FIBRONECTIN IS REQUIRED FOR PLATELET ADHESION AND FOR THROMBUS FORMATION ON SUBENDOTHELIUM AND COLLAGEN SURFACES

1987 ◽  
Author(s):  
E Bastida ◽  
G Escolar ◽  
R Castillo ◽  
A Ordinas ◽  
J J Sixma
Keyword(s):  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Fibrillar Collagen ◽  
Flow Conditions ◽  
Shear Rates ◽  
Human Collagen ◽  
Platelet Thrombus ◽  
Computerized System ◽  
Total Coverage

Fibronectin (FN) plays a role in several adhesion mediated functions including the interaction of platelets with subendothelium.We investigated the role of plasma FN in platelet adhesion and platelet thrombus formation under flow conditions.To do this we used two different perfusion models:1)the annular chamber with α -chymotrypsin-treated rabbit vessel segments and 2)the flat chamber with coverslips coated with fibrillar purified human collagen type III.Perfusates consisted of washed platelets, and washed red blood celIs,suspended in normal or FN-depleted plasma.Perfusions were carried out for 10 min at shear rates of 300 or 1300 sec™1 Platelet deposition and thrombus dimensions were morphometrically evaluated by a computerized system. We found that depletion of plasma FN significantly reduced the percentage of total coverage surface and percentage of platelet thrombus, at both shear rates studied, and in both perfusion systems (p < 0.01)(p < 0.01).The dimensions of the platelet thrombi formed in perfusions at high shear rate were also significantly reduced in perfusions carried out with FN-depleted plasma.(p < 0.01). Addition of purified FN to FN-depleted perfusates restored all the values to those measured in the control perfusions.These results indicate that, in addition to supporting platelet adhesion to the subendothelium and to fibrillar collagen, FN contributes to platelet thrombus formation under flow conditions.

scholarly journals Fibronectin is required for platelet adhesion and for thrombus formation on subendothelium and collagen surfaces

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1437-1442 ◽  
Author(s):  
E Bastida ◽  
G Escolar ◽  
A Ordinas ◽  
JJ Sixma
Keyword(s):  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Flow Conditions ◽  
Shear Rates ◽  
Platelet Aggregate ◽  
Human Collagen ◽  
Platelet Thrombus ◽  
Computerized System ◽  
Total Coverage

Abstract Fibronectin (FN) plays a role in several adhesion mediated functions including the interaction of platelets with subendothelium. We investigated the role of plasma FN in platelet adhesion and platelet thrombus formation under flow conditions. We used two different perfusion models: the annular chamber with alpha-chymotrypsin-treated rabbit vessel segments, and the flat chamber with coverslips coated with fibrillar purified human collagen type III. Perfusates consisted of washed platelets and washed RBCs, suspended in normal or FN-depleted plasma. Perfusions were carried out for ten minutes at shear rates of 300 or 1,300 s-1. Platelet deposition and thrombus dimensions were evaluated morphometrically by a computerized system. We found that depletion of plasma fibronectin significantly reduced the percentage of total coverage surface and percentage of platelet thrombus, at both shear rates studied, and in both perfusion systems (P less than .01) (P less than .01). The dimensions of the platelet thrombi formed in perfusions at high shear rate were also significantly reduced in perfusions carried out with FN depleted plasma (P less than .01). Addition of purified FN to FN-depleted perfusates restored all values to those measured in the control perfusions. These results indicate that plasma FN is required for platelet aggregate and thrombus formation following adhesion under flow conditions.

scholarly journals Fibronectin is required for platelet adhesion and for thrombus formation on subendothelium and collagen surfaces

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1437-1442
Author(s):  
E Bastida ◽  
G Escolar ◽  
A Ordinas ◽  
JJ Sixma
Keyword(s):  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Flow Conditions ◽  
Shear Rates ◽  
Platelet Aggregate ◽  
Human Collagen ◽  
Platelet Thrombus ◽  
Computerized System ◽  
Total Coverage

Fibronectin (FN) plays a role in several adhesion mediated functions including the interaction of platelets with subendothelium. We investigated the role of plasma FN in platelet adhesion and platelet thrombus formation under flow conditions. We used two different perfusion models: the annular chamber with alpha-chymotrypsin-treated rabbit vessel segments, and the flat chamber with coverslips coated with fibrillar purified human collagen type III. Perfusates consisted of washed platelets and washed RBCs, suspended in normal or FN-depleted plasma. Perfusions were carried out for ten minutes at shear rates of 300 or 1,300 s-1. Platelet deposition and thrombus dimensions were evaluated morphometrically by a computerized system. We found that depletion of plasma fibronectin significantly reduced the percentage of total coverage surface and percentage of platelet thrombus, at both shear rates studied, and in both perfusion systems (P less than .01) (P less than .01). The dimensions of the platelet thrombi formed in perfusions at high shear rate were also significantly reduced in perfusions carried out with FN depleted plasma (P less than .01). Addition of purified FN to FN-depleted perfusates restored all values to those measured in the control perfusions. These results indicate that plasma FN is required for platelet aggregate and thrombus formation following adhesion under flow conditions.

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.

Revised Model for Platelet Adhesion to Collagen.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2999-2999
Author(s):  
Lucia Stefanini ◽  
Moritz Stolla ◽  
Sean F Maloney ◽  
Timothy Daniel Ouellette ◽  
Claire Roden ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Mapk Signaling ◽  
Low Flow ◽  
Flow Conditions ◽  
Integrin Activation ◽  
Shear Rates ◽  
Slow Kinetics ◽  
Rap1 Activation

Abstract Abstract 2999 Poster Board II-968 The Gi-coupled ADP receptor, P2Y12, is the target of clopidogrel bisulfate (Plavix), currently the most successful anti-platelet strategy used in the clinic. In a recent study, we have shown that the Ca2+-sensing nucleotide exchange factor, CalDAG-GEFI, and P2Y12 represent the major signaling pathways leading to Rap1 and integrin activation in platelets (Cifuni et al., 2008, Blood). In the present study, we have further evaluated the importance of CalDAG-GEFI signaling and Rap1 activation for various aspects of platelet activation, and we have compared thrombus formation of CalDAG-GEFI−/− and WT/clopidogrel platelets under static and flow conditions in vitro. Our studies establish a revised model for platelet activation by collagen. In platelets activated with threshold concentrations of GPVI agonists, CalDAG-GEFI serves as a highly sensitive response element to Ca2+ that allows for the rapid activation of Rap1. CalDAG-GEFI-mediated Rap1 activation triggers a first wave of integrin activation and ERK (MAPK) signaling, followed by TxA2 release. TxA2 provides crucial feedback for the activation of PKC and granule/ADP release. ADP in turn triggers the second, P2Y12-dependent wave of Rap1-mediated signaling events, leading to the sustained activation of integrins and further release of TxA2. Higher concentrations of GPVI agonists lead to the concomitant activation of CalDAG-GEFI and PKC, facilitating platelet aggregation independent of feedback by endogenous TxA2. Under physiological flow conditions, CalDAG-GEFI-dependent platelet activation (clopidogrel-treated WT platelets) allowed for the formation of small but unstable thrombi, which rapidly disintegrated at high shear rates. In contrast, CalDAG-GEFI−/− platelets (P2Y12-dependent platelet activation) in anticoagulated blood firmly adhered to the thrombogenic surface but failed to form thrombi, even at high concentrations of collagen. Addition of exogenous TxA2 to anticoagulated CalDAG-GEFI−/− blood did not restore thrombus formation under flow. However, small thrombi were observed with non-anticoagulated CalDAG-GEFI−/− blood perfused at venous but not arterial shear rates, suggesting that a) locally generated thrombin facilitates the recruitment of free flowing CalDAG-GEFI−/− platelets to already adherent platelets, and b) the slow kinetics of P2Y12-dependent Rap1 activation only supports thrombin-induced platelet-platelet cohesion at low shear conditions. In conclusion, our studies demonstrate that CalDAG-GEFI/Rap1 signaling plays a critical role for the first wave of integrin activation and TxA2 generation important for platelet adhesion to a thrombogenic surface. Signaling by P2Y12/Rap1 is essential for sustained platelet activation/thrombus stabilization and partially compensates for CalDAG-GEFI/Rap1-mediated platelet adhesion under low flow conditions. Disclosures: No relevant conflicts of interest to declare.

CRGT Peptide In Cytoplasm Regulates Thrombus Formation Via Dissociating c-Src-β3 Interaction

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3501-3501
Author(s):  
Jiansong Huang ◽  
Xiaofeng Shi ◽  
Wenda Xi ◽  
Ping Liu ◽  
Xiaodong Xi
Keyword(s):  
Molecular Mechanisms ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Flow Conditions ◽  
Cho Cell ◽  
Integrin Αiibβ3 ◽  
Shear Rates ◽  
Platelet Adhesion And Aggregation

Abstract The RGT sequences of the integrin β3 tail directly and constitutively bind the inactive c-Src, regulating integrin αIIbβ3 signaling and platelet function. Previous work has shown that disrupting the interaction of c-Src with β3 via myristoylated RGT peptide or deletion of the RGT sequences in β3 selectively inhibits integrin αIIbβ3 outside-in signaling in platelets. However, the precise molecular mechanisms by which the Src-β3 association regulates integrin αIIbβ3 signaling need to be clarified. We found that active c-Src phosphoylated the Y747 and Y759 residues of β3 directly at the in vitro protein/protein level or in CHO cell models bearing Tac-β3 chimeras, which were devoid of the intact β3 signal transduction. Furthermore, data from mass spectrometry, [γ-32P] ATP incorporation assays and CHO cell/Tac-β3 chimeras demonstrated that the direct phosphorylation of Y747 and Y759 by active c-Src did not depend on the binding of c-Src to the RGT sequences of the β3 tail. To further investigate the biological functions of Src-β3 association in signal transduction we employed a cell-permeable and reduction-sensitive peptide (myr-AC∼CRGT), which disrupted the Src-β3 association in platelets independent of membrane-anchorage, and found that when platelets were stimulated by thrombin the c-Src activation and the phosphorylation of the tyrosine residues of the β3 tail were substantially inhibited by the presence of the peptide. These results suggest that one of the crucial biological functions of Src-β3 association is to serve as a “bridge” linking integrin signaling with the c-Src full activation and phosphorylation of the tyrosines of the β3 tail. To answer whether the RGT peptide binding to Src is able to alter the enzymatic activity of c-Src, we examined the Src-Csk association, the phosphorylation status of Y416 and Y527 of c-Src and the c-Src kinase catalytic activity. Results showed that myr-AC∼CRGT did not dissociate Csk from c-Src in resting platelets and the phosphorylation level of Y416 and Y527 of c-Src remained unaltered. Consistent data were also obtained from in vitro analysis of the c-Src kinase catalytic activity in the presence of CRGT peptide. These results suggest that myr-AC∼CRGT peptide per se does not fully activate c-Src. Myr-AC∼CRGT was also found to inhibit integrin αIIbβ3 outside-in signaling in human platelets. To examine the effect of the myr-AC∼CRGT on platelet adhesion and aggregation under flow conditions, we measured the platelet thrombus formation under different shear rates. Myr-AC∼CRGT did not affect the platelet adhesion at a wall shear rate of 125 s-1. The inability of myr-AC∼CRGT to affect platelet adhesion and aggregation remained at 500 s-1 shear rates. At 1,500 s-1, or 5,000 s-1 rates, myr-AC∼CRGT partially inhibited platelet adhesion and aggregation. These observations indicate that the Src-regulated outside-in signaling plays a pivotal role in the stable thrombus formation and the thrombus growth under flow conditions. The present study reveals novel insights into the molecular mechanisms by which c-Src regulates integrin αIIbβ3 signaling, particularly the phorsphorylation of the β3 cytoplasmic tyrosines, and provides first evidence in human platelets that the RGT peptide or derivatives regulate thrombus formation through dissociating the Src-β3 interaction. The data of this work allow us to anticipate that intracellular delivery of the RGT peptide or its analogues may have potential in the development of a new antithrombotic strategy where only the Src-β3 interaction is specifically interrupted so as to provide an effective inhibition on thrombosis together with a decent hemostasis. Disclosures: No relevant conflicts of interest to declare.

Just a Drop of Blood: Exploration of Shear-Dependent Platelet Adhesion and Activation in Microfluidic Devices.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3650-3650 ◽  
Author(s):  
Ana Kasirer-Friede ◽  
Edgar Gutierrez ◽  
Brian Petrich ◽  
Sanford J. Shattil ◽  
Mark H. Ginsberg ◽  
...  
Keyword(s):  
Shear Rate ◽  
Shear Flow ◽  
Blood Sample ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Microfluidic Devices ◽  
Extracellular Matrices ◽  
Wild Type ◽  
Shear Rates ◽  
Platelet Thrombus

Abstract Interactions of platelets and their adhesion receptors with extracellular matrices are essential for hemostasis. Platelets experience different shear rates as they circulate through the vasculature. Conventional studies of platelets in shear flow are performed in simple flow chambers with relatively large volumes of cells and reagents, limiting testing when these are in short supply. Microfluidics technology should enable the concurrent study of multiple small volume samples across a wide range of shear rates, thereby allowing trends to emerge that might be difficult to detect otherwise. To achieve this goal, we fabricated PDMS microfluidic devices that permit testing of dynamic platelet adhesion over a 100-fold span of shear rates from a single 200μl blood sample. Alternate device design permits synchronous monitoring of platelet adhesion from two genetically distinct blood samples or treatment groups. We have used this technology to gain new insights into integrin αIIbβ3 function in mepacrine-labeled platelets under shear flow. In whole blood, the adhesion of wild-type mouse platelets to a fibrinogen-coated substrate was shear rate-dependent, similarly to human platelets. In contrast, adhesion of αIIbβ3-deficient (β3−/−) platelets was virtually absent above a shear rate of 100 s−1. To distinguish between requirements for the presence of an intact extracellular αIIbβ3 ligand binding domain versus an intact integrin activation process, we used mice with a Y/A mutation at residue 747 in the β3 cytoplasmic tail (β3Y747A), that selectively blocks talin interaction with β3, agonist-induced αIIbβ3 activation, and platelet thrombus formation in vivo. When compared to wild-type and β3−/− platelets, a normal αIIbβ3 extracellular domain on β3Y747A platelets partially rescued dynamic adhesion to fibrinogen by 50–80% at ≤130 s−1, but by only 25% at 250 s−1. Treatment of wild-type platelets with PGE1 to inhibit platelet activation similarly reduced adhesion to fibrinogen at higher shear rates. On fibrillar type I collagen, wild-type platelets formed an initial monolayer and progressively larger thrombi over time. In addition, platelets supported rolling and firm adhesion of granulocytes in a manner dependent on shear rate, platelet P-selectin and granulocyte PSGL1. In contrast, no platelet thrombus growth on collagen was observed with αIIbβ3-deficient or β3Y747A platelets, or with wild-type platelets treated with PGE1. Furthermore, even the initial adhesion of αIIbβ3-deficient and β3Y747A platelets to collagen rapidly declined at increasing shear rates (120%, 30% and 7% of wild-type platelets at 70, 1000 and 4000 s−1, respectively (p &lt; 0.01)). Taken together, these studies establish that microfluidics provides an efficient and high-throughput platform to study mechanisms of dynamic platelet adhesion, activation and thrombus formation on extracellular matrices. Furthermore, they demonstrate a role for talin-dependent αIIbβ3 activation in all of these processes. This platform will be particularly useful under conditions where blood sample volumes or reagents are limiting, such as in neonates, and genetically-modified model organisms.

scholarly journals Correction of the platelet adhesion defect in delta-storage pool deficiency at elevated hematocrit--possible role of adenosine diphosphate

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4214-4222 ◽  
Author(s):  
HJ Weiss ◽  
B Lages ◽  
T Hoffmann ◽  
VT Turitto
Keyword(s):  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Adenosine Diphosphate ◽  
Dense Granule ◽  
High Shear ◽  
Shear Rates ◽  
Storage Pool ◽  
Corrective Effect ◽  
High Shear Rates

Previous studies on patients with storage pool deficiency (SPD) who are specifically deficient in platelet dense granules (delta-SPD) have suggested a role for dense granule substances, in all likelihood adenosine diphosphate (ADP), in mediating thrombus formation on subendothelium at high shear rates. The role of dense granule substances in mediating platelet adhesion appears to be more complicated Previous studies in delta-SPD suggested an adhesion defect that was strongly influenced by the patient's hematocrit (Hct) value. To explore further the possibility that red blood cells (RBCs) may influence the role that platelet storage granules play in mediating adhesion at high shear rates, we have measured adhesion (and thrombus formation) throughout a preselected range of Hct values (30% to 60%) in normal subjects and in patients with delta-SPD. The present studies confirm the defect in platelet adhesion in patients with delta-SPD, most significantly at Hct values of 30% to 40%. This defect (but not that of thrombus formation) can be completely corrected by the addition of RBCs. The correction of the platelet adhesion defect by RBCs was specific for delta-SPD; it was not observed in either von Willebrand's disease or thrombasthenia. Studies performed on normal blood under conditions that could be expected to block any effect of ADP on adhesion and an analysis of the type of adhesion defect in delta-SPD suggest that ADP may be involved in the process required for platelet spreading on the subendothelium. The corrective effect of RBCs on platelet adhesion in delta-SPD appears to be chemical rather than physical in nature, possibly due to shear-induced release of RBC ADP or to other recently described properties of RBCs that enhance collagen- induced platelet interactions.

scholarly journals Calin from Hirudo medicinalis, an inhibitor of von Willebrand factor binding to collagen under static and flow conditions

Blood ◽  
1995 ◽  
Vol 85 (3) ◽  
pp. 705-711 ◽  
Author(s):  
J Harsfalvi ◽  
JM Stassen ◽  
MF Hoylaerts ◽  
E Van Houtte ◽  
RT Sawyer ◽  
...  
Keyword(s):  
Shear Stress ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
High Shear ◽  
High Shear Stress ◽  
Flow Conditions ◽  
Shear Rates ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Low Shear

Calin from the saliva of the medicinal leech, Hirudo medicinalis, is a potent inhibitor of collagen mediated platelet adhesion and activation. In addition to inhibition of the direct platelet-collagen interaction, we presently demonstrate that binding of von Willebrand to coated collagen can be prevented by Calin, both under static and flow conditions in agreement with the occurrence of binding of Calin to collagen, confirmed by Biospecific Interaction Analysis. To define whether Calin acted by inhibiting the platelet-collagen or the platelet- von Willebrand factor (vWF)-collagen-mediated thrombus formation, platelet adhesion to different types of collagens was studied in a parallel-plate flow chamber perfused with whole blood at different shear rates. Calin dose-dependently prevented platelet adhesion to the different collagens tested both at high- and low-shear stress. The concentration of Calin needed to cause 50% inhibition of platelet adhesion at high-shear stress was some fivefold lower than that needed for inhibition of vWF-binding under similar conditions, implying that at high-shear stress, the effect of Calin on the direct platelet- collagen interactions, suffices to prevent thrombus formation. Platelet adhesion to extracellular matrix (ECM) of cultured human umbilical vein endothelial cells was only partially prevented by Calin, and even less so at a high-shear rather than a low-shear rate, whereas the platelet binding to coated vWF and fibrinogen were minimally affected at both shear rates. Thus, Calin interferes with both the direct platelet- collagen interaction and the vWF-collagen binding. Both effects may contribute to the inhibition of platelet adhesion in flowing conditions, although the former seems to predominate.

ASIALO VON WILLEBRAND FACTOR ENHANCES PLATELET ADHESION TO VASCULAR SUBENDOTHELIUM

1987 ◽  
Author(s):  
A Ordinas ◽  
E Bastida ◽  
M Garrido ◽  
J Monteagudo ◽  
L de Marco ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Human Albumin ◽  
Flow Conditions ◽  
Shear Rates ◽  
High Shear Rates ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Adhesion Of Platelets

Native Von Willebrand factor (NvWF) binds to platelets activated by thrombin, ADP or ristocetin, and also supports the adhesion of platelets to subendothelium at high shear rates. In contrast, asialo von Willebrand factor (AvWF) induces platelet aggregation in absence of platelet activators. We investigated the role of AvWF in supporting the adhesion of platelets to rabbit vessel subendothelium under flow conditions at a shear rate of 2000 sec-1 for 5 min using the Baumgartner perfusion system. We also studied the effects of blockage of platelet GPIb or GPIIb/IIIa on platelet adhesion using monoclonal antibodies (Mabs),and we measured the rate of binding of 111I-labeled NvWF and AvWF to subendothelium. Perfusates consisted of washed platelts and red cells resuspended in a 4% human albumin solution to which increasing concentrations of NvWF or AvWF had been added. Platelets interacting with the perfused vessels were evaluated morphometrically using a computerized system. At a concentration of 1.2 /ig/ml the percentage of total coverage surface was 21.3 ± 4.8% and 40.0±14.6%, for NvWF and AvWF, respectively (p<0.01). Addition of either Mab against GPIb (LJlbl) or against GPIIb/IIIa (CP8) to the perfusates, reduced platelet deposition (p <0.01). The rates of binding of 111I-labeled NvWF and AvWF to perfused vessel subendothelium were similar (0.83±0.1μg and 0.95±0.1 μg ,respectively).Our results indicate that AvWF enhances the interaction of washed platelets with the vessel subendothelium under flow conditions. Furthermore, they suggest that this effect is related to the interaction of AvWF with platelets and not to an increased affinity of AvWF for subendothelium.

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