Carboxyl-Terminal Thrombospondin-Type 1 (TSP-1) Repeats of ADAMTS13 Inhibits Platelet Adhesion to Collagen-Coated Surface Under Shear Stress, Independent of Proteolytic Activity

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 195-195 ◽  
Author(s):  
Juan (Jenny) Xiao ◽  
X. Long Zheng
Keyword(s):  
Shear Stress ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Surface Coverage ◽  
Thrombus Formation ◽  
Microfluidic System ◽  
Dependent Manner ◽  
C Terminus ◽  
Coated Surface

Abstract Abstract 195 ADAMTS13 contains multiple free thiols on its surface, which may form disulfide bonds with surface-exposed free thiols on plasma-derived von Willebrand factor (VWF). This interaction may prevent lateral association of apposed VWF under arterial shear stress. However, the functional consequence of ADAMTS13-VWF interaction without proteolysis is not known. We hypothesize that the interaction between the C-terminus of ADAMTS13 and the C-terminus of VWF inhibits thrombus formation under shear stress. Using a BioFlux microfluidic system, we showed that under arterial shear stress, 10 dyn/cm2, fluorescein-labeled platelets from PPACK (thrombin inhibitor) anti-coagulated human whole blood adhered to collagen (type I)-coated surface in a time-dependent manner. Addition of human recombinant full-length ADAMTS13 (10 nM) into whole blood dramatically reduced the surface coverage of fluorescein-labeled platelets. Conversely, addition of an inhibitory polyclonal anti-ADAMTS13 IgGs (150 ug/ml) to whole blood dramatically accelerated the accumulation of fluorescein-labeled platelets. These results suggest that this microfluidic system is highly sensitive for the assessment of anti-thrombotic function of ADAMTS13. Under the same conditions, we were able to further show that addition of recombinant C-terminal fragment of ADAMTS13 comprising of the 5th to 8th thrombospondin type 1 (TSP1) repeats and two CUB domains (T5C) or the 2nd to 8th TSP1 repeats and two CUB domains (T2C) into whole blood also inhibited the surface coverage of fluorescein-labeled platelets on collagen-coated surface in a concentration-dependent manner. In the presence of 0.1 μM and 0.5 μM of recombinant T2C or T5C, the surface coverage of fluorescein-labeled platelets was reduced by ∼40% and ∼60%, respectively. The inhibitory activity of these recombinant C-terminal fragments was nearly abolished if pre-treated with 40 mM of N-ethylmaleimide which blocked surface-exposed free thiols. Moreover, recombinant CUB domains at the highest concentration tested (1.0 μM) did not appear to alter the surface coverage of fluorecein-labeled platelets under the same conditions. These results suggest that the C-terminal TSP1 repeats of ADAMTS13 inhibit platelet adhesion and aggretion or thrombus formation through thiol-thiol interactions between ADAMTS13 and VWF (or other proteins). We conclude that the C-terminal TSP1 repeats may modulate thrombus formation independent of proteolytic activity. Disclosures: No relevant conflicts of interest to declare.

Neutrophil Extracellular Traps Induce Platelet Adhesion and Thrombus Formation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1345-1345 ◽  
Author(s):  
Tobias Fuchs ◽  
Alexander Brill ◽  
Daniel Dürschmied ◽  
Daphne Schatzberg ◽  
John H. Hartwig ◽  
...  
Keyword(s):  
Whole Blood ◽  
Platelet Adhesion ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Neutrophil Extracellular Traps ◽  
Thrombin Inhibitor ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Extracellular Traps ◽  
Deep Vein

Abstract Abstract 1345 Introduction Thrombus stability is provided by very large polymers adhering to platelets and anchoring the thrombus to the vessel wall. The best described polymers are fibrin and von Willebrand Factor (VWF). Activated neutrophils and other leukocytes can form an extracellular fibrous network which is composed of DNA, histones, and granular proteins. These neutrophil extracellular traps (NETs) are present in various inflammatory diseases. In deep vein thrombosis (DVT) inflammation closely cooperates with thrombosis. Here we examine whether NETs provide a new means to support the adhesion and recruitment of platelets and whether NETs are present in DVT. Methods and Results: To study the interaction of platelets with NETs, we isolated human neutrophils, induced NET formation and perfused over the NETs human platelets in plasma or whole blood anticoagulated with the thrombin inhibitor PPACK. Microscopic analysis revealed that under flow platelets adhere avidly to NETs. Perfusion of whole blood at physiological shear resulted in formation of thrombi on NETs in a time dependent manner. Addition of DNase1 degraded NETs and removed all platelets and thrombi demonstrating their adhesion to NETs. Thrombus formation on NETs was absent if blood was supplemented with EDTA indicating the requirement for divalent cations. Perfusion of NETs with heparinized blood dismantled NETs and prevented thrombus formation. Incubation of NETs with heparin alone released histones from NETs, indicating that heparin destroys the chromatin backbone of NETs. Furthermore, immunocytochemistry revealed that NETs were able to bind platelet adhesion molecules VWF and fibronectin from human plasma. Immunohistochemical analysis of a baboon deep vein thrombus showed abundant extracellular chromatin which co-localized with fibronectin and VWF. Conclusions: We show that extracellular traps are able to promote thrombosis in vitro and are abundant in vivo in DVT. We propose that extracellular chromatin provides a new type of scaffold that promotes platelet adhesion, activation, and aggregation and may be important for thrombus initiation or stability. Disclosures No relevant conflicts of interest to declare.

scholarly journals Platelet-Delivered Recombinant ADAMTS13 Inhibits Platelet Adhesion and Aggregation on Collagen Surface Under Flow in the Presence of Autoantibodies from Acquired Thrombotic Thrombocytopenic Purpura

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1374-1374
Author(s):  
Mohammad S. Abdelgawwad ◽  
Jenny K. McDaniel ◽  
Wenjing Cao ◽  
Huy P. Pham ◽  
Lance A. Williams ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Thrombotic Thrombocytopenic Purpura ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Effective Therapy ◽  
Confocal Imaging ◽  
Dependent Manner ◽  
Thrombocytopenic Purpura ◽  
Platelet Adhesion And Aggregation

Abstract Background: Acquired thrombotic thrombocytopenic purpura (TTP) is a rare but potentially fatal syndrome. It is primarily caused by severe deficiency of plasma ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity. ADAMTS13 is a plasma metalloprotease that cleaves von Willebrand factor (VWF), thereby reducing the platelet adhesion and aggregation under flow. Plasma exchange is the only effective initial therapy for acquired autoimmune TTP. However, in hospital mortality rate remains as high as 10-20% in addition to the concern about the complications associated with the placement of central catheter and the use of plasma products. Therefore, a better and more effective therapy is urgently needed. Objectives: To develop a more effective therapy, we determined the uptake and package of recombinant ADAMTS13 (rA13) in platelets and assessed the efficacy of platelets-delivered rA13 in inhibiting platelet adhesion and aggregation and thrombus formation under arterial shear. Methods: Human platelets were isolated from whole blood, washed, and incubated with various concentrations of rA13 (1-100 microgram/ml) at 25 and 37 degree Celsius for various times. The amount of rA13 uptake by platelets was determined in cell lysate by Western blotting and FRETS-VWF73 assays and in fixed cells by immunofluoresent microscopy and flow cytometry. The function of platelet delivered rA13 was also determined by shear-induced platelet adhesion and aggregation on collagen surface using a microfluidic system. Results: Freshly isolated and blood bank stored platelets were able to update rA13 in a temperature, concentration, and time-dependent manner. When rA13 (5 microgram/ml) was incubated with platelets, nearly all platelets were positive for rA13, assessed by immunofluoresent microscopy and flow cytometry. The rA13 inside platelets remained intact and was proteolytically active in cleaving FRETS-VWF73. Confocal imaging revealed that rA13 was partially co-localized with VWF in alpha granules of platelets. Microfluidic assay demonstrated that platelet-delivered rA13 was able to dramatically inhibit platelet adhesion and aggregation on collagen-coated surface under arterial shear (100 dyne/cm2) in the absence and presence of a human monoclonal antibody against ADAMTS13 (scFv4-20) that was isolated from a patient with acquired autoimmune TTP. These results were consistent with the inhibitory effects observed with mouse transgenic platelets expressing rA13 under the same conditions. When 1/3 of whole blood Adamts13-/- platelets were replaced with transgenic platelets (containing rA13), the coverage of all fluorescent platelets onto a VWF-collagen surface was dramatically reduced (data not shown). Conclusion: Our results demonstrate that platelets uptake and deliver rA13 to the site of thrombus formation under flow and the platelet-delivered rA13 may be efficacious for treating acquired TTP with inhibitors. Disclosures Zheng: Ablynx: Consultancy; Alexion: Research Funding.

Fibrin-incorporated vitronectin is involved in platelet adhesion and thrombus formation through homotypic interactions with platelet-associated vitronectin

Blood ◽  
2004 ◽  
Vol 104 (4) ◽  
pp. 1034-1041 ◽  
Author(s):  
Ya-Ping Wu ◽  
Haiko J. Bloemendal ◽  
Emile E. Voest ◽  
Ton Logtenberg ◽  
Philip G. de Groot ◽  
...  
Keyword(s):  
Whole Blood ◽  
Platelet Adhesion ◽  
Surface Coverage ◽  
Thrombus Formation ◽  
Blood Platelets ◽  
Blood Clot ◽  
Human Monoclonal Antibody ◽  
Fold Increase ◽  
Platelet Surface ◽  
Fibrin Network

AbstractWhen a blood clot is formed, vitronectin (VN) is incorporated. Here we studied the consequence of VN incorporation for platelet interactions under flow. Perfusion of whole blood over a fibrin network, formed from purified fibrinogen, resulted in approximately 20% surface coverage with blood platelets. Incorporation of purified multimeric VN into the fibrin network resulted in a 2-fold increase in surface coverage with platelets and in enhancement of platelet aggregate formation. A human monoclonal antibody (huMab VN18), directed against the multimeric form of VN, inhibited platelet adhesion to the combined fibrin/VN matrix to the level of adhesion on fibrin alone. This inhibition was also shown when whole blood was perfused over a plasma-derived clot. Surprisingly, the inhibitory action of the antibody was not directed toward VN incorporated into the fibrin network but toward VN released from the platelets. We conclude that VN-potentiated platelet-clot interaction requires VN in the clot and multimeric VN bound to the platelet surface. Our results provide evidence that homotypic VN interactions contribute to platelet adhesion and aggregation to a blood clot. This report demonstrates for the first time that self-assembly of VN may provide a physiologically relevant contribution to platelet aggregation on a blood clot.

Abstract 56: Coagulation Factor XI Promotes Distal Platelet Activation and Single Platelet Consumption in the Bloodstream Under Shear Flow

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Jenya Zilberman-Rudenko ◽  
Chantal Wiesenekker ◽  
Asako Itakura ◽  
Owen J McCarty
Keyword(s):  
Shear Flow ◽  
Platelet Activation ◽  
Platelet Adhesion ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Coagulation Factor ◽  
Thrombin Inhibitor ◽  
Dependent Manner ◽  
Factor Xi ◽  
Primate Model

Objective: Coagulation factor XI (FXI) has been shown to contribute to thrombus formation on collagen or tissue factor (TF)-coated surfaces in vitro and in vivo by enhancing thrombin generation. Whether the role of the intrinsic pathway of coagulation is restricted to the local site of thrombus formation is unknown. This study was designed to determine whether FXI could promote both proximal and distal platelet activation and aggregate formation in the bloodstream. Approach and Results: Pharmacological blockade of FXI activation or thrombin activity in blood did not affect local platelet adhesion, yet reduced local platelet aggregation, thrombin localization and fibrin formation on immobilized collagen and TF under shear flow, ex vivo . Downstream of the thrombus formed on immobilized collagen or collagen and 10 pM TF, platelet CD62P expression and microaggregate formation and progressive platelet consumption were significantly reduced in the presence of FXI-function blocking antibodies or a thrombin inhibitor in a shear rate- and time-dependent manner. In a non-human primate model of thrombus formation, we found that inhibition of FXI reduced single platelet consumption in the bloodstream distal to a site of thrombus formation. Conclusions: This study demonstrates that the FXI-thrombin axis contributes to distal platelet activation and procoagulant microaggregate formation in the blood flow downstream of the site of thrombus formation. Our data highlights FXI as a novel therapeutic target for inhibiting distal platelet activation without affecting proximal platelet adhesion.

scholarly journals The Deglycosylated Form of 1E12, a Monoclonal Anti-PF4 IgG, Strongly Inhibits Antibody-Triggered Cellular Activation in Vaccine-Induced Thrombotic Thrombocytopenia, and Is a Potential New Treatment for Vιττ

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 582-582
Author(s):  
Caroline Vayne ◽  
Raghavendra Palankar ◽  
Sandra Billy ◽  
Stefan Handtke ◽  
Thomas Thiele ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Whole Blood ◽  
Research Funding ◽  
Thrombus Formation ◽  
Serotonin Release ◽  
Dependent Manner ◽  
Igg Antibodies ◽  
Cellular Activation ◽  
Cellular Effects ◽  
Dna Release

Abstract Introduction Vaccine-induced thrombotic thrombocytopenia (VITT) is a severe complication of recombinant adenoviral vector vaccines used to prevent COVID-19, likely due to anti-platelet factor 4 (PF4) IgG antibodies. The specificity and platelet-activating activity of VITT antibodies strikingly resemble that of antibodies detected in "autoimmune" heparin-induced thrombocytopenia (HIT), but their features remain poorly characterized. In particular, a better knowledge of these antibodies should help to understand the mechanisms leading to hypercoagulability and the particular thrombotic events observed in VITT, but rarely in typical HIT. We have recently developed a chimeric IgG1 anti-PF4 antibody, 1E12, which strongly mimics "autoimmune" HIT antibodies in terms of specificity and cellular effects. Therefore, we assessed whether 1E12 could mimic VITT antibodies. We then evaluated the capability of DG-1E12, a deglycosylated form of 1E12 unable to bind FcγR, to inhibit cellular activation induced by VITT antibodies. Methods and Results Using a PF4-sensitized serotonin release assay (PF4-SRA) (Vayne C, New Engl J Med, 2021), we demonstrated that 1E12 (5 and 10 μg/mL) strongly activated platelets, with a pattern similar to that obtained with human VITT samples (n=7), i.e. in a PF4-dependent manner and without heparin. This platelet activation was inhibited by low heparin concentration (0.5 IU/mL), an effect also observed with VITT samples. Serotonin release induced by 1E12 was also fully inhibited by IV-3, a monoclonal antibody blocking FcγRIIa, or by IdeS, a bacterial protease that cleaves IgG and strongly inhibits the binding of IgG antibodies to FcγRIIa. This inhibitory effect of IV-3 and IdeS strongly supports that interactions between pathogenic anti-PF4 IgG and FcγRIIa play a central role in VITT. Incubation of 1E12 or VITT samples with isolated neutrophils (PMN) and platelets with PF4 (10 µg/mL) strongly induced DNA release and NETosis, supporting that PMN are involved in the processes leading to thrombosis in VITT. Furthermore, when whole blood from healthy donors incubated with 1E12 or VITT plasma was perfused in capillaries coated with von Willebrand Factor, numerous large platelet/leukocyte aggregates containing fibrin(ogen) were formed. To investigate whether 1E12 and VITT antibodies recognize overlapping epitopes on PF4, we then performed competitive assays with a deglycosylated form of 1E12 (DG-1E12), still able to bind PF4 but not to interact with Fcγ receptors. In PF4-SRA, pre-incubation of DG-1E12 (50 µg/mL) dramatically reduced platelet activation induced by VITT antibodies, which was fully abrogated for 9 of the 14 VITT samples tested. Additional experiments using a whole blood PF4-enhanced flow cytometry assay recently designed for VITT diagnosis (Handtke et al, Blood 2021), confirmed that DG-1E12 fully prevented platelet activation induced by VITT antibodies. Moreover, when platelets and neutrophils were pre-incubated with DG-1E12 (100 µg/mL), NETosis and thus DNA release, nuclear rounding, and DNA decondensation induced by VITT antibodies were completely inhibited. Finally, DG-1E12 (100 µg/mL) also fully abolished VITT antibody-mediated thrombus formation in whole blood in vitro under vein flow conditions. Comparatively, DG-1E12 did not inhibit ALB6, a murine monoclonal anti-CD9 antibody, which also strongly activates platelets in a FcγRIIa-dependent manner. Conclusions Our results show that 1E12 exhibits features similar to those of human VITT antibodies in terms of specificity, affinity and cellular effects, and could therefore be used as a model antibody to study the pathophysiology of VITT. Our data also demonstrate that DG-1E12 prevents blood cell activation and thrombus formation induced by VITT antibodies, likely due to the competitive effect of its Fab fragment on antibody binding to PF4. DG-1E12 may allow the development of a new drug neutralizing the pathogenic effect of autoimmune anti-PF4 antibodies, such as those associated with VITT. Disclosures Thiele: Bristol Myers Squibb: Honoraria, Other; Pfizer: Honoraria, Other; Bayer: Honoraria; Chugai Pharma: Honoraria, Other; Novo Nordisk: Other; Novartis: Honoraria; Daichii Sankyo: Other. Pouplard: Stago: Research Funding. Greinacher: Macopharma: Honoraria; Biomarin/Prosensa: Other, Research Funding; Sagent: Other, Research Funding; Rovi: Other, Research Funding; Gore inc.: Other, Research Funding; Bayer Healthcare: Other, Research Funding; Paringenix: Other, Research Funding; BMS: Honoraria, Other, Research Funding; MSD: Honoraria, Other, Research Funding; Boehringer Ingelheim: Honoraria, Other, Research Funding; Aspen: Honoraria, Other, Research Funding; Portola: Other; Ergomed: Other; Instrument Laboratory: Honoraria; Chromatec: Honoraria. Gruel: Stago: Other: symposium fees, Research Funding. Rollin: Stago: Research Funding.

scholarly journals Recognition of PF4-VWF complexes by heparin-induced thrombocytopenia antibodies contributes to thrombus propagation

Blood ◽  
2020 ◽  
Vol 135 (15) ◽  
pp. 1270-1280 ◽  
Author(s):  
Ian Johnston ◽  
Amrita Sarkar ◽  
Vincent Hayes ◽  
Gavin T. Koma ◽  
Gowthami M. Arepally ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Complex Formation ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Microfluidic System ◽  
Microfluidic Channels ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by complexes between platelet factor 4 (PF4) and heparin or other polyanions, but the risk of thrombosis extends beyond exposure to heparin implicating other PF4 partners. We recently reported that peri-thrombus endothelium is targeted by HIT antibodies, but the binding site(s) has not been identified. We now show that PF4 binds at multiple discrete sites along the surface of extended strings of von Willebrand factor (VWF) released from the endothelium following photochemical injury in an endothelialized microfluidic system under flow. The HIT-like monoclonal antibody KKO and HIT patient antibodies recognize PF4-VWF complexes, promoting platelet adhesion and enlargement of thrombi within the microfluidic channels. Platelet adhesion to the PF4-VWF-HIT antibody complexes is inhibited by antibodies that block FcγRIIA or the glycoprotein Ib-IX complex on platelets. Disruption of PF4-VWF-HIT antibody complexes by drugs that prevent or block VWF oligomerization attenuate thrombus formation in a murine model of HIT. Together, these studies demonstrate assembly of HIT immune complexes along VWF strings released by injured endothelium that might propagate the risk of thrombosis in HIT. Disruption of PF4-VWF complex formation may provide a new therapeutic approach to HIT.

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.

scholarly journals New Insights into the Prothrombotic State of Heparin-Induced Thrombocytopenia (HIT) Using a Novel Microfluidic System: The Endothelial Lining and “Rolling Barrage” of Activation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 574-574
Author(s):  
Vincent Hayes ◽  
Ian Johnston ◽  
Douglas B. Cines ◽  
Lubica Rauova ◽  
Mortimer Poncz
Keyword(s):  
Vascular Injury ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Blood Platelets ◽  
Microfluidic System ◽  
Confocal Imaging ◽  
Prothrombotic State ◽  
Platelet Factor ◽  
Sequence Of Events ◽  
Local Sequence

Abstract The most feared feature of HIT is antibody-mediated thrombosis. We have shown that this prothrombotic state is related to binding of platelet factor 4 (PF4), a chemokine densely packaged into platelet alpha-granules, to surface glycosaminoglycans (GAGs) expressed on hematopoietic and vascular cells. PF4/GAG surface complexes are recognized by HIT antibodies, activating the targeted cells. Unlike platelets that express only low-affinity chondroitin sulfate surface GAGs, endothelial cells (EC) express a glycocalyx enriched in heparan sulfate, which has higher affinity for PF4, potentially increasing their propensity to become a target for immune injury leading to thrombosis. We examined the details of the development of in situ thrombi using the cremaster arteriole laser injury model beginning with transgenic mice expressing only human PF4 (hPF4+), but lacking FcγRIIA. These mice do not develop thrombocytopenia or thrombosis when injected with the HIT-like monoclonal antibody KKO or IgGs isolated from patients with HIT. In these mice, antigenic PF4/GAG complexes were recognized by KKO at sites of vascular injury even in the absence of infused heparin. In fact, infusion of sufficient heparin dissociated PF4 from sites of injury, consistent with its higher affinity for PF4 than cell surface GAGs. This suggests that antigenic PF4/GAG complexes normally develop intravascularly whenever thrombus formation occurs, yet these complexes do not typically initiate antibody-mediated thrombosis. Real-time confocal imaging of injured vessels revealed that PF4 first bound almost exclusively to the peri-injury endothelium. This was especially evident immediately upstream of the thrombus where turbulent blood flow may lead to platelet degranulation and subsequent adherence of the released PF4 to the glycocalyx. Beginning approximately two minutes post-injury, binding of KKO, presumably to PF4/GAG complexes on platelets, is seen at the interface between the shell and core of the thrombus. We then repeated these same studies in hPF4+/FcγRIIA+ mice, where infused KKO or HIT IgGs leads to significant thrombocytopenia and widespread development of thrombi as in HIT. Similar adherence of PF4 to the peri-injury EC and then to the core/shell interface of the thrombus as seen in the hPF4+ mice, but the changes were more extensive in hPF4+/FcγRIIA+ mice after KKO infusion and often lead to vascular occlusion. To further define the basis of the prothrombotic state in HIT and to extend our studies to a human system, we examined thrombus formation in HIT in a novel microfluidic system in which vascular injury was induced in an upstream portion of a human umbilical vein EC-lined channel by reactive oxygen species generated through excitation of infused hematoporphyrin by blue light (490 nm). Following infusion of human blood, platelets accumulated and released PF4, which bound the injured endothelium, while the downstream endothelium remained quiescent. Addition of KKO to the infused whole blood lead to a HIT-like state with marked increase in platelet adhesion and binding of PF4 to the injured endothelium, but binding of PF4 now spread downstream of the boundary between injured and uninjured endothelium. This was followed by downstream platelet adhesion and often occlusion of the channel. We proposed that this spread in EC injury was a result of a “rolling barrage” of PF4 released from platelets binding to the injured patch of EC complexing to the downstream glycocalyx on the non-injured endothelium followed by KKO binding and subsequent endothelial activation. The newly activated ECs bound additional platelets and the process repeats, rolling downstream and extending thrombus growth. Thus, these studies provide important new insights into the local sequence of events that propagate clots in HIT: Targeting of the endothelial glycocalyx by HIT antibodies is a major contributor to the prothrombotic state. Platelets adherent to the site of original injury release PF4, which then binds to downstream EC glycocalyx and initiates repetitive cycles of PF4 binding, EC activation and platelet adherence, and further release of PF4 that propagates growth of thrombi to previously uninvolved vasculature. Disclosures No relevant conflicts of interest to declare.

STAT3 Regulates Collagen-Induced Platelet Aggregation Independent of Its Transcription Factor Activity

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 157-157
Author(s):  
Zhou Zhou ◽  
Francisca C. Gushiken ◽  
Angela Bergeron ◽  
Vinod K. Vijayan ◽  
Rolando Rumbaut ◽  
...  
Keyword(s):  
Shear Stress ◽  
Transcription Factor ◽  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Dependent Manner ◽  
Transcription Factor Activity ◽  
Factor Activity ◽  
Dose Dependent

Abstract Abstract 157 Signal Transducer and Activator of Transcription 3 (STAT3) serves as a transcription factor activated by cytokine-induced intracellular signals, which are critical in megakaryopoiesis. This signaling pathway may also be active in anucleated platelets that are primed by proinflammatory cytokines, suggesting that STAT3 plays a role in platelet hyperactivity associated with inflammation. We have recently found that three different classes of STAT3 inhibitors each selectively inhibited collagen-induced aggregation of human platelets by ∼50%. They also blocked thrombus formation (∼80%) on immobilized collagen under an arterial shear stress of 62.5 dyn/cm2. These STAT3 inhibitors also blocked platelet aggregation induced by collagen-related peptide, suggesting that they acted on GP VI-mediated intracellular signaling in platelets. These in vitro results were further verified in two sets of experiments in mouse models. First, an oligonucleotide G-quartet STAT3 inhibitor (1 mg/ml) or a scrambled control oligonucleotide were infused into C57/BJ6 mice daily for three days. Collagen-induced platelet aggregation was then induced and found to be reduced by up to 60% in mice infused with the STAT3 inhibitor, but not with the control oligonucleotide. Photochemical injury-induced thrombosis in the cremaster arterioles was also significantly delayed in the inhibitor-infused mice as compared to control mice. Second, infusing STAT3 inhibitor could result in platelet inhibitor indirectly by acting endothelial cells. To address this concern, we have generated platelet-specific STAT3 null mice that have developed normally and have normal platelet counts. The collagen-, but not TRAP-induced platelet aggregation in the platelet STAT3 KO mice was reduced as compared to their littermates. Platelets from the platelet-specific STAT3 KO mice were also significantly defective in thrombus formation on immobilized collagen under 62.5 dyn/cm2 of fluid shear stress that was generated in a parallel-plate flow chamber system. Consistent with results from these functional assays, collagen induced rapid (peaked at 5 min after stimulation) and dose-dependent tyrosine phosphorylation of STAT3, but not of STAT1 or STAT5 in washed human platelets. The phosphorylation was blocked dose-dependently by two STAT3 inhibitors. Syk inhibitors also blocked collagen-induced STAT3 phosphorylation in a dose-dependent manner, but STAT3 inhibitors had no effect on Syk phosphorylation, suggesting that Syk acts upstream of STAT3. Furthermore, STAT3 inhibitors also dose-dependently reduced collagen-induced tyrosine phosphorylation of PLCγ2, which is a known substrate of Syk. Consistent with this temporal interaction among STAT3, Syk and PLCγ2, activated STAT3 co-immunoprecipitated phosphorylated Syk and PLCγ2 in collagen-activated human platelets. The tri-molecular complex was also immunoprecipitated by an antibody to PLCγ2. Taken together, these data suggest that STAT3 regulates collagen-induced platelet aggregation, independent of its transcription factor activity. The regulation is potentially achieved by STAT3 serving as a protein scaffold linking the kinase Syk with its substrate PLCγ2 to enhance the signal relay in collagen-activated platelets. This cross-talk between collagen and cytokine signaling pathways provides a mechanism for how proinflammatory mediators could prime platelets for activation by hemostatic ligands. Disclosures: No relevant conflicts of interest to declare.

Microfluidic and Flow Cytometric Studies Support a Role for Monocytes and Coated Platelets in the Prothrombotic State in Heparin-Induced Thrombocytopenia (HIT)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 539-539
Author(s):  
Valerie Tutwiler ◽  
Hyun Sook Ahn ◽  
Douglas B. Cines ◽  
Rodney M. Camire ◽  
Mortimer Poncz ◽  
...  
Keyword(s):  
Whole Blood ◽  
Platelet Adhesion ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Passive Immunization ◽  
Annexin V ◽  
Thrombin Inhibitor ◽  
Prothrombotic State ◽  
Flow Cytometric ◽  
Activated State

Abstract Abstract 539 HIT is an immune thrombocytopenia associated with a high risk of developing thrombosis. A passive immunization murine model of this disorder has provided important insights into the underlying pathogenesis of this disease, but is limited by its inability to study human cells and limited ability to define the contribution of various hematopoeitic and vascular cells to the prothrombotic state. We used a microfluidic system in conjunction with flow cytometry to further our understanding of the prothrombotic nature of HIT. Platelet adhesion and aggregation was studied in whole blood labeled with Calcein AM, perfused through a microfluidic channel (BioFlux 200 system, Fluxion) coated with von Willebrand factor (vWf) at shear stress of 20 dyne/cm2 at 37°C. A 40–60% increase in platelet adhesion (relative area covered by platelets) with up to a 4 fold increase in average aggregate size was seen in the presence of the pathogenic HIT-like monoclonal antibody (moAb) KKO (50 μg/ml) in conjunction with PF4 (10 μg/ml) when compared to control samples with PF4 only or with PF4 plus a non-pathogenic anti-PF4 moAb RTO (p <0.01). Monocyte-depletion decreased platelet aggregation by 20 – 40% relative to whole blood or after monocyte-repletion (P<0.0001). In HIT, thrombin plays a key role in the formation of platelet aggregates. Addition of thrombin inhibitor PPACK to the whole blood stimulated by KKO and PF4 decreased thrombus formation in the microfluidic chamber by 40% (p<0.001). Coated platelets are prothrombotic and characterized by phosphatidylserine (PS) exposure and binding of FVa and FXa. This activated state requires dual stimulation via thrombin and ITAM receptors. Flow cytometric studies of annexin V and FXa binding showed extensive induction of coated platelets in whole blood by KKO plus PF4 in contrast to PF4 or PF4 plus RTO (annexin V: p<0.0001; Factor Xa p<0.01). These new studies, focused on human blood, support our finding in the passive murine HIT model as to the importance of monocytes to thrombus formation and suggest that the prothrombotic nature of HIT may also be promoted by the generation of coated platelets. Identification of coated platelets may also lead to new diagnostic tests and new therapeutic interventions in HIT. Disclosures: No relevant conflicts of interest to declare.

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