Biomechanical thrombosis: the dark side of force and dawn of mechano-medicine

Arterial thrombosis is in part contributed by excessive platelet aggregation, which can lead to blood clotting and subsequent heart attack and stroke. Platelets are sensitive to the haemodynamic environment. Rapid haemodynamcis and disturbed blood flow, which occur in vessels with growing thrombi and atherosclerotic plaques or is caused by medical device implantation and intervention, promotes platelet aggregation and thrombus formation. In such situations, conventional antiplatelet drugs often have suboptimal efficacy and a serious side effect of excessive bleeding. Investigating the mechanisms of platelet biomechanical activation provides insights distinct from the classic views of agonist-stimulated platelet thrombus formation. In this work, we review the recent discoveries underlying haemodynamic force-reinforced platelet binding and mechanosensing primarily mediated by three platelet receptors: glycoprotein Ib (GPIb), glycoprotein IIb/IIIa (GPIIb/IIIa) and glycoprotein VI (GPVI), and their implications for development of antithrombotic ‘mechano-medicine’ .

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Glycoprotein Ibα inhibition and ADP receptor antagonists, but not aspirin, reduce platelet thrombus formation in flowing blood exposed to atherosclerotic plaques

Thrombosis and Haemostasis ◽

10.1160/th06-07-0415 ◽

2007 ◽

Vol 97 (03) ◽

pp. 435-443 ◽

Cited By ~ 32

Author(s):

Sandra Penz ◽

Armin Reininger ◽

Orsolya Toth ◽

Hans Deckmyn ◽

Richard Brandi ◽

...

Keyword(s):

Platelet Aggregation ◽

Thrombus Formation ◽

Arterial Flow ◽

Atherosclerotic Plaques ◽

Aggregate Formation ◽

Receptor Antagonists ◽

P2y12 Antagonists ◽

Platelet Thrombus ◽

Adp Receptor ◽

Adp Receptor Antagonists

SummaryAnti-platelet drugs are used to prevent intra-arterial thrombus formation after rupture of atherosclerotic plaques. Until now, the inhibitory effect of present and future anti-platelet drugs such as aspirin, ADP receptor P2Y1/P2Y12 antagonists and glycoprotein (GP) Ibα inhibitors on the interaction of platelets with human plaques is not known. To study those effects we obtained human atherosclerotic plaques by surgical endarterectomy. Plaques induced maximal platelet aggregation in hirudinized platelet-rich plasma (PRP) and blood that was effectively inhibited by aspirin, the P2Y1 antagonist MRS2179 and the P2Y12 antagonist AR-C69931 MX, but not by GPIbα blockade with the mAB 6B4. Inhibition of platelet aggregation by MRS2179 was 74 ± 37% and 68 ± 20%, by AR-C69931 MX 94 ± 7% and 80 ± 6%, and by aspirin 88 ± 19% and 64 ± 28%, in PRP and blood, respectively (mean ± SD; n6–12 with plaques from 6 patients). The combination of both ADP receptor antagonists completely inhibited plaque-induced platelet aggregation in hirudinized PRP and blood. Under arterial flow conditions (1,500s-1), blockade of platelet GPIbα– resulted in a strong decrease of plaque-stimulated platelet adhesion/aggregate formation of 77 ± 5% (mean ± SD; n=4). Furthermore, MRS2I79, AR-C6993IMX and their combination reduced plaque-dependent platelet aggregate formation by 35 ± 14%, 32 ± 13% and 58 ± 12% (mean ± SD; n=5), respectively. Aspirin was without significant effect. In conclusion, a GPIbα-blocking antibody, as well as P2Y1 and P2Y12 receptor antagonists, alone or in combination, reduce in contrast to aspirin human plaque-induced platelet thrombus formation under arterial flow. Although these new anti-platelet agents inhibit platelet thrombus formation after plaque rupture, more efficient platelet blockers are required.

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Characterization of GPVI- or GPVI-CD39-Coated Nanoparticles and Their Impact on In Vitro Thrombus Formation

International Journal of Molecular Sciences ◽

10.3390/ijms23010011 ◽

2021 ◽

Vol 23 (1) ◽

pp. 11

Author(s):

Jeremy A. Nestele ◽

Anne-Katrin Rohlfing ◽

Valerie Dicenta ◽

Alexander Bild ◽

Daniela Eißler ◽

...

Keyword(s):

Platelet Aggregation ◽

Antithrombotic Therapy ◽

Light Transmission ◽

Thrombus Formation ◽

Bleeding Risk ◽

Significant Inhibition ◽

Site Specific ◽

Glycoprotein Vi ◽

Coated Nanoparticles

Traditional antithrombotic agents commonly share a therapy-limiting side effect, as they increase the overall systemic bleeding risk. A novel approach for targeted antithrombotic therapy is nanoparticles. In other therapeutic fields, nanoparticles have enabled site-specific delivery with low levels of toxicity and side effects. Here, we paired nanotechnology with an established dimeric glycoprotein VI-Fc (GPVI-Fc) and a GPVI-CD39 fusion protein, thereby combining site-specific delivery and new antithrombotic drugs. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles, NP-BSA, NP-GPVI and NP-GPVI-CD39 were characterized through electron microscopy, atomic force measurements and flow cytometry. Light transmission aggregometry enabled analysis of platelet aggregation. Thrombus formation was observed through flow chamber experiments. NP-GPVI and NP-GPVI-CD39 displayed a characteristic surface coating pattern. Fluorescence properties were identical amongst all samples. NP-GPVI and NP-GPVI-CD39 significantly impaired platelet aggregation. Thrombus formation was significantly impaired by NP-GPVI and was particularly impaired by NP-GPVI-CD39. The receptor-coated nanoparticles NP-GPVI and the bifunctional molecule NP-GPVI-CD39 demonstrated significant inhibition of in vitro thrombus formation. Consequently, the nanoparticle-mediated antithrombotic effect of GPVI-Fc, as well as GPVI-CD39, and an additive impact of CD39 was confirmed. In conclusion, NP-GPVI and NP-GPVI-CD39 may serve as a promising foundation for a novel therapeutic approach regarding targeted antithrombotic therapy.

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Antithrombotic effect of a monoclonal antibody to the platelet glycoprotein IIb/IIIa receptor in an experimental animal model

Blood ◽

10.1182/blood.v68.3.783.bloodjournal683783 ◽

1986 ◽

Vol 68 (3) ◽

pp. 783-786 ◽

Cited By ~ 1

Author(s):

BS Coller ◽

JD Folts ◽

LE Scudder ◽

SR Smith

Keyword(s):

Monoclonal Antibody ◽

Animal Model ◽

Platelet Aggregation ◽

Ex Vivo ◽

Thrombus Formation ◽

Platelet Glycoprotein ◽

Antithrombotic Effect ◽

Antithrombotic Agent ◽

Antithrombotic Effects ◽

Platelet Thrombus

A murine monoclonal antibody directed at the platelet glycoprotein IIb/IIIa complex, which blocks platelet aggregation ex vivo, was tested for its antithrombotic effects in an established animal model of acute platelet thrombus formation in partially stenosed arteries. Infusion of 0.7 to 0.8 mg/kg of the F(ab')2 fragment of the antibody completely blocked new thrombus formation despite multiple provocations, making it the most potent antithrombotic agent tested in this model.

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Identification of a Binding Site for Glycoprotein Ibα in the Apple 3 Domain of Factor XI

Journal of Biological Chemistry ◽

10.1074/jbc.m406727200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 45470-45476 ◽

Cited By ~ 40

Author(s):

Frank A. Baglia ◽

David Gailani ◽

José A. López ◽

Peter N. Walsh

Keyword(s):

Amino Acids ◽

Binding Site ◽

Thrombus Formation ◽

Platelet Glycoprotein ◽

Heparin Binding ◽

Factor Xi ◽

Platelet Binding ◽

Factor Xia ◽

Consolidation Phase ◽

Platelet Thrombus

Factor XI (FXI) is a homodimeric plasma zymogen that is cleaved at two internal Arg369–Ile370bonds by thrombin, factor XIIa, or factor XIa. FXI circulates as a complex with the glycoprotein high molecular weight kininogen (HK). FXI binds to specific sites (Kd= ∼10 nm,Bmax= ∼1,500/platelet) on the surface of stimulated platelets, where it is efficiently activated by thrombin. The FXI Apple 3 (A3) domain mediates binding to platelets in the presence of HK and zinc ions (Zn2+) or prothrombin and calcium ions. The platelet glycoprotein (GP) Ib-IX-V complex is the receptor for FXI (Baglia, F. A., Badellino, K. O., Li, C. Q., Lopez, J. A., and Walsh, P. N. (2002)J. Biol. Chem.277, 1662–1668). Using surface plasmon resonance, we determined that FXI binds specifically to glycocalicin, the extracellular domain of GPIbα, in a Zn2+-dependent fashion (Kd= ∼52 nm). We now show that recombinant FXI A3 domain inhibits FXI inbinding to glycocalicin in the presence of Zn2+, whereas the recombinant FXI A1, A2, or A4 domains have no effect. Experiments with full-length recombinant FXI mutants show that, in the presence of Zn2+, glycocalicin binds FXI at a heparin-binding site in A3 (Lys252and Lys253) and not by amino acids previously shown to be required for platelet binding (Ser248, Arg250, Lys255, Phe260, and Gln263). However, binding in the presence of HK and Zn2+requires Ser248, Arg250, Lys255, Phe260, and GLn263and not Lys252and Lys253. Thus, binding of FXI to GPIbα is mediated by amino acids in the A3 domain in the presence or absence of HK. This interaction is important for the initiation of the consolidation phase of blood coagulation and the generation of thrombin at sites of platelet thrombus formation.

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Endotoxin enhances microvascular thrombosis in mouse cremaster venules via a TLR4-dependent, neutrophil-independent mechanism

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00305.2005 ◽

2006 ◽

Vol 290 (4) ◽

pp. H1671-H1679 ◽

Cited By ~ 48

Author(s):

Rolando E. Rumbaut ◽

Ricardo V. Bellera ◽

Jaspreet K. Randhawa ◽

Corie N. Shrimpton ◽

Swapan K. Dasgupta ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Adhesion ◽

Ex Vivo ◽

Thrombus Formation ◽

Mouse Strains ◽

Microvascular Endothelium ◽

Microvascular Thrombosis ◽

Platelet Thrombus ◽

Adhesive Interactions

Endotoxemia promotes adhesive interactions between platelets and microvascular endothelium in vivo. We sought to determine whether endotoxin (lipopolysaccharide, LPS) modified platelet thrombus formation in mouse cremaster venules and whether Toll-like receptor 4 (TLR4) and neutrophils were involved in the response. Intravital videomicroscopy was performed in the cremaster microcirculation of pentobarbital-anesthetized mice; venular platelet thrombi were induced with a light/dye endothelial injury model. C57BL/6 mice treated with Escherichia coli endotoxin had enhanced rates of venular platelet thrombus formation: the time to microvessel occlusion was reduced by ∼50% ( P < 0.005) compared with saline-treated animals. Enhanced microvascular thrombosis was evident as early as 2 h after LPS administration. LPS had no effect on thrombosis in either of two mouse strains with altered TLR4 signaling (C57BL/10ScNJ or C3H/HeJ), whereas it enhanced thrombosis in the control strains (C57BL/10J and C3H/HeN). LPS also enhanced platelet adhesion to endothelium in the absence of light/dye injury. Platelet adhesion, but not enhanced thrombosis, was inhibited by depletion of circulating neutrophils. LPS failed to enhance platelet aggregation ex vivo and did not influence platelet P-selectin expression, a marker of platelet activation. These findings support the notion that endotoxemia promotes platelet thrombus formation independent of neutrophils and without enhancement of platelet aggregation, via a TLR4-dependent mechanism.

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Identification of novel downstream targets of platelet glycoprotein VI activation by differential proteome analysis: implications for thrombus formation

Blood ◽

10.1182/blood-2009-07-230268 ◽

2010 ◽

Vol 115 (20) ◽

pp. 4102-4110 ◽

Cited By ~ 46

Author(s):

Christian Schulz ◽

Nina V. Leuschen ◽

Thomas Fröhlich ◽

Michael Lorenz ◽

Susanne Pfeiler ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Membrane Trafficking ◽

Thrombus Formation ◽

Multivesicular Body ◽

Platelet Glycoprotein ◽

Body Protein ◽

Glycoprotein Vi ◽

Differentially Abundant Proteins ◽

Platelet Proteins

Abstract Platelets play a key role in hemostasis and various diseases including arterial thrombosis. Glycoprotein VI (GPVI) mediates adhesion to collagen structures exposed at sites of vascular injury and subsequent platelet activation. We determined the effects of specific activation of GPVI on the human platelet proteome. Isolated human platelets were stimulated with an activating monoclonal antibody specific for GPVI. Platelet proteins were analyzed by 2-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry. We identified 8 differentially abundant proteins associated with cell signaling, metabolism, organization and rearrangement of the cytoskeleton, and membrane trafficking. Differentially abundant proteins included aldose reductase (AR), beta-centractin, charged multivesicular body protein 3, Src substrate cortactin, ERp57, and pleckstrin. Importantly, GPVI-modulated protein abundance was functionally relevant. Correspondingly, AR enzyme activity significantly increased upon GPVI activation and inhibition of AR resulted in reduced platelet aggregation. Furthermore, ERp57 was released upon ligation of platelet GPVI and increased the activity of tissue factor, a major initiator of blood coagulation. In summary, GPVI activation results in differential changes in abundance of platelet proteins, including AR and ERp57, which support platelet aggregation and platelet-dependent coagulation. These results provide further insight into the mechanisms that underlie platelet activation through the GPVI receptor and may help to identify novel pharmacologic targets.

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Effect of Shear Rate on Thrombus Growth

ASME 2010 8th International Conference on Nanochannels, Microchannels, and Minichannels: Parts A and B ◽

10.1115/fedsm-icnmm2010-31090 ◽

2010 ◽

Author(s):

Elham Tolouei ◽

Andreas Fouras ◽

Josie Carberry

Keyword(s):

Platelet Aggregation ◽

Shear Rate ◽

Blood Clotting ◽

Thrombus Formation ◽

Measurement Techniques ◽

Clotting Factors ◽

Critical Height ◽

Local Shear ◽

Blood Clotting Factors

In this study micro and nano scale measurement techniques are applied to platelet studies and determination of factors in platelet aggregation and thrombus formation. Conventionally it has been assumed that platelets are stimulated by blood clotting factors and platelet activators to aggregate and form a thrombus at sites of vascular injury. We have recently shown that a primary factor in initiating platelet aggregation is hemodynamic shear. This paper presents the effect of shear rate on the time evolution of thrombus formation and the final geometry of a mature thrombus. A relationship between maximum mature thrombus height and local shear rate is formulated. We have shown that the shear rate is not only an important factor in initiating platelet aggregation but is also one of the main inhibitors of platelet aggregation and thrombus formation. We propose that when the platelets reach a critical height, they encounter a specific local hemodynamic range, which prevents further thrombus growth.

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Effects Of Hemostatic Agents On Platelet Aggregation

10.1055/s-0038-1652366 ◽

1981 ◽

Author(s):

P N Sawyer ◽

G Schwann ◽

B Stanczewski ◽

M T Jones

Keyword(s):

Platelet Aggregation ◽

Blood Clotting ◽

Thrombus Formation ◽

Coagulation System ◽

Thrombin Time ◽

Hemostatic Agents ◽

Adp Release ◽

Enzymatic Mechanisms ◽

Derived Data ◽

Primary Platelet

Several new collagen derived hemostatic agents have been introduced into the surgical armamentarium. The presumption is they produce platelet aggregation, ADP release and blood clotting through the intrinsic system.Studies of interreaction of these new hemostatic agents with both blood and platelets suggests that the type of reaction with whole blood is not known or due to primary platelet activation. Avitene interreacts in part by platelet aggregation but also by interreacting with other components in blood including fibrinogen.The available evidence with a new dissolving collagenous hemostat, “Superstat”™ indicates probable activation of the extrinsic coagulation system, directly converting prothrombin to thrombin, and fibrinogen to fibrin. Thrombin time ratios (experimental vs. control) are Superstat 1% - 0.81 ± 0.16/0.93 ± 0.06, Superstat 2% - 0.84 ± 0.11/0.93 ± 0.06, Superstat 3% - 0.61 ± 0.15/0.71 ± 0.02. Comparative studies have been completed on several other agents, among them, Kollagen Haemo. Vlies - 0.66 ± 0.1/0.71 ± 0.02, Gelfoam - 0.75 ± 0.15/0.71 ± 0.02, Surgicel - 0.73 ± 0.4/ 0.71 ± 0.02, Avitene - 0.75 ± 0.3/0.71 ± 0.02, Collatamp - 0.73 ± 0.4/0.71 ± 0.02. Derived data from thrombin times, clotting times and, when possible, platelet aggregation displays widely different rates of thrombus formation. We believe the enzymatic mechanisms for these reactions are currently only partially known.The fact that clinically useful hemostatic agents each appear to produce hemostasis by several routes, and do not all use the same route, is of extreme interest in basic thrombosis studies as well as in clinical surgery.

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Synergism between platelet collagen receptors defined using receptor-specific collagen-mimetic peptide substrata in flowing blood

Blood ◽

10.1182/blood-2010-01-260778 ◽

2010 ◽

Vol 115 (24) ◽

pp. 5069-5079 ◽

Cited By ~ 79

Author(s):

Nicholas Pugh ◽

Anna M. C. Simpson ◽

Peter A. Smethurst ◽

Philip G. de Groot ◽

Nicolas Raynal ◽

...

Keyword(s):

Thrombus Formation ◽

Confocal Imaging ◽

Peptide Substrates ◽

Shear Rates ◽

Glycoprotein Vi ◽

Collagen Receptors ◽

Platelet Binding ◽

Von Willebrand ◽

Willebrand Factor

AbstractExposed subendothelial collagen acts as a substrate for platelet adhesion and thrombus formation after vascular injury. Synthetic collagen-derived triple-helical peptides, designated collagen-related peptide (CRP), GFOGER, and VWF-III, can specifically engage the platelet collagen receptors, glycoprotein VI and integrin α2β1, and plasma von Willebrand factor (VWF), respectively. Hitherto, the role of these 3 collagen-binding axes has been studied indirectly. Use of these uniform peptide substrates, rather than collagen fibers, provides independent control of each axis. Here, we use confocal imaging and novel image analysis techniques to investigate the effects of receptor-ligand engagement on platelet binding and activation during thrombus formation under flow conditions. At low shear (100s−1 and 300s−1), both GFOGER and CRP are required for thrombus formation. At 1000s−1, a combination of either CRP or GFOGER with VWF-III induces comparable thrombus formation, and VWF-III increases thrombus deposition at all shear rates, being indispensable at 3000s−1. A combination of CRP and VWF-III is sufficient to support extensive platelet deposition at 3000s−1, with slight additional effect of GFOGER. Measurement of thrombus height after specific receptor blockade or use of altered proportions of peptides indicates a signaling rather than adhesive role for glycoprotein VI, and primarily adhesive roles for both α2β1 and the VWF axis.

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