scholarly journals The Proteoglycan Biglycan Modulates Platelet Adhesion and Thrombus Formation in a GPVI-Dependent Manner

2021 ◽  
Vol 22 (22) ◽  
pp. 12168
Author(s):  
Henrike Hoermann ◽  
Irena Krueger ◽  
Nadine Maurus ◽  
Friedrich Reusswig ◽  
Yi Sun ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Vessel Wall ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Three Dimensional ◽  
Dependent Manner ◽  
Prolonged Bleeding ◽  
Vessel Injury

Background: Vascular injury induces the exposure of subendothelial extracellular matrix (ECM) important to serve as substrate for platelets to adhere to the injured vessel wall to avoid massive blood loss. Different ECM proteins are known to initiate platelet adhesion and activation. In atherosclerotic mice, the small, leucine-rich proteoglycan biglycan is important for the regulation of thrombin activity via heparin cofactor II. However, nothing is known about the role of biglycan for hemostasis and thrombosis under nonatherosclerotic conditions. Methods: The role of biglycan for platelet adhesion and thrombus formation was investigated using a recombinant protein and biglycan knockout mice. Results: The present study identified biglycan as important ECM protein for the adhesion and activation of platelets, and the formation of three-dimensional thrombi under flow conditions. Platelet adhesion to immobilized biglycan induces the reorganization of the platelet cytoskeleton. Mechanistically, biglycan binds and activates the major collagen receptor glycoprotein (GP)VI, because reduced platelet adhesion to recombinant biglycan was observed when GPVI was blocked and enhanced tyrosine phosphorylation in a GPVI-dependent manner was observed when platelets were stimulated with biglycan. In vivo, the deficiency of biglycan resulted in reduced platelet adhesion to the injured carotid artery and prolonged bleeding times. Conclusions: Loss of biglycan in the vessel wall of mice but not in platelets led to reduced platelet adhesion at the injured carotid artery and prolonged bleeding times, suggesting a crucial role for biglycan as ECM protein that binds and activates platelets via GPVI upon vessel injury.

The contributions of the α2β1 integrin to vascular thrombosis in vivo

Blood ◽  
2003 ◽  
Vol 102 (10) ◽  
pp. 3652-3657 ◽  
Author(s):  
Li He ◽  
Loretta K. Pappan ◽  
David G. Grenache ◽  
Zhengzhi Li ◽  
Douglas M. Tollefsen ◽  
...  
Keyword(s):  
Vascular Injury ◽  
Vessel Wall ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Critical Role ◽  
Flow Conditions ◽  
In Vivo Models ◽  
Vascular Thrombosis ◽  
Α2β1 Integrin

AbstractThe α2β1 integrin serves as a receptor for collagens, laminin, and several other nonmatrix ligands. Many studies have suggested that the α2β1 integrin is a critical mediator of platelet adhesion to collagen within the vessel wall after vascular injury and that the interactions of the platelet α2β1 integrin with subendothelial collagen after vascular injury are required for proper hemostasis. We have used the α2β1 integrin-deficient mouse to evaluate the contributions of the α2β1 integrin in 2 in vivo models of thrombosis. Studies using a model of endothelial injury to the carotid artery reveal that the α2β1 integrin plays a critical role in vascular thrombosis at the blood-vessel wall interface under flow conditions. In contrast, the α2β1 integrin is not required for the formation of thrombi and pulmonary emboli following intravascular injection of collagen. Our results are the first to document a critical in vivo role for the α2β1 integrin in thrombus formation at the vessel wall under conditions of shear following vascular injury. (Blood. 2003;102:3652-3657)

Cadherin-6 Forms a Functional Adhesion Complex with Alpha-Catenin and Beta-Catenin in Platelets and Is Required for Platelet Aggregation and Thrombus Formation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2232-2232 ◽  
Author(s):  
Michele Mumaw ◽  
Maria de la Fuente ◽  
Carolyn Aldana ◽  
Wei Li ◽  
Marvin T Nieman
Keyword(s):  
Platelet Aggregation ◽  
Adhesion Molecules ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Bleeding Complications ◽  
Severe Bleeding ◽  
Beta Catenin ◽  
Adhesion Complex

Abstract The regulation of hemostasis and thrombus formation is a tightly controlled event that has catastrophic consequences when it is deregulated. One of the hallmarks of the thrombus is aggregated platelets. Upon platelet stimulation, adhesion molecules become activated and mediate multiple cell-cell interactions. Therapeutically, blocking platelet adhesion is a proven method for preventing pathological arterial thrombus formation. However, targeting the primary adhesion receptor, integrin αIIbβ3, results in severe bleeding complications. Therefore, identifying novel proteins or uncovering novel functions for known proteins in platelets is a necessary first step to facilitate the development of safer anti-platelet therapeutics. We have identified that the cell adhesion molecule cadherin-6 forms a functional adhesion complex with α-catenin and β-catenin in platelets. The goal of our project was to determine the mechanism of cadherin-6 mediated adhesion in platelets. Our initial experiments demonstated that cadherin-6 and β-catenin co-localize at the plasma membrane in platelets using confocal immunofluorescence microscopy. We determined that α-catenin and β-catenin co-immunoprecipitate with cadherin-6 from platelet lysates. To examine the functional role of cadherin-6 on platelet aggregation we used a cadherin-6 blocking antibody (10 μg/ml). Blocking cadherin-6 inhibited mouse platelet aggregation induced by PAR4 peptide. We next determined the role of cadherin-6 in vivo by examining carotid artery thrombosis after 7.5% FeCl3 treatment. C57Bl6 mice were injected with cadherin-6 antibody IV and labeled with rhodamine 6G by jugular vein injection. Thrombus formation was imaged in real time by fluorescent intravital microscopy. Blocking cadherin-6 prevented thrombosis for the duration of the experiment (30 min). To verify that the effects that we observed were specific to cadherin-6 expressed on platelets, we isolated platelets from donor mice and treated with cadherin-6 antibody or control IgG ex vivo. The treated platelets were perfused into recipient mice that were irradiated with 11 Gy to make the animals thrombocytopenic. The cadherin-6 antibody treated platelets formed an occlusion at 26.4 ± 3.6 min vs. 13.7 ± 2.0 min for the IgG (p=0.03). Importantly, the cadherin-6 antibody did not affect platelet counts compared to IgG controls 2.97 ± 0.40 (×108) vs. 3.02 ± 0.20 (×108). These combined studies show that caderhin-6 forms a complex with the necessary proteins required to mediate adhesion in platelets. Our results demonstrate that platelet cadherin-6 has a physiologically important role during platelet activation and thrombus formation in vivo. In summary, we have identified a novel adhesion complex in platelets that may provide a mechanism to limit platelet aggregation therapeutically. On going studies will determine the regulation of the cadherin-6/catenin complex and how cadherin-6 cooperates with other platelet adhesion molecules. Disclosures No relevant conflicts of interest to declare.

scholarly journals Vascular protective effect of aspirin and rivaroxaban upon endothelial denudation of the mouse carotid artery

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
T. G. Mastenbroek ◽  
M. F. A. Karel ◽  
M. Nagy ◽  
W. Chayoua ◽  
E. I. J. Korsten ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Vessel Wall ◽  
Thrombus Formation ◽  
Positive Trend ◽  
Atherosclerotic Cardiovascular Disease ◽  
Protective Effects ◽  
Endothelial Denudation ◽  
Vascular Stiffening

Abstract While in recent trials the dual pathway inhibition with aspirin plus rivaroxaban has shown to be efficacious in patients with atherosclerotic cardiovascular disease, little is known about the effects of this combination treatment on thrombus formation and vascular remodelling upon vascular damage. The aim of this study was to examine the effects of aspirin and/or rivaroxaban on injury-induced murine arterial thrombus formation in vivo and in vitro, vessel-wall remodelling, and platelet-leukocyte aggregates. Temporary ligation of the carotid artery of C57BL/6 mice, fed a western type diet, led to endothelial denudation and sub-occlusive thrombus formation. At the site of ligation, the vessel wall stiffened and the intima-media thickened. Aspirin treatment antagonized vascular stiffening and rivaroxaban treatment led to a positive trend towards reduced stiffening. Local intima-media thickening was antagonized by both aspirin or rivaroxaban treatment. Platelet-leukocyte aggregates and the number of platelets per leukocyte were reduced in aspirin and/or rivaroxaban treatment groups. Furthermore, rivaroxaban restricted thrombus growth and height in vitro. In sum, this study shows vascular protective effects of aspirin and rivaroxaban, upon vascular injury of the mouse artery.

Hematopoietic cell-derived microparticle tissue factor contributes to fibrin formation during thrombus propagation

Blood ◽  
2004 ◽  
Vol 104 (10) ◽  
pp. 3190-3197 ◽  
Author(s):  
Janet Chou ◽  
Nigel Mackman ◽  
Glenn Merrill-Skoloff ◽  
Brian Pedersen ◽  
Barbara C. Furie ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tissue Factor ◽  
Vessel Wall ◽  
Thrombus Formation ◽  
Hematopoietic Cell ◽  
Chimeric Mice ◽  
Wild Type ◽  
Vessel Injury ◽  
Living Mouse

Abstract Tissue factor (TF) is expressed on nonvascular cells and cells within the vessel wall and circulates in blood associated with microparticles. Although blood-borne TF accumulates into the developing thrombus during thrombus formation, the contribution of blood-borne TF and vessel wall TF to thrombin generation in vivo following vessel injury is unknown. To determine the source and role of blood-borne microparticle TF, we studied arterial thrombus formation in a living mouse using intravital microscopy. Platelet, TF, and fibrin accumulation in the developing thrombus was compared in wild-type and low TF mice. Compared to wild-type mice, low TF mice formed very small platelet thrombi lacking TF or fibrin. Wild-type and low TF mice received transplants of bone marrow from wild-type and low TF mice. Arterial thrombi in low TF bone marrow/wild-type chimeric mice had decreased size and decreased TF and fibrin levels. Arterial thrombi in wild-type bone marrow/low TF chimeric mice showed decreased platelet thrombus size but normal TF and fibrin levels. This demonstrates that blood-borne TF associated with hematopoietic cell-derived microparticles contributes to thrombus propagation.

scholarly journals A Crucial Role of Glycoprotein VI for Platelet Recruitment to the Injured Arterial Wall In Vivo

2002 ◽  
Vol 197 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Steffen Massberg ◽  
Meinrad Gawaz ◽  
Sabine Grüner ◽  
Valerie Schulte ◽  
Ildiko Konrad ◽  
...  
Keyword(s):  
Vascular Injury ◽  
Vessel Wall ◽  
Thrombus Formation ◽  
Arterial Occlusion ◽  
Arterial Injury ◽  
Platelet Glycoprotein ◽  
Glycoprotein Vi ◽  
Strict Requirement

Platelet adhesion and aggregation at sites of vascular injury is crucial for hemostasis but may lead to arterial occlusion in the setting of atherosclerosis and precipitate diseases such as myocardial infarction. A current hypothesis suggests that platelet glycoprotein (GP) Ib interaction with von Willebrand factor recruits flowing platelets to the injured vessel wall, where subendothelial fibrillar collagens support their firm adhesion and activation. However, so far this hypothesis has not been tested in vivo. Here, we demonstrate by intravital fluorescence microscopy of the mouse carotid artery that inhibition or absence of the major platelet collagen receptor, GPVI, abolishes platelet–vessel wall interactions after endothelial denudation. Unexpectedly, inhibition of GPVI by the monoclonal antibody JAQ1 reduced platelet tethering to the subendothelium by ∼89%. In addition, stable arrest and aggregation of platelets was virtually abolished under these conditions. Using different models of arterial injury, the strict requirement for GPVI in these processes was confirmed in GPVI-deficient mice, where platelets also failed to adhere and aggregate on the damaged vessel wall. These findings reveal an unexpected role of GPVI in the initiation of platelet attachment at sites of vascular injury and unequivocally identify platelet–collagen interactions (via GPVI) as the major determinant of arterial thrombus formation.

Importance of GPVI in Platelet Activation and Thrombus Formation In Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 842-842 ◽  
Author(s):  
Christophe Dubois ◽  
Laurence Panicot-Dubois ◽  
Bruce Furie ◽  
Barbara C. Furie
Keyword(s):  
Ferric Chloride ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Wild Type ◽  
Laser Injury ◽  
Vessel Injury ◽  
Arterial Thrombus ◽  
Platelet Accumulation ◽  
Chloride Treatment

Abstract Collagen is one of the major components of the vessel wall responsible for platelet adhesion and activation at sites of vascular injury. In vitro studies have shown that α2β1 and GPVI directly and αIIbβ3 and GPIb-IX-V indirectly, via vWF, are all involved in the adhesion of platelets to collagen. However, the importance of GPVI on the adhesion and activation of platelets in vivo is still controversial. Here we show that in vivo GPVI plays an important role in platelet adhesion and activation when collagen is exposed to blood. To determine the role of GPVI on thrombus formation, we compared thrombus formation in FcRγ null mice, which do not express GPVI on the platelet surface, and in wild type mice after vessel injury induced by ferric chloride or by a nitrogen dye laser. We studied arterial thrombus formation in the microcirculation of the cremaster and the mesentery muscles using high speed multi channel intravital fluorescence widefield microscopy. Real time platelet accumulation in the developing thrombus was detected using a fluorescent antibody directed against αIIb. After an injury induced by the ferric chloride, we observed a significant delay in both the time to formation of an initial thrombus and the time to vessel occlusion in FcRγ null mice in comparison with wild type mice. When activated platelets isolated from FcRγ null mice were injected into a recipient FcRγ null mouse, we were able to restore the formation of a thrombus. This effect was abolished by injection of Lamifiban, an inhibitor of activated αIIbβ3. These results indicate that GPVI is not only involved in vivo in the adhesion of platelets to collagen but also plays an important role in the activation of the platelet fibrinogen receptor αIIbβ3. In contrast, platelet accumulation after laser-induced injury in the FcRγ null and the wild type mice was comparable. No difference in the kinetics of platelet accumulation into the laser induced growing thrombus was observed. To understand the different pathways leading to thrombus formation in vivo after ferric chloride or laser induced injury, we examined collagen exposure after vessel injury and the accumulation of tissue factor (TF) in the developing arterial thrombus of FcRγ null and wild type mice using antibodies directed against mouse collagen type I and mouse TF. We observed a significant exposure of collagen at sites of thrombus formation after ferric chloride treatment. In contrast, we did not observe any collagen exposure on blood vessels after laser-induced injury. Furthermore, the ratio of TF/platelets present into the thrombus after injury was 5 fold greater after laser injury than after ferric chloride treatment. These results suggest that TF but not collagen plays an important role in thrombus formation induced by laser injury of the vessel wall. Altogether, our results indicate that the GPVI receptor is involved in vivo in platelet adhesion when collagen is exposed to blood and plays an important role in the activation of other platelet integrins such as αIIbβ3 leading to the formation of a stable thrombus.

Role of Thiol Isomerase ERp5 in Thrombus Formation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 370-370
Author(s):  
Freda H. Passam ◽  
Lin Lin ◽  
Mingdong Huang ◽  
Jonathan M. Gibbins ◽  
Bruce Furie ◽  
...  
Keyword(s):  
Cho Cells ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Dependent Manner ◽  
Wild Type ◽  
In Vivo Models ◽  
Platelet Accumulation

Abstract Abstract 370 Protein disulfide isomerase is required for thrombus formation in various in vivo models of thrombosis. Another member of the thiol isomerase family, endoplasmic reticulum protein 5 (ERp5), is released from activated platelets and co-immunoprecipitates with beta 3 integrin (Jordan et al, 2005). We further investigated the association of ERp5 with the platelet fibrinogen receptor alpha IIb beta 3 and the significance of ERp5 release in thrombus formation in vivo. Recombinant purified ERp5 was labeled with Alexa 488 and used in direct binding assays to CHO cells expressing wild type (WT) alpha IIb beta 3, CHO cells expressing mutant alpha IIb beta 3 (containing an Asp119Tyr substitution in the beta 3 subunit) and to control CHO cells. The mutant alpha IIb beta 3 does not bind fibrinogen. ERp5 bound to CHO cells expressing wild type (WT) alpha IIb beta 3 in a dose-dependent manner but did not bind to CHO cells expressing mutant alpha IIb beta 3 or to control CHO cells. The relative increase in the geomean of Alexa 488-labeled ERp5 binding to 0.5 ×106 WT alpha IIb beta 3 CHO cells over that bound to control CHO cells was 20, 45 and 85% for ERp5 concentrations of 80, 160 and 400 nM respectively. Binding of ERp5 (160 nM) to WT alpha IIb beta 3 expressing CHO cells was further increased by 75% when the integrin was activated with 2 mM Mn2+ compared to non-activated WT alpha IIb beta 3 CHO cells. A role for ERp5 in thrombus formation was studied in the laser injury model of thrombosis in mouse cremaster arterioles using a rabbit polyclonal anti-ERp5 antibody, immunoaffinity purified against recombinant ERp5. This antibody detected ERp5 in the releasate of thrombin-activated mouse platelets in vitro by Western blot and on the surface of thrombin-activated mouse platelets by flow cytometry. Dylight 649-labeled anti-CD42b was infused into the mouse circulation to detect platelet accumulation and Alexa 488-labeled anti-ERp5 antibody at 0.05 ug/g, a dose that does not inhibit thrombus formation, was infused to detect ERp5. The fluorescent anti-ERp5 signal detected at the thrombus site was compared to the signal produced by a non-specific IgG labeled with Alexa 488 infused into a control mouse. Anti-ERp5 fluorescence was detected in the thrombus with kinetics that followed platelet accumulation whereas there was minimal signal from the control IgG. We examined whether higher doses of anti-ERp5 affect thrombus formation. Platelet and fibrin accumulation were detected using fluorescently labeled anti-CD42b antibody and monoclonal anti-fibrin-specific antibody respectively before or after the injection of unlabeled anti-ERp5 antibody or pre-immune IgG at 2.5 ug/g. Platelet and fibrin accumulation, expressed as area under the curve of the median integrated fluorescence over time, was obtained from 14 thrombi in 6 mice formed before infusion of antibody, 18 thrombi in 2 mice formed after infusion of control IgG and 29 thrombi in 3 mice formed after infusion of anti-ERp5. Anti-ERp5 infusion caused a 70% decrease in the deposition of platelets and a 62% decrease in fibrin accumulation compared to infusion of control antibody (p<0.01). There was no difference in platelet and fibrin accumulation before infusion of antibody and after infusion of control antibody. These results provide evidence for a role of a second thiol isomerase, ERp5, in thrombus formation, a function which may be mediated through its association with alpha IIb beta 3. Disclosures: No relevant conflicts of interest to declare.

Multiple integrin-ligand interactions synergize in shear-resistant platelet adhesion at sites of arterial injury in vivo

Blood ◽  
2003 ◽  
Vol 102 (12) ◽  
pp. 4021-4027 ◽  
Author(s):  
Sabine Grüner ◽  
Miroslava Prostredna ◽  
Valerie Schulte ◽  
Thomas Krieg ◽  
Beate Eckes ◽  
...  
Keyword(s):  
Vessel Wall ◽  
Platelet Adhesion ◽  
Arterial Injury ◽  
Β1 Integrins ◽  
Ligand Interactions ◽  
Integrin Ligand ◽  
Aggregation Of Platelets ◽  
Αiibβ3 Integrin

Abstract Damage to the integrity of the vessel wall results in exposure of the subendothelial extracellular matrix (ECM), which triggers integrin-dependent adhesion and aggregation of platelets. The role of platelet β1 integrins in these processes remains mostly undefined. Here, we demonstrate by intravital fluorescence microscopy that platelet adhesion and thrombus growth on the exposed ECM of the injured carotid artery is not significantly altered in α2-null mice and even in mice with a Cre/loxP-mediated loss of all β1 integrins on their platelets. In contrast, inhibition of αIIbβ3 integrin on platelets in wild-type mice blocked aggregate formation and reduced platelet adhesion by 60.0%. Strikingly, αIIbβ3 inhibition had a comparable effect in α2-null mice, demonstrating that other receptors mediate shear-resistant adhesion in the absence of functional α2β1 and αIIbβ3. These were identified to be α5β1 and/or α6β1 as αIIbβ3 inhibition abrogated platelet adhesion in β1-null mice. We conclude that shear-resistant platelet adhesion on the injured vessel wall in vivo is a highly integrated process involving multiple integrin-ligand interactions, none of which by itself is essential. (Blood. 2003;102:4021-4027)

Effects of NO-Donors on Thrombus Formation and Microcirculation in Cerebral Vessels of the Rat

1996 ◽  
Vol 76 (01) ◽  
pp. 111-117 ◽  
Author(s):  
Yasuto Sasaki ◽  
Junji Seki ◽  
John C Giddings ◽  
Junichiro Yamamoto
Keyword(s):  
Blood Flow ◽  
Thrombus Formation ◽  
Dependent Manner ◽  
Red Cell ◽  
Cell Velocity ◽  
Red Cell Velocity ◽  
The Mean ◽  
Wall Shear ◽  
Dose Dependent

SummarySodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), are known to liberate nitric oxide (NO). In this study the effects of SNP and SIN-1 on thrombus formation in rat cerebral arterioles and venules in vivo were assessed using a helium-neon (He-Ne) laser. SNP infused at doses from 10 Μg/kg/h significantly inhibited thrombus formation in a dose dependent manner. This inhibition of thrombus formation was suppressed by methylene blue. SIN-1 at a dose of 100 Μg/kg/h also demonstrated a significant antithrombotic effect. Moreover, treatment with SNP increased vessel diameter in a dose dependent manner and enhanced the mean red cell velocity measured with a fiber-optic laser-Doppler anemometer microscope (FLDAM). Blood flow, calculated from the mean red cell velocity and vessel diameters was increased significantly during infusion. In contrast, mean wall shear rates in the arterioles and venules were not changed by SNP infusion. The results indicated that SNP and SIN-1 possessed potent antithrombotic activities, whilst SNP increased cerebral blood flow without changing wall shear rate. The findings suggest that the NO released by SNP and SIN-1 may be beneficial for the treatment and protection of cerebral infarction

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