Elevated Plasma Levels of Soluble Platelet Glycoprotein VI (GPVI) in Patients with Thrombotic Patients

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5145-5145
Author(s):  
Hideo Wada ◽  
Tsutomu Nobori ◽  
Katsuki Naitoh
Keyword(s):  
Healthy Volunteers ◽  
Plasma Levels ◽  
Vascular Endothelial Cells ◽  
Early Stage ◽  
Platelet Glycoprotein ◽  
Vascular Endothelial ◽  
Glycoprotein Vi ◽  
Th 16 ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 5145 Introduction Thrombotic microangiopathy (TMA) is caused by various conditions, such as decreased a ADAMTS13 level, activated or injured vascular endothelial cells or activated platelets and it must be diagnosed at the early stage. This study, examined the activation of platelets by measuring of soluble platelet glycoprotein VI (sGPVI) level in patients with TMA. Materials and Methods The plasma levels of sGPVI, ADAMTS13 activity, von Willebrand factor (VWF) and VWF propeptide (VWFpp) were measured in 40 healthy volunteers, 46 patients without thrombosis (TH), 15 postoperative patients, 13 with disseminated intravascular coagulation (DIC) and 70 with TMA. The plasma levels of GPVI and VWFpp were measured by ELISA and ADAMTS13 was measured by the FRETS assay. There were 27 TMA with markedly decreased ADAMTS13 levels (TMA-ADAMTS13) and 43 TMA without (TMA-Other). Results The plasma levels of sGPVI (median; 25. 0–75. 0%tile) were significantly higher in patients without TH (16. 2 ng/mL 12. 6–22. 5 ng/mL), postoperative patients (31. 6 ng/mL; 28. 3–35. 1 ng/mL), DIC (44. 5 ng/mL; 36. 6– 60. 8 ng/mL), TMA (40. 8 ng/mL; 32. 9–56. 7 ng/mL) than in healthy volunteers (11. 4 ng/mL; 9. 1–14. 8 ng/mL). In addition, the plasma levels of sGPVI post in postoperative patients were significantly higher than in patients without TH (p< 0. 001), and these levels were also significantly higher in those with TMA and DIC than in without TH (p< 0. 001, respectively). The plasma levels of sGPVI were significantly higher in patients with TTP-ADAMTS13 (36. 8 ng/mL; 30. 5–49. 0 ng/mL) and TMA-Other (51. 9 ng/mL; 37. 4–66. 3 ng/mL) than in patients without TH (p< 0. 001, respectively). Conclusion The measurement of sGPVI is therefore considered to be important for the diagnosis of TMA. Disclosures: Wada: Mochida pharmaceutical CO., LTD: Suporting GPVI assay Other.

Crotalin, a vWF and GP Ib Cleaving Metalloproteinase from Venom of Crotalus atrox

2001 ◽  
Vol 86 (12) ◽  
pp. 1501-1511 ◽  
Author(s):  
Wen-Bin Wu ◽  
Hui-Chin Peng ◽  
Tur-Fu Huang
Keyword(s):  
Platelet Adhesion ◽  
Early Stage ◽  
Gel Filtration ◽  
Platelet Glycoprotein ◽  
Antithrombotic Effect ◽  
Binding Motifs ◽  
Sds Page ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryBinding of von Willebrand factor (vWF) to a variety of extracellular matrix (ECM) components and to platelet glycoprotein (GP) Ib-IX-V complex is important in mediating platelet adhesion and aggregation in the early stage of hemostasis. We previously purified a potent antithrombotic protein, named crotalin, functionally acting as a GP Ib antagonist (1). In this study, we further characterized crotalin as a P-I metalloproteinase with a molecular mass of 25 kDa as determined by gel filtration and two-dimensional SDS-PAGE. Crotalin is a vWF binding and cleaving metalloproteinase. In addition, crotalin cleaved platelet GP Ib as judged by flow cytometry and Western blotting. The multiple effects of crotalin on vWF and platelet GP Ib antagonized ristocetin-, but not collagen and thrombin-induced platelet aggregation, suggesting that its effect is specific. We also found that crotalin auto-proteolytically degraded to ~14 and ~10 kDa fragments in the presence of SDS. Interestingly, both degradation fragments, intact and reduced crotalin were able to bind vWF, suggesting the binding of crotalin to vWF is conformation-independent. In conclusion, the results presented further explain the potent antithrombotic effect of crotalin in vivo. In addition, the multiple effects of crotalin may be used as a tool to determine the binding motifs that are responsible for the vWF-ECMs or vWF-GP Ib interaction.

Perturbation of cultured human vascular endothelial cells by phorbol ester or thrombin alters the cellular von Willebrand factor distribution

1987 ◽  
Vol 133 (1) ◽  
pp. 79-87 ◽  
Author(s):  
Jan Hendrik Reinders ◽  
Richard C. Vervoorn ◽  
Cornelis L. Verweij ◽  
Jan A. Van Mourik ◽  
Philip G. De Groot
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Phorbol Ester ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Von Willebrand ◽  
Willebrand Factor

scholarly journals Nonredundant Roles of Platelet Glycoprotein VI and Integrin αIIbβ3 in Fibrin-Mediated Microthrombus Formation

2020 ◽  
Author(s):  
Gina Perrella ◽  
Jingnan Huang ◽  
Isabella Provenzale ◽  
Frauke Swieringa ◽  
Floor C.J.I. Heubel-Moenen ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Factor Xiiia ◽  
Platelet Glycoprotein ◽  
Platelet Suspension ◽  
Integrin Αiibβ3 ◽  
Glycoprotein Vi ◽  
Von Willebrand ◽  
Willebrand Factor

Objective: Fibrin is considered to strengthen thrombus formation via integrin αIIbβ3, but recent findings indicate that fibrin can also act as ligand for platelet glycoprotein VI. Approach and Results: To investigate the thrombus-forming potential of fibrin and the roles of platelet receptors herein, we generated a range of immobilized fibrin surfaces, some of which were cross-linked with factor XIIIa and contained VWF-BP (von Willebrand factor-binding peptide). Multicolor microfluidics assays with whole-blood flowed at high shear rate (1000 s −1 ) indicated that the fibrin surfaces, regardless of the presence of factor XIIIa or VWF-BP, supported platelet adhesion and activation (P-selectin expression), but only microthrombi were formed consisting of bilayers of platelets. Fibrinogen surfaces produced similar microthrombi. Markedly, tiggering of coagulation with tissue factor or blocking of thrombin no more than moderately affected the fibrin-induced microthrombus formation. Absence of αIIbβ3 in Glanzmann thrombasthenia annulled platelet adhesion. Blocking of glycoprotein VI with Fab 9O12 substantially, but incompletely reduced platelet secretion, Ca 2+ signaling and aggregation, while inhibition of Syk further reduced these responses. In platelet suspension, glycoprotein VI blockage or Syk inhibition prevented fibrin-induced platelet aggregation. Microthrombi on fibrin surfaces triggered only minimal thrombin generation, in spite of thrombin binding to the fibrin fibers. Conclusions: Together, these results indicate that fibrin fibers, regardless of their way of formation, act as a consolidating surface in microthrombus formation via nonredundant roles of platelet glycoprotein VI and integrin αIIbβ3 through signaling via Syk and low-level Ca 2+ rises.

scholarly journals Surgery for gastric cancer increases plasma levels of vascular endothelial growth factor and von Willebrand factor

2002 ◽  
Vol 5 (3) ◽  
pp. 137-141 ◽  
Author(s):  
Masataka Ikeda ◽  
Hiroshi Furukawa ◽  
Hiroshi Imamura ◽  
Jyunzo Shimizu ◽  
Hideyuki Ishida ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Von Willebrand Factor ◽  
Plasma Levels ◽  
Vascular Endothelial ◽  
Von Willebrand ◽  
Endothelial Growth Factor ◽  
Willebrand Factor

Biosynthesis and Apical Secretion of ADAMTS13 Metalloprotease in Vascular Endothelial Cells and in Madin-Darby Canine Kidney Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2652-2652 ◽  
Author(s):  
Dezhi Shang ◽  
X. Wu Zheng ◽  
Jihui Ai ◽  
X. Long Zheng
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Mdck Cells ◽  
Vascular Endothelial ◽  
Rt Pcr ◽  
Madin Darby Canine Kidney ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Darby Canine Kidney ◽  
Canine Kidney

Abstract ADAMTS13, a reprolysin-like metalloprotease, limits platelet aggregation by cleaving von Willebrand factor. Deficiency of plasma ADAMTS13 activity can lead to a life-threatening complication, thrombotic thrombocytopenic purpura (TTP). It has been shown that ADAMTS13 is synthesized in human and mouse hepatic stellate cells. But detection of ADAMTS13 mRNA by a reverse transcriptase polymerase chain reaction (RT-PCR) in many other tissues (brain, heart, lungs, kidneys, pancreas, adrenal gland, prostate, uterus and placenta) suggests that the vascular endothelial cells may also produce low level of ADAMTS13 protease. We showed that full-length ADAMTS13 mRNA and protein were detected in human umbilical cord vein endothelial cells (HUVEC), human aortic endothelial cells (HAEC), and microvascular endothelial cells (ECV304) by RT-PCR, SDS-polyacrylamide gel electrophoresis and Western blot. The ADAMTS13 in the cell lysates and the serum-free conditioned media of HUVEC, HAEC and ECV304 cleaved a peptidyl substrate, FRETS-VWF73, specifically, and such a proteolytic cleavage could be completely abolished by 10 mM EDTA. Immunohistochemistry showed that both endogenous and recombinant ADAMTS13 in HUVEC and HAEC appeared to be co-localized with von Willebrand factor in the endoplasmic reticulum and/or Golgi apparatus, but not in the Weibel-Parade bodies, suggesting that ADAMTS13 is readily secreted rather than stored inside the cells. To determine whether ADAMTS13 is secreted into the lumen of the vessels, we assessed the polarity of ADAMTS13 secretion in ECV304 and Madin-Darby canine kidney (MDCK) cells, two well-characterized polarized endothelial and epithelial cell lines for studying protein sorting. Both endogenous ADAMTS13 and recombinant ADAMTS13-V5-His expressed in ECV304 was predominantly secreted into the apical domain of the cells. Similarly, the recombinant ADAMTS13-V5-His in MDCK cells was also secreted predominantly into the apical domain. Apical secretion of ADAMTS13-V5-His appeared to depend on at least a transient association of ADAMTS13-V5-His with Triton X-100 insoluble glycosphingolipid and cholesterol-enriched microdomains, namely rafts. Deletion of the distal carboxyl terminal CUB (Complement C1r/C1s, Urinary EGF, and Bone morphogenic protein) domains or removal of membrane cholesterol by treatment of MDCK cells expressing recombinant ADAMTS13-V5-His with lovastatin and methyl-β-cyclodextrin completely abolished the raft association and apical secretion of ADAMTS13-V5-His in MDCK cells. Moreover, an ADAMTS13 mutation (4143insA), which naturally occurs in a patient with TTP that deletes the second CUB domain not only significantly impaired the amount of total ADAMTS13 protein secreted into the conditioned medium, but also reversed the polarity of ADAMTS13 secretion in MDCK cells. Our data demonstrate that: 1) active full-length ADAMTS13 protease is produced in human vascular endothelial cells; 2) the CUB domains are critical for apical secretion via their interaction with lipid rafts; 3) mutations in ADAMTS13 gene that disrupt sorting signals may result in congenital ADAMTS13 deficiency, leading to TTP in some cases. Considering a large surface area of the vascular endothelial cells, apically secreted ADAMTS13 from these cells may contribute significantly to plasma pool of ADAMTS13 protease.

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