stabilization, and the specific contribution of each depends upon the nature of the thrombogenic surface, i.e. of the causative lesion. The marked effect of factor XIIa inhibition in preventing carotid artery occlusion suggests that a functional link between contact phase activation and tissue factor pathway leading to thrombin generation may be operative under defined conditions in vivo.

scholarly journals Alpha-Dystroglycan Supports Platelet Aggregation and Thrombus Formation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 11-11
Author(s):  
Yiming Wang ◽  
Reid C. Gallant ◽  
Miguel A. D. Neves ◽  
Xi Lei ◽  
Sahil Gupta ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Vital Role ◽  
Platelet Surface ◽  
Glycoprotein Complex ◽  
Dystrophin Glycoprotein Complex ◽  
Β3 Integrin ◽  
Dystrophin Glycoprotein ◽  
Αiibβ3 Integrin

Introduction: Fibrinogen (Fg) and von Willebrand factor (VWF) have been considered essential for platelet adhesion and aggregation. However, platelet aggregation still occurs in mice lacking Fg and/or VWF or plasma fibronectin but not β3 integrin (JCI, 2000; JTH, 2006; Blood, 2009; JCI, 2014). This suggests that other non-classical αIIbβ3 integrin ligand(s) mediate platelet aggregation. α-dystroglycan (α-DG) is a component of the dystrophin-glycoprotein complex that binds extracellular matrix proteins containing laminin-G like domains via unique heteropolysaccharide [-GlcA-β1,3-Xyl-α1,3-]n called matriglycan, which can be targeted specifically with monoclonal antibody IIH6C4. Although α-DG was identified in a recent proteomic study of platelet releasate, its membrane expression and function in platelets have never been investigated. Methods and Results: Using the anti-α-DG monoclonal antibody IIH6C4, we found expression of α-DG in mouse and human resting platelets in Western blots. α-DG expression was also identified on the non-permeabilized mouse and human resting platelets by flow cytometry, indicating that α-DG is constitutively expressed on the platelet surface. We next examined whether disruption of the integrity of the dystrophin-glycoprotein complex affects the platelet aggregation. In a dystrophin-deficient mouse model of Duchenne muscular dystrophy with reduced α-DG expression (mdx mice), we found that ADP induced platelet aggregation in platelet-rich plasma (PRP) decreased 50%, suggesting that the integrity of the dystrophin-glycoprotein complex is required for normal platelet aggregation. To test whether inhibition of platelet aggregation can be achieved by targeting α-DG, we applied the well-established polyclonal (H300) and monoclonal (IIH6C4 and VIA4) anti-α-DG antibodies. Mouse gel-filtered platelet aggregation induced by thrombin was significantly inhibited by all three antibodies. Mouse platelet aggregation in PRP was also inhibited by H300. For platelets from healthy human donors, the inhibitive effect was more profound. Using a lower concentration of H300 (1 µg/mL in human vs. 2 µg/mL in mouse), ADP induced human platelet aggregation in PRP was inhibited to less than 50% of control and quickly de-aggregated within 5 minutes, while no de-aggregation was observed in the controls. Human gel-filtered platelet aggregation was also inhibited in a dose-dependent manner by these antibodies. Our results thus revealed a vital role of α-DG in platelet aggregation. In an ex vivo perfusion chamber model, human platelet adhesion and thrombus formation on collagen were markedly decreased at an arterial shear rate of 1800/s by anti-α-DG antibodies. Interestingly, although α-DG was found to be a ligand of laminin, platelet adhesion on laminin was not significantly altered by these antibodies, suggesting that contribution of α-DG to thrombus formation is not through its classical ligand laminin but other previously unidentified mechanisms. Next, we tested the role of α-DG in thrombus formation in vivo. Using a mouse cremaster artery laser-injury intravital microscopy model, we found that the anti-α-DG antibodies significantly delayed and decreased thrombus formation. To investigate the underlying mechanism of the surprisingly profound impact of α-DG on platelet aggregation and thrombus formation, we performed the co-immunoprecipitation assay and found that α-DG interacts with both β3 integrin and fibronectin, even in the absence of Fg and VWF, suggesting that α-DG may directly or form an α-DG-fibronectin complex to bind αIIbβ3 integrin, contributing to platelet aggregation and thrombus formation. Conclusion: Our data demonstrated that α-DG and likely other components of the dystrophin-glycoprotein complex are expressed on the platelet surface, and play a vital role in platelet aggregation and thrombus formation. α-DG may contribute to platelet aggregation independent of VWF and Fg through direct or indirect interaction with αIIbβ3 integrin. It is likely that patients with muscular dystrophies, such as those with Duchenne muscular dystrophy, are protected from thrombosis. More importantly, our data established α-DG, and potentially other components of the dystrophin-glycoprotein complex, as novel targets for the treatment of thrombotic disorders. Disclosures No relevant conflicts of interest to declare.

scholarly journals In Silico Hemostasis Modeling and Prediction

2020 ◽  
Vol 40 (04) ◽  
pp. 524-535
Author(s):  
Dmitry Y. Nechipurenko ◽  
Aleksey M. Shibeko ◽  
Anastasia N. Sveshnikova ◽  
Mikhail A. Panteleev
Keyword(s):  
In Silico ◽  
Physiological Response ◽  
Platelet Adhesion ◽  
State Of The Art ◽  
Thrombus Formation ◽  
The State ◽  
Modeling And Prediction ◽  
Hemostatic System ◽  
Single Enzyme

AbstractComputational physiology, i.e., reproduction of physiological (and, by extension, pathophysiological) processes in silico, could be considered one of the major goals in computational biology. One might use computers to simulate molecular interactions, enzyme kinetics, gene expression, or whole networks of biochemical reactions, but it is (patho)physiological meaning that is usually the meaningful goal of the research even when a single enzyme is its subject. Although exponential rise in the use of computational and mathematical models in the field of hemostasis and thrombosis began in the 1980s (first for blood coagulation, then for platelet adhesion, and finally for platelet signal transduction), the majority of their successful applications are still focused on simulating the elements of the hemostatic system rather than the total (patho)physiological response in situ. Here we discuss the state of the art, the state of the progress toward the efficient “virtual thrombus formation,” and what one can already get from the existing models.

Signaling through GP Ib-IX-V activates αIIbβ3 independently of other receptors

Blood ◽  
2004 ◽  
Vol 103 (9) ◽  
pp. 3403-3411 ◽  
Author(s):  
Ana Kasirer-Friede ◽  
Maria Rita Cozzi ◽  
Mario Mazzucato ◽  
Luigi De Marco ◽  
Zaverio M. Ruggeri ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Signaling Proteins ◽  
Phosphatidylinositol 3 Kinase ◽  
Kinase C ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Pharmacologic Inhibition

Abstract Platelet adhesion to von Willebrand factor (VWF) activates αIIbβ3, a prerequisite for thrombus formation. However, it is unclear whether the primary VWF receptor, glycoprotein (GP) Ib-IX-V, mediates αIIbβ3 activation directly or through other signaling proteins physically associated with it (eg, FcR γ-chain), possibly with the contribution of other agonist receptors and of VWF signaling through αIIbβ3. To resolve this question, human and GP Ibα transgenic mouse platelets were plated on dimeric VWF A1 domain (dA1VWF), which engages only GP Ib-IX-V, in the presence of inhibitors of other agonist receptors. Platelet adhesion to dA1VWF induced Src kinase-dependent tyrosine phosphorylation of the FcR γ-chain and the adapter molecule, ADAP, and triggered intracellular Ca2+ oscillations and αIIbβ3 activation. Inhibition of Ca2+ oscillations with BAPTA-AM prevented αIIbβ3 activation but not tyrosine phosphorylation. Pharmacologic inhibition of protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI 3-kinase) prevented αIIbβ3 activation but not Ca2+ oscillations. Inhibition of Src with 2 distinct compounds blocked all responses downstream of GP Ib-IX-V under static or flow conditions. However, dA1VWF-induced responses were reduced only slightly in GP Ibα transgenic platelets lacking FcR γ-chain. These data establish that GP Ib-IX-V itself can signal to activate αIIbβ3, through sequential actions of Src kinases, Ca2+ oscillations, and PI 3-kinase/PKC. (Blood. 2004;103:3403-3411)

scholarly journals The Small GTPase Rap1b: A Bidirectional Regulator of Platelet Adhesion Receptors

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Gianni Francesco Guidetti ◽  
Mauro Torti
Keyword(s):  
Cell Adhesion ◽  
Signaling Pathways ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Complex Pattern ◽  
Adhesive Properties ◽  
Adhesion Receptors ◽  
Inside Out

Integrins and other families of cell adhesion receptors are responsible for platelet adhesion and aggregation, which are essential steps for physiological haemostasis, as well as for the development of thrombosis. The modulation of platelet adhesive properties is the result of a complex pattern of inside-out and outside-in signaling pathways, in which the members of the Rap family of small GTPases are bidirectionally involved. This paper focuses on the regulation of the main Rap GTPase expressed in circulating platelets, Rap1b, downstream of adhesion receptors, and summarizes the most recent achievements in the investigation of the function of this protein as regulator of platelet adhesion and thrombus formation.

Levels of von Willebrand factor and ADAMTS13 determine clinical outcome after cardioversion for atrial fibrillation

2011 ◽  
Vol 105 (03) ◽  
pp. 435-443 ◽  
Author(s):  
Veronika Bruno ◽  
Rudolf Jarai ◽  
Susanne Gruber ◽  
Thomas Höchtl ◽  
Ivan Brozovic ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Independent Predictor ◽  
Thrombus Formation ◽  
Critical Role ◽  
Inverse Correlation ◽  
Von Willebrand ◽  
Rhythm Stability ◽  
Willebrand Factor

SummaryVon Willebrand factor (vWF) plays an essential role in platelet adhesion and thrombus formation. Patients with atrial fibrillation (AF) exhibit higher plasma vWF and lower ADAMTS13 antigen levels compared to controls. Little is known about vWF and ADAMTS13 in AF patients treated with cardioversion (CV). Thus we investigated the alterations of plasma vWF and ADAMTS13 after CV and evaluated the predictive value of these parameters for recurrence of AF. In this observational study we determined plasma levels of vWF and ADAMTS13 in 77 patients before and immediately after CV, as well as 24 hours (h) and six weeks thereafter, by means of commercially available assays. The vWF/ ADAMTS13-ratio was significantly elevated immediately after CV (p=0.02) and 24 h after CV (p=0.002) as compared to baseline levels. ADAMTS13, 24 h after CV, exhibited a significant association with recurrence of AF (HR: 0.97; p=0.037). Accordingly, tertiles of ADAMTS13 showed a stepwise inverse correlation with the risk of recurrent AF (HR: 0.50; p=0.009). After adjustment for confounders, ADAMTS13 remained significant as an independent predictor of recurrent AF (HR: 0.61; p=0.047). Similarly, the vWF/ADAMTS13-ratio, 24 h after CV, was associated with rhythm stability and remained an independent predictor of recurrent AF (HR: 1.88; p=0.028). The regulation of vWF and its cleaving protease ADAMTS13 after CV might play a critical role in producing a pro-thrombotic milieu immediately after CV for AF. Since ADAMTS13 plasma concentration and the vWF/ADAMTS13-ratio are independently associated with rhythm stability, these indexes might be used for prediction of recurrence of AF.

scholarly journals SIN-1 reduces platelet adhesion and platelet thrombus formation in a porcine model of balloon angioplasty.

Circulation ◽  
1993 ◽  
Vol 87 (2) ◽  
pp. 590-597 ◽  
Author(s):  
P H Groves ◽  
M J Lewis ◽  
H A Cheadle ◽  
W J Penny
Keyword(s):  
Balloon Angioplasty ◽  
Platelet Adhesion ◽  
Porcine Model ◽  
Thrombus Formation ◽  
Platelet Thrombus

Tyrosine Kinase Inhibitor Sunitinib Delays Platelet-Induced Coagulation: Additive Effects of Aspirin

2021 ◽  
Author(s):  
Delia I. Fernández ◽  
Alicia Veninga ◽  
Bibian M. E. Tullemans ◽  
Constance C. F. M. J. Baaten ◽  
Linsey J. F. Peters ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Cancer Patients ◽  
Kinase Inhibitor ◽  
Synergistic Effects ◽  
Thrombus Formation ◽  
Type I ◽  
Glycoprotein Vi ◽  
Fibrin Formation ◽  
Increased Risk

Abstract Background Sunitinib is a multitarget tyrosine kinase inhibitor (TKI) used for cancer treatment. In platelets, sunitinib affects collagen-induced activation under noncoagulating conditions. We investigated (1) the effects of sunitinib on thrombus formation induced by other TK-dependent receptors, and (2) the effects under coagulating conditions. Cardiovascular disease is a comorbidity in cancer patients, resulting in possible aspirin treatment. Sunitinib and aspirin are associated with increased bleeding risk, and therefore we also investigated (3) the synergistic effects of these compounds on thrombus and fibrin formation. Methods Blood or isolated platelets from healthy volunteers or cancer patients were incubated with sunitinib and/or aspirin or vehicle. Platelet activation was determined by TK phosphorylation, flow cytometry, changes in [Ca2+]i, aggregometry, and whole blood perfusion over multiple surfaces, including collagen with(out) tissue factor (TF) was performed. Results Sunitinib reduced thrombus formation and phosphatidylserine (PS) exposure under flow on collagen type I and III. Also, sunitinib inhibited glycoprotein VI-induced TK phosphorylation and Ca2+ elevation. Upon TF-triggered coagulation, sunitinib decreased PS exposure and fibrin formation. In blood from cancer patients more pronounced effects of sunitinib were observed in lung and pancreatic as compared to neuroglioblastoma and other cancer types. Compared to sunitinib alone, sunitinib plus aspirin further reduced platelet aggregation, thrombus formation, and PS exposure on collagen under flow with(out) coagulation. Conclusion Sunitinib suppresses collagen-induced procoagulant activity and delays fibrin formation, which was aggravated by aspirin. Therefore, we urge for awareness of the combined antiplatelet effects of TKIs with aspirin, as this may result in increased risk of bleeding.

Abstract 56: Coagulation Factor XI Promotes Distal Platelet Activation and Single Platelet Consumption in the Bloodstream Under Shear Flow

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Jenya Zilberman-Rudenko ◽  
Chantal Wiesenekker ◽  
Asako Itakura ◽  
Owen J McCarty
Keyword(s):  
Shear Flow ◽  
Platelet Activation ◽  
Platelet Adhesion ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Coagulation Factor ◽  
Thrombin Inhibitor ◽  
Dependent Manner ◽  
Factor Xi ◽  
Primate Model

Objective: Coagulation factor XI (FXI) has been shown to contribute to thrombus formation on collagen or tissue factor (TF)-coated surfaces in vitro and in vivo by enhancing thrombin generation. Whether the role of the intrinsic pathway of coagulation is restricted to the local site of thrombus formation is unknown. This study was designed to determine whether FXI could promote both proximal and distal platelet activation and aggregate formation in the bloodstream. Approach and Results: Pharmacological blockade of FXI activation or thrombin activity in blood did not affect local platelet adhesion, yet reduced local platelet aggregation, thrombin localization and fibrin formation on immobilized collagen and TF under shear flow, ex vivo . Downstream of the thrombus formed on immobilized collagen or collagen and 10 pM TF, platelet CD62P expression and microaggregate formation and progressive platelet consumption were significantly reduced in the presence of FXI-function blocking antibodies or a thrombin inhibitor in a shear rate- and time-dependent manner. In a non-human primate model of thrombus formation, we found that inhibition of FXI reduced single platelet consumption in the bloodstream distal to a site of thrombus formation. Conclusions: This study demonstrates that the FXI-thrombin axis contributes to distal platelet activation and procoagulant microaggregate formation in the blood flow downstream of the site of thrombus formation. Our data highlights FXI as a novel therapeutic target for inhibiting distal platelet activation without affecting proximal platelet adhesion.

Simulation of platelet adhesion and aggregation regulated by fibrinogen and von Willebrand factor

2008 ◽  
Vol 99 (01) ◽  
pp. 108-115 ◽  
Author(s):  
Koichiro Yano ◽  
Ken-ichi Tsubota ◽  
Takuji Ishikawa ◽  
Shigeo Wada ◽  
Takami Yamaguchi ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Simple Shear Flow ◽  
Stokesian Dynamics ◽  
General Observation ◽  
Significant Development ◽  
Platelet Adhesion And Aggregation ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryWe propose a method to analyze platelet adhesion and aggregation computationally, taking into account the distinct properties of two plasma proteins, vonWillebrand factor (vWF) and fibrinogen (Fbg). In this method, the hydrodynamic interactions between platelet particles under simple shear flow were simulated using Stokesian dynamics based on the additivity of velocities. The binding force between particles mediated by vWF and Fbg was modeled using the Voigt model. Two Voigt models with different properties were introduced to consider the distinct behaviors of vWF and Fbg. Our results qualitatively agreed with the general observation of a previous in-vitro experiment, thus demonstrating that the significant development of thrombus formation in height requires not only vWF, but also Fbg. This agreement of simulation and experimental results qualitatively validates our model and suggests that consideration of the distinct roles of vWF and Fbg is essential to investigate the physiological and pathophysiological mechanisms of thrombus formation using a computational approach.

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