Mice That Lack RhoA In Their Platelets Have Normal Actin Dynamics, but Have Macrothrombocytopenia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 549-549
Author(s):  
Aae Suzuki ◽  
Lurong Lian ◽  
Liang Zhao ◽  
Sang H. Min ◽  
Yuhuan Wang ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Platelet Adhesion ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Adhesion Formation ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Tissue Culture Cell ◽  
Platelet Factor ◽  
Gtp Binding

Abstract Abstract 549 In response to agonist stimulation, platelets undergo a rapid reorganization of their actin cytoskeleton. This process involves simultaneous disassembly and assembly of filamentous actin, and is one of the earliest phenomena seen in platelet activation. Ex vivo flow models suggest that the platelet cytoskeleton is required for platelet adhesion that can withstand the shear conditions found within the arterial vascular system. The signaling pathways that link external stimuli with actin assembly are believed to include polyphosphoinositides, small GTP-binding proteins, and actin binding proteins. Extrapolations of data, mostly derived from tissue culture cell lines, suggest that a central component of this signaling cascade is the small GTP binding protein, RhoA. A few studies using a RhoA-specific pharmacologic inhibitor, C3 exotoxin, suggest that RhoA is essential for platelet spreading and focal adhesion formation. These findings support the hypothesis that RhoA within platelets is critical for the cytoskeletal dependent processes that contribute to platelet plug formation. To determine the true in vivo role of RhoA within platelets, we utilized a murine genetic approach. Mice were genetically modified to contain conditional RhoA null mutation by inserting LoxP sites flanking exon 3. This exon encodes the P-loop and Switch 1 domains within this protein. RhoA fl/fl mice were crossed with Platelet factor 4 (PF4) expressing Cre mice. The PF4 promotor leads to Cre expression exclusively in platelets and megakaryocytes, thereby producing homologous recombination at the LoxP sites, and deletion the critical exon only within these cells. This end result of this breeding strategy produced RhoA fl/fl PF4 Cre+ mice that specifically lacked RhoA only in their platelets and megakaryocytes. RhoA fl/fl PF4 Cre+ mice were compared with their RhoA fl/fl PF4 Cre- littermates. RhoA fl/fl PF4 Cre+ mice appeared normal, but had platelet counts that were 30% +/− 3% lower than normal. The mean platelet volume was also increased by 25 % +/− 7 % in the RhoA-null platelets. Review of the peripheral blood smears confirmed that the mice had macrothrombocytopenia, but did not reveal any abnormalities in the erythrocytes or leukocytes of the mice. Examination of the bone marrows from RhoA fl/fl PF4 Cre+ mice demonstrated that they had at least as many megakaryocytes as RhoA fl/fl PF4 Cre- mice. But compared to the control cells, the RhoA-null megakaryocytes were larger, more lobulated, and had more cytoplasm. Furthermore, the thromobocytopenia is probably not due to splenic sequestration because the spleens of RhoA fl/fl PF4 Cre- mice were only minimally larger (less than 10%) than those of the control mice. These results suggest that the mechanism for thrombocytopenia is due to peripheral destruction. Platelets derived from RhoA fl/fl PF4 Cre+ mice were studied ex vivo, and were found to undergo shape change and aggregate normally in response to thrombin, collagen, and the thromboxane A2 analog, U46619. Surprisingly, platelet adhesion and cell spreading was also unaffected by the loss of RhoA. It is also remarkable that total F-actin (as assessed by phalloidin staining) was identical in the platelets derived from RhoA fl/fl PF4 Cre+ and RhoA fl/fl PF4 Cre- mice. Our results definitively refute the model that RhoA is an essential component of platelet actin dynamics and platelet adhesion. Instead our findings surprisingly indicate that loss of the platelet RhoA causes macrothrombocytopenia. Our data suggests that the development of macrothrombocytopenia is due to an intrinsic platelet abnormality that leads to a shortened platelet lifespan. Disclosures: No relevant conflicts of interest to declare.

Role Of Monocyte Fcγ Receptors In The Prothrombotic State Of Heparin Induced Thrombocytopenia (HIT)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 570-570
Author(s):  
Lubica Rauova ◽  
Daria Madeeva ◽  
Valerie Tutwiler ◽  
Shahara Bailey ◽  
Vincent M. Hayes ◽  
...  
Keyword(s):  
Whole Blood ◽  
Platelet Adhesion ◽  
Conflicts Of Interest ◽  
Adhesion Formation ◽  
Microfluidic Channel ◽  
Selective Inhibition ◽  
Prothrombotic State ◽  
Platelet Factor ◽  
Selective Blockade

Abstract HIT is an antibody-mediated disorder characterized by thrombocytopenia and thrombosis. Activation of platelet via FcγRIIA by HIT antibodies complexed to surface-bound platelet factor 4 (PF4) contributes to the prothrombotic state. However, using an in vitro microfluidic whole human blood model of HIT, we have found through depletion and repletion studies that activation of monocytes through Syk-kinase is a key step in generating a prothrombotic milieu. Here we define the Fcγ receptor(s) upstream of Syk that mediate the thrombotic signal in monocytes. Monocytes isolated from normal healthy volunteers were incubated with 50 µg/ml of blocking antibodies against FcγRIIA (CD32, clone IV.3) and/or against FcγRI (CD64, clone 10.1) or against FcγRIII (CD16, clone 3G8), washed to remove unbound antibody and added to monocyte depleted-whole blood. These monocyte-repleted samples were then stimulated with PF4 (10 µg/ml) and the HIT like monoclonal antibody KKO (50 µg/ml) for 30 min. After recalcification (5 mmol CaCl2), samples were perfused over a von Willebrand factor-coated BioFlux microfluidic channel at a shear stress of 20 dyne/cm2 at 37°C for 15 min. Platelet adhesion was quantified by fluorescence microscopy after adding Calcein-AM (3 µM), and fibrin was visualized by adding Alexa 561-labeled fibrinogen (1.5 μg/ml) to the whole blood prior to the perfusion. Selective inhibition of FcγRIIA on monocytes led to an ∼40% inhibition of fibrin deposition and comparable reduction in the formation of “coated” platelets relative to samples repleted with non-inhibited monocytes. Flow cytometry studies showed that these observations were not due to leaching of IV.3 antibody off the monocytes with subsequent blocking of platelet FcγRIIA. In contrast, selective blockade of FcγRI or FcγRIII in depletion/repletion studies or in whole blood did not significantly reduce platelet adhesion, formation of coated platelets, or fibrin accumulation. However, blocking FcγRI did decrease tissue factor (TF) activity on monocytes stimulated with PF4 and KKO and this effect was additive to FcγRIIA inhibition. In summary, our data show that monocyte-bound HIT IgG antibodies contribute to the prothrombotic state by activating FcγRIIA directly and indirectly by generating TF though FcγRI. We speculate that this activation of monocytes contributes to platelet activation and monocyte microparticle formation demonstrated previously. Drugs that target FcγRI and FcγRIIA on monocytes may help prevent thrombosis in HIT without causing platelet dysfunction or increasing the risk of bleeding. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Platelet adhesion: Structural and functional diversity of short dystrophin and utrophins in the formation of dystrophinassociated-protein complexes related to actin dynamics

2005 ◽  
Vol 94 (12) ◽  
pp. 1203-1212 ◽  
Author(s):  
Doris Cerecedo ◽  
Dalila Martínez-Rojas ◽  
Oscar Chávez ◽  
Francisco Martínez-Pérez ◽  
Francisco García-Sierra ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Platelet Adhesion ◽  
Protein Complexes ◽  
Human Platelets ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Dynamic Cell ◽  
Associated Proteins ◽  
Coated Surfaces ◽  
Dynamic Structures

SummaryPlatelets are dynamic cell fragments that modify their shape during activation. Utrophin and dystrophins are minor actin-binding proteins present in muscle and non-muscle cytoskeleton. In the present study, we characterised the pattern of Dp71 isoforms and utrophin gene products by immunoblot in human platelets. Two new dystrophin isoforms were found, Dp71f and Dp71d, as well as the Up71 isoform and the dystrophin-associated proteins, α and β-dystrobrevins. Distribution of Dp71d/Dp71Δ110 m, Up400/Up71 and dystrophin-associated proteins in relation to the actin cytoskeleton was evaluated by confocal microscopy in both resting and platelets adhered on glass. Formation of two dystrophin-associated protein complexes (Dp71d/Dp71Δ110 m ~DAPC and Up400/Up71~DAPC) was demonstrated by co-immunoprecipitation and their distribution in relation to the actin cytoskeleton was characterised during platelet adhesion. The Dp71d/Dp71Δ110 m ~DAPC is maintained mainly at the granulomere and is associated with dynamic structures during activation by adhesion to thrombin-coated surfaces. Participation of both Dp71d/Dp71Δ110 m ~DAPC and Up400/Up71~DAPC in the biological roles of the platelets is discussed.

scholarly journals Inherited abnormalities in platelet organelles and platelet formation and associated altered expression of low molecular weight guanosine triphosphate-binding proteins in the mouse pigment mutant gunmetal

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2626-2635 ◽  
Author(s):  
RT Swank ◽  
SY Jiang ◽  
M Reddington ◽  
J Conway ◽  
D Stephenson ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Binding Proteins ◽  
Low Molecular Weight ◽  
Guanosine Triphosphate ◽  
Platelet Factor ◽  
Altered Expression ◽  
Gtp Binding Proteins ◽  
Dense Granules ◽  
Gtp Binding ◽  
Platelet Formation

Abstract Gunmetal (gm/gm) is a recessively inherited mouse pigment dilution mutant that has high mortality and poor reproductive rates. In these studies, several hematologic defects were found associated with the mutation, including prolonged bleeding times, together with thrombocytopenia and increased platelet size. A unique feature is the presence of simultaneous abnormalities in two platelet organelles, dense granules and alpha-granules. The dense granule component serotonin is present at about half the normal concentration, as are visible dense granules. Three alpha-granule components (fibrinogen, platelet factor 4, and von Willebrand factor) are also significantly reduced. Thus, in several respects the gunmetal mutant resembles the human gray platelet syndrome. A novel abnormality in expression of low molecular weight guanosine triphosphate (GTP)-binding proteins occurs in platelets of gunmetal. In Western blot assays, two additional GTP- binding proteins of 28.5 and 25 Kd were detected. The abnormal expression of GTP-binding proteins is, like the hematologic defects, genetically recessive and is tissue specific. Liver, kidney, brain, spleen, macrophages, and neutrophils have normal GTP-binding protein expression. The additional GTP-binding proteins are soluble. The data indicate that platelet formation and platelet organelle biogenesis are under common genetic control and that abnormal regulation of GTP- binding proteins may affect one or both processes.

scholarly journals STIP1/HOP Regulates the Actin Cytoskeleton through Interactions with Actin and Changes in Actin-Binding Proteins Cofilin and Profilin

2020 ◽  
Vol 21 (9) ◽  
pp. 3152 ◽  
Author(s):  
Samantha Joy Beckley ◽  
Morgan Campbell Hunter ◽  
Sarah Naulikha Kituyi ◽  
Ianthe Wingate ◽  
Abantika Chakraborty ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Binding Proteins ◽  
Major Effect ◽  
Vital Role ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Nuclear Accumulation ◽  
Actin Binding Proteins ◽  
Hsp90 Chaperone

Cell migration plays a vital role in both health and disease. It is driven by reorganization of the actin cytoskeleton, which is regulated by actin-binding proteins cofilin and profilin. Stress-inducible phosphoprotein 1 (STIP1) is a well-described co-chaperone of the Hsp90 chaperone system, and our findings identify a potential regulatory role of STIP1 in actin dynamics. We show that STIP1 can be isolated in complex with actin and Hsp90 from HEK293T cells and directly interacts with actin in vitro via the C-terminal TPR2AB-DP2 domain of STIP1, potentially due to a region spanning two putative actin-binding motifs. We found that STIP1 could stimulate the in vitro ATPase activity of actin, suggesting a potential role in the modulation of F-actin formation. Interestingly, while STIP1 depletion in HEK293T cells had no major effect on total actin levels, it led to increased nuclear accumulation of actin, disorganization of F-actin structures, and an increase and decrease in cofilin and profilin levels, respectively. This study suggests that STIP1 regulates the cytoskeleton by interacting with actin, or via regulating the ratio of proteins known to affect actin dynamics.

scholarly journals Actin-binding proteins: the long road to understanding the dynamic landscape of cellular actin networks

2016 ◽  
Vol 27 (16) ◽  
pp. 2519-2522 ◽  
Author(s):  
Pekka Lappalainen
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Three Dimensional ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Binding Proteins ◽  
Vast Number ◽  
Actin Regulatory Proteins ◽  
The Individual

The actin cytoskeleton supports a vast number of cellular processes in nonmuscle cells. It is well established that the organization and dynamics of the actin cytoskeleton are controlled by a large array of actin-binding proteins. However, it was only 40 years ago that the first nonmuscle actin-binding protein, filamin, was identified and characterized. Filamin was shown to bind and cross-link actin filaments into higher-order structures and contribute to phagocytosis in macrophages. Subsequently many other nonmuscle actin-binding proteins were identified and characterized. These proteins regulate almost all steps of the actin filament assembly and disassembly cycles, as well as the arrangement of actin filaments into diverse three-dimensional structures. Although the individual biochemical activities of most actin-regulatory proteins are relatively well understood, knowledge of how these proteins function together in a common cytoplasm to control actin dynamics and architecture is only beginning to emerge. Furthermore, understanding how signaling pathways and mechanical cues control the activities of various actin-binding proteins in different cellular, developmental, and pathological processes will keep researchers busy for decades.

scholarly journals Inherited abnormalities in platelet organelles and platelet formation and associated altered expression of low molecular weight guanosine triphosphate-binding proteins in the mouse pigment mutant gunmetal

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2626-2635 ◽  
Author(s):  
RT Swank ◽  
SY Jiang ◽  
M Reddington ◽  
J Conway ◽  
D Stephenson ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Binding Proteins ◽  
Low Molecular Weight ◽  
Guanosine Triphosphate ◽  
Platelet Factor ◽  
Altered Expression ◽  
Gtp Binding Proteins ◽  
Dense Granules ◽  
Gtp Binding ◽  
Platelet Formation

Gunmetal (gm/gm) is a recessively inherited mouse pigment dilution mutant that has high mortality and poor reproductive rates. In these studies, several hematologic defects were found associated with the mutation, including prolonged bleeding times, together with thrombocytopenia and increased platelet size. A unique feature is the presence of simultaneous abnormalities in two platelet organelles, dense granules and alpha-granules. The dense granule component serotonin is present at about half the normal concentration, as are visible dense granules. Three alpha-granule components (fibrinogen, platelet factor 4, and von Willebrand factor) are also significantly reduced. Thus, in several respects the gunmetal mutant resembles the human gray platelet syndrome. A novel abnormality in expression of low molecular weight guanosine triphosphate (GTP)-binding proteins occurs in platelets of gunmetal. In Western blot assays, two additional GTP- binding proteins of 28.5 and 25 Kd were detected. The abnormal expression of GTP-binding proteins is, like the hematologic defects, genetically recessive and is tissue specific. Liver, kidney, brain, spleen, macrophages, and neutrophils have normal GTP-binding protein expression. The additional GTP-binding proteins are soluble. The data indicate that platelet formation and platelet organelle biogenesis are under common genetic control and that abnormal regulation of GTP- binding proteins may affect one or both processes.

Loss of PIP5KIβ Causes a Defect in Lamellipodia Formation and Shear Resistant Adhesion.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 141-141
Author(s):  
Xinsheng Chen ◽  
Yanfeng Wang ◽  
Edward K. Williamson ◽  
Timothy J. Stalker ◽  
Lawrence F. Brass ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Ex Vivo ◽  
Intracellular Localization ◽  
Second Messenger ◽  
Arterial System ◽  
Actin Binding ◽  
Wild Type ◽  
Differential Interference Contrast Microscopy ◽  
Actin Binding Proteins ◽  
Lamellipodia Formation

Abstract Phosphatidylinositol 4,5-bisphosphate (PIP2) is widely known for the production of lipid second messengers after its hydrolysis by phospholipase C or phosphorylation by phosphatidylinositol 3-kinase. PIP2 also regulates cytoskeletal dynamics by directly interacting with actin-binding proteins. Three isoforms of PIP5KI (α, β, and γ) are all capable of phosphorylating PI4P to synthesize PIP2. However, these isoforms have different primary structures, expression levels in various tissues, and intracellular localization. Our previous studies have demonstrated that PIP5KIβ and PIP5KIγ are the dominant isoforms present in platelets. We generated and bred mice heterozygous for a null mutation into the murine PIP5KIβ gene, and crossed these mice to determine the phenotype of mice lacking this protein. PIP5KIβ-null mice were born, appeared developmentally normal, had normal platelet counts, and exhibited no spontaneous hemorrhage. Compared to platelets derived from wild type littermates, platelets lacking PIP5KIβ had PIP2 concentrations that were 61% of normal under basal conditions (p<0.01), and 51% of normal 45 seconds following thrombin stimulation (p<0.01). Similarly, maximum IP3 levels were only 65% of normal in the knockout platelets (p<0.01). Consistent with this second messenger defect, PIP5KIβ −/− platelets had impaired aggregation in response to submaximal doses of thrombin, ADP, collagen, and a thromboxane analogue (U46619). PIP5KIβ-null platelets exhibited disaggregation suggesting that sustained second messenger formation is critical for a sustained aggregation response. Since PIP2 can directly associate with, and thereby regulate actin-binding proteins, we analyzed platelet spreading upon fibrinogen. PIP5KIβ knockout platelets start to spread, but ultimately spread less well than platelets derived from wild type littermates. Imaging this process with real time differential interference contrast microscopy, we found that PIP5KIβ-null platelets extend filopodia as efficiently as wild type platelets, but have difficulty anchoring down these extended membranes. When a filopod on a PIP5KIβ −/− platelet does ultimately adhere to the matrix, a normal lamellipod is rapidly formed. The cytoskeletal organization of PIP5KIβ knockout platelets spread upon fibrinogen was further studied in the electron microscope. This higher resolution analysis verified the profound defect in lamellipodia formation. We speculated that this process of lamellipodia formation is critical for adhesion under the shear conditions found within the arterial system. To test this hypothesis, we analyzed the ability of PIP5KIβ knockout platelets to adhere to collagen in a flow chamber. At all shear conditions between 200 and 1100/s, platelets lacking PIP5KIβ consistently adhered less than wild type platelets. To further analyze the necessity of PIP5KIβ in adhesion of platelets under conditions of arterial shear, we compared PIP5KIβ −/− and PIP5KIβ +/+ mice in a ferric chloride carotid injury model. Under conditions that induced thrombosis in 75% of wild type mice (n=4), we only detected thrombi in 20% of PIP5KIβ-null mice (n=5). Together, these data demonstrate that PIP5KIβ is required for sustained PIP2 and second messenger synthesis, the formation of actin-rich lamellipodia, and stable ex vivo and in vivo platelet adhesion under shear.

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