Non-Catalytic Domains of ADAMTS13 Can Redirect the Substrate Specificity of ADAMTS5

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2211-2211
Author(s):  
Weiqiang Gao ◽  
Elodee Tuley ◽  
J. Evan Sadler
Keyword(s):  
Western Blotting ◽  
Initial Rate ◽  
Conflicts Of Interest ◽  
Cleavage Sites ◽  
Catalytic Domains ◽  
Substrate Cleavage ◽  
A Site ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Synthesis And Degradation

Abstract Abstract 2211 The von Willebrand factor (VWF) cleaving protease, ADAMTS13, and the aggrecanase, ADAMTS5, have a modular structure that includes metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), cysteine-rich (C), and spacer (S) domains. Both enzymes utilize some combination of DTCS domains to bind substrates and facilitate proteolysis, thereby maintaining the homeostatic balance between the synthesis and degradation of their substrates. We constructed chimeric metalloproteinases and substrates to examine the activity of ADAMTS13 and ADAMTS5 towards their respective physiological cleavage sites at VWF Tyr1605-Met1606 and aggrecan interglobular domain (IGD) Glu373-Ala374 (as shown in table). Cleavage rates were determined by ELISA, and C-terminal products were sequenced to confirm specificity. ADAMTS13 truncated after the S domain (MDTCS13) cleaved recombinant VWF Asp1596-Arg1668 (VWF73) much more rapidly than did constructs MDT13/CS5 and MD13/TCS5, in which distal ADAMTS13 domains were replaced by those of ADAMTS5. Similarly, replacement of the C-terminal 52 residues of VWF73 by aggrecan IGD residues Glu394-Gly458 (VWF/IGD) markedly impaired cleavage by MDTCS13, MDT13/CS5, and MD13/TCS5.Therefore, optimal cleavage of VWF73 depends on interactions between ADAMTS13 TCS domains and C-terminal sites of VWF73. In contrast, MDTCS5 was inactive toward VWF73 or VWF/IGD, whereas MD5/TCS13 efficiently cleaved VWF73 (at Glu1615-Ile1616) but not VWF/IGD. Thus, the TCS domains of ADAMTS13 interact with the Lys1617-Arg1668 segment of VWF73. In the context of ADAMTS13, this interaction accelerates the physiological cleavage of VWF. In the context of the chimeric MD5/TCS13, this interaction allows the ADAMTS5 active site to recognize and cleave a new site in VWF73, which is otherwise resistant to ADAMTS5. As expected, MDTCS5 readily cleaved recombinant IGD residues Thr331-Gly458 (IGD). Replacing the C-terminal 65 residues of IGD with VWF Lys1617-Arg1668 (IGD/VWF) minimally altered the rate of cleavage by MDTCS5, and replacement of the ADAMTS5 TCS domains by those of ADAMTS13 (MD5/TCS13) had little effect on the cleavage of either IGD or IGD/VWF. MDTCS5 and MD5/TCS13 also cleaved the same Glu373-Ala374 site in purified aggrecan with similar efficiency (not shown in table), indicating that recognition of the major cleavage site in the IGD domain does not depend strongly on specific TCS domains. In contrast, the other major site of aggrecan proteolysis by MDTCS5, at Glu1480-Ala1481, was completely resistant to MD5/TCS13, indicating that the TCS domains of ADAMTS13 cannot substitute for those of ADAMTS5. These results show that non-catalytic domains, particularly TCS domains, are principal modifiers of physiological substrate recognition and cleavage by ADAMTS5 and ADAMTS13, and these domains may have a similar function in other members of the ADAMTS family. Values (mean ± SD) for kcat/Km (×105 M-1s-1) were calculated from the initial rate of substrate cleavage determined by ELISA of products. Cleavage of aggrecan at the Glu1480-Ala1481 bond was analyzed by Western blotting with a site-specific antibody. N/A (no activity) indicates no significant cleavage identified by Western blotting or ELISA. Disclosures: No relevant conflicts of interest to declare.

Identification of a Novel Exosite (Glu634-Arg639) in the Spacer Domain of ADAMTS13 Required for Recognition of Von Willebrand Factor.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2214-2214 ◽  
Author(s):  
Veronica Casina ◽  
Hayley Hanby ◽  
Anastasia Lyalenko ◽  
X. Long Zheng
Keyword(s):  
Proteolytic Activity ◽  
Conflicts Of Interest ◽  
Specific Activity ◽  
Substrate Recognition ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Evolutionarily Conserved ◽  
A Site ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 2214 Exosite binding plays a key role in cleavage of VWF by ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats, 13). Two exosites that are evolutionarily conserved from zebra fish to mammals have been identified in the spacer domain by sequence alignment. Previous studies have shown that exosite 3 in the spacer domain plays a critical role for substrate recognition (Blood 115:2300–10, 2010), and modification of this exosite generates ADAMTS13 variants with improved specific activity but reduced autoantibody binding (Blood 119:3836–43, 2012). In the present study, using a site-directed mutagenesis approach, we identified a novel exosite near exosite 3 in the spacer domain, termed exosite 4, a region between residues Glu634 and Arg639. A partial (ΔEx4a:deletion of Leu632-Asp635 or ΔEx4b:deletion of Arg636-Arg639) or complete deletion of the exosite (ΔEx4) significantly impaired proteolytic activity towards peptidyl VWF73 and multimeric VWF. Moreover, substitution of all surface exposed residues in Ex4A (LTED/AAAA) or Ex4b (RLPR/AAAA) with alanine had a similarly detrimental effect on proteolytic activity. Further studies demonstrated that the residues Asp635 and Arg636 in exosite 4 play a critical role for substrate recognition. We conclude that the region between residues Glu634 and Arg639 is a novel exosite necessary for recognition and cleavage of VWF. Disclosures: No relevant conflicts of interest to declare.

CARBOHYDRATE PREVENTS LOSS OF LARGE VON WILLEBRAND FACTOR MULTIMERS BY PROTECTING AGAINST AMINO TERMINAL PROTEOLYTIC CLEAVAGE

1987 ◽  
Author(s):  
A B Federici ◽  
S D Berkowitz
Keyword(s):  
Von Willebrand Factor ◽  
Proteinase Inhibitors ◽  
Enzymatic Digestion ◽  
Terminal Region ◽  
Cleavage Sites ◽  
Amino Terminal ◽  
Carboxy Terminal ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor

We have previously shown that carbohydrate (CHO) protects von Willebrand factor (vWF) from proteolytic degradation. We have now shown that removal of CHO from the vWF subunit exposes additional cleavage sites in the amino terminal region and that cleavages in this region are associated with loss of large multimers. We examined and compared the extent of large multimer loss with sites of subunit cleavage of native and GHO-modified vWF after treatment with plasmin, chymotrypsin, and trypsin. Highly purified vWF was treated with neuraminidase and β-galactosidase in the presence of proteinase inhibitors to remove 90-95% of the sialic acid and 45-50% of the D-galactose without loss of large multimers or diminution of the ristocetin cofactor activity. The extent and approximate location of subunit cleavage was determined by immunoblotting and monoclonal antibody epitope mapping. Multimeric analysis revealed an increasingly greater loss of large multimers when native vWF was digested with plasmin, chymotrypsin, and trypsin, respectively. Large multimer loss was more extensive with each enzyme after CHO-modification of vWF. On subunit analysis, plasmin, chymotrypsin, and trypsin were shown to produce both amino and carboxy terminal fragments. The number, location, and relative quantities of carboxy terminal fragments produced by these enzymes were unchanged after CHO modification. However, digestion of the amino terminal region was considerably more extensive as judged by a marked decrease or absence of the larger fragments seen when native vWF was digested, and by the appearance of new smaller molecular weight species. Thus, enzymatic digestion of vWF after removal of carbohydrate produced new cleavages in the amino terminal region but did not alter the location or extent of carboxy terminal cleavages. Therefore, the greater loss of large multimers that occurs after CHO modification is likely to be the result of cleavages in the amino terminal region of the molecule. It appears that by protecting the vWF subunit against amino terminal cleavage, carbohydrate inhibits the loss of large multimers.

scholarly journals Covalent crosslinking of von Willebrand factor to fibrin

Blood ◽  
1986 ◽  
Vol 68 (1) ◽  
pp. 95-101 ◽  
Author(s):  
M Hada ◽  
M Kaminski ◽  
P Bockenstedt ◽  
J McDonagh
Keyword(s):  
Von Willebrand Factor ◽  
Initial Rate ◽  
Factor Xiiia ◽  
Covalent Bonds ◽  
Covalent Crosslinking ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Plasmin Inhibitor

Abstract Factor XIIIa crosslinks a limited number of substrates via epsilon(gamma-glutamyl)-lysyl bond formation. It crosslinks fibrin to itself, alpha 2-plasmin inhibitor and fibronectin to fibrin, and fibronectin to collagen. Results presented here show that plasma von Willebrand factor (vWF) is a substrate for factor XIIIa and can be crosslinked to fibrin during gel formation. vWF-fibrin crosslinking was studied in purified systems and in plasma with 125I-vWF and 131I- fibrinogen. vWF incorporation into fibrin increased with time or increasing factor XIIIa. After electrophoresis of dissolved clots, distribution of 125I and 131I was measured and showed that vWF was crosslinked to the alpha chain of fibrin and entered the high-mol-wt alpha polymer. vWF-fibrin crosslinking decreased the initial rate of alpha polymer formation. Crosslinking of vWF polymer to itself could not be demonstrated under physiologic conditions but occurred if vWF was reduced first. Factor XIIIa catalyzed incorporation of putrescine into both monomeric and polymeric vWF. Altogether, these studies indicate that factor XIIIa can readily form covalent bonds between glutamine in vWF and lysine in fibrin alpha chains. This reaction occurs readily in vitro when plasma clotting is slow and may occur in vivo under similar conditions.

The Interaction of Von Willebrand Factor with GPIb-IX Initiates Platelet Apoptosis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3002-3002
Author(s):  
Suping Li ◽  
Zhicheng Wang ◽  
Yi Liao ◽  
Guanglei Liu ◽  
Weilin Zhang ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Intracellular Signaling ◽  
Conflicts Of Interest ◽  
Cytoplasmic Domain ◽  
Shear Stresses ◽  
Apoptotic Signaling ◽  
Platelet Apoptosis ◽  
Signaling Events ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 3002 Poster Board II-979 Background: The interaction of glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) initiates the adherence of platelets to sites of vascular injury, simultaneously triggers intracellular signaling events such as elevation of cytoplasmic calcium and activations of multiple protein kinase pathways which result in the activation of the ligand binding function of GPIIb/IIIa, leading to platelet activation and thrombus formation. The intracellular signaling protein 14-3-3ζ and the membrane skeleton protein filamin A have been confirmed to interact with the intracellular domain of GPIbalpha and play important roles in the regulation of platelet function. The signaling events elicited by GPIbalpha-VWF interaction, such as calcium mobilization and phosphatidylserine (PS) exposure are similar to those occurring during apoptosis. Particularly, the 14-3-3ζ binding domain of GPIbalpha has been reported to involve in the regulation of cell proliferations. However, it is still unclear whether the GPIbalpha-VWF interaction induces platelet apoptosis. Objectives: To investigate whether the GPIbalpha-VWF interaction induces platelet apoptosis and the role of 14-3-3ζ in apoptotic signaling. Methods: Apoptotic events were assessed in platelets or Chinese hamster ovary (CHO) cells expressing GPIb-IX (1b9) interacted with VWF by flow cytometry or Western blotting. Results: Ristocetin induced GPIbalpha-VWF interaction elicited apoptotic events in platelets, including phosphatidylserine exposure, elevations of Bax and Bak, gelsolin cleavage, and depolarization of mitochondrial inner transmembrane potential. Apoptotic events were also elicited in platelets exposed to pathologic shear stresses in the presence of VWF, however, the shear-induced apoptosis was eliminated by anti-GPIbalpha antibody AK-2. Furthermore, apoptotic events occurred in 1b9 cells stimulated with VWF and ristocetin, but were significantly diminished in two CHO cell lines expressing mutant GPIb-IX with truncation of the cytoplasmic domain of GPIbalpha or a serine-to-alanine mutation at 14-3-3ζ binding site in GPIbalpha. Conclusions: This study demonstrates that the GPIbalpha-VWF interaction induces apoptotic events in platelets and association of 14-3-3ζ with the cytoplasmic domain of GPIbalpha is essential for apoptotic signaling. The finding may suggest a novel mechanism for platelet clearance or some thrombocytopenic diseases. The reagents that block 14-3-3ζ-GPIbalpha interaction might be potentially useful in platelet storage or anti-thrombocytopenia. Disclosures: No relevant conflicts of interest to declare.

Association of Quantitative and Qualitative Abnormalities of Von Willebrand Factor and Risk of Death In Hemodialysis Patients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2217-2217
Author(s):  
Rachel Holden ◽  
Angie Tuttle ◽  
Francis MacLeod ◽  
Toni Burbidge ◽  
Carol Hegadorn ◽  
...  
Keyword(s):  
Chronic Kidney Disease ◽  
Kidney Disease ◽  
Vascular Disease ◽  
Von Willebrand Factor ◽  
Functional Activity ◽  
Conflicts Of Interest ◽  
Proteolytic Processing ◽  
Risk Of Death ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 2217 In order to evaluate the possible role of abnormalities of von Willebrand factor in the hemostatic defects seen in indivdiuals with chronic kidney disease (CKD), a cohort study was performed evaluating pre- and post-dialysis levels of von Willebrand factor (VWF), VWF multimer profiles and levels of its cleaving protease, ADAMTS-13. There were 57 subjects (31 males, 26 females) enrolled with CKD with a mean age of 75 years (range 60 – 90). Subjects with known vascular disease were recruited; 49 (86%) had documented ischemic heart disease, 16 (29%) had cerebrovascular disease and 17 (31%) had peripheral vascular disease. A little over half had diabetes mellitus (30 subjects or 54%), 37 (67%) were on antiplatelet therapy and 7 (13%) were chronically anticoagulated with warfarin. Blood samples were drawn immediately pre- and again post-dialysis and all results were compared with a group of age-matched normal controls (Table 1). As has been previously reported, VWF antigen levels (VWF:Ag) and VWF functional activity as measured by the ristocetin cofactor assay (VWF:RCo) were higher in the pre-dialysis samples compared with controls, and both levels were increased even further following dialysis. Additionally, the percentage of high molecular weight VWF multimers (% HMWM) were significantly increased in the pre-dialysis samples compared with controls. This is a novel finding, and the level of % HMWM seen in the subjects is similar to what has been reported in individuals with Thrombotic Thrombocytopenic Purpura (TTP). This difference decreased following dialysis, potentially due to the effect of shear stress on VWF and the resultant proteolytic processing, however still remained significantly higher when compared with controls. ADAMTS-13 functional activity was lower in the subjects compared with controls, providing a possibly explanation for the increase in % HMWM. IL-6 levels are higher in subjects compared with controls. IL-6, which is an inflammatory cytokine known to be increased in patients with CKD, has been previously reported as a marker of inactivation of ADAMTS-13. Two years after enrollment, follow up of the subjects revealed that 22 had died, 17 from documented cardiovascular events. Higher VWF levels at the time of enrollment significantly correlated with risk of death (p=0.041) during the study period. The increase in % HMWM suggests that a “TTP-like phenotype” may also be playing a role. Taken together, these data suggest that both quantitative and qualitative abnormalities of VWF contribute to the risk of thrombotic death in chronic kidney disease. Disclosures: No relevant conflicts of interest to declare.

ADAMTS13 In 4 Different VWF/VIII Concentrates and Its Impact on Therapy.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3677-3677
Author(s):  
Mirjeta Qorraj ◽  
Tanja Falter ◽  
Sarah Steinemann ◽  
Thomas Vigh ◽  
Inge Scharrer
Keyword(s):  
Von Willebrand Factor ◽  
Gel Electrophoresis ◽  
Conflicts Of Interest ◽  
Von Willebrand Disease ◽  
Relative Reduction ◽  
Hemorrhagic Diathesis ◽  
Adamts13 Activity ◽  
Kinetic Measurements ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 3677 Introduction: The hemostatic activity of von Willebrand Factor (VWF) is mainly controlled by the plasma metalloprotease ADAMTS13, which cleaves ultralarge VWF multimers. A qualitative or quantitative deficiency of VWF induces the most common hemorrhagic diathesis, the von Willebrand Disease (VWD). The current classification graduates the VWD in three major types. Depending on severity and the type of VWD the treatment with VWF/FVIII concentrates may by necessary. The commercially available VWF/FVIII concentrates differ in their multimer structure and furthermore also in their pharmacokinetics. We investigated commercial VWF concentrates with respect to their ADAMTS 13 activity and antigen levels with the newest available methods. Moreover, to detect a possible correlation, we analysed the VWF multimer structure of the concentrates. Methods: We analysed 4 human derived VWF/VIII-concentrates (over all 7charges) after reconstitution according to the manufacturer's instructions in different dilutions. Following methods were used: BCS Method according to Böhm detects the capacity of the concentrates for autoproteolysis. The VWF solutions were diluted with 5mol/l urea and then incubated for 14–16h at 37°C in low ionic TRIS buffer containing BaCl2 and different plasma samples: pool plasma; plasma from patients with TTP with neutralizing ADAMTS13 auto-antibodies; plasma from patients with TTP without auto-antibodies. The residual VWF:Ristocetin Cofactor (VWF:RCo) activity was subsequently measured using the BC von Willebrand Reagent from Dade Behring. ELISA Technozym®ADAMTS13 and Actifluor TM ADAMTS13 are based on the kinetic measurements of the activity with fluorescence resonance energy transfer (FRET). ADAMTS13 antigen was measured by use of the Technozym ELISA kit. SDS-Gel electrophoresis in 1% Agarose Gel was used to investigate the structure of VWF multimers. Results: The BCS Method according to Böhm is an indirect measurement for endogenous ADAMTS13 activity in the investigated concentrate. Important is the loss of the residual VWF:RCo in the concentrates in presence of TTP-plasma without antibodies and pool plasma compared to the residual VWF:RCo in presence of TTP-plasma with antibodies. All concentrates show some ADAMTS13 activity, however product 1 contains more ADAMTS13 than the other concentrates. The results of the two FRETS-assays correspond very well to the BCS-method results; in addition the assays detect directly the ADAMTS13 activity also in very low measurement range. In a dilution of 16U VWF per ml concentrate the ADAMTS13 activity in product 1 with 4.3% was the highest compared to product 2: 3.2%, product 3: 2.6% and product 4: 2%. The great variability of the test results in higher concentrations may be caused by interferences between some constituents of the concentrates and the analysis. In the same sample set and dilution the ADAMTS13 antigen values correlate very well with ADAMTS13 activity values. The SDS gel electrophoresis reveals the different VWF structure of product1; it has less large and ultralarge multimers. There could be a correlation to the relatively higher ADAMTS13 activity and antigen level. Conclusion: All the investigated VWF/VIII concentrates contain some ADAMTS13 activity and antigen. This was found especially by FRETs assay due to the high sensitivity. Because of the correlation between ADAMTS13 activity and modified VWF multimer structure we like to conclude that ADAMTS13 has influence on stability and therefore also on quality of the concentrates. This might have a therapeutic consequence especially for VWD type 2A. Type 2A is characterized by a relative reduction of intermediate and large VWF multimer. The multimeric abnormalities are commonly the result of in vivo proteolytic degradation of the von Willebrand factor caused by ADAMTS13. Disclosures: No relevant conflicts of interest to declare.

Reduced Von Willebrand Factor Secretion Is Associated with Loss of Weibel-Palade Formation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 541-541
Author(s):  
Giancarlo Castaman ◽  
Sofia Helene Giacomelli ◽  
Paula M. Jacobi ◽  
Tobias Obser ◽  
Reinhard Schneppenheim ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Conflicts Of Interest ◽  
Negative Impact ◽  
Von Willebrand Disease ◽  
Hek293 Cells ◽  
Wild Type ◽  
And Function ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 541 Background. Von Willebrand Disease (VWD) is caused by mutations in von Willebrand factor (VWF) that have different pathophysiologic effect in causing low plasma VWF levels. Type 1 VWD includes patients with quantitative plasma VWF deficiency with normal VWF structure and function. Aim of the study. We report three different novel type 1 VWF mutations (A1716P, C2190Y and R2663C) which although located in different VWF domains are associated with reduced secretion and lack of formation of Weibel-Palade body-like granules. Methods. Transient expression of recombinant mutant full-length VWF in 293 EBNA cells was performed and secretion, collagen binding, and GpIb binding assessed in comparison to wild-type VWF. Furthermore, expression was also examined in HEK293 cells that form Weibel-Palade body (WPB)-like granules when transfected with wt VWF. Results. The multimer analysis of plasma VWF was compatible with type 1 VWD. The results of 3 different expression experiments showed a slightly reduced VWF synthesis and drastically impaired secretion into the medium with homozygous expression. In HEK293 cells, homozygous A1716P and C2190Y VWF variants failed to form WPB-like granules, while R2663C was capable of forming granules, but had fewer cells with granules and more with ER-localized VWF. Heterozygous expression of A1716P and C2160Y VWF variants had a negative impact on wild-type VWF and WPB-like granules were observed in transfected cells. Conclusions. Our results demonstrate that homozygous and heterozygous quantitative VWF deficiency caused by missense VWF mutations can be associated with inability to form endothelial Weibel-Palade-like granules and mutations in different VWF domains can affect the formation of these organelles. Disclosures: No relevant conflicts of interest to declare.

A Novel Mutation of ADAMTS13 Gene in Congenital Thrombotic Thrombocytopenic Purpura

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4674-4674
Author(s):  
Xia Bai ◽  
Jian Su ◽  
Minghua Jiang ◽  
Zhaoyue Wang ◽  
Changgeng Ruan
Keyword(s):  
Conflicts Of Interest ◽  
Novel Mutation ◽  
Gene Mutations ◽  
Type I ◽  
Adamts13 Activity ◽  
Intron Boundary ◽  
Congenital Thrombotic Thrombocytopenic Purpura ◽  
Peripheral Leukocytes ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 4674 Congenital thrombotic thrombocytopenic purura (TTP) is caused by gene mutations of von Willebrand factor-cleaving protease (a disintegrin and metalloprotease with thrombospondin type I domains 13, ADAMTS13). In this study, one novel mutation in the ADAMTS13 gene was found in a woman whose presents are first cousins. Thrombocytopenia occurred during the second trimesters in her first pregnancy, and she died of recurrent attacks after diagnosis of TTP. The ADAMTS13 activity measured using the recombinant FRET-VWF73 during her acute episode was less than 5%. ADAMTS13 inhibitor was negative measured by 9:1 mixture of patient and pooled normal plasma followed by ADAMTS13 activity assay using the VWF multimer electrophoresis. The 29 exons and exon-intron boundary sites of ADAMTS13 gene was analyzed using the human genomic DNA extracted from peripheral leukocytes of the patient. The results demonstrated she was homozygous for R498C. This novel mutant was constructed using the expression plasmid pSectag containing ADAMTS13 cDNA, and the vector was introduced by linpofectamine 2000 to Hela cells. Western Blot revealed that rADAMTS13-wide type (WT) was synthesized as a single band with molecular mass close to 190 Kda in the conditioned media, however, no detectable ADAMTS13 of this mutant existed. The lysates of cells expressing the mutant showed the same protein amounts compared to the rADAMTS13-WT. The immunofluorescence study demonstrated that mutant had the same localization pattern at Endoplasmic Reticulum(ER)and Golgi-compartments compared to the rADAMTS13-WT. The results imply that this mutant may be retained in the cellular ER and Golgi-comparments, but rapidly degraded or insufficiently secreted. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Extensive contacts between ADAMTS13 exosites and von Willebrand factor domain A2 contribute to substrate specificity

Blood ◽  
2008 ◽  
Vol 112 (5) ◽  
pp. 1713-1719 ◽  
Author(s):  
Weiqiang Gao ◽  
Patricia J. Anderson ◽  
J. Evan Sadler
Keyword(s):  
Von Willebrand Factor ◽  
Plasma Proteins ◽  
Scissile Bond ◽  
Substrate Cleavage ◽  
Α Helix ◽  
Substrate Binding Sites ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor ◽  
Specific Distance

Abstract The metalloprotease ADAMTS13 efficiently cleaves only the Tyr1605-Met1606 bond in the central A2 domain of multimeric von Willebrand factor (VWF), even though VWF constitutes only 0.02% of plasma proteins. This remarkable specificity depends in part on binding of the noncatalytic ADAMTS13 spacer domain to the C-terminal α-helix of VWF domain A2. By kinetic analysis of recombinant ADAMTS13 constructs, we show that the first thrombospondin-1, Cys-rich, and spacer domains of ADAMTS13 interact with segments of VWF domain A2 between Gln1624 and Arg1668, and together these exosite interactions increase the rate of substrate cleavage by at least approximately 300-fold. Internal deletion of Gln1624-Arg1641 minimally affected the rate of cleavage, indicating that ADAMTS13 does not require a specific distance between the scissile bond and auxiliary substrate binding sites. Smaller deletions of the P2-P9 or the P4′-P18′ residues on either side of the Tyr1605-Met1606 bond abolished cleavage, indicating that the metalloprotease domain interacts with additional residues flanking the cleavage site. Thus, specific recognition of VWF depends on cooperative, modular contacts between several ADAMTS13 domains and discrete segments of VWF domain A2.

scholarly journals Inflammation Drives Coagulopathies in Sars-Cov-2 Patients

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 34-35
Author(s):  
Martha MS Sim ◽  
Meenakshi Banerjee ◽  
Melissa Hollifield ◽  
Hammodah Alfar ◽  
Xian Li ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Conflicts Of Interest ◽  
Similar Process ◽  
Aids Patients ◽  
Thrombotic Risk ◽  
S Protein ◽  
Von Willebrand ◽  
Willebrand Factor

Background:A hypercoagulable state has been consistently reported in patients with severe Coronavirus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), characterized by elevated D-dimer, prolonged PT, and mild thrombocytopenia, though the mechanism is unclear. We have previously shown that human immunodeficiency virus (HIV) infection causes depletion of the anticoagulant protein S and virus-mediated platelet activation. Based on early reports, we hypothesized that a similar process contributed to COVID-19-associated thrombosis. Aim:To probe platelet activation and coagulation factor activity in SARS-CoV-2-infected patients. Methods:Blood was collected from consenting patients with differing COVID-19 severity: outpatients (15), hospitalized inpatients (15), and healthy controls (8). Platelet-leukocyte aggregate (PLA) formation and monocyte profiling were measured by flow cytometry. Coagulation factors were assessed by enzymatic assays. PS, von Willebrand Factor (vWF), PC, cytokines, and anti-S-Protein (viral spike protein) IgG were measured by ELISAs. Results:Ninety percent of SARS-CoV-2+ out-patients and in-patients had circulating anti-S-Protein IgG, but plasma IL-6 and TNFα were only elevated in three in-patients, consistent with reports that systemic inflammation is relatively rare in this population. Immune response did not correlate with disease severity. Unlike in HIV1+/AIDS patients, total PS was not reduced in SARS-CoV-2+ patients. However, the anticoagulant pool of PS ("free PS") was reduced in plasma samples from in-patients compared to controls (47.2%±23.3% vs. 100.8±42.6%, p=0057), while out-patients had an intermediate concentration (73.1%±28.9%). Specific loss of free PS is likely mediated by an increase in C4-binding protein (C4bp), which binds PS. In-patients also had a trend toward elevated plasma tissue factor (TF) compared to controls (79.5±121.4 fM vs. 37.8±39.7 fM, p = 0.32). Endothelial cells and monocytes can express TF under inflammatory conditions. We evaluated endothelial damage and dysfunction by measuring E-Selectin, which was unchanged in either in-patients or out-patients, and von Willebrand Factor (vWF), which was elevated in in-patients compared to controls (143±29.8 ng/mL vs. 56.2±41.9 ng/mL, p=0.0023). Plasma from in-patients also had elevated myeloperoxidase (524±187 ng/mL vs. 127±35 ng/mL, p=0.0026) and had a trend toward increased platelet-leukocyte aggregates (14.6±11.7% vs. 5.2±3.7%, p=0.24), indicating platelet and leukocyte stimulation. Unlike in the HIV1+/AIDS patients, no virus was detectable in any of the SARS-CoV-2+ patient plasmas. Consistent with a lack of direct platelet-virus interaction, plasma PF4 and platelet Akt phosphorylation were unchanged in the patient samples. We also observed a trend toward increased TF on TF+/CD64+/CD11b+ monocytes from in-patients compared to controls (MFI = 3244±2340 vs. 1741±382, p=0.18). Two inpatients were followed until they were SARS-CoV-2-negative. In both, PLAs, IL-6, vWF, and plasma TF remained elevated out to 28 days and PS remained reduced, suggesting that hemostatic dysregulation persists after SARS-CoV-2 is undetectable. Conclusions:We propose that localized inflammation in SARS-CoV-2+ patients results in a decrease in anticoagulant PS, through a shift of the free and C4bp-bound forms. At the same time, this inflammation causes stimulation of endothelial cells, which secrete procoagulant vWF, monocytes, which express TF and release it into plasma on microvesicles, and platelets, which form platelet-leukocyte aggregates. These changes may not return to baseline post-infection, suggesting that long-term monitoring of thrombotic risk may be necessary for SARS-CoV-2+ patients. Disclosures No relevant conflicts of interest to declare.

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