A novel flow cytometric analysis for platelet activation on immobilized von Willebrand factor or fibrillar collagen

Abstract Acute thrombocytopenia has been consistently reported following IV administration of adenoviral vectors (Ad) but the mechanism responsible for this phenomenon has not been elucidated. Thrombocytopenia appears 24 hours after IV administration of Ad and is vector dose dependent. In this study, we have assessed the potential roles of the adhesive proteins P-selectin and von Willebrand Factor (VWF) on the aggregation and clearance of platelets following virus administration. We have addressed the question of whether the thrombocytopenia is due to a direct effect of the virus on platelets or an indirect effect related to interaction of platelets with other proteins or cells modified by the virus. We assessed platelet count in a group of Balb/c and C57Bl/6 mice over 1 week period following Ad administration and performed a detailed examination of the events within the first 24 h after Ad injection, the period that precedes the appearance of thrombocytopenia. We examined the effect of Ad on expression of the platelet activation marker P-selectin and the formation of platelet leukocyte aggregates (PLA) by means of flowcytometry after incubation of adenovirus with mouse platelets in vitro, and following Ad administration in vivo. To assess the role of VWF in Ad-induced thrombocytopenia we measured plasma VWF levels one hour after injection of Ad. Further investigations involved comparison of platelet counts, platelet activation, and the formation of PLA in a group of VWF KO mice. All studies have been performed with a replication deficient E1/E3-deleted Ad 1x 1011 viral particles/mouse. Our in vitro studies have shown that Ad directly activates mouse platelets as shown by increased expression of P-selectin. The average index of platelet activation for platelets stimulated by Ad was 2519.4 compared to 128.2 for resting platelets (n=5, p<0.02). Flow cytometric analysis of CD41 (platelets) and CD45 (leucocytes) double stained positive events indicated that Ad stimulation induced PLA when compared to the unstimulated samples. Our in vivo studies have confirmed the development of significant thrombocytopenia in both Balb/c as well as C57Bl/6 WT mice (n=8, p=0.00001, n= 6, p=0.002) 24 hours following Ad administration. Significant P-selectin expression was documented in both strains (n=4,p=0.0003; n=3, p=0.0008 respectively) as well as significant PLA one hour following Ad (n=4, p=0.01; n=3, p=0.007). The VWF KO mice showed non-significant thrombocytopenia (n= 6, p=0.063) at 24 hours following Ad, significant P-selectin expression (n=3, p=0.0003), but no significant PLA formation at one hour (n=3 p=0.12) relative to pre-injection levels. Plasma VWF levels were significantly elevated in both Balb/c and C57Bl/6 WT mice one hour following administration of the virus (n= 3, p=0.02; n= 3, p= 0.001). The average plasma VWF levels were 48.1 U/mL at 1h compared to 5.7 U/mL pre injection in Balb /c mice and 85.9 U/mL compared to 6.1 U/mL in C57Bl/6 mice. These studies have shown that Ad can act as an inducer of mouse platelet activation and as a promoter for platelet-leukocyte association both in vitro and in vivo. We have demonstrated a role for Ad in stimulating VWF release from the endothelium, and have shown that VWF has a critical role in platelet activation and clearance following Ad administration. We conclude that P-selectin and VWF proteins are directly involved in interactions between endothelial cells, platelets and leukocytes, a complex interaction that can explain at least in part the mechanisms underlying Ad-mediated thrombocytopenia.

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Pathological von Willebrand factor fibers resist tissue plasminogen activator and ADAMTS13 while promoting the contact pathway and shear-induced platelet activation

Journal of Thrombosis and Haemostasis ◽

10.1111/jth.13044 ◽

2015 ◽

Vol 13 (9) ◽

pp. 1699-1708 ◽

Cited By ~ 26

Author(s):

B. A. Herbig ◽

S. L. Diamond

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Platelet Activation ◽

Von Willebrand Factor ◽

Tissue Plasminogen ◽

Contact Pathway ◽

Von Willebrand ◽

Willebrand Factor

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N-terminal Flanking Region of A1 Domain in von Willebrand Factor Stabilizes Structure of A1A2A3 Complex and Modulates Platelet Activation under Shear Stress

Journal of Biological Chemistry ◽

10.1074/jbc.m112.348573 ◽

2012 ◽

Vol 287 (18) ◽

pp. 14579-14585 ◽

Cited By ~ 28

Author(s):

Matthew Auton ◽

Katie E. Sowa ◽

Molly Behymer ◽

Miguel A. Cruz

Keyword(s):

Shear Stress ◽

Platelet Activation ◽

Von Willebrand Factor ◽

Flanking Region ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor

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von Willebrand factor bound to glycoprotein Ib is cleared from the platelet surface after platelet activation by thrombin

Blood ◽

10.1182/blood.v79.8.2011.2011 ◽

1992 ◽

Vol 79 (8) ◽

pp. 2011-2021 ◽

Cited By ~ 30

Author(s):

P Hourdille ◽

HR Gralnick ◽

E Heilmann ◽

A Derlon ◽

AM Ferrer ◽

...

Keyword(s):

Flow Cytometry ◽

Platelet Activation ◽

Von Willebrand Factor ◽

Platelet Rich Plasma ◽

Surface Expression ◽

Von Willebrand Disease ◽

Platelet Surface ◽

Immunogold Staining ◽

Von Willebrand ◽

Willebrand Factor

Abstract We recently reported that after activation of human platelets by thrombin, glycoprotein (GP) Ib-IX complexes are translocated to the surface-connected canalicular system (SCCS) (Blood 76:1503, 1990). As GPIb is a major receptor for von Willebrand factor (vWF) in platelet adhesion, we have now examined the consequences of thrombin activation on the organization of vWF bound to GPIb on the platelet surface. Studies were performed using monoclonal or polyclonal antibodies in either immunogold staining and electron microscopy (Au-EM) or in flow cytometry. When unstirred platelet-rich plasma was incubated with ristocetin, bound vWF was located by Au-EM as discrete masses regularly distributed over the cell surface. Platelets from a patient with Glanzmann's thrombasthenia, lacking GPIIb-IIIa complexes, gave a similar pattern, confirming that this represented binding to GPIb. That ristocetin was not precipitating vWF before their binding to the platelets was shown by the detection of similar masses on the surface of platelets of a patient with type IIB von Willebrand disease. Experiments were continued using washed normal platelets incubated in Tyrode-EDTA, the purpose of the EDTA being to limit the surface expression of endogenous vWF after platelet stimulation. Under these conditions, platelets were treated with ristocetin for 5 minutes at 37 degrees C in the presence of increasing amounts of purified vWF. This was followed by incubation with thrombin (0.5 U/mL) for periods of up to 10 minutes. Flow cytometry showed a time-dependent loss in the surface expression of vWF bound to GPIb and these changes were confirmed by Au-EM. In particular, immunogold staining performed on ultrathin sections showed that the bulk of the vWF was being cleared to internal membrane systems. Surface clearance of vWF during thrombin- induced platelet activation is a potential mechanism for regulating platelet adhesivity.

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Recurrent Arterial Thrombosis Linked to Autoimmune Antibodies Enhancing von Willebrand Factor Binding to Platelets and Inducing FcγRII Receptor-Mediated Platelet Activation

Blood ◽

10.1182/blood.v91.8.2810.2810_2810_2817 ◽

1998 ◽

Vol 91 (8) ◽

pp. 2810-2817 ◽

Cited By ~ 15

Author(s):

Marc F. Hoylaerts ◽

Chantal Thys ◽

Jef Arnout ◽

Jos Vermylen

Keyword(s):

Platelet Activation ◽

Von Willebrand Factor ◽

Low Dose ◽

Antithyroid Drug ◽

Fetal Loss ◽

Thyroid Peroxidase ◽

Spontaneous Aggregation ◽

Von Willebrand ◽

Induced Aggregation ◽

Willebrand Factor

A patient with a history of recurrent late fetal loss associated with multiple placental infarcts and cerebrovascular ischemia at the age of 36, followed a year later by a myocardial infarction, was referred for further investigation. Coronary angiography was normal. Antinuclear factor, lupus anticoagulant, anticardiolipin antibodies, and other thrombophilia parameters were negative, but there was moderate hyperthyroidism with positive thyroid peroxidase antibodies. Platelet numbers and von Willebrand factor (vWF) were normal. Her platelets showed spontaneous aggregation that disappeared with aspirin intake. However, aggregation still was induced by low levels of ristocetin (0.3 to 0.5 mg/mL). The low-dose ristocetin aggregation in patient platelet-rich plasma (PRP) was completely blocked by neutralizing antiglycoprotein Ib (GPIb) and anti-vWF antibodies. The monoclonal anti-FcγRII receptor antibody IV.3 inhibited partly, which suggests that PRP aggregation by low-dose ristocetin was elicited by vWF-immunoglobulin (Ig) complexes. Upon addition to washed human platelets, with vWF (10 μg/mL), purified patient Igs dose-dependently enhanced ristocetin (0.15 mg/mL)-induced aggregation between 0 and 500 μg/mL, an effect that disappeared again above 1 mg/mL. Aggregation was dependent on the vWF concentration and was blocked by IV.3 or neutralizing anti-GPIb or anti-vWF antibodies. The spontaneous aggregation of normal platelets resuspended in patient plasma could be inhibited totally by IV.3 and partially by neutralizing anti-GPIb or anti-vWF antibodies. Perfusion with normal anticoagulated blood, enriched with 10% of control or patient plasma, over surfaces coated with vWF showed increased platelet adhesion and activation in the presence of patient antibodies. Treatment of the patient with the antithyroid drug thiamazol and temporary corticosteroids, aspirin, and ticlopidine did not correct the platelet hypersensitivity to ristocetin. These observations suggest that some autoantibodies to vWF may both enhance vWF binding to platelets and cause platelet activation through binding to the FcγRII receptor, and thereby may be responsible for a new form of antibody-mediated thrombosis.

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A role for p38 MAP kinase in platelet activation by von Willebrand Factor

Thrombosis and Haemostasis ◽

10.1160/th03-02-0083 ◽

2004 ◽

Vol 91 (01) ◽

pp. 102-110 ◽

Cited By ~ 41

Author(s):

Ilaria Canobbio ◽

Stefania Reineri ◽

Fabiola Sinigaglia ◽

Cesare Balduini ◽

Mauro Torti

Keyword(s):

Arachidonic Acid ◽

Phospholipase A2 ◽

Map Kinase ◽

Platelet Activation ◽

Von Willebrand Factor ◽

P38 Map Kinase ◽

Cyclooxygenase Inhibitors ◽

Von Willebrand ◽

Willebrand Factor ◽

Stimulation Of

SummaryPlatelet activation induced by von Willebrand factor (VWF) binding to the membrane GPIb-IX-V receptor involves multiple signal transduction pathways. Among these, recruitment and activation of the FcγRIIA and stimulation of phospholipase A2 represent independent events equally essential to support a complete platelet response. Phospholipase A2 is activated by calcium and by phosphorylation through MAP kinases. In this work, we found that VWF stimulated the rapid and sustained phosphorylation of p38 MAP kinase (p38MAPK). In vitro kinase assay revealed that VWF-stimulated phosphorylation of p38MAPK was associated with increased kinase activity. Binding of VWF to GPIb-IX-V, but not to integrin αIIbβ3, was required to support phosphorylation of p38MAPK. Neither the blockade of the membrane FcγRIIA by a specific monoclonal antibody or the prevention of thromboxane A2 synthesis by cyclooxygenase inhibitors affected VWF-induced p38MAPK activation. However, phosphorylation of p38MAPK was prevented by the tyrosine kinase Syk inhibitor piceatannol. Treatment of platelets with the p38MAPK inhibitor SB203580 totally prevented VWFstimulated platelet aggregation. Moreover, release of arachidonic acid induced by VWF was strongly impaired by inhibition of p38MAPK. We also found thatVWF induced phosphorylation of cytosolic phospholipase A2, and that this process was prevented by the p38MAPK inhibitor SB203580.These results demonstrate that p38MAPK is a key element in the FcγRIIA-independent pathway for VWF-induced platelet activation, and is involved in the stimulation of phospholipase A2 and arachidonic acid release.

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Selective induction of a glycoprotein IIIa ligand-induced binding site by fibrinogen and von Willebrand factor

Blood ◽

10.1182/blood.v88.10.3824.bloodjournal88103824 ◽

1996 ◽

Vol 88 (10) ◽

pp. 3824-3830 ◽

Cited By ~ 25

Author(s):

TH Mondoro ◽

CD Wall ◽

MM White ◽

LK Jennings

Keyword(s):

Platelet Aggregation ◽

Von Willebrand Factor ◽

Binding Sites ◽

Flow Cytometric Analysis ◽

Clot Retraction ◽

Recognition Sequence ◽

Ligand Interaction ◽

Selective Ligand ◽

Von Willebrand ◽

Willebrand Factor

Ligand-induced binding sites (LIBS) are neoantigenic regions of glycoprotein (GP)IIb-IIIa that are exposed upon interaction of the receptor with the ligand fibrinogen or the ligand recognition sequence (RGDS). LIBS have been suggested to contribute to postreceptor occupancy events such as full-scale platelet aggregation, adhesion to collagen, and clot retraction. This study examined the induction requirements of a GPIIIa LIBS with regard to ligand specificity. Through the use of the anti-LIBS D3, we report that this complex- activating antibody induces fibrinogen-and von Willebrand factor-binding to GPIIb-IIIa on intact platelets. Bound ligand was detected by flow cytometric analysis and platelet aggregation assays. These bound ligands increased the number of D3-binding sites and altered the affinity of D3 for GPIIb-IIIa on platelets. In contrast, activation of platelet GPIIb-IIIa by D3 did not increase the binding of another RGD- containing ligand, vitronectin. Furthermore, bound vitronectin on thrombin-stimulated platelets did not cause the expression of the D3 LIBS epitope. We conclude direct activation of GPIIb-IIIa in the absence of platelet activation results in selective ligand interaction and that D3 LIBS induction requires the binding of the multivalent ligands, fibrinogen or von Willebrand factor. Thus, the region of GPIIIa recognized by D3 may be an important regulatory domain in ligand- receptor interactions that directly mediate platelet aggregation.

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