GP-VI Disruption Confers Additional Protection against Arterial Thrombosis in PAR-4 Deficient Mice

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3932-3932
Author(s):  
Ivo Cornelissen ◽  
Erica De Candia ◽  
Shaun R Coughlin
Keyword(s):  
Thrombus Formation ◽  
Thrombin Inhibitor ◽  
Thrombosis Model ◽  
Abdominal Bleeding ◽  
Additional Protection ◽  
Platelet Signaling ◽  
Thrombin Activation ◽  
Platelet Thrombus ◽  
Activation Pathways ◽  
Adhesive Proteins

Abstract Following vascular injury, platelets are recruited and activated by adhesive proteins exposed on the subendothelium (including collagen), released agonists and locally generated thrombin. In mice, protease activated receptor 4 (PAR-4) mediates the platelet signaling response to thrombin. Previous studies showed that platelets from PAR-4 null mice do not respond to thrombin and while platelet thrombi do form at the site of injury in PAR-4 deficient animals, they do not propagate and extend into the lumen like thrombi in wild-type mice. The activation pathway responsible for this juxtamural platelet accumulation remains unknown. The collagen exposed on the subendothelium activates and recruites platelets at the site of injury by interacting with membrane glycoprotein (GP) VI. Therefore, the collagen-GP-VI pathway is a putative candidate for the platelet thrombus formation near the vessel wall. Using hirudin as a thrombin inhibitor in mice with disrupted GP-VI expression, it has been previously shown that GP-VI signaling and thrombin activation pathways may cooperate during thrombus growth. To more precisely evaluate the interplay between the thrombin/PAR-4 and collagen/GP-VI signaling pathways, we crossed mice lacking PAR-4 and GP-VI, and subsequently compared bleeding time and arterial thrombus formation in PAR-4:GP-VI double or single deficient animals. All genetic combinations were born at the expected mendelian distribution. Double deficient females carried offspring to term and no bleeding was observed during parturition. Pups lacking both receptors demonstrated transient perinatal abdominal bleeding which resolved rapidly. However, we observed prolonged tail bleeding times in double deficient animals compared to their single deficient littermates. Furthermore in a FeCl3-induced carotid thrombosis model, mice lacking both receptors were completely protected compared to their littermates. This study suggests that there is an interaction between the thrombin and collagen activating pathways in the setting of hemostasis and thrombosis. Figure Figure Figure Figure

Absence of Fibrinogen in Afibrinogenemia Results in Large but Loosely Packed Thrombi under Flow Conditions

2001 ◽  
Vol 85 (04) ◽  
pp. 736-742 ◽  
Author(s):  
Ya-Ping Wu ◽  
Martin IJsseldijk ◽  
Jaap Zwaginga ◽  
Jan Sixma ◽  
Philip de Groot ◽  
...  
Keyword(s):  
Morphological Analysis ◽  
Surface Coverage ◽  
Thrombus Formation ◽  
Flow Conditions ◽  
Thrombotic Complications ◽  
Platelet Thrombus ◽  
The Difference ◽  
Adhesive Proteins ◽  
Lamellipodia Formation

SummaryWe studied the role of fibrinogen in platelet thrombus formation under flow on adhesive proteins using afibrinogenemic blood (LMWH anticoagulated) in a perfusion system. Perfusions with afibrinogenemic blood showed strong increased surface coverage and thrombus volume that normalized upon addition of fibrinogen. Similar studies using citrate anticoagulated blood showed that this was due to fibrinogen and not fibrin. Morphological analysis showed that afibrinogenemic thrombi were loosely packed and consisted mainly of dendritic platelets that contacted one another through filopodia. However, in the presence of fibrinogen, platelets formed lamellipodia and spread out on top of one another. Studies with radiolabeled platelets showed similar numbers of platelets in both conditions demonstrating that the difference is one of packing and the larger size is due to absence of lamellipodia formation and spreading. The found increased thrombus size and loosely packed platelets might help to understand thrombotic complications sometimes seen in afibrinogenemia patients.

scholarly journals A novel nucleotide-based thrombin inhibitor inhibits clot-bound thrombin and reduces arterial platelet thrombus formation

Blood ◽  
1994 ◽  
Vol 83 (3) ◽  
pp. 677-682 ◽  
Author(s):  
WX Li ◽  
AV Kaplan ◽  
GW Grant ◽  
JJ Toole ◽  
LL Leung
Keyword(s):  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Shear Rates ◽  
High Shear Rates ◽  
Dependent Inhibition ◽  
Platelet Thrombus

A novel thrombin inhibitor based on single-stranded (ss) deoxynucleotides with the sequence GGTTGGTGTGGTTGG (thrombin aptamer) has been recently discovered. In this study, we tested its efficacy in inhibiting clot-bound thrombin activity and platelet thrombus formation in an ex vivo whole artery angioplasty model. The thrombin aptamer showed a specific dose-dependent inhibition of thrombin-induced platelet aggregation (0.5 U/mL) in human platelet-rich plasma, with an IC50 of approximately 70 to 80 nmol/L. In an in vitro clot-bound thrombin assay system, heparin, used at clinically relevant concentrations of 0.2 U/mL and 0.4 U/mL, was ineffective in inhibiting clot-bound thrombin (6.5% and 34.9% inhibition at 0.2 U/mL and 0.4 U/mL, respectively). In contrast, the thrombin aptamer at an equivalent anticoagulant concentration inhibited clot-bound thrombin (79.7% inhibition). In an ex vivo whole artery angioplasty model, the thrombin aptamer markedly suppressed the generation of fibrinopeptide A (FPA), whereas heparin at 2 U/mL was ineffective. Compared with a scrambled ssDNA control, the thrombin aptamer reduced platelet deposition by 34.5% +/- 5% (mean +/- SEM, n = 4, P = .09) at low shear rates (approximately 200 s-1) and 61.3% +/- 11% (mean +/- SEM, n = 4, P = .05) at high shear rates (approximately 850 s-1). Thrombin aptamers based on ssDNA molecules represent a new class of thrombin inhibitors with potent anticoagulant and antithrombotic properties.

scholarly journals The P2Y12 Receptor Antagonist Selatogrel Dissolves Preformed Platelet Thrombi In Vivo

2021 ◽  
Vol 10 (22) ◽  
pp. 5349
Author(s):  
Lydie Crescence ◽  
Markus Kramberg ◽  
Martine Baumann ◽  
Markus Rey ◽  
Sebastien Roux ◽  
...  
Keyword(s):  
Blood Flow ◽  
Guinea Pigs ◽  
Thrombus Formation ◽  
P2y12 Receptor ◽  
Early Application ◽  
Acute Thrombosis ◽  
Thrombosis Model ◽  
Platelet Thrombus ◽  
Platelet Thrombi

Selatogrel, a potent and reversible antagonist of the P2Y12 receptor, inhibited FeCl3-induced thrombosis in rats. Here, we report the anti-thrombotic effect of selatogrel after subcutaneous applications in guinea pigs and mice. Selatogrel inhibited platelet function only 10 min after subcutaneous application in mice. In addition, in a modified Folts thrombosis model in guinea pigs, selatogrel prevented a decrease in blood-flow, indicative of the inhibition of ongoing thrombosis, approximately 10 min after subcutaneous injection. Selatogrel fully normalised blood flow; therefore, we speculate that it may not only prevent, but also dissolve, platelet thrombi. Thrombus dissolution was investigated using real-time intravital microscopy in mice. The infusion of selatogrel during ongoing platelet thrombus formation stopped growth and induced the dissolution of the preformed platelet thrombus. In addition, platelet-rich thrombi were given 30 min to consolidate in vivo. The infusion of selatogrel dissolved the preformed and consolidated platelet thrombi. Dissolution was limited to the disintegration of the occluding part of the platelet thrombi, leaving small mural platelet aggregates to seal the blood vessel. Therefore, our experiments uncovered a novel advantage of selatogrel: the dissolution of pre-formed thrombi without the disintegration of haemostatic seals, suggesting a bipartite benefit of the early application of selatogrel in patients with acute thrombosis.

Synergistic Antithrombotic Properties of G4120, a RGD-Containing Synthetic Peptide, and Argatroban, a Synthetic Thrombin Inhibitor, in a Hamster Femoral Vein Platelet-Rich Thrombosis Model

1992 ◽  
Vol 68 (03) ◽  
pp. 336-340 ◽  
Author(s):  
Yoshimi Imura ◽  
Jean-Marie Stassen ◽  
Ingrid Vreys ◽  
Emmanuel Lesaffre ◽  
Herman K Gold ◽  
...  
Keyword(s):  
Confidence Interval ◽  
Thrombus Formation ◽  
Femoral Vein ◽  
Arterial Occlusion ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Rgd Peptides ◽  
Thrombosis Model ◽  
Dependent Inhibition ◽  
Dose Dependent

SummaryThe synergistic antithrombotic properties of G4120, a synthetic Arg-Gly-Asp (RGD) containing peptide which strongly inhibits platelet aggregation, and of Argatroban, a synthetic thrombin inhibitor, were examined in a reproducible quantitative hamster femoral vein platelet-rich mural thrombosis model. Bolus injections of G4120 and Argatroban inhibit thrombus formation in a dose-dependent way; 50% inhibition (ID50) is obtained with 11 µg/kg G4120 and with 2 mg/kg Argatroban. Combined bolus injections of 3 µg/kg G4120 with 0.5, 0.75 or 1 mg/kg Argatroban and of 1 mg/kg Argatroban with 1.5 or 3 µg/kg G4120 caused linear dose-dependent inhibition of thrombus formation, whereas 3 µg/kg G4120 or 1 mg/kg Argatroban alone had very little effect (<20% inhibition). ID50 was obtained with the combination of 3 µg/kg G4120 and 0.5 mg/kg Argatroban, corresponding to an equi-effective fractional combination of 0.62 with a 95% confidence interval of 0.50 to 0.74. Alternatively the ID50 was obtained with the combination of 1 mg/kg Argatroban and 1.3 µg/kg G4120, corresponding to an equi-effective fractional combination of 0.52 with a 95% confidence interval of 0.18 to 0.86. In both instances these results are indicative of a significant synergistic interaction. Bolus injection of 10 mg/kg aspirin, 100 U/kg heparin or the combination did not inhibit thrombus formation.The synergistic effect of the combination of platelet inhibiting RGD-peptides and synthetic thrombin inhibitors could be useful in the prevention of arterial occlusion with platelet-rich thrombus in patients with ischemic heart disease following thrombolytic therapy or angioplasty, although this combination is not expected to reverse platelet thrombus formation.

scholarly journals A novel nucleotide-based thrombin inhibitor inhibits clot-bound thrombin and reduces arterial platelet thrombus formation

Blood ◽  
1994 ◽  
Vol 83 (3) ◽  
pp. 677-682 ◽  
Author(s):  
WX Li ◽  
AV Kaplan ◽  
GW Grant ◽  
JJ Toole ◽  
LL Leung
Keyword(s):  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Shear Rates ◽  
High Shear Rates ◽  
Dependent Inhibition ◽  
Platelet Thrombus

Abstract A novel thrombin inhibitor based on single-stranded (ss) deoxynucleotides with the sequence GGTTGGTGTGGTTGG (thrombin aptamer) has been recently discovered. In this study, we tested its efficacy in inhibiting clot-bound thrombin activity and platelet thrombus formation in an ex vivo whole artery angioplasty model. The thrombin aptamer showed a specific dose-dependent inhibition of thrombin-induced platelet aggregation (0.5 U/mL) in human platelet-rich plasma, with an IC50 of approximately 70 to 80 nmol/L. In an in vitro clot-bound thrombin assay system, heparin, used at clinically relevant concentrations of 0.2 U/mL and 0.4 U/mL, was ineffective in inhibiting clot-bound thrombin (6.5% and 34.9% inhibition at 0.2 U/mL and 0.4 U/mL, respectively). In contrast, the thrombin aptamer at an equivalent anticoagulant concentration inhibited clot-bound thrombin (79.7% inhibition). In an ex vivo whole artery angioplasty model, the thrombin aptamer markedly suppressed the generation of fibrinopeptide A (FPA), whereas heparin at 2 U/mL was ineffective. Compared with a scrambled ssDNA control, the thrombin aptamer reduced platelet deposition by 34.5% +/- 5% (mean +/- SEM, n = 4, P = .09) at low shear rates (approximately 200 s-1) and 61.3% +/- 11% (mean +/- SEM, n = 4, P = .05) at high shear rates (approximately 850 s-1). Thrombin aptamers based on ssDNA molecules represent a new class of thrombin inhibitors with potent anticoagulant and antithrombotic properties.

Clues for Understanding the Structure and Function of a Prototypic Human Integrin: The Platelet Glycoprotein IIb/IIIa Complex

1994 ◽  
Vol 72 (01) ◽  
pp. 001-015 ◽  
Author(s):  
Juan J Calvete
Keyword(s):  
Structure Function ◽  
Thrombus Formation ◽  
Platelet Glycoprotein ◽  
Primary Role ◽  
Clot Retraction ◽  
Inhibitory Peptide ◽  
Rgd Sequence ◽  
Platelet Receptor ◽  
Adhesive Proteins ◽  
And Function

SummaryThe glycoprotein (GP) IIb/IIIa, a Ca2+-dependent heterodimer, is the major integrin on the platelet plasma membrane. On resting platelets GPIIb/IIIa is maintained in an inactive conformation and serves as a low affinity adhesion receptor for surface-coated fibrinogen, whereas upon platelet activation signals within the cytoplasma alter the receptor function of GPIIb/IIIa (inside-out signalling), which undergoes a measurable conformational change within its exoplasmic domains, and becomes a competent receptor for soluble fibrinogen and some other RGD sequence-containing plasma adhesive proteins. Upon ligand binding, further structural alterations trigger the association of receptor-occupied GPIIb/IIIa complexes with themselves within the plane of the membrane. The simultaneous binding of dimeric fibrinogen molecules to GPIIb/IIIa clusters on adjacent platelets leads to platelet aggregation, which promotes attachment of fibrinogen-GPIIb/IIIa clusters to the cytoskeleton (outside-in signalling). This, in turn, provides the necessary physical link for clot retraction to occur, and generates a cascade of intracellular biochemical reactions which result in the formation of a multiprotein signalling complex at the cytoplasmic domains of GPIIb/IIIa. Glycoprotein IMIIa, also called αIIbβ3 in the integrin nomenclature, plays thus a primary role in both platelet adhesion and thrombus formation at the site of vascular injury. In addition, the human glycoprotein Ilb/IIIa complex is the most thoroughly studied integrin receptor, its molecular biology and major features of its primary structure having been elucidated mainly during the last six years. Furthermore, localization of functionally relevant monoclonal antibody epitopes, determination of the cross-linking sites of inhibitory peptide ligands, proteolytic dissection of the isolated integrin, and analysis of natural and artificial GPIIb/IIIa mutants have recently provided a wealth of information regarding structure-function relationships of human GPIIb/IIIa. The aim of this review is to summarize these many structural and functional data in the perspective of an emerging model. Although most of the interpretations based on structural elements of this initial biochemical model require independent confirmation, they may help us to understand the structure-function relationship of this major platelet receptor, and of other members of the integrin superfamily, as well as to perform further investigations in order to test current hypotheses.

An Electron Microscopic Study of Platelet Thrombus Formation in the Rabbit with Particular Regard to 5-Hydroxytryptamine Release

1967 ◽  
Vol 18 (03/04) ◽  
pp. 592-604 ◽  
Author(s):  
H. R Baumgartner ◽  
J. P Tranzer ◽  
A Studer
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Vessel Wall ◽  
Thrombus Formation ◽  
Endothelial Damage ◽  
Electron Microscopic ◽  
Rabbit Ear ◽  
Basement Lamina ◽  
Platelet Thrombus

SummaryElectron microscopic and histologic examination of rabbit ear vein segments 4 and 30 min after slight endothelial damage have yielded the following findings :1. Platelets do not adhere to damaged endothelial cells.2. If the vessel wall is denuded of the whole endothelial cell, platelets adhere to the intimai basement lamina as do endothelial cells.3. The distance between adherent platelets as well as endothelial cells and intimai basement lamina measures 10 to 20 mµ, whereas the distance between aggregated platelets is 30 to 60 mµ.4. 5-hydroxytryptamine (5-HT) is released from platelets during viscous metamorphosis at least in part as 5-HT organelles.It should be noted that the presence of collagen fibers is not necessary for platelet thrombus formation in vivo.

Antithrombotic Effect of Two Low Molecular Weight Thrombin Inhibitors and a Low-Molecular Weight Heparin in a Caval Vein Thrombosis Model in the Rat

1997 ◽  
Vol 78 (05) ◽  
pp. 1404-1407 ◽  
Author(s):  
B I Eriksson ◽  
S Carlsson ◽  
M Halvarsson ◽  
B Risberg ◽  
C Mattsson
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Clinical Situation ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Low Molecular Weight ◽  
Surgical Trauma ◽  
Antithrombotic Effect ◽  
Caval Vein ◽  
Thrombosis Model

SummaryA sensitive thrombosis model with a high reproducibility was developed in the rat, utilizing stasis of the caval vein and a standardized surgical trauma as the only thrombogenic stimuli. Since no procoagulant substances were used, the results of the present study might be relevant in a clinical situation. The antithrombotic effect of two recently synthesized low-molecular-weight thrombin inhibitors have been compared to dalteparin, (Fragmin) a low-molecular-weight heparin fragment. Each compound was studied at 8 different doses with 10 rats in each group. On a gravimetric basis, the thrombin inhibitor melagatran was twice as potent as dalteparin (ED50 16 and 33 µ/kg per h, respectively). The second thrombin inhibitor, inogatran, had an intermediate effect, with an ED50 of 24 µLg/kg per h. No differences in antithrombotic effect were, however, found when the compounds were compared at anticoagulant equivalent doses (same APTT prolongation). A 50% reduction in the mean thrombus weight was obtained when APTT was prolonged to 1.2 to 1.3 times the pretreatment value.

The Effect of Agents which Modify Platelet Behaviour and of Magnesium Ions on Thrombus Formation In Vivo

1979 ◽  
Vol 42 (02) ◽  
pp. 603-610 ◽  
Author(s):  
J H Adams ◽  
J R A Mitchell
Keyword(s):  
Thrombus Formation ◽  
Cerebral Arteries ◽  
Magnesium Ions ◽  
Body Formation ◽  
Inflammatory Agent ◽  
Platelet Thrombus ◽  
Magnesium Salts ◽  
Higher Doses ◽  
Do So

SummaryThe ability of potential anti-thrombotic agents to modify platelet-thrombus formation in injured cerebral arteries in the rabbit was tested. Low doses of heparin were without effect, while higher doses produced variable suppression of white body formation but at the expense of bleeding. Aspirin did not inhibit white body formation but another non-steroid anti-inflammatory agent, flurbiprofen was able to do so, as was the anti-gout agent, sulphinpyrazone. Magnesium salts both topically and parenterally, suppressed thrombus formation and increased the concentration of ADP which was required to initiate thrombus production at minor injury sites.

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