Control of thrombus embolization and fibronectin internalization by integrin αIIbβ3 engagement of the fibrinogen γ chain

AbstractFibrin(ogen) deficiency (Fg-/-) was shown previously to be compatible with rapid thrombus growth within injured arterioles, but platelet fibronectin content was increased and newly formed thrombi were unstable. To further define the role of fibrin(ogen) in thrombus formation and stabilization, platelet biology was examined in mice expressing a form of fibrinogen that clots normally but lacks the γ chain C-terminal binding site for αIIbβ3 (FgγΔ5). Thrombus growth within the arterioles of FgγΔ5 mice appeared faster than in wild-type mice despite a far greater emboli formation. Unlike Fg-/- mice, the emboli were relatively small and released from the top of thrombi, rather than by fracture at the vessel wall. The fibronectin content in FgγΔ5 platelets was also dramatically increased through a β3 integrin-dependent mechanism. The following has been concluded: (1) Fibrin formation contributes to, but is not sufficient for, the stabilization of arterial thrombi. Platelet receptor engagement of the C-terminal of the Fg γ chain contributes to the stable incorporation of platelets into thrombi. (2) Alternative ligands to fibrinogen can support efficient thrombus growth. (3) Fibrinogen is internalized through αIIbβ3 engagement of the fibrinogen γ chain element, and this interaction secondarily controls the fibronectin content of platelets. (Blood. 2003;102: 3609-3614)

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Differential Role of Components of the Fibrinolytic System in the Formation and Removal of Thrombus Induced by Endothelial Injury

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614532 ◽

1999 ◽

Vol 81 (04) ◽

pp. 601-604 ◽

Cited By ~ 55

Author(s):

Hiroyuki Matsuno ◽

Osamu Kozawa ◽

Masayuki Niwa ◽

Shigeru Ueshima ◽

Osamu Matsuo ◽

...

Keyword(s):

Plasminogen Activator ◽

Thrombus Formation ◽

Endothelial Injury ◽

Fibrinolytic System ◽

Tissue Type ◽

Clot Lysis ◽

Wild Type ◽

Type Plasminogen Activator ◽

Pai 1

SummaryThe role of fibrinolytic system components in thrombus formation and removal in vivo was investigated in groups of six mice deficient in urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), or plasminogen activator inhibitor-1 (PAI-1) (u-PA-/-, t-PA-/- or PAI-1-/-, respectively) or of their wild type controls (u-PA+/+, t-PA+/+ or PAI-1+/+). Thrombus was induced in the murine carotid artery by endothelial injury using the photochemical reaction between rose bengal and green light (540 nm). Blood flow was continuously monitored for 90 min on day 0 and for 20 min on days 1, 2 and 3. The times to occlusion after the initiation of endothelial injury in u-PA+/+, t-PA+/+ or PAI-1+/+ mice were 9.4 ± 1.3, 9.8 ± 1.1 or 9.7 ± 1.6 min, respectively. u-PA-/- and t-PA-/- mice were indistinguishable from controls, whereas that of PAI-1-/- mice were significantly prolonged (18.4 ± 3.7 min). Occlusion persisted for the initial 90 min observation period in 10 of 18 wild type mice and was followed by cyclic reflow and reocclusion in the remaining 8 mice. At day 1, persistent occlusion was observed in 1 wild type mouse, 8 mice had cyclic reflow and reocclusion and 9 mice had persistent reflow. At day 2, all injured arteries had persistent reflow. Persistent occlusion for 90 min on day 0 was observed in 3 u-PA-/-, in all t-PA-/- mice at day 1 and in 2 of the t-PA-/-mice at day 2 (p <0.01 versus wild type mice). Persistent patency was observed in all PAI-1-/- mice at day 1 and in 5 of the 6 u-PA-/- mice at day 2 (both p <0.05 versus wild type mice). In conclusion, t-PA increases the rate of clot lysis after endothelial injury, PAI-1 reduces the time to occlusion and delays clot lysis, whereas u-PA has little effect on thrombus formation and spontaneous lysis.

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The Two ADF-H Domains of Twinfilin Play Functionally Distinct Roles in Interactions with Actin Monomers

Molecular Biology of the Cell ◽

10.1091/mbc.e02-03-0157 ◽

2002 ◽

Vol 13 (11) ◽

pp. 3811-3821 ◽

Cited By ~ 55

Author(s):

Pauli J. Ojala ◽

Ville O. Paavilainen ◽

Maria K. Vartiainen ◽

Roman Tuma ◽

Alan G. Weeds ◽

...

Keyword(s):

Binding Site ◽

Binding Activity ◽

Size Exclusion ◽

Actin Dynamics ◽

Actin Binding ◽

Actin Monomer ◽

Wild Type ◽

Native Page ◽

Exclusion Chromatography

Twinfilin is a ubiquitous and abundant actin monomer–binding protein that is composed of two ADF-H domains. To elucidate the role of twinfilin in actin dynamics, we examined the interactions of mouse twinfilin and its isolated ADF-H domains with G-actin. Wild-type twinfilin binds ADP-G-actin with higher affinity (K D = 0.05 μM) than ATP-G-actin (K D = 0.47 μM) under physiological ionic conditions and forms a relatively stable (k off = 1.8 s−1) complex with ADP-G-actin. Data from native PAGE and size exclusion chromatography coupled with light scattering suggest that twinfilin competes with ADF/cofilin for the high-affinity binding site on actin monomers, although at higher concentrations, twinfilin, cofilin, and actin may also form a ternary complex. By systematic deletion analysis, we show that the actin-binding activity is located entirely in the two ADF-H domains of twinfilin. Individually, these domains compete for the same binding site on actin, but the C-terminal ADF-H domain, which has >10-fold higher affinity for ADP-G-actin, is almost entirely responsible for the ability of twinfilin to increase the amount of monomeric actin in cosedimentation assays. Isolated ADF-H domains associate with ADP-G-actin with rapid second-order kinetics, whereas the association of wild-type twinfilin with G-actin exhibits kinetics consistent with a two-step binding process. These data suggest that the association with an actin monomer induces a first-order conformational change within the twinfilin molecule. On the basis of these results, we propose a kinetic model for the role of twinfilin in actin dynamics and its possible function in cells.

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Abstract 487: Systemic Administration of Target-Seeking, Vessel Penetrating Recombinant Decorin Fusion Protein, Car-DCN, Reduces Severity of Abdominal Aortic Aneurysm in Mice

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.487 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Yue (Steve) Shen ◽

Valerio Russo ◽

Matthew Zeglinski ◽

Stephanie Sellers ◽

Zhengguo Wu ◽

...

Keyword(s):

Abdominal Aortic Aneurysm ◽

Aortic Aneurysm ◽

Fusion Protein ◽

Vessel Wall ◽

C Terminus ◽

Abdominal Aortic ◽

Homing Peptide ◽

Apoe Ko Mice ◽

Dependent Mechanism

Objective: Decorin (DCN) is a small leucine-rich proteoglycan that mediates collagen fibrollogenesis, organization, and tensile strength. DCN is reduced in abdominal aortic aneurysm (AAA) through a Granzyme B-dependent mechanism resulting in vessel wall instability and aneurysm formation. A recombinant decorin fusion protein CAR-DCN was engineered with an extended C-terminus comprised of CAR homing peptide that recognizes inflammatory blood vessels and penetrates deep into the vessel wall. In the present study, we sought to evaluate the role of systemically administered CAR-DCN in AAA progression and rupture rate in a murine model. Approach and Results: To induce aneurysm, apolipoprotein E knockout (ApoE-KO) mice were infused with 28 days of angiotensin II (AngII). CAR-DCN or vehicle was systemically administrated until day 15. We observed a significant increase in the survival of CAR-DCN-treated mice (93%) compared to vehicle controls (60%). Although the incidence of AAA onset was similar between vehicle and CAR-DCN groups, the severity of aneurysm in the CAR-DCN group was significantly reduced. Furthermore, histological analysis revealed that CAR-DCN treatment significantly increased DCN and collagen levels in aortic walls compares to vehicle controls. Conclusions: CAR-DCN administration attenuated the severity of Ang II-induced AAA in mice by reinforcing the vessel wall. CAR-DCN may represent a novel therapeutic strategy to attenuate AAA progression and rupture.

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Role of eNOS-derived NO in the postischemic anti-inflammatory effects of antecedent ethanol ingestion in murine small intestine

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00282.2006 ◽

2007 ◽

Vol 292 (3) ◽

pp. H1435-H1442 ◽

Cited By ~ 19

Author(s):

Taiji Yamaguchi ◽

Kazuhiro Kamada ◽

Catherine Dayton ◽

F. Spencer Gaskin ◽

Mozow Yusof ◽

...

Keyword(s):

Ethanol Exposure ◽

Ethanol Ingestion ◽

Leukocyte Rolling ◽

Wild Type ◽

No Donor ◽

Nos Inhibitors ◽

Low Levels ◽

Ethanol Preconditioning ◽

Dependent Mechanism

Ingestion of low levels of ethanol 24 h before [ethanol preconditioning (EPC)] ischemia and reperfusion (I/R) prevents postischemic leukocyte rolling (LR) and adhesion (LA), effects that were abolished by adenosine A2 receptor (ADO-A2R) antagonists or nitric oxide (NO) synthase (NOS) inhibitors. The aims of this study were to determine whether NO derived from endothelial NOS (eNOS) during the period of ethanol exposure triggered entrance into this preconditioned state and whether these events were initiated by an ADO-A2R-dependent mechanism. Ethanol or distilled water vehicle was administered to C57BL/6J [wild type (WT)] or eNOS-deficient (eNOS−/−) mice by gavage. Twenty-four hours later, the superior mesenteric artery was occluded for 45 min. LR and LA were quantified by intravital microscopy after 30 and 60 min of reperfusion. I/R increased LR and LA in WT mice, effects that were abolished by EPC or NO donor preconditioning (NO-PC). NO-PC was not attenuated by coincident administration of an ADO-A2R antagonist. I/R increased LR and LA in eNOS−/− mice to levels comparable with those noted in WT animals. However, EPC only slightly attenuated postischemic LR and LA, whereas NO-PC remained effective as a preconditioning stimulus in eNOS−/− mice. Preconditioning with an ADO-A2R agonist (which we previously demonstrated prevents I/R-induced LR and LA in WT animals) failed to attenuate these postischemic adhesive responses in eNOS−/− mice. Our results indicate that EPC is triggered by NO formed secondary to ADO-A2R-dependent eNOS activation during the period of ethanol exposure 24 h before I/R.

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Characterization of Integrin αIIbβ3-Mediated Outside-in Signaling by Protein Kinase Cδ in Platelets

International Journal of Molecular Sciences ◽

10.3390/ijms21186563 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6563

Author(s):

Preeti Kumari Chaudhary ◽

Sanggu Kim ◽

Youngheun Jee ◽

Seung-Hun Lee ◽

Soochong Kim

Keyword(s):

Protein Kinase ◽

Kinase Inhibitor ◽

Wild Type ◽

Clot Retraction ◽

Integrin Αiibβ3 ◽

Tyrosine Kinase 2 ◽

Platelet Spreading ◽

Protein Kinase Cδ

Engagement of integrin αIIbβ3 promotes platelet–platelet interaction and stimulates outside-in signaling that amplifies activation. Protein kinase Cδ (PKCδ) is known to play an important role in platelet activation, but its role in outside-in signaling has not been established. In the present study, we determined the role of PKCδ and its signaling pathways in integrin αIIbβ3-mediated outside-in signaling in platelets using PKCδ-deficient platelets. Platelet spreading to immobilized fibrinogen resulted in PKCδ phosphorylation, suggesting that αIIbβ3 activation caused PKCδ activation. αIIbβ3-mediated phosphorylation of Akt was significantly inhibited in PKCδ -/- platelets, indicating a role of PKCδ in outside-in signaling. αIIbβ3-mediated PKCδ phosphorylation was inhibited by proline-rich tyrosine kinase 2 (Pyk2) selective inhibitor, suggesting that Pyk2 contributes to the regulation of PKCδ phosphorylation in outside-in signaling. Additionally, Src-family kinase inhibitor PP2 inhibited integrin-mediated Pyk2 and PKCδ phosphorylation. Lastly, platelet spreading was inhibited in PKCδ -/- platelets compared to the wild-type (WT) platelets, and clot retraction from PKCδ -/- platelets was markedly delayed, indicating that PKCδ is involved in the regulation of αIIbβ3-dependent interactivities with cytoskeleton elements. Together, these results provide evidence that PKCδ plays an important role in outside-in signaling, which is regulated by Pyk2 in platelets.

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Macrovascular thrombosis is driven by tissue factor derived primarily from the blood vessel wall

Blood ◽

10.1182/blood-2004-06-2225 ◽

2005 ◽

Vol 105 (1) ◽

pp. 192-198 ◽

Cited By ~ 212

Author(s):

Sharlene M. Day ◽

Jennifer L. Reeve ◽

Brian Pedersen ◽

Diana M Farris ◽

Daniel D. Myers ◽

...

Keyword(s):

Blood Vessel ◽

Tissue Factor ◽

Vessel Wall ◽

Thrombus Formation ◽

Vena Cava ◽

Vascular Wall ◽

Blood Vessel Wall ◽

Wild Type

Abstract Leukocytes and leukocyte-derived microparticles contain low levels of tissue factor (TF) and incorporate into forming thrombi. Although this circulating pool of TF has been proposed to play a key role in thrombosis, its functional significance relative to that of vascular wall TF is poorly defined. We tested the hypothesis that leukocyte-derived TF contributes to thrombus formation in vivo. Compared to wild-type mice, mice with severe TF deficiency (ie, TF–/–, hTF-Tg+, or “low-TF”) demonstrated markedly impaired thrombus formation after carotid artery injury or inferior vena cava ligation. A bone marrow transplantation strategy was used to modulate levels of leukocyte-derived TF. Transplantation of low-TF marrow into wild-type mice did not suppress arterial or venous thrombus formation. Similarly, transplantation of wild-type marrow into low-TF mice did not accelerate thrombosis. In vitro analyses revealed that TF activity in the blood was very low and was markedly exceeded by that present in the vessel wall. Therefore, our results suggest that thrombus formation in the arterial and venous macrovasculature is driven primarily by TF derived from the blood vessel wall as opposed to leukocytes.

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Endobrevin/VAMP-8-Mediated Dense Granule Release Is Required for Efficient Thrombus Formation in Vivo.

Blood ◽

10.1182/blood.v112.11.1836.1836 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1836-1836

Author(s):

Price S. Blair ◽

Qiansheng Ren ◽

Gwenda J. Graham ◽

James R. Dilks ◽

Sidney W. Whiteheart ◽

...

Keyword(s):

Vascular Injury ◽

Thrombus Formation ◽

Dense Granule ◽

Wild Type ◽

Platelet Accumulation ◽

Induced Aggregation ◽

Kinetics Of ◽

Granule Release

Abstract Individuals whose platelets lack dense core or alpha-granules suffer varying degrees of abnormal bleeding, implying that granule cargo contributes to hemostasis. Despite these clinical observations, little is known regarding the effects of impaired platelet granule secretion on thrombus formation in vivo. The release of cargo from platelet granules requires a group of membrane proteins called SNAREs (Soluble NSF Attachment Protein Receptors) that mediate fusion of granule membranes to the plasma membrane and open canalicular system. Endobrevin/VAMP-8 is the primary vesicular-SNARE (v-SNARE) responsible for efficient release of dense core and a-granule contents. To evaluate the importance of VAMP-8-mediated secretion on the kinetics of thrombus formation in vivo, we measured platelet accumulation following laser-induced vascular injury in VAMP-8−/− mice. Three different phases of thrombus formation - initiation, maximal accumulation, and stabilized platelet accumulation - were tested. Analysis of initial thrombus formation from wild-type and VAMP-8−/− mice showed that average platelet accumulation in VAMP- 8−/− mice was 23% of accumulation in wild-type mice (P=0.009) at 30 sec following injury. There was a trend towards smaller maximal thrombus size in VAMP-8−/− mice, but the difference was not statistically significant (P=0.1). Average stabilized platelet accumulation at 180 sec in VAMP-8−/− mice was 40% of wild-type mice (P=0.05). Thus, thrombus formation is delayed and decreased in VAMP-8−/− mice, but not absent. Dense granule release occurs more rapidly than alpha-granule release, which does not occur for 2–3 min following laser-induced vascular injury. Agonist-induced dense granule release from VAMP-8−/− platelets is defective. To directly evaluate the role of dense granule release on the kinetics of thrombus formation, we assessed thrombus formation in the mouse model of Hermansky-Pudlak syndrome, ruby-eye, which lack dense granules. Thrombus formation following laser-induced vascular injury was nearly abolished in ruby-eye mice such that maximal platelet accumulation was 15% that of wild-type mice. In vitro, the thrombin doses required to induce irreversible aggregation in wild-type, VAMP-8−/−, and ruby-eye platelets were 25 mU, 50 mU, and 150 mU, respectively. Incubation with apyrase had little effect on thrombin-induced aggregation of VAMP-8−/− or ruby-eye platelets. In contrast, incubation of wild-type platelets with apyrase reduced their thrombin sensitivity compared to that of ruby-eye platelets. Supplementation with a substimulatory ADP concentration reversed the thrombin-induced aggregation defect in VAMP-8−/− and ruby-eye mice. Thus, defective ADP release is the primary abnormality leading to impaired aggregation in VAMP-8−/− and ruby-eye mice. Tail bleeding times were assessed in VAMP- 8−/− mice to evaluate the role of VAMP-8 in hemostasis. In contrast to ruby-eye mice, which have a markedly prolonged bleeding time, tail bleeding times in VAMP-8−/− mice were not significantly prolonged compared to those in wild-type mice. These results demonstrate the importance of VAMP-8 and dense granule release in the initial phases of thrombus formation and validate the distal platelet secretory machinery as a potential target for anti-platelet therapies.

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Agonist-Induced Kindlin-3 Phsphorylation Regulates αIIbβ3 Integrin Activation In HEL Cells and Platelets

Blood ◽

10.1182/blood.v122.21.22.22 ◽

2013 ◽

Vol 122 (21) ◽

pp. 22-22

Author(s):

Katarzyna Bialkowska ◽

Eugene Podrez ◽

Tatiana V. Byzova ◽

Edward F. Plow

Keyword(s):

Conflicts Of Interest ◽

Focal Adhesions ◽

Human Platelets ◽

Phosphorylation Sites ◽

Integrin Activation ◽

Suspension Cells ◽

Integrin Αiibβ3 ◽

Hel Cells ◽

Β3 Integrin

Abstract The contributions of integrins to platelet responses depend upon the dynamic regulation of their activation status, which in turn depends on engagement of binding partners by their cytoplasmic tails. It is well-established that not only talin but also kindlin family members are essential for integrin activation, and both must present for optimal integrin function. Recent studies in humans have specifically emphasized the vital role of kindlin-3 in integrin functions in hematopoietic cells, including platelets, where kindlin-3 deficiency can lead to episodic bleeding, frequent infections and osteopetrosis, consequences of an inability to activate β1, β2 and β3 integrins. Despite this evidence, little is known about kindlin-3 structure-function relationship. Here, we used human platelets and human erythroleukemic HEL cell line that expresses integrin αIIbβ3 to investigate whether posttranslational modification(s) of kindlin-3 occurs and can influence its integrin activity. Non-stimulated HEL cells are suspension cells, and they do not adhere to fibrinogen or bind soluble fibrinogen and PAC-1 antibody (specific for activated αIIbβ3) readily. Thrombopoietin or PMA stimulation activated αIIbβ3 such that the cells adhered and spread on fibrinogen and increased their binding of PAC-1 and soluble fibrinogen. β3 integrin and kindlin-3 colocalized in focal adhesions in the adherent cells, and there was enhanced β3 integrin-kindlin-3 association as detected by coimmunoprecipitation. Kindlin-3 knockdown impaired agonist-stimulated adhesion and spreading on fibrinogen. Since, as we have shown previously, β3 integrin phosphorylation regulates kindlin and integrin interaction, we sought to determine whether kindlin-3 is also phosphorylated. Human platelets were stimulated with thrombin and HEL cells with PMA, and kindlin-3 was immunoprecipitated from lysates of control and stimulated cells. A kindlin-3 peptide showing significant increase in phosphorylation upon agonist stimulation was identified in both platelets and HEL cells by mass spectrometry. T482 or S484 were identified as phosphorylation sites in sequence that resides in the kindlin-3 variable region, which is not present either in kindlin-1 or kindlin-2 but is conserved across all species in which kindlin-3 has been sequenced. When expressed in HEL cells, TS/AA kindlin-3 mutant displayed decreased soluble fibrinogen binding and cell spreading on immobilized fibrinogen when compared to wild-type kindlin-3. Membrane-permeable, poly-arginine tagged kindlin-3 peptide containing the candidate phosphorylation sites kindlin-3 was introduced into HEL cells and platelets. HEL cell adhesion and spreading was blunted by the kindlin-3 peptide when compared to a scramble poly-arginine control peptide. Moreover, thrombin-induced platelet aggregation was inhibited by kindlin-3 peptide but not by the scramble peptide. Thus, our data emphasizes a role of previously unknown, agonist-induced kindlin-3 phosphorylation, in integrin αIIbβ3 activation in HEL cells and platelets and provides a basis for functional differences between kindlin-3 and its other two paralogs, kindlin-1 and kindlin-2. Disclosures: No relevant conflicts of interest to declare.

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Platelet-Mediated Mechanical Tensile Force Influences ADAMTS13 Localization and Regulation Of Thrombus Development At The Site Of Platelet Accumulation

Blood ◽

10.1182/blood.v122.21.454.454 ◽

2013 ◽

Vol 122 (21) ◽

pp. 454-454

Author(s):

Yasuaki Shida ◽

Laura L. Swystun ◽

Christine Brown ◽

Jeff Mewburn ◽

Kate Sponagle ◽

...

Keyword(s):

Thrombus Formation ◽

Tensile Force ◽

Flow Chamber ◽

High Shear ◽

Wild Type ◽

Hydrodynamic Shear ◽

Chamber Model ◽

Mechanical Tensile ◽

Very High

Background The multimeric glycoprotein von Willebrand factor (VWF) mediates platelet adhesion and aggregation at the site of vessel injury. The adhesive property of VWF is regulated by its multimer length, such that ultra large VWF (ULVWF) multimers, newly released from the endothelium, have greater hemostatic activity. multimer size is regulated by the metalloprotease ADAMTS13, which cleaves the A2 domain to reduce VWF multimer size and functional activity. static conditions, VWF maintains a globular conformation and the ADAMTS13 cleavage site is inaccessible. However, the exposure of endothelial-anchored VWF to tensile forces mediated by platelets and hydrodynamic shear enhance the cleavage of VWF by ADAMTS13. releases VWF of optimal hemostatic length from the endothelium into the plasma. We have previously reported using a flow chamber model which demonstrates that in addition to regulating VWF length and activity at the site of release, ADAMTS13 also associates with VWF at the site of thrombus formation. observed that under conditions of high and very high shear, ADAMTS13 reduced the size of thrombus volume., multi-coloured immunostaining revealed that ADAMTS13 co-localized with VWF and platelets at the top and middle layers of the thrombus, in the presence of very high shear. Aim To better understand the mechanism by which ADAMTS13 regulates thrombus size in our flow chamber model, we assessed the contribution of platelet tensile force to the localization of ADAMTS13 at the site of the thrombus. this model, the contributions of platelet GPIb, GPIIbIIIa, and P-selectin to ADAMTS13 localization were observed. Method Full length mouse VWF and ADAMTS13 cDNA were cloned into pCIneo and pcDNA3.1 plasmid, respectively. The gain of platelet GPIb binding mutation V1316M, and loss of GPIIbIIIa binding mutation (RGD to RGG) were introduced by site-directed-mutagenesis. mCherry was cloned at the C terminus of ADAMTS13 with a 12AA linker. Recombinant mVWF and mADAMTS13-mCherry proteins were produced via HEK293T cells by calcium phosphate transient transfection. mADAMTS13-mCherry (2 U/mL) and wild type or mutant mVWF (4 U/mL) was added to whole blood obtained from VWF-/-/ADAMTS13-/- double knockout mice. Whole blood containing DiOC6-labeled platelets was perfused over a collagen coated flow chamber at very high shear (7500s-1). The role of P-selectin was also analyzed by adding a P-selectin blocking antibody to blood obtained from ADAMTS13-/-knockout mice prior to the flow chamber experiment. After the perfusion, thrombi were fixed and immunostaining was performed to further analyze the distribution of platelets, VWF and ADAMTS13. Result As previously reported, ADAMTS13 localization was observed in the top and middle layers of the thrombus in the presence of wild type mVWF. The GPIb gain-of-function mutation V1316M increased both platelet (126%, p<0.0001) and VWF (190% and p<0.0001) accumulation at the thrombus site. ADAMTS13 localization was also increased (135%, p<0.001) relative to the binding to wild type VWF. Interestingly, with this gain-of-function VWF mutant, ADAMTS13 localization was found throughout the entire thrombus. In contrast, the GPIIbIIIa RGD binding mutant demonstrated decreased VWF (56%, p<0.01), and ADAMTS13 (82%, p<0.05) intensity, although platelet intensity was unaffected. to wild type, ADAMTS13 localized to the middle and top layers of the thrombus. Finally, inhibition of P-selectin significantly decreased VWF (46%, p<0.01) and ADAMTS13 (34%, p<0.01) localization to the thrombus, but again did not significantly alter platelet binding. Conclusion These studies demonstrate the central role of platelet-mediated mechanical tensile force on the regulation of thrombus growth at the site of platelet accumulation. Enhanced tensile force induced by increased GPIb binding resulted in increased ADAMTS13 localization, while reduced tensile force through loss of GPIIbIIIa or P-selectin binding decreased ADAMTS13 localization. This suggests that ADAMTS13 activity at the site of thrombus formation is maintained by the combination of hydrodynamic shear force and platelet tethering. aggregate, these studies suggest that under conditions of shear, ADAMTS13 regulates thrombus size by preserving the hemostatic function of the thrombus, and preventing dysregulated thrombus growth. Disclosures: No relevant conflicts of interest to declare.

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Extracellular Protein Disulfide Isomerase Regulates Vitronectin during the Initiation of Thrombus Formation

Blood ◽

10.1182/blood.v128.22.15.15 ◽

2016 ◽

Vol 128 (22) ◽

pp. 15-15

Author(s):

Chao Fang ◽

Sheryl R. Bowley ◽

Barbara C. Furie ◽

Bruce Furie

Keyword(s):

Endothelial Cells ◽

Protein Disulfide Isomerase ◽

Vessel Wall ◽

Plasma Sample ◽

Thrombus Formation ◽

Disulfide Isomerase ◽

Platelet Accumulation ◽

Protein Disulfide ◽

Kinetic Trapping

Abstract Protein disulfide isomerase (PDI), secreted by platelets and endothelial cells upon vascular injury, is required for thrombus formation. However, the precise mechanism by which PDI regulates thrombosis remains elusive. Using PDI variants that form stable mixed disulfide complexes with their substrates, we performed kinetic trapping experiment in platelet rich plasma and identified multiple substrate proteins for PDI, including vitronectin. Importantly, when using variants of endoplasmic reticulum protein 57 (ERp57), a thiol isomerase that has a similar domain structure as PDI and is also important for thrombus formation, the trapping mutants of ERp57 do not interact with vitronectin. This result has demonstrated the substrate specificity of PDI during our kinetic trapping experiment. Further study using polyethylene glycol (PEG)-based gel mobility shift assay combined with mass spectrometry has identified the redox reaction between PDI and vitronectin occurs on two disulfide bonds Cys 137-161 and Cys 274-453 in the hemopexin-like domains of plasma vitronectin. Vitronectin, as a substrate of extracellular PDI, has been shown to be important for thrombus formation. Vitronectin null mice have reduced platelet accumulation and fibrin deposition in the cremaster arterioles following laser injury. Vitronectin null mice also have significantly prolonged large-vessel thrombosis in the carotid artery using the ferric chloride thrombosis model. Using intravital microscopy we showed that vitronectin rapidly accumulates in a growing thrombus following vessel injury. When mice are treated with eptifibatide to eliminate platelet accumulation, we still observe significant amount of vitronectin accumulation on the vessel wall in the absence of platelet thrombus. This observation was further confirmed using confocal intravital microscopy. After 3D reconstruction of a growing thrombus in mouse cremaster arteriole, vitronectin was identified to locate primarily on the CD31 stained vessel wall. These combined studies suggest that plasma-derived vitronectin and not platelet-derived vitronectin is the primary substrate of PDI. Our study further showed that the indispensable role of vitronectin to a growing thrombus depends on extracellular PDI. Native plasma vitronectin does not bind to αvβ3 or αIIbβ3-integrins on endothelial cells and platelets. On solid phase binding assay, plasma sample pre-treated with wild-type PDI showed significantly increased binding of vitronectin to its ligand αvβ3 or αIIbβ3-integrins. However, this increase was not observed in plasma pre-treated with dead-mutant PDI or ERp57. In addition, using immunofluorescent staining, PDI treated plasma sample also showed significantly increased binding of vitronectin to activated human umbilical vein endothelial cells (HUVECs) and this binding was abrogated by RGD peptides or an αvβ3 blocking antibody. The critical role of extracellular PDI for the regulation of vitronectin in a growing thrombus was further confirmed in our in vivo studies. When mice were treated with quecetin-3-rutinoside or two different inhibitory antibodies that selectively block PDI activity, the accumulation of vitronectin and platelets was significantly reduced. These combined results demonstrate that extracellular PDI regulates vitronectin in a growing thrombus to promote platelet accumulation and fibrin generation. In summary, our studies have revealed a novel regulatory mechanism during the initiation of thrombus formation. Under normal physiologic conditions in the absence of secreted PDI, thrombus formation is suppressed and maintains a quiescent, patent vasculature. The release of PDI during vascular injury serves as a novel regulatory switch that allows activation of proteins, including vitronectin, which are critical for the following platelet accumulation and fibrin generation. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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