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.

Specific Pharmacological Targeting of the Syk Kinase Activity in Platelets: A Novel, Safe Anti-Thrombotic Strategy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 409-409 ◽  
Author(s):  
Suzanne Delaney ◽  
Uma Sinha ◽  
Nisha Nanda ◽  
Yibing Yan ◽  
Anjali Pandey ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Arterial Thrombosis ◽  
Kinase Activity ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Wild Type ◽  
Primary Hemostasis ◽  
Syk Kinase ◽  
Perfusion Chamber

Abstract Studies of the Syk −/− mouse have implicated spleen tyrosine kinase (Syk), a signaling protein with both kinase and scaffolding activities, in platelet signaling following engagement of GPVI and αIIbβ3 by collagen and fibrinogen, respectively. The present study was designed to determine whether specific inhibition of the kinase activity of Syk, without targeting the Syk scaffolding function, affected in vivo arterial thrombosis. In preliminary experiments, blood from wild-type and Syk−/− mice was perfused through collagen-coated capillaries under arterial shear rates to study ex vivo thrombosis. While blood from wild-type mice formed robust thrombi (37±4.7 μm3/μm2), none was observed in Syk−/− mice. Thrombi intermediate in size (16±3.9 μm3/μm2) developed in Syk+/− mice. To achieve specific pharmacological targeting of the kinase activity of Syk, P142-76, a potent (IC50 = 4 nM) and selective Syk kinase inhibitor was utilized. P142-76 was screened against a broad panel of 139 purified kinases at 50 nM. While Syk kinase was inhibited by 92%, all other kinases retained more than 70% of their activity. In washed human platelets, P142-76 inhibited convulxin (CVX)-induced phosphorylation of LAT (linker for activation of T-cells; IC50 = 111 nM) and intracellular calcium increases (IC50 = 31 nM). The GPVI/Syk-specificity of P142-76 activity was confirmed by its inability to inhibit intracellular calcium increases induced by the PAR1 thrombin receptor agonist TRAP. P142-76 also inhibited CVX-induced aggregation of both human washed platelets (IC50 = 87 nM) and platelet-rich plasma (IC50 = 2.5 μM). Considering the controversial data in respect to the participation of GPVI in arterial thrombosis in murine models, the dependence of arterial thrombosis on Syk function was studied in vivo in pigs. Cross-species activity of P142-76 was confirmed in vitro (CVX-induced PRP aggregation IC50= 350 nM; 5 μM P142-76 completely inhibited thrombosis triggered by collagen in the perfusion chamber assay). At a plasma concentration which abolished ex vivo CVX-induced but not ADP-induced pig platelet aggregation, P142-76 significantly inhibited the deposition of [111In]-labeled platelets in a carotid artery crush swine thrombosis model, without compromising primary hemostasis. % aggregation Swine (n=3) Platelet Deposition % inhibition Plasma Conc (ng/ml) Bleed Time (min) Activated Clotting Time (sec) ADP (20 μM) CVX (250 ng/ml) Control Artery 0 0 3±0.9 133±22 100 100 Treated Artery 76±6.5 1343±304 3.5±0.3 130±13 100 0 To clarify further the contribution of the kinase activity of Syk to arterial thrombosis, effects of P142-76 on human blood were evaluated in real time in the collagen-coated perfusion chamber. Low concentrations of P142-76 (0.3 μM) affected thrombus stability, while increasing concentrations (1–5 μM) delayed and then completely inhibited thrombus formation. Furthermore, P142-76 destabilized pre-formed thrombi, indicating a critical role for Syk in conferring strength to platelet-platelet interactions, i.e. αIIbβ3-mediated cohesion. Our data indicate that the kinase activity of Syk acts in arterial thrombosis through at least two distinct mechanisms. First, Syk kinase confers stability to platelet-platelet interactions downstream of αIIbβ3. Second, it initiates thrombus formation on collagen surfaces. This dual activity of the kinase activity of Syk makes it a preferred target for inhibition of arterial thrombosis, as it does not compromise primary hemostasis.

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.

Plasmin Generation Plays different Roles in the Formation and Removal of Arterial and Venous Thrombus in Mice

2002 ◽  
Vol 87 (01) ◽  
pp. 98-104 ◽  
Author(s):  
Osamu Kozawa ◽  
Kiyotaka Okada ◽  
Shigeru Ueshima ◽  
Osamu Matsuo ◽  
Toshihiko Uematsu ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Bleeding Time ◽  
Jugular Vein ◽  
Thrombus Formation ◽  
Endothelial Injury ◽  
Wild Type ◽  
Venous Thrombus ◽  
Vascular Patency ◽  
The Times

SummaryThe role of plasminogen (Plg) and α2-antiplasmin (α2-AP) in vascular thrombolysis in vivo was investigated in mice deficient in plasminogen (Plg−/−) or α2-AP (α2-AP−/−) or their wild type (PAI-1+/+, α2AP+/+). A thrombus was induced in the murine carotid artery or the internal jugular vein by endothelial injury. Blood flow was continuously monitored for 90 min and for 6 h 30 min after the initiation of endothelial injury. The times to occlusion by the developing thrombus in the carotid artery and the jugular vein of wild type mice were 12 ± 1.8 and 7.2 ± 1.9 min, respectively. The arterial thrombus formation in α2AP−/− mice was indistinguishable from the one in wild type mice, whereas the time to occlusion in Plg−/− was significantly shortened to 5.9 ± 1.7 min. Vascular patency after spontaneous reperfusion was markedly improved in α2-AP−/− mice. On the contrary, arterial patency in Plg−/− mice was aggravated. In venous thrombus formation, the time to occlusion in α2-AP−/− mice was significantly prolonged (27.1 ± 5.2 min), whereas in Plg−/− it was slightly shortened to 6.5 ± 2.5 min. Vascular patency after spontaneous reperfusion was also improved in α2-AP−/− mice, but not in Plg−/− mice. Histological observations using SEM indicated that fibrin nets were firmly fixed on the injured area in Plg−/− mice, but not in α2-AP−/− mice. The tail bleeding time was not different in any type of mice. However, re-bleeding time using a template bleeding device was significantly prolonged in α2-AP−/− as compared with that of wild type mice. In conclusion, lack of plasminogen markedly reduces the antithrombotic activities in vivo, whereas α2-AP plays a more important role in the formation and removal of venous thrombus in mice. Consequently, the inhibition of α2-AP could be a useful tool for the therapy of venous thrombosis and the prevention of re-thrombus formation.

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 Platelets Loaded with Recombinant ADAMTS13 Are Efficacious in Inhibiting Arterial Thrombosis Under Flow: A Potential Novel Therapeutic for Acquired TTP

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 763-763
Author(s):  
Mohammad Abdelgawwad ◽  
Liang Zheng ◽  
X. Long Zheng
Keyword(s):  
Ferric Chloride ◽  
Arterial Thrombosis ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Dependent Manner ◽  
Novel Therapy ◽  
Concentration Dependent Manner ◽  
Novel Therapeutic

Abstract Background: Thrombotic thrombocytopenic purpura (TTP), a potentially life-threatening blood clotting disorder, is resulted from severe deficiency of ADAMTS13 activity. Plasma exchange is the only effective initial therapy for acquired TTP, which replenishes ADAMTS13 while removing autoantibodies. However, the in-hospital mortality rate is still high, suggesting that another more effective therapy is urgently needed. Objectives: The present study aims to develop a novel therapy using platelets as carriers to deliver recombinant ADAMTS13 (rADAMTS13) to the sites of vascular injury, where platelets are activated and de-granulated to release rADAMTS13 from the storage granules. Our specific aims are: 1) determine the in vitro uptake of rADAMTS13 by platelets; 2) determine the efficacy of platelet-delivered rADAMTS13 in anti-arterial thrombosis under flow and in vivo. Methods: Freshly isolated human platelets from whole blood were incubated with an increasing concentration of rADAMTS13 (0, 5, 20, 50, and 100 µg/ml) at 25°C and 37°C for hours. Platelets were then washed and lysed with 1% Triton X-100 to assess the rADAMTS13 activity via a FRETS-VWF73 assay. Western blotting and confocal microscopy determined the amounts of rADAMTS13 protein and localization in platelets, respectively. The anti-thrombotic activity of the released rADAMTS13 from activated platelets was determined by a microfluidic system (BioFlux) and by a ferric chloride induced thrombus formation in mesenteric arterioles/venules in Adamts13-/- mice. Results: Human washed platelets were able to uptake rADAMTS13 in a temperature- and concentration-dependent manner. The rADAMTS13 protein packed inside human platelets remained intact and active towards the FRET-VWF73 substrate. Confocal microscopic analysis demonstrated its localization of uptaken rADAMTS13 in the alpha granules, partially overlapped with VWF. The rADAMTS13 released from activated human platelets under shear (100 dyne/cm2) was capable of cleaving VWF and inhibiting platelet agglutination and aggregation on a collagen-coated surface in a concentration-dependent manner. Transfusion of platelets expressing rADAMTS13 into Adamts13-/- mice dramatically reduced the rate of thrombus formation in the mesenteric arterioles/venules after ferric chloride injury. Conclusions: Our results demonstrate that human platelets can be reengineered in vitro to contain sufficient amounts of rADAMTS13 in the alpha granules. The rADAMTS13 inside platelets is releasable and proteolytically functional under shear stress or in vivo under flow. Our findings suggest that the transfusion of platelets loaded with rADAMTS13 may represent a novel therapeutic strategy for acquired TTP with inhibitors. Disclosures Zheng: Alexion: Speakers Bureau; Ablynx: Consultancy.

Antibodies to Human Factor XII with Antithrombotic Properties

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1106-1106
Author(s):  
Anton Matafonov ◽  
Adam E. Gailani ◽  
Stephanie L. Grach ◽  
Philberta Y Leung ◽  
Qiufang Cheng ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Thrombus Formation ◽  
Arterial Occlusion ◽  
Clot Formation ◽  
Type I ◽  
Wild Type ◽  
Contact Activation ◽  
Thrombotic Occlusion ◽  
Deficient Mice

Abstract Abstract 1106 The plasma protease factor XIIa (FXIIa) contributes to vascular occlusion in murine thrombosis models, at least partly through activation of factor XI (FXI). While there is good correlation between plasma FXI levels and thrombotic events in humans, the situation is not as clear for FXII (the precursor of FXIIa), suggesting fundamental differences in thrombus formation in mice and humans. To facilitate studies on the effects of FXII/XIIa on thrombus formation, we developed novel inhibitory antibodies to human FXII, designated 9A2 and 15H8, by immunizing FXII-deficient mice with human FXII. Using recombinant human FXII molecules that lack various domains, and chimeras in which specific domains in FXII are replaced with those from the related protein hepatocyte growth factor activator, we determined that 9A2 and 15H8 bind to the FXII/XIIa non-catalytic heavy chain at different sites. 9A2 binds on or near the EGF2 domain, while 15H8 binds to the fibronectin type I and/or kringle domain. These areas have been implicated in FXII binding to polyanionic surfaces. Saturating concentrations of 9A2 or 15H8 reduced FXII activity by 50% and 90%, respectively, in an aPTT assay using normal plasma, while combining the antibodies resulted in >95% inhibition. However, in assays in which clot formation was triggered by adding FXIIa directly to plasma, preincubation of FXIIa with either antibody did not prolong the clotting time. Furthermore, neither antibody had a strong effect in a chromogenic assay of FXI activation by FXIIa, indicating the antibodies interfere with the aPTT assay primarily by inhibiting FXII activation. FXII activation in the aPTT assay is initiated by addition of a polyanion such as silica to the plasma to induce contact activation. In vivo, polymers of inorganic phosphate (polyP) may serve a similar function. Contact activation is triggered in plasma when FXII bound to the polyanion is activated, probably by trace amounts of FXIIa or another protease present in the plasma. Once formed, FXIIa converts the zymogens prekallikrein and FXI to the proteases kallikrein and FXIa, both of which can activate additional FXIIa to amplify the process. In the presence of 9A2 or 15H8, activation of pure FXII in the presence of either silica or polyP was significantly reduced. Interestingly, the antibodies actually potentiated FXII activation by kallikrein or FXIa in the absence of a polyanion. Taken as a whole, these results suggest that binding of 9A2 or 15H8 to FXII results in conformational changes that make FXII a better substrate for kallikrein and FXIa, possibly by mimicking the effect of FXII binding to a polyanion, but that prevent activation of FXII by FXIIa (autoactivation), blunting the overall rate of activation. We tested the effects of 9A2 and 15H8 in a mouse model in which thrombotic occlusion of the carotid artery is induced by exposing the vessel to a 3.5% solution of ferric chloride. Wild type C57Bl/6 mice develop arterial occlusion within 5 to 10 minutes, while FXII-deficient mice are resistant to arterial occlusion. Infusion of human FXII into FXII-deficient mice restores the wild type phenotype. 15H8 prevented thrombus formation in mice reconstituted with human FXII, while 9A2 reduced the rate of thrombotic occlusion by 50%. In an ex vivo flow model, perfusion of human blood through collagen-coated tubes at a shear rate of 300 sec−1 results in tube occlusion by platelet and fibrin rich clot in ∼15 minutes. 15H8 effectively blocked fibrin formation and reduced platelet accumulation, preventing tube occlusion. 9A2 was also effective at preventing clot formation, but there was evidence of some fibrin accumulation over time. In summary, the monoclonal anti-human FXII IgGs 9A2 and 15H8 prevent thrombus formation in whole blood in vivo and ex vivo by interfering with FXII activation. Our data support the hypothesis that pharmacologic inhibition of FXII activation may have therapeutic utility in disorders that are driven or aggravated by the blood contact system. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Vascular dermatan sulfate regulates the antithrombotic activity of heparin cofactor II

Blood ◽  
2008 ◽  
Vol 111 (8) ◽  
pp. 4118-4125 ◽  
Author(s):  
Li He ◽  
Tusar K. Giri ◽  
Cristina P. Vicente ◽  
Douglas M. Tollefsen
Keyword(s):  
Carotid Artery ◽  
Dermatan Sulfate ◽  
Thrombus Formation ◽  
Wild Type ◽  
Heparin Cofactor Ii ◽  
Arterial Endothelium ◽  
Carotid Arterial ◽  
Deficient Mice

AbstractHeparin cofactor II (HCII)–deficient mice form occlusive thrombi more rapidly than do wild-type mice following injury to the carotid arterial endothelium. Dermatan sulfate (DS) and heparan sulfate (HS) increase the rate of inhibition of thrombin by HCII in vitro, but it is unknown whether vascular glycosaminoglycans play a role in the antithrombotic effect of HCII in vivo. In this study, we found that intravenous injection of either wild-type recombinant HCII or a variant with low affinity for HS (K173H) corrected the abnormally short thrombosis time of HCII-deficient mice, while a variant with low affinity for DS (R189H) had no effect. When HCII was incubated with frozen sections of the mouse carotid artery, it bound specifically to DS in the adventitia. HCII was undetectable in the wall of the uninjured carotid artery, but it became concentrated in the adventitia following endothelial injury. These results support the hypothesis that HCII interacts with DS in the vessel wall after disruption of the endothelium and that this interaction regulates thrombus formation in vivo.

Abstract 341: The Role of Nucleotidase in Arterial Thrombosis

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Roman Covarrubias ◽  
Elena Chepurko ◽  
Tatiana Novitskaya ◽  
Karen M Dwyer ◽  
Simon C Robson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Carotid Artery ◽  
Ferric Chloride ◽  
Arterial Thrombosis ◽  
Ex Vivo ◽  
Wild Type ◽  
Circulating Cells ◽  
Clodronate Liposome

Objective: To determine how leukocyte nucleotidase affects arterial thrombosis. Approach and Results: Ectonucleoside triphosphate diphosphohydrolase-1 (CD39) is expressed on circulating cells, endothelium and smooth muscle cells where it hydrolyzes extracellular ATP or ADP to AMP. We have demonstrated that transgenic mice with a global overexpression of human CD39 (hCD39-Tg) are protected against ferric chloride-induced carotid artery thrombosis. Furthermore, transplant of hCD39-Tg bone marrow into WT recipient mice increases the time to thrombosis when compared to recipient mice (wild-type or hCD39-Tg) receiving wild-type bone marrow. Based upon these data and previously published work, we hypothesized that CD39 expression on leukocytes is responsible for the prolongation of the time to thrombosis measured in hCD39-Tg mice. To test this hypothesis, we first performed ex vivo mixing experiments. Addition of hCD39-Tg monocytes to WT blood inhibits the expression of activated glycoprotein IIb/IIIa on platelets in response to ADP as measured by FACS analysis (Baseline: 1224 ± 94.9 MFI vs hCD39-Tg monocytes: 663.5 ± 61.5 activated glycoprotein IIb/IIIa MFI: n=4; p< 0.001). Subsequently, in vivo we demonstrated that monocytes with increased CD39 contribute to extending the time to thrombosis. Clodronate liposome depletion of monocytes (WT: 69% decrease; hCD39-Tg: 63% decrease) resulted in a normalization of the time to thrombosis in hCD39-Tg mice (8.0 ± 1.07 minutes, n = 10) when compared to control loaded liposomes (120.0 ± 0.0, n = 14). No changes in the time to thrombosis were detected in wild-type mice treated with clodronate (8.6 ± 1.35 minutes, n = 8) or control liposomes (7.8 ± 0.80 minutes, n=8). Conclusion: Increased expression of CD39 on monocytes can inhibit platelet activation and extend the time to thrombosis following ferric chloride-induced carotid artery injury.

The Modification of Experimental Occlusive Arterial Thrombosis in the Rat by Malayan Pit Viper Venom

1968 ◽  
Vol 19 (01/02) ◽  
pp. 242-247 ◽  
Author(s):  
K. E Chan
Keyword(s):  
Arterial Thrombosis ◽  
Thrombus Formation ◽  
Arterial Occlusion ◽  
Control Group ◽  
Antithrombotic Effect ◽  
Daily Dose

SummaryThe effect of Malayan pit viper (Ancistrodon rhodostoma) venom on the fate of experimental arterial thrombosis was studied in rats. A suitable daily dose of venom (500 μg) was used to induce hypofibrinogenaemia in the treated rats for the greater part of each of three consecutive post-operative days.The treated animals showed a statistically significant overall reduction in the incidence of both red thrombus formation and thrombotic arterial occlusion when compared to a control group. This antithrombotic effect of the venom could be observed in the 7-day period following the cessation of the treatment.

scholarly journals Accumulation of Tissue Factor into Developing Thrombi In Vivo Is Dependent upon Microparticle P-Selectin Glycoprotein Ligand 1 and Platelet P-Selectin

2003 ◽  
Vol 197 (11) ◽  
pp. 1585-1598 ◽  
Author(s):  
Shahrokh Falati ◽  
Qingde Liu ◽  
Peter Gross ◽  
Glenn Merrill-Skoloff ◽  
Janet Chou ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Thrombus Formation ◽  
Recipient Mouse ◽  
Wild Type ◽  
Activated Platelets ◽  
Injury Model ◽  
Platelet Thrombus ◽  
Flowing Blood

Using a laser-induced endothelial injury model, we examined thrombus formation in the microcirculation of wild-type and genetically altered mice by real-time in vivo microscopy to analyze this complex physiologic process in a system that includes the vessel wall, the presence of flowing blood, and the absence of anticoagulants. We observe P-selectin expression, tissue factor accumulation, and fibrin generation after platelet localization in the developing thrombus in arterioles of wild-type mice. However, mice lacking P-selectin glycoprotein ligand 1 (PSGL-1) or P-selectin, or wild-type mice infused with blocking P-selectin antibodies, developed platelet thrombi containing minimal tissue factor and fibrin. To explore the delivery of tissue factor into a developing thrombus, we identified monocyte-derived microparticles in human platelet–poor plasma that express tissue factor, PSGL-1, and CD14. Fluorescently labeled mouse microparticles infused into a recipient mouse localized within the developing thrombus, indicating that one pathway for the initiation of blood coagulation in vivo involves the accumulation of tissue factor– and PSGL-1–containing microparticles in the platelet thrombus expressing P-selectin. These monocyte-derived microparticles bind to activated platelets in an interaction mediated by platelet P-selectin and microparticle PSGL-1. We propose that PSGL-1 plays a role in blood coagulation in addition to its known role in leukocyte trafficking.

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