Mechanisms of Static and Shear-Induced Platelet Adhesion: Differences in the Adhesion Supported by MMRN1 Comparisons with Other β3 Integrin Ligands.

Abstract Platelet adhesion and aggregation at the site of vascular injury are key events in hemostasis and thrombosis. These processes are supported by interactions between platelet glycoprotein (GP) receptors (including integrin αIIbβ3, GP Ib-IX-V and GPVI) and ligands that include von Willebrand factor (VWF), collagen, and fibrinogen (Fg). Recently, the polymeric protein multimerin 1 (MMRN1) was identified to bind β3 integrins. Normally, MMRN1 is sequestered within secretion granules of platelets, megakaryocytes, and endothelium until its release. In static adhesion assays, MMRN1 supports platelet adhesion to integrins αIIbβ3 and αvβ3 by an RGD-dependent mechanism. Study goals and methods: To further determine the mechanisms of MMRN1 binding to platelets, we investigated (i) the importance of platelet activation in MMRN1 binding to platelets, (ii) the ability of MMRN1 to support platelet adhesion compared to other adhesive ligands (Fg, VWF), and (iii) the role of β3 integrins in MMRN1 binding to platelets at low and high shear. Results: The binding of secreted platelet MMRN1 to thrombin activated platelets was significantly reduced by antibody inhibitors of ligand binding to αIIbβ3 and αvβ3. Thrombasthenic platelets (GT), deficient in αIIbβ3 and αvβ3 integrins, stored normal quantities of MMRN1 but after activation, they retained 34% less MMRN1 than normal platelets. Platelet adhesion experiments confirmed that αIIbβ3/αvβ3 and other binding sites supported MMRN1 binding to platelets. MMRN1 supported platelet adhesion at both low (150 s-1) and high (1500 s-1) shear rates. Like platelet adhesion to VWF, platelet adhesion to MMRN1 was greater at high shear rates. Platelet stimulation by agonists was essential to induce platelet binding to MMRN1 in static and shear adhesion assays, but not to induce platelet adhesion to Fg and vWF. While platelets activated by ADP and TRAP showed similar adhesion to Fg and VWF, TRAP induced more platelet adhesion to MMRN1 at high shear rates than ADP. Inhibitors of ligand binding to αIIbβ3 and αvβ3 had greater effects on high compared to low shear platelet adhesion to MMRN1. Conclusions: These data indicate interesting differences in the mechanisms that support platelet adhesion to MMRN1 and other β3 ligands, which could be important for molecular events in hemostasis and thrombosis. The activation-dependent binding of MMRN1 to platelets, augmented by high shear flow, may reflect the unique recognition properties of platelet β3 integrins and/or exposure of other binding sites (e.g. phosphatidylserine) on activated platelets that promote adhesive interactions with MMRN1.

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Platelet von Willebrand factor: evidence for its involvement in platelet adhesion to collagen

Blood ◽

10.1182/blood.v70.4.1214.bloodjournal7041214 ◽

1987 ◽

Vol 70 (4) ◽

pp. 1214-1217

Author(s):

E Fressinaud ◽

D Baruch ◽

C Rothschild ◽

HR Baumgartner ◽

D Meyer

Keyword(s):

Shear Rate ◽

Von Willebrand Factor ◽

Platelet Adhesion ◽

Von Willebrand Disease ◽

High Shear ◽

Shear Rates ◽

High Shear Rates ◽

Von Willebrand ◽

Willebrand Factor

Although it is well established that plasma von Willebrand Factor (vWF) is essential to platelet adhesion to subendothelium at high shear rates, the role of platelet vWF is less clear. We studied the respective role of both plasma and platelet vWF in mediating platelet adhesion to fibrillar collagen in a parallel-plate perfusion chamber. Reconstituted blood containing RBCs, various mixtures of labeled washed platelets and plasma from controls or five patients with severe von Willebrand disease (vWD), was perfused through the chamber for five minutes at a shear rate of 1,600 s-1. Platelet-collagen interactions were estimated by counting the radioactivity in deposited platelets and by quantitative morphometry. When the perfusate consisted of normal platelets suspended in normal plasma, platelet deposition on the collagen was 24.7 +/- 3.6 X 10(6)/cm2 (mean +/- SEM, n = 6). Significantly less deposition (16 +/- 2.3) was observed when vWD platelets were substituted for normal platelets. In mixtures containing vWD plasma, significantly greater deposition (9 +/- 2.2) was obtained with normal than with vWD platelets (1 +/- 0.4) demonstrating a role for platelet vWF in mediating the deposition of platelets on collagen. Morphometric analysis confirmed these data. Our findings indicate that platelet, as well as plasma, vWF mediates platelet-collagen interactions at a high shear rate.

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

Blood ◽

10.1182/blood.v87.10.4214.bloodjournal87104214 ◽

1996 ◽

Vol 87 (10) ◽

pp. 4214-4222 ◽

Cited By ~ 18

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.

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Platelet adhesion at high shear rates: The roles of von Willebrand factor/GPIb and the β1 integrin α2β1

Thrombosis Research ◽

10.1016/0049-3848(95)00219-7 ◽

1996 ◽

Vol 81 (1) ◽

pp. 113-119 ◽

Cited By ~ 18

Author(s):

Harvey R. Gralnicks ◽

Wendy S. Kramer ◽

Laurie P. McKeown ◽

Leonard Garfinkel ◽

Amos Pinot ◽

...

Keyword(s):

Von Willebrand Factor ◽

Platelet Adhesion ◽

Β1 Integrin ◽

High Shear ◽

Shear Rates ◽

High Shear Rates ◽

Von Willebrand ◽

Willebrand Factor

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Platelet von Willebrand factor: evidence for its involvement in platelet adhesion to collagen

Blood ◽

10.1182/blood.v70.4.1214.1214 ◽

1987 ◽

Vol 70 (4) ◽

pp. 1214-1217 ◽

Cited By ~ 36

Author(s):

E Fressinaud ◽

D Baruch ◽

C Rothschild ◽

HR Baumgartner ◽

D Meyer

Keyword(s):

Shear Rate ◽

Von Willebrand Factor ◽

Platelet Adhesion ◽

Von Willebrand Disease ◽

High Shear ◽

Shear Rates ◽

High Shear Rates ◽

Von Willebrand ◽

Willebrand Factor

Abstract Although it is well established that plasma von Willebrand Factor (vWF) is essential to platelet adhesion to subendothelium at high shear rates, the role of platelet vWF is less clear. We studied the respective role of both plasma and platelet vWF in mediating platelet adhesion to fibrillar collagen in a parallel-plate perfusion chamber. Reconstituted blood containing RBCs, various mixtures of labeled washed platelets and plasma from controls or five patients with severe von Willebrand disease (vWD), was perfused through the chamber for five minutes at a shear rate of 1,600 s-1. Platelet-collagen interactions were estimated by counting the radioactivity in deposited platelets and by quantitative morphometry. When the perfusate consisted of normal platelets suspended in normal plasma, platelet deposition on the collagen was 24.7 +/- 3.6 X 10(6)/cm2 (mean +/- SEM, n = 6). Significantly less deposition (16 +/- 2.3) was observed when vWD platelets were substituted for normal platelets. In mixtures containing vWD plasma, significantly greater deposition (9 +/- 2.2) was obtained with normal than with vWD platelets (1 +/- 0.4) demonstrating a role for platelet vWF in mediating the deposition of platelets on collagen. Morphometric analysis confirmed these data. Our findings indicate that platelet, as well as plasma, vWF mediates platelet-collagen interactions at a high shear rate.

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Soluble Monomeric Von Willebrand Factor A1A2A3 (R1450E) Mutant Fragment Enhances Shear-Induced Platelet Aggregation and Platelet Adhesion to Fibrin(ogen) Via αIIbβ3 Under High Shear Stress.

Blood ◽

10.1182/blood.v114.22.4025.4025 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4025-4025

Author(s):

Miguel A. Cruz ◽

Katie E. Sowa ◽

Scott M. Smith

Keyword(s):

Shear Stress ◽

Platelet Aggregation ◽

Von Willebrand Factor ◽

Platelet Adhesion ◽

Surface Coverage ◽

High Shear ◽

Shear Rates ◽

High Shear Rates ◽

Von Willebrand ◽

Willebrand Factor

Abstract Abstract 4025 Poster Board III-961 Recently, we described that the gain of function mutation R1450E in the A1 domain of von Willebrand factor (VWF) eliminates the formation of catch bond with glycoprotein (GP)Ibα, prolonging the bond lifetimes at low forces. Because those studies were performed with the mutant immobilized on a plastic surface, we further characterize the effect of this mutant on platelet function in solution and under shear stress. Both wild type (WT) and mutant A1A2A3 proteins were expressed in HEK293 cells and purified to homogeneity. The monomeric state of A1A2A3 proteins were assessed by gel filtration chromatography and neither of the proteins had formed dimers or any higher order aggregates. The recombinant A1A2A3 mutant bound spontaneously to GPIbα without the modulator ristocetin with a half-maximal binding observed at 65 ± 8 nM. This apparent dissociation constant was comparable to that of WT (50 ± 10 nM) in the presence of ristocetin. The mutant failed to induce spontaneous platelet aggregation under stirring conditions, and blocked 100% ristocetin-induced platelet agglutination (RIPA) at concentration of 250 nM. At the same concentration, the mutant increased shear-induced platelet aggregation (SIPA) at 500s-1 and 5000s-1 shear rates, reaching 42% and 66%, respectively, while SIPA did not exceed 18% in the presence of WT. The anti-αIIbβ3 antibody 7E3 blocked the effect of the mutant on SIPA. Blood was then incubated with the mutant (250 nM) and perfused over a surface coated with fibrin(ogen) at different shear rates. Blood containing WT resulted in <10% surface coverage by platelets after 1.5 minutes while platelets from blood containing the mutant rapidly bound covering 100% of the fibrin(ogen) surface area at 1500s-1. At shear rate of 2500s-1, surface coverage was 20% for the mutant and 0% for WT fragment. EDTA and antibodies 6D1 (GPIbα) and 10E5 (αIIbβ3) effectively blocked mutant-mediated platelet adhesion and thrombus formation under high shear rates. The addition of ristocetin (0.5 mg/ml) to whole blood prior perfusion reproduced the effect of the mutant. Here, we describe an A1A2A3 mutant that bound spontaneously to GPIbα but affected differently RIPA and SIPA. These results suggest that hydrodynamic forces directly act on the GPIbα-mutant A1A2A3 complex, regulating signaling. In addition, platelet activation induced by the binding of soluble mutant A1A2A3 or plasma VWF results in αIIbβ3-mediated platelet adhesion to fibrin(ogen) under high shear rates. Disclosures: No relevant conflicts of interest to declare.

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VWF Concentrates - Does Size Really Matter - Von Willebrand Factor Function under Physiological Flow Conditions.

Blood ◽

10.1182/blood.v110.11.3138.3138 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3138-3138

Author(s):

Bruce A. Schwartz ◽

Birte Fuchs ◽

C. Fisseau ◽

Craig M. Kessler ◽

Christoph Kannicht

Keyword(s):

Platelet Adhesion ◽

Flow Chamber ◽

High Shear ◽

Flow Conditions ◽

Shear Rates ◽

Physiological Flow ◽

High Shear Rates ◽

Platelet Binding ◽

Von Willebrand ◽

Willebrand Factor

Abstract Introduction: The Von Willebrand Factor (VWF), is a very large glycoprotein present in human plasma essential for normal thrombus formation at sites of vascular injury. Blood coagulation is initiated by exposure of vessel wall components, e.g. collagen, followed by platelet tethering, activation and adhesion leading to the formation of a stable clot. All steps of blood coagulation occur in flowing blood under various conditions depending on vessel size. It is important to assess the function of a VWF concentrate under physiologic conditions. Under static or low shear conditions, platelets can bind directly to collagen without assistance of VWF, while the VWF is essential for mediation of platelet adhesion under high flow occurring in the arterial circulation. It has been assumed that multimer size plays an important role in this binding and in subsequent platelet adhesion. In this study we evaluated the binding of VWF of different multimeric structures to collagen and determined VWF-mediated platelet binding under low to high shear rates in a flow chamber model. Methods: A flow-chamber coated with human collagen was developed to mimic physiological flow conditions. A high purity VWF/factor VIII (FVIII) concentrate (Wilate®) and two other VWF/FVIII concentrates were tested at shear rates of 400 1700 and 4000 s−1 reproducing shear rates occurring in veins, small arteries and capillaries. Collagen-bound VWF was characterized by antigen determination (VWF:Ag) and multimer (MM) analysis. Binding of labeled platelets was visualized by a fluorescence microscope and surface coverage was quantified. Results: All VWF MMs independent of MM size were found to bind to collagen even under high shear rates. The amount of collagen-bound VWF:Ag and VWF-mediated platelet adhesion at 1700 s−1 differed significantly between the tested concentrates, when equal VWF:Ag amounts were applied. Conclusion: Binding of VWF to collagen does not depend on VWF MM size in this model even when measured under high shear rates. The differences in collagen-bound VWF:Ag and VWF-mediated platelet binding do not seem to depend on the VWF MM distribution of the concentrates. Other structural features than VWF MM size may likely be caused by the differences seen in the binding levels of the different concentrates and should be further explored. Possible differences between VWF activity assays performed under static or flow conditions and their appropriate use for VWD diagnosis and quantification of in-vivo activity need to be further investigated.

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Examining platelet adhesion via Stokes flow simulations and microfluidic experiments

Soft Matter ◽

10.1039/c4sm01450b ◽

2015 ◽

Vol 11 (2) ◽

pp. 355-367 ◽

Cited By ~ 13

Author(s):

Sean Fitzgibbon ◽

Jonathan Cowman ◽

Antonio J. Ricco ◽

Dermot Kenny ◽

Eric S. G. Shaqfeh

Keyword(s):

Platelet Function ◽

Stokes Flow ◽

Platelet Adhesion ◽

High Shear ◽

Shear Rates ◽

Flow Simulations ◽

High Shear Rates

While critically important, the platelet function at the high shear rates typical of the microcirculation is relatively poorly understood.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646346 ◽

1992 ◽

Vol 68 (06) ◽

pp. 694-700 ◽

Cited By ~ 25

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.

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Asialo von Willebrand Factor Enhances Platelet Adhesion to Vessel Subendothelium

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647629 ◽

1988 ◽

Vol 60 (01) ◽

pp. 030-034 ◽

Cited By ~ 9

Author(s):

Eva Bastida ◽

Juan Monteagudo ◽

Antonio Ordinas ◽

Luigi De Marco ◽

Ricardo Castillo

Keyword(s):

Von Willebrand Factor ◽

Platelet Adhesion ◽

Human Albumin ◽

Membrane Receptors ◽

Shear Rates ◽

High Shear Rates ◽

Platelet Deposition ◽

Platelet Spreading ◽

Von Willebrand ◽

Willebrand Factor

SummaryNative von Willebrand factor (N-vWF) binds to platelets activated by thrombin, ADP or ristocetin. Asialo vWF (As-vWF) induces platelet aggregation in absence of platelet activators. N-vWF mediates platelet adhesion to vessel subendothelium at high shear rates. We have investigated the role of As-vWF in supporting platelet deposition to rabbit vessel subendothelium at a shear rate of 2,000 sec-1, using the Baumgartner perfusion system. We have studied the effects of the addition of As-vWF (from 2 to 12 μg/ml) to perfusates consisting of washed red blood cells, 4% human albumin and washed platelets. Our results show a significant increase in platelet deposition on subendothelium (p <0.01) in perfusions to which As-vWF had been added. Blockage of the platelet glycoproteins Ib and IIb/IIIa (GPIb and GPIIb/IIIa) by specific monoclonal antibodies (LJIb1 and LJCP8, respectively) resulted in a decrease of platelet deposition in both types of perfusates prepared with N-vWF and As-vWF. Our results indicate that As-vWF enhances platelet deposition to vessel subendothelium under flow conditions. Furthermore, they suggest that this effect is mediated by the binding of As-vWF to platelet membrane receptors, which in turn, promote platelet spreading and adhesion to the subendothelium.

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