Importance of GPVI in Platelet Activation and Thrombus Formation In Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 842-842 ◽  
Author(s):  
Christophe Dubois ◽  
Laurence Panicot-Dubois ◽  
Bruce Furie ◽  
Barbara C. Furie
Keyword(s):  
Ferric Chloride ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Wild Type ◽  
Laser Injury ◽  
Vessel Injury ◽  
Arterial Thrombus ◽  
Platelet Accumulation ◽  
Chloride Treatment

Abstract Collagen is one of the major components of the vessel wall responsible for platelet adhesion and activation at sites of vascular injury. In vitro studies have shown that α2β1 and GPVI directly and αIIbβ3 and GPIb-IX-V indirectly, via vWF, are all involved in the adhesion of platelets to collagen. However, the importance of GPVI on the adhesion and activation of platelets in vivo is still controversial. Here we show that in vivo GPVI plays an important role in platelet adhesion and activation when collagen is exposed to blood. To determine the role of GPVI on thrombus formation, we compared thrombus formation in FcRγ null mice, which do not express GPVI on the platelet surface, and in wild type mice after vessel injury induced by ferric chloride or by a nitrogen dye laser. We studied arterial thrombus formation in the microcirculation of the cremaster and the mesentery muscles using high speed multi channel intravital fluorescence widefield microscopy. Real time platelet accumulation in the developing thrombus was detected using a fluorescent antibody directed against αIIb. After an injury induced by the ferric chloride, we observed a significant delay in both the time to formation of an initial thrombus and the time to vessel occlusion in FcRγ null mice in comparison with wild type mice. When activated platelets isolated from FcRγ null mice were injected into a recipient FcRγ null mouse, we were able to restore the formation of a thrombus. This effect was abolished by injection of Lamifiban, an inhibitor of activated αIIbβ3. These results indicate that GPVI is not only involved in vivo in the adhesion of platelets to collagen but also plays an important role in the activation of the platelet fibrinogen receptor αIIbβ3. In contrast, platelet accumulation after laser-induced injury in the FcRγ null and the wild type mice was comparable. No difference in the kinetics of platelet accumulation into the laser induced growing thrombus was observed. To understand the different pathways leading to thrombus formation in vivo after ferric chloride or laser induced injury, we examined collagen exposure after vessel injury and the accumulation of tissue factor (TF) in the developing arterial thrombus of FcRγ null and wild type mice using antibodies directed against mouse collagen type I and mouse TF. We observed a significant exposure of collagen at sites of thrombus formation after ferric chloride treatment. In contrast, we did not observe any collagen exposure on blood vessels after laser-induced injury. Furthermore, the ratio of TF/platelets present into the thrombus after injury was 5 fold greater after laser injury than after ferric chloride treatment. These results suggest that TF but not collagen plays an important role in thrombus formation induced by laser injury of the vessel wall. Altogether, our results indicate that the GPVI receptor is involved in vivo in platelet adhesion when collagen is exposed to blood and plays an important role in the activation of other platelet integrins such as αIIbβ3 leading to the formation of a stable thrombus.

scholarly journals Glycoprotein VI–dependent and –independent pathways of thrombus formation in vivo

Blood ◽  
2006 ◽  
Vol 107 (10) ◽  
pp. 3902-3906 ◽  
Author(s):  
Christophe Dubois ◽  
Laurence Panicot-Dubois ◽  
Glenn Merrill-Skoloff ◽  
Bruce Furie ◽  
Barbara C. Furie
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
Severe Injury ◽  
Wild Type ◽  
Vessel Occlusion ◽  
Platelet Surface ◽  
Glycoprotein Vi ◽  
Laser Injury ◽  
Platelet Accumulation

The role of the collagen receptor glycoprotein VI (GPVI) in arteriolar thrombus formation was studied in FcRγ-null mice (FcRγ–/–) lacking platelet surface GPVI. Thrombi were induced with severe or mild FeCl3 injury. Collagen exposure was significantly delayed and diminished in mild compared with severe FeCl3 injury. Times to initial thrombus formation and vessel occlusion were delayed in FcRγ–/– compared with wild-type mice after severe injury. Platelet accumulation in wild-type mice was decreased after mild compared with severe injury. However, there was little difference between platelet accumulation after severe or mild injury in FcRγ–/–. These data indicate a significant role for GPVI in FeCl3-induced thrombus formation. Pretreatment of wild-type mice with lepirudin further impaired mild FeCl3-induced thrombus formation, demonstrating a role for thrombin. Laser-induced thrombus formation in wild-type and FcRγ–/– was comparable. Collagen exposure to circulating blood was undetectable after laser injury. Normalized for thrombus size, thrombus-associated tissue factor was 5-fold higher in laser-induced thrombi than in severe FeCl3-induced thrombi. Thus, platelet activation by thrombin appears to be more important after laser injury than platelet activation by GPVI-collagen. It may thus be important when considering targets for antithrombotic therapy to use multiple animal models with diverse pathways to thrombus formation.

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

Blood ◽  
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.

Endothelium but Not Platelet-Derived Protein Disulfide Isomerase Is Required for Fibrin Generation during Thrombus Formation in Vivo.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 691-691 ◽  
Author(s):  
Reema Jasuja ◽  
Jaehyung Cho ◽  
Bruce Furie ◽  
Barbara Furie
Keyword(s):  
Endothelial Cells ◽  
Protein Disulfide Isomerase ◽  
Thrombus Formation ◽  
Fibrin Deposition ◽  
Wild Type ◽  
Disulfide Isomerase ◽  
Laser Injury ◽  
Injury Model ◽  
Protein Disulfide

Abstract We have previously reported that protein disulfide isomerase is required in wild-type mice for platelet thrombus formation and fibrin generation in an in vivo laser injury model of thrombosis (Cho et al. J. Clin. Invest., 2008; 118:1123–31). Fibrin deposition after laser injury to the vessel wall in Par4−/− mice, lacking the G protein-coupled platelet thrombin receptor, is independent of platelets or requires minimal platelet activation or accumulation (Vandendries et al. Proc. Natl. Acad. Sci., 2007; 104:288–92). However, protein disulfide isomerase inhibitors have a dramatic effect on fibrin accumulation in Par4− mice, suggesting that these inhibitors may function by a platelet independent mechanism. Here, we compare the contributions of endothelium and platelet-derived protein disulfide isomerase to fibrin generation in the mouse laser injury model of thrombosis. In vitro studies using cultured human umbilical vein endothelial cells and human aortic endothelial cells show that protein disulfide isomerase can be secreted rapidly into the culture medium from these cells upon thrombin stimulation. Using intravital microscopy, we observe that protein disulfide isomerase is not detectable on the vessel wall prior to laser injury but can be detected on the injured cremaster arteriolar wall and in the developing thrombus very rapidly after laser induced injury in the live mouse. The median integrated fluorescence intensity for protein disulfide isomerase in wild-type mice was compared to wild-type mice injected with 10ug/g mouse of Integrilin, an inhibitor of platelet activation and platelet thrombus formation, and thus, an inhibitor of the contribution of platelet derived protein disulfide isomerase to thrombus formation. Protein disulfide isomerase expression was similar in both treated and untreated animals upto 30 seconds post-laser injury. After 30 seconds, the expression of protein disulfide isomerase in integrilin treated mice was significantly decreased compared to that in untreated mice, indicating that the initial protein disulfide isomerase was derived from the endothelium and later additional protein disulfide isomerase was derived from the platelets following their accumulation in the developing thrombus. Fibrin deposition, a measure of thrombin generation was comparable in wild-type mice that had been treated with Integrilin or treated with a control buffer, suggesting that endothelial-derived protein disulfide isomerase was sufficient for fibrin generation. The rate and amount of fibrin generation was indistinguishable in both groups. Furthermore, inhibition of the protein disulfide isomerase with the function blocking monoclonal antibody RL-90 (3ug/g mouse) eliminated any fibrin deposition in wild-type mice that had been treated with Integrilin. Taken together, these data indicate that endothelium-derived protein disulfide isomerase is necessary to support fibrin deposition in vivo in our laser injury model of thrombus formation. The initial protein disulfide isomerase expressed at the site of injury is derived from endothelial cells but platelets activated at the site of thrombus formation contribute, amplify and sustain protein disulfide isomerase expression.

GPIbα Is Essential for Platelet Adhesion during Thrombus Formation: Studies with Mutant Mice Deficient in the Extracellular Domain of GPIbα.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3659-3659
Author(s):  
Wolfgang Bergmeier ◽  
Crystal L. Piffath ◽  
Zaverio M. Ruggeri ◽  
Jerry L. Ware ◽  
Denisa D. Wagner
Keyword(s):  
Ferric Chloride ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Mutant Mice ◽  
Interleukin 4 ◽  
Wild Type ◽  
Platelet Surface ◽  
Surface Receptor ◽  
A Site ◽  
Von Willebrand

Abstract GPIbα binding to von Willebrand factor (vWf) exposed at a site of vascular injury is the first step in the formation of a hemostatic plug. In addition, GPIbα binding to platelet-bound vWf has previously been shown to play a key role in the incorporation of circulating platelets into a growing thrombus. However, studies in vWf-deficient mice demonstrated delayed but not absent arterial thrombus formation, suggesting that GPIbα may bind a ligand other than vWf to facilitate platelet adhesion, or that a platelet surface receptor other than GPIbα can mediate platelet adhesion in arterioles in the absence of vWf. Here we studied thrombus formation in transgenic mice expressing GPIbα in which the extracytoplasmic sequence has been replaced by an isolated domain of the human interleukin 4 receptor (IL4R-tg mice). Early platelet adhesion to ferric chloride-treated mesenteric arterioles in IL4R-tg mice was decreased by >98% when compared with controls. As a consequence, thrombus formation was completely inhibited in all of the mutant mice. To study the role of GPIbα in platelet adhesion to already adherent platelets, we studied thrombus formation in wild-type mice infused with wild-type platelets labeled with calcein-green and IL4R-tg platelets labeled with calcein-orange/red. Upon ferric chloride-induced injury, wild-type but not IL4R-tg platelets incorporated into the growing thrombus. A similar result was observed with wild-type platelets treated with O-sialoglycoprotein endopeptidase to remove the 45 kD N-terminal domain of GPIbα. In summary, our studies in GPIbα mutant mice provide additional evidence that under arterial flow conditions GPIbα is the only receptor expressed on the platelet surface that mediates initial platelet adhesion to the subendothelium as well as to already adherent platelets. Our data further suggest that a ligand other than vWf may contribute to GPIbα-dependent platelet adhesion to subendothelium and to a growing thrombus.

The Role of Platelet Adhesion Receptor GPIb α Far Exceeds That of Its Main Ligand von Willebrand Factor in Arterial Thrombosis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1797-1797 ◽  
Author(s):  
Wolfgang Bergmeier ◽  
Crystal L. Piffath ◽  
Tobias Goerge ◽  
Stephen M. Cifuni ◽  
Zaverio M. Ruggeri ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Arterial Thrombosis ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Interleukin 4 ◽  
Arterial Thrombus ◽  
A Site ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract GPIbα binding to von Willebrand factor (VWF) exposed at a site of vascular injury is thought to be the first step in the formation of a hemostatic plug. However, our previous studies in VWF-deficient mice demonstrated delayed but not absent arterial thrombus formation suggesting that, under these conditions, GPIbα may bind other ligands or that a receptor other than GPIbα can mediate platelet adhesion. Here we studied thrombus formation in transgenic mice expressing GPIbα in which the extracellular domain was replaced by that of the human interleukin-4 receptor (IL4Rα/GPIbα-tg mice). Platelet adhesion to ferric chloride-treated mesenteric arterioles in IL4Rα/GPIbα-tg mice was virtually absent in contrast to avid adhesion in wild-type (WT) mice. As a consequence, arterial thrombus formation was completely inhibited in the mutant mice. Our studies further show that, when infused into WT recipient mice, IL4Rα/GPIbα-tg platelets or WT platelets lacking the 45 kD N-terminal domain of GPIbα failed to incorporate into growing arterial thrombi, even if the platelets were activated prior to infusion. Surprisingly, platelets lacking β3 integrins, which are unable to form thrombi on their own, incorporated efficiently into WT thrombi. Our studies provide in vivo evidence that GPIbα is absolutely required for recruitment of platelets to both exposed subendothelium and thrombi under arterial flow conditions. Thus, GPIbα contributes to arterial thrombosis by important adhesion mechanisms independent of the binding to VWF.

Procoagulant Role Of Necrotic Platelets Demonstrated Using Novel Platelet Necrosis Marker

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3512-3512
Author(s):  
Minh Hua ◽  
Leonardo Pasalic ◽  
Robert Lindeman ◽  
Philip Hogg ◽  
Vivien M Chen
Keyword(s):  
Platelet Activation ◽  
Ferric Chloride ◽  
Cyclosporine A ◽  
Thrombus Formation ◽  
Laser Injury ◽  
Agonist Stimulation ◽  
Injury Model ◽  
Exogenous Calcium

Abstract Strong agonist stimulation generates a platelet subpopulation characterized by phosphatidylserine (PS) exposure, loss of mitochondrial membrane potential and high fibrinogen retention. This population is proposed to be procoagulant, dependent on formation of the mitochondrial permeability transition pore (mPTP) with a distinct role from activated aggregratory platelets. These platelets have features of necrosis. The functional relevance of necrotic platelets in vivo is unknown due to lack of a suitable marker for these platelets. We show that a novel small molecule cellular necrosis marker, GSAO1, labels a procoagulant platelet subpopulation with features of necrosis and use it to explore the functional role of these platelets. We demonstrated using flow cytometry analysis of washed human platelets that fluorescently tagged GSAO labels a subpopulation of P-selectin positive platelets after thrombin and collagen stimulation with features of necrosis: high annexin V binding, calcein loss and dependence on exogenous calcium. This population is not dependent on the intrinsic apoptosis pathway as there was no change with pancaspase inhibition using ZVADFMK prior to dual agonist stimulation (p=0.567, n=5). In contrast, inhibition of mPTP formation through cyclophilin-D inhibition with cyclosporine A significantly inhibited GSAO+ve platelet generation (p<0.001, n=5), confirming dependence on the mitochondrial necrosis pathway. Mass spectrometry analysis of biotin-GSAO labelled proteins from platelets after streptavidin pull down identified thromboxane A synthase (TBXAS-1) as the major binding ligand after dual stimulation. Binding to TBXAS-1 was abrogated by dithiol alkylation, showing the mechanism of retention of GSAO in necrotic platelets is via covalent cross linking of closely-spaced cysteine thiols in the ligand. This allows persistent signal from the probe within the necrotic platelet with no evidence of washout. GSAO+ve platelets correlated with procoagulant potential as measured by peak and endogenous thrombin potential in the calibrated automated thrombogram (CAT) assay. Linear regression analysis showed a significant relationship between % change in GSAO+ve platelets and % change in peak thrombin after treatment with cyclosporine A or in absence of exogenous calcium (R2=0.648, p<0.01), indicating that GSAO identifies a procoagulant subpopulation. In contrast, no relationship was seen between P-selectin and peak thrombin values (R2=0.002). Inhibition of platelet activation by aspirin had no effect on the generation of GSAO+ve platelets indicating a potential uncoupling between platelet activation and necrosis pathways. After establishing that the imaging compound does not affect platelet function and coagulation in vitro, or thrombus formation in vivo, we went on to investigate the presence of GSAO+ve necrotic platelets in thrombus formation in a collagen dependent (ferric chloride) and collagen independent (laser injury) murine model of thrombosis. Confocal intravital imaging of the cremaster arterioles with fluorescent GSAO and tagged-CD42b demonstrated GSAO+ve platelets in the occlusive platelet aggregate after initiation with 10% ferric chloride. The GSAO+ve aggregating platelets specifically colabeled with calcium sensing dye rhodamine 2 indicating high sustained intracellular calcium, consistent with a necrotic phenotype. There was no signal with active site replaced control GSCA. In contrast, the laser injury model showed minimal staining with GSAO three minutes post laser injury. Using a novel platelet necrosis marker, we are able to demonstrate that necrotic platelets are procoagulant and present in the occlusive ferric chloride model and not in the non-occlusive laser injury model of thrombosis. This suggests excess platelet necrosis may be a key driving factor underlying pathological occlusive thrombi. GSAO is a promising tool for understanding factors that potentiate platelet necrosis which may offer attractive anti-thrombotic targets. 1. Park D, Don AS, Massamiri T, et al. Noninvasive imaging of cell death using an hsp90 ligand. J Am Chem Soc. 2011;133(9):2832-2835. Disclosures: No relevant conflicts of interest to declare.

Granules and thrombus formation

Blood ◽  
2009 ◽  
Vol 114 (5) ◽  
pp. 932-933 ◽  
Author(s):  
Walter H. A. Kahr
Keyword(s):  
Thrombus Formation ◽  
Dense Granule ◽  
Snare Protein ◽  
Laser Injury ◽  
Platelet Accumulation ◽  
Dense Granule Secretion ◽  
Granule Secretion

In this issue of Blood, Graham and colleagues demonstrate the importance of platelet dense granule secretion for in vivo platelet accumulation following laser injury, which is mediated by the SNARE protein Endobrevin/VAMP-8.

scholarly journals The Proteoglycan Biglycan Modulates Platelet Adhesion and Thrombus Formation in a GPVI-Dependent Manner

2021 ◽  
Vol 22 (22) ◽  
pp. 12168
Author(s):  
Henrike Hoermann ◽  
Irena Krueger ◽  
Nadine Maurus ◽  
Friedrich Reusswig ◽  
Yi Sun ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Vessel Wall ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Three Dimensional ◽  
Dependent Manner ◽  
Prolonged Bleeding ◽  
Vessel Injury

Background: Vascular injury induces the exposure of subendothelial extracellular matrix (ECM) important to serve as substrate for platelets to adhere to the injured vessel wall to avoid massive blood loss. Different ECM proteins are known to initiate platelet adhesion and activation. In atherosclerotic mice, the small, leucine-rich proteoglycan biglycan is important for the regulation of thrombin activity via heparin cofactor II. However, nothing is known about the role of biglycan for hemostasis and thrombosis under nonatherosclerotic conditions. Methods: The role of biglycan for platelet adhesion and thrombus formation was investigated using a recombinant protein and biglycan knockout mice. Results: The present study identified biglycan as important ECM protein for the adhesion and activation of platelets, and the formation of three-dimensional thrombi under flow conditions. Platelet adhesion to immobilized biglycan induces the reorganization of the platelet cytoskeleton. Mechanistically, biglycan binds and activates the major collagen receptor glycoprotein (GP)VI, because reduced platelet adhesion to recombinant biglycan was observed when GPVI was blocked and enhanced tyrosine phosphorylation in a GPVI-dependent manner was observed when platelets were stimulated with biglycan. In vivo, the deficiency of biglycan resulted in reduced platelet adhesion to the injured carotid artery and prolonged bleeding times. Conclusions: Loss of biglycan in the vessel wall of mice but not in platelets led to reduced platelet adhesion at the injured carotid artery and prolonged bleeding times, suggesting a crucial role for biglycan as ECM protein that binds and activates platelets via GPVI upon vessel injury.

Multiple Carboxyl-Terminal Domains of ADAMTS13 Are Required for Systemic Anti-Arterial Thrombosis In Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 24-24 ◽  
Author(s):  
Juan (Jenny) Xiao ◽  
Sheng-Yu Jin ◽  
X. Long Zheng
Keyword(s):  
Ferric Chloride ◽  
Arterial Thrombosis ◽  
Thrombus Formation ◽  
Arterial Occlusion ◽  
Final Concentration ◽  
Wild Type ◽  
The Times ◽  
Carotid Arterial ◽  
Terminal Domains

Abstract Abstract 24 A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS)-13 cleaves newly released ultra large (UL) von Willebrand factor (VWF), thereby inhibiting excessive platelet aggregation and thrombus formation. Inability to cleave ULVWF due to deficiency of plasma ADAMTS13 activity may result in thrombotic thrombocytopenic purpura (TTP), a potentially fatal illness, and other arterial thrombotic diseases (i.e. myocardial and cerebral infarctions). However, little is known about the structural components of ADAMTS13 required for systemic anti-arterial thromboses in vivo. In this study, we determined the biological effect of ADAMTS13 and variants on arterial thromboses in a murine model using two different assays. First, using a ferric chloride-induced carotid arterial occlusion assay, we demonstrated that the times to a complete occlusion (TCO) of carotid artery after topical application of 10% ferric chloride (soaked in a filter paper 1×2 min, for 2 min) in wild-type (C57BL/6) mice and Adamts13-/- mice were 10.0 ± 1.0 min (mean ± SEM) (n=9) and 5.3 ± 0.4 min (n=10), respectively. The difference was statistically highly significant (p<0.0001). An infusion of 10 nM of recombinant human full-length ADATMS13 (FL), a variant truncated after the 8th TSP1 repeat (T8) and after the spacer domain (S) into the Adamts13-/- mice restored the TCO to 12.7 ± 1.7 min (n=12), 8.0 ± 1.8 (n=7), and 22.0 ± 2.1 min (n=12), respectively. These results suggest that the N-terminal fragment of ADAMTS13 up to the spacer domain is sufficient for protection against ferric chloride induced arterial thrombosis. Moreover, an infusion of an ADAMTS13 mutant lacking 6 amino acid residues between Arg659 and Glu664 (d6a) into Adamts13-/-mice did not restore the TCO (5.9 ± 0.6 min, n=11), suggesting the critical role of the spacer domain in anti-arterial thrombosis in vivo. Paradoxically, however, an infusion of a recombinant C-terminal fragment of ADAMTS13 consisting of the TSP1 5–8 repeats and CUB domains (T5C) at the final concentration of 50 nM (10x endogenous murine plasma ADAMTS13 concentration) into wild-type mice significantly shortened the TCO (5.9 ± 1.9 min) (n=11), similar to that in the Adamts13-/- mice receiving injection of PBS alone (p=0.440). These results indicate that the middle and distal C-terminal domains of ADAMTS13 in the context of the whole molecule may also participate in substrate recognition and are required for anti-arterial thrombotic function in vivo. Second, the results from intravital microscopy by visualizing a real-time thrombus formation in the mesenteric arterioles nearly mirrored the data obtained by the carotid arterial occlusion assay. In these experiments, the times to an initial thrombus formation (Ti) (defined as the time to form a thrombus >30 μ m) and the times to a complete occlusion of blood vessel (Tc) were determined. We showed that the Ti and Tc in wild-type C57BL6 mice were 8.8 ± 0.6 min (mean ± SEM) and 13.7 ± 1.0 min (n=12), respectively. The Ti and Tc in the Adamts13-/- mice (same genetic background) were 5.2 ± 0.6 min (n=15) and 9.6 ± 0.8 min (n=15), respectively. The differences in both Ti and Tc between wild-type mice and Adamts13-/- mice were statistically highly significant (p<0.001). An infusion of recombinant human FL, T8, and S, but not d6a at a final concentration of 10 nM significantly prolonged the Ti (FL: 9.7 ± 0.9 min, n=10; T8: 10.1 ± 1.5 min, n=10; S: 11.5 ± 1.5 min, n=10; d6a: 5.3 ± 0.57 min, n=11) and the Tc (FL: 15.3 ± 1.4 min; T8: 21.9 ± 2.2 min; S: 16.2 ± 1.6 min; d6a:10.0 ± 1.1 min). The differences in both Ti and Tc between the control (buffer injected) and experimental groups were all statistically highly significant. We conclude that multiple C-terminal domains of ADAMTS13 are required for systemic anti-arterial/arteriolar thrombosis under (patho) physiological conditions. Our findings may shed more light on pathogenesis of TTP and provide molecular basis for a rational design of novel therapies for TTP and perhaps other arterial thrombotic disorders because of dysfunction of VWF/ADAMTS13 axis. Disclosures: No relevant conflicts of interest to declare.

The Interaction of Coagulation Factor XI with Polyphosphate

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 498-498
Author(s):  
Yipeng Geng ◽  
Ingrid M. Verhamme ◽  
Qiufang Cheng ◽  
Anton Matafonov ◽  
Stephen B. Smith ◽  
...  
Keyword(s):  
Ferric Chloride ◽  
Conformational Changes ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Coagulation Factor ◽  
Wild Type ◽  
Zymogen Activation ◽  
Factor Xi ◽  
Dimeric Structure

Abstract Abstract 498 The plasma zymogen factor XI (FXI) is a homodimer of 80 kDa subunits. During blood coagulation, each subunit is activated by cleavage of the Arg369-Ile370 bond by factor XIIa (FXIIa) or thrombin. Initially, one subunit of the FXI dimer is activated to create the species 1/2FXIa, followed by activation of the second subunit, generating FXIa. Initial rates of activation of the first subunit are relatively low (∼8000 M−1.sec−1 for FXIIa and 120 M−1.sec−1 for thrombin). Rates for activation of the second subunit are even lower (1200 M−1.sec−1 for FXIIa and 35 M−1.sec−1 for thrombin), suggesting that conformational changes accompanying activation of a subunit reduce the efficiency of activation of its partner. The slow rate of FXI activation in solution strongly suggests that a cofactor is required for protease activation in vivo. FXI activation by FXIIa or thrombin is enhanced by polyanions such as dextran sulfate (DS). In addition, polyanions induce FXI activation by FXIa (autoactivation). Polymers of inorganic phosphate (polyP) released from platelet dense granules accelerate FXI activation by thrombin or FXIa (Blood 2011;118:6963), and likely represent the physiologic counterpart to DS. PolyP (4 μM) increased the initial rate of FXI activation by FXIIa ∼30-fold (300,000 M−1.sec−1), and by a-thrombin ∼3600-fold (440,000 M−1.sec−1). Furthermore, polyP induced FXI autoactivation in a manner similar to DS. Each FXI subunit contains two polyanion binding sites (residues Arg250, Lys252, Lys253, Lys255 on the A3 domain, and Lys529, Arg530, Arg532 on the catalytic domain). Both sites bind heparin, and are required for normal heparin-mediated enhancement of FXIa inhibition by antithrombin. FXI lacking the A3 domain site (FXIΔA3), but not FXI lacking the protease domain site (FXIΔCD), is activated slowly in the presence of DS compared to wild type FXI (FXIWT). Interestingly, both FXIΔA3 and FXIΔCD are activated slowly compared to FXIWT in the presence of polyP, and a species lacking both sites (FXIΔA3/CD) has an even greater defect, indicating FXI's interaction with polyP is different from its interaction with DS. The FXI gene arose from a duplication of the gene for the monomeric protein prekallikrein (PK). The observations that the dimeric structure of FXI is highly conserved across species, and that the ancestral molecule is a monomer, strongly indicate that the dimer is important for a specific aspect of FXI function. It was recently reported that monomeric forms of FXI are activated slowly compared to dimeric FXI in solution or in the presence of DS (J Biol Chem 2008;283:18655). FXI dimer formation is mediated through a hydrophobic interface involving Leu284, Ile290, and Tyr329, and an interchain disulfide bond involving Cys321. A Ser substitution for Cys321 in combination with an Ala substitution for Leu284 or Ile290 results in the monomeric species FXIC321S, L284A and FXIC321S, I290A. Rates of FXIC321S, L284A or FXIC321S, I290A activation by FXIIa were significantly lower than for FXIWT in the presence of polyP. However, this defect was not observed during activation by thrombin or FXIa, demonstrating that the dimeric structure is not a prerequisite for zymogen activation on polyP. FXI-deficient (FXI−/−) mice are more resistant to arterial thrombus formation induced by vessel injury with ferric chloride than are wild type mice. FXIWT, FXIC321S, L284A and FXIC321S, I290A were transiently expressed in FXI−/− mice by hydrodynamic tail vein injection. While the three proteins were expressed at comparable levels, only FXIWT completely reconstituted the wild type phenotype in the ferric chloride thrombosis model. In summary, polyP is a strong candidate for a cofactor to support FXI activation in vivo. The interaction of FXI with this polyanion differs from its interaction with DS. The dimeric structure of FXI appears to be required for normal protease function in vivo, and for FXIIa-mediated FXI activation, but not for thrombin- or FXIa-mediated activation in the presence of polyP. Considering that the FXI homolog PK is a monomer that is activated efficiently by FXIIa, and that FXII deficiency is not associated with a significant phenotype, our results suggest that the FXI dimeric structure is required for a function distinct from zymogen activation. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document