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.

scholarly journals Gain-of-function ADAMTS13 variants that are resistant to autoantibodies against ADAMTS13 in patients with acquired thrombotic thrombocytopenic purpura

Blood ◽  
2012 ◽  
Vol 119 (16) ◽  
pp. 3836-3843 ◽  
Author(s):  
Cui Jian ◽  
Juan Xiao ◽  
Lingjie Gong ◽  
Christopher G. Skipwith ◽  
Sheng-Yu Jin ◽  
...  
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Specific Activity ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Gain Of Function ◽  
Thrombocytopenic Purpura ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Thrombotic thrombocytopenic purpura (TTP) is primarily caused by immunoglobulin G (IgG) autoantibodies against A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats, 13 (ADAMTS13). Nearly all adult idiopathic TTP patients harbor IgGs, which bind the spacer domain of ADAMTS13, a region critical for recognition and proteolysis of von Willebrand factor (VWF). We hypothesize that a modification of an exosite in the spacer domain may generate ADAMTS13 variants with reduced autoantibody binding while preserving or enhancing specific activity. Site-directed mutagenesis was used to generate a series of ADAMTS13 variants, and their functional properties were assessed. Of 24 novel ADAMTS13 variants, 2 (ie, M4, R660K/F592Y/R568K/Y661F and M5, R660K/F592Y/R568K/Y661F/Y665F) exhibited increased specific activity approximately 4- to 5-fold and approximately 10- to 12-fold cleaving a peptide VWF73 substrate and multimeric VWF, respectively. More interestingly, the gain-of-function ADAMTS13 variants were more resistant to inhibition by anti-ADAMTS13 autoantibodies from patients with acquired idiopathic TTP because of reduced binding by anti-ADAMTS13 IgGs. These results shed more light on the critical role of the exosite in the spacer domain in substrate recognition. Our findings also help understand the pathogenesis of acquired autoimmune TTP. The autoantibody-resistant ADAMTS13 variants may be further developed as a novel therapeutic for acquired TTP with inhibitors.

Gain-of-Function ADAMTS13 Variants Resistant to Anti-ADAMTS13 Autoantibody Inhibition: A Potential Novel Therapy for Acquired Autoimmune Thrombotic Thrombocytopenic Purpura

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1188-1188
Author(s):  
Cui Jian ◽  
Juan (Jenny) Xiao ◽  
Lingjie Gong ◽  
Christopher G. Skipwith ◽  
Sheng-Yu Jin ◽  
...  
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Conflicts Of Interest ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Binding Interaction ◽  
Novel Therapy ◽  
Thrombocytopenic Purpura ◽  
Von Willebrand ◽  
First Time ◽  
Willebrand Factor

Abstract Abstract 1188 Spacer domain of ADAMTS13 is essential for recognition and proteolytic cleavage of von Willebrand factor. It also contains major antigenic epitope targeted by anti-ADAMTS13 autoantibodies in patients with acquired (idiopathic) thrombotic thrombocytopenic purpura (TTP). Through site-directed mutagenesis and recombinant protein strategy, we identified two novel ADAMTS13 variants, which exhibited enhanced specific activity, but reduced inhibition by anti-ADAMTS13 autoantibodies. Among twenty-three ADAMTS13 variants tested, ADAMTS13-M4 (R660K/F592Y/R568K /Y661F) and ADAMTS13-M5 (R660K/F592Y/R568K /Y661F/Y665F) exhibited increased specific activity by 4∼5 fold and 12∼16 fold in cleavage of FRETS-VWF73 and plasma-derived multimeric VWF under denaturing conditions, respectively. Moreover, these two novel ADAMTS13 variants (i.e. M4 and M5) were resistant to inhibition by anti-ADAMTS13 IgG autoantibodies from 10 out of 12 acquired TTP patients tested. In contrast, proteolytic activity of wild-type ADAMTS13 and several other mutants (i.e. M1: R660K, M2: R660K/F592Y, and M3: R660K/F592Y/R568K) were significantly inhibited by same amount of anti-ADAMTS13 IgGs in all 12 patients' plasmas. The resistance to inhibition of these variants was the result of reduced binding interaction between the ADAMTS13 variants (M4 and M5) and the anti-ADAMTS13 IgGs. These findings demonstrate for the first time that an exosite modification in the spacer domain may be a viable strategy for identification of novel ADAMTS13 variants with more desirable enzymatic properties. The results provide a potential tool for treatment of acquired (autoimmune) TTP and perhaps other arterial thrombotic disorders. Disclosures: No relevant conflicts of interest to declare.

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.

Oxidation of Key Methionine Residues in the N-Terminal Domains of ADAMTS13 Correlates with Loss of Its Proteolytic Activity,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3276-3276
Author(s):  
Yi Wang ◽  
Junmei Chen ◽  
Minhua Ling ◽  
José A. López ◽  
Dominic W Chung ◽  
...  
Keyword(s):  
Proteolytic Activity ◽  
Von Willebrand Factor ◽  
Hypochlorous Acid ◽  
Conflicts Of Interest ◽  
Human Neutrophils ◽  
Adamts13 Activity ◽  
Oxidative Inactivation ◽  
Rich Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 3276 Deficiency of ADAMTS13 is associated with a life-threatening thrombotic microangiopathic disease—thrombotic thrombocytopenic purpura (TTP), which is characterized by the accumulation of aggregates of hyper-reactive ultra-large von Willebrand factor (VWF) and platelets in the microvasculature. In addition to TTP, systemic inflammatory syndromes such as acute systemic inflammation caused by endotoxin, acute pancreatitis, and severe sepsis demonstrate reduced ADAMTS13 activity. The cause of low ADAMTS13 activity is not known. One possible cause is oxidative inactivation of the enzyme by hypochlorous acid (HOCl), a potent oxidant released from activated neutrophils that is known to damage proteins. In this study, we exposed ADAMTS13 to HOCl produced by a myeloperoxidase (MPO)-H2O2-Cl− system and measured the proteolytic activity of oxidized ADAMTS13 using a small VWF A2 peptide and plasma VWFas substrates. ADAMTS13 activity decreased as a function of oxidant concentration. Treatment with 25 nM MPO plus 50 μM H2O2 reduced ADAMTS13 activity by over 85%. Such concentrations of MPO and H2O2 are routinely found in vivo at sites of inflammation. ADAMTS13 contains a series of structural domains: a metalloprotease domain (M), a disintegrin-like domain (D), a thrombospondin type 1 repeat (TSP1,T), a Cys-rich domain (C), a spacer domain (S), 7 additional TSP1 repeats, and 2 CUB domains. The MDTCS domains are essential for its proteolytic activity, while the C-terminal TSP1 and CUB domains may act as modulators. The MDTCS region contains 10 Met residues, Met being the amino acid residue most vulnerable to oxidation by HOCl. Using mass spectrometry, we identified 7 Met-containing peptides in this region after proteolytic digestion of ADAMTS13. Three of these 7 methionines are highly sensitive to oxidation by HOCl, including M249, M331 and M496. M249 is situated in the ”Met-turn” at the catalytic center of the metalloprotease domain. M331 and M496 are located in the disintegrin-like domain and the Cys-rich domain, respectively. The extent of oxidation of these Met residues was proportional to the HOCl concentration, and strongly correlated with loss of enzymatic activity. The same three Met residues were also oxidized after exposure of ADAMTS13 to activated human neutrophils. These observations suggest an oxidative mechanism for ADAMTS13 inactivation in systemic inflammatory syndromes and that the oxidation-sensitive Mets may serve as biomarkers for this effect. Coupled with our earlier observation that HOCl oxidation of von Willebrand factor enhances its adhesive function and renders it resistant to cleavage by ADAMTS13, these findings indicate that pathological situations during which neutrophils are activated produce extremely prothrombotic conditions, perhaps explaining why many inflammatory syndromes are associated with thrombosis in small and large blood vessels. Disclosures: No relevant conflicts of interest to declare.

Levels of von Willebrand factor and ADAMTS13 determine clinical outcome after cardioversion for atrial fibrillation

2011 ◽  
Vol 105 (03) ◽  
pp. 435-443 ◽  
Author(s):  
Veronika Bruno ◽  
Rudolf Jarai ◽  
Susanne Gruber ◽  
Thomas Höchtl ◽  
Ivan Brozovic ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Independent Predictor ◽  
Thrombus Formation ◽  
Critical Role ◽  
Inverse Correlation ◽  
Von Willebrand ◽  
Rhythm Stability ◽  
Willebrand Factor

SummaryVon Willebrand factor (vWF) plays an essential role in platelet adhesion and thrombus formation. Patients with atrial fibrillation (AF) exhibit higher plasma vWF and lower ADAMTS13 antigen levels compared to controls. Little is known about vWF and ADAMTS13 in AF patients treated with cardioversion (CV). Thus we investigated the alterations of plasma vWF and ADAMTS13 after CV and evaluated the predictive value of these parameters for recurrence of AF. In this observational study we determined plasma levels of vWF and ADAMTS13 in 77 patients before and immediately after CV, as well as 24 hours (h) and six weeks thereafter, by means of commercially available assays. The vWF/ ADAMTS13-ratio was significantly elevated immediately after CV (p=0.02) and 24 h after CV (p=0.002) as compared to baseline levels. ADAMTS13, 24 h after CV, exhibited a significant association with recurrence of AF (HR: 0.97; p=0.037). Accordingly, tertiles of ADAMTS13 showed a stepwise inverse correlation with the risk of recurrent AF (HR: 0.50; p=0.009). After adjustment for confounders, ADAMTS13 remained significant as an independent predictor of recurrent AF (HR: 0.61; p=0.047). Similarly, the vWF/ADAMTS13-ratio, 24 h after CV, was associated with rhythm stability and remained an independent predictor of recurrent AF (HR: 1.88; p=0.028). The regulation of vWF and its cleaving protease ADAMTS13 after CV might play a critical role in producing a pro-thrombotic milieu immediately after CV for AF. Since ADAMTS13 plasma concentration and the vWF/ADAMTS13-ratio are independently associated with rhythm stability, these indexes might be used for prediction of recurrence of AF.

scholarly journals Asialo-von Willebrand factor inhibits platelet adherence to human arterial subendothelium: discrepancy between ristocetin cofactor activity and primary hemostatic function

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1084-1089 ◽  
Author(s):  
JB Lawrence ◽  
HR Gralnick
Keyword(s):  
Von Willebrand Factor ◽  
Whole Blood ◽  
Specific Activity ◽  
Human Platelets ◽  
Primary Hemostasis ◽  
Platelet Adherence ◽  
Wall Shear ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor

Abstract Platelet adherence at high wall shear rates requires plasma von Willebrand factor (vWF). Clinically, the ristocetin cofactor (RCof) activity is the only widely available assay for vWF function. When purified vWF is treated with neuraminidase to yield asialo-vWF (AS- vWF), its RCof activity is increased by 20% to 40%. AS-vWF binds to normal human platelets independently of ristocetin and induces platelet aggregation in the presence of fibrinogen. To determine whether AS-vWF also shows an enhanced capacity to support platelet adherence to subendothelium, we used the Baumgartner technique. Intact vWF, AS-vWF, or AS-vWF treated with beta-galactosidase (asialo, agalacto-vWF; AS,AG- vWF) was added to normal citrated whole blood before perfusion over human umbilical artery segments (wall shear rate, 2,600 sec-1). Four micrograms per milliliter AS-vWF caused a 69% reduction in total platelet adherence compared with citrated whole blood (P less than .001), and 4 micrograms/mL AS,AG-vWF led to a 48% reduction (P less than .005). With 4 micrograms/mL intact vWF, the platelet adherence values were not significantly different from the controls. No significant differences in subendothelial platelet thrombi or postperfusion platelet counts were evident among any of the groups. In reconstituted afibrinogenemic perfusates, 4 micrograms/mL AS-vWF caused a 42% reduction in platelet adherence (P less than .05). Thus, AS-vWF is a potent inhibitor of platelet adherence, despite its enhanced RCof specific activity. Abnormalities in vWF carbohydrate may play a role in impaired primary hemostasis in some patients with von Willebrand's disease.

Identification and analysis of copine/BONZAI proteins among evolutionarily diverse plant species

Genome ◽  
2016 ◽  
Vol 59 (8) ◽  
pp. 565-573 ◽  
Author(s):  
Baohong Zou ◽  
Xuexue Hong ◽  
Yuan Ding ◽  
Xiang Wang ◽  
He Liu ◽  
...  
Keyword(s):  
Plant Species ◽  
Protein Localization ◽  
Calcium Dependent ◽  
Functional Studies ◽  
Phylogeny Analysis ◽  
Genome Wide ◽  
Evolutionarily Conserved ◽  
Von Willebrand ◽  
Diverse Plant Species ◽  
Willebrand Factor

Copines are evolutionarily conserved calcium-dependent membrane-binding proteins with potentially critical biological functions. In plants, the function of these proteins has not been analyzed except for in Arabidopsis thaliana where they play critical roles in development and disease resistance. To facilitate functional studies of copine proteins in crop plants, genome-wide identification, curation, and phylogeny analysis of copines in 16 selected plant species were conducted. All the identified 32 plant copines have conserved features of the two C2 domains (C2A and C2B) and the von Willebrand factor A (vWA) domain. Different from animal and protozoa copines, plant copines have glycine at the second residue potentially acquiring a unique protein myristoylation modification. Phylogenetic analysis suggests that copine was present as one copy when evolving from green algae to basal flowering plants, and duplicated before the divergence of monocots and dicots. In addition, gene expression and protein localization study of rice copines suggests both conserved and different properties of copines in dicots and monocots. This study will contribute to uncovering the role of copine genes in different plant species.

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.

A Novel Platelet Small-Molecule Agonist Targeting Glycoprotein Ibalpha.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4010-4010
Author(s):  
Jianfeng Yang ◽  
Zhi Chen ◽  
Weiliang Zhu ◽  
Changgeng Ruan
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Small Molecule ◽  
Platelet Adhesion ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Structural Basis ◽  
Terminal Fragment ◽  
Von Willebrand

Abstract Abstract 4010 Poster Board III-946 Introduction Interaction of glycoprotein (GP) Ibα with Von Willebrand factor (VWF) plays a critical role in platelet adhesion and signal transduction for αIIbβ3 activation under condition of high shear stress. Methods Based on the crystal structure of platelet GPIbα (PDB:1P9A), virtual screening was employed to identify active compounds. Compounds in SPECS database were docked to VWF binding site on the surface of GPIbα. The screening was carried out with the DOCK4.0 program. The 150 highest-scoring compounds were obtained for further bioassay and those with inhibitory activity of VWF binding to GPIbα were investigated the effect on platelet activation and aggregation. Results We found one compound, designated it as YC148, blocked ristocetin-induced plasma VWF binding to recombinant N-terminal fragment GPIbα (H1-V289) by ELISA method. More interestingly, YC148 did not inhibit ristocetin-induced platelet aggregation, on the contrary, it induced platelet aggregation itself in the absence of exogenous modulators such as ristocetin and botrocetin. A VWF A1 blocking antibody could not block platelet aggregation induced by YC148 despite it completely inhibited ristocetin-induced platelet agglutination. And YC148 also stimulated washed platelet aggregation where VWF was absent in the resuspension buffer. These indicated that the aggregation stimulated by YC148 could not the result from VWF binding. Flow cytomety also showed that YC148 increased P-selectin expression on platelet membrane and promoted monoclonal antibody PAC-1 binding to platelet. The platelet aggregation stimulated by YC148 was inhibited by anti-GPIbα monoclonal antibody AN51 and 6D1. Conclusion A novel exogenous small-molecule agonist was found to activate platelet through binding to GPIbα. It provides us a new tool for investigating platelet GPIb outside-in signaling pathway in platelet adhesion and aggregation. Furthermore, the structure of YC148 may provide a structural basis for developing new hemostatic drugs based on the inhibition of VWF-GPIb interaction. The effect of YC148 on platelet from Bernard-Soulier syndrome or GPIbα N-terminal fragment deficient platelet after in vitro cleavage will be further investigated. Disclosures: No relevant conflicts of interest to declare.

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