The Influence of Shear Rate and Red Cell Concentration on Platelet Adhesion and Thrombus Formation in Human Blood

1979 ◽  
Author(s):  
V.T. Turitto ◽  
H.J. Weiss ◽  
H. R. Baumgartner
Keyword(s):  
Platelet Adhesion ◽  
Cell Concentration ◽  
Thrombus Formation ◽  
Size Variation ◽  
Arrival Rate ◽  
Red Cells ◽  
Steady Increase ◽  
Shear Rates ◽  
Perfusion Chamber ◽  
Diffusion Controlled

The interaction of platelets with subendothelium requires the transport of platelets to the vicinity of the surface, as well as the basic cell-surface reaction. Exposure of subendothelium to human citrated blood flowing in an annular perfusion chamber at wall shear rates (α) of 50-10,000 sec-1 indicates that a diffusion controlled (DC) transport regime exists below 650 sec- l in which platelet adhesion (C+S) was strongly dependent on α, and thrombus formation (T) was absent . Above 800 sec-1, an apparently reaction controlled (RC) regime predominates in which C+S was independent of α, and T increased in both extent and size. Variation of hematocrit (H) from 0-701. in the RC regime (2600 sec-1) lead to a steady increase of C+S with H, and an exponential increase in T as H increased from 30 to 70%. In the DC regime (200 sec-1) virtually no thrombi were formed for all H, and C+S increased as H increased to 40%; above 40%, C+S became independent of H. Thus, at low α (venous), the platelet-subendothelial reaction is controlled primarily by the arrival rate of platelets at the surface and the red cells increase this transport for H up to 40%. At high α (microcirculatory) , the platelet-vessel wall reactivity becomes more dominant and red cells increase the ability of platelets to attach to the subendothelium.

Anthocyanins Inhibit Platelet Activation and Attenuate Thrombus Growth In Both Human and Murine Thrombosis Models

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3197-3197 ◽  
Author(s):  
Yan Yang ◽  
Zhenyin Shi ◽  
Adili Reheman ◽  
Wuxun Jin ◽  
Conglei Li ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Intravital Microscopy ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Vessel Occlusion ◽  
Shear Rates ◽  
Perfusion Chamber ◽  
Platelet Adhesion And Aggregation

Abstract Abstract 3197 Background: Thrombosis and cardiovascular diseases (CVDs) result from blood vessel occlusion by inappropriately activated platelets. They are the leading causes of morbidity and mortality worldwide. Anthocyanins are major phytochemicals abundant in plant food and have been shown to play a protective role against CVDs. Our previous studies have demonstrated that anthocyanins are antioxidative and prevent inflammation (J Biol Chem. 2005; 280:36792-01; Arterioscler Thromb Vasc Biol. 2007; 27:519-24), which may indirectly affect platelet function. It has also been reported that anthocyanins affect platelet activities in whole blood and platelet rich plasma (PRP). However, the direct effects of anthocyanins on platelet function and thrombus formation have not been studied. Methods: Here we investigated the effects of anthocyanins on thrombosis using purified platelets as well as several thrombosis models in vitro and in vivo. Cyaniding-3-gulucoside (Cy-3-g) and delphinidin-3-glucoside (Dp-3-g), the two predominantly bioactive compounds of anthocyanin preparations, were prepared from Polyphenol AS Company in Norway. Purified gel-filtered platelets and PRP from healthy human volunteers and C57BL/6J mice were incubated at 37°C for 10 minutes with different concentrations (0.5μM, 5μM and 50μM) of Cy-3-g, Dp-3-g or PBS buffer as a control. Platelet aggregation was assessed by aggregometry using 5μM ADP, 10μg/ml collagen, or 100μM thrombin receptor activating peptide (TRAP; AYPGKF) as agonists. Platelet adhesion and aggregation were assessed in response to an immobilized collagen matrix in an ex vivo perfusion chamber at both high (1800 s-1) and low (600 s-1) shear rates. The expression of activated GPIIbIIIa was determined via PAC-1 monoclonal antibody in flow cytometry. Lastly, the effects of anthocyanins on thrombus formation in C57BL/6J mice were assessed using a FeCl3-induced intravital microscopy thrombosis model. Results: Both Cy-3-g and Dp-3-g significantly inhibited platelet aggregation induced by collagen and TRAP in gel-filtered platelets, and inhibited aggregation induced by ADP, TRAP and collagen in human and mouse PRP. These inhibitory functions were observed at Cy-3-g and Dp-3-g doses as low as 0.5μM. Cy-3-g and Dp-3-g also reduced the surface expression of activated GPIIbIIIa on resting human platelets in a dose-dependent manner. These compounds also markedly reduced platelet adhesion and aggregation in perfusion chamber assays at both low and high shear rates. Using intravital microscopy, we further demonstrated that Cy-3-g and Dp-3-g decreased platelet deposition, destabilized thrombi, and prolonged the time required for thrombus formation and vessel occlusion. Conclusions: our data clearly demonstrated for the first time that anthocyanin compounds directly inhibited platelet activation, adhesion and aggregation, as well as attenuated thrombus growth at both arterial and veinous shear stresses. These effects on platelets likely contribute to the protective effects of anthocyanins against thrombosis and CVDs. Disclosures: No relevant conflicts of interest to declare.

Inhibition of Platelet Adhesion to Fibrin(ogen) in Flowing Whole Blood by Arg-Gly-Asp and Fibrinogen γ-Chain Carboxy Terminal Peptides

1992 ◽  
Vol 68 (06) ◽  
pp. 694-700 ◽  
Author(s):  
Roy R Hantgan ◽  
Silvia C Endenburg ◽  
I Cavero ◽  
Gérard Marguerie ◽  
André Uzan ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Integrin Receptor ◽  
Shear Rates ◽  
Perfusion Chamber ◽  
Receptor Recognition ◽  
Adhesive Interactions ◽  
Coated Surfaces ◽  
Carboxy Terminal

SummaryWe have employed synthetic peptides with sequences corresponding to the integrin receptor-recognition regions of fibrinogen as inhibitors of platelet aggregation and adhesion to fibrinogen-and fibrin-coated surfaces in flowing whole blood, using a rectangular perfusion chamber at wall shear rates of 300 s–1 and 1,300 s–1. D-RGDW caused substantial inhibition of platelet aggregation and adhesion to fibrinogen and fibrin at both shear rates, although it was least effective at blocking platelet adhesion to fibrin at 300 s–1. RGDS was a weaker inhibitor, and produced a biphasic dose-response curve; SDRG was inactive. HHLGGAK-QAGDV partially inhibited platelet aggregation and adhesion to fibrin(ogen) at both shear rates. These results support the identification of an RGD-specific receptor, most likely the platelet integrin glycoprotein IIb: III a, as the primary receptor responsible for platelet: fibrin(ogen) adhesive interactions under flow conditions, and indicate that platelet adhesion to surface bound fibrin(ogen) is stabilized by multivalent receptor-ligand contacts.

scholarly journals Prostacyclin (prostaglandin I2, PGI2) inhibits platelet adhesion and thrombus formation on subendothelium

Blood ◽  
1979 ◽  
Vol 53 (2) ◽  
pp. 244-250 ◽  
Author(s):  
HJ Weiss ◽  
VT Turitto
Keyword(s):  
Blood Vessels ◽  
Human Blood ◽  
Platelet Adhesion ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Rabbit Aorta ◽  
Prostaglandin I2 ◽  
Shear Rates ◽  
Platelet Spreading ◽  
Aggregation Of Platelets

Abstract Prostaglandin I2 (prostacyclin, PGI2), a substance synthesized in the wall of blood vessels, has been previously shown to inhibit the aggregation of platelets in stirred platelet-rich plasma. We used a method in which segments of deendothelialized rabbit aorta are perfused at arterial shear rates with human blood and found that both platelet adhesion and thrombus formation on subendothelium was inhibited in blood containing 10 nM PGI2. PGI2 appears to reduce adhesion by inhibiting platelet spreading. These findings suggest that PGI2 could regulate the deposition of platelets on vascular surfaces.

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.

scholarly journals Glycoprotein Ib, von Willebrand factor, and glycoprotein IIb:IIIa are all involved in platelet adhesion to fibrin in flowing whole blood

Blood ◽  
1990 ◽  
Vol 76 (2) ◽  
pp. 345-353 ◽  
Author(s):  
RR Hantgan ◽  
G Hindriks ◽  
RG Taylor ◽  
JJ Sixma ◽  
PG de Groot
Keyword(s):  
Platelet Aggregation ◽  
Shear Rate ◽  
Von Willebrand Factor ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Shear Rates ◽  
Wall Shear ◽  
Von Willebrand ◽  
Willebrand Factor

We have investigated the molecular basis of thrombus formation by measuring the extent of platelet deposition from flowing whole blood onto fibrin-coated glass coverslips under well-defined shear conditions in a rectangular perfusion chamber. Platelets readily and specifically adhered to fibrin-coated coverslips in 5 minute perfusion experiments done at either low (300 s-1) or high (1,300 s-1) wall shear rates. Scanning electron microscopic examination of fibrin-coated coverslips after perfusions showed surface coverage by a monolayer of adherent, partly spread platelets. Platelet adhesion to fibrin was effectively inhibited by a monoclonal antibody (MoAb) specific for glycoprotein (GP) IIb:IIIa. The dose-response curve for inhibition of adhesion by anti-GPIIb:IIIa at both shear rates paralleled that for inhibition of platelet aggregation. Platelet aggregation and adhesion to fibrin were also blocked by low concentrations of prostacyclin. In contrast, anti- GPIb reduced adhesion by 40% at 300 s-1 and by 70% at 1,300 s-1. A similar pattern of shear rate-dependent, incomplete inhibition resulted with a MoAb specific for the GPIb-recognition region of von Willebrand factor (vWF). Platelets from an individual with severe von Willebrand's disease, whose plasma and platelets contained essentially no vWF, exhibited defective adhesion to fibrin, especially at the higher shear rate. Addition of purified vWF restored adhesion to normal values. These results are consistent with a two-site model for platelet adhesion to fibrin, in which the GPIIb:IIIa complex is the primary receptor, with GPIb:vWF providing a secondary adhesion pathway that is especially important at high wall shear rates.

Ethanol Inhibition of Thrombus Formation on Collagen-Coated Glass

1990 ◽  
Vol 63 (03) ◽  
pp. 510-516 ◽  
Author(s):  
Charlene K Owens ◽  
Larry V Mclntire ◽  
Andrew Lasslo
Keyword(s):  
Parallel Plate ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Flow Chamber ◽  
Mural Thrombus ◽  
Parallel Plate Flow Chamber ◽  
Shear Rates ◽  
Coated Glass ◽  
Normal Platelet ◽  
Platelet Accumulation

SummaryEpi-fluorescent video microscopy was used to evaluate the effect of ethanol on platelet mural thrombus formation. Whole blood, treated with ethanol, was perfused over collagen coated glass in a parallel-plate flow chamber at a shear rate of 1,000/s. Digital image processing and photodiode measurements were used to analyze the dynamics of thrombus growth on this surface. Ethanol concentrations as low as 0.02% v/v were found to inhibit 45 + 33% (± S.D.) of normal platelet accumulation on the slide while 0.2% v/v ethanol effected an 82 ± 15% inhibition of mural thrombus formation. While platelet adhesion to the collagen surface appeared unaffected by ethanol concentrations up to 0.1% v/v, 0.2% v/v ethanol had an effect on adhesion as well as aggregation. These results imply that low ethanol concentrations inhibit the formation of mural thrombi in a model of a damaged blood vessel at physiological shear rates. This inhibition would not be detected in systems which measure bulk aggregation, e.g. in aggregometric determinations.

scholarly journals Shear Rate Dependence of Platelet Adhesion to Collagenous Surfaces in Willebrand Factor-Depleted Blood

1977 ◽  
Author(s):  
H. R. Baumgartner ◽  
Th. B. Tschopp ◽  
D. Meyer
Keyword(s):  
Shear Rate ◽  
Human Blood ◽  
Platelet Adhesion ◽  
Platelet Rich Plasma ◽  
Shear Rates ◽  
Small Vessels ◽  
Perfusion Chamber ◽  
Function Defect ◽  
Induced Aggregation ◽  
Willebrand Factor

A platelet function defect similar to that in von Willebrand’s disease, was produced by adding to human blood an antibody raised in rabbits against human factor Vlll/Willebrand factor. The effect of this antibody (F VIII Ab) on platelet adhesion was tested in an annular perfusion chamber. Only a small adhesion defect was observed at a shear rate corresponding to that in large arteries (830 s-1) . Since bleeding usually occurs from small vessels, platelet adhesion to collagenous surfaces was investigated at higher shear rates corresponding to those in small vessels. The surfaces were exposed to human blood (l5mM citrate) at 1, 2 and 4x103 s-1 shear rate for 6, and 3 min, respectively. Different exposure times were chosen in order to obtain in control perfusions 50 – 70 % coverage of subendothelium with platelets at each shear rate. Platelet adhesion and adhesion-induced aggregation were measured morphometrically. As compared to control, F VIII Ab inhibited adhesion to subendothelium by 15±6, 32±21, 40±10 and 90±2 % (mean ± SE) at 0.83, 1, 2 and 4x103 s-1 shear rate, respectively. The corresponding inhibition by F VIII Ab observed on the fibrillar collagen of α-chymotrypsin-digested subendothelium was 62±11, 75±20, 99±1 and 100 %. In platelet rich plasma, F VIII Ab abolished Ristocetin-, inhibited collagen- and had no effect on ADP-induced aggregation.Thus the defect in platelet adhesion to collagenous surfaces observed in Willebrand factor-depleted blood is minimal at low (venous) and maximal at high (small vessel) shear rates.

The Effect of Platelet PlA Polymorphism on Experimental Thrombus Formation in Man Depends on Blood Flow and Thrombogenic Substrate

2001 ◽  
Vol 85 (06) ◽  
pp. 1097-1103 ◽  
Author(s):  
Kjell Sakariassen ◽  
Hélène Grandjean ◽  
Claire Thalamas ◽  
Bernard Boneu ◽  
Pierre Sié ◽  
...  
Keyword(s):  
Blood Flow ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Flow Properties ◽  
Shear Rates ◽  
Perfusion Chamber ◽  
Platelet Receptor ◽  
Platelet Deposition ◽  
Coronary Syndromes ◽  
Platelet Thrombus

SummaryA number of studies have reported conflicting data on the association of the PlA1/PlA2 polymorphism of the GPIIIa gene and coronary syndromes. We have investigated the effect of this polymorphism on experimental platelet thrombus formation in man. Forty healthy male volunteers were genotyped for the PlA1/PlA2 polymorphism. Thrombus formation was induced ex vivo by exposing a tissue factor (TF) or a collagencoated coverslip in a parallel plate perfusion chamber to native blood for 2 and 4 min. The shear rates at these surfaces were 650 and 2,600 s–1. Platelet and fibrin deposition was quantified by immunoenzymatic methods. The frequencies of PlA1/PlA1 and PlA1/PlA2 genotypes were 52.5% and 47.5%, respectively. Ex vivo deposition of fibrin on TF was not affected by the PlA1/PlA2 polymorphism. However, the ex vivo platelet deposition at 650 s–1 was higher in blood from PlA1/PlA1 individuals than in PlA1/PlA2 individuals (P = 0.008 at 4 min). On collagen, neither fibrin nor platelet deposition was significantly affected by the PlA1/PlA2 polymorphism. Platelet thrombus formation is significantly influenced by genetic variations in the GPIIIa platelet receptor. This effect depends on the blood flow properties and the nature of the thrombogenic stimulus.

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 < 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.

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