Enhanced Thrombotic Responses Are Associated With Striatin Deficiency and Aldosterone

Background In addition to its role on blood pressure, aldosterone (ALDO) also affects the hemostatic system leading to increased experimental thrombosis. Striatin is an intermediate in the rapid, nongenomic actions of ALDO. Striatin heterozygote knockout ( Strn +/‐ ) mice have salt sensitivity of blood pressure and mildly chronically increased ALDO levels. In addition, in humans, striatin polymorphic gene variants are associated with increased salt sensitivity of blood pressure. Thus, we hypothesized that striatin deficiency would be associated with an increased prothrombotic response. Methods and Results Strn +/ ‐ mice and wild‐type littermates were maintained on a liberal sodium diet (1.6%). We measured in vivo thrombus formation following laser‐induced injury in cremaster arterioles using intravital microscopy. Mice were randomized to intravenous administration of ALDO or its vehicle. Acutely, ALDO increased thrombotic responses in wild‐type mice ( P <0.01) versus controls within minutes as determined by increased platelet accumulation and fibrin deposition at the site of laser injury. We then compared thrombus formation without ALDO administration in Strn +/‐ and wild‐type mice. Strn +/‐ mice showed highly significant increases in laser‐induced thrombosis ( P <0.001), as shown by increased platelet accumulation and fibrin deposition. Interestingly, the response in the Strn +/‐ mice basally was far greater than the wild‐type mice with ALDO administration, and ALDO administration produced no additional effect on thrombus responses in Strn +/‐ mice. Conclusions These results demonstrate a novel protective role of striatin in experimental thrombosis. Such a protective effect may be reduced in human striatin risk allele carriers, given the similar salt sensitivity of blood pressure in these individuals and Strn +/‐ mice.

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Protease-Activator Receptor 4 Is Required for Maximal Thrombus Growth, but Not for Fibrin Generation in Thrombi after Laser Injury.

Blood ◽

10.1182/blood.v104.11.624.624 ◽

2004 ◽

Vol 104 (11) ◽

pp. 624-624 ◽

Cited By ~ 3

Author(s):

Erik R. Vandendries ◽

Justin R. Hamilton ◽

Shaun R. Coughlin ◽

Barbara C. Furie ◽

Bruce Furie

Keyword(s):

Platelet Activation ◽

High Speed ◽

Thrombin Generation ◽

Thrombus Formation ◽

Shape Change ◽

Fluorescent Antibody ◽

Fibrin Deposition ◽

Wild Type ◽

Platelet Accumulation

Abstract The serine protease, thrombin, is necessary for the conversion of fibrinogen to fibrin and is a potent activator of platelets. Thrombin-induced platelet activation, as measured by shape change, calcium mobilization, and ATP secretion, requires the protease-activator receptor 4 (PAR4). Platelets isolated from PAR4 knock-out mice are unresponsive to thrombin, and PAR4 null mice appear to be protected from thrombosis in a ferric chloride-induced injury model of thrombosis and a thromboplastin model of pulmonary embolism. To examine further the role of thrombin-induced platelet activation in developing thrombi, we have examined the in vivo kinetics of thrombus formation in living mice lacking PAR4 using high-speed widefield digital microscopy. In this study, platelets were labeled using anti-CD41 Fab fragments conjugated to Alexa-488. Thrombi were generated by laser-induced injury of the cremaster arteriolar vessel wall, and the total fluorescent antibody accumulation was monitored and quantitated for 5 minutes after injury. In PAR4 null mice, the thrombi generated were significantly smaller with an early arrest of thrombus growth when compared to thrombi generated in wild-type mice. The maximum thrombus platelet accumulation in PAR4 null mice (median of 30 thrombi in 3 mice) was 75% less than that seen in wild-type mice (median of 33 thrombi in 4 mice)(P<0.001). The time to half-maximal and the time to maximal thrombus formation in PAR4 null mice is approximately 5.5 seconds and 16 seconds, respectively, compared to 45 seconds and 74 seconds in wild-type mice (P<0.001). The shortened time to maximal platelet accumulation appears to be secondary to an early termination of thrombus growth. Fibrin generation was monitored using Alexa-647 conjugated to an anti-fibrin antibody that does not recognize fibrinogen in mice simultaneously infused with anti-CD41 Fab conjugated to Alexa-488. No difference in total fibrin accumulation was seen during the first 4 minutes of thrombus formation in PAR4 null mice (median of 23 thrombi in 3 mice) compared to thrombi generated in wild-type mice (median of 26 thrombi in 4 mice) despite a significant decrease in platelet accumulation in PAR4 null thrombi. Most of the fibrin deposition in both wild-type and PAR4 null thrombi was observed at the vascular wall/thrombus interface. In summary, thrombin-induced platelet activation via PAR4 is required for normal thrombus growth. However, in this model of thrombosis, neither PAR4 nor maximal thrombus growth appears to be necessary for fibrin deposition. This suggests that a platelet-independent mechanism of thrombin generation may exist. Alternatively, the amount of platelet accumulation and activation in PAR4 null mice may be sufficient for normal thrombin generation and subsequent fibrin deposition.

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

Blood ◽

10.1182/blood.v112.11.691.691 ◽

2008 ◽

Vol 112 (11) ◽

pp. 691-691 ◽

Cited By ~ 1

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.

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Factor XI Deficient Mice Have Reduced Platelet Accumulation and Fibrin Deposition after Laser Injury.

Blood ◽

10.1182/blood.v104.11.218.218 ◽

2004 ◽

Vol 104 (11) ◽

pp. 218-218

Author(s):

T. Regan Baird ◽

David Gailani ◽

Bruce Furie ◽

Barbara C. Furie

Keyword(s):

Tissue Factor ◽

Factor Viia ◽

Thrombus Formation ◽

Factor Xa ◽

Fibrin Deposition ◽

Wild Type ◽

Factor Xi ◽

Factor Xia ◽

Tissue Factor Pathway ◽

Platelet Accumulation

Abstract Tissue factor exposure at sites of vascular injury results in the generation of factor Xa and thrombin. A current model of blood coagulation suggests that the amount of thrombin generated through this pathway is limited by the inhibition of the factor VIIa-tissue factor complex by tissue factor pathway inhibitor in the presence of factor Xa. The initial thrombin activates a number of hemostatic proteins including factor XI. Factor XIa then activates factor IX leading to generation of the tenase complex to maintain the thrombin flux. While in vitro studies support this hypothesis the importance of factor XI for thrombus formation in vivo remains unclear. We have examined thrombus formation upon laser injury to the arterioles (30–50 μm diameter) of the cremaster muscle in living mice lacking factor XI using digital multi-channel fluorescence intravital microscopy. Platelets were labeled with Alexa 488 conjugated murine CD41 Fab fragments by systemic infusion of the antibody. Maximum platelet accumulation in factor XI null mice (median of 35 thrombi in 4 mice) is only 25% of that of wild type mice (median of 40 thrombi in 4 mice) after injury (p<0.03). The time course of platelet accumulation is similar between both genotypes. Maximum platelet accumulation occurs in approximately 90 seconds (p<0.2). Fibrin deposition was observed simultaneously using an Alexa 660 conjugated anti-fibrin antibody that does not recognize fibrinogen. Maximum fibrin deposition in factor XI null mice is 50% that of wild type mice (p<0.001) and the rate of fibrin generation is slower in factor XI null mice. However, the time to achieve half maximal fibrin deposition is approximately the same in factor XI null mice (77 sec) compared to wild type mice (63.5 sec, p<0.09). These data suggest that the primary difference in response to laser induced injury between the factor XI null mice and wild type mice is the level of thrombin generated and supports the hypothesis that factor XI participates in maintaining thrombin flux after inhibition of the factor VII-tissue factor. The model above postulates a single source of tissue factor, the vessel wall, and further, that the tissue factor-factor VIIa complex formed from the exposed tissue factor is rapidly inactivated by tissue factor pathway inhibitor after the appearance of the initial factor Xa formed. In addition it has been suggested that a rapidly growing thrombus blocks access to vascular wall tissue factor. However we have recently observed that there is a P-selectin and P-selectin glycoprotein ligand 1 dependent pathway of blood coagulation that recruits blood borne tissue factor into a growing thrombus at sites of laser-induced vessel injury. Both vessel wall and blood borne tissue factor are required for normal thrombus formation. Our data suggest that although tissue factor is continuously recruited to the growing thrombus, factor XIa plays a significant role in thrombin generation.

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Deficiency of MicroRNA-181a Results in Transcriptome-Wide Cell Specific Changes in the Kidney that Lead to Elevated Blood Pressure and Salt Sensitivity

10.1101/2021.03.18.436092 ◽

2021 ◽

Author(s):

Madeleine R. Paterson ◽

Kristy L. Jackson ◽

Malathi I. Dona ◽

Gabriella E. Farrugia ◽

Bruna Visniauskas ◽

...

Keyword(s):

Blood Pressure ◽

Knockout Mice ◽

Molecular Mechanisms ◽

Salt Sensitivity ◽

Juxtaglomerular Cells ◽

Renal Cells ◽

Wild Type ◽

Knockout Mouse Model

AbstractMicroRNA miR-181a is down-regulated in the kidneys of hypertensive patients and hypertensive mice. In vitro, miR-181a is a posttranslational inhibitor of renin expression, but pleiotropic mechanisms by which miR-181a may influence blood pressure (BP) are unknown. Here we determined whether deletion of miR-181a/b-1 in vivo changes BP and the molecular mechanisms involved at the single-cell level. We developed a knockout mouse model lacking miR-181a/b-1 genes using CRISPR/Cas9 technology. Radio-telemetry probes were implanted in twelve-week-old C57BL/6J wild-type and miR-181a/b-1 knockout mice. Systolic and diastolic BP were 4-5mmHg higher in knockout compared with wild-type mice over 24-hours (P<0.01). Compared with wild-type mice, renal renin was higher in the juxtaglomerular cells of knockout mice. BP was similar in wild-type mice on a high (3.1%) versus low (0.3%) sodium diet (+0.4±0.8mmHg) but knockout mice showed salt sensitivity (+3.3±0.8mmHg, P<0.001). Since microRNAs can target several mRNAs simultaneously, we performed single-nuclei RNA-sequencing in 6,699 renal cells. We identified 12 distinct types of renal cells, all of which had genes that were dysregulated. This included genes involved in renal fibrosis and inflammation such as Stat4, Col4a1, Cd81, Flt3l, Cxcl16, Smad4. We observed up-regulation of pathways related to the immune system, inflammatory response, reactive oxygen species and nerve development, consistent with higher tyrosine hydroxylase. In conclusion, downregulation of the miR-181a gene led to increased BP and salt sensitivity in mice. This is likely due to an increase in renin expression in juxtaglomerular cells, as well as microRNA-driven pleiotropic effects impacting renal pathways associated with hypertension.

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Regulation of Protein Disulfide Isomerase By S-Nitrosylation Controls Its Function during Thrombus Formation

Blood ◽

10.1182/blood.v124.21.93.93 ◽

2014 ◽

Vol 124 (21) ◽

pp. 93-93

Author(s):

Roelof H Bekendam ◽

Gopal Srila ◽

Pavan K Bendapudi ◽

James R Dilks ◽

Lin Lin ◽

...

Keyword(s):

Platelet Aggregation ◽

Vascular Injury ◽

Protein Disulfide Isomerase ◽

Thrombus Formation ◽

Reductase Activity ◽

Wild Type ◽

Disulfide Isomerase ◽

Platelet Accumulation ◽

Protein Disulfide

Abstract Protein disulfide isomerase (PDI) is an oxidoreductase that is essential for thrombus formation following vascular injury. Clinical trials testing the efficacy and safety of PDI inhibition in the setting of thrombotic disease are currently underway. Yet while preclinical and clinical trials of PDI in thrombosis have progressed rapidly, the mechanisms by which PDI is regulated in the vasculature and how it mediates thrombosis remain unknown. PDI has an a-b-b'-x-a' domain structure, where the a and a' domains contain a CGHC motif responsible for cleaving and forming disulfide bonds. The active site cysteines within the catalytic CGHC motif that perform oxidoreductive reactions can also undergo S-nitrosylation. We have evaluated the hypothesis that nitric oxide (NO) converts PDI into a nitrosylase and regulates PDI oxidoreductase activity in the vasculature during thrombus formation. Initial studies demonstrated that incubation of recombinant PDI with the NO donor, SNAP, resulted in an 83±1.4% decrease in its reductase activity. A transnitrosylase assay using the NO indicator DAF-FM showed that S-nitrosylated PDI (SNO-PDI) transferred NO into platelets and inhibited platelet aggregation. To define the molecular determinants of PDI nitrosylation activity, we evaluated mutant PDIs containing Cys -> Ala mutations of the CGHC (a domain)/CGHC (a' domain) motifs in the platelet-based transnitrosylase assay. Wild-type PDI (CGHC/CGHC) demonstrated full reductase and nitrosylase activity and the enzymatically dead mutant (AGHA/AGHA) showed neither activity. In contrast, the CGHA/CGHA mutant maintained nitrosylase activity (41±0.23%), but had no reductase activity. This observation suggested that reductase and nitrosylase activities were separable. To further evaluate this supposition, we screened a series of PDI mutants in which intervening sequences of the CGHC domain had been modified. The screen identified CGPC/CGPC as a nitrosylase-biased mutant that showed a 59±2.31% decrease in reductase activity, but a 72±1.83% increase in nitrosylase activity compared to wild-type PDI. Another nitrosylase-biased mutant, CGRC/CGRC, showed a similar activity pattern. Since PDI is prothrombotic and SNO-PDI is antithrombotic, we compared the activity of nitrosylase-biased mutants with wild-type PDI in platelet aggregation studies in the presence of physiological concentrations of GSNO. While wild-type PDI had little effect on platelet aggregation, nitrosylase-biased PDIs such as the CGPC/CGPC and CGRC/CGRC mutant completely inhibited platelet aggregation. These studies show that the prothrombotic oxidoreductase activities of PDI are separable from their antithrombotic nitrosylase activities and that nitrosylase-biased PDI mutants have antiplatelet activity. We next evaluated the effect of PDI nitrosylation on thrombus formation in vivo. Infusion of SNO-PDI into mice inhibited thrombus formation following laser-induced vascular injury of cremaster arterioles. Mice deficient in glutathione-S-nitrosyl reductase (GSNOR) were used to assess the role of endogenous NO in thrombus formation. GSNOR enzymatically reduces GSNO, the main storage form of NO in cells. Platelet accumulation and fibrin formation were hardly detectable in GSNOR-/- mice. Infusion of recombinant WT PDI, but not an enzymatically dead PDI, reversed the defect in platelet accumulation and fibrin generation to levels of WT mice. In order to visualize NO during thrombus formation, the NO-sensitive dye DAF-FM was infused into mice and NO signal in endothelium monitored following laser-induced injury. DAF-FM signal decreased rapidly following laser injury of cremaster arterioles, indicating an activation-induced reduction in endothelial NO in vivo. In conclusion, our studies show that oxidoreductase and nitrosylase activities of PDI are separable and support a model whereby high endothelial NO levels maintain vascular quiescence in part by maintaining PDI as a nitrosylase and blocking its prothrombotic PDI activity. We propose that the reduction of NO levels that occurs with vascular injury or endothelial dysfunction contributes to the conversion of PDI from an anti-thrombotic nitrosylase to a prothrombotic reductase. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood-2005-09-3687 ◽

2006 ◽

Vol 107 (10) ◽

pp. 3902-3906 ◽

Cited By ~ 158

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.

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

Blood ◽

10.1182/blood.v104.11.842.842 ◽

2004 ◽

Vol 104 (11) ◽

pp. 842-842 ◽

Cited By ~ 3

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.

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Thrombin-Dependent Platelet Activation Is VWF-Independent during Thrombus Formation In Vivo.

Blood ◽

10.1182/blood.v108.11.1510.1510 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1510-1510

Author(s):

Christophe Dubois ◽

Laurence Panicot-Dubois ◽

Justin F. Gainor ◽

Barbara C. Furie ◽

Bruce Furie

Keyword(s):

Platelet Activation ◽

Thrombus Formation ◽

Specific Inhibitor ◽

Calcium Mobilization ◽

Wild Type ◽

Platelet Surface ◽

Platelet Thrombus ◽

Platelet Accumulation ◽

Kinetics Of

Abstract Adhesion to and activation of platelets at an injured vessel wall are critical events in the formation of a thrombus. Calcium mobilization is one marker of platelet activation. Of different agonists capable of activating platelets in vitro, thrombin, collagen and vWF have been described to induce calcium mobilization, leading to the formation of aggregates. Using high speed digital multichannel intravital microscopy, we characterized calcium mobilization during platelet activation and thrombus formation in genetically modified mice. The kinetics of platelet activation and accumulation after laser-induced injury in cremaster muscle arterioles of living mice were analyzed. In wild type mice, platelets adhered and accumulated rapidly at the site of laser-induced injury. Thrombi increased in size, reached a maximum size at 90–120 sec, decreased in size and then stabilized within 3 to 4 min post-injury. In vWF−/− mice, the kinetics of platelet accumulation followed the same pattern as in wild type mice. However, a significant albeit modest reduction in the size of each thrombus was observed in these genetically deficient mice in comparison with wild type mice. By ranking the thrombi by size, we observed that 40% of the thrombi formed in vWF−/− mice were present in the quadrant containing the smallest thrombi versus 18% for the wild type mice. Only 8% of the thrombi formed in vWF−/− mice were distributed in the quadrant containing the largest thrombi versus 32% for the wild type mice. In wild type mice treated with lepirudin, a specific inhibitor of thrombin activity, a small early accumulation of platelets was observed at about 16 sec whereas in untreated wild type mice this early accumulation is often obscured by subsequent thrombin-mediated platelet accumulation and activation. However, at the time of maximal thrombus size in wild-type mice, platelet accumulation in wild type mice was more than ten-fold greater than in wild type mice treated with lepirudin. The kinetics of platelet accumulation were similar in FcRγ−/− mice lacking GPVI, GPVI-depleted mice and wild type mice. Furthermore, depletion of GPVI from the platelet surface of vWF−/− mice or platelets of wild type mice treated with lepirudin did not alter the kinetics of platelet accumulation in those mice. By monitoring calcium mobilization per platelet engaged in the growing thrombus, we observed that elevated calcium levels in each platelet were similar in FcRγ−/−, GPVI depleted, vWF−/− and wild type mice. However in wild type mice treated with lepirudin, platelet calcium mobilization was almost completely inhibited in comparison with those observed in wild type mice. Our results indicate that thrombin is the major agonist leading to platelet activation after laser-induced injury. Collagen, as previously reported (Dubois, Blood.2006;107:3902) does not play a role in platelet thrombus formation after laser injury and, based on data reported here, does not play a role in platelet activation in this model. vWF is important for the growth of the platelet thrombus but is not required for initial platelet accumulation or platelet activation in vivo in this thrombosis model. The platelet agonist or ligand responsible for initial early platelet accumulation after laser-induced injury is unknown, and does not require GPVI, thrombin or vWF.

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Role of Thiol Isomerase ERp5 in Thrombus Formation

Blood ◽

10.1182/blood.v118.21.370.370 ◽

2011 ◽

Vol 118 (21) ◽

pp. 370-370

Author(s):

Freda H. Passam ◽

Lin Lin ◽

Mingdong Huang ◽

Jonathan M. Gibbins ◽

Bruce Furie ◽

...

Keyword(s):

Cho Cells ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Dependent Manner ◽

Wild Type ◽

In Vivo Models ◽

Platelet Accumulation

Abstract Abstract 370 Protein disulfide isomerase is required for thrombus formation in various in vivo models of thrombosis. Another member of the thiol isomerase family, endoplasmic reticulum protein 5 (ERp5), is released from activated platelets and co-immunoprecipitates with beta 3 integrin (Jordan et al, 2005). We further investigated the association of ERp5 with the platelet fibrinogen receptor alpha IIb beta 3 and the significance of ERp5 release in thrombus formation in vivo. Recombinant purified ERp5 was labeled with Alexa 488 and used in direct binding assays to CHO cells expressing wild type (WT) alpha IIb beta 3, CHO cells expressing mutant alpha IIb beta 3 (containing an Asp119Tyr substitution in the beta 3 subunit) and to control CHO cells. The mutant alpha IIb beta 3 does not bind fibrinogen. ERp5 bound to CHO cells expressing wild type (WT) alpha IIb beta 3 in a dose-dependent manner but did not bind to CHO cells expressing mutant alpha IIb beta 3 or to control CHO cells. The relative increase in the geomean of Alexa 488-labeled ERp5 binding to 0.5 ×106 WT alpha IIb beta 3 CHO cells over that bound to control CHO cells was 20, 45 and 85% for ERp5 concentrations of 80, 160 and 400 nM respectively. Binding of ERp5 (160 nM) to WT alpha IIb beta 3 expressing CHO cells was further increased by 75% when the integrin was activated with 2 mM Mn2+ compared to non-activated WT alpha IIb beta 3 CHO cells. A role for ERp5 in thrombus formation was studied in the laser injury model of thrombosis in mouse cremaster arterioles using a rabbit polyclonal anti-ERp5 antibody, immunoaffinity purified against recombinant ERp5. This antibody detected ERp5 in the releasate of thrombin-activated mouse platelets in vitro by Western blot and on the surface of thrombin-activated mouse platelets by flow cytometry. Dylight 649-labeled anti-CD42b was infused into the mouse circulation to detect platelet accumulation and Alexa 488-labeled anti-ERp5 antibody at 0.05 ug/g, a dose that does not inhibit thrombus formation, was infused to detect ERp5. The fluorescent anti-ERp5 signal detected at the thrombus site was compared to the signal produced by a non-specific IgG labeled with Alexa 488 infused into a control mouse. Anti-ERp5 fluorescence was detected in the thrombus with kinetics that followed platelet accumulation whereas there was minimal signal from the control IgG. We examined whether higher doses of anti-ERp5 affect thrombus formation. Platelet and fibrin accumulation were detected using fluorescently labeled anti-CD42b antibody and monoclonal anti-fibrin-specific antibody respectively before or after the injection of unlabeled anti-ERp5 antibody or pre-immune IgG at 2.5 ug/g. Platelet and fibrin accumulation, expressed as area under the curve of the median integrated fluorescence over time, was obtained from 14 thrombi in 6 mice formed before infusion of antibody, 18 thrombi in 2 mice formed after infusion of control IgG and 29 thrombi in 3 mice formed after infusion of anti-ERp5. Anti-ERp5 infusion caused a 70% decrease in the deposition of platelets and a 62% decrease in fibrin accumulation compared to infusion of control antibody (p<0.01). There was no difference in platelet and fibrin accumulation before infusion of antibody and after infusion of control antibody. These results provide evidence for a role of a second thiol isomerase, ERp5, in thrombus formation, a function which may be mediated through its association with alpha IIb beta 3. Disclosures: No relevant conflicts of interest to declare.

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