scholarly journals Role of platelets in regulating activated coagulation factor XI activity

2021 ◽  
Author(s):  
Stephanie E. Reitsma ◽  
Jiaqing Pang ◽  
Vikram Raghunathan ◽  
Joseph J. Shatzel ◽  
Christina U. Lorentz ◽  
...  
Keyword(s):  
Binding Sites ◽  
Critical Role ◽  
Coagulation Factor ◽  
Platelet Membrane ◽  
Anion Binding ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Protease Nexin ◽  
Binding Mechanisms

Factor (F)XI has been shown to bind platelets, but the functional significance of this observation remains unknown. Platelets are essential for hemostasis and play a critical role in thrombosis, while FXI is not essential for hemostasis, but promotes thrombosis. An apparent functional contradiction, platelets are known to support thrombin generation, yet, platelet granules release protease inhibitors, including those of activated FXI (FXIa). We aim to investigate the secretory and binding mechanisms by which platelets could support or inhibit FXIa activity. The presence of platelets enhanced FXIa activity in purified system and increased FIX activation by FXIa and fibrin generation in human plasma. In contrast, platelets reduced the activation of FXI by FXIIa and the activation of FXII by kallikrein. Incubation of FXIa with the platelet secretome, which contains FXIa inhibitors, such as protease nexin-II, abolished FXIa activity, yet in the presence of activated platelets the secretome was not able to block the activity of FXIa. FXIa variants lacking the anion-binding sites did not alter the effect of platelets on FXIa activity or interaction. Western blot analysis of bound FXIa (by FXI(a)-platelet membrane immunoprecipitation) showed that the interaction with platelets is zinc-dependent and, unlike FXI binding to platelets, not dependent on glycoprotein Ib (GPIb). FXIa binding to the platelet membrane increases its capacity to activate FIX in plasma likely by protecting it from inhibition by inhibitors secreted by activated platelets. Our findings suggest that an interaction of FXIa with the platelet surface may induce an allosteric modulation of FXIa.

PSGL-1 regulates platelet P-selectin-mediated endothelial activation and shedding of P-selectin from activated platelets

2007 ◽  
Vol 98 (10) ◽  
pp. 806-812 ◽  
Author(s):  
Vandana Dole ◽  
Wolfgang Bergmeier ◽  
Ian Patten ◽  
Junichi Hirahashi ◽  
Tanya Mayadas ◽  
...  
Keyword(s):  
Endothelial Activation ◽  
Leukocyte Rolling ◽  
Wild Type ◽  
Platelet Surface ◽  
Activated Platelets ◽  
And Function ◽  
Body Secretion

SummaryWe have previously shown that activated platelets in circulation stimulate release of endothelial Weibel-Palade bodies thus increasing leukocyte rolling in venules. P-selectin on the activated platelets mediates adhesion to leukocytes via PSGL-1 and is rapidly shed into plasma. We were interested in studying the role of PSGL-1 in regulating expression and function of platelet P-selectin. We show here that PSGL-1 is critical for the activation of endothelial cells in venules of mice infused with activated platelets. The interaction of platelet P-selectin with PSGL-1 is also required for P-selectin shedding, as P-selectin was retained significantly longer on the surface of activated platelets infused into PSGL-1-/- compared to wild-type mice. The leukocyte integrin αMβ2 (Mac-1) was not required for P-selectin shedding. In addition to shedding, P-selectin can be downregulated from the platelet surface through internalization and this is the predominant mechanism in the absence of PSGL-1. We demonstrate that leukocyte- neutrophil elastase,known to cleave P-selectin in vitro, is not the major sheddase for P-selectin in vivo. In conclusion, interaction of platelet P-selectin with PSGL-1 is crucial for activation of the endothelium andWeibel-Palade body secretion. The interaction with PSGL-1 also results in rapid shedding of P-selectin thus downregulating the inflammatory potential of the platelet.

Extracellular Calcium And Platelets

1981 ◽  
Author(s):  
P M Taylor ◽  
S Heptinstall
Keyword(s):  
Divalent Cation ◽  
Binding Sites ◽  
Divalent Cations ◽  
Extracellular Calcium ◽  
Time Dependent ◽  
Aggregation Process ◽  
Platelet Surface ◽  
Intracellular Pool ◽  
Continuous Movement

To gain more information on the role of extracellular Ca in platelet behaviour, the movement of 45Ca between plasma and platelets has been studied. Ttoo experimental procedures have been used: platelets were either studied in plasma that contained near-physiological levels of divalent cations or were studied in divalent cation-depleted plasma.There was a continuous movement of Ca from plasma into platelets when the latter were suspended in plasma that contained near-physiological levels of divalent cations. The iptake was linear with time (2.0 to 2.5 ng ion Ca/109 platelets/60 mins) and was faster at 37°C than at 25°C. The amount of Ca taken up by the platelets increased as the extracellular Ca level was increased and was markedly inhibited by Mg. Sr did not affect the uptake. EGTA displaced only a small amount of the Ca that associated with the plater lets which indicated that Ca was taken up into an intracellular pool rather than sinply bound to the platelet surface. The relevance of this movement of Ca into the cells to platelet behaviour has not been established.Studies using platelets suspended in divalent cation- depleted plasma shewed that extracellular Ca was in equilibrium with Ca bound at or near the platelet surface. The binding of Ca was time-dependent but saturable (0.30 to 0.50 ng ion Ca/109 platelets/30 mins), and the majority was readily displaced by EGTA. The amount of Ca bound to the cells increased as the extracellular Ca level was increased but was little affected by an excess of either Mg or Sr. Mare Ca bound to platelets when they were incubated at 25°C than at 37°C. This was because platelets lost their ability to bind Ca when they were incubated at 37°C in divalent cation-depleted plasma. This phenomenon was time-dependent and irreversible and was paralleled by a loss in the ability of the platelets to aggregate. These Ca binding sites would seem to be relevant to the aggregation process.

REDISTRIBUTION OF PLATELET GLYCOPROTEINS INDUCED BY ALLO- AND AUTOANTIBODIES

1987 ◽  
Author(s):  
S Santoso ◽  
V Kiefel ◽  
C Mueller-Eckhardt
Keyword(s):  
Binding Sites ◽  
Total Radioactivity ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Platelet Surface ◽  
In Vitro Effects ◽  
Immunoblot Technique ◽  
Antigen Antibody

It is now well established that two of the major membrane glycoproteins (GP) of human platelets, GP lb and Ilb/IIIa, are functionally prominent for adhesion, aggregation and carry the binding sites for allknown types of human platelet specific antibodies (ab). Although a number of in vitro effects of ab on platelet function have been described, the role of the GP specificity of the various ab with regard to membrane mobility and redistribution phenomena is asyet unknown.In this work, we studied the effect on platelet membrane redistribution of allo- ab, auto-aband a quinidine-dependent ab directed against various epitopes on GP lb, lib and Ilia using immunofluorescence and a quantitative radioimmunoassay. The platelet GP's carrying the corresponding epitopes were determined using immunoblot technique or radioimmuno-precipitation. When unfixed platelets were incubated with alio- or auto-ab against epitopes on GP liborGP IlIa cap formation and internalization of antigenantibody complexes were visualized by fluorescence. In contrast, no changes of antigen distribution were seen with auto-ab or quinidine- dependent ab directed against GP lb. To quantitate antigen-antibody complexes internalization a specially designed radioimmunoassay was employed. If unfixed platelets weretreated with allo- or auto-ab against GP lib or GP Ilia precipitous reduction of external radioactivity was found, whereas the total radioactivity remainedessentially unchanged. This indicated that a portionof approximately 50-70% of GP lib or GP Ilia had been removed from the platelet surface and had been internalized. Internalization could not be induced with auto-ab or quinidine dependent ab against GP lb.We conclude that membrane redistribution of human platelets can be induced by various human ab with specificity for GP lib and/or Ilia and is a function of the target GP rather than the source of therespective abSupported by Deutsche Forschungsgemeinschaft (Mu 277/9-6)

Roles of Platelets and Factor XI in the Initiation of Blood Coagulation by Thrombin

2001 ◽  
Vol 86 (07) ◽  
pp. 75-82 ◽  
Author(s):  
Peter Walsh
Keyword(s):  
Binding Site ◽  
Tissue Factor ◽  
Vascular Injury ◽  
Factor Xi ◽  
Platelet Surface ◽  
Proteolytic Activation ◽  
Activated Platelets ◽  
Tissue Factor Pathway ◽  
Protease Nexin ◽  
Normal Hemostasis

SummaryTo account for the variable hemostatic defect in patients with factor XI (FXI) deficiency, with normal hemostasis in contact factor deficiencies, a coagulation paradigm is presented whereby trace quantities of thrombin, generated transiently by exposure of tissue factor at sites of vascular injury, activates FXI bound to the platelet surface in the presence of prothrombin or high Mr kininogen (HK). Tissue factor pathway inhibitor (TFPI) limits the flux of thrombin generated by the tissue factor pathway, and protease nexin II (PNII), released from activated platelets, inhibits solution phase FXIa and localizes FIX activation to the platelet surface where FXIa is protected from inactivation by PNII. Either prothrombin or HK binds to the Apple 1 (A1) domain of FXI, thereby exposing a platelet-binding site in the FXI A3 domain. Dimeric FXI binds to activated platelets directly through the A3 domain of one monomer. After proteolytic activation of platelet-bound FXI by thrombin (or FXIIa), a substrate binding site for FIX is exposed in the opposite monomer that promotes FIX activation on the platelet surface resulting in the local explosive generation of thrombin and the formation of hemostatic thrombi at sites of vascular injury.

Interaction of Recombinant Factor VIIa with Rehydrated, Lyophilized Platelets.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3994-3994
Author(s):  
Thomas H. Fischer ◽  
Alisa S. Wolberg ◽  
Arthur P. Bode ◽  
Kevin J. Ramer ◽  
Timothy C. Nichols
Keyword(s):  
Surface Modification ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Equilibrium Constants ◽  
Fluorescent Microscopy ◽  
Coagulation Factor ◽  
Platelet Membrane ◽  
Dependent Manner ◽  
Platelet Surface ◽  
Equilibrium Binding

Abstract The experiments presented here were undertaken to determine if factor VIIa (rFVIIa, the Novo Nordisk product NovoSeven™) will directly bind to rehydrated, lyophilized (RL) platelets (Stasix™ platelets, Entegrion, Inc. trade) for the formation of a catalytic surface with an enhanced ability to generate thrombin. The relationship of rFVIIa to the RL platelet surface was examined by measuring equilibrium and non-equilibrium binding of the coagulation factor to the cells, by studying the subcellular localization of the coagulation factor on RL platelets, and by following the effects of the surface modification on the kinetics of thrombin generation. The association of rFVIIa with RL platelets occurred with an on rate of 3.6x103 sec−1moles−1. Saturation occurred in minutes and was calcium dependent. Disassociation (in plasma or citrated saline) was slow, with over half of the coagulation factor remaining bound after two hours (with slow and fast rate constants of 5.0x10−5 and 4.1x10−4 sec−5 respectively). These results define a binding site with an apparent equilibrium constants of 110 nM. Equilibrium binding of rFVIIa to RL platelets was analyzed with flow cytometry and Western analysis. The rFVIIa was bound to RL platelets in a dose-dependent manner when incubated at concentrations of 0.3 to 10.0 uM rFVIIa and 3x104 to 106 RL platelets/ul in citrated saline. When high concentrations of rFVIIa were bound to RL platelets densities of over one million molecules of rFVIIa per RL platelet was obtained. Fluorescent microscopy analysis revealed that the rFVIIa was localized to the surface membrane and that some rFVIIa localized internally to the outer surface of the surface connected open canalicular system and/or sites of internal trafficking. Flow cytometric analysis with annexin V demonstrated that considerable quantities of phosphatidylserine were present on the external surface of the RL platelet membrane for potential facilitation of rFVIIa binding. The effect of RL platelet surface modification by rFVIIa on thrombin generation was investigated by following the hydrolysis of the thrombin-specific fluorogenic substate D-phe-pro-arg-ANSNHin plasma. rFVIIa and RL platelets accelerated thrombin generation in this system with rFVIIa being approximately twice as effective (per molecule of the recombinant protein) when added to the assay system pre-bound to RL platelets as compared to being initially free in the plasma. Similar results were obtained when free and RL platelet bound rFVIIa were tested in factor IX-deficient plasma. These experiments show that rFVIIa retains activity when super-saturated on the RL platelet membrane. The results of the studies presented here suggest that RL platelets can be used to concentrate rFVIIa at sites of vascular injury.

Critical Role of Calcium in the Regulation of the ER-to-Golgi Transport of FV and FVIII by the LMAN1-MCFD2 Cargo Receptor.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2140-2140
Author(s):  
Chunlei Zheng ◽  
Huihui Liu ◽  
David Ginsburg ◽  
Bin Zhang
Keyword(s):  
Factor Viii ◽  
Binding Sites ◽  
Critical Role ◽  
Carbohydrate Recognition Domain ◽  
Factor V ◽  
Receptor Complex ◽  
Carbohydrate Recognition ◽  
Golgi Transport ◽  
Ef Hand

Abstract Abstract 2140 Poster Board II-117 Coagulation factor V (FV) and factor VIII (FVIII) play key roles in hemostasis and thrombosis. The LMAN1 (ERGIC-53)-MCFD2 complex is a mammalian cargo receptor for efficient transport of FV and FVIII from the endoplasmic reticulum (ER) to the Golgi. Mutations in either LMAN1 or MCFD2 cause a bleeding disorder, combined deficiency of factor V and factor VIII. LMAN1 is a type-1 transmembrane protein with a Ca2+-dependent carbohydrate recognition domain homologous to leguminous lectins. MCFD2 is a small soluble protein with an N-terminal sequence of unknown structure and two Ca2+-binding EF-hand domains at the C terminus. LMAN1 and MCFD2 form a Ca2+-dependent protein complex in the ER-Golgi intermediate compartment (ERGIC), an organelle between the ER and Golgi that is unique to higher eukaryotic cells. FV and FVIII interact with the LMAN1-MCFD2 complex in a Ca2+ -dependent manner. To elucidate the role of Ca2+ in regulating the ER-to-Golgi transport of FV and FVIII, we determined the structural features important for the organization of the receptor complex and the interaction of this complex with its client cargo FV and FVIII. We show that the C-terminal Ca2+-binding EF hand domains of MCFD2 are both necessary and sufficient for interaction with LMAN1. The EF hand domains also mediate the interaction with FV and FVIII. All MCFD2 missense mutants identified in F5F8D patients are localized to the EF hand domains and fail to bind LMAN1. However, these mutants still retain the FV and FVIII binding activities. Circular dichroism spectroscopy studies on missense mutations localized to different structural elements of the EF hand domains suggest that Ca2+-induced folding of MCFD2 is important for LMAN1 interaction, but not essential for FV and FVIII binding. We also demonstrate that the carbohydrate recognition domain (CRD) of LMAN1 contains separate binding sites for MCFD2 and FV/FVIII. Mutations in the Ca2+ and sugar binding sites of CRD disrupt FV and FVIII interaction, without affecting MCFD2 binding, suggesting that the Ca2+ binding sites in LMAN1 are primarily required for the recognition of sugar residues in FV and FVIII. These results support a model in which Ca2+ plays a critical role in regulating the binding in the ER and the subsequent release in the ERGIC of FV and FVIII. Ca2+ concentration is higher in the ER than in the ERGIC and the Golgi. In the ER lumen, FV and FVIII loading is initiated by a flexible interaction with MCFD2 and stabilized by the follow-up interaction of sugar side chains of FV and FVIII with the carbohydrate binding site of LMAN1. The LMAN1-FV/FVIII interaction is more sensitive to Ca2+ concentration than the LMAN1-MCFD2 interaction, so that the lower Ca2+/pH in the ERGIC triggers the release of FV and FVIII but not the dissociation of the LMAN1-MCFD2 receptor complex. The empty receptor complex is subsequently recycled back to the ER for the next round of cargo loading. Disclosures: No relevant conflicts of interest to declare.

scholarly journals AUF1 Promotes Proliferation and Invasion of Thyroid Cancer via Downregulation of ZBTB2 and Subsequent TRIM58

2021 ◽  
Vol 11 ◽  
Author(s):  
Xin Du ◽  
Jia-Mei Wang ◽  
Da-Lin Zhang ◽  
Tong Wu ◽  
Xiao-Yan Zeng ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Cancer Cells ◽  
Binding Sites ◽  
Critical Role ◽  
Common Type ◽  
Migration And Invasion ◽  
Papillary Thyroid ◽  
Thyroid Cancer Cells ◽  
Proliferation And Invasion

The pathogenesis of papillary thyroid cancer (PTC), the most common type of thyroid cancer, is not yet fully understood. This limits the therapeutic options for approximately 7% of invasive PTC patients. The critical role of AUF1 in the progression of thyroid cancer was first reported in 2009, however, its molecular mechanism remained unclear. Our study used CRISPR/Cas 9 system to knockdown AUF1 in IHH4 and TPC1 cells. We noticed that the expression of TRIM58 and ZBTB2 were increased in the AUF1 knockdown IHH4 and TPC1 cells. When TRIM58 and ZBTB2 were inhibited by small hairpin RNAs (shRNAs) against TRIM58 (shTRIM58) and ZBTB2 (shZBTB2), respectively, the proliferation, migration, and invasion ability of the AUF1-knockdown IHH4 and TPC1 cells were increased. In addition, two ZBTB2 binding sites (-719~-709 and -677~-668) on TRIM58 promoter and two AUF1 binding sites (1250-1256 and 1258-1265) on ZBTB2 3’-UTR were identified. These results suggested that AUF1 affecting thyroid cancer cells via regulating the expression of ZBTB2 and TRIM58.

Effect of Pyridoxal 5'-Phosphate (PALP) on Human Platelet Aggregation, Dense Granule Release and Thromboxane B2 Generation - Role of Schiff Base Formation

1982 ◽  
Vol 48 (02) ◽  
pp. 136-141 ◽  
Author(s):  
S Krishnamurthi ◽  
J Westwick ◽  
V V Kakkar
Keyword(s):  
Schiff Base ◽  
Direct Interaction ◽  
Dense Granule ◽  
Platelet Membrane ◽  
Thromboxane B2 ◽  
Platelet Surface ◽  
Schiff Base Formation ◽  
Induced Aggregation ◽  
Base Formation

SummaryPyridoxal 5’-phosphate (PALP) inhibited ADP, thrombin, adrenaline, PAF and AA induced aggregation and 14C-5HT release. Thromboxane B2 (TxB2) generation induced by all the above agents except AA was also inhibited indicating that PALP may be inhibiting AA release via phospholipase A2 activation rather than AA metabolism. PALP inhibited ristocetin induced aggegation in PRP and agglutination in formaldehyde-treated washed platelets (FWP). Inhibition of ADP, adrenaline, PAF and AA-induced aggregation and 14C-5HT release by PALP was found in resuspended platelets pretreated with PALP and sodium borohydride suggesting that inhibition was mediated by Schiff base formation with platelet surface amino groups.Irreversible fixation of PALP to the platelet membrane by borohydride reduction also inhibited thrombin induced 14C-5HT release and TxB2 generation but not thrombin induced primary aggregation or ristocetin induced agglutination in FWP. This suggests that PALP may interact with specific glycoproteins on the platelet membrane involved in ADP, adrenaline and PAF induced primary aggregation and that PALP could be inhibiting ristocetin induced agglutination by direct interaction with ristocetin or FVIII RCoF.

Abstract 51: A Critical Role for Outside-In Signaling of Integrin AlphalIIIb/Beta3 in Shear-Dependent Platelet Microvesicle Formation and Phosphatidylerine Exposure

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Aiming Pang ◽  
Yujie Cui ◽  
Michael K Delaney ◽  
Aleksandra Stojanovic-Terpo ◽  
Xiaoping Du
Keyword(s):  
Flow Cytometry ◽  
Shear Stress ◽  
Critical Role ◽  
Selective Inhibitor ◽  
Wild Type ◽  
Activated Platelets ◽  
Binding Function ◽  
Integrin Antagonists ◽  
Dependent Signaling Pathway

Platelets promote coagulation mainly by exposing membrane phosphatidylserine (PS) and releasing PS-expressing microvesicles (MV). We have recently shown that PS exposure and MV release induced by platelet agonists requires shear stress. To identify the receptor responsible for the shear-dependent signaling leading to PS exposure and MV release, we compared platelets from β 3 -/- mice and wild-type mice in MV release and PS exposure under defined shear stress introduced using a cone-plate rheometer. MV release and PS exposure were determined using flow cytometry. Shear-dependent PS exposure and MV release were significantly suppressed in β 3 -/- platelets. Similarly, Wild type platelets treated with integrin antagonists also showed defective PS exposure and MV release. These data indicate an important role for the ligand binding function of integrin αιιb/β3 in shear-dependent MV release and PS exposure. To determine whether the role of integrin αιιb/β3 is due to its outside-in signaling, β 3 -/- platelets were transplanted with wild type β 3 or a mutant β 3 with the critical Gα13 binding site of the β 3 cytoplasmic domain (EEE) changed to alanines (AAA), which was previously shown to selectively abolish outside-in signaling of αIIb/β 3 . Transplantation of wild type β 3 rescued the defective MVs release and PS exposure of β 3 -/- platelets. In contrast, AAA mutant failed to rescue these defects. Consistently, wild type platelets treated with the selective inhibitor of Gα13-integrin interaction, inhibited integrin outside-in signaling and also PS exposure and MV release under shear stress. Furthermore, we also showed that the inhibition of Src, which is important in outside-in signaling downtream of Gα13, also abolished shear-dependent MV release and PS exposure. These data suggest that integrin outside-in signaling mediated by the Gα13-β 3 interaction and Src-dependent signaling pathway plays an important role in transmitting shear-induced mechanical signals leading to MV release and PS exposure in activated platelets.

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