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

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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Distinct Localization of Coagulation Factor VIII, Von Willebrand Factor and Factor VIIIa-Mimetic Bispecific Antibody Contributing to Thrombus Formation Under Whole Blood Flow Conditions

Blood ◽

10.1182/blood.v124.21.1483.1483 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1483-1483

Author(s):

Yasuaki Shida ◽

Keiji Nogami ◽

Hiroaki Minami ◽

Hiroaki Yaoi ◽

Tomoko Matsumoto ◽

...

Keyword(s):

Shear Flow ◽

Research Funding ◽

Hemophilia A ◽

Thrombus Formation ◽

Flow Chamber ◽

High Shear ◽

Flow Conditions ◽

Von Willebrand ◽

Low Shear

Abstract Background Factor VIII (FVIII) is an essential factor for coagulation system in the intrinsic pathway. Due to the short survival of FVIII in the plasma circulation, it requires von Willebrand factor (VWF) as a carrier protein to maintain the optimal level for hemostasis. VWF also plays an important role in primary hemostasis by bridging platelets to exposed subendothelial collagens, especially under high shear flow environment. Since VWF carries FVIII, it is conceivable that VWF takes FVIII to the sites of vascular injury. However, the role of FVIII at the local sites under flow conditions is not fully understood despite of the fact that increased level of FVIII is associated with the risk of venous thrombosis and the deficiency of FVIII is the pathology of the bleeding disorder, hemophilia A. The treatment of hemophilia A largely depends on the infusion of FVIII concentrates, which is often complicated by the development of the inhibitor. Recently, bispecific antibody（ACE910）that mimics the role of FVIIIa by recognizing FIXa and FX has been developed and is currently under clinical trial. This antibody theoretically works regardless of the presence of devastating inhibitors against FVIII. Furthermore, it could also improve the clinical outcome of the other bleeding disorders, such as von Willebrand disease (VWD). Aim To analyze the role of FVIII and VWF, and impact of ACE910 at the sites of vascular injury under various shear conditions, we have developed the flow-mediated thrombosis model using flow chamber system. Method Whole blood obtained from healthy donors, hemophilia A and VWD patients were perfused into the collagen coated flow chamber under high (2,500s-1) or low shear (50s-1) flow conditions with/without FVIII concentrate, FVIII/VWF concentrate and ACE910. Formed thrombus was fixed and immunostaining was performed with phalloidin (Platelet), anti-FVIII antibody (FVIII) and anti-thrombin antibody (Thrombin). For the detection of ACE910, anti-human IgG or anti-ACE antibody (rAQ8 or rAJ540) were used. Size of thrombi and distribution of platelet, FVIII, thrombin and ACE910 were analyzed. Result 1) Under high shear flow, thrombus formation of VWD blood was significantly impaired while blood from Hemophilia A demonstrated nearly normal thrombus formation. Addition of FVIII/VWF but not FVIII concentrate to the blood of these patients rescued the impaired thrombus formation. ACE910 enhanced the thrombus formation of blood from both VWD and hemophilia A. Under low shear flow, blood from both hemophilia A and VWD demonstrated decreased thrombus formation. FVIII, FVIII/VWF concentrates and ACE910 improved the size of thrombus. 2) Localization of FVIII was evaluated with thrombin as a marker for the activation of coagulation. Platelets and thrombin demonstrated complete co-localization and intensity of thrombin staining was associated with thrombus size. VWF localized mainly outer layer of thrombus and FVIII localized in and around thrombus. At high shear condition, FVIII and VWF mostly existed with platelets. By contrast, FVIII and VWF demonstrated less co-localization with platelets under low shear condition. ACE910 demonstrated similar tendency to FVIII localization although ACE910 did not appear around thrombus. Conclusion We have developed the flow chamber system to evaluate the extent of thrombogenesis under various shear environment. VWF showed dominant role under high shear conditions while FVIII plays a key role under low shear conditions. FVIII, VWF and ACE910 demonstrated distinct localization. Interestingly, the distribution of FVIII was broader than VWF and platelet. FVIII localized to platelets presumably prior to its activation and contributed for the subsequent thrombin generation at local sites. Finally, ACE910 demonstrated consistent enhancement of thrombus formation of blood from both hemophilia A and VWD and, therefore, is prompted for the treatment of these bleeding disorders. Disclosures Shida: Chugai Pharmaceutical Co., Ltd.: Research Funding. Nogami:Chugai Pharmaceutical Co., Ltd.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Minami:Chugai Pharmaceutical Co., Ltd.: Research Funding. Yaoi:Chugai Pharmaceutical Co., Ltd.: Research Funding. Matsumoto:Chugai Pharmaceutical Co., Ltd.: Research Funding. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Hattori:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Shima:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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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 chemokine CXCL14 mediates platelet function and migration via direct interaction with CXCR4

Cardiovascular Research ◽

10.1093/cvr/cvaa080 ◽

2020 ◽

Cited By ~ 2

Author(s):

Alexander Witte ◽

Anne-Katrin Rohlfing ◽

Benjamin Dannenmann ◽

Valerie Dicenta ◽

Masoud Nasri ◽

...

Keyword(s):

Thrombus Formation ◽

Direct Interaction ◽

Flow Chamber ◽

Wild Type ◽

Autocrine Factors ◽

Inflammatory Processes ◽

And Migration ◽

Induced Pluripotent ◽

Human Ipsc ◽

Platelet Functions

Abstract Aims Beyond classical roles in thrombosis and haemostasis, it becomes increasingly clear that platelets contribute as key players to inflammatory processes. The involvement of platelets in these processes is often mediated through a variety of platelet-derived chemokines which are released upon activation and act as paracrine and autocrine factors. In this study, we investigate CXCL14, a newly described platelet chemokine and its role in thrombus formation as well as monocyte and platelet migration. In addition, we examine the chemokine receptor CXCR4 as a possible receptor for CXCL14 on platelets. Furthermore, with the use of artificially generated platelets derived from induced pluripotent stem cells (iPSC), we investigate the importance of CXCR4 for CXCL14-mediated platelet functions. Methods and results In this study, we showed that CXCL14 deficient platelets reveal reduced thrombus formation under flow compared with wild-type platelets using a standardized flow chamber. Addition of recombinant CXCL14 normalized platelet-dependent thrombus formation on collagen. Furthermore, we found that CXCL14 is a chemoattractant for platelets and mediates migration via CXCR4. CXCL14 promotes platelet migration of platelets through the receptor CXCR4 as evidenced by murine CXCR4-deficient platelets and human iPSC-derived cultured platelets deficient in CXCR4. We found that CXCL14 directly interacts with the CXCR4 as verified by immunoprecipitation and confocal microscopy. Conclusions Our results reveal CXCL14 as a novel platelet-derived chemokine that is involved in thrombus formation and platelet migration. Furthermore, we identified CXCR4 as principal receptor for CXCL14, an interaction promoting platelet migration.

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Effect of AR-H067637, the Active Metabolite of the Oral Anticoagulant AZD0837, on Thrombus Formation Using Human Whole Blood in an In Vitro Flow Chamber Model

Blood ◽

10.1182/blood.v112.11.4049.4049 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4049-4049

Author(s):

Margareta Elg ◽

Helen Zachrisson

Keyword(s):

Active Metabolite ◽

Thrombus Formation ◽

Plasma Concentrations ◽

Flow Chamber ◽

Direct Thrombin Inhibitor ◽

Thrombin Inhibitor ◽

Antithrombotic Effect ◽

Chamber Model ◽

Platelet Content ◽

D Dimer

Abstract AR-H067637 is a direct thrombin inhibitor derived in vivo from the orally available prodrug AZD0837. AR-H067637 is a reversible and selective direct thrombin inhibitor, which has an inhibition constant, Ki, of 2–4 nmol/L against human alpha-thrombin. During early development of a new anticoagulant drug, data on antithrombotic doses from animal studies may help guide selection of the dose to use in initial human studies. In the present study, using a flow chamber model developed by Badimon and colleagues (Badimon L, et al. J Lab Clin Med1987;110:706–718), the antithrombotic effect of AR-H067637 was evaluated using pig aorta and human whole blood. Following collection of informed consent, blood was taken from 11 healthy subjects into citrate-containing (10%, 0.109 M) tubes. Subjects were not permitted to receive medication for 7 days prior to blood collection. Blood was also collected in EDTA-containing tubes for cell counting. Denuded pig aorta pieces were used as the thrombogenic surface in the flow chamber. AR-H067637 was added to the blood at final blood concentrations ranging from 0.01 to 10 μmol/L, corresponding to plasma concentrations of 0.02 to 16 μmol/L. The blood was drawn for 5 minutes through the flow chamber with a shear of 220−s which is comparable with venous flow rate. The thrombus formed inside the chamber was degraded by plasmin, and platelets attached to the thrombus were lysed. The degradation product of fibrin, D-dimer, and the expression of the platelet cell adhesion molecule P-selectin were used as indirect measures of fibrin and platelet content in the thrombus, respectively. The anticoagulant effect of AR-H067637 was determined using the activated partial thromboplastin time (APTT) and prothrombin time (PT) assays. When using D-dimer levels as a measure of thrombus size, 25%, 50% and 75% thrombus inhibition was estimated to occur at AR-H067637 plasma concentrations of 0.21, 0.48 and 1.32 μmol/L, respectively. A significant inhibition of P-selectin expression by AR-H067637 was seen only at the highest concentration. APTT and PT were shown to be prolonged in a concentration-dependent manner; 50% inhibition of thrombus formation on the pig aorta was obtained at 1.8 and 1.2 times prolongations of APTT and PT, respectively. Hematological parameters such as WBC, RBC, HCT and platelets were all within the normal range. In conclusion, this study demonstrates that AR-H067637, the active metabolite of the oral prodrug AZD0837, has antithrombotic effects, causing concentration-dependent inhibition of thrombus formation measured as fibrin degradation products on the denuded pig aorta. Only a small effect at the highest concentration was observed on inhibition of platelet content in the thrombus, measured by P-selectin. This is in accordance with thrombin being a very potent platelet agonist. Therefore, higher concentrations of a thrombin inhibitor are needed to totally prevent platelet activation and aggregation, compared to those needed to prevent fibrin formation. APTT and PT prolongation correlated with the antithrombotic effect of AR-H067637 with >75% inhibition of fibrin formation at APTT and PT prolongations of 2.4 and 1.7, respectively.

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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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Familial Overexpression of β Antithrombin Caused by an Asn135Thr Substitution

Blood ◽

10.1182/blood.v93.12.4242.412k02_4242_4247 ◽

1999 ◽

Vol 93 (12) ◽

pp. 4242-4247

Author(s):

T.A. Bayston ◽

A. Tripodi ◽

P.M. Mannucci ◽

E. Thompson ◽

H. Ireland ◽

...

Keyword(s):

Direct Sequencing ◽

Consensus Sequence ◽

Wild Type ◽

Antithrombin Deficiency ◽

Free System ◽

Heparin Interaction ◽

A Cell ◽

Variant Protein ◽

Very High

We have investigated the basis of antithrombin deficiency in an asymptomatic individual (and family) with borderline levels (≈70% antigen and activity) of antithrombin. Direct sequencing of amplified DNA showed a mutation in codon 135, AAC to ACC, predicting a heterozygous Asn135Thr substitution. This substitution alters the predicted consensus sequence for glycosylation, Asn-X-Ser, adjacent to the heparin interaction site of antithrombin. The antithrombin isolated from plasma of the proband by heparin-Sepharose chromatography contained amounts of β antithrombin (the very high affinity fraction) greatly increased (≈20% to 30% of total) above the trace levels found in normals. Expression of the residue 135 variant in both a cell-free system and COS-7 cells confirmed altered glycosylation arising as a consequence of the mutation. Wild-type and variant protein were translated and exported from COS-7 cells with apparently equal efficiency, in contrast to the reduced level of variant observed in plasma of the affected individual. This case represents a novel cause of antithrombin deficiency, removal of glycosylation concensus sequence, and highlights the potentially important role of β antithrombin in regulating coagulation.

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Platelet JNK1 is involved in secretion and thrombus formation

Blood ◽

10.1182/blood-2009-07-233932 ◽

2010 ◽

Vol 115 (20) ◽

pp. 4083-4092 ◽

Cited By ~ 72

Author(s):

Frédéric Adam ◽

Alexandre Kauskot ◽

Paquita Nurden ◽

Eric Sulpice ◽

Marc F. Hoylaerts ◽

...

Keyword(s):

Platelet Aggregation ◽

Thrombus Formation ◽

Blood Perfusion ◽

Collagen Matrix ◽

In Vivo Model ◽

Wild Type ◽

Platelet Secretion

Abstract The role of c-Jun NH2-terminal kinase 1 (JNK1) in hemostasis and thrombosis remains unclear. We show here, with JNK1-deficient (JNK1−/−) mice, that JNK1 plays an important role in platelet biology and thrombus formation. In tail-bleeding assays, JNK1−/− mice exhibited longer bleeding times than wild-type mice (396 ± 39 seconds vs 245 ± 32 seconds). We also carried out in vitro whole-blood perfusion assays on a collagen matrix under arterial shear conditions. Thrombus formation was significantly reduced for JNK1−/− platelets (51%). In an in vivo model of thrombosis induced by photochemical injury to cecum vessels, occlusion times were 4.3 times longer in JNK1−/− arterioles than in wild-type arterioles. Moreover, in vitro studies carried out in platelet aggregation conditions demonstrated that, at low doses of agonists, platelet secretion was impaired in JNK1−/− platelets, leading to altered integrin αIIbβ3 activation and reduced platelet aggregation, via a mechanism involving protein kinase C. JNK1 thus appears to be essential for platelet secretion in vitro, consistent with its role in thrombus growth in vivo. Finally, we showed that ERK2 and another isoform of JNK affect platelet aggregation through 2 pathways, one dependent and another independent of JNK1.

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Familial Overexpression of β Antithrombin Caused by an Asn135Thr Substitution

Blood ◽

10.1182/blood.v93.12.4242 ◽

1999 ◽

Vol 93 (12) ◽

pp. 4242-4247 ◽

Cited By ~ 9

Author(s):

T.A. Bayston ◽

A. Tripodi ◽

P.M. Mannucci ◽

E. Thompson ◽

H. Ireland ◽

...

Keyword(s):

Direct Sequencing ◽

Consensus Sequence ◽

Wild Type ◽

Antithrombin Deficiency ◽

Free System ◽

Heparin Interaction ◽

A Cell ◽

Variant Protein ◽

Very High

Abstract We have investigated the basis of antithrombin deficiency in an asymptomatic individual (and family) with borderline levels (≈70% antigen and activity) of antithrombin. Direct sequencing of amplified DNA showed a mutation in codon 135, AAC to ACC, predicting a heterozygous Asn135Thr substitution. This substitution alters the predicted consensus sequence for glycosylation, Asn-X-Ser, adjacent to the heparin interaction site of antithrombin. The antithrombin isolated from plasma of the proband by heparin-Sepharose chromatography contained amounts of β antithrombin (the very high affinity fraction) greatly increased (≈20% to 30% of total) above the trace levels found in normals. Expression of the residue 135 variant in both a cell-free system and COS-7 cells confirmed altered glycosylation arising as a consequence of the mutation. Wild-type and variant protein were translated and exported from COS-7 cells with apparently equal efficiency, in contrast to the reduced level of variant observed in plasma of the affected individual. This case represents a novel cause of antithrombin deficiency, removal of glycosylation concensus sequence, and highlights the potentially important role of β antithrombin in regulating coagulation.

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The Role of the VWF A1 and A2 Domains in Murine Models of TTP and Thrombosis.

Blood ◽

10.1182/blood.v114.22.3060.3060 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3060-3060

Author(s):

Jennifer Barr ◽

Justin Barr ◽

David Motto

Keyword(s):

Thrombus Formation ◽

Platelet Glycoprotein ◽

Cysteine Residues ◽

Wild Type ◽

Domain Sequence ◽

Initial Adhesion ◽

A1 Domain ◽

A2 Domain

Abstract Abstract 3060 Poster Board II-1036 von Willebrand Factor (VWF) is a large multimeric plasma glycoprotein synthesized in endothelial cells and megakaryocytes. In humans and mice, VWF dysfunction is associated both with defects in hemostasis, and with the systemic blood clotting disease thrombotic thrombocytopenic purpura (TTP). The initial adhesion of platelets to sites of vascular injury in large part involves binding of the VWF A1 domain to the platelet glycoprotein receptor GPIb alpha. This VWF A1-GPIb alpha interaction, along with deficiency of the ADAMTS13 plasma metalloprotease, is thought to be required for the pathogenesis of TTP. Deficiency of ADAMTS13 results in the failure to cleave the Y1605-M1606 sissile bond within the A2 domain of VWF. The structure of VWF is strongly influenced by its high content of cysteine residues, all of which are involved in inter-or intra-chain disulfide bonds. The location of these cysteine residues within the A domains leads to the formation of disulfide loops within the A1 (Cys 1272-1458) and A3 (Cys 1686-1872) domains, but not within the A2 domain. The lack of a disulfide loop allows the A2 domain to assume a “flexible” conformation that is thought to “open” in response to fluid shear stress, exposing the Y1605-M1606 bond to cleavage by ADAMTS13. To investigate specifically the role of the VWF A1-Gp1b alpha interaction in the context of otherwise functional VWF in vivo, we generated a chimeric murine VWF expression construct in which the murine A1 domain sequence is replaced with the corresponding sequence from human VWF (the human VWF A1 domain is known to not interact appreciably with murine GPIb alpha). Additionally, we engineered a VWF construct in which paired cysteine residues analogous to those in the A1 and A3 domains were introduced into the A2 domain sequence, with the goal being to “lock” the A2 domain closed and prevent cleavage by ADAMTS13. Hydrodynamic tail vein injection of both the VWF-hA1 and the VWF-A2 lock constructs into VWF-deficient mice resulted in plasma VWF levels up to 20-fold higher than observed in wild-type mice, dependent on the amount of plasmid injected. Importantly, the degree of VWF multimerization appeared nearly identical both to that observed in wild-type mice, and to mice injected with wild-type murine VWF, and expression persisted for approximately 30 days. Functionally, unlike WT murine VWF, expression of VWF-hA1 failed to restore thrombus formation in a ferric chloride-induced injury model, demonstrating the crucial importance of the VWF A1-GP1b apha interaction in thrombus formation. Currently we are investigating whether expression of VWF-hA1 can support disease pathogenesis in a mouse model of TTP. Similarly, we are determining whether expression of VWF-A2 lock leads to development of TTP, even in the presence of ADAMTS13. The ultimate goal of these studies is to completely “humanize” the VWF A1-GP1b alpha interaction in mice by replacing the murine GP1b alpha sequence with that from humans. These resulting animals will be used to further investigate the role of the VWF A1 domain-GPIb alpha interaction in vivo, and should prove useful for identifying compounds to effectively inhibit this interaction in humans. In addition, the expression of a VWF construct that is unable to be cleaved by ADAMTS13 should help to elucidate the role of VWF cleavage in TTP pathogenesis. Disclosures No relevant conflicts of interest to declare.

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Apolipoprotein E receptor 2 is involved in the thrombotic complications in a murine model of the antiphospholipid syndrome

Blood ◽

10.1182/blood-2010-07-299099 ◽

2011 ◽

Vol 117 (4) ◽

pp. 1408-1414 ◽

Cited By ~ 74

Author(s):

Zurina Romay-Penabad ◽

Renan Aguilar-Valenzuela ◽

Rolf T. Urbanus ◽

Ronald H. W. M. Derksen ◽

Maarten T. T. Pennings ◽

...

Keyword(s):

Apolipoprotein E ◽

Thrombus Formation ◽

Convincing Evidence ◽

Wild Type ◽

Glycoprotein I ◽

Thrombotic Complications ◽

Deficient Mice ◽

Pathogenic Effects

Abstract Antiphospholipid (aPL)/anti-β2 glycoprotein I (anti-β2GPI) antibodies stimulates tissue factor (TF) expression within vasculature and in blood cells, thereby leading to increased thrombosis. Several cellular receptors have been proposed to mediate these effects, but no convincing evidence for the involvement of a specific one has been provided. We investigated the role of Apolipoprotein E receptor 2 (ApoER2′) on the pathogenic effects of a patient-derived polyclonal aPL IgG preparation (IgG-APS), a murine anti-β2GPI monoclonal antibody (E7) and of a constructed dimeric β2GPI I (dimer), which in vitro mimics β2GPI-antibody immune complexes, using an animal model of thrombosis, and ApoER2-deficient (−/−) mice. In wild type mice, IgG-APS, E7 and the dimer increased thrombus formation, carotid artery TF activity as well as peritoneal macrophage TF activity/expression. Those pathogenic effects were significantly reduced in ApoER2 (−/−) mice. In addition, those effects induced by the IgG-APS, by E7 and by the dimer were inhibited by treatment of wild-type mice with soluble binding domain 1 of ApoER2 (sBD1). Altogether these data show that ApoER2 is involved in pathogenesis of antiphospholipids antibodies.

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